CN116457472A - Rna核酸适配体传感器 - Google Patents

Rna核酸适配体传感器 Download PDF

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CN116457472A
CN116457472A CN202180070122.6A CN202180070122A CN116457472A CN 116457472 A CN116457472 A CN 116457472A CN 202180070122 A CN202180070122 A CN 202180070122A CN 116457472 A CN116457472 A CN 116457472A
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莱恩·弗拉驰
里贾纳·马赫
格奥尔格·肖曼
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Sens Epp Co ltd
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Abstract

本发明涉及一种用于优化细胞中感兴趣的RNA序列的产生的方法，包括以下步骤：a)将能够表达感兴趣的异源RNA的载体引入多个细胞，其中所述的RNA用RNA标签标记，所述的RNA标签包含能够稳定荧光团的核酸适配体和能够稳定核酸适配体的支架；b)在允许感兴趣的异源RNA表达的条件下，在培养基中培养细胞；c)将所述的荧光团加入所述的培养基中；以及d)鉴定那些显示出最高荧光强度并因此显示出感兴趣的RNA的高表达的细胞。

Description

RNA核酸适配体传感器
技术领域
本发明涉及用于优化异源RNA在细胞中表达的方法。同样，本发明涉及能够定量表达RNA的细胞。
背景技术
近年来的研究使人们意识到，RNA不仅仅是一种过渡分子，它还具有多种调节和酶促功能。例如，已经描述了许多小RNA在基因表达的调节中起关键作用，并且RNA序列中的高阶结构，如核糖开关或核酶，充当mRNA表达的调节因子(Breaker，R.R.Natural andengined nucleaned acids as tools to explored biology.Nature 2004:432838-845.Nellen，W.,and C.Hammann.Small RNAs:analysis and regulatoryfunctions.Nucleic acids and molecular biology.Springer-Verlag2005,Heidelberg,Germany)。基于其作为过渡分子(mRNA)、人工小干扰RNA分子(siRNAs)、催化活性RNA分子(核酶)、调节或相互作用RNA分子(RNA核酸适配体)的功能，RNA在治疗、生物农药和其他应用中具有作为活性成分的多用途潜力(Khan，A.U.Ribozyme:a clinictool.Clin.Chim.Acta 2006:367,20-27.Fletcher,S.J.,Reeves,P.T.,Hoang,B.T.&Mitter,N.A基于RNAi的生物农药展望.Front.Plant Sci.11,(2020).Cagliari,D.et al.非转化RNAi对害虫和植物病害的管理.Frontiers in Plant Science vol.10(2019).Zotti,M.et al.RNA干扰技术在作物防治节肢动物害虫、病原体和线虫中的应用.PestManagement Science vol.74 1239-1250(2018).Vallazza,B.et al.重组信使RNA技术及其在癌症免疫治疗、转录替代治疗、多能干细胞诱导等方面的应用.WileyInterdisciplinary Reviews:RNAvol.6 471 -499(2015).Sahin,U.,Karikó,K.&Türeci,O.基于信使核糖核酸的疗法开发一类新药.Nature Reviews Drug Discovery vol.13759-780(2014).Pardi,N.,Hogan,M.J.&Weissman,D.信使核糖核酸疫苗技术的最新进展.Current Opinion in Immunology vol.65 14-20(2020).)。为了进行进一步阐明这些功能所需的测定，并为了提供用于应用的基于RNA的活性成分，需要能够产生大量给定RNA的技术。
产生RNA最直接的方法之一是体外转录RNA。这种方法是基于对所有生命形式中控制RNA合成的酶过程的模仿。用于此目的的一个常见***是T7 RNA聚合酶，它从DNA模板开始，可以在几个小时的反应时间内产生高达毫克量的RNA。然而，并不是所有的DNA序列都同样适合通过T7聚合酶进行转录。此外，在3'末端非特异性添加核苷酸的问题导致了不均匀性，这在检查调节功能时可能是至关重要的。此外，这种方法也是劳动密集型的。此外，体外转录反应的可扩展性以及给定RNA的最大可实现量是有限的，这使得在合理的时间内大量提供给定RNA具有挑战性且成本高昂。
化学合成目前通常用于生产小于10个核苷酸和最多80个核苷酸的RNA寡核苷酸。合成是在固体载体如聚苯乙烯或可控孔玻璃上进行的，并涉及在3’至5’方向上添加相应的核苷酸。由于反应条件可以优化，化学合成可以产生任何给定的RNA，而不考虑其序列。然而，该方法本身不适合生产具有超过100个核苷酸的较大RNA分子，并且成本也很高。
相反，大RNA分子可以通过重组表达***有效地产生，类似于重组蛋白的工业生产。大肠杆菌已被用于这一目的，但并非没有困难：细胞内核糖核酸酶的降解、转录物的大的3′端和5′端异质性以及低RNA滴度阻碍了广泛的应用(Ponchon L，DardelF.Recombinant RNA technology:the tRNA scaffold.Nat Methods.2007；4:571-6.)。替代宿主可能提供更好的解决方案(Suzuki H，Ando T,Umekage S,Tanaka T,KikuchiY.Extracellular production of an RNA aptamer by ribonuclease-free marinebacteria harboring engineered plasmids:a proposal for industrial RNA drugproduction.Appl Environ Microbiol.2010；76:786-93.)。然而，到目前为止，它们尚未开发到为现有***提供可行替代品的阶段(Baronti，L.,Karlsson,H.,M.et al.Aguide to large-scale RNA sample preparation.Anal Bioanal Chem.2018:410,3239-3252.)。
发明内容
为了优化重组RNA表达***，需要一种快速可靠的方法来鉴定对给定RNA分子表现出高表达的细胞。
该问题通过一种用于优化细胞中感兴趣的异源RNA序列的生产的方法来解决，该方法包括以下步骤：
a)将能够表达感兴趣的异源RNA的载体引入多个细胞中，其中所述的RNA用RNA标签标记，所述的RNA标签包含
-能够稳定荧光团的核酸适配体，以及
-能够稳定所述的核酸适配体的支架；
b)在允许感兴趣的异源RNA表达的条件下，在培养基中培养细胞；
c)将所述的荧光团加入所述的培养基中；
d)鉴定那些显示出最高荧光强度并因此显示出感兴趣的RNA的高表达的细胞。
同样，该问题通过一种生产感兴趣的异源RNA的方法来解决，该方法包括以下步骤：
a)将能够表达感兴趣的异源RNA的载体引入多个细胞，其中所述的RNA用RNA标签标记，所述的RNA标签包含能够稳定荧光团的核酸适配体和能够稳定所述的核酸适配体的支架；
b)在允许感兴趣的异源RNA表达的条件下，在培养基中培养细胞；
c)将所述的荧光团加入所述的培养基中；
d)鉴定和分离那些显示出最高荧光强度并因此显示出感兴趣的RNA的高表达的细胞；
e)从在步骤d)中分离的细胞中去除步骤a)的第一载体；
f)将能够在没有RNA标签的情况下表达感兴趣的异源RNA的第二载体引入步骤e)获得的细胞中；
g)通过培养在步骤f)中获得的细胞来产生感兴趣的RNA。
该问题还通过一种用于比较不同细胞对感兴趣的异源RNA序列的生产能力的方法来解决，该方法包括以下步骤：
a)将能够表达感兴趣的异源RNA的载体引入多个细胞中，其中所述的RNA用RNA标签标记，所述的RNA标签包含
-能够稳定荧光团的核酸适配体，以及
-能够稳定所述的核酸适配体的支架；
b)在允许感兴趣的RNA表达的条件下，在培养基中培养细胞；
c)将所述的荧光团加入所述的培养基中；
比较所述的多个细胞之间的荧光强度。
此外，该问题通过携带能够表达感兴趣的异源RNA的载体的微生物细胞来解决，其中所述的RNA用RNA标签标记，所述的RNA标签包含
-能够稳定荧光团的核酸适配体，以及
-能够稳定核酸适配体的RNA支架。
附图说明
图1显示未经(左图)和经(右图)N-甲基-N’-硝基-N-亚硝基胍(MNNG)处理的谷氨酸棒状杆菌ATCC 13032Δcg2273 pJC1-PF1-U1A-F30::西兰花/UUCG-TF1的100000个代表性细胞的前向散射特性(x轴)和530nm荧光特性(y轴)的细胞仪采集。矩形表示用于分离高荧光细胞的分选门。(参见实施例1d)。
图2显示添加荧光团DFHBI后，谷氨酸棒状杆菌ATCC 13032_Δcg2273_16S rRNA-西兰花的荧光增加了x倍。所描述的荧光诱导通过细胞术分析记录为中值荧光(488ex/530-30em)，并通过菌株谷氨酸棒状杆菌ATCC 13032_Δcg2273_16S rRNA-西兰花的未染色培养物的中值荧光(48ex/530-30m)进行归一化。
图3显示通过逆转录定量PCR确定的与对照菌株谷氨酸棒状杆菌ATCC 13032_Δcg2273相比，菌株谷氨酸棒菌ATCC 13032_Δcg2273_16S rRNA-西兰花中F30::西兰花的相对转录水平。
图4显示添加荧光团DFHBI或TO1后，菌株谷氨酸棒状杆菌ATCC 13032_Δcg2273_pJC1_PF1-U1A-TF1，谷氨酸棒状杆菌ATCC 13032_Δcg2273_pJC1_PF1-U1A-F30::西兰花/UUCG-TF1和谷氨酸棒状杆菌ATCC 13032_Δcg2273_pJC1_PF1-U1A-F30::芒果3-TF1的荧光增加x倍。所描述的荧光诱导通过细胞术分析记录为中值荧光(488ex/530-30em)，并分别通过未染色培养物的中值荧光(488ex/530-30mm)进行归一化。
图5显示在T7启动子的控制下表达α微管蛋白正义RNA与F30::西兰花的融合以及相应的α微管蛋白反义RNA的DNA构建体的设计原理。此外，还显示所得dsRNA的方案和相应的序列长度。箭头表示RNase A的活性，导致图7中可见的片段，L3。
图6显示添加荧光团DFHBI后，菌株谷氨酸棒状杆菌ATCC 13032_Δcg2273_pJC1和谷氨酸棒状杆菌ATCC 13032_Δcg2273_pJC1_dsRNA_PT7-α微管蛋白-F30::西兰花的荧光增加了x倍。所描述的荧光诱导通过细胞术分析记录为中值荧光(488ex/530-30em)，并分别通过未染色培养物的中值荧光(488ex/530-30mm)进行归一化。
图7显示dsRNA梯(美国马萨诸塞州伊普斯维奇新英格兰生物实验室)，谷氨酸棒状杆菌ATCC 13032(DE3)_Δcg2273 pJC1总RNA(L1)，谷氨酸棒状杆菌ATCC 13032(DE3)_Δcg2273 pJC1_dsRNA_PT7-α微管蛋白-F30::西兰花总RNA(L2)以及用核糖核酸酶A处理的谷氨酸棒状杆菌ATCC 130322(DE3)_Δcg2273 pJC1_dsRNA_PT7-α微管蛋白-F30::西兰花总RNA(L3)的毛细管电泳分析的数字凝胶表示。在配备有DNF-471RNA试剂盒(安捷伦科技公司，加利福尼亚州圣克拉拉，美国)的片段分析仪***上进行分析。LM描述了DNF-471RNA试剂盒的较低标记物。
图8显示添加荧光团DFHBI后，菌株谷氨酸棒状杆菌ATCC 13032_Δcg2273_pJC1，谷氨酸棒状杆菌ATCC 13032_Δcg2273_pJC1_PT7-luc2(Et)-西兰花-T7和谷氨酸棒状杆菌ATCC 13032_Δcg2273_pJC1_PT7-egfp-西兰花-TT7的荧光增加了x倍。所描述的荧光诱导通过细胞术分析记录为中值荧光(488ex/530-30em)，并分别通过未染色培养物的中值荧光(488ex/530-30mm)进行归一化。
图9显示在1％琼脂糖凝胶上体内产生的RNA片段egfp-F30::西兰花和luc2-F30::西兰花。菌株谷氨酸棒状杆菌ATCC 13032(DE3)_Δcg2273_pJC1-PT7-egfp-西兰花-TT7和谷氨酸棒状杆菌ATC C 13032(DE3)_Δcg2273_pJC1-PT7-luc2-西兰花-TT7的制备RNA样品用于通过ProtoScriptII逆转录酶和合适引物将RNA逆转录为cDNA，然后通过OneTaq热启动DNA聚合酶扩增片段egfp-F30::西兰花和luc2-F30::西兰花(泳道3和6)。作为阳性对照，使用相同的DNA聚合酶和引物扩增制备的质粒DNA(泳道1和4)，作为阴性对照，使用同样的DNA聚合酶和引物扩增制得的RNA样品，而不将RNA逆转录成cDNA(泳道2和5)。
具体实施方式
如本文所用，术语“包括”和“包含”应理解为“包含/包含”和“组成/构成”。
在一个方面，本发明涉及一种用于优化细胞中感兴趣的异源RNA序列的生产的方法，包括以下步骤：
a)将能够表达感兴趣的异源RNA的载体引入多个细胞中，其中所述的RNA用RNA标签标记，所述的RNA标签包含
·能够稳定荧光团的核酸适配体以及
·能够稳定核酸适配体的支架；
b)在允许感兴趣的异源RNA表达的条件下，在培养基中培养细胞；
c)将所述的荧光团加入所述的培养基中；
d)鉴定那些显示出最高荧光强度并因此显示出感兴趣的RNA的高表达的细胞。
在本发明的上下文中，细胞可以是真核细胞或原核细胞。在一个实施方案中，所述的细胞是微生物细胞。微生物细胞或微生物可用于产生分子如DNA、RNA或蛋白质。它们可以分化为革兰氏阳性和革兰氏阴性微生物。优选地，根据本发明的微生物细胞是革兰氏阳性微生物细胞。在本发明的一个方面，根据本发明的微生物细胞来自棒状杆菌属，特别是谷氨酸棒状杆菌。根据本发明并用于本发明的细胞在本文中也称为宿主细胞。
首先，将能够表达感兴趣的异源RNA的载体引入细胞中。载体可以是适合于在宿主细胞中表达RNA的任何载体，例如质粒；病毒载体，例如逆转录病毒、慢病毒、腺病毒、腺相关病毒或Lambda噬菌体；或人工染色体，例如BAC、YAC或HAC。
本发明中使用的载体能够表达感兴趣的RNA。感兴趣的RNA的表达可以是组成的或有条件的。例如，可以通过添加乙酸盐、脱水四环素、***糖、葡萄糖酸盐、异丙基β-D-1-硫代吡喃半乳糖苷(IPTG)、光、麦芽糖、甲醇、丙酸盐或通过提高培养温度来诱导感兴趣的RNA的表达。
除了下面列出的元素之外，载体可以包括表达、分子克隆和复制所必需或增强的额外元件。例如，载体可以包含选择或标记基因，例如编码β-半乳糖苷酶的lacZ、编码荧光素酶的luc、编码氯霉素转移酶的cat或其他表达对抗生素耐药性的抗性基因。载体还可以包括复制起点、多克隆位点和/或转录终止子。
由载体表达的感兴趣的RNA是异源RNA，即在宿主细胞的野生型中不表达的RNA分子。感兴趣的RNA的序列不受限制，可以是任何天然或人工产生的RNA序列。在一个实施方案中，感兴趣的RNA是mRNA、病毒RNA、逆转录病毒RNA、反义RNA、复制子RNA、双顺反子或多顺反子RNA、小干扰RNA或免疫刺激RNA。在一个实施方案中，感兴趣的RNA具有20-10000个核苷酸的长度。
用RNA标签标记感兴趣的RNA。本文中的“标记”是指RNA标签与感兴趣的RNA连接，并且该标签与感兴趣的RNA一起表达。在从载体表达异源RNA时，不从感兴趣的RNA中去除RNA标签。根据本发明，标签不必直接连接到感兴趣的RNA，尽管这是优选的实施方案。但是RNA标签和感兴趣的RNA也可以通过核苷酸间隔序列分离。
本发明中使用的RNA标签包括核酸适配体。“核酸适配体”在本文中被理解为指能够结合小分子荧光团的RNA寡核苷酸。通常，核酸适配体是10-100个碱基的核酸寡核苷酸，其与小分子以高亲和力结合以诱导其荧光。在结合事件发生之前，核酸适配体和靶小分子都不是强荧光的，但核酸适配体与其靶分子的结合激活了靶分子的荧光。能够结合感兴趣分子的核酸适配体可以通过指数富集(SELEX)通过配体的***进化产生(Paige，J.S.，Wu，K.Y.and Jaffrey，S.R.RNA mimics of green fluorescent protein.Science；2011；333,642-6.)。文献中描述了各种核酸适配体(Ouellet，J.(2016)RNA fluorescence withlight-Up aptamers.Front.Chem.4,1-12.Bouhedda,F.,Autour,A.,and Ryckelynck,M.(2017)Light-Up RNA Aptamers and Their Cognate Fluorogens:From TheirDevelopment to Their Applications.Int.J.Mol.Sci.19,44.)。本发明中使用的优选核酸适配体序列包括：
SEQ ID NO:1
核酸适配体II-mini3-4:
AGGUUCGAAGCUUUUGCUUGGACGAACCG
SEQ ID NO:2
芒果:
GAAGGGACGGUGCGGAGAGGAGA
SEQ ID NO:3
芒果2:
GAAGGAGAGGAGAGGAAGAGGAGA
SEQ ID NO:4
芒果3:
GGAAGGAUUGGUAUGUGGUAUA
SEQ ID NO:5
芒果4:
CGAGGGAGUGGUGAGGAUGAGGCGA
SEQ ID NO:6
菠菜:
GACGCAACUGAAUGAAAUGGUGAAGGACGGGUCCAGGUGUGGCUGCUUCGGCA GUGCAGCUUGUUGAGUAGAGUGUGAGCUCCGUAACUAGUCGCGUC
SEQ ID NO:7
菠菜2:
GAUGUAACUGAAUGAAAUGGUGAAGGACGGGUCCAGUAGGCUGCUUCGGCAGC CUACUUGUUGAGUAGAGUGUGAGCUCCGUAACUAGUUACAUC
SEQ ID NO:8
西兰花:
AGACGGUCGGGUCCAGAUAUUCGUAUCUGUCGAGUAGAGUGUGGGCU
SEQ ID NO:13
玉米:
CGAGGAAGGAGGUCUGAGGAGGUCACUG
SEQ ID NO:14
橙色西兰花:
GAGACGCAACUGAAUGAAAUGGUGAAGGAGACGGUCGGGUCCAGGUGCACAAA UGUGGCCUGUUGAGUAGCGUGUGGGCUCCGUAACUAGUCGCGUCAC
SEQ ID NO:65
橙色西兰花短款:
AGACGGUCGGGUCCAGGUGCACAAAUGUGGCCUGUUGAGUAGCGUGUGGGCU
SEQ ID NO:15
红色西兰花:
GAGACGCAACUGAAUGAAAUGUUUUCGGAGACGGUCGGGUCCAGUCCCAACGA UGUUGGCUGUUGAGUAGUGUGUGGGCUCCGUAACUAGUCGCGUCAC
SEQ ID NO:66
红色西兰花短款:
AGACGGUCGGGUCCAGUCCCAACGAUGUUGGCUGUUGAGUAGUGUGUGGGCU
SEQ ID NO:16
嫩菠菜:
AAGGACGGGUCCGUUGAGUAGAGUGUGAG
本发明中使用的RNA标签中包含的核酸适配体能够结合荧光团，即在光激发时发出荧光的有机分子。发光强度基本上取决于荧光团与核酸适配体的结合，因为荧光团的结构在与核酸适配体结合时是稳定的。这种稳定性导致激发能优选地离解为荧光。这意味着根据本发明，荧光团的发光在与核酸适配体结合后强烈增加。
本发明中使用的荧光团是可以容易地进入细胞的无毒小分子。先前已经描述了能够与这些核酸适配体结合的核酸适配体和荧光团对。可被核酸适配体结合的荧光团包括(Z)-4-(2-羟基亚苄基)-1,2-二甲基-1H-咪唑-5(4H)-酮(2-HBI)，(5Z)-5-[(3,5-二氟-4-羟基苯基)亚甲基]-3,5-二氢-2,3-二甲基-4H-咪唑-4-酮(DFHBI)，(5Z)-5-[(3,5-二氟-4-羟基苯基)亚甲基]-3,5-二氢-2-甲基-3-(2,2,2-三氟乙基)-4H-咪唑-4-酮(DFHBI-1T)，DFHBI-2T，2-(4-(二甲基氨基)亚苄基)-1H-茚-1,3(2H)-二酮(DMABI)，(Z)-4-(3,5-二甲基-4-羟基亚苄基)-1,2-二甲基-1H-咪唑-5(4H)-酮(DMHBI)，3,5-二氟-4-羟基亚苄基咪唑啉-2-肟(DFHO)，2-(2-甲基苯并[d]噻唑-3-鎓-3-基)乙酸盐(2-(2-Methylbenzo[d]thiazol-3-ium-3-yl)acetate)(TO1)，4-[(E)-2-(乙酰基苯基氨基)乙烯基]-1-甲基喹啉碘化物(TO3)和(N-(6-氨基己基)-2-(2,6-二叔丁基-4-(5-(4-甲基哌嗪-1-基)-1H，1'H-2,5'-二苯并[d]咪唑-2'-基)苯氧基)乙酰胺)(Hoechst 1C)。
在优选的实施方案中，核酸适配体包含SEQ ID NO:1，并且荧光团是Hoechst 1C。在另一个优选实施方案中，核酸适配体包含SEQ ID NO:2，并且荧光团是TO1或TO3。在另一个优选实施方案中，核酸适配体包含SEQ ID NO:3，并且荧光团是TO1或TO3。在另一个优选实施方案中，核酸适配体包含SEQ ID NO:4，并且荧光团是TO1或TO3。在另一个优选实施方案中，核酸适配体包含SEQ ID NO:5，并且荧光团是TO1或TO3。在另一个优选实施方案中，核酸适配体包含SEQ ID NO:6，并且荧光团是2-HBI、DFHBI、DFHBI-1T、DFHBI-2T、DMABI或DMHBI中的任何一种。在另一个优选实施方案中，核酸适配体包含SEQ ID NO:7，并且荧光团是2-HBI、DFHBI、DFHBI-1T、DFHBI-2T、DMABI或DMHBI中的任何一种。在另一个优选实施方案中，核酸适配体包含SEQ ID NO:8，并且荧光团是2-HBI、DFHBI、DFHBI-1T、DFHBI-2T、DMABI或DMHBI中的任何一种。在另一个优选实施方案中，核酸适配体包含SEQ ID No:13、14、15、65或66，并且荧光团是DFHO。在另一个优选实施方案中，核酸适配体包含SEQ ID No:16，并且荧光团是DHFBI或DHFBI-1T。
本发明中使用的RNA标签进一步包括RNA支架。RNA支架可以是能够稳定核酸适配体、支持功能性核酸适配体结构的形成和减少核酸适配体降解的任何核苷酸序列。根据本发明的RNA支架包括至少一个由NNNN表示的***位点。在本发明中使用的载体中，NNNN被相应的核酸适配体取代。
在一个实施方案中，支架包括两个***位点，例如SEQ ID NO:11和12。根据本发明，可以将一个或两个核酸适配体***这样的支架中。如果***两个核酸适配体，则可以将相同或不同的核酸适配体***两个***位点。这方面的例子是SEQ ID NO:77和78。如果将相同的核酸适配体***两个***位点，则在添加荧光团之后发射的荧光信号将比仅存在一个核酸适配体时更强。使用两种不同的核酸适配体具有这样的优点，即表达感兴趣的RNA的细胞将发出两个不同的荧光信号。这可能有利于排除误报。
如果只有一个核酸适配体被***到具有两个***位点的支架中，则第二个***位点可以被任何间隔序列取代。在一个优选的实施方案中，间隔序列包括四个核苷酸。在一个特别优选的实施方案中，间隔序列是UUCG或TTCG。
在优选的实施方案中，RNA支架包含以下序列之一：
SEQ ID NO:9
tRNALys:
GCCCGGAUAGCUCAGUCGGUAGAGCAGNNNNCGGGUCCAGGGUUCAAGUCCCU GUUCGGGCGCCA
SEQ ID NO:10
V5:
UGCCUGGCGACCAUAGCGAUUGNNNNCAAUUAGCGCCGAUGGUAGUGUGGGGU UUCCCCAUGUGAGAGUAGGACAUCGCCAGGCAU
SEQ ID NO:11
F29:
UUGUCACGUGUAUGUGGGNNNNCCCACAUACUUUGUUGAUCCNNNNGGAUCAAUCAUGGCAA
SEQ ID NO:12
F30:
UUGCCAUGUGUAUGUGGGNNNNCCCACAUACUCUGAUGAUCCNNNNGGAUCAU UCAUGGCAA
在一个实施方案中，RNA标签包含SEQ ID NO:17至64，69，77或78中的任何一个。在一个优选的实施方案中，RNA标签包含SEQ ID NO:69。
SEQ ID NO:17
tRNALys::核酸适配体II-mini3-4
GCCCGGAUAGCUCAGUCGGUAGAGCAGAGGUUCGAAGCUUUUGCUUGGACGAA CCGCGGGUCCAGGGUUCAAGUCCCUGUUCGGGCGCCA
SEQ ID NO:18
tRNALys::芒果
GCCCGGAUAGCUCAGUCGGUAGAGCAGGAAGGGACGGUGCGGAGAGGAGACGGGUCCAGGGUUCAAGUCCCUGUUCGGGCGCCA
SEQ ID NO:19
tRNALys::芒果2
GCCCGGAUAGCUCAGUCGGUAGAGCAGGAAGGAGAGGAGAGGAAGAGGAGACG GGUCCAGGGUUCAAGUCCCUGUUCGGGCGCCA
SEQ ID NO:20
tRNALys::芒果3
GCCCGGAUAGCUCAGUCGGUAGAGCAGGGAAGGAUUGGUAUGUGGUAUACGGGUCCAGGGUUCAAGUCCCUGUUCGGGCGCCA
SEQ ID NO:21
tRNALys::芒果
4GCCCGGAUAGCUCAGUCGGUAGAGCAGCGAGGGAGUGGUGAGGAUGAGGCGA CGGGUCCAGGGUUCAAGUCCCUGUUCGGGCGCCA
SEQ ID NO:22
tRNALys::菠菜
GCCCGGAUAGCUCAGUCGGUAGAGCAGGACGCAACUGAAUGAAAUGGUGAAGGACGGGUCCAGGUGUGGCUGCUUCGGCAGUGCAGCUUGUUGAGUAGAGUGUGAGCUCCGUAACUAGUCGCGUCCGGGUCCAGGGUUCAAGUCCCUGUUCGGGCGCCA
SEQ ID NO:23
tRNALys::菠菜2
GCCCGGAUAGCUCAGUCGGUAGAGCAGGAUGUAACUGAAUGAAAUGGUGAAGGACGGGUCCAGUAGGCUGCUUCGGCAGCCUACUUGUUGAGUAGAGUGUGAGCUCCGUAACUAGUUACAUCCGGGUCCAGGGUUCAAGUCCCUGUUCGGGCGCCA
SEQ ID NO:24
tRNALys::西兰花
GCCCGGAUAGCUCAGUCGGUAGAGCAGAGACGGUCGGGUCCAGAUAUUCGUAU CUGUCGAGUAGAGUGUGGGCUCGGGUCCAGGGUUCAAGUCCCUGUUCGGGCGCCASEQ ID NO:25
tRNALys::玉米
GCCCGGAUAGCUCAGUCGGUAGAGCAGCGAGGAAGGAGGUCUGAGGAGGUCAC UGCGGGUCCAGGGUUCAAGUCCCUGUUCGGGCGCCA
SEQ ID NO:26
tRNALys::橙色西兰花
GCCCGGAUAGCUCAGUCGGUAGAGCAGAGACGGUCGGGUCCAGGUGCACAAAUGUGGCCUGUUGAGUAGCGUGUGGGCUCGGGUCCAGGGUUCAAGUCCCUGUUCGGGCGCCA
SEQ ID NO:27
tRNALys::红色西兰花
GCCCGGAUAGCUCAGUCGGUAGAGCAGAGACGGUCGGGUCCAGUCCCAACGAUGUUGGCUGUUGAGUAGUGUGUGGGCUCGGGUCCAGGGUUCAAGUCCCUGUUCGGGCGCCA
SEQ ID NO:28
tRNALys::嫩菠菜
GCCCGGAUAGCUCAGUCGGUAGAGCAGAAGGACGGGUCCGUUGAGUAGAGUGU GAGCGGGUCCAGGGUUCAAGUCCCUGUUCGGGCGCCA
SEQ ID NO:29
V5::核酸适配体II-mini3-4
UGCCUGGCGACCAUAGCGAUUGAGGUUCGAAGCUUUUGCUUGGACGAACCGCAAUUAGCGCCGAUGGUAGUGUGGGGUUUCCCCAUGUGAGAGUAGGACAUCGCCAGGCAU
SEQ ID NO:30
V5::芒果
UGCCUGGCGACCAUAGCGAUUGGAAGGGACGGUGCGGAGAGGAGACAAUUAGC GCCGAUGGUAGUGUGGGGUUUCCCCAUGUGAGAGUAGGACAUCGCCAGGCAU
SEQ ID NO:31
V5::芒果2
UGCCUGGCGACCAUAGCGAUUGGAAGGAGAGGAGAGGAAGAGGAGACAAUUAG CGCCGAUGGUAGUGUGGGGUUUCCCCAUGUGAGAGUAGGACAUCGCCAGGCAU
SEQ ID NO:32
V5::芒果3
UGCCUGGCGACCAUAGCGAUUGGGAAGGAUUGGUAUGUGGUAUACAAUUAGCGCCGAUGGUAGUGUGGGGUUUCCCCAUGUGAGAGUAGGACAUCGCCAGGCAU
SEQ ID NO:33
V5::芒果4
UGCCUGGCGACCAUAGCGAUUGCGAGGGAGUGGUGAGGAUGAGGCGACAAUUAGCGCCGAUGGUAGUGUGGGGUUUCCCCAUGUGAGAGUAGGACAUCGCCAGGCAU
SEQ ID NO:34
V5::菠菜
UGCCUGGCGACCAUAGCGAUUGGACGCAACUGAAUGAAAUGGUGAAGGACGGGUCCAGGUGUGGCUGCUUCGGCAGUGCAGCUUGUUGAGUAGAGUGUGAGCUCCGUAACUAGUCGCGUCCAAUUAGCGCCGAUGGUAGUGUGGGGUUUCCCCAUGUGAGAGUAGGACAUCGCCAGGCAU
SEQ ID NO:35
V5::菠菜2
UGCCUGGCGACCAUAGCGAUUGGAUGUAACUGAAUGAAAUGGUGAAGGACGGGUCCAGUAGGCUGCUUCGGCAGCCUACUUGUUGAGUAGAGUGUGAGCUCCGUAACUAGUUACAUCCAAUUAGCGCCGAUGGUAGUGUGGGGUUUCCCCAUGUGAGAGUAGGACAUCGCCAGGCAU
SEQ ID NO:36
V5::西兰花
UGCCUGGCGACCAUAGCGAUUGAGACGGUCGGGUCCAGAUAUUCGUAUCUGUCGAGUAGAGUGUGGGCUCAAUUAGCGCCGAUGGUAGUGUGGGGUUUCCCCAUGUGAGAGUAGGACAUCGCCAGGCAU
SEQ ID NO:37
V5::玉米
UGCCUGGCGACCAUAGCGAUUGCGAGGAAGGAGGUCUGAGGAGGUCACUGCAAUUAGCGCCGAUGGUAGUGUGGGGUUUCCCCAUGUGAGAGUAGGACAUCGCCAGGCAU
SEQ ID NO:38
V5::橙色西兰花
UGCCUGGCGACCAUAGCGAUUGAGACGGUCGGGUCCAGGUGCACAAAUGUGGCCUGUUGAGUAGCGUGUGGGCUCAAUUAGCGCCGAUGGUAGUGUGGGGUUUCCCCAUGUGAGAGUAGGACAUCGCCAGGCAU
SEQ ID NO:39
V5::红色西兰花
UGCCUGGCGACCAUAGCGAUUGAGACGGUCGGGUCCAGUCCCAACGAUGUUGGCUGUUGAGUAGUGUGUGGGCUCAAUUAGCGCCGAUGGUAGUGUGGGGUUUCCCCAUGUGAGAGUAGGACAUCGCCAGGCAU
SEQ ID NO:40
V5::嫩菠菜
UGCCUGGCGACCAUAGCGAUUGAAGGACGGGUCCGUUGAGUAGAGUGUGAGCAAUUAGCGCCGAUGGUAGUGUGGGGUUUCCCCAUGUGAGAGUAGGACAUCGCCAGGCAU
SEQ ID NO:41
F29::核酸适配体II-mini3-4
UUGUCACGUGUAUGUGGGAGGUUCGAAGCUUUUGCUUGGACGAACCGCCCACA UACUUUGUUGAUCCNNNNGGAUCAAUCAUGGCAA
SEQ ID NO:42
F29::芒果
UUGUCACGUGUAUGUGGGGAAGGGACGGUGCGGAGAGGAGACCCACAUACUUUGUUGAUCCNNNNGGAUCAAUCAUGGCAA
SEQ ID NO:43
F29::芒果2
UUGUCACGUGUAUGUGGGGAAGGAGAGGAGAGGAAGAGGAGACCCACAUACUUUGUUGAUCCNNNNGGAUCAAUCAUGGCAA
SEQ ID NO:44
F29::芒果3
UUGUCACGUGUAUGUGGGGGAAGGAUUGGUAUGUGGUAUACCCACAUACUUUGUUGAUCCNNNNGGAUCAAUCAUGGCAA
SEQ ID NO:45
F29::芒果4
UUGUCACGUGUAUGUGGGCGAGGGAGUGGUGAGGAUGAGGCGACCCACAUACUUUGUUGAUCCNNNNGGAUCAAUCAUGGCAA
SEQ ID NO:46
F29::菠菜
UUGUCACGUGUAUGUGGGGACGCAACUGAAUGAAAUGGUGAAGGACGGGUCCAGGUGUGGCUGCUUCGGCAGUGCAGCUUGUUGAGUAGAGUGUGAGCUCCGUAACUAGUCGCGUCCCCACAUACUUUGUUGAUCCNNNNGGAUCAAUCAUGGCAA
SEQ ID NO:47
F29::菠菜2
UUGUCACGUGUAUGUGGGGAUGUAACUGAAUGAAAUGGUGAAGGACGGGUCCAGUAGGCUGCUUCGGCAGCCUACUUGUUGAGUAGAGUGUGAGCUCCGUAACUAGUUACAUCCCCACAUACUUUGUUGAUCCNNNNGGAUCAAUCAUGGCAA
SEQ ID NO:48
F29::西兰花
UUGUCACGUGUAUGUGGGAGACGGUCGGGUCCAGAUAUUCGUAUCUGUCGAGU AGAGUGUGGGCUCCCACAUACUUUGUUGAUCCNNNNGGAUCAAUCAUGGCAA
SEQ ID NO:49
F29::玉米
UUGUCACGUGUAUGUGGGCGAGGAAGGAGGUCUGAGGAGGUCACUGCCCACAU ACUUUGUUGAUCCNNNNGGAUCAAUCAUGGCAA
SEQ ID NO:50
F29::橙色西兰花
UUGUCACGUGUAUGUGGGAGACGGUCGGGUCCAGGUGCACAAAUGUGGCCUGUUGAGUAGCGUGUGGGCUCCCACAUACUUUGUUGAUCCNNNNGGAUCAAUCAUGGCAA
SEQ ID NO:51
F29::红色西兰花
UUGUCACGUGUAUGUGGGAGACGGUCGGGUCCAGUCCCAACGAUGUUGGCUGUUGAGUAGUGUGUGGGCUCCCACAUACUUUGUUGAUCCNNNNGGAUCAAUCAUGGCAA
SEQ ID NO:52
F29::嫩菠菜
UUGUCACGUGUAUGUGGGAAGGACGGGUCCGUUGAGUAGAGUGUGAGCCCACA UACUUUGUUGAUCCNNNNGGAUCAAUCAUGGCAA
SEQ ID NO:53
F30::核酸适配体II-mini3-4
UUGCCAUGUGUAUGUGGGAGGUUCGAAGCUUUUGCUUGGACGAACCGCCCACA UACUCUGAUGAUCCNNNNGGAUCAUUCAUGGCAA
SEQ ID NO:54
F30::芒果:
UUGCCAUGUGUAUGUGGGGAAGGGACGGUGCGGAGAGGAGACCCACAUACUCUGAUGAUCCNNNNGGAUCAUUCAUGGCAA
SEQ ID NO:55
F30::芒果2:
UUGCCAUGUGUAUGUGGGGAAGGAGAGGAGAGGAAGAGGAGACCCACAUACUCUGAUGAUCCNNNNGGAUCAUUCAUGGCAA
SEQ ID NO:56
F30::芒果3:
UUGCCAUGUGUAUGUGGGGGAAGGAUUGGUAUGUGGUAUACCCACAUACUCUGAUGAUCCNNNNGGAUCAUUCAUGGCAA
SEQ ID NO:57
F30::芒果4
UUGCCAUGUGUAUGUGGGCGAGGGAGUGGUGAGGAUGAGGCGACCCACAUACUCUGAUGAUCCNNNNGGAUCAUUCAUGGCAA
SEQ ID NO:58
F30::菠菜
UUGCCAUGUGUAUGUGGGGACGCAACUGAAUGAAAUGGUGAAGGACGGGUCCAGGUGUGGCUGCUUCGGCAGUGCAGCUUGUUGAGUAGAGUGUGAGCUCCGUAACUAGUCGCGUCCCCACAUACUCUGAUGAUCCNNNNGGAUCAUUCAUGGCAA
SEQ ID NO:59
F30::菠菜2
UUGCCAUGUGUAUGUGGGGAUGUAACUGAAUGAAAUGGUGAAGGACGGGUCCAGUAGGCUGCUUCGGCAGCCUACUUGUUGAGUAGAGUGUGAGCUCCGUAACUAGUUACAUCCCCACAUACUCUGAUGAUCCNNNNGGAUCAUUCAUGGCAA
SEQ ID NO:60
F30::西兰花
UUGCCAUGUGUAUGUGGGAGACGGUCGGGUCCAGAUAUUCGUAUCUGUCGAGUAGAGUGUGGGCUCCCACAUACUCUGAUGAUCCNNNNGGAUCAUUCAUGGCAA
SEQ ID NO:61
F30::玉米
UUGCCAUGUGUAUGUGGGCGAGGAAGGAGGUCUGAGGAGGUCACUGCCCACAUACUCUGAUGAUCCNNNNGGAUCAUUCAUGGCAA
SEQ ID NO:62
F30::橙色西兰花
UUGCCAUGUGUAUGUGGGAGACGGUCGGGUCCAGGUGCACAAAUGUGGCCUGUUGAGUAGCGUGUGGGCUCCCACAUACUCUGAUGAUCCNNNNGGAUCAUUCAUGGCAA
SEQ ID NO:63
F30::红色西兰花
UUGCCAUGUGUAUGUGGGAGACGGUCGGGUCCAGUCCCAACGAUGUUGGCUGUUGAGUAGUGUGUGGGCUCCCACAUACUCUGAUGAUCCNNNNGGAUCAUUCAUGGCAA
SEQ ID NO:64
F30::嫩菠菜
UUGCCAUGUGUAUGUGGGAAGGACGGGUCCGUUGAGUAGAGUGUGAGCCCACA UACUCUGAUGAUCCNNNNGGAUCAUUCAUGGCAA
SEQ ID NO:77
F30-西兰花-西兰花
UUGCCAUGUGUAUGUGGGAGACGGUCGGGUCCAGAUAUUCGUAUCUGUCGAGUAGAGUGUGGGCUCCCACAUACUCUGAUGAUCCAGACGGUCGGGUCCAGAUAUUCGUAUCUGUCGAGUAGAGUGUGGGCUGGAUCAUUCAUGGCAA
SEQ ID NO:78
F30-西兰花-芒果
UUGCCAUGUGUAUGUGGGAGACGGUCGGGUCCAGAUAUUCGUAUCUGUCGAGUAGAGUGUGGGCUCCCACAUACUCUGAUGAUCCGAAGGGACGGUGCGGAGAGGAGAGGAUCAUUCAUGGCAA
本发明中使用的载体可以通过本领域已知的任何方法引入细胞中，例如通过转化、转染或病毒转导。本领域技术人员知道如何可以进一步优化这些方法以确保载体以足够的量存在于每个细胞中。本发明设想，一旦载体被引入细胞，就可以将其整合到染色体中。这种整合可能不一定包括完整的载体序列，而是在给定的遗传环境中表达靶RNA所需的载体片段。
根据本发明的方法旨在通过鉴定增加RNA产生的细胞或培养条件来优化RNA产生。因此，在一个实施方案中，本发明方法中使用的细胞携带可能影响RNA表达的染色体遗传突变。可以使用本领域已知的任何技术对细胞进行诱变，例如，通过UV辐射的随机诱变或定点诱变，例如通过CRISPR-Cas。
在另一个实施方案中，细胞携带不同的载体，这些载体都能够表达相同的感兴趣的RNA，但载体中包含的其他元素不同，因此来自载体的RNA产量不同。
一旦将载体引入细胞中，就在允许异源RNA表达的条件下培养细胞。在感兴趣的异源RNA的表达是有条件的情况下，可以首先在不诱导表达的情况下培养细胞，并在一段时间后诱导表达。
在一个实施方案中，细胞之间的培养条件是变化的，例如关于温度、溶解氧水平、搅拌速度、压力或培养基。该实施方案允许鉴定用于表达异源RNA的最佳培养条件。因此，本发明的方法也可用于优化生产感兴趣的特定RNA的培养条件。
在细胞培养足够长的时间以表达来自载体的异源RNA后，将能够结合标记有异源RNA的核酸适配体的荧光团添加到培养基中，并允许其进入细胞，在那里它可以结合核酸适配体。本领域已知如何确定培养基中荧光团的合适浓度。
在那些具有高浓度感兴趣的异源RNA的细胞中，标签以更高的量存在，因此更高量的荧光团将被结合并发出荧光。相反，那些仅显示出低浓度的包括标签的感兴趣RNA的细胞将携带较少活化的荧光团，即与核酸适配体结合的荧光团并因此发射较少的荧光。重要的是，存在于细胞中但未与核酸适配体结合的荧光团将仅表现出非常弱或没有荧光。
每个细胞发出的荧光程度可以使用本领域已知的任何技术来确定。在一个实施方案中，通过光谱法评估细胞。在优选实施方案中，通过流式细胞术根据细胞的荧光水平对细胞进行分选。这允许识别并同时分离那些显示出高水平荧光的细胞。随后可以分析分离的细胞携带的遗传改变或载体中的染色体遗传改变。同样，可以确定产生最高数量荧光细胞的那些培养条件。
在另一个实施方案中，本发明涉及一种用于比较不同细胞对感兴趣的异源RNA序列的生产能力的方法，包括以下步骤：
d)将能够表达感兴趣的异源RNA的载体引入多个细胞中，其中所述的RNA用RNA标签标记，包含
·能够稳定荧光团的核酸适配体以及
·能够稳定核酸适配体的支架；
e)在允许感兴趣的RNA表达的条件下，在培养基中培养细胞；
f)将所述的荧光团加入所述的培养基中；
g)比较所述的多个细胞之间的荧光强度。
在另一个方面，本发明涉及一种生产感兴趣的RNA的方法，包括以下步骤：
a)将能够表达感兴趣的异源RNA的第一载体引入多个细胞，其中所述的RNA用RNA标签标记，所述的RNA标签包含能够稳定荧光团的核酸适配体和能够稳定所述的核酸适配体的支架；
b)在允许感兴趣的异源RNA表达的条件下，在培养基中培养细胞；
c)将所述荧光团加入所述培养基中；
d)鉴定和分离那些显示出最高荧光强度并因此显示出感兴趣的RNA的高表达的细胞；
e)从在步骤d)中分离的细胞中去除步骤a)的第一载体；
f)将能够在没有RNA标签的情况下表达感兴趣的异源RNA的第二载体引入步骤e)获得的细胞中；
g)通过培养在步骤f)中获得的细胞来产生感兴趣的RNA。
根据本发明的方法生产感兴趣的RNA包括第一识别细胞，在RNA标签的帮助下显示出感兴趣的RNA的高表达，然后使用这些细胞生产感兴趣RNA。
因为希望最终产物不包括任何不必要的序列，所以一旦鉴定出合适的细胞，就在RNA生产之前移除能够表达感兴趣的标记RNA的载体。载体的去除可以通过制备电活性细胞来实现，如前所述(Tauch，A.，Kirchner，O.，B.，/>S.，Pühler，A.,andKalinowski，J.Efficient Electrotransformation of Corynebacterium diphtheriaewith a Mini-Replicon Derived from the Corynebacterium glutamicum PlasmidpGA.Curr.Microbiol.2002；45,362-367)和在不添加DNA模板的情况下对这些细胞进行电穿孔。这增加了自发载体丢失的可能性。随后用与第一载体相同的第二载体转化细胞，不同之处在于第二载体不包含RNA标签。然后将这些细胞用于产生感兴趣的RNA。
所产生的RNA的提取和纯化可以根据本领域已知的方法进行。同样，在提取后可以使用已知的技术对所产生的RNA的量进行定量。
本发明的方法允许通过特异性选择表现出感兴趣RNA高表达的细胞来最大化感兴趣RNA的产生。因为本发明中使用的载体可以很容易地被工程化以携带任何感兴趣的RNA，所以本发明提供了一种快速、有效和普遍适用的方法，以在生产某种RNA分子时节省成本和时间。
在第三个方面，本发明涉及携带能够表达感兴趣的异源RNA的载体的微生物细胞，其中所述的RNA用RNA标签标记，所述的RNA标签包含
-能够稳定荧光团的核酸适配体，以及
-能够稳定核酸适配体的RNA支架。
根据本发明的细胞携带能够表达感兴趣的异源RNA的载体。“携带”在本文中的定义是指细胞含有或包含载体，该载体作为染色体外质粒或整合到其一条或几条染色体中。
因为本发明的细胞表达的异源RNA被标记，所以一旦将相应的荧光团添加到细胞中，就可以对其进行检测和定量。因此，根据本发明的细胞可以基于它们产生的异源RNA的量而容易且方便地进行分类和分离(例如通过流式细胞术)。
实施例
在下文中，参考附图和实施例更详细地描述本发明，然而这些附图和实施方案并不旨在限制本发明。
实施例1
a)载体pJC1-PF1-U1A-F30::西兰花/UUCG-TF1和pJC1-PF1-U1A-TF1的构建
质粒载体的构建是通过化学合成合成DNA片段(pJC1-PF1-U1A-TF1的SEQ ID NO:72和pJC1-PF1-U1A-F30::西兰花/UUCG-TF1的SEQ ID NO:71)的方式并将其连接到pJC1中(Cremer,J.,Treptow,C.,Eggeling,L.,and Sahm,H.Regulation of Enzymes of LysineBiosynthesis in Corynebacterium glutamicum.Microbiol.1988；134,3221-3229)。SEQID NO:71含有启动子PF1(SEQ ID NO:67)，即先前描述的非编码重组U1A*-RNA(SEQ ID NO:68)(Hashiro,S.,Mitsuhashi,M.,and Yasueda,H.Overexpression system forrecombinant RNA in Corynebacterium glutamicum using a strong promoter derivedfrom corynephage BFK20.J.Biosci.Bioeng.2019；128,255-263)，F30支架(SEQ ID NO:69)，在第一整合点具有西兰花核酸适配体(SEQ ID NO：8)，在第二整合点具有“UUCG间隔”，以及终止子序列TF1(SEQ ID NO:70)。SEQ ID NO:72包含启动子PF1(SEQ ID NO:67)、非编码重组U1A*-RNA(SEQ ID NO:68)和终止子序列TF1(SEQ ID NO:70)，但既没有支架也没有核酸适配体序列。
在用限制性内切酶XbaI和SalI切割合成的DNA片段并随后纯化反应混合物之后，将切下的DNA片段用于与载体pJC1的单独连接反应，该载体pJC1也用XbaI或SalI线性化并去磷酸化。将连接混合物直接用于转化大肠杆菌XL1-蓝，并在含有50μg/ml卡那霉素的LB板上进行转化子的选择。将生长在这些板上并因此对卡那霉素具有抗性的16个菌落用于菌落PCR。用引物pJC1_check_f(SEQ ID NO:73)和pJC1_check_rev(SEQ ID NO:74)进行菌落PCR，以分析合成片段是否***载体pJC1中。在琼脂糖凝胶中对菌落PCR产物的分析显示，在所分析的样品中，预期的PCR产物具有521bp(pJC1-PF1-U1A-TF1)和626bp(pJC1-PF1-U1A-F30::西兰花/UUCG-TF1)的大小，于是培养四个菌落用于更大规模的质粒制备。培养16小时后，通过离心收集这些培养物并制备质粒DNA。用菌落PCR中使用的引物对其中两种质粒制剂进行测序。***物的序列分析显示与预期序列100％一致。所得质粒分别命名为pJC1-PF1-U1A-TF1(SEQ ID NO:76)和pJC1-PF1-U1A-F30::西兰花/UUCG-TF1(SEQ ID NO:75)。
SEQ ID No:67
PF1:
CTCGAGCGGGACGGTCGAACCAGCTTCAAGCGACCGGATGAGTATGTTACAGTAGA TAGCG
SEQ ID NO:68
靶RNA:
AGCGGGAGACCGCTCGACCTTAGTTCTCCTGTTGCGGGGGAGTTCATGGGATCCAG GTACCCAGGGCGAGGCTTATCCATTGCACTCCGGATGTGCTGACCCCTG
SEQ ID NO:69
F30，***位点1为西兰花，***位点2为“TTCG”:
TTGCCATGTGTATGTGGGAGACGGTCGGGTCCAGATATTCGTATCTGTCGAGTAGAG TGTGGGCTCCCACATACTCTGATGATCCTTCGGGATCATTCATGGCAA
SEQ ID NO:70
TF1:
CTAGCATAGCATAAAATAACGCCCCACCTTCTTAACGGGAGGTGGGGCGTTATTTTTACGGGGATCGCT
SEQ ID NO:71
SEQ ID NO:67-70组合，在5'-端具有XbaI识别位点，在3'-端具有SalI识别位点：
CTGTCTCTAGACTCGAGCGGGACGGTCGAACCAGCTTCAAGCGACCGGATGAGTATGTTACAGTAGATAGCGAGCGGGAGACCGCTCGACCTTAGTTCTCCTGTTGCGGGGGAGTTCATGGGATCCAGGTACCCAGGGCGAGGCTTATCCATTGCACTCCGGATGTGCTGACCCCTGTTGCCATGTGTATGTGGGAGACGGTCGGGTCCAGATATTCGTATCTGTCGAGTAGAGTGTGGGCTCCCACATACTCTGATGATCCTTCGGGATCATTCATGGCAAGCTAGCATAGCATAAAATAACGCCCCACCTTCTTAACGGGAGGTGGGGCGTTATTTTTACGGTCGACCTGTCSEQ ID NO:72
SEQ ID NO:67，68和70，在5'-端具有XbaI识别位点，在3'-端具有SalI识别位点：
CTGTCTCTAGACTCGAGCGGGACGGTCGAACCAGCTTCAAGCGACCGGATGAGTATGTTACAGTAGATAGCGAGCGGGAGACCGCTCGACCTTAGTTCTCCTGTTGCGGGGGAGTTCATGGGATCCAGGTACCCAGGGCGAGGCTTATCCATTGCACTCCGGATGTGCTGACCCCTGGCTAGCATAGCATAAAATAACGCCCCACCTTCTTAACGGGAGGTGGGGCGTTATTTTTACGGTCGACCTGTC
SEQ ID NO:73
pJC1_check_f:
TGAAGACCGTCAACCAAAGG
SEQ ID NO:74
pJC1_check_rev:
TGCCGGGAAGCTAGAGTAAG
SEQ ID NO:75
pJC1-PF1-U1A-F30::西兰花/UUCG-TF1
CCGAGAAGTTTTTTACAAAAGGCAAAAACTTTTTCGGGATCGACAGAAATAAAACGATCGACGGTACGCAACAAAAAAGCGTCAGGATCGCCGTAGAGCGATTGAAGACCGTCAACCAAAGGGGAAGCCTCCAATCGACGCGACGCGCGCTCTACGGCGATCCTGACGCAGATTTTTAGCTATCTGTCGCAGCGCCCTCAGGGACAAGCCACCCGCACAACGTCGCGAGGGCGATCAGCGACGCCGCAGGGGGATCCTCTAGACTCGAGCGGGACGGTCGAACCAGCTTCAAGCGACCGGATGAGTATGTTACAGTAGATAGCGAGCGGGAGACCGCTCGACCTTAGTTCTCCTGTTGCGGGGGAGTTCATGGGATCCAGGTACCCAGGGCGAGGCTTATCCATTGCACTCCGGATGTGCTGACCCCTGTTGCCATGTGTATGTGGGAGACGGTCGGGTCCAGATATTCGTATCTGTCGAGTAGAGTGTGGGCTCCCACATACTCTGATGATCCTTCGGGATCATTCATGGCAAGCTAGCATAGCATAAAATAACGCCCCACCTTCTTAACGGGAGGTGGGGCGTTATTTTTACGGTCGACCTGCAGCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAACTATGGATGAACGAAATAGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTAACTGTCAGACCAAGTTTACTCATATATACTTTAGATTGATTTAAAACTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCTTAATAAGATGATCTTCTTGAGATCGTTTTGGTCTGCGCGTAATCTCTTGCTCTGAAA
ACGAAAAAACCGCCTTGCAGGGCGGTTTTTCGAAGGTTCTCTGAGCTACCAACTCTTTG
AACCGAGGTAACTGGCTTGGAGGAGCGCAGTCACCAAAACTTGTCCTTTCAGTTTAGCC
TTAACCGGCGCATGACTTCAAGACTAACTCCTCTAAATCAATTACCAGTGGCTGCTGCCA
GTGGTGCTTTTGCATGTCTTTCCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGC
AGCGGTCGGACTGAACGGGGGGTTCGTGCATACAGTCCAGCTTGGAGCGAACTGCCTA
CCCGGAACTGAGTGTCAGGCGTGGAATGAGACAAACGCGGCCATAACAGCGGAATGAC
ACCGGTAAACCGAAAGGCAGGAACAGGAGAGCGCACGAGGGAGCCGCCAGGGGGAA
ACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCACTGATTTGAGCGTCAGATTTC
GTGATGCTTGTCAGGGGGGCGGAGCCTATGGAAAAACGGCTTTGCCGCGGCCCTCTCAC
TTCCCTGTTAAGTATCTTCCTGGCATCTTCCAGGAAATCTCCGCCCCGTTCGTAAGCCATT
TCCGCTCGCCGCAGTCGAACGACCGAGCGTAGCGAGTCAGTGAGCGAGGAAGCGGAAT
ATATCCTGTATCACATATTCTGCTGACGCACCGGTGCAGCCTTTTTTCTCCTGCCACATGA
AGCACTTCACTGACACCCTCATCAGTGCCAACATAGTAAGCCAGTATACACTCCGCTAGC
GCTGAGGTCTGCCTCGTGAAGAAGGTGTTGCTGACTCATACCAGGCCTGAATCGCCCCA
TCATCCAGCCAGAAAGTGAGGGAGCCACGGTTGATGAGAGCTTTGTTGTAGGTGGACC
AGTTGGTGATTTTGAACTTTTGCTTTGCCACGGAACGGTCTGCGTTGTCGGGAAGATGC
GTGATCTGATCCTTCAACTCAGCAAAAGTTCGATTTATTCAACAAAGCCACGTTGTGTCT
CAAAATCTCTGATGTTACATTGCACAAGATAAAAATATATCATCATGAACAATAAAACTGT
CTGCTTACATAAACAGTAATACAAGGGGTGTTATGAGCCATATTCAACGGGAAACGTCTT
GCTCGAGGCCGCGATTAAATTCCAACATGGATGCTGATTTATATGGGTATAAATGGGCTC
GCGATAATGTCGGGCAATCAGGTGCGACAATCTATCGATTGTATGGGAAGCCCGATGCGC
CAGAGTTGTTTCTGAAACATGGCAAAGGTAGCGTTGCCAATGATGTTACAGATGAGATG
GTCAGACTAAACTGGCTGACGGAATTTATGCCTCTTCCGACCATCAAGCATTTTATCC
GTACTCCTGATGATGCATGGTTACTCACCACTGCGATCCCCGGGAAAACA
GCATTCCAGGTATTAGAAGAATATCCTGATTCAGGTGAAAATATTGTTGA
TGCGCTGGCAGTGTTCCTGCGCCGGTTGCATTCGATTCCTGTTTGTAATT
GTCCTTTTAACAGCGATCGCGTATTTCGTCTCGCTCAGGCGCAATCACGA
ATGAATAACGGTTTGGTTGATGCGAGTGATTTTGATGACGAGCGTAATGG
CTGGCCTGTTGAACAAGTCTGGAAAGAAATGCATAAGCTTTTGCCATTCT
CACCGGATTCAGTCGTCACTCATGGTGATTTCTCACTTGATAACCTTATT
TTTGACGAGGGGAAATTAATAGGTTGTATTGATGTTGGACGAGTCGGAAT
CGCAGACCGATACCAGGATCTTGCCATCCTATGGAACTGCCTCGGTGAGT
TTTCTCCTTCATTACAGAAACGGCTTTTTCAAAAATATGGTATTGATAAT
CCTGATATGAATAAATTGCAGTTTCATTTGATGCTCGATGAGTTTTTCTA
ATCAGAATTGGTTAATTGGTTGTAACACTGGCAGAGCATTACGCTGACTT
GACGGGACGGCGGCTTTGTTGAATAAATCGAACTTTTGCTGAGTTGAAGG
ATCAGATCACGCATCTTCCCGACAACGCAGACCGTTCCGTGGCAAAGCAA
AAGTTCAAAATCACCAACTGGTCCACCTACAACAAAGCTCTCATCAACCG
TGGCTCCCTCACTTTCTGGCTGGATGATGGGGCGATTCAGGCCTGGTATG
AGTCAGCAACACCTTCTTCACGAGGCAGACCTCAGCGCTCAAAGATGCAG
GGGTAAAAGCTAACCGCATCTTTACCGACAAGGCATCCGGCAGTTCAACA
GATCGGGAAGGGCTGGATTTGCTGAGGATGAAGGTGGAGGAAGGTGATGT
CATTCTGGTGAAGAAGCTCGACCGTCTTGGCCGCGACACCGCCGACATGA
TCCAACTGATAAAAGAGTTTGATGCTCAGGGTGTAGCGGTTCGGTTTATT
GACGACGGGATCAGTACCGACGGTGATATGGGGCAAATGGTGGTCACCAT
CCTGTCGGCTGTGGCACAGGCTGAACGCCGGAGGATCAAGTCGGTCAAGC
CAAGCGCAACCAGCGGCACCGCCGCGAGCAACGTCGCAAGGGCGATCAGG
GGACGATTTTTGCGAAGAATTTCCACGGTAAGAATCCAATCTCTCGAATT
TAGGGTGAAAGAAGCTTGGCATAGGGGTGTGCACGAACTCGGTGGAGGAA
ATTTCCGCGGGGCAAGGCTTCGCGAAGCGGAGTCGCGGCAGTGGCTTTGA
AGATCTTTGGGAGCAGTCCTTGTGCGCTTACGAGGTGAGCCGGTGGGGAA
CCGTTATCTGCCTATGGTGTGAGCCCCCCTAGAGAGCTTCAAGAGCAATC
AGCCCGACCTAGAAAGGAGGCCAAGAGAGAGACCCCTACGGGGGGAACCG
TTTTCTGCCTACGAGATGGCACATTTACTGGGAAGCTTTACGGCGTCCTC
GTGGAAGTTCAATGCCCGCAGACTTAAGTGCTCTATTCACGGTCTGACGT
GACACGCTAAATTCAGACATAGCTTCATTGATTGTCGGCCACGAGCCAGT
CTCTCCCTCAACAGTCATAAACCAACCTGCAATGGTCAAGCGATTTCCTT
TAGCTTTCCTAGCTTGTCGTTGACTGGACTTAGCTAGTTTTTCTCGCTGT
GCTCGGGCGTACTCACTGTTTGGGTCTTTCCAGCGTTCTGCGGCCTTTTT
ACCGCCACGTCTTCCCATAGTGGCCAGAGCTTTTCGCCCTCGGCTGCTCT
GCGTCTCTGTCTGACGAGCAGGGACGACTGGCTGGCCTTTAGCGACGTAG
CCGCGCACACGTCGCGCCATCGTCTGGCGGTCACGCATCGGCGGCAGATC
AGGCTCACGGCCGTCTGCTCCGACCGCCTGAGCGACGGTGTAGGCACGCT
CGTAGGCGTCGATGATCTTGGTGTCTTTTAGGCGCTCACCAGCCGCTTTT
AACTGGTATCCCACAGTCAAAGCGTGGCGAAAAGCCGTCTCATCACGGGC
GGCACGCCCTGGAGCAGTCCAGAGGACACGGACGCCGTCGATCAGCTCTC
CAGACGCTTCAGCGGCGCTCGGCAGGCTTGCTTCAAGCGTGGCAAGTGCT
TTTGCTTCCGCAGTGGCTTTTCTTGCCGCTTCGATACGTGCCCGTCCGCT
AGAAAACTCCTGCTCATAGCGTTTTTTAGGTTTTTCTGTGCCTGAGATCA
TGCGAGCAACCTCCATAAGATCAGCTAGGCGATCCACGCGATTGTGCTGG
GCATGCCAGCGGTACGCGGTGGGATCGTCGGAGACGTGCAGTGGCCACCG
GCTCAGCCTATGTGAAAAAGCCTGGTCAGCGCCGAAAACGCGGGTCATTT
CCTCGGTCGTTGCAGCCAGCAGGCGCATATTCGGGCTGCTCATGCCTGCT
GCGGCATACACCGGATCAATGAGCCAGATGAGCTGGCATTTCCCGCTCAG
TGGATTCACGCCGATCCAAGCTGGCGCTTTTTCCAGGCGTGCCCAGCGCT
CCAAAATCGCGTAGACCTCGGGGTTTACGTGCTCGATTTTCCCGCCGGCC
TGGTGGCTCGGCACATCAATGTCCAGGACAAGCACGGCTGCGTGCTGCGC
GTGCGTCAGAGCAACATACTGGCACCGGGCAAGCGATTTTGAACCAACTC
GGTATAACTTCGGCTGTGTTTCTCCCGTGTCCGGGTCTTTGATCCAAGCG
CTGGCGAAGTCGCGGGTCTTGCTGCCCTGGAAATTTTCTCTGCCCAGGTG
AGCGAGGAATTCGCGGCGGTCTTCGCTCGTCCAGCCACGTGATCGCAGCG
CGAGCTCGGGATGGGTGTCGAACAGATCAGCGGAAAATTTCCAGGCCGGT
GTGTCAATGTCTCGTGAATCCGCTAGAGTCATTTTTGAGCGCTTTCTCCC
AGGTTTGGACTGGGGGTTAGCCGACGCCCTGTGAGTTACCGCTCACGGGG
CGTTCAACATTTTTCAGGTATTCGTGCAGCTTATCGCTTCTTGCCGCCTG
TGCGCTTTTTCGACGCGCGACGCTGCTGCCGATTCGGTGCAGGTGGTGGC
GGCGCTGACACGTCCTGGGCGGCCACGGCCACACGAAACGCGGCATTTAC
GATGTTTGTCATGCCTGCGGGCACCGCGCCACGATCGCGGATAATTCTCG
CTGCCGCTTCCAGCTCTGTGACGACCATGGCCAAAATTTCGCTCGGGGGA
CGCACTTCCAGCGCCATTTGCGACCTAGCCGCCTCCAGCTCCTCGGCGTG
GCGTTTGTTGGCGCGCTCGCGGCTGGCTGCGGCACGACACGCATCTGAGC
AATATTTTGCGCGCCGTCCTCGCGGGTCAGGCCGGGGAGGAATCAGGCCA
CCGCAGTAGGCGCAACTGATTCGATCCTCCACTACTGTGCGTCCTCCTGG
CGCTGCCGAGCACGCAGCTCGTCAGCCAGCTCCTCAAGATCCGCCACGAG
AGTTTCTAGGTCGCTCGCGGCACTGGCCCAGTCTCGTGATGCTGGCGCGT
CCGTCGTATCGAGAGCTCGGAAAAATCCGATCACCGTTTTTAAATCGACG
GCAGCATCGAGCGCGTCGGACTCCAGCGCGACATCAGAGAGATCCATAGC
TGATGATTCGGGCCAATTTTGGTACTTCGTCGTGAAGGTCATGACACCAT
TATAACGAACGTTCGTTAAAGTTTTTGGCGGAAAATCACGCGGCACGAAA
ATTTTCACGAAGCGGGACTTTGCGCAGCTCAGGGGTGCTAAAAATTTTGT
ATCGCACTTGATTTTTCCGAAAGACAGATTATCTGCAAACGGTGTGTCGT
ATTTCTGGCTTGGTTTTTAAAAAATCTGGAATCGAAAATTTGCGGGGCGASEQ ID NO:76
pJC1-PF1-U1A-TF1:
CCGAGAAGTTTTTTACAAAAGGCAAAAACTTTTTCGGGATCGACAGAAATAAAACGATCGACGGTACGCAACAAAAAAGCGTCAGGATCGCCGTAGAGCGATTGAAGACCGTCAACCAAAGGGGAAGCCTCCAATCGACGCGACGCGCGCTCTACGGCGATCCTGACGCAGATTTTTAGCTATCTGTCGCAGCGCCCTCAGGGACAAGCCACCCGCACAACGTCGCGAGGGCGATCAGCGACGCCGCAGGGGGATCCTCTAGACTCGAGCGGGACGGTCGAACCAGCTTCAAGCGACCGGATGAGTATGTTACAGTAGATAGCGAGCGGGAGACCGCTCGACCTTAGTTCTCCTGTTGCGGGGGAGTTCATGGGATCCAGGTACCCAGGGCGAGGCTTATCCATTGCACTCCGGATGTGCTGACCCCTGGCTAGCATAGCATAAAATAACGCCCCACCTTCTTAACGGGAGGTGGGGCGTTATTTTTACGGGGATCGCTTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGGTCGACCTGCAGCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAACTATGGATGAACGAAATAGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTAACTGTCAGACCAAGTTTACTCATATATACTTTAGATTGATTTAAAACTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCTTAATAAGATGATCTTCTTGAGATCGTTTTGGTCTGCGCGTAATCTCTTGCTCTGAAAACGAAAAAACCGCCTTGCAGGGCGGTTTTTCGAAGGTTCTCTGAGCTACCAACTCTTTGAACCGAGGTAACTGGCTTGGAGGAGCGCAGTCACCAAAACTTGTCCTTTCAGTTTAGCCTTAACCGGCGCATGACTTCAAGACTAACTCCTCTAAATCAATTACCAGTGGCTGCTGCCAGTGGTGCTTTTGCATGTCTTTCCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGACTGAACGGGGGGTTCGTGCATACAGTCCAGCTTGGAGCGAACTGCCTACCCGGAACTGAGTGTCAGGCGTGGAATGAGACAAACGCGGCCATAACAGCGGAATGACACCGGTAAACCGAAAGGCAGGAACAGGAGAGCGCACGAGGGAGCCGCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCACTGATTTGAGCGTCAGATT
TCGTGATGCTTGTCAGGGGGGCGGAGCCTATGGAAAAACGGCTTTGCCGC
GGCCCTCTCACTTCCCTGTTAAGTATCTTCCTGGCATCTTCCAGGAAATC
TCCGCCCCGTTCGTAAGCCATTTCCGCTCGCCGCAGTCGAACGACCGAGC
GTAGCGAGTCAGTGAGCGAGGAAGCGGAATATATCCTGTATCACATATTC
TGCTGACGCACCGGTGCAGCCTTTTTTCTCCTGCCACATGAAGCACTTCA
CTGACACCCTCATCAGTGCCAACATAGTAAGCCAGTATACACTCCGCTAG
CGCTGAGGTCTGCCTCGTGAAGAAGGTGTTGCTGACTCATACCAGGCCTG
AATCGCCCCATCATCCAGCCAGAAAGTGAGGGAGCCACGGTTGATGAGAG
CTTTGTTGTAGGTGGACCAGTTGGTGATTTTGAACTTTTGCTTTGCCACG
GAACGGTCTGCGTTGTCGGGAAGATGCGTGATCTGATCCTTCAACTCAGC
AAAAGTTCGATTTATTCAACAAAGCCACGTTGTGTCTCAAAATCTCTGAT
GTTACATTGCACAAGATAAAAATATATCATCATGAACAATAAAACTGTCT
GCTTACATAAACAGTAATACAAGGGGTGTTATGAGCCATATTCAACGGGA
AACGTCTTGCTCGAGGCCGCGATTAAATTCCAACATGGATGCTGATTTAT
ATGGGTATAAATGGGCTCGCGATAATGTCGGGCAATCAGGTGCGACAATC
TATCGATTGTATGGGAAGCCCGATGCGCCAGAGTTGTTTCTGAAACATGG
CAAAGGTAGCGTTGCCAATGATGTTACAGATGAGATGGTCAGACTAAACT
GGCTGACGGAATTTATGCCTCTTCCGACCATCAAGCATTTTATCCGTACT
CCTGATGATGCATGGTTACTCACCACTGCGATCCCCGGGAAAACAGCATT
CCAGGTATTAGAAGAATATCCTGATTCAGGTGAAAATATTGTTGATGCGC
TGGCAGTGTTCCTGCGCCGGTTGCATTCGATTCCTGTTTGTAATTGTCCT
TTTAACAGCGATCGCGTATTTCGTCTCGCTCAGGCGCAATCACGAATGAA
TAACGGTTTGGTTGATGCGAGTGATTTTGATGACGAGCGTAATGGCTGGC
CTGTTGAACAAGTCTGGAAAGAAATGCATAAGCTTTTGCCATTCTCACCG
GATTCAGTCGTCACTCATGGTGATTTCTCACTTGATAACCTTATTTTTGA
CGAGGGGAAATTAATAGGTTGTATTGATGTTGGACGAGTCGGAATCGCAG
ACCGATACCAGGATCTTGCCATCCTATGGAACTGCCTCGGTGAGTTTTCT
CCTTCATTACAGAAACGGCTTTTTCAAAAATATGGTATTGATAATCCTGA
TATGAATAAATTGCAGTTTCATTTGATGCTCGATGAGTTTTTCTAATCAG
AATTGGTTAATTGGTTGTAACACTGGCAGAGCATTACGCTGACTTGACGG
GACGGCGGCTTTGTTGAATAAATCGAACTTTTGCTGAGTTGAAGGATCAG
ATCACGCATCTTCCCGACAACGCAGACCGTTCCGTGGCAAAGCAAAAGTT
CAAAATCACCAACTGGTCCACCTACAACAAAGCTCTCATCAACCGTGGCT
CCCTCACTTTCTGGCTGGATGATGGGGCGATTCAGGCCTGGTATGAGTCA
GCAACACCTTCTTCACGAGGCAGACCTCAGCGCTCAAAGATGCAGGGGTA
AAAGCTAACCGCATCTTTACCGACAAGGCATCCGGCAGTTCAACAGATCG
GGAAGGGCTGGATTTGCTGAGGATGAAGGTGGAGGAAGGTGATGTCATTC
TGGTGAAGAAGCTCGACCGTCTTGGCCGCGACACCGCCGACATGATCCAA
CTGATAAAAGAGTTTGATGCTCAGGGTGTAGCGGTTCGGTTTATTGACGA
CGGGATCAGTACCGACGGTGATATGGGGCAAATGGTGGTCACCATCCTGT
CGGCTGTGGCACAGGCTGAACGCCGGAGGATCAAGTCGGTCAAGCCAAGC
GCAACCAGCGGCACCGCCGCGAGCAACGTCGCAAGGGCGATCAGGGGACG
ATTTTTGCGAAGAATTTCCACGGTAAGAATCCAATCTCTCGAATTTAGGG
TGAAAGAAGCTTGGCATAGGGGTGTGCACGAACTCGGTGGAGGAAATTTC
CGCGGGGCAAGGCTTCGCGAAGCGGAGTCGCGGCAGTGGCTTTGAAGATC
TTTGGGAGCAGTCCTTGTGCGCTTACGAGGTGAGCCGGTGGGGAACCGTT
ATCTGCCTATGGTGTGAGCCCCCCTAGAGAGCTTCAAGAGCAATCAGCCC
GACCTAGAAAGGAGGCCAAGAGAGAGACCCCTACGGGGGGAACCGTTTTC
TGCCTACGAGATGGCACATTTACTGGGAAGCTTTACGGCGTCCTCGTGGA
AGTTCAATGCCCGCAGACTTAAGTGCTCTATTCACGGTCTGACGTGACAC
GCTAAATTCAGACATAGCTTCATTGATTGTCGGCCACGAGCCAGTCTCTC
CCTCAACAGTCATAAACCAACCTGCAATGGTCAAGCGATTTCCTTTAGCT
TTCCTAGCTTGTCGTTGACTGGACTTAGCTAGTTTTTCTCGCTGTGCTCG
GGCGTACTCACTGTTTGGGTCTTTCCAGCGTTCTGCGGCCTTTTTACCGC
CACGTCTTCCCATAGTGGCCAGAGCTTTTCGCCCTCGGCTGCTCTGCGTC
TCTGTCTGACGAGCAGGGACGACTGGCTGGCCTTTAGCGACGTAGCCGCG
CACACGTCGCGCCATCGTCTGGCGGTCACGCATCGGCGGCAGATCAGGCT
CACGGCCGTCTGCTCCGACCGCCTGAGCGACGGTGTAGGCACGCTCGTAG
GCGTCGATGATCTTGGTGTCTTTTAGGCGCTCACCAGCCGCTTTTAACTG
GTATCCCACAGTCAAAGCGTGGCGAAAAGCCGTCTCATCACGGGCGGCAC
GCCCTGGAGCAGTCCAGAGGACACGGACGCCGTCGATCAGCTCTCCAGAC
GCTTCAGCGGCGCTCGGCAGGCTTGCTTCAAGCGTGGCAAGTGCTTTTGC
TTCCGCAGTGGCTTTTCTTGCCGCTTCGATACGTGCCCGTCCGCTAGAAA
ACTCCTGCTCATAGCGTTTTTTAGGTTTTTCTGTGCCTGAGATCATGCGA
GCAACCTCCATAAGATCAGCTAGGCGATCCACGCGATTGTGCTGGGCATG
CCAGCGGTACGCGGTGGGATCGTCGGAGACGTGCAGTGGCCACCGGCTCA
GCCTATGTGAAAAAGCCTGGTCAGCGCCGAAAACGCGGGTCATTTCCTCG
GTCGTTGCAGCCAGCAGGCGCATATTCGGGCTGCTCATGCCTGCTGCGGC
ATACACCGGATCAATGAGCCAGATGAGCTGGCATTTCCCGCTCAGTGGAT
TCACGCCGATCCAAGCTGGCGCTTTTTCCAGGCGTGCCCAGCGCTCCAAA
ATCGCGTAGACCTCGGGGTTTACGTGCTCGATTTTCCCGCCGGCCTGGTG
GCTCGGCACATCAATGTCCAGGACAAGCACGGCTGCGTGCTGCGCGTGCG
TCAGAGCAACATACTGGCACCGGGCAAGCGATTTTGAACCAACTCGGTAT
AACTTCGGCTGTGTTTCTCCCGTGTCCGGGTCTTTGATCCAAGCGCTGGC
GAAGTCGCGGGTCTTGCTGCCCTGGAAATTTTCTCTGCCCAGGTGAGCGA
GGAATTCGCGGCGGTCTTCGCTCGTCCAGCCACGTGATCGCAGCGCGAGC
TCGGGATGGGTGTCGAACAGATCAGCGGAAAATTTCCAGGCCGGTGTGTC
AATGTCTCGTGAATCCGCTAGAGTCATTTTTGAGCGCTTTCTCCCAGGTT
TGGACTGGGGGTTAGCCGACGCCCTGTGAGTTACCGCTCACGGGGCGTTC
AACATTTTTCAGGTATTCGTGCAGCTTATCGCTTCTTGCCGCCTGTGCGC
TTTTTCGACGCGCGACGCTGCTGCCGATTCGGTGCAGGTGGTGGCGGCGC
TGACACGTCCTGGGCGGCCACGGCCACACGAAACGCGGCATTTACGATGT
TTGTCATGCCTGCGGGCACCGCGCCACGATCGCGGATAATTCTCGCTGCC
GCTTCCAGCTCTGTGACGACCATGGCCAAAATTTCGCTCGGGGGACGCAC
TTCCAGCGCCATTTGCGACCTAGCCGCCTCCAGCTCCTCGGCGTGGCGTT
TGTTGGCGCGCTCGCGGCTGGCTGCGGCACGACACGCATCTGAGCAATAT
TTTGCGCGCCGTCCTCGCGGGTCAGGCCGGGGAGGAATCAGGCCACCGCA
GTAGGCGCAACTGATTCGATCCTCCACTACTGTGCGTCCTCCTGGCGCTG
CCGAGCACGCAGCTCGTCAGCCAGCTCCTCAAGATCCGCCACGAGAGTTT
CTAGGTCGCTCGCGGCACTGGCCCAGTCTCGTGATGCTGGCGCGTCCGTC
GTATCGAGAGCTCGGAAAAATCCGATCACCGTTTTTAAATCGACGGCAGC
ATCGAGCGCGTCGGACTCCAGCGCGACATCAGAGAGATCCATAGCTGATG
ATTCGGGCCAATTTTGGTACTTCGTCGTGAAGGTCATGACACCATTATAA
CGAACGTTCGTTAAAGTTTTTGGCGGAAAATCACGCGGCACGAAAATTTT
CACGAAGCGGGACTTTGCGCAGCTCAGGGGTGCTAAAAATTTTGTATCGC
ACTTGATTTTTCCGAAAGACAGATTATCTGCAAACGGTGTGTCGTATTTC
TGGCTTGGTTTTTAAAAAATCTGGAATCGAAAATTTGCGGGGCGA
b)pJC1-PF1-U1A-F30::西兰花/UUCG-TF1和pJC1-PF1-U1A-TF1转化谷氨酸棒状杆菌
根据Tauch等人，2002(Tauch,A.,Kirchner,O.,B.,/>S.,Pühler,A.,and Kalinowski,J.Efficient Electrotransformation of Corynebacteriumdiphtheriae with a Mini-Replicon Derived from the Corynebacterium glutamicumPlasmid pGA 1.Curr.Microbiol.2002；45,362-367T)制备谷氨酸棒状杆菌菌株ATCC13032Δcg2273的感受态细胞，并用pJC1 PF1 U1A F30::西兰花/UUCG-TF1和pJC1-PF1-U1A-TF1转化。转化子的选择是在有15μg/ml卡那霉素的CGIII(Menkel，E.,Thierbach,G.,Eggeling,L.,and Sahm,H.Influence of increased aspartate availability onlysine formation by a recombinant strain of Corynebacterium glutamicum andutilization of fumarate.Appl.Environ.Microbiol.1989；55,684-688)琼脂(1％)板上进行的。由此获得的克隆被命名为谷氨酸棒状杆菌ATCC 13032Δcg2273 pJC1-PF1-U1A-F30::西兰花/UUCG-TF1或谷氨酸棒状杆菌ATCC 13032△cg2273 pJC1-PF1-U1A-TF1，这取决于用于转化的质粒。
c)谷氨酸棒状杆菌ATCC 13032Δcg2273 pJC1-PF1-U1A-F30::西兰花/UUCG-TF1的突变
将产生的菌株谷氨酸棒状杆菌ATCC 13032Δcg2273 pJC1-PF1-U1A-F30::西兰花/UUCG-TF1在CGIII培养基中培养过夜，温度为30℃，转速为120rpm，总体积为10mL，培养基中加入15μg/mL卡那霉素。使用来自该预培养物的细胞制备在5mL总体积的磷酸盐缓冲盐水(PBS)中OD600为0.5的细胞悬浮液。将N-甲基-N’-硝基-N-亚硝基胍(MNNG)添加至最终浓度为25μg/mL。孵育20分钟后，取1.5mL样品，离心(2000rpm，2分钟)并重悬于2mL PBS中。该洗涤步骤重复两次，然后在1.5mL PBS中最终重悬，并转移到含有15μg/mL卡那霉素的15mL新鲜CGIII培养基中。将培养物在无挡板摇瓶中30℃、120rpm下培养16小时。
d)通过荧光激活细胞分选(FACS)鉴定和分离显示最高荧光强度并因此高表达感兴趣RNA的细胞
使用最终浓度为500μM DFHBI的PBS将来自c)的再生培养物稀释至OD为0.6。孵育10m后，在配备有70μm喷嘴的AriaIII高速细胞分选机(BD Biosciences，Franklin Lakes，NJ，USA)中分析细胞悬浮液，并在70psi的鞘压下运行。使用488nm蓝色固体激光器进行激发。将前向散射特性(FSC)记录为小角度散射，将侧散射特性(SSC)记录为488nm激光器的正交散射。502nm长通和530/30nm带通滤波器的组合用于荧光检测。FACSDiva 8.0.1(BDBiosciences，San Jose，USA)用于FACS控制和数据分析。在数据采集之前，通过FSC-H与SSC-H图中的电子门控，将碎片和电子噪声排除在分析之外。对FSC-H对照FSC-W图中得到的群体进行另一个门控步骤，以排除双联。用这种两步选通产生的群体进行荧光采集(图1)。从培养液中分离出96个与未经MNNG处理的谷氨酸棒状杆菌ATCC 13032Δcg2273pJC1-PF1-U1A-F30::西兰花/UUCG-TF1相比荧光增强的细胞，并将其置于含有15μg/mL卡那霉素的CGIII琼脂平板(1％)上。
e)培养d)中分离的细胞用于表型验证
在分离的细胞中，19个在30℃下培养的接下来48小时内生长成菌落，并用于接种10mL CGIII培养基。分离的细胞在30℃、120rpm的无挡板摇瓶中进行培养。培养20小时后，使用最终浓度为500μM DFHBI的PBS将培养液稀释至OD 0.6。孵育10分钟后，使用d)中列出的设置在AriaIII高速细胞分选机中分析细胞悬浮液。在培养的19个培养物中，4个显示出与起始菌株谷氨酸棒状杆菌ATCC 13032Δcg2273 pJC1-PF1-U1A-F30::西兰花/UUCG-TF1相比增加1.5至2倍的荧光。
f)从d)中分离的细胞中提取感兴趣的RNA
使用e)中分析的培养基，使用Monarch总RNA试剂盒(New England Biolabs，Ipswich，MA，USA)将来自四个表现最好的菌株的1x109细胞用于RNA提取。使用安捷伦生物分析仪2100(安捷伦科技公司，Santa Clara，CA，USA)分析分离的RNA。增加的荧光对应于每提取的总RNA增加的靶RNA丰度。
表1
g)使用分离菌株生产无标签的靶RNA
使用Tauch等人，2002(Tauch,A.,Kirchner,O.,B.,/>S.,Pühler,A.,and Kalinowski,J.Efficient Electrotransformation of Corynebacteriumdiphtheriae with a Mini-Replicon Derived from the Corynebacterium glutamicumPlasmid pGA 1.Curr.Microbiol.2002；45,362-367)改造版本的转化方案从分离菌株中去除质粒pJC1-PF1-U1A-F30::西兰花/UUCG-TF1。根据Tauch等人，2002年，菌株被鉴定为有能力，并在不添加质粒DNA的情况下进行电穿孔。根据原始方案再生后，将细胞稀释并扩散到非选择性CGIII琼脂上。生长的菌落在含有15μg/mL卡那霉素的CGIII琼脂和非选择性CGIII琼脂上划线。通过菌落PCR证实从卡那霉素敏感细胞中成功去除pJC1-PF1-U1A-F30::西兰花/UUCG-TF1(没有引物组合pJC1_check_f和pJC1_check_rev的产物)。如b)所述，用pJC1-PF1-U1A-TF1转化由此产生的无质粒菌株，以能够在没有由F30支架和西兰花组成的标签的情况下产生靶RNA。与对照菌株谷氨酸棒状杆菌ATCC 13032Δcg2273 pJC1-PF1-U1A-TF1相比，根据e)中所述的菌株的培养以及如f)中所描述的所产生的RNA的提取和分析证实了靶RNA的产生增加。
该实验表明，本发明通过将迄今为止不显著的表型(RNA产生)与荧光输出连接，能够从诱变的细胞培养基中分离出具有改进的目的异源RNA产生的细胞。
实施例2
a)pK19msB_16S rRNA-西兰花载体的构建
通过化学合成的方式合成的DNA片段(16S rRNA-西兰花的SEQ ID NO:79)，并将其***pK19mobsacB的限制性位点EcoRI和HindIII中，得到质粒pK19msB_16S rRNB-西兰花(SEQ ID NO:80)(从美国南旧金山Twist Bioscience订购)，从而实现质粒的构建。SEQ IDNO:79包含核酸适配体整合位点(SEQ ID NO:81)上游601bp、用于验证正整合的限制性位点(SEQ ID NO:122:tctaga)、***位点中具有西兰花核酸适配体的F30支架(SEQ ID NO:69)和靶整合位点下游479bp(SEQ ID NO:83)。
SEQ ID NO:79
SEQ ID NO:81,82,83and 69组合:
CGCACAAGCGGCGGAGCATGTGGATTAATTCGATGCAACGCGAAGAACCTTACCTGGGCTTGACATGGACCGGATCGGCGTAGAGATACGTTTTCCCTTGTGGTCGGTTCACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGTCTTATGTTGCCAGCACATTGTGGTGGGTACTCATGAGAGACTGCCGGGGTTAACTCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGTCCAGGGCTTCACACATGCTACAATGGTCGGTACAGCGAGTTGCCACACCGTGAGGTGGAGCTAATCTCTTAAAGCCGGCCTCAGTTCGGATTGGGGTCTGCAACTCGACCCCATGAAGTCGGAGTCGCTAGTAATCGCAGATCAGCAACGCTGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACGTCATGAAAGTTGGTAACACCCGAAGCCAGTGGCCCAACCTTTTAGGGGGGAGCTGTCGAAGGTGGGATCGGCGATTGGGACGAAGTCGTAACAAGGTAGCCGTACCGGAAGGTGCGGCTGGATCTAGATTGCCATGTGTATGTGGGAGACGGTCGGGTCCAGATATTCGTATCTGTCGAGTAGAGTGTGGGCTCCCACATACTCTGATGATCCTTCGGGATCATTCATGGCAACCTCCTTTCTAAGGAGCTTTATTAACCCACATCAGACTGTGTCTGGTTGGTGGGTTGTTGGTGTTGGAACCCGTATGTGGTTGCCATCAACATATTTTTAATCGGGTGGAGATGACCCCTCGGGTGACAACAACACAGCAAACAGTGCTGTGATTAATAGGTGGCATGCTGTTGGGTGTCTGGAATGACATCGCAAGCATCACCTTTTGGTGGTGTGTGTGGGTTGTTTCTAACATCGAGCATCGTCAACACGGGTAGAGAATGTTGTGTTCTTTGGTTGTGGTGGGGGTGGTGTGTTGTGTGAGAACTGTATAGTGGACGCGAGCATCTTTATTTTTTTGTTTTTTGTTGTGTGATACCGAACGCGCCCGCACTTTGTGTGTGGGTTATAGTATTTTGTTTGTTGTTTTGTAGGGCACACGGTGGATGCCTTGGCATATCAAGCCGATGAAGGACGTGAGAGGCTGCGTTATGCCTCG
SEQ ID NO:80
pK19msB_16S rRNA-西兰花:
CGCACAAGCGGCGGAGCATGTGGATTAATTCGATGCAACGCGAAGAACCTTACCTGGGCTTGACATGGACCGGATCGGCGTAGAGATACGTTTTCCCTTGTGGTCGGTTCACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGTCTTATGTTGCCAGCACATTGTGGTGGGTACTCATGAGAGACTGCCGGGGTTAACTCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGTCCAGGGCTTCACACATGCTACAATGGTCGGTACAGCGAGTTGCCACACCGTGAGGTGGAGCTAATCTCTTAAAGCCGGCCTCAGTTCGGATTGGGGTCTGCAACTCGACCCCATGAAGTCGGAGTCGCTAGTAATCGCAGATCAGCAACGCTGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACGTCATGAAAGTTGGTAACACCCGAAGCCAGTGGCCCAACCTTTTAGGGGGGAGCTGTCGAAGGTGGGATCGGCGATTGGGACGAAGTCGTAACAAGGTAGCCGTACCGGAAGGTGCGGCTGGATCTAGATTGCCATGTGTATGTGGGAGACGGTCGGGTCCAGATATTCGTATCTGTCGAGTAGAGTGTGGGCTCCCACATACTCTGATGATCCTTCGGGATCATTCATGGCAACCTCCTTTCTAAGGAGCTTTATTAACCCACATCAGACTGTGTCTGGTTGGTGGGTTGTTGGTGTTGGAACCCGTATGTGGTTGCCATCAACATATTTTTAATCGGGTGGAGATGACCCCTCGGGTGACAACAACACAGCAAACAGTGCTGTGATTAATAGGTGGCATGCTGTTGGGTGTCTGGAATGACATCGCAAGCATCACCTTTTGGTGGTGTGTGTGGGTTGTTTCTAACATCGAGCATCGTCAACACGGGTAGAGAATGTTGTGTTCTTTGGTTGTGGTGGGGGTGGTGTGTTGTGTGAGAACTGTATAGTGGACGCGAGCATCTTTATTTTTTTGTTTTTTGTTGTGTGATACCGAACGCGCCCGCACTTTGTGTGTGGGTTATAGTATTTTGTTTGTTGTTTTGTAGGGCACACGGTGGATGCCTTGGCATATCAAGCCGATGAAGGACGTGAGAGGCTGCGTTATGCCTCGAAGCTTGGCGTAATCATGGTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCACACAACATACGAGCCGGAAGCATAAAGTGTAAAGCCTGGGGTGCCTAATGAGTGAGCTAACTCACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTAT
TGGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGC
GAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAAC
GCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCC
GCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACG
CTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTG
GAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCT
TTCTCCCTTCGGGAAGCGTGGCGCTTTCTCAATGCTCACGCTGTAGGTATCTCAGTTCGG
TGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGC
TGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCA
CTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAG
AGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGGACAGTATTTGGTATCTGCG
CTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAA
ACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAA
AGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAA
ACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTT
TGGGGTGGGCGAAGAACTCCAGCATGAGATCCCCGCGCTGGAGGATCATCCAGCCCTG
ATAGAAACAGAAGCCACTGGAGCACCTCAAAAACACCATCATACACTAAATCAGTAAGT
TGGCAGCATCACCCGACGCACTTTGCGCCGAATAAATACCTGTGACGGAAGATCACTTC
GCAGAATAAATAAATCCTGGTGTCCCTGTTGATACCGGGAAGCCCTGGGCCAACTTTTG
GCGAAAATGAGACGTTGATCGGCACGTAAGAGGTTCCAACTTTCACCATAATGAAATAA
GATCACTACCGGGCGTATTTTTTGAGTTATCGAGATTTTCAGGAGCTGATAGAAACAGAA
GCCACTGGAGCACCTCAAAAACACCATCATACACTAAATCAGTAAGTTGGCAGCATCAC
CCGACGCACTTTGCGCCGAATAAATACCTGTGACGGAAGATCACTTCGCAGAATAAATA
AATCCTGGTGTCCCTGTTGATACCGGGAAGCCCTGGGCCAACTTTTGGCGAAAATGAGA
CGTTGATCGGCACGTAAGAGGTTCCAACTTTCACCATAATGAAATAAGATCACTACCGGG
CGTATTTTTTGAGTTATCGAGATTTTCAGGAGCTCTTTGGCATCGTCTCTCGCCTGTCCCC
TCAGTTCAGTAATTTCCTGCATTTGCCTGTTTCCAGTCGGTAGATATTCCACAAAACAGC
AGGGAAGCAGCGCTTTTCCGCTGCATAACCCTGCTTCGGGGTCATTATAGCGATTTTTTC
GGTATATCCATCCTTTTTCGCACGATATACAGGATTTTGCCAAAGGGTTCGTGTAGACTTT
CCTTGGTGTATCCAACGGCGTCAGCGGGGCAGGATAGGTGAAGTAGGCCCACCCGCGA
GCGGGTGTTCCTTCTTCACTGTCCCTTATTCGCACCTGGCGGTGCTCAACGGGAATCCTG
CTCTGCGAGGCTGGCCGGCTACCGCCGGCGTAACAGATGAGGGCAAGCGGATGGCTGA
TGAAACCAAGCCAACCAGGAAGGGCAGCCCACCTATCAAGGTGTACTGCCTTCCAGAC
GAACGAAGAGCGATTGAGGAAAAGGCGGCGGCGGCCGGCATGAGCCTGTCGGCCTACC
TGCTGGCCGTCGGCCAGGGCTACAAAATCACGGGCGTCGTGGACTATGAGCACGTCCGC
GAGGGCGTCCCGGAAAACGATTCCGAAGCCCAACCTTTCATAGAAGGCGGCGGTGGAA
TCGAAATCTCGTGATGGCAGGTTGGGCGTCGCTTGGTCGGTCATTTCGCTCGGTACCCAT
CGGCATTTTCTTTTGCGTTTTTATTTGTTAACTGTTAATTGTCCTTGTTCAAGGATGCTGTC
TTTGACAACAGATGTTTTCTTGCCTTTGATGTTCAGCAGGAAGCTCGGCGCAAACGTTG
ATTGTTTGTCTGCGTAGAATCCTCTGTTTGTCATATAGCTTGTAATCACGACATTGTTTCCT
TTCGCTTGAGGTACAGCGAAGTGTGAGTAAGTAAAGGTTACATCGTTAGGATCAAGATC
CATTTTTAACACAAGGCCAGTTTTGTTCAGCGGCTTGTATGGGCCAGTTAAAGAATTAGA
AACATAACCAAGCATGTAAATATCGTTAGACGTAATGCCGTCAATCGTCATTTTTGATCCG
CGGGAGTCAGTGAACAGGTACCATTTGCCGTTCATTTTAAAGACGTTCGCGCGTTCAATT
TCATCTGTTACTGTGTTAGATGCAATCAGCGGTTTCATCACTTTTTTCAGTGTGTAATCAT
CGTTTAGCTCAATCATACCGAGAGCGCCGTTTGCTAACTCAGCCGTGCGTTTTTTATCGC
TTTGCAGAAGTTTTTGACTTTCTTGACGGAAGAATGATGTGCTTTTGCCATAGTATGCTTT
GTTAAATAAAGATTCTTCGCCTTGGTAGCCATCTTCAGTTCCAGTGTTTGCTTCAAATACT
AAGTATTTGTGGCCTTTATCTTCTACGTAGTGAGGATCTCTCAGCGTATGGTTGTCGCCTG
AGCTGTAGTTGCCTTCATCGATGAACTGCTGTACATTTTGATACGTTTTTCCGTCACCGTC
AAAGATTGATTTATAATCCTCTACACCGTTGATGTTCAAAGAGCTGTCTGATGCTGATACG
TTAACTTGTGCAGTTGTCAGTGTTTGTTTGCCGTAATGTTTACCGGAGAAATCAGTGTAG
AATAAACGGATTTTTCCGTCAGATGTAAATGTGGCTGAACCTGACCATTCTTGTGTTTGG
TCTTTTAGGATAGAATCATTTGCATCGAATTTGTCGCTGTCTTTAAAGACGCGGCCAGCG
TTTTTCCAGCTGTCAATAGAAGTTTCGCCGACTTTTTGATAGAACATGTAAATCGATGTGT
CATCCGCATTTTTAGGATCTCCGGCTAATGCAAAGACGATGTGGTAGCCGTGATAGTTTG
CGACAGTGCCGTCAGCGTTTTGTAATGGCCAGCTGTCCCAAACGTCCAGGCCTTTTGCA
GAAGAGATATTTTTAATTGTGGACGAATCAAATTCAGAAACTTGATATTTTTCATTTTTTT
GCTGTTCAGGGATTTGCAGCATATCATGGCGTGTAATATGGGAAATGCCGTATGTTTCCTT
ATATGGCTTTTGGTTCGTTTCTTTCGCAAACGCTTGAGTTGCGCCTCCTGCCAGCAGTGC
GGTAGTAAAGGTTAATACTGTTGCTTGTTTTGCAAACTTTTTGATGTTCATCGTTCATGTC
TCCTTTTTTATGTACTGTGTTAGCGGTCTGCTTCTTCCAGCCCTCCTGTTTGAAGATGGCA
AGTTAGTTACGCACAATAAAAAAAGACCTAAAATATGTAAGGGGTGACGCCAAAGTATA
CACTTTGCCCTTTACACATTTTAGGTCTTGCCTGCTTTATCAGTAACAAACCCGCGCGATT
TACTTTTCGACCTCATTCTATTAGACTCTCGTTTGGATTGCAACTGGTCTATTTTCCTCTTT
TGTTTGATAGAAAATCATAAAAGGATTTGCAGACTACGGGCCTAAAGAACTAAAAAATC
TATCTGTTTCTTTTCATTCTCTGTATTTTTTATAGTTTCTGTTGCATGGGCATAAAGTTGCC
TTTTTAATCACAATTCAGAAAATATCATAATATCTCATTTCACTAAATAATAGTGAACGGC
AGGTATATGTGATGGGTTAAAAAGGATCGATCCTCTAGCGAACCCCAGAGTCCCGCTCA
GAAGAACTCGTCAAGAAGGCGATAGAAGGCGATGCGCTGCGAATCGGGAGCGGCGATA
CCGTAAAGCACGAGGAAGCGGTCAGCCCATTCGCCGCCAAGCTCTTCAGCAATATCACG
GGTAGCCAACGCTATGTCCTGATAGCGGTCCGCCACACCCAGCCGGCCACAGTCGATGA
ATCCAGAAAAGCGGCCATTTTCCACCATGATATTCGGCAAGCAGGCATCGCCATGGGTC
ACGACGAGATCCTCGCCGTCGGGCATCCGCGCCTTGAGCCTGGCGAACAGTTCGGCTG
GCGCGAGCCCCTGATGCTCTTCGTCCAGATCATCCTGATCGACAAGACCGGCTTCCATCC
GAGTACGTGCTCGCTCGATGCGATGTTTCGCTTGGTGGTCGAATGGGCAGGTAGCCGGA
TCAAGCGTATGCAGCCGCCGCATTGCATCAGCCATGATGGATACTTTCTCGGCAGGAGCA
AGGTGAGATGACAGGAGATCCTGCCCCGGCACTTCGCCCAATAGCAGCCAGTCCCTTCC
CGCTTCAGTGACAACGTCGAGCACAGCTGCGCAAGGAACGCCCGTCGTGGCCAGCCAC
GATAGCCGCGCTGCCTCGTCTTGGAGTTCATTCAGGGCACCGGACAGGTCGGTCTTGAC
AAAAAGAACCGGGCGCCCCTGCGCTGACAGCCGGAACACGGCGGCATCAGAGCAGCC
GATTGTCTGTTGTGCCCAGTCATAGCCGAATAGCCTCTCCACCCAAGCGGCCGGAGAAC
CTGCGTGCAATCCATCTTGTTCAATCATGCGAAACGATCCTCATCCTGTCTCTTGATCAG
ATCTTGATCCCCTGCGCCATCAGATCCTTGGCGGCAAGAAAGCCATCCAGTTTACTTTGC
AGGGCTTCCCAACCTTACCAGAGGGCGCCCCAGCTGGCAATTCCGGTTCGCTTGCTGTC
CATAAAACCGCCCAGTCTAGCTATCGCCATGTAAGCCCACTGCAAGCTACCTGCTTTCTC
TTTGCGCTTGCGTTTTCCCTTGTCCAGATAGCCCAGTAGCTGACATTCATCCGGGGTCAG
CACCGTTTCTGCGGACTGGCTTTCTACGTGTTCCGCTTCCTTTAGCAGCCCTTGCGCCCT
GAGTGCTTGCGGCAGCGTGAAGCTAGCTTATCGCGCCATTCGCCATTCAGGCTGCGCAA
CTGTTGGGAAGGGCGATCGGTGCGGGCCTCTTCGCTATTACGCCAGCTGGCGAAAGGGG
GATGTGCTGCAAGGCGATTAAGTTGGGTAACGCCAGGGTTTTCCCAGTCACGACGTTGT
AAAACGACGGCCAGTGAATT
SEQ ID NO:81
靶整合地点(上游):
CGCACAAGCGGCGGAGCATGTGGATTAATTCGATGCAACGCGAAGAACCTTACCTGGGCTTGACATGGACCGGATCGGCGTAGAGATACGTTTTCCCTTGTGGTCGGTTCACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGTCTTATGTTGCCAGCACATTGTGGTGGGTACTCATGAGAGACTGCCGGGGTTAACTCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGTCCAGGGCTTCACACATGCTACAATGGTCGGTACAGCGAGTTGCCACACCGTGAGGTGGAGCTAATCTCTTAAAGCCGGCCTCAGTTCGGATTGGGGTCTGCAACTCGACCCCATGAAGTCGGAGTCGCTAGTAATCGCAGATCAGCAACGCTGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACGTCATGAAAGTTGGTAACACCCGAAGCCAGTGGCCCAACCTTTTAGGGGGGAGCTGTCGAAGGTGGGATCGGCGATTGGGACGAAGTCGTAACAAGGTAGCCGTACCGGAAGGTGCGGCTGGA
SEQ ID NO:82
用于验证正整合的限制位点
TCTAGA
SEQ ID NO:83
靶整合位点(下游)：
CCTCCTTTCTAAGGAGCTTTATTAACCCACATCAGACTGTGTCTGGTTGGTGGGTTGTTGGTGTTGGAACCCGTATGTGGTTGCCATCAACATATTTTTAATCGGGTGGAGATGACCCCTCGGGTGACAACAACACAGCAAACAGTGCTGTGATTAATAGGTGGCATGCTGTTGGGTGTCTGGAATGACATCGCAAGCATCACCTTTTGGTGGTGTGTGTGGGTTGTTTCTAACATCGAGCATCGTCAACACGGGTAGAGAATGTTGTGTTCTTTGGTTGTGGTGGGGGTGGTGTGTTGTGTGAGAACTGTATAGTGGACGCGAGCATCTTTATTTTTTTGTTTTTTGTTGTGTGATACCGAACGCGCCCGCACTTTGTGTGTGGGTTATAGTATTTTGTTTGTTGTTTTGTAGGGCACACGGTGGATGCCTTGGCATATCAAGCCGATGAAGGACGTGAGAGGCTGCGTTATGCCTCG
b)通过使用质粒pK19msB_16S rRNA-西兰花的转化和选择，在谷氨酸棒状杆菌ATCC 13032Δcg2273的16S rRNA的3'端整合F30::西兰花
根据Tauch等人，2002(Tauch,A.,Kirchner,O.,B.,/>S.,Pühler,A.,and Kalinowski,J.Efficient Electrotransformation of Corynebacteriumdiphtheriae with a Mini-Replicon Derived from the Corynebacterium glutamicumPlasmid pGA 1.Curr.Microbiol.2002；45,362-367T)制备谷氨酸棒状杆菌菌株ATCC13032Δcg2273的感受态细胞，并通过电穿孔用pK19msB_16S rRNA-西兰花转化。转化子的选择是在有25μg/ml卡那霉素的BHI(脑心输注)琼脂(1％)板上进行的。第一次和第二次重组如Niebisch和Bott，2001(Niebisch and Bott.Molecular analysis of thecytochrome bc1-aa3 branch of the Corynebacterium glutamicum respiratory chaincontaining an unusual diheme cytochrome c1.Arch.Microbiol.2001；175,282-294)先前所述进行。使用引物16S rRNA-西兰花_for(SEQ ID NO:84)和16S rRNA-西兰花_rev(SEQID NO:85)通过菌落PCR验证得到的克隆。所得到的PCR产物被限制性内切酶XbaI消化，因为只有成功整合核酸适配体的克隆才能被XbaI吸收。谷氨酸棒状杆菌ATCC 13032Δcg2273的基因组包含六个rrn簇拷贝(rrnA、rrnB、rrnC、rrnD、rrnE、rrnF)，每个包含16SrRNA、23SrRNA和5S rRNA(Martin，Barreiro，Gonzalez-Lavado，Barriuso.Ribosomal RNA andribosomal proteins in corynebacteria.2003.J Biotechnol.4；104(1-3):41-53).由于所有rrn簇具有很高的序列相似性，所有六个簇都是F30::西兰花的潜在整合位点。为此，使用通用引物(16SrRNA-西兰花_rev，SEQ ID NO:85)和簇特异性引物(rrnA_rev SEQ ID NO:86，rrnB_rev SEQ ID NO:87，rrnC_rev SEQ ID NO:88，rrnD_rev SEQ ID NO:89，rrnE_revSEQ ID NO:90，rrnF_rev SEQ ID NO:91)，通过菌落PCR再次测试在第一次菌落PCR和消化中显示阳性整合的菌株。为了进一步的研究，使用了一个在rrnA簇的3'端含有16S rRNA-F30::西兰花融合物的克隆。由此产生的菌株被命名为谷氨酸棒状杆菌ATCC 13032Δcg2273_16S rRNA-西兰花。
SEQ ID NO:84
16S rRNA-西兰花_for:
GTCTTCCACGACTTCTGTGC
SEQ ID NO:85
16S rRNA-西兰花_rev:
GTGTAAACCTCCACACCAGC
SEQ ID NO:86
rrnA_rev:
CTTGACCTGGGAAGTTGCG
SEQ ID NO:87
rrnB_rev:
GCAAGATTGCTTGCTACCAC
SEQ ID NO:88
rrnC_rev:
AGAAACTCGGAGCGACCATC
SEQ ID NO:89
rrnD_rev:
CGTCACACATCGCTCTACAG
SEQ ID NO:90
rrnE_rev:
GAAGCCTTCCCATCAAGCATC
SEQ ID NO:91
rrnF_rev:
CACATCAAGGTGACACGGAG
c)使用荧光活化细胞分选(FACS)培养和验证细胞
将产生的菌株谷氨酸棒状杆菌ATCC 13032Δcg2273_16S rRNA-西兰花在BHI琼脂平板上划线，在30℃下培养过夜。将生长的细胞重悬于CGIII培养基中，并在含有2mL CGIII培养液的管中将OD600调节至0.75。将细胞在30℃和120rpm下孵育4小时。随后，使用最终浓度为500μM DFHBI的PBS将细胞稀释至OD600为0.6。使用实施例1d)中已经描述的AriaIII高速细胞分选器对细胞进行分析。与未染色的细胞相比，DFHBI染色的细胞显示出显著增加的荧光输出(参见图2)。
d)RNA的提取和16S rRNA-西兰花的逆转录定量PCR定量
根据实施例1f)分离RNA，每个样品使用1.38×109个细胞。根据Wolf等人的方案进行逆转录定量PCR(qPCR)(Wolf，Timo et al.(2017)The MalR type regulator AcrC is atranscriptional repressor of acarbose biosynthetic genes in Actinoplanessp.SE50/110,BMC Genomics)，在罗氏公司(德国曼海姆)的96***中的96孔板(Sarstedt，Nümbrecht，德国)中使用SensiFast SYBR No-Rox一步试剂盒(Bioline，London，United Kingdom)，使用/>96SW 1.1(罗氏，曼海姆，德国)。相对RNA量计算为2-ΔCq。ΔCq计算为菌株谷氨酸棒状杆菌ATCC 13032Δcg2273_16S rRNA-西兰花与基因组中没有F30::西兰花整合的对照菌株谷氨酸棒状杆菌ATCC 13032Δcg2273的平均Cq的差异。对于qPCR，引物对qPCR_broc_fw(SEQ ID NO:92)和qPCR_broc_rev(SEQ IDNO:93)用于扩增结合F30::西兰花片段的233bp片段。结果显示了相对转录物水平，并验证了菌株谷氨酸棒状杆菌ATCC 13032Δcg2273_16S rRNA-西兰花制备的RNA样品中16SrRNA-F30::西兰花转录物的存在(参见图3)。
SEQ ID NO:92
qPCR_broc_fw:
tcatgaaagttggtaacacccgaag
SEQ ID NO:93
qPCR_broc_rev:
ttgccatgaatgatcccgaaggat
该实验显示了迄今为止不显著的表型(RNA产生)与荧光输出的成功连接。在这个实验中，产生的RNA长度为1545个核苷酸，是从***细胞的染色体转录而来。因此，根据实施例1中所示的程序，使用本发明可以优化染色体基因座中编码的长RNA的发酵生产。
实施例3
a)载体pUC18_PT7-U1A-F30::西兰花/UUCG-T7的构建
通过化学合成合成的DNA片段(PT7-U1A-F30::西兰花/UUCG-TT7的SEQ ID NO:94)并将其连接到pUC18中得到质粒pUC18-PT7-U1A-F30::西兰花/UUCG-TT7(SEQ ID NO：95，从美国南旧金山Twist Bioscience订购)进行质粒的构建(Norrander J,Kempe T,MessingJ.Construction of improved M13 vectors using oligodeoxynucleotidedirectedmutagenesis.Gene.1983Oec；26(1):1 01-6.)。SEQ ID NO:94包含启动子PT7(SEQ ID NO:66)、感兴趣的RNA(SEQ ID NO:68)、在第一整合点具有西兰花核酸适配体和在第二整合点具有“UUCG间隔”的F30支架(SEQ ID NO:69)和终止子序列TT7(SEQ ID NO:97)。
在用限制性内切酶EcoRI和HindIII切割合成的DNA片段并随后纯化反应混合物之后，将切下的DNA片段用于与载体pUC18的连接反应，该载体pUC18也已用EcoRI和HindIII线性化并去磷酸化。将连接混合物直接用于转化大肠杆菌DH5α，并在含有100μg/ml羧苄青霉素的LB板上进行转化子的筛选。16个菌落生长在这些平板上，因此对羧苄青霉素具有耐药性，用于菌落PCR。用引物pUC18_check_f(SEQ ID NO:98)和pUC18_check_rev(SEQ ID NO:99)进行菌落PCR，以分析合成的片段是否***载体pUC18中。在琼脂糖凝胶上对菌落PCR产物的分析显示预期的PCR产物具有428bp的大小(pUC18-PT7-U1A-F30::西兰花/UUCG-T7)，于是培养三个菌落用于更大规模的质粒制备。培养16小时后，通过离心收集这些培养物，并制备质粒DNA。用菌落PCR中使用的引物对其中两种质粒制剂进行测序，***物的序列显示与预期序列100％一致。所得质粒命名为pUC18-PT7-U1A-F30::西兰花/UUCG-T7(SEQ IDNO:95)。
SEQ ID NO:94
SEQ ID NO:68、69、96和97组合，在5'-端具有EcoRI识别位点，在3'-端具有Hindll识别位点：
GAATTCTAATACGACTCACTATAGAGCGGGAGACCGCTCGACCTTAGTTCTCCTGTTGCGGGGGAGTTCATGGGATCCAGGTACCCAGGGCGAGGCTTATCCATTGCACTCCGGATGTGCTGACCCCTGTTGCCATGTGTATGTGGGAGACGGTCGGGTCCAGATATTCGTATCTGTCGAGTAGAGTGTGGGCTCCCACATACTCTGATGATCCTTCGGGATCATTCATGGCAACTAGCATAACCCCTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTGAAGCTT
SEQ ID NO:95
pUC18-PT7-U1A-F30::西兰花/UUCG-TT7:
AATTCTAATACGACTCACTATAGAGCGGGAGACCGCTCGACCTTAGTTCTCCTGTTGCGGGGGAGTTCATGGGATCCAGGTACCCAGGGCGAGGCTTATCCATTGCACTCCGGATGTGCTGACCCCTGTTGCCATGTGTATGTGGGAGACGGTCGGGTCCAGATATTCGTATCTGTCGAGTAGAGTGTGGGCTCCCACATACTCTGATGATCCTTCGGGATCATTCATGGCAACTAGCATAACCCCTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTGAAGCTTGGCACTGGCCGTCGTTTTACAACGTCGTGACTGGGAAAACCCTGGCGTTACCCAACTTAATCGCCTTGCAGCACATCCCCCTTTCGCCAGCTGGCGTAATAGCGAAGAGGCCCGCACCGATCGCCCTTCCCAACAGTTGCGCAGCCTGAATGGCGAATGGCGCCTGATGCGGTATTTTCTCCTTACGCATCTGTGCGGTATTTCACACCGCATATGGTGCACTCTCAGTACAATCTGCTCTGATGCCGCATAGTTAAGCCAGCCCCGACACCCGCCAACACCCGCTGACGCGCCCTGACGGGCTTGTCTGCTCCCGGCATCCGCTTACAGACAAGCTGTGACCGTCTCCGGGAGCTGCATGTGTCAGAGGTTTTCACCGTCATCACCGAAACGCGCGAGACGAAAGGGCCTCGTGATACGCCTATTTTTATAGGTTAATGTCATGATAATAATGGTTTCTTAGACGTCAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAGACAATAACCCTGATAAATGCTTCAATAATATTGAAAAAGGAAGAGTATGAGTATTCAACATTTCCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCACGAGTGGGTTACATCGAACTGGATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTATTGACGCCGGGCAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCACCAGTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAGAATTATGCAGTGCTGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGACAACGATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCATACCAAACGACGAGCGTGACACCACGATGCCTGTAGCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAACTATGGATGAACGAAATAGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTAACTGTCAGACCAAGTTTACTCATATATACTTTAGATTGATTTAAAACTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTCCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGAAGAGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATGCAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGAGCGCAACGCAATTAATGTGAGTTAGCTCACTCATTAGGCACCCCAGGCTTTACACTTTATGCTTCCGGCTCGTATGTTGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGCTATGACCATGATTACG
SEQ ID NO:96
PT7:
TAATACGACTCACTATAG
SEQ ID NO:97
TT7:
CTAGCATAACCCCTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTG
SEQ ID NO:98
pUC18_check_f:
TTCCGGCTCGTATGTTGTG
SEQ ID NO:99
pUC18_check_rev:
AGGCGATTAAGTTGGGTAACG
b)用pUC18 PT7 U1A F30::西兰花/UUCG-TT7转化大肠杆菌HT115
为了在大肠杆菌HT115细胞中转化质粒((Timmons,Court,Fire(2001)Ingestionof bacterially expressed dsRNAs can produce specific and potent geneticinterference in Caenorhabditis elegans.Gene 263(2001)103-112)，使用根据Chung等人，1989(Chung,Niemela,Miller(1989).One-step preparation of competentEscherichia coli:Transformation and storage of bacterial cells in the samesolution.Proc Natl Acad Sci USA 86,2172-2175).)的转化和储存溶液(TSS)转化方案。将目标菌株的单个菌落接种在含有3mL LB培养基的试管中，并生长直到OD600达到0.3和0.8之间。随后，将培养物在冰上冷却10分钟。加入等体积(3mL)的冰冷的2x TSS(8g/L细菌-胰蛋白酶、5g/L酵母提取物、5g/L NaCl、200g/L PEG 8000)，并通过避免加热细胞来彻底涡旋管。将细菌悬浮液在冰上再孵育10分钟。向1mL感受态细胞中加入至少10ng质粒DNA，并通过涡旋混合。然后将悬浮液在冰上放置30分钟，并将200μL培养物接种在含有100μg/mL羧苄青霉素的LB琼脂板(1％)上。
c)使用荧光激活细胞分选(FACS)对大肠杆菌HT115_pUC18_PT7-U1A-F30::西兰花/UUCG-TT7细胞的培养和表型验证
将大肠杆菌HT115_pUC18_PT7-U1A-F30::西兰花/UUCG-TT7细胞从单个菌落接种到含有2mL 2x YT培养基(16g/L胰蛋白酶、10g/L酵母提取物和5g/L NaCl)和100μg/mL羧苄青霉素的试管中，并在37℃和120rpm下培养过夜。第二天，使用预培养物接种2mL含有100μg/mL羧苄青霉素的新鲜2x YT培养基，OD600为0.1。细胞在37℃和120rpm下生长，三小时后，通过添加0.4mM IPTG诱导T7 RNA聚合酶的表达。继续培养四个小时。然后，使用最终浓度为50μM DFHBI的PBS将细胞稀释至OD600为0.6。孵育10分钟后，如实施例1d)中所述，通过FACS分析细胞悬浮液。DFHBI染色的大肠杆菌HT115_pUC18_PT7-U1A-F30::西兰花/UUCG-TT7细胞与未染色的细胞相比显示出显著增加的荧光。
该实验显示了迄今为止不显著的表型(RNA产生)与荧光输出的成功连接。在这个实验中，产生的RNA是从革兰氏阴性细菌细胞中的载体转录出来的。因此，根据实施例1中所示的程序，使用本发明可以使用革兰氏阴性细菌细胞优化RNA的发酵生产。
实施例4
a)载体pJC1-PF1-U1A-F30::芒果3-TF1的构建
质粒的构建是通过化学合成的方式合成的DNA片段(PF1-U1A-F30::芒果3-TF1的SEQ ID NO:100，从美国旧金山南部的Twist Bioscience订购)及其连接到pJC1(CREMER,J.,TREPTOW,C.,EGGELlNG,L.,and SAHM,H.Regulation of Enzymes of LysineBiosynthesis in Corynebacterium glutamicum.Microbiol.1988；134,3221-3229)。SEQID NO:100包含启动子PF1(SEQ ID NO:67)、感兴趣的RNA(SEQ ID NO:68)、在整合点具有芒果3核酸适配体的F30支架(SEQ ID NO:101)和终止子序列TF1(SEQ ID NO:70)。
在用限制性内切酶XbaI和SalI切割合成的DNA片段并随后纯化反应混合物之后，将切下的DNA片段用于与载体pJC1的连接反应中，该载体也已用XbaI或SalI线性化并去磷酸化。将连接混合物直接用于转化大肠杆菌DH5α，并在含有50μg/ml卡那霉素的LB板上进行转化子的筛选。16个菌落生长在这些平板上，因此对卡那霉素具有抗性，用于菌落PCR。用引物pJC1_check_f(SEQ ID NO:73)和pJC1_check_rev(SEQ ID NO:74)进行菌落PCR，以分析合成的片段是否***载体pJC1中。在琼脂糖凝胶上对菌落PCR产物的分析显示预期的PCR产物具有682bp的大小(pJC1-PF1-U1A-F30::芒果3-TF1)，于是培养三个菌落用于更大规模的质粒制备。培养16小时后，通过离心收集这些培养物，并制备质粒DNA。用菌落PCR中使用的引物对其中两种质粒制剂进行测序，***物的序列显示与预期序列100％一致。所得质粒命名为pJC1-PF1-U1A-F30::芒果3-TF1(SEQ ID NO:102)。
SEQ ID NO:100
SEQ ID NO:67，68，101，70组合，5'-端的XbaI识别位点、3'-端的SalI识别位点和序列101上游的SacI识别位点：
CTGTCTCTAGACTCGAGCGGGACGGTCGAACCAGCTTCAAGCGACCGGATGAGTATGTTACAGTAGATAGCGAGCGGGAGACCGCTCGACCTTAGTTCTCCTGTTGCGGGGGAGTTCATGGGATCCAGGTACCCAGGGCGAGGCTTATCCATTGCACTCCGGATGTGCTGACCCCTGGGAGCTCTTGCCATGTGTATGTGGGGGAAGGATTGGTATGTGGTATACCCACATACTCTGATGATCCTTCGGGATCATTCATGGCAAGCTAGCATAGCATAAAATAACGCCCCACCTTCTTAACGGGAGGTGGGGCGTTATTTTTACGTCGAC
SEQ ID NO:101
F30，芒果3在其***位点1和“TTCG”在***位点2：
TTGCCATGTGTATGTGGGGGAAGGATTGGTATGTGGTATACCCACATACTCTGATGATCCTTCGGGATCATTCATGGCAA
SEQ ID NO:102
pJC1-PF1-U1A-F30::芒果3-TF1:
CTAGACTCGAGCGGGACGGTCGAACCAGCTTCAAGCGACCGGATGAGTATGTTACAGTAGATAGCGAGCGGGAGACCGCTCGACCTTAGTTCTCCTGTTGCGGGGGAGTTCATGGGATCCAGGTACCCAGGGCGAGGCTTATCCATTGCACTCCGGATGTGCTGACCCCTGGGAGCTCTTGCCATGTGTATGTGGGGGAAGGATTGGTATGTGGTATACCCACATACTCTGATGATCCTTCGGGATCATTCATGGCAAGCTAGCATAGCATAAAATAACGCCCCACCTTCTTAACGGGAGGTGGGGCGTTATTTTTACGTCGACCTGCAGCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAACTATGGATGAACGAAATAGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTAACTGTCAGACCAAGTTTACTCATATATACTTTAGATTGATTTAAAACTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCTTAATAAGATGATCTTCTTGAGATCGTTTTGGTCTGCGCGTAATCTCTTGCTCTGAAAACGAAAAAACCGCCTTGCAGGGCGGTTTTTCGAAGGTTCTCTGAGCTACCAACTCTTTGAACCGAGGTAACTGGCTTGGAGGAGCGCAGTCACCAAAACTTGTCCTTTCAGTTTAGCCTTAACCGGCGCATGACTTCAAGACTAACTCCTCTAAATCAATTACCAGTGGCTGCTGCCAGTGGTGCTTTTGCATGTCTTTCCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGACTGAACGGGGGGTTCGTGCATACAGTCCAGCTTGGAGCGAACTGCCTACCCGGAACTGAGTGTCAGGCGTGGAATGAGACAAACGCGGCCATAACAGCGGAATGACACCGGTAAACCGAAAGGCAGGAACAGGAGAGCGCACGAGGGAGCCGCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCACTGATTTGAGCGTCAGATTTCGTGATGCTTGTCAGGGGGGCGGAGCCTATGGAAAAACGGCTTTGCCGCGGCCCTCTCACTTCCCTGTTAAGTATCTTCCTGGCATCTTCCAGGAAATCTCCGCCCCGTTCGTAAGCCATTTCCGCTCGCCGCAGTCGAACGACCGAGCGTAGCGAGTCAGTGAGCGAGGAAGCGGAATATATCCTGTATCACATATTCTGCTGACGCACCGGTGCAGCCTTTTTTCTCCTGCCACATGAAGCACTTCACTGACACCCTCATCAGTGCCAACATAGTAAGCCAGTATACACTCCGCTAGCGCTGAGGTCTGCCTCGTGAAGAAGGTGTTGCTGACTCATACCAGGCCTGAATCGCCCCATCATCCAGCCAGAAAGTGAGGGAGCCACGGTTGATGAGAGC
TTTGTTGTAGGTGGACCAGTTGGTGATTTTGAACTTTTGCTTTGCCACGGAACGGTCTGC
GTTGTCGGGAAGATGCGTGATCTGATCCTTCAACTCAGCAAAAGTTCGATTTATTCAACA
AAGCCACGTTGTGTCTCAAAATCTCTGATGTTACATTGCACAAGATAAAAATATATCATC
ATGAACAATAAAACTGTCTGCTTACATAAACAGTAATACAAGGGGTGTTATGAGCCATAT
TCAACGGGAAACGTCTTGCTCGAGGCCGCGATTAAATTCCAACATGGATGCTGATTTATA
TGGGTATAAATGGGCTCGCGATAATGTCGGGCAATCAGGTGCGACAATCTATCGATTGTA
TGGGAAGCCCGATGCGCCAGAGTTGTTTCTGAAACATGGCAAAGGTAGCGTTGCCAATG
ATGTTACAGATGAGATGGTCAGACTAAACTGGCTGACGGAATTTATGCCTCTTCCGACCA
TCAAGCATTTTATCCGTACTCCTGATGATGCATGGTTACTCACCACTGCGATCCCCGGGA
AAACAGCATTCCAGGTATTAGAAGAATATCCTGATTCAGGTGAAAATATTGTTGATGCGC
TGGCAGTGTTCCTGCGCCGGTTGCATTCGATTCCTGTTTGTAATTGTCCTTTTAACAGCG
ATCGCGTATTTCGTCTCGCTCAGGCGCAATCACGAATGAATAACGGTTTGGTTGATGCGA
GTGATTTTGATGACGAGCGTAATGGCTGGCCTGTTGAACAAGTCTGGAAAGAAATGCAT
AAGCTTTTGCCATTCTCACCGGATTCAGTCGTCACTCATGGTGATTTCTCACTTGATAACC
TTATTTTTGACGAGGGGAAATTAATAGGTTGTATTGATGTTGGACGAGTCGGAATCGCAG
ACCGATACCAGGATCTTGCCATCCTATGGAACTGCCTCGGTGAGTTTTCTCCTTCATTACA
GAAACGGCTTTTTCAAAAATATGGTATTGATAATCCTGATATGAATAAATTGCAGTTTCAT
TTGATGCTCGATGAGTTTTTCTAATCAGAATTGGTTAATTGGTTGTAACACTGGCAGAGC
ATTACGCTGACTTGACGGGACGGCGGCTTTGTTGAATAAATCGAACTTTTGCTGAGTTG
AAGGATCAGATCACGCATCTTCCCGACAACGCAGACCGTTCCGTGGCAAAGCAAAAGT
TCAAAATCACCAACTGGTCCACCTACAACAAAGCTCTCATCAACCGTGGCTCCCTCACT
TTCTGGCTGGATGATGGGGCGATTCAGGCCTGGTATGAGTCAGCAACACCTTCTTCACG
AGGCAGACCTCAGCGCTCAAAGATGCAGGGGTAAAAGCTAACCGCATCTTTACCGACA
AGGCATCCGGCAGTTCAACAGATCGGGAAGGGCTGGATTTGCTGAGGATGAAGGTGGA
GGAAGGTGATGTCATTCTGGTGAAGAAGCTCGACCGTCTTGGCCGCGACACCGCCGAC
ATGATCCAACTGATAAAAGAGTTTGATGCTCAGGGTGTAGCGGTTCGGTTTATTGACGAC
GGGATCAGTACCGACGGTGATATGGGGCAAATGGTGGTCACCATCCTGTCGGCTGTGGC
ACAGGCTGAACGCCGGAGGATCAAGTCGGTCAAGCCAAGCGCAACCAGCGGCACCGC
CGCGAGCAACGTCGCAAGGGCGATCAGGGGACGATTTTTGCGAAGAATTTCCACGGTA
AGAATCCAATCTCTCGAATTTAGGGTGAAAGAAGCTTGGCATAGGGGTGTGCACGAACT
CGGTGGAGGAAATTTCCGCGGGGCAAGGCTTCGCGAAGCGGAGTCGCGGCAGTGGCTT
TGAAGATCTTTGGGAGCAGTCCTTGTGCGCTTACGAGGTGAGCCGGTGGGGAACCGTTA
TCTGCCTATGGTGTGAGCCCCCCTAGAGAGCTTCAAGAGCAATCAGCCCGACCTAGAAA
GGAGGCCAAGAGAGAGACCCCTACGGGGGGAACCGTTTTCTGCCTACGAGATGGCACA
TTTACTGGGAAGCTTTACGGCGTCCTCGTGGAAGTTCAATGCCCGCAGACTTAAGTGCT
CTATTCACGGTCTGACGTGACACGCTAAATTCAGACATAGCTTCATTGATTGTCGGCCAC
GAGCCAGTCTCTCCCTCAACAGTCATAAACCAACCTGCAATGGTCAAGCGATTTCCTTTA
GCTTTCCTAGCTTGTCGTTGACTGGACTTAGCTAGTTTTTCTCGCTGTGCTCGGGCGTAC
TCACTGTTTGGGTCTTTCCAGCGTTCTGCGGCCTTTTTACCGCCACGTCTTCCCATAGTG
GCCAGAGCTTTTCGCCCTCGGCTGCTCTGCGTCTCTGTCTGACGAGCAGGGACGACTGG
CTGGCCTTTAGCGACGTAGCCGCGCACACGTCGCGCCATCGTCTGGCGGTCACGCATCG
GCGGCAGATCAGGCTCACGGCCGTCTGCTCCGACCGCCTGAGCGACGGTGTAGGCACG
CTCGTAGGCGTCGATGATCTTGGTGTCTTTTAGGCGCTCACCAGCCGCTTTTAACTGGTA
TCCCACAGTCAAAGCGTGGCGAAAAGCCGTCTCATCACGGGCGGCACGCCCTGGAGCA
GTCCAGAGGACACGGACGCCGTCGATCAGCTCTCCAGACGCTTCAGCGGCGCTCGGCA
GGCTTGCTTCAAGCGTGGCAAGTGCTTTTGCTTCCGCAGTGGCTTTTCTTGCCGCTTCGA
TACGTGCCCGTCCGCTAGAAAACTCCTGCTCATAGCGTTTTTTAGGTTTTTCTGTGCCTG
AGATCATGCGAGCAACCTCCATAAGATCAGCTAGGCGATCCACGCGATTGTGCTGGGCA
TGCCAGCGGTACGCGGTGGGATCGTCGGAGACGTGCAGTGGCCACCGGCTCAGCCTAT
GTGAAAAAGCCTGGTCAGCGCCGAAAACGCGGGTCATTTCCTCGGTCGTTGCAGCCAG
CAGGCGCATATTCGGGCTGCTCATGCCTGCTGCGGCATACACCGGATCAATGAGCCAGAT
GAGCTGGCATTTCCCGCTCAGTGGATTCACGCCGATCCAAGCTGGCGCTTTTTCCAGGC
GTGCCCAGCGCTCCAAAATCGCGTAGACCTCGGGGTTTACGTGCTCGATTTTCCCGCCG
GCCTGGTGGCTCGGCACATCAATGTCCAGGACAAGCACGGCTGCGTGCTGCGCGTGCG
TCAGAGCAACATACTGGCACCGGGCAAGCGATTTTGAACCAACTCGGTATAACTTCGGC
TGTGTTTCTCCCGTGTCCGGGTCTTTGATCCAAGCGCTGGCGAAGTCGCGGGTCTTGCT
GCCCTGGAAATTTTCTCTGCCCAGGTGAGCGAGGAATTCGCGGCGGTCTTCGCTCGTCC
AGCCACGTGATCGCAGCGCGAGCTCGGGATGGGTGTCGAACAGATCAGCGGAAAATTT
CCAGGCCGGTGTGTCAATGTCTCGTGAATCCGCTAGAGTCATTTTTGAGCGCTTTCTCCC
AGGTTTGGACTGGGGGTTAGCCGACGCCCTGTGAGTTACCGCTCACGGGGCGTTCAAC
ATTTTTCAGGTATTCGTGCAGCTTATCGCTTCTTGCCGCCTGTGCGCTTTTTCGACGCGCG
ACGCTGCTGCCGATTCGGTGCAGGTGGTGGCGGCGCTGACACGTCCTGGGCGGCCACG
GCCACACGAAACGCGGCATTTACGATGTTTGTCATGCCTGCGGGCACCGCGCCACGATC
GCGGATAATTCTCGCTGCCGCTTCCAGCTCTGTGACGACCATGGCCAAAATTTCGCTCGG
GGGACGCACTTCCAGCGCCATTTGCGACCTAGCCGCCTCCAGCTCCTCGGCGTGGCGTT
TGTTGGCGCGCTCGCGGCTGGCTGCGGCACGACACGCATCTGAGCAATATTTTGCGCGC
CGTCCTCGCGGGTCAGGCCGGGGAGGAATCAGGCCACCGCAGTAGGCGCAACTGATTC
GATCCTCCACTACTGTGCGTCCTCCTGGCGCTGCCGAGCACGCAGCTCGTCAGCCAGCT
CCTCAAGATCCGCCACGAGAGTTTCTAGGTCGCTCGCGGCACTGGCCCAGTCTCGTGAT
GCTGGCGCGTCCGTCGTATCGAGAGCTCGGAAAAATCCGATCACCGTTTTTAAATCGAC
GGCAGCATCGAGCGCGTCGGACTCCAGCGCGACATCAGAGAGATCCATAGCTGATGATT
CGGGCCAATTTTGGTACTTCGTCGTGAAGGTCATGACACCATTATAACGAACGTTCGTTA
AAGTTTTTGGCGGAAAATCACGCGGCACGAAAATTTTCACGAAGCGGGACTTTGCGCA
GCTCAGGGGTGCTAAAAATTTTGTATCGCACTTGATTTTTCCGAAAGACAGATTATCTGC
AAACGGTGTGTCGTATTTCTGGCTTGGTTTTTAAAAAATCTGGAATCGAAAATTTGCGGG
GCGACCGAGAAGTTTTTTACAAAAGGCAAAAACTTTTTCGGGATCGACAGAAATAAAA
CGATCGACGGTACGCAACAAAAAAGCGTCAGGATCGCCGTAGAGCGATTGAAGACCGT
CAACCAAAGGGGAAGCCTCCAATCGACGCGACGCGCGCTCTACGGCGATCCTGACGCA
GATTTTTAGCTATCTGTCGCAGCGCCCTCAGGGACAAGCCACCCGCACAACGTCGCGAG
GGCGATCAGCGACGCCGCAGGGGGATCCT
b)质粒pJC1-PF1-U1A-F30::芒果3-TF1转化谷氨酸棒状杆菌ATCC 13032Δcg2273
根据Tauch等人，2002(Tauch,A.,Kirchner,O.,B.,/>S.,Pühler,A.,and Kalinowski,J.Efficient Electrotransformation of Corynebacteriumdiphtheriae with a Mini-Replicon Derived from the Corynebacterium glutamicumPlasmid pGA 1.Curr.Microbiol.2002；45,362-367)制备谷氨酸棒杆菌菌株ATCC 13032Δcg2273的感受态细胞，并用pJC1-PF1-U1A-F30::芒果3-TF1转化。转化子的选择是在含有25μG/ml卡那霉素的CGIII(Menkel,E.,Thierbach,G.,Eggeling,L.,and Sahm,H.Influenceof increased aspartate availability on Iysine formation by arecombinantstrain of Corynebacterium glutamicum and utilization of fumarate.Appl.Environ.Microbiol.1989；55,684-688)琼脂(1％)板上进行的。由此获得的克隆命名为谷氨酸棒状杆菌ATCC 13032Δcg2273 pJC1-PF1-U1A-F30::芒果3-TF1。
c)使用荧光激活细胞分选(FACS)的细胞培养和表型验证
产生的菌株谷氨酸棒状杆菌ATCC 13032Δcg2273_pJC1-PF1-U1A-F30:芒果3-TF1以及菌株谷氨酸棒状杆菌ATCC 13032Δcg2273_pJC1-PF1-U1A-F30:西兰花/UUCG-TF1和谷氨酸棒状杆菌ATCC 13032Δcg2273_pJC1-PF1-U1A-FT1(参见实施例1)在含有25μg/mL卡那霉素的BHI琼脂板上划线，并在30℃下培养。将生长的细胞重悬于含有25μg/mL卡那霉素的CGIII培养基中，并在含2mL有25μmg/mL卡那菌素的培养基的试管中将OD600调节至0.75。将细胞在30℃和120rpm下孵育18小时。随后，使用最终浓度为500μM DFHBI或0.1μM TO1的PBS将细胞稀释至OD600为0.6。使用实施例1d)中已经描述的AriaIII高速细胞分选机分析染色和未染色的细胞。DFHBI染色的谷氨酸棒状杆菌ATCC 13032Δcg2273_pJC1-PF1-U1A-F30::西兰花/UUCG-TF1与未染色的细胞相比，荧光输出增加了约六倍(见图4)。谷氨酸棒状杆菌ATCC 13032Δcg2273 pJC1-PF1-U1A-F30::芒果3-TF1和谷氨酸棒状杆菌ATCC 13032Δcg2273_pJC1-PF1-U1A-TF1在添加荧光团DFHBI后没有显示出增加的荧光输出。TO1染色的谷氨酸棒状杆菌ATCC 13032Δcg2273 pJC1-PF1-U1A-F30::芒果3-TF1细胞显示，与未染色的细胞相比，荧光输出增加了约六倍(见图4)。添加TO1后，与对照菌株相比，菌株谷氨酸棒状杆菌ATCC 13032Δcg2273_pJC1-PF1-U1A-F30::西兰花/UUCG-TF1和谷氨酸棒状杆菌ATCC 13032△cg2273_pJC1-PF1-U1A-TF1的细胞显示出约1.2倍的荧光轻微增加，这表明使用荧光团TO1对细胞染色的特异性略有降低。
该实验显示了迄今为止不显著的表型(RNA产生)与荧光输出的成功连接。在这个实验中，将产生的RNA与F30支架和两种不同的核酸适配体，西兰花或芒果3融合。分别通过补充荧光团DFHBI或TO1来诱导荧光发射。根据实施例1中所示的程序，因此，使用本发明，使用两种核酸适配体中的任一种及其各自的荧光团来优化RNA的发酵生产是可能的。
实施例5
a)载体pJC1-PF1-U1A-F30::玉米-TF1的构建
质粒的构建是通过化学合成的方式合成的DNA片段(PF1-U1A-F30::玉米-TF1的SEQ ID NO:103，从美国旧金山南部的Twist Bioscience订购)及其连接到pJC1(Cremer,J.,Treptow,C.,Eggeling,L.,and Sahm,H.Regulation of Enzymes of LysineBiosynthesis in Corynebacterium glutamicum.Microbiol.1988；134,3221-3229)，得到质粒pJC1-PF1-U1A-F30::玉米-TF1(SEQ ID NO:cc3)。SEQ ID NO:103包含启动子PF1(SEQID NO:67)、感兴趣的RNA(SEQ ID NO:68)、在第一整合点具有玉米核酸适配体且在第二整合点(SEQ ID NO:104)具有UUCG的F30支架和终止子序列TF1(SEQ ID NO:70)。
在用限制性内切酶XbaI和SalI切割合成的DNA片段并随后纯化反应混合物之后，将切下的DNA片段用于与载体pJC1的连接反应中，该载体也已用XbaI或SalI线性化并去磷酸化。将连接混合物直接用于转化大肠杆菌DH5α，并在含有50μg/ml卡那霉素的LB板上进行转化子的筛选。16个菌落生长在这些板上，因此对卡那霉素具有抗性，用于菌落PCR。用引物pJC1_check_f(SEQ ID NO:73)和pJC1_check_rev(SEQ ID NO:74)进行菌落PCR，以分析合成的片段是否***载体pJC1中。在琼脂糖凝胶上对菌落PCR产物的分析显示预期的PCR产物具有682bp的大小(pJC1-PF1-U1A-F30::玉米-TF1)，于是培养三个菌落用于更大规模的质粒制备。培养16小时后，通过离心收集这些培养物，并制备质粒DNA。用菌落PCR中使用的引物对其中两种质粒制剂进行测序，***物的序列显示与预期序列100％一致。所得质粒命名为pJC1-PF1-U1A-F30::玉米-TF1(SEQ ID NO:105)。
SEQ ID NO:103
SEQ ID NO:67，68，cc2，70组合，5'-端的XbaI识别位点、3'-端的SalI识别位点：
CTGTCTCTAGACTCGAGCGGGACGGTCGAACCAGCTTCAAGCGACCGGATGAGTATGTTACAGTAGATAGCGAGCGGGAGACCGCTCGACCTTAGTTCTCCTGTTGCGGGGGAGTTCATGGGATCCAGGTACCCAGGGCGAGGCTTATCCATTGCACTCCGGATGTGCTGACCCCTGGGAGCTCTTGCCATGTGTATGTGGGCGAGGAAGGAGGTCTGAGGAGGTCACTGCCCACATACTCTGATGATCCTTCGGGATCATTCATGGCAAGCTAGCATAGCATAAAATAACGCCCCACCTTCTTAACGGGAGGTGGGGCGTTATTTTTACGTCGAC
SEQ ID NO:104
F30，玉米在其***位点1和“TTCG”在***位点2：
TTGCCATGTGTATGTGGGCGAGGAAGGAGGTCTGAGGAGGTCACTGCCCACATA
CTCTGATGATCCTTCGGGATCATTCATGGCAA
SEQ ID NO:105
pJC1-PF1-U1A-F30::玉米-TF1
CTAGACTCGAGCGGGACGGTCGAACCAGCTTCAAGCGACCGGATGAGTATGTTACAGTAGATAGCGAGCGGGAGACCGCTCGACCTTAGTTCTCCTGTTGCGGGGGAGTTCATGGGATCCAGGTACCCAGGGCGAGGCTTATCCATTGCACTCCGGATGTGCTGACCCCTGGGAGCTCTTGCCATGTGTATGTGGGCGAGGAAGGAGGTCTGAGGAGGTCACTGCCCACATACTCTGATGATCCTTCGGGATCATTCATGGCAAGCTAGCATAGCATAAAATAACGCCCCACCTTCTTAACGGGAGGTGGGGCGTTATTTTTACGTCGACCTGCAGCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAACTATGGATGAACGAAATAGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTAACTGTCAGACCAAGTTTACTCATATATACTTTAGATTGATTTAAAACTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCTTAATAAGATGATCTTCTTGAGATCGTTTTGGTCTGCGCGTAATCTCTTGCTCTGAAAACGAAAAAACCGCCTTGCAGGGCGGTTTTTCGAAGGTTCTCTGAGCTACCAACTCTTTGAACCGAGGTAACTGGCTTGGAGGAGCGCAGTCACCAAAACTTGTCCTTTCAGTTTAGCCTTAACCGGCGCATGACTTCAAGACTAACTCCTCTAAATCAATTACCAGTGGCTGCTGCCAGTGGTGCTTTTGCATGTCTTTCCGGGTTGGACTCAAG
ACGATAGTTACCGGATAAGGCGCAGCGGTCGGACTGAACGGGGGGTTCGTGCATACAGT
CCAGCTTGGAGCGAACTGCCTACCCGGAACTGAGTGTCAGGCGTGGAATGAGACAAAC
GCGGCCATAACAGCGGAATGACACCGGTAAACCGAAAGGCAGGAACAGGAGAGCGCA
CGAGGGAGCCGCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCAC
CACTGATTTGAGCGTCAGATTTCGTGATGCTTGTCAGGGGGGCGGAGCCTATGGAAAAA
CGGCTTTGCCGCGGCCCTCTCACTTCCCTGTTAAGTATCTTCCTGGCATCTTCCAGGAAA
TCTCCGCCCCGTTCGTAAGCCATTTCCGCTCGCCGCAGTCGAACGACCGAGCGTAGCGA
GTCAGTGAGCGAGGAAGCGGAATATATCCTGTATCACATATTCTGCTGACGCACCGGTGC
AGCCTTTTTTCTCCTGCCACATGAAGCACTTCACTGACACCCTCATCAGTGCCAACATAG
TAAGCCAGTATACACTCCGCTAGCGCTGAGGTCTGCCTCGTGAAGAAGGTGTTGCTGAC
TCATACCAGGCCTGAATCGCCCCATCATCCAGCCAGAAAGTGAGGGAGCCACGGTTGAT
GAGAGCTTTGTTGTAGGTGGACCAGTTGGTGATTTTGAACTTTTGCTTTGCCACGGAAC
GGTCTGCGTTGTCGGGAAGATGCGTGATCTGATCCTTCAACTCAGCAAAAGTTCGATTTA
TTCAACAAAGCCACGTTGTGTCTCAAAATCTCTGATGTTACATTGCACAAGATAAAAATA
TATCATCATGAACAATAAAACTGTCTGCTTACATAAACAGTAATACAAGGGGTGTTATGA
GCCATATTCAACGGGAAACGTCTTGCTCGAGGCCGCGATTAAATTCCAACATGGATGCTG
ATTTATATGGGTATAAATGGGCTCGCGATAATGTCGGGCAATCAGGTGCGACAATCTATCG
ATTGTATGGGAAGCCCGATGCGCCAGAGTTGTTTCTGAAACATGGCAAAGGTAGCGTTG
CCAATGATGTTACAGATGAGATGGTCAGACTAAACTGGCTGACGGAATTTATGCCTCTTC
CGACCATCAAGCATTTTATCCGTACTCCTGATGATGCATGGTTACTCACCACTGCGATCCC
CGGGAAAACAGCATTCCAGGTATTAGAAGAATATCCTGATTCAGGTGAAAATATTGTTGA
TGCGCTGGCAGTGTTCCTGCGCCGGTTGCATTCGATTCCTGTTTGTAATTGTCCTTTTAAC
AGCGATCGCGTATTTCGTCTCGCTCAGGCGCAATCACGAATGAATAACGGTTTGGTTGAT
GCGAGTGATTTTGATGACGAGCGTAATGGCTGGCCTGTTGAACAAGTCTGGAAAGAAAT
GCATAAGCTTTTGCCATTCTCACCGGATTCAGTCGTCACTCATGGTGATTTCTCACTTGAT
AACCTTATTTTTGACGAGGGGAAATTAATAGGTTGTATTGATGTTGGACGAGTCGGAATC
GCAGACCGATACCAGGATCTTGCCATCCTATGGAACTGCCTCGGTGAGTTTTCTCCTTCA
TTACAGAAACGGCTTTTTCAAAAATATGGTATTGATAATCCTGATATGAATAAATTGCAGT
TTCATTTGATGCTCGATGAGTTTTTCTAATCAGAATTGGTTAATTGGTTGTAACACTGGCA
GAGCATTACGCTGACTTGACGGGACGGCGGCTTTGTTGAATAAATCGAACTTTTGCTGA
GTTGAAGGATCAGATCACGCATCTTCCCGACAACGCAGACCGTTCCGTGGCAAAGCAA
AAGTTCAAAATCACCAACTGGTCCACCTACAACAAAGCTCTCATCAACCGTGGCTCCCT
CACTTTCTGGCTGGATGATGGGGCGATTCAGGCCTGGTATGAGTCAGCAACACCTTCTTC
ACGAGGCAGACCTCAGCGCTCAAAGATGCAGGGGTAAAAGCTAACCGCATCTTTACCG
ACAAGGCATCCGGCAGTTCAACAGATCGGGAAGGGCTGGATTTGCTGAGGATGAAGGT
GGAGGAAGGTGATGTCATTCTGGTGAAGAAGCTCGACCGTCTTGGCCGCGACACCGCC
GACATGATCCAACTGATAAAAGAGTTTGATGCTCAGGGTGTAGCGGTTCGGTTTATTGAC
GACGGGATCAGTACCGACGGTGATATGGGGCAAATGGTGGTCACCATCCTGTCGGCTGT
GGCACAGGCTGAACGCCGGAGGATCAAGTCGGTCAAGCCAAGCGCAACCAGCGGCAC
CGCCGCGAGCAACGTCGCAAGGGCGATCAGGGGACGATTTTTGCGAAGAATTTCCACG
GTAAGAATCCAATCTCTCGAATTTAGGGTGAAAGAAGCTTGGCATAGGGGTGTGCACGA
ACTCGGTGGAGGAAATTTCCGCGGGGCAAGGCTTCGCGAAGCGGAGTCGCGGCAGTGG
CTTTGAAGATCTTTGGGAGCAGTCCTTGTGCGCTTACGAGGTGAGCCGGTGGGGAACCG
TTATCTGCCTATGGTGTGAGCCCCCCTAGAGAGCTTCAAGAGCAATCAGCCCGACCTAG
AAAGGAGGCCAAGAGAGAGACCCCTACGGGGGGAACCGTTTTCTGCCTACGAGATGGC
ACATTTACTGGGAAGCTTTACGGCGTCCTCGTGGAAGTTCAATGCCCGCAGACTTAAGT
GCTCTATTCACGGTCTGACGTGACACGCTAAATTCAGACATAGCTTCATTGATTGTCGGC
CACGAGCCAGTCTCTCCCTCAACAGTCATAAACCAACCTGCAATGGTCAAGCGATTTCC
TTTAGCTTTCCTAGCTTGTCGTTGACTGGACTTAGCTAGTTTTTCTCGCTGTGCTCGGGC
GTACTCACTGTTTGGGTCTTTCCAGCGTTCTGCGGCCTTTTTACCGCCACGTCTTCCCATA
GTGGCCAGAGCTTTTCGCCCTCGGCTGCTCTGCGTCTCTGTCTGACGAGCAGGGACGAC
TGGCTGGCCTTTAGCGACGTAGCCGCGCACACGTCGCGCCATCGTCTGGCGGTCACGCA
TCGGCGGCAGATCAGGCTCACGGCCGTCTGCTCCGACCGCCTGAGCGACGGTGTAGGC
ACGCTCGTAGGCGTCGATGATCTTGGTGTCTTTTAGGCGCTCACCAGCCGCTTTTAACTG
GTATCCCACAGTCAAAGCGTGGCGAAAAGCCGTCTCATCACGGGCGGCACGCCCTGGA
GCAGTCCAGAGGACACGGACGCCGTCGATCAGCTCTCCAGACGCTTCAGCGGCGCTCG
GCAGGCTTGCTTCAAGCGTGGCAAGTGCTTTTGCTTCCGCAGTGGCTTTTCTTGCCGCTT
CGATACGTGCCCGTCCGCTAGAAAACTCCTGCTCATAGCGTTTTTTAGGTTTTTCTGTGC
CTGAGATCATGCGAGCAACCTCCATAAGATCAGCTAGGCGATCCACGCGATTGTGCTGG
GCATGCCAGCGGTACGCGGTGGGATCGTCGGAGACGTGCAGTGGCCACCGGCTCAGCC
TATGTGAAAAAGCCTGGTCAGCGCCGAAAACGCGGGTCATTTCCTCGGTCGTTGCAGCC
AGCAGGCGCATATTCGGGCTGCTCATGCCTGCTGCGGCATACACCGGATCAATGAGCCA
GATGAGCTGGCATTTCCCGCTCAGTGGATTCACGCCGATCCAAGCTGGCGCTTTTTCCAG
GCGTGCCCAGCGCTCCAAAATCGCGTAGACCTCGGGGTTTACGTGCTCGATTTTCCCGC
CGGCCTGGTGGCTCGGCACATCAATGTCCAGGACAAGCACGGCTGCGTGCTGCGCGTG
CGTCAGAGCAACATACTGGCACCGGGCAAGCGATTTTGAACCAACTCGGTATAACTTCG
GCTGTGTTTCTCCCGTGTCCGGGTCTTTGATCCAAGCGCTGGCGAAGTCGCGGGTCTTG
CTGCCCTGGAAATTTTCTCTGCCCAGGTGAGCGAGGAATTCGCGGCGGTCTTCGCTCGT
CCAGCCACGTGATCGCAGCGCGAGCTCGGGATGGGTGTCGAACAGATCAGCGGAAAAT
TTCCAGGCCGGTGTGTCAATGTCTCGTGAATCCGCTAGAGTCATTTTTGAGCGCTTTCTC
CCAGGTTTGGACTGGGGGTTAGCCGACGCCCTGTGAGTTACCGCTCACGGGGCGTTCAA
CATTTTTCAGGTATTCGTGCAGCTTATCGCTTCTTGCCGCCTGTGCGCTTTTTCGACGCGC
GACGCTGCTGCCGATTCGGTGCAGGTGGTGGCGGCGCTGACACGTCCTGGGCGGCCAC
GGCCACACGAAACGCGGCATTTACGATGTTTGTCATGCCTGCGGGCACCGCGCCACGAT
CGCGGATAATTCTCGCTGCCGCTTCCAGCTCTGTGACGACCATGGCCAAAATTTCGCTCG
GGGGACGCACTTCCAGCGCCATTTGCGACCTAGCCGCCTCCAGCTCCTCGGCGTGGCGT
TTGTTGGCGCGCTCGCGGCTGGCTGCGGCACGACACGCATCTGAGCAATATTTTGCGCG
CCGTCCTCGCGGGTCAGGCCGGGGAGGAATCAGGCCACCGCAGTAGGCGCAACTGATT
CGATCCTCCACTACTGTGCGTCCTCCTGGCGCTGCCGAGCACGCAGCTCGTCAGCCAGC
TCCTCAAGATCCGCCACGAGAGTTTCTAGGTCGCTCGCGGCACTGGCCCAGTCTCGTGA
TGCTGGCGCGTCCGTCGTATCGAGAGCTCGGAAAAATCCGATCACCGTTTTTAAATCGA
CGGCAGCATCGAGCGCGTCGGACTCCAGCGCGACATCAGAGAGATCCATAGCTGATGAT
TCGGGCCAATTTTGGTACTTCGTCGTGAAGGTCATGACACCATTATAACGAACGTTCGTT
AAAGTTTTTGGCGGAAAATCACGCGGCACGAAAATTTTCACGAAGCGGGACTTTGCGC
AGCTCAGGGGTGCTAAAAATTTTGTATCGCACTTGATTTTTCCGAAAGACAGATTATCTG
CAAACGGTGTGTCGTATTTCTGGCTTGGTTTTTAAAAAATCTGGAATCGAAAATTTGCGG
GGCGACCGAGAAGTTTTTTACAAAAGGCAAAAACTTTTTCGGGATCGACAGAAATAAA
ACGATCGACGGTACGCAACAAAAAAGCGTCAGGATCGCCGTAGAGCGATTGAAGACCG
TCAACCAAAGGGGAAGCCTCCAATCGACGCGACGCGCGCTCTACGGCGATCCTGACGC
AGATTTTTAGCTATCTGTCGCAGCGCCCTCAGGGACAAGCCACCCGCACAACGTCGCGA
GGGCGATCAGCGACGCCGCAGGGGGATCCT
b)质粒pJC1-PF1-U1A-F30::玉米-TF1转化谷氨酸棒状杆菌ATCC 13032Δcg2273
根据Tauch等人，2002(Tauch,A.,Kirchner,O.,B.,/>S.,Pühler,A.,and Kalinowski,J.Efficient Electrotransformation of Corynebacteriumdiphtheriae with a Mini-Replicon Derived from the Corynebacterium glutamicumPlasmid pGA 1.Curr.Microbiol.2002；45,362-367)制备谷氨酸棒杆菌菌株ATCC 13032Δcg2273的感受态细胞，并用pJC1-PF1-U1A-F30::玉米-TF1转化。转化子的选择是在CGIII(Menkel,E.,Thierbach,G.,Eggeling,L.,and Sahm,H.Influence of increasedaspartate availability on Iysine formation by a recombinant strain ofCorynebacterium glutamicum and utilization of fumarate.Appl.Environ.Microbiol.1989；55,684-688)琼脂(1％)板上用25μG/ml卡那霉素进行的。由此获得的克隆命名为谷氨酸棒状杆菌ATCC 13032Δcg2273 pJC1-PF1-U1A-F30::玉米-TF1。
c)使用荧光活化细胞分选(FACS)进行细胞培养和表型验证
将产生的菌株谷氨酸棒状杆菌ATCC 13032Δcg2273_pJC1-PF1-U1A-F30::玉米-TF1在含有25μg/mL卡那霉素的BHI琼脂平板上划线，并在30℃下培养。将生长的细胞重悬于含有25μg/mL卡那霉素的CGIII培养基中，并在含2mL有25μmg/mL卡那菌素的培养基的试管中将OD600调节至0.75。将细胞在30℃和120rpm下孵育18小时。随后，使用最终浓度为500μMDFHO的PBS将细胞稀释至OD600为0.6。使用实施例1d)中已经描述的AriaIII高速细胞分选机分析染色和未染色的细胞。与未染色的细胞相比，DFHO染色的谷氨酸棒状杆菌ATCC 13032Δcg2273_pJC1-PF1-U1A-F30::玉米-TF1细胞显示出显著增加的荧光输出。
该实验显示了迄今为止不显著的表型(RNA产生)与荧光输出的成功连接。在这个实验中，将产生的RNA与F30支架和两种不同的核酸适配体(西兰花或玉米)融合。分别通过补充荧光团DFHBI或DFHO来诱导荧光发射。根据实施例1中所示的程序，因此，使用本发明，使用两种核酸适配体中的任一种及其各自的荧光团来优化RNA的发酵生产是可能的。
实施例6
a)载体pJC1_dsRNA_PT7-α微管蛋白-F30::西兰花和pJC1_dsRNA_PT7-CYP3-F30::西兰花的构建
质粒的构建是通过化学合成的方式合成DNA片段(dsRNA_PT7-α微管蛋白-F30::西兰花的SEQ ID NO:106)及其连接到载体pJC1(SEQ ID NO:107)的限制性位点BamHI和EcoRI(Cremer,J.,Treptow,C.,Eggeling,L.,and Sahm,H.Regulation of Enzymes of LysineBiosynthesis in Corynebacterium glutamicum.Microbiol.1988；134,3221-3229)，制成质粒pJC1_dsRNA_PT7-α微管蛋白-F30::西兰花(SEQ ID NO:108)(从美国南旧金山TwistBioscience订购)。SEQ ID NO:106包含启动子PT7(SEQ ID NO:96)，一个编码来自瓦罗阿破坏菌(Varroa destructor)的411bpα微管蛋白RNA的核苷酸序列(SEQ ID NO:110)(Garbianet al.,2012,Bidirectional transfer of RNAi between Honey bee and Varroadestructor:Varroa gene silencing reduces Varroa population,PLOS Pathogens)，F30支架，在第一整合点具有西兰花核酸适配体，在第二整合点具有“UUCG间隔”(SEQ IDNO:69)，终止子序列TT7(SEQ ID NO:97)，第二T7启动子(SEQ ID NO:96)，编码411bpα微管蛋白反义RNA的核苷酸序列(SEQ ID NO:111)和终止子序列TF1(SEQ ID NO:70)。设计的一般原理如图5所示。
SEQ ID NO:106
dsRNA_PT7-α微管蛋白-F30::西兰花:
TAATACGACTCACTATAGGGCGAATGGAGAACATCGCACAGGACTTCGGTAAAAAGTGCCGATTGGGCTTCGCCATCTACCCGGCTCCGCAGGTTTCCACTGCCGTTGTCGAACCATACAACTCGGTTTTGACGACACATGCCACCCTCGAACACGCTGACTGCGTATTCATGATGGATAATGAGGCGATCTATCAGATCTGTCGTCGGAATCTTGGAGTTGAACGACCGGCGTATCAAAATCTCAATCGACTGATTAGCCAGGCCGTTTCGGCGATAACCGCTTCTCTACGTTTTTCCGGAGCGTTGAATGTTGACCTCAACGAATTTCAGACGAATCTCGTCCCCTACCCGCGAATCCATTTCCCGCTCGTCACTTATGCTCCGATTATTTCGGCTGAGAAGGCTCATCACGAGCAACATAACGTACTGGAATACGTATTGCCATGTGTATGTGGGAGACGGTCGGGTCCAGATATTCGTATCTGTCGAGTAGAGTGTGGGCTCCCACATACTCTGATGATCCTTCGGGATCATTCATGGCAAGTCGACCTAGCATAACCCCTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTGCTGAAAGTATCATCCACATATGAGTACTCTAATACGACTCACTATAGGGCGATTCCAGTACGTTATGTTGCTCGTGATGAGCCTTCTCAGCCGAAATAATCGGAGCATAAGTGACGAGCGGGAAATGGATTCGCGGGTAGGGGACGAGATTCGTCTGAAATTCGTTGAGGTCAACATTCAACGCTCCGGAAAAACGTAGAGAAGCGGTTATCGCCGAAACGGCCTGGCTAATCAGTCGATTGAGATTTTGATACGCCGGTCGTTCAACTCCAAGATTCCGACGACAGATCTGATAGATCGCCTCATTATCCATCATGAATACGCAGTCAGCGTGTTCGAGGGTGGCATGTGTCGTCAAAACCGAGTTGTATGGTTCGACAACGGCAGTGGAAACCTGCGGAGCCGGGTAGATGGCGAAGCCCAATCGGCACTTTTTACCGAAGTCCTGTGCGATGTTCTCCATATAGCATAAAATAACGCCCCACCTTCTTAACGGGAGGTGGGGCGTTATTTTTA
SEQ ID NO:107
pJC1
CCGAGAAGTTTTTTACAAAAGGCAAAAACTTTTTCGGGATCGACAGAAATAAAACGATCGACGGTACGCAACAAAAAAGCGTCAGGATCGCCGTAGAGCGATTGAAGACCGTCAACCAAAGGGGAAGCCTCCAATCGACGCGACGCGCGCTCTACGGCGATCCTGACGCAGATTTTTAGCTATCTGTCGCAGCGCCCTCAGGGACAAGCCACCCGCACAACGTCGCGAGGGCGATCAGCGACGCCGCAGGGGGATCCTCTAGAGTCGACCTGCAGCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAACTATGGATGAACGAAATAGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTAACTGTCAGACCAAGTTTACTCATATATACTTTAGATTGATTTAAAACTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCTTAATAAGATGATCTTCTTGAGATCGTTTTGGTC
TGCGCGTAATCTCTTGCTCTGAAAACGAAAAAACCGCCTTGCAGGGCGGTTTTTCGAAG
GTTCTCTGAGCTACCAACTCTTTGAACCGAGGTAACTGGCTTGGAGGAGCGCAGTCACC
AAAACTTGTCCTTTCAGTTTAGCCTTAACCGGCGCATGACTTCAAGACTAACTCCTCTAA
ATCAATTACCAGTGGCTGCTGCCAGTGGTGCTTTTGCATGTCTTTCCGGGTTGGACTCAA
GACGATAGTTACCGGATAAGGCGCAGCGGTCGGACTGAACGGGGGGTTCGTGCATACA
GTCCAGCTTGGAGCGAACTGCCTACCCGGAACTGAGTGTCAGGCGTGGAATGAGACAA
ACGCGGCCATAACAGCGGAATGACACCGGTAAACCGAAAGGCAGGAACAGGAGAGCG
CACGAGGGAGCCGCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCC
ACCACTGATTTGAGCGTCAGATTTCGTGATGCTTGTCAGGGGGGCGGAGCCTATGGAAA
AACGGCTTTGCCGCGGCCCTCTCACTTCCCTGTTAAGTATCTTCCTGGCATCTTCCAGGA
AATCTCCGCCCCGTTCGTAAGCCATTTCCGCTCGCCGCAGTCGAACGACCGAGCGTAGC
GAGTCAGTGAGCGAGGAAGCGGAATATATCCTGTATCACATATTCTGCTGACGCACCGGT
GCAGCCTTTTTTCTCCTGCCACATGAAGCACTTCACTGACACCCTCATCAGTGCCAACAT
AGTAAGCCAGTATACACTCCGCTAGCGCTGAGGTCTGCCTCGTGAAGAAGGTGTTGCTG
ACTCATACCAGGCCTGAATCGCCCCATCATCCAGCCAGAAAGTGAGGGAGCCACGGTTG
ATGAGAGCTTTGTTGTAGGTGGACCAGTTGGTGATTTTGAACTTTTGCTTTGCCACGGAA
CGGTCTGCGTTGTCGGGAAGATGCGTGATCTGATCCTTCAACTCAGCAAAAGTTCGATT
TATTCAACAAAGCCACGTTGTGTCTCAAAATCTCTGATGTTACATTGCACAAGATAAAAA
TATATCATCATGAACAATAAAACTGTCTGCTTACATAAACAGTAATACAAGGGGTGTTATG
AGCCATATTCAACGGGAAACGTCTTGCTCGAGGCCGCGATTAAATTCCAACATGGATGCT
GATTTATATGGGTATAAATGGGCTCGCGATAATGTCGGGCAATCAGGTGCGACAATCTATC
GATTGTATGGGAAGCCCGATGCGCCAGAGTTGTTTCTGAAACATGGCAAAGGTAGCGTT
GCCAATGATGTTACAGATGAGATGGTCAGACTAAACTGGCTGACGGAATTTATGCCTCTT
CCGACCATCAAGCATTTTATCCGTACTCCTGATGATGCATGGTTACTCACCACTGCGATCC
CCGGGAAAACAGCATTCCAGGTATTAGAAGAATATCCTGATTCAGGTGAAAATATTGTTG
ATGCGCTGGCAGTGTTCCTGCGCCGGTTGCATTCGATTCCTGTTTGTAATTGTCCTTTTAA
CAGCGATCGCGTATTTCGTCTCGCTCAGGCGCAATCACGAATGAATAACGGTTTGGTTGA
TGCGAGTGATTTTGATGACGAGCGTAATGGCTGGCCTGTTGAACAAGTCTGGAAAGAAA
TGCATAAGCTTTTGCCATTCTCACCGGATTCAGTCGTCACTCATGGTGATTTCTCACTTGA
TAACCTTATTTTTGACGAGGGGAAATTAATAGGTTGTATTGATGTTGGACGAGTCGGAAT
CGCAGACCGATACCAGGATCTTGCCATCCTATGGAACTGCCTCGGTGAGTTTTCTCCTTC
ATTACAGAAACGGCTTTTTCAAAAATATGGTATTGATAATCCTGATATGAATAAATTGCAG
TTTCATTTGATGCTCGATGAGTTTTTCTAATCAGAATTGGTTAATTGGTTGTAACACTGGC
AGAGCATTACGCTGACTTGACGGGACGGCGGCTTTGTTGAATAAATCGAACTTTTGCTG
AGTTGAAGGATCAGATCACGCATCTTCCCGACAACGCAGACCGTTCCGTGGCAAAGCA
AAAGTTCAAAATCACCAACTGGTCCACCTACAACAAAGCTCTCATCAACCGTGGCTCCC
TCACTTTCTGGCTGGATGATGGGGCGATTCAGGCCTGGTATGAGTCAGCAACACCTTCTT
CACGAGGCAGACCTCAGCGCTCAAAGATGCAGGGGTAAAAGCTAACCGCATCTTTACC
GACAAGGCATCCGGCAGTTCAACAGATCGGGAAGGGCTGGATTTGCTGAGGATGAAGG
TGGAGGAAGGTGATGTCATTCTGGTGAAGAAGCTCGACCGTCTTGGCCGCGACACCGC
CGACATGATCCAACTGATAAAAGAGTTTGATGCTCAGGGTGTAGCGGTTCGGTTTATTGA
CGACGGGATCAGTACCGACGGTGATATGGGGCAAATGGTGGTCACCATCCTGTCGGCTG
TGGCACAGGCTGAACGCCGGAGGATCAAGTCGGTCAAGCCAAGCGCAACCAGCGGCA
CCGCCGCGAGCAACGTCGCAAGGGCGATCAGGGGACGATTTTTGCGAAGAATTTCCAC
GGTAAGAATCCAATCTCTCGAATTTAGGGTGAAAGAAGCTTGGCATAGGGGTGTGCACG
AACTCGGTGGAGGAAATTTCCGCGGGGCAAGGCTTCGCGAAGCGGAGTCGCGGCAGTG
GCTTTGAAGATCTTTGGGAGCAGTCCTTGTGCGCTTACGAGGTGAGCCGGTGGGGAACC
GTTATCTGCCTATGGTGTGAGCCCCCCTAGAGAGCTTCAAGAGCAATCAGCCCGACCTA
GAAAGGAGGCCAAGAGAGAGACCCCTACGGGGGGAACCGTTTTCTGCCTACGAGATGG
CACATTTACTGGGAAGCTTTACGGCGTCCTCGTGGAAGTTCAATGCCCGCAGACTTAAG
TGCTCTATTCACGGTCTGACGTGACACGCTAAATTCAGACATAGCTTCATTGATTGTCGG
CCACGAGCCAGTCTCTCCCTCAACAGTCATAAACCAACCTGCAATGGTCAAGCGATTTC
CTTTAGCTTTCCTAGCTTGTCGTTGACTGGACTTAGCTAGTTTTTCTCGCTGTGCTCGGGC
GTACTCACTGTTTGGGTCTTTCCAGCGTTCTGCGGCCTTTTTACCGCCACGTCTTCCCATA
GTGGCCAGAGCTTTTCGCCCTCGGCTGCTCTGCGTCTCTGTCTGACGAGCAGGGACGAC
TGGCTGGCCTTTAGCGACGTAGCCGCGCACACGTCGCGCCATCGTCTGGCGGTCACGCA
TCGGCGGCAGATCAGGCTCACGGCCGTCTGCTCCGACCGCCTGAGCGACGGTGTAGGC
ACGCTCGTAGGCGTCGATGATCTTGGTGTCTTTTAGGCGCTCACCAGCCGCTTTTAACTG
GTATCCCACAGTCAAAGCGTGGCGAAAAGCCGTCTCATCACGGGCGGCACGCCCTGGA
GCAGTCCAGAGGACACGGACGCCGTCGATCAGCTCTCCAGACGCTTCAGCGGCGCTCG
GCAGGCTTGCTTCAAGCGTGGCAAGTGCTTTTGCTTCCGCAGTGGCTTTTCTTGCCGCTT
CGATACGTGCCCGTCCGCTAGAAAACTCCTGCTCATAGCGTTTTTTAGGTTTTTCTGTGC
CTGAGATCATGCGAGCAACCTCCATAAGATCAGCTAGGCGATCCACGCGATTGTGCTGG
GCATGCCAGCGGTACGCGGTGGGATCGTCGGAGACGTGCAGTGGCCACCGGCTCAGCC
TATGTGAAAAAGCCTGGTCAGCGCCGAAAACGCGGGTCATTTCCTCGGTCGTTGCAGCC
AGCAGGCGCATATTCGGGCTGCTCATGCCTGCTGCGGCATACACCGGATCAATGAGCCA
GATGAGCTGGCATTTCCCGCTCAGTGGATTCACGCCGATCCAAGCTGGCGCTTTTTCCAG
GCGTGCCCAGCGCTCCAAAATCGCGTAGACCTCGGGGTTTACGTGCTCGATTTTCCCGC
CGGCCTGGTGGCTCGGCACATCAATGTCCAGGACAAGCACGGCTGCGTGCTGCGCGTG
CGTCAGAGCAACATACTGGCACCGGGCAAGCGATTTTGAACCAACTCGGTATAACTTCG
GCTGTGTTTCTCCCGTGTCCGGGTCTTTGATCCAAGCGCTGGCGAAGTCGCGGGTCTTG
CTGCCCTGGAAATTTTCTCTGCCCAGGTGAGCGAGGAATTCGCGGCGGTCTTCGCTCGT
CCAGCCACGTGATCGCAGCGCGAGCTCGGGATGGGTGTCGAACAGATCAGCGGAAAAT
TTCCAGGCCGGTGTGTCAATGTCTCGTGAATCCGCTAGAGTCATTTTTGAGCGCTTTCTC
CCAGGTTTGGACTGGGGGTTAGCCGACGCCCTGTGAGTTACCGCTCACGGGGCGTTCAA
CATTTTTCAGGTATTCGTGCAGCTTATCGCTTCTTGCCGCCTGTGCGCTTTTTCGACGCGC
GACGCTGCTGCCGATTCGGTGCAGGTGGTGGCGGCGCTGACACGTCCTGGGCGGCCAC
GGCCACACGAAACGCGGCATTTACGATGTTTGTCATGCCTGCGGGCACCGCGCCACGAT
CGCGGATAATTCTCGCTGCCGCTTCCAGCTCTGTGACGACCATGGCCAAAATTTCGCTCG
GGGGACGCACTTCCAGCGCCATTTGCGACCTAGCCGCCTCCAGCTCCTCGGCGTGGCGT
TTGTTGGCGCGCTCGCGGCTGGCTGCGGCACGACACGCATCTGAGCAATATTTTGCGCG
CCGTCCTCGCGGGTCAGGCCGGGGAGGAATCAGGCCACCGCAGTAGGCGCAACTGATT
CGATCCTCCACTACTGTGCGTCCTCCTGGCGCTGCCGAGCACGCAGCTCGTCAGCCAGC
TCCTCAAGATCCGCCACGAGAGTTTCTAGGTCGCTCGCGGCACTGGCCCAGTCTCGTGA
TGCTGGCGCGTCCGTCGTATCGAGAGCTCGGAAAAATCCGATCACCGTTTTTAAATCGA
CGGCAGCATCGAGCGCGTCGGACTCCAGCGCGACATCAGAGAGATCCATAGCTGATGAT
TCGGGCCAATTTTGGTACTTCGTCGTGAAGGTCATGACACCATTATAACGAACGTTCGTT
AAAGTTTTTGGCGGAAAATCACGCGGCACGAAAATTTTCACGAAGCGGGACTTTGCGC
AGCTCAGGGGTGCTAAAAATTTTGTATCGCACTTGATTTTTCCGAAAGACAGATTATCTG
CAAACGGTGTGTCGTATTTCTGGCTTGGTTTTTAAAAAATCTGGAATCGAAAATTTGCGG
GGCGA
SEQ ID NO:108
pJC1_dsRNA_PT7-α-微管蛋白-F30::西兰花:
AACGTCTTGCTCGAGGCCGCGATTAAATTCCAACATGGATGCTGATTTATATGGGTATAAATGGGCTCGCGATAATGTCGGGCAATCAGGTGCGACAATCTATCGATTGTATGGGAAGCCCGATGCGCCAGAGTTGTTTCTGAAACATGGCAAAGGTAGCGTTGCCAATGATGTTACAGATGAGATGGTCAGACTAAACTGGCTGACGGAATTTATGCCTCTTCCGACCATCAAGCATTTTATCCGTACTCCTGATGATGCATGGTTACTCACCACTGCGATCCCCGGGAAAACAGCATTCCAGGTATTAGAAGAATATCCTGATTCAGGTGAAAATATTGTTGATGCGCTGGCAGTGTTCCTGCGCCGGTTGCATTCGATTCCTGTTTGTAATTGTCCTTTTAACAGCGATCGCGTATTTCGTCTCGCTCAGGCGCAATCACGAATGAATAACGGTTTGGTTGATGCGAGTGATTT
TGATGACGAGCGTAATGGCTGGCCTGTTGAACAAGTCTGGAAAGAAATGCATAAGCTTT
TGCCATTCTCACCGGATTCAGTCGTCACTCATGGTGATTTCTCACTTGATAACCTTATTTT
TGACGAGGGGAAATTAATAGGTTGTATTGATGTTGGACGAGTCGGAATCGCAGACCGAT
ACCAGGATCTTGCCATCCTATGGAACTGCCTCGGTGAGTTTTCTCCTTCATTACAGAAAC
GGCTTTTTCAAAAATATGGTATTGATAATCCTGATATGAATAAATTGCAGTTTCATTTGATG
CTCGATGAGTTTTTCTAATCAGAATTGGTTAATTGGTTGTAACACTGGCAGAGCATTACG
CTGACTTGACGGGACGGCGGCTTTGTTGAATAAATCGAACTTTTGCTGAGTTGAAGGAT
CAGATCACGCATCTTCCCGACAACGCAGACCGTTCCGTGGCAAAGCAAAAGTTCAAAA
TCACCAACTGGTCCACCTACAACAAAGCTCTCATCAACCGTGGCTCCCTCACTTTCTGG
CTGGATGATGGGGCGATTCAGGCCTGGTATGAGTCAGCAACACCTTCTTCACGAGGCAG
ACCTCAGCGCTCAAAGATGCAGGGGTAAAAGCTAACCGCATCTTTACCGACAAGGCATC
CGGCAGTTCAACAGATCGGGAAGGGCTGGATTTGCTGAGGATGAAGGTGGAGGAAGGT
GATGTCATTCTGGTGAAGAAGCTCGACCGTCTTGGCCGCGACACCGCCGACATGATCCA
ACTGATAAAAGAGTTTGATGCTCAGGGTGTAGCGGTTCGGTTTATTGACGACGGGATCA
GTACCGACGGTGATATGGGGCAAATGGTGGTCACCATCCTGTCGGCTGTGGCACAGGCT
GAACGCCGGAGGATCAAGTCGGTCAAGCCAAGCGCAACCAGCGGCACCGCCGCGAGC
AACGTCGCAAGGGCGATCAGGGGACGATTTTTGCGAAGAATTTCCACGGTAAGAATCCA
ATCTCTCGAATTTAGGGTGAAAGAAGCTTGGCATAGGGGTGTGCACGAACTCGGTGGAG
GAAATTTCCGCGGGGCAAGGCTTCGCGAAGCGGAGTCGCGGCAGTGGCTTTGAAGATC
TTTGGGAGCAGTCCTTGTGCGCTTACGAGGTGAGCCGGTGGGGAACCGTTATCTGCCTA
TGGTGTGAGCCCCCCTAGAGAGCTTCAAGAGCAATCAGCCCGACCTAGAAAGGAGGCC
AAGAGAGAGACCCCTACGGGGGGAACCGTTTTCTGCCTACGAGATGGCACATTTACTGG
GAAGCTTTACGGCGTCCTCGTGGAAGTTCAATGCCCGCAGACTTAAGTGCTCTATTCAC
GGTCTGACGTGACACGCTAAATTCAGACATAGCTTCATTGATTGTCGGCCACGAGCCAG
TCTCTCCCTCAACAGTCATAAACCAACCTGCAATGGTCAAGCGATTTCCTTTAGCTTTCC
TAGCTTGTCGTTGACTGGACTTAGCTAGTTTTTCTCGCTGTGCTCGGGCGTACTCACTGT
TTGGGTCTTTCCAGCGTTCTGCGGCCTTTTTACCGCCACGTCTTCCCATAGTGGCCAGAG
CTTTTCGCCCTCGGCTGCTCTGCGTCTCTGTCTGACGAGCAGGGACGACTGGCTGGCCT
TTAGCGACGTAGCCGCGCACACGTCGCGCCATCGTCTGGCGGTCACGCATCGGCGGCAG
ATCAGGCTCACGGCCGTCTGCTCCGACCGCCTGAGCGACGGTGTAGGCACGCTCGTAGG
CGTCGATGATCTTGGTGTCTTTTAGGCGCTCACCAGCCGCTTTTAACTGGTATCCCACAG
TCAAAGCGTGGCGAAAAGCCGTCTCATCACGGGCGGCACGCCCTGGAGCAGTCCAGAG
GACACGGACGCCGTCGATCAGCTCTCCAGACGCTTCAGCGGCGCTCGGCAGGCTTGCTT
CAAGCGTGGCAAGTGCTTTTGCTTCCGCAGTGGCTTTTCTTGCCGCTTCGATACGTGCCC
GTCCGCTAGAAAACTCCTGCTCATAGCGTTTTTTAGGTTTTTCTGTGCCTGAGATCATGC
GAGCAACCTCCATAAGATCAGCTAGGCGATCCACGCGATTGTGCTGGGCATGCCAGCGG
TACGCGGTGGGATCGTCGGAGACGTGCAGTGGCCACCGGCTCAGCCTATGTGAAAAAG
CCTGGTCAGCGCCGAAAACGCGGGTCATTTCCTCGGTCGTTGCAGCCAGCAGGCGCATA
TTCGGGCTGCTCATGCCTGCTGCGGCATACACCGGATCAATGAGCCAGATGAGCTGGCA
TTTCCCGCTCAGTGGATTCACGCCGATCCAAGCTGGCGCTTTTTCCAGGCGTGCCCAGC
GCTCCAAAATCGCGTAGACCTCGGGGTTTACGTGCTCGATTTTCCCGCCGGCCTGGTGG
CTCGGCACATCAATGTCCAGGACAAGCACGGCTGCGTGCTGCGCGTGCGTCAGAGCAA
CATACTGGCACCGGGCAAGCGATTTTGAACCAACTCGGTATAACTTCGGCTGTGTTTCTC
CCGTGTCCGGGTCTTTGATCCAAGCGCTGGCGAAGTCGCGGGTCTTGCTGCCCTGGAAA
TTTTCTCTGCCCAGGTGAGCGAGGAATTCGCGGCGGTCTTCGCTCGTCCAGCCACGTGA
TCGCAGCGCGAGCTCGGGATGGGTGTCGAACAGATCAGCGGAAAATTTCCAGGCCGGT
GTGTCAATGTCTCGTGAATCCGCTAGAGTCATTTTTGAGCGCTTTCTCCCAGGTTTGGAC
TGGGGGTTAGCCGACGCCCTGTGAGTTACCGCTCACGGGGCGTTCAACATTTTTCAGGT
ATTCGTGCAGCTTATCGCTTCTTGCCGCCTGTGCGCTTTTTCGACGCGCGACGCTGCTGC
CGATTCGGTGCAGGTGGTGGCGGCGCTGACACGTCCTGGGCGGCCACGGCCACACGAA
ACGCGGCATTTACGATGTTTGTCATGCCTGCGGGCACCGCGCCACGATCGCGGATAATTC
TCGCTGCCGCTTCCAGCTCTGTGACGACCATGGCCAAAATTTCGCTCGGGGGACGCACT
TCCAGCGCCATTTGCGACCTAGCCGCCTCCAGCTCCTCGGCGTGGCGTTTGTTGGCGCG
CTCGCGGCTGGCTGCGGCACGACACGCATCTGAGCAATATTTTGCGCGCCGTCCTCGCG
GGTCAGGCCGGGGAGGAATCAGGCCACCGCAGTAGGCGCAACTGATTCGATCCTCCAC
TACTGTGCGTCCTCCTGGCGCTGCCGAGCACGCAGCTCGTCAGCCAGCTCCTCAAGATC
CGCCACGAGAGTTTCTAGGTCGCTCGCGGCACTGGCCCAGTCTCGTGATGCTGGCGCGT
CCGTCGTATCGAGAGCTCGGAAAAATCCGATCACCGTTTTTAAATCGACGGCAGCATCG
AGCGCGTCGGACTCCAGCGCGACATCAGAGAGATCCATAGCTGATGATTCGGGCCAATT
TTGGTACTTCGTCGTGAAGGTCATGACACCATTATAACGAACGTTCGTTAAAGTTTTTGG
CGGAAAATCACGCGGCACGAAAATTTTCACGAAGCGGGACTTTGCGCAGCTCAGGGGT
GCTAAAAATTTTGTATCGCACTTGATTTTTCCGAAAGACAGATTATCTGCAAACGGTGTG
TCGTATTTCTGGCTTGGTTTTTAAAAAATCTGGAATCGAAAATTTGCGGGGCGACCGAGA
AGTTTTTTACAAAAGGCAAAAACTTTTTCGGGATCGACAGAAATAAAACGATCGACGGT
ACGCAACAAAAAAGCGTCAGGATCGCCGTAGAGCGATTGAAGACCGTCAACCAAAGGG
GAAGCCTCCAATCGACGCGACGCGCGCTCTACGGCGATCCTGACGCAGATTTTTAGCTAT
CTGTCGCAGCGCCCTCAGGGACAAGCCACCCGCACAACGTCGCGAGGGCGATCAGCGA
CGCCGCAGGGGGATCCGGATCCTCTAGACTAATACGACTCACTATAGGGCGAATGGAGA
ACATCGCACAGGACTTCGGTAAAAAGTGCCGATTGGGCTTCGCCATCTACCCGGCTCCG
CAGGTTTCCACTGCCGTTGTCGAACCATACAACTCGGTTTTGACGACACATGCCACCCT
CGAACACGCTGACTGCGTATTCATGATGGATAATGAGGCGATCTATCAGATCTGTCGTCG
GAATCTTGGAGTTGAACGACCGGCGTATCAAAATCTCAATCGACTGATTAGCCAGGCCG
TTTCGGCGATAACCGCTTCTCTACGTTTTTCCGGAGCGTTGAATGTTGACCTCAACGAAT
TTCAGACGAATCTCGTCCCCTACCCGCGAATCCATTTCCCGCTCGTCACTTATGCTCCGAT
TATTTCGGCTGAGAAGGCTCATCACGAGCAACATAACGTACTGGAATACGTATTGCCATG
TGTATGTGGGAGACGGTCGGGTCCAGATATTCGTATCTGTCGAGTAGAGTGTGGGCTCCC
ACATACTCTGATGATCCTTCGGGATCATTCATGGCAAGTCGACCTAGCATAACCCCTTGG
GGCCTCTAAACGGGTCTTGAGGGGTTTTTTGCTGAAAGTATCATCCACATATGAGTACTC
TAATACGACTCACTATAGGGCGATTCCAGTACGTTATGTTGCTCGTGATGAGCCTTCTCAG
CCGAAATAATCGGAGCATAAGTGACGAGCGGGAAATGGATTCGCGGGTAGGGGACGAG
ATTCGTCTGAAATTCGTTGAGGTCAACATTCAACGCTCCGGAAAAACGTAGAGAAGCGG
TTATCGCCGAAACGGCCTGGCTAATCAGTCGATTGAGATTTTGATACGCCGGTCGTTCAA
CTCCAAGATTCCGACGACAGATCTGATAGATCGCCTCATTATCCATCATGAATACGCAGTC
AGCGTGTTCGAGGGTGGCATGTGTCGTCAAAACCGAGTTGTATGGTTCGACAACGGCAG
TGGAAACCTGCGGAGCCGGGTAGATGGCGAAGCCCAATCGGCACTTTTTACCGAAGTCC
TGTGCGATGTTCTCCATATAGCATAAAATAACGCCCCACCTTCTTAACGGGAGGTGGGGC
GTTATTTTTAGGTACCGATATCGATATCCAATGGCAACAACGTTGCGCAAACTATTAACTG
GCGAACTACTTACTCTAGCTTCCCGGCAACAATTAATAGACTGGATGGAGGCGGATAAA
GTTGCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAATCT
GGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCC
CTCCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAACTATGGATGAACGAAATAG
ACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTAACTGTCAGACCAAGTTTA
CTCATATATACTTTAGATTGATTTAAAACTTCATTTTTAATTTAAAAGGATCTAGGTGAAGA
TCCTTTTTGATAATCTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTC
AGACCCCTTAATAAGATGATCTTCTTGAGATCGTTTTGGTCTGCGCGTAATCTCTTGCTCT
GAAAACGAAAAAACCGCCTTGCAGGGCGGTTTTTCGAAGGTTCTCTGAGCTACCAACT
CTTTGAACCGAGGTAACTGGCTTGGAGGAGCGCAGTCACCAAAACTTGTCCTTTCAGTT
TAGCCTTAACCGGCGCATGACTTCAAGACTAACTCCTCTAAATCAATTACCAGTGGCTGC
TGCCAGTGGTGCTTTTGCATGTCTTTCCGGGTTGGACTCAAGACGATAGTTACCGGATAA
GGCGCAGCGGTCGGACTGAACGGGGGGTTCGTGCATACAGTCCAGCTTGGAGCGAACT
GCCTACCCGGAACTGAGTGTCAGGCGTGGAATGAGACAAACGCGGCCATAACAGCGGA
ATGACACCGGTAAACCGAAAGGCAGGAACAGGAGAGCGCACGAGGGAGCCGCCAGGG
GGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCACTGATTTGAGCGTCAG
ATTTCGTGATGCTTGTCAGGGGGGCGGAGCCTATGGAAAAACGGCTTTGCCGCGGCCCT
CTCACTTCCCTGTTAAGTATCTTCCTGGCATCTTCCAGGAAATCTCCGCCCCGTTCGTAA
GCCATTTCCGCTCGCCGCAGTCGAACGACCGAGCGTAGCGAGTCAGTGAGCGAGGAAG
CGGAATATATCCTGTATCACATATTCTGCTGACGCACCGGTGCAGCCTTTTTTCTCCTGCC
ACATGAAGCACTTCACTGACACCCTCATCAGTGCCAACATAGTAAGCCAGTATACACTCC
GCTAGCGCTGAGGTCTGCCTCGTGAAGAAGGTGTTGCTGACTCATACCAGGCCTGAATC
GCCCCATCATCCAGCCAGAAAGTGAGGGAGCCACGGTTGATGAGAGCTTTGTTGTAGGT
GGACCAGTTGGTGATTTTGAACTTTTGCTTTGCCACGGAACGGTCTGCGTTGTCGGGAA
GATGCGTGATCTGATCCTTCAACTCAGCAAAAGTTCGATTTATTCAACAAAGCCACGTTG
TGTCTCAAAATCTCTGATGTTACATTGCACAAGATAAAAATATATCATCATGAACAATAAA
ACTGTCTGCTTACATAAACAGTAATACAAGGGGTGTTATGAGCCATATTCAACGGGA
SEQ ID NO:110
来自Varroa destructor的α-微管蛋白RNA：
ATGGAGAACATCGCACAGGACTTCGGTAAAAAGTGCCGATTGGGCTTCGCCATCTACCCGGCTCCGCAGGTTTCCACTGCCGTTGTCGAACCATACAACTCGGTTTTGACGACACATGCCACCCTCGAACACGCTGACTGCGTATTCATGATGGATAATGAGGCGATCTATCAGATCTGTCGTCGGAATCTTGGAGTTGAACGACCGGCGTATCAAAATCTCAATCGACTGATTAGCCAGGCCGTTTCGGCGATAACCGCTTCTCTACGTTTTTCCGGAGCGTTGAATGTTGACCTCAACGAATTTCAGACGAATCTCGTCCCCTACCCGCGAATCCATTTCCCGCTCGTCACTTATGCTCCGATTATTTCGGCTGAGAAGGCTCATCACGAGCAACATAACGTACTGGAA
SEQ ID NO:111
α-微管蛋白反义RNA:
TTCCAGTACGTTATGTTGCTCGTGATGAGCCTTCTCAGCCGAAATAATCGGAGCATAAGTGACGAGCGGGAAATGGATTCGCGGGTAGGGGACGAGATTCGTCTGAAATTCGTTGAGGTCAACATTCAACGCTCCGGAAAAACGTAGAGAAGCGGTTATCGCCGAAACGGCCTGGCTAATCAGTCGATTGAGATTTTGATACGCCGGTCGTTCAACTCCAAGATTCCGACGACAGATCTGATAGATCGCCTCATTATCCATCATGAATACGCAGTCAGCGTGTTCGAGGGTGGCATGTGTCGTCAAAACCGAGTTGTATGGTTCGACAACGGCAGTGGAAACCTGCGGAGCCGGGTAGATGGCGAAGCCCAATCGGCACTTTTTACCGAAGTCCTGTGCGATGTTCTCCATb)谷氨酸棒状杆菌ATCC 13032Δcg2273的cg1121-cg1122中lambda DE3区域的整合
在lacUV5启动子控制下的T7 RNA聚合酶由λDE3噬菌体构建体表达(Moffat eral.(1984)Nucleotide sequence of the gene for bacteriophage T7 RNA polymerase,J Mol Biol 173 265-269)。Kortmann及其同事使用DE3片段构建质粒pK18mobsacB-DE3(SEQ ID NO:115)，用于整合到谷氨酸棒状杆菌的cg1121和cg1122的基因间区域((Kortmann,Kuhl,Klaffl,Bott.2015.A chromosomally encoded T7 RNApolymerasedependent gene expression system for Corynebacterium glutamicum_construction and comparative evaluation at the single-cell level.MicrobialBiotechnology,8(2)253-265)。根据Tauch等人，2002(Tauch,A.,Kirchner,O.,B.,S.,Pühler,A.,and Kalinowski,J.Efficient Electrotransformation ofCorynebacterium diphtheriae with a Mini-Replicon Derived from theCorynebacterium glutamicum Plasmid pGA 1.Curr.Microbiol.2002；45,362-367)制备谷氨酸棒杆菌菌株ATCC 13032Δcg2273的感受态细胞，并用pK18mobsacB-OE3通过电穿孔转化。转化子的选择是在含有25μg/ml卡那霉素的BHI(脑心输注)琼脂(1％)平板上进行的。第一次和第二次重组如Niebisch和Bott，2001先前所述进行(Niebisch andBott.Molecular analysis of the cytochrome bc1-aa3 branch of theCorynebacterium glutamicum respiratory chain containing an unusual dihemecytochrome c1.Arch.Microbiol.2001；175,282-294).使用引物DE3_for(SEQ ID NO:116)和DE3_rev(SEQ ID NO:117)通过菌落PCR验证得到的克隆。由此产生的菌株命名为谷氨酸棒状杆菌ATCC 13032(DE3)_Δcg2273。
SEQ ID NO:115
pK18mobsacB-DE3:
CTAGCTTCACGCTGCCGCAAGCACTCAGGGCGCAAGGGCTGCTAAAGGAAGCGGAACACGTAGAAAGCCAGTCCGCAGAAACGGTGCTGACCCCGGATGAATGTCAGCTACTGGGCTATCTGGACAAGGGAAAACGCAAGCGCAAAGAGAAAGCAGGTAGCTTGCAGTGGGCTTACATGGCGATAGCTAGACTGGGCGGTTTTATGGACAGCAAGCGAACCGGAATTGCCAGCTGGGGCGCCCTCTGGTAAGGTTGGGAAGCCCTGCAAAGTAAACTGGATGGCTTTCTTGCCGCCAAGGATCTGATGGCGCAGGGGATCAAGATCTGATCAAGAGACAGGATGAGGATCGTTTCGCATGATTGAACAAGATGGATTGCACGCAGGTTCTCCGGCCGCTTGGGTGGAGAGGCTATTCGGCTATGACTGGGCACAACAGACAATCGGCTGCTCTGATGCCGCCGTGTTCCGGCTGTCAGCGCAGGGGCGCCCGGTTCTTTTTGTCAAGACCGACCTGTCCGGTGCCCTGAATGAACTCCAAGACGAGGCAGCGCGGCTATCGTGGCTGGCCACGACGGGCGTTCCTTGCGCAGCTGTGCTCGACGTTGTCACTGAAGCGGGAAGGGACTGGCTGCTATTGGGCGAAGTGCCGGGGCAGGATCTCCTGTCATCTCACCTTGCTCCTGCCGAGAAAGTATCCATCATGGCTGATGCAATGCGGCGGCTGCATACGCTTGATCCGGCTACCTGCCCATTCGACCACCAAGCGAAACATCGCATCGAGCGAGCACGTACTCGGATGGAAGCCGGTCTTGTCG
ATCAGGATGATCTGGACGAAGAGCATCAGGGGCTCGCGCCAGCCGAACTGTTCGCCAG
GCTCAAGGCGCGGATGCCCGACGGCGAGGATCTCGTCGTGACCCATGGCGATGCCTGCT
TGCCGAATATCATGGTGGAAAATGGCCGCTTTTCTGGATTCATCGACTGTGGCCGGCTGG
GTGTGGCGGACCGCTATCAGGACATAGCGTTGGCTACCCGTGATATTGCTGAAGAGCTT
GGCGGCGAATGGGCTGACCGCTTCCTCGTGCTTTACGGTATCGCCGCTCCCGATTCGCA
GCGCATCGCCTTCTATCGCCTTCTTGACGAGTTCTTCTGAGCGGGACTCTGGGGTTCGCT
AGAGGATCGATCCTTTTTAACCCATCACATATACCTGCCGTTCACTATTATTTAGTGAAAT
GAGATATTATGATATTTTCTGAATTGTGATTAAAAAGGCAACTTTATGCCCATGCAACAGA
AACTATAAAAAATACAGAGAATGAAAAGAAACAGATAGATTTTTTAGTTCTTTAGGCCCG
TAGTCTGCAAATCCTTTTATGATTTTCTATCAAACAAAAGAGGAAAATAGACCAGTTGCA
ATCCAAACGAGAGTCTAATAGAATGAGGTCGAAAAGTAAATCGCGCGGGTTTGTTACTG
ATAAAGCAGGCAAGACCTAAAATGTGTAAAGGGCAAAGTGTATACTTTGGCGTCACCCC
TTACATATTTTAGGTCTTTTTTTATTGTGCGTAACTAACTTGCCATCTTCAAACAGGAGGG
CTGGAAGAAGCAGACCGCTAACACAGTACATAAAAAAGGAGACATGAACGATGAACAT
CAAAAAGTTTGCAAAACAAGCAACAGTATTAACCTTTACTACCGCACTGCTGGCAGGAG
GCGCAACTCAAGCGTTTGCGAAAGAAACGAACCAAAAGCCATATAAGGAAACATACGG
CATTTCCCATATTACACGCCATGATATGCTGCAAATCCCTGAACAGCAAAAAAATGAAAA
ATATCAAGTTTCTGAATTTGATTCGTCCACAATTAAAAATATCTCTTCTGCAAAAGGCCTG
GACGTTTGGGACAGCTGGCCATTACAAAACGCTGACGGCACTGTCGCAAACTATCACGG
CTACCACATCGTCTTTGCATTAGCCGGAGATCCTAAAAATGCGGATGACACATCGATTTA
CATGTTCTATCAAAAAGTCGGCGAAACTTCTATTGACAGCTGGAAAAACGCTGGCCGCG
TCTTTAAAGACAGCGACAAATTCGATGCAAATGATTCTATCCTAAAAGACCAAACACAA
GAATGGTCAGGTTCAGCCACATTTACATCTGACGGAAAAATCCGTTTATTCTACACTGAT
TTCTCCGGTAAACATTACGGCAAACAAACACTGACAACTGCACAAGTTAACGTATCAGC
ATCAGACAGCTCTTTGAACATCAACGGTGTAGAGGATTATAAATCAATCTTTGACGGTGA
CGGAAAAANCGTATCAAAATGTACAGCAGTTCATCGATGAAGGCAACTACAGCTCAGGC
GACAACCATACGCTGAGAGATCCTCACTACGTAGAAGATAAAGGCCACAAATACTTAGT
ATTTGAAGCAAACACTGGAACTGAAGATGGCTACCAAGGCGAAGAATCTTTATTTAACA
AAGCATACTATGGCAAAAGCACATCATTCTTCCGTCAAGAAAGTCAAAAACTTCTGCAA
AGCGATAAAAAACGCACGGCTGAGTTAGCAAACGGCGCTCTCGGTATGATTGAGCTAAA
CGATGATTACACACTGAAAAAAGTGATGAAACCGCTGATTGCATCTAACACAGTAACAG
ATGAAATTGAACGCGCGAACGTCTTTAAAATGAACGGCAAATGGTACCTGTTCACTGAC
TCCCGCGGATCAAAAATGACGATTGACGGCATTACGTCTAACGATATTTACATGCTTGGT
TATGTTTCTAATTCTTTAACTGGCCCATACAAGCCGCTGAACAAAACTGGCCTTGTGTTA
AAAATGGATCTTGATCCTAACGATGTAACCTTTACTTACTCACACTTCGCTGTACCTCAA
GCGAAAGGAAACAATGTCGTGATTACAAGCTATATGACAAACAGAGGATTCTACGCAGA
CAAACAATCAACGTTTGCGCCGAGCTTCCTGCTGAACATCAAAGGCAAGAAAACATCT
GTTGTCAAAGACAGCATCCTTGAACAAGGACAATTAACAGTTAACAAATAAAAACGCA
AAAGAAAATGCCGATGGGTACCGAGCGAAATGACCGACCAAGCGACGCCCAACCTGCC
ATCACGAGATTTCGATTCCACCGCCGCCTTCTATGAAAGGTTGGGCTTCGGAATCGTTTT
CCGGGACGCCCTCGCGGACGTGCTCATAGTCCACGACGCCCGTGATTTTGTAGCCCTGG
CCGACGGCCAGCAGGTAGGCCGACAGGCTCATGCCGGCCGCCGCCGCCTTTTCCTCAAT
CGCTCTTCGTTCGTCTGGAAGGCAGTACACCTTGATAGGTGGGCTGCCCTTCCTGGTTG
GCTTGGTTTCATCAGCCATCCGCTTGCCCTCATCTGTTACGCCGGCGGTAGCCGGCCAGC
CTCGCAGAGCAGGATTCCCGTTGAGCACCGCCAGGTGCGAATAAGGGACAGTGAAGAA
GGAACACCCGCTCGCGGGTGGGCCTACTTCACCTATCCTGCCCCGCTGACGCCGTTGGA
TACACCAAGGAAAGTCTACACGAACCCTTTGGCAAAATCCTGTATATCGTGCGAAAAAG
GATGGATATACCGAAAAAATCGCTATAATGACCCCGAAGCAGGGTTATGCAGCGGAAAA
GCGCTGCTTCCCTGCTGTTTTGTGGAATATCTACCGACTGGAAACAGGCAAATGCAGGA
AATTACTGAACTGAGGGGACAGGCGAGAGACGATGCCAAAGAGCTCCTGAAAATCTCG
ATAACTCAAAAAATACGCCCGGTAGTGATCTTATTTCATTATGGTGAAAGTTGGAACCTC
TTACGTGCCGATCAACGTCTCATTTTCGCCAAAAGTTGGCCCAGGGCTTCCCGGTATCAA
CAGGGACACCAGGATTTATTTATTCTGCGAAGTGATCTTCCGTCACAGGTATTTATTCGGC
GCAAAGTGCGTCGGGTGATGCTGCCAACTTACTGATTTAGTGTATGATGGTGTTTTTGAG
GTGCTCCAGTGGCTTCTGTTTCTATCAGCTCCTGAAAATCTCGATAACTCAAAAAATACG
CCCGGTAGTGATCTTATTTCATTATGGTGAAAGTTGGAACCTCTTACGTGCCGATCAACG
TCTCATTTTCGCCAAAAGTTGGCCCAGGGCTTCCCGGTATCAACAGGGACACCAGGATT
TATTTATTCTGCGAAGTGATCTTCCGTCACAGGTATTTATTCGGCGCAAAGTGCGTCGGG
TGATGCTGCCAACTTACTGATTTAGTGTATGATGGTGTTTTTGAGGTGCTCCAGTGGCTTC
TGTTTCTATCAGGGCTGGATGATCCTCCAGCGCGGGGATCTCATGCTGGAGTTCTTCGCC
CACCCCAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCCTTAA
CGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTG
AGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAG
CGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTC
AGCAGAGCGCAGATACCAAATACTGTCCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTC
AAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCT
GCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAA
GGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAAC
GACCTACACCGAACTGAGATACCTACAGCGTGAGCATTGAGAAAGCGCCACGCTTCCCG
AAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCA
CGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCAC
CTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAA
CGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTT
CTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAGTGAGCTGAT
ACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGAA
GAGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATGCAGCTG
GCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGAGCGCAACGCAATTAATGTGAGT
TAGCTCACTCATTAGGCACCCCAGGCTTTACACTTTATGCTTCCGGCTCGTATGTTGTGTG
GAATTGTGAGCGGATAACAATTTCACACAGGAAACAGCTATGACCATGATTACGAATTG
GCTCCGTTGTTCACGTCTACTACGACGGCGACGAGAACGACAAGGAAACCTTCCTCATC
GGTACCCGTGCTGGCGCTTCCGAGAACCCAGATCTTGAGACCTACTCTGAGCAGTCCCC
ACTCGGCGCTGCAATTCTCGGAGCTCAGGAAGGCGACACCCGTCAGTACACCGCTCCA
AATGGTTCCGTTATCTCCGTAACTGTTGTTTCTGCAGAACCATACAACTCAGCAAAAGCC
GCGACACTCCGCGGCAAAAACTAACCAAGGATTTAAAAGTCTTCAAAATGACAACTCTT
TCACGTAAGTTCTTCGTTTCTGCTACCACAGCCCTGGCGGCAGTCGCACTGGTTGCGTG
TTCCCCTAATGAGATTGATTCTGAACTGAAGGTGCCAACGGCAACTGGCGTTTCTTTACC
TTCGAAGAACGTTTCCGCGACCTCAACTGCTACTACAGATGAGGATGCGCCTGGCTACA
TTGATTGCGTAGCCGCACCAACTCAGCAACCTGCTGAAATCTCACTAAACTGTGCAATG
GATATTGATCGGCTCACGGATATTTCTTGGAGCGAATGGGATACTGATTCCGCAACTGGA
ACCGGTACCCGCATCGTAACCGCTGCAAATGGTCAAGAGACCGAAACCGAAGATATTGA
GGTGAAGCTTTCCTTCCCCACCGAGTCTTCCCAAGGCCTAGTGTTCACTCAGGTCACCG
TCGATGGACAGGTTCTCTTCCTCTAATCCTCCATAATTAGAGAGCGTAAGGCCCCTACTT
CCTGTTTTAGGAAATAGGGGCCTTTTGTTGTCTTCTCCTGGAGGCTATTTAAGAAGTTTA
AATTGTGTCCATGAGTTCGCTCGAGAACTGCGCAACTCGTGAAAGGTAGGCGGATCCAG
ATCCCGGACACCATCGAATGGCGCAAAACCTTTCGCGGTATGGCATGATAGCGCCCGGA
AGAGAGTCAATTCAGGGTGGTGAATGTGAAACCAGTAACGTTATACGATGTCGCAGAGT
ATGCCGGTGTCTCTTATCAGACCGTTTCCCGCGTGGTGAACCAGGCCAGCCACGTTTCT
GCGAAAACGCGGGAAAAAGTGGAAGCGGCGATGGCGGAGCTGAATTACATTCCCAACC
GCGTGGCACAACAACTGGCGGGCAAACAGTCGTTGCTGATTGGCGTTGCCACCTCCAG
TCTGGCCCTGCACGCGCCGTCGCAAATTGTCGCGGCGATTAAATCTCGCGCCGATCAAC
TGGGTGCCAGCGTGGTGGTGTCGATGGTAGAACGAAGCGGCGTCGAAGCCTGTAAAGC
GGCGGTGCACAATCTTCTCGCGCAACGCGTCAGTGGGCTGATCATTAACTATCCGCTGG
ATGACCAGGATGCCATTGCTGTGGAAGCTGCCTGCACTAATGTTCCGGCGTTATTTCTTG
ATGTCTCTGACCAGACACCCATCAACAGTATTATTTTCTCCCATGAAGACGGTACGCGAC
TGGGCGTGGAGCATCTGGTCGCATTGGGTCACCAGCAAATCGCGCTGTTAGCGGGCCCA
TTAAGTTCTGTCTCGGCGCGTCTGCGTCTGGCTGGCTGGCATAAATATCTCACTCGCAAT
CAAATTCAGCCGATAGCGGAACGGGAAGGCGACTGGAGTGCCATGTCCGGTTTTCAAC
AAACCATGCAAATGCTGAATGAGGGCATCGTTCCCACTGCGATGCTGGTTGCCAACGAT
CAGATGGCGCTGGGCGCAATGCGCGCCATTACCGAGTCCGGGCTGCGCGTTGGTGCGGA
TATCTCGGTAGTGGGATACGACGATACCGAAGACAGCTCATGTTATATCCCGCCGTTAAC
CACCATCAAACAGGATTTTCGCCTGCTGGGGCAAACCAGCGTGGACCGCTTGCTGCAAC
TCTCTCAGGGCCAGGCGGTGAAGGGCAATCAGCTGTTGCCCGTCTCACTGGTGAAAAG
AAAAACCACCCTGGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCAT
TAATGCAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGAGCGCAACGCAA
TTAATGTAAGTTAGCTCACTCATTAGGCACCCCAGGCTTTACACTTTATGCTTCCGGCTCG
TATAATGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGCTATGACCATGA
TTACGGATTCACTGGCCGTCGTTTTACAACGTCGTGACTGGGAAAACCCTGGCGTTACC
CAACTTAATCGCCTTGCAGCACATCCCCCTTTCGCCAGCTGGCGTAATAGCGAAGAGGC
CCGCACCGATCGCCCTTCCCAACAGTTGCGCAGCCTGAATGGCGAATGGCGCTTTGCCT
GGTTTCCGGCACCAGAAGCGGTGCCGGAAAGCTGGCTGGAGTGCGATCTTCCTGAGGC
CGATACTGTCGTCGTCCCCTCAAACTGGCAGATGCACGGTTACGATGCGCCCATCTACAC
CAACGTGACCTATCCCATTACGGTCAATCCGCCGTTTGTTCCCACGGAGAATCCGACGG
GTTGTTACTCGCTCACATTTAATGTTGATGAAAGCTGGCTACAGGAAGGCCAGACGCGA
ATTATTTTTGATGGCGTCGGGATCTGATCCGGATTTACTAACTGGAAGAGGCACTAAATG
AACACGATTAACATCGCTAAGAACGACTTCTCTGACATCGAACTGGCTGCTATCCCGTTC
AACACTCTGGCTGACCATTACGGTGAGCGTTTAGCTCGCGAACAGTTGGCCCTTGAGCA
TGAGTCTTACGAGATGGGTGAAGCACGCTTCCGCAAGATGTTTGAGCGTCAACTTAAAG
CTGGTGAGGTTGCGGATAACGCTGCCGCCAAGCCTCTCATCACTACCCTACTCCCTAAG
ATGATTGCACGCATCAACGACTGGTTTGAGGAAGTGAAAGCTAAGCGCGGCAAGCGCC
CGACAGCCTTCCAGTTCCTGCAAGAAATCAAGCCGGAAGCCGTAGCGTACATCACCATT
AAGACCACTCTGGCTTGCCTAACCAGTGCTGACAATACAACCGTTCAGGCTGTAGCAAG
CGCAATCGGTCGGGCCATTGAGGACGAGGCTCGCTTCGGTCGTATCCGTGACCTTGAAG
CTAAGCACTTCAAGAAAAACGTTGAGGAACAACTCAACAAGCGCGTAGGGCACGTCTA
CAAGAAAGCATTTATGCAAGTTGTCGAGGCTGACATGCTCTCTAAGGGTCTACTCGGTG
GCGAGGCGTGGTCTTCGTGGCATAAGGAAGACTCTATTCATGTAGGAGTACGCTGCATC
GAGATGCTCATTGAGTCAACCGGAATGGTTAGCTTACACCGCCAAAATGCTGGCGTAGT
AGGTCAAGACTCTGAGACTATCGAACTCGCACCTGAATACGCTGAGGCTATCGCAACCC
GTGCAGGTGCGCTGGCTGGCATCTCTCCGATGTTCCAACCTTGCGTAGTTCCTCCTAAGC
CGTGGACTGGCATTACTGGTGGTGGCTATTGGGCTAACGGTCGTCGTCCTCTGGCGCTG
GTGCGTACTCACAGTAAGAAAGCACTGATGCGCTACGAAGACGTTTACATGCCTGAGGT
GTACAAAGCGATTAACATTGCGCAAAACACCGCATGGAAAATCAACAAGAAAGTCCTA
GCGGTCGCCAACGTAATCACCAAGTGGAAGCATTGTCCGGTCGAGGACATCCCTGCGAT
TGAGCGTGAAGAACTCCCGATGAAACCGGAAGACATCGACATGAATCCTGAGGCTCTC
ACCGCGTGGAAACGTGCTGCCGCTGCTGTGTACCGCAAGGACAAGGCTCGCAAGTCTC
GCCGTATCAGCCTTGAGTTCATGCTTGAGCAAGCCAATAAGTTTGCTAACCATAAGGCCA
TCTGGTTCCCTTACAACATGGACTGGCGCGGTCGTGTTTACGCTGTGTCAATGTTCAACC
CGCAAGGTAACGATATGACCAAAGGACTGCTTACGCTGGCGAAAGGTAAACCAATCGGT
AAGGAAGGTTACTACTGGCTGAAAATCCACGGTGCAAACTGTGCGGGTGTCGATAAGGT
TCCGTTCCCTGAGCGCATCAAGTTCATTGAGGAAAACCACGAGAACATCATGGCTTGCG
CTAAGTCTCCACTGGAGAACACTTGGTGGGCTGAGCAAGATTCTCCGTTCTGCTTCCTT
GCGTTCTGCTTTGAGTACGCTGGGGTACAGCACCACGGCCTGAGCTATAACTGCTCCCTT
CCGCTGGCGTTTGACGGGTCTTGCTCTGGCATCCAGCACTTCTCCGCGATGCTCCGAGAT
GAGGTAGGTGGTCGCGCGGTTAACTTGCTTCCTAGTGAAACCGTTCAGGACATCTACGG
GATTGTTGCTAAGAAAGTCAACGAGATTCTACAAGCAGACGCAATCAATGGGACCGATA
ACGAAGTAGTTACCGTGACCGATGAGAACACTGGTGAAATCTCTGAGAAAGTCAAGCT
GGGCACTAAGGCACTGGCTGGTCAATGGCTGGCTTACGGTGTTACTCGCAGTGTGACTA
AGCGTTCAGTCATGACGCTGGCTTACGGGTCCAAAGAGTTCGGCTTCCGTCAACAAGTG
CTGGAAGATACCATTCAGCCAGCTATTGATTCCGGCAAGGGTCTGATGTTCACTCAGCCG
AATCAGGCTGCTGGATACATGGCTAAGCTGATTTGGGAATCTGTGAGCGTGACGGTGGT
AGCTGCGGTTGAAGCAATGAACTGGCTTAAGTCTGCTGCTAAGCTGCTGGCTGCTGAGG
TCAAAGATAAGAAGACTGGAGAGATTCTTCGCAAGCGTTGCGCTGTGCATTGGGTAACT
CCTGATGGTTTCCCTGTGTGGCAGGAATACAAGAAGCCTATTCAGACGCGCTTGAACCT
GATGTTCCTCGGTCAGTTCCGCTTACAGCCTACCATTAACACCAACAAAGATAGCGAGAT
TGATGCACACAAACAGGAGTCTGGTATCGCTCCTAACTTTGTACACAGCCAAGACGGTA
GCCACCTTCGTAAGACTGTAGTGTGGGCACACGAGAAGTACGGAATCGAATCTTTTGCA
CTGATTCACGACTCCTTCGGTACCATTCCGGCTGACGCTGCGAACCTGTTCAAAGCAGT
GCGCGAAACTATGGTTGACACATATGAGTCTTGTGATGTACTGGCTGATTTCTACGACCA
GTTCGCTGACCAGTTGCACGAGTCTCAATTGGACAAAATGCCAGCACTTCCGGCTAAAG
GTAACTTGAACCTCCGTGACATCTTAGAGTCGGACTTCGCGTTCGCGTAACGAATTCGC
GTATGGCAATGACAGTTTGAGACGGCCACAGGCGATTCTGAGAAGCCATTTTCTTTGGG
CGCCGTGGCAGTTTTTATTGGGTCCCACCGCCGAACTGCATATTCGAACCAAGGAGCCT
CAAAAATCGAGCTCGCTTTGGTCTCAAACGCACATTTATCGCGCGTTGAAGTGTGCGTTT
GAGACCAAAGAGCCCTCCACAACGCACGTCTTTGGTTTGGATATGACAGGTGCCCAAG
AACTCACCCCGCCCCATGCTCACAGAGCCCCCATCAGAAGCCAAAAGACCCCTTCCCTG
CCCAAGAAGAACAGGATGAAGGGGTCTTGTGCTGCGTAAACTAGCGGTTTTGGAAGTA
GCTAAGCAGACGTAGGATTTCGGTGTAGAGCCAGACCAAGGTCACTGCAAGACCAAGC
GCAACGCCCCATGCCATCTTGGAAGGTGCACCTTCGCGGACGAGGCGGTCAGCTGCATC
GAAGTCGGAGAGGAAGCTGAATGCTGCCAGGCCGATGCAGAAGAGGGAGAAGATAATC
GCGATGATTCCACCGTCACGCAGTGGGCTTGCGCCACCAGTGAACAGTGCCCATACAAC
GTTGCCCAGGACAAGAACCAGGACGCCAACCATCATGCCGGTGAGGATGCGGTTGAAC
TTAGGAGTGACCTTGATAGCGCCAGTCTTGTATACAAACAGCATGCCAATGAATACACCG
ATGGTGCCAAGGACTGCCTGGCCAATGAGGCCACCTGCGTTGGCGTTACCAACTGTGAA
GCCGGACAGCAGAAGGGAAATTCCGCCGACGAAGAGGCCTTCGAATACTGCGTAAATC
AAAGTGACTGCCGCAGAT
SEQ ID NO:116
DE3_for
TTTGGCGTGTGGTTGGTTAG
SEQ ID NO:117
DE3_rev
TCTCTGAGCTGCTGGCCAAC
c)谷氨酸棒状杆菌ATCC 13032(DE3)_Δcg2273与pJC1_dsRNA_PT7-α微管蛋白-F30::西兰花或pJC1_dsRNA_PT7-CYP3-F30::西兰花的转化
根据Tauch等人，2002(Tauch,A.,Kirchner,O.,B.,/>S.,Pühler,A.,and Kalinowski,J.Efficient Electrotransformation of Corynebacteriumdiphtheriae with a Mini-Replicon Derived from the Corynebacterium glutamicumPlasmid pGA 1.Curr.Microbiol.2002；45,362-367)制备谷氨酸棒杆菌菌株ATCC 13032(DE3)_Δcg2273的感受态细胞，并用pJC1or pJC1_dsRNA_PT7-α微管蛋白-F30::西兰花转化。转化子的选择是在含有25μg/ml卡那霉素的BHI琼脂(1％)平板上进行的((Menkel,E.,Thierbach,G.,Eggeling,L.,and Sahm,H.Influence of increased aspartateavailability on Iysine formation by a recombinant strain of Corynebacteriumglutamicum and utilization of fumarate.Appl.Environ.Microbiol.1989；55,684-688)。由此获得的克隆被命名为谷氨酸棒状杆菌ATCC 13032(DE3)_Δcg2273 pJC1或谷氨酸棒状杆菌ATCC13032(DE3)_Δcg2273 pJC1_dsRNA_PT7-α微管蛋白-F30::西兰花，这取决于用于转化的质粒。
d)使用荧光激活细胞分选(FACS)培养用于表型验证的细胞
将产生的菌株谷氨酸棒状杆菌ATCC 13032(DE3)_Δcg2273 pJC1和谷氨酸棒状杆菌ATCC 13032(DE3)_Δcg2273 pJC1_dsRNA_PT7-α微管蛋白-F30::西兰花，在含有25μg/mL卡那霉素的BHI琼脂平板上划线，并在30℃下培养。将生长的细胞重悬于含有25μg/mL卡那霉素的CGIII培养基中，并在含有2mL含抗生素的培养基的试管中将OD600调节至0.75。细胞在30℃和120rpm下孵育，培养6小时后用1.5mM IPTG诱导。之后，继续培养6小时。随后，使用最终浓度为500μM DFHBI的PBS将细胞稀释至OD600为0.6。使用实施例1d)中已经描述的AriaIII高速细胞分选器对细胞进行分析。与未染色的细胞相比，DFHBI染色的谷氨酸棒状杆菌ATCC 13032(DE3)_Δcg2273 pJC1_dsRNA_PT7-α微管蛋白-F30::西兰花显示出约5倍的荧光输出增加，而DFHBI染的谷氨酸棒菌ATCC 13032(DE3)_Δcg2273 pJC1显示出约1，2倍的荧光产量增加(见图6)。
e)从d)中分离的细胞中提取感兴趣的RNA
使用c)中分析的培养基，使用Monarch总RNA试剂盒(New England Biolabs，Ipswich，MA，USA)将来自培养物的1.38×109个细胞用于RNA提取，如实施例1中所述。
f)通过RNaseIII和RNaseA消化形成双链RNA产物的验证
通过RNase A消化形成双链RNA产物的验证。
根据制造商的建议(AppliChem GmbH，Darmstadt，Germany)，将从谷氨酸棒状杆菌ATCC 13032(DE3)_Δcg2273 pJC1_dsRNA_PT7-α微管蛋白-F30::西兰花的培养物中分离的总共2μgRNA用50ngRNase A在室温、300mM NaCl存在下处理一个小时，以去除mRNA，rRNA并去除靶RNA的单链部分(参见图5)。RNase A处理后，使用T2030 Monarch RNA清理试剂盒(New England Biolabs，Ipswich，MA，USA)纯化反应物。dsRNA Ladder(New EnglandBiolabs，Ipswich，MA，USA)，e)中提取的分离的总RNA和RNase A处理的RNA使用配备有DNF-471RNA试剂盒的安捷伦片段分析仪(安捷伦科技公司，Santa Clara，CA，USA)进行分析。仅在谷氨酸棒状杆菌ATCC 13032(DE3)_Δcg2273 pJC1_dsRNA_PT7-α微管蛋白-F30::西兰花的总RNA提取中可见的两条带，类似于靶RNA片段的计算片段大小(522nt，较低片段)和包括α微管蛋白asRNA链末端的F1终止子(575nt，较大片段)的靶RNA的计算片段大小(参见图7)。RNase A反应后，可以看到一个与α微管蛋白dsRNA大小完全相同的片段，所有的mRNA和rRNA都被降解。
该实验显示了迄今为止不显著的表型(dsRNA产生)与荧光输出的成功连接。在这个实验中，产生的dsRNA具有411个核苷酸的长度，并从载体上转录。因此，根据实施例1中所示的程序，使用本发明可以优化dsRNA的发酵生产。
实施例7
a)载体pJC1-PT7-egfp-西兰花-TT7和pJC1-PT7-luc2-西兰花-TT7的构建
质粒的构建是通过化学合成的方式合成DNA片段(PT7-egfp-西兰花-TT7的SEQ IDNO:118和PT7-luc2-西兰花-TT7的SEQ ID NO:119)来实现的，并将其***pJC1的限制性位点BamHI和EcoRV中，从而产生质粒pJC1_PT7-egfp-西兰花-TT7(SEQ ID NO:120)和pJC1-PT7-luc2-西兰花-TT7(SEQ ID NO:121)(订购自美国旧金山南部的Twist Bioscience)。SEQ ID NO:118包含T7启动子(SEQ ID NO:96)、编码增强型绿色荧光蛋白(由维多利亚绿锥虫修饰)的基因egfp(SEQ ID NO:112)、在***位点具有西兰花核酸适配体的F30支架(SEQID NO:69)和T7终止子(SEQ ID NO:97)。SEQ ID NO:119包含T7启动子(SEQ ID NO:96)、编码火绒萤光素酶(Photinus pyralis)的基因luc2(SEQ ID NO:82)、在***位点具有西兰花核酸适配体的F30支架(SEQ ID NO:69)和T7终止子(SEQ ID NO:97)。
SEQ ID NO:118
PT7-egfp-西兰花-TT7(SEQ ID NO:96,112,69和97组合):
TAATACGACTCACTATAGGGAGACCCAAGCTGGCTAGCGTTTAAACTTAAGCTTCGAATTCTGCAGTCGACGGTACCGCGGGCCCGGGATCCACCGGTCGCCACCATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAGTAATACGTATTGCCATGTGTATGTGGGAGACGGTCGGGTCCAGATATTCGTATCTGTCGAGTAGAGTGTGGGCTCCCACATACTCTGATGATCCTTCGGGATCATTCATGGCAAGTCGACCTAGCATAACCCCTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTG
SEQ ID NO:119
PT7-luc2-西兰花-TT7(SEQ ID NO:96,82,69和97组合):
TAATACGACTCACTATAGGGAGACCCAAGCTGGCTAGCGTTTAAACTTAAGCTTGGCAATCCGGTACTGTTGGTAAAGCCACCATGGAAGATGCCAAAAACATTAAGAAGGGCCCAGCGCCATTCTACCCACTCGAAGACGGGACCGCCGGCGAGCAGCTGCACAAAGCCATGAAGCGCTACGCCCTGGTGCCCGGCACCATCGCCTTTACCGACGCACATATCGAGGTGGACATTACCTACGCCGAGTACTTCGAGATGAGCGTTCGGCTGGCAGAAGCTATGAAGCGCTATGGGCTGAATACAAACCATCGGATCGTGGTGTGCAGCGAGAATAGCTTGCAGTTCTTCATGCCCGTGTTGGGTGCCCTGTTCATCGGTGTGGCTGTGGCCCCAGCTAACGACATCTACAACGAGCGCGAGCTGCTGAACAGCATGGGCATCAGCCAGCCCACCGTCGTATTCGTGAGCAAGAAAGGGCTGCAAAAGATCCTCAACGTGCAAAAGAAGCTACCGATCATACAAAAGATCATCATCATGGATAGCAAGACCGACTACCAGGGCTTCCAAAGCATGTACACCTTCGTGACTTCCCATTTGCCACCCGGCTTCAACGAGTACGACTTCGTGCCCGAGAGCTTCGACCGGGACAAAACCATCGCCCTGATCATGAACAGTAGTGGCAGTACCGGATTGCCCAAGGGCGTAGCCCTACCGCACCGCACCGCTTGTGTCCGATTCAGTCATGCCCGCGACCCCATCTTCGGCAACCAGATCATCCCCGACACCGCTATCCTCAGCGTGGTGCCATTTCACCACGGCTTCGGCATGTTCACCACGCTGGGCTACTTGATCTGCGGCTTTCGGGTCGTGCTCATGTACCGCTTCGAGGAGGAGCTATTCTTGCGCAGCTTGCAAGACTATAAGATTCAATCTGCCCTGCTGGTGCCCACACTATTTAGCTTCTTCGCTAAGAGCACTCTCATCGACAAGTACGACCTAAGCAACTTGCACGAGATCGCCAGCGGCGGGGCGCCGCTCAGCAAGGAGGTAGGTGAGGCCGTGGCCAAACGCTTCCACCTACCAGGCATCCGCCAGGGCTACGGCCTGACAGAAACAACCAGCGCCATTCTGATCACCCCCGAAGGGGACGACAAGCCTGGCGCAGTAGGCAAGGTGGTGCCCTTCTTCGAGGCTAAGGTGGTGGACTTGGACACCGGTAAGACACTGGGTGTGAACCAGCGCGGCGAGCTGTGCGTCCGTGGCCCCATGATCATGAGCGGCTACGTTAACAACCCCGAGGCTACAAACGCTCTCATCGACAAGGACGGCTGGCTGCACAGCGGCGACATCGCCTACTGGGACGAGGACGAGCACTTCTTCATCGTGGACCGGCTGAAGAGCCTGATCAAATACAAGGGCTACCAGGTAGCCCCAGCCGAACTGGAGAGCATCCTGCTGCAACACCCCAACATCTTCGACGCCGGGGTCGCCGGCCTGCCCGACGACGATGCCGGCGAGCTGCCCGCCGCAGTCGTCGTGCTGGAACACGGTAAAACCATGACCGAGAAGGAGATCGTGGACTATGTGGCCAGCCAGGTTACAACCGCCAAGAAGCTGCGCGGTGGTGTTGTGTTCGTGGACGAGGTGCCTAAAGGACTGACCGGCAAGTTGGACGCCCGCAAGATCCGCGAGATTCTCATTAAGGCCAAGAAGGGCGGCAAGATCGCCGTGTAATAATTCTAGATACGTATTGCCATGTGTATGTGGGAGACGGTCGGGTCCAGATATTCGTATCTGTCGAGTAGAGTGTGGGCTCCCACATACTCTGATGATCCTTCGGGATCATTCATGGCAAGTCGACCTAGCATAACCCCTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTG
SEQ ID NO:120
pJC1-PT7-egfp-西兰花-TT7:
CCGAGAAGTTTTTTACAAAAGGCAAAAACTTTTTCGGGATCGACAGAAATAAAACGATCGACGGTACGCAACAAAAAAGCGTCAGGATCGCCGTAGAGCGATTGAAGACCGTCAACCAAAGGGGAAGCCTCCAATCGACGCGACGCGCGCTCTACGGCGATCCTGACGCAGATTTTTAGCTATCTGTCGCAGCGCCCTCAGGGACAAGCCACCCGCACAACGTCGCGAGGGCGATCAGCGACGCCGCAGGGTAGTACTCAGCTGTAATACGACTCACTATAGGGAGACCCAAGCTGGCTAGCGTTTAAACTTAAGCTTCGAATTCTGCAGTCGACGGTACCGCGGGCCCGGGATCCACCGGTCGCCACCATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAGTAATACGTATTGCCATGTGTATGTGGGAGACGGTCGGGTCCAGATATTCGTATCTGTCGAGTAGAGTGTGGGCTCCCACATACTCTGATGATCCTTCGGGATCATTCATGGCAAGTCGACCTAGCATAACCCCTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTGCTGAAAGATATCATCCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAACTATGGATGAACGAAATAGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTAAC
TGTCAGACCAAGTTTACTCATATATACTTTAGATTGATTTAAAACTTCATTTTTAATTTAAA
AGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCCTTAACGTGAGTTT
TCGTTCCACTGAGCGTCAGACCCCTTAATAAGATGATCTTCTTGAGATCGTTTTGGTCTG
CGCGTAATCTCTTGCTCTGAAAACGAAAAAACCGCCTTGCAGGGCGGTTTTTCGAAGGT
TCTCTGAGCTACCAACTCTTTGAACCGAGGTAACTGGCTTGGAGGAGCGCAGTCACCAA
AACTTGTCCTTTCAGTTTAGCCTTAACCGGCGCATGACTTCAAGACTAACTCCTCTAAAT
CAATTACCAGTGGCTGCTGCCAGTGGTGCTTTTGCATGTCTTTCCGGGTTGGACTCAAGA
CGATAGTTACCGGATAAGGCGCAGCGGTCGGACTGAACGGGGGGTTCGTGCATACAGTC
CAGCTTGGAGCGAACTGCCTACCCGGAACTGAGTGTCAGGCGTGGAATGAGACAAACG
CGGCCATAACAGCGGAATGACACCGGTAAACCGAAAGGCAGGAACAGGAGAGCGCAC
GAGGGAGCCGCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACC
ACTGATTTGAGCGTCAGATTTCGTGATGCTTGTCAGGGGGGCGGAGCCTATGGAAAAAC
GGCTTTGCCGCGGCCCTCTCACTTCCCTGTTAAGTATCTTCCTGGCATCTTCCAGGAAAT
CTCCGCCCCGTTCGTAAGCCATTTCCGCTCGCCGCAGTCGAACGACCGAGCGTAGCGAG
TCAGTGAGCGAGGAAGCGGAATATATCCTGTATCACATATTCTGCTGACGCACCGGTGCA
GCCTTTTTTCTCCTGCCACATGAAGCACTTCACTGACACCCTCATCAGTGCCAACATAGT
AAGCCAGTATACACTCCGCTAGCGCTGAGGTCTGCCTCGTGAAGAAGGTGTTGCTGACT
CATACCAGGCCTGAATCGCCCCATCATCCAGCCAGAAAGTGAGGGAGCCACGGTTGATG
AGAGCTTTGTTGTAGGTGGACCAGTTGGTGATTTTGAACTTTTGCTTTGCCACGGAACG
GTCTGCGTTGTCGGGAAGATGCGTGATCTGATCCTTCAACTCAGCAAAAGTTCGATTTAT
TCAACAAAGCCACGTTGTGTCTCAAAATCTCTGATGTTACATTGCACAAGATAAAAATAT
ATCATCATGAACAATAAAACTGTCTGCTTACATAAACAGTAATACAAGGGGTGTTATGAG
CCATATTCAACGGGAAACGTCTTGCTCGAGGCCGCGATTAAATTCCAACATGGATGCTGA
TTTATATGGGTATAAATGGGCTCGCGATAATGTCGGGCAATCAGGTGCGACAATCTATCGA
TTGTATGGGAAGCCCGATGCGCCAGAGTTGTTTCTGAAACATGGCAAAGGTAGCGTTGC
CAATGATGTTACAGATGAGATGGTCAGACTAAACTGGCTGACGGAATTTATGCCTCTTCC
GACCATCAAGCATTTTATCCGTACTCCTGATGATGCATGGTTACTCACCACTGCGATCCCC
GGGAAAACAGCATTCCAGGTATTAGAAGAATATCCTGATTCAGGTGAAAATATTGTTGAT
GCGCTGGCAGTGTTCCTGCGCCGGTTGCATTCGATTCCTGTTTGTAATTGTCCTTTTAAC
AGCGATCGCGTATTTCGTCTCGCTCAGGCGCAATCACGAATGAATAACGGTTTGGTTGAT
GCGAGTGATTTTGATGACGAGCGTAATGGCTGGCCTGTTGAACAAGTCTGGAAAGAAAT
GCATAAGCTTTTGCCATTCTCACCGGATTCAGTCGTCACTCATGGTGATTTCTCACTTGAT
AACCTTATTTTTGACGAGGGGAAATTAATAGGTTGTATTGATGTTGGACGAGTCGGAATC
GCAGACCGATACCAGGATCTTGCCATCCTATGGAACTGCCTCGGTGAGTTTTCTCCTTCA
TTACAGAAACGGCTTTTTCAAAAATATGGTATTGATAATCCTGATATGAATAAATTGCAGT
TTCATTTGATGCTCGATGAGTTTTTCTAATCAGAATTGGTTAATTGGTTGTAACACTGGCA
GAGCATTACGCTGACTTGACGGGACGGCGGCTTTGTTGAATAAATCGAACTTTTGCTGA
GTTGAAGGATCAGATCACGCATCTTCCCGACAACGCAGACCGTTCCGTGGCAAAGCAA
AAGTTCAAAATCACCAACTGGTCCACCTACAACAAAGCTCTCATCAACCGTGGCTCCCT
CACTTTCTGGCTGGATGATGGGGCGATTCAGGCCTGGTATGAGTCAGCAACACCTTCTTC
ACGAGGCAGACCTCAGCGCTCAAAGATGCAGGGGTAAAAGCTAACCGCATCTTTACCG
ACAAGGCATCCGGCAGTTCAACAGATCGGGAAGGGCTGGATTTGCTGAGGATGAAGGT
GGAGGAAGGTGATGTCATTCTGGTGAAGAAGCTCGACCGTCTTGGCCGCGACACCGCC
GACATGATCCAACTGATAAAAGAGTTTGATGCTCAGGGTGTAGCGGTTCGGTTTATTGAC
GACGGGATCAGTACCGACGGTGATATGGGGCAAATGGTGGTCACCATCCTGTCGGCTGT
GGCACAGGCTGAACGCCGGAGGATCAAGTCGGTCAAGCCAAGCGCAACCAGCGGCAC
CGCCGCGAGCAACGTCGCAAGGGCGATCAGGGGACGATTTTTGCGAAGAATTTCCACG
GTAAGAATCCAATCTCTCGAATTTAGGGTGAAAGAAGCTTGGCATAGGGGTGTGCACGA
ACTCGGTGGAGGAAATTTCCGCGGGGCAAGGCTTCGCGAAGCGGAGTCGCGGCAGTGG
CTTTGAAGATCTTTGGGAGCAGTCCTTGTGCGCTTACGAGGTGAGCCGGTGGGGAACCG
TTATCTGCCTATGGTGTGAGCCCCCCTAGAGAGCTTCAAGAGCAATCAGCCCGACCTAG
AAAGGAGGCCAAGAGAGAGACCCCTACGGGGGGAACCGTTTTCTGCCTACGAGATGGC
ACATTTACTGGGAAGCTTTACGGCGTCCTCGTGGAAGTTCAATGCCCGCAGACTTAAGT
GCTCTATTCACGGTCTGACGTGACACGCTAAATTCAGACATAGCTTCATTGATTGTCGGC
CACGAGCCAGTCTCTCCCTCAACAGTCATAAACCAACCTGCAATGGTCAAGCGATTTCC
TTTAGCTTTCCTAGCTTGTCGTTGACTGGACTTAGCTAGTTTTTCTCGCTGTGCTCGGGC
GTACTCACTGTTTGGGTCTTTCCAGCGTTCTGCGGCCTTTTTACCGCCACGTCTTCCCATA
GTGGCCAGAGCTTTTCGCCCTCGGCTGCTCTGCGTCTCTGTCTGACGAGCAGGGACGAC
TGGCTGGCCTTTAGCGACGTAGCCGCGCACACGTCGCGCCATCGTCTGGCGGTCACGCA
TCGGCGGCAGATCAGGCTCACGGCCGTCTGCTCCGACCGCCTGAGCGACGGTGTAGGC
ACGCTCGTAGGCGTCGATGATCTTGGTGTCTTTTAGGCGCTCACCAGCCGCTTTTAACTG
GTATCCCACAGTCAAAGCGTGGCGAAAAGCCGTCTCATCACGGGCGGCACGCCCTGGA
GCAGTCCAGAGGACACGGACGCCGTCGATCAGCTCTCCAGACGCTTCAGCGGCGCTCG
GCAGGCTTGCTTCAAGCGTGGCAAGTGCTTTTGCTTCCGCAGTGGCTTTTCTTGCCGCTT
CGATACGTGCCCGTCCGCTAGAAAACTCCTGCTCATAGCGTTTTTTAGGTTTTTCTGTGC
CTGAGATCATGCGAGCAACCTCCATAAGATCAGCTAGGCGATCCACGCGATTGTGCTGG
GCATGCCAGCGGTACGCGGTGGGATCGTCGGAGACGTGCAGTGGCCACCGGCTCAGCC
TATGTGAAAAAGCCTGGTCAGCGCCGAAAACGCGGGTCATTTCCTCGGTCGTTGCAGCC
AGCAGGCGCATATTCGGGCTGCTCATGCCTGCTGCGGCATACACCGGATCAATGAGCCA
GATGAGCTGGCATTTCCCGCTCAGTGGATTCACGCCGATCCAAGCTGGCGCTTTTTCCAG
GCGTGCCCAGCGCTCCAAAATCGCGTAGACCTCGGGGTTTACGTGCTCGATTTTCCCGC
CGGCCTGGTGGCTCGGCACATCAATGTCCAGGACAAGCACGGCTGCGTGCTGCGCGTG
CGTCAGAGCAACATACTGGCACCGGGCAAGCGATTTTGAACCAACTCGGTATAACTTCG
GCTGTGTTTCTCCCGTGTCCGGGTCTTTGATCCAAGCGCTGGCGAAGTCGCGGGTCTTG
CTGCCCTGGAAATTTTCTCTGCCCAGGTGAGCGAGGAATTCGCGGCGGTCTTCGCTCGT
CCAGCCACGTGATCGCAGCGCGAGCTCGGGATGGGTGTCGAACAGATCAGCGGAAAAT
TTCCAGGCCGGTGTGTCAATGTCTCGTGAATCCGCTAGAGTCATTTTTGAGCGCTTTCTC
CCAGGTTTGGACTGGGGGTTAGCCGACGCCCTGTGAGTTACCGCTCACGGGGCGTTCAA
CATTTTTCAGGTATTCGTGCAGCTTATCGCTTCTTGCCGCCTGTGCGCTTTTTCGACGCGC
GACGCTGCTGCCGATTCGGTGCAGGTGGTGGCGGCGCTGACACGTCCTGGGCGGCCAC
GGCCACACGAAACGCGGCATTTACGATGTTTGTCATGCCTGCGGGCACCGCGCCACGAT
CGCGGATAATTCTCGCTGCCGCTTCCAGCTCTGTGACGACCATGGCCAAAATTTCGCTCG
GGGGACGCACTTCCAGCGCCATTTGCGACCTAGCCGCCTCCAGCTCCTCGGCGTGGCGT
TTGTTGGCGCGCTCGCGGCTGGCTGCGGCACGACACGCATCTGAGCAATATTTTGCGCG
CCGTCCTCGCGGGTCAGGCCGGGGAGGAATCAGGCCACCGCAGTAGGCGCAACTGATT
CGATCCTCCACTACTGTGCGTCCTCCTGGCGCTGCCGAGCACGCAGCTCGTCAGCCAGC
TCCTCAAGATCCGCCACGAGAGTTTCTAGGTCGCTCGCGGCACTGGCCCAGTCTCGTGA
TGCTGGCGCGTCCGTCGTATCGAGAGCTCGGAAAAATCCGATCACCGTTTTTAAATCGA
CGGCAGCATCGAGCGCGTCGGACTCCAGCGCGACATCAGAGAGATCCATAGCTGATGAT
TCGGGCCAATTTTGGTACTTCGTCGTGAAGGTCATGACACCATTATAACGAACGTTCGTT
AAAGTTTTTGGCGGAAAATCACGCGGCACGAAAATTTTCACGAAGCGGGACTTTGCGC
AGCTCAGGGGTGCTAAAAATTTTGTATCGCACTTGATTTTTCCGAAAGACAGATTATCTG
CAAACGGTGTGTCGTATTTCTGGCTTGGTTTTTAAAAAATCTGGAATCGAAAATTTGCGG
GGCGA
SEQ ID NO:121
pJC1-PT7-luc2-西兰花-TT7:
GGTACGCAACAAAAAAGCGTCAGGATCGCCGTAGAGCGATTGAAGACCGTCAACCAAAGGGGAAGCCTCCAATCGACGCGACGCGCGCTCTACGGCGATCCTGACGCAGATTTTTAGCTATCTGTCGCAGCGCCCTCAGGGACAAGCCACCCGCACAACGTCGCGAGGGCGATCAGCGACGCCGCAGGGGGATCCAGTACTCAGCTGGCTAACTAGAGAACCCACTGCTTACTGGCTTATCGAAATTAATACGACTCACTATAGGGAGACCCAAGCTGGCTAGCGTTTAAA
CTTAAGCTTGGCAATCCGGTACTGTTGGTAAAGCCACCATGGAAGATGCCAAAAACATT
AAGAAGGGCCCAGCGCCATTCTACCCACTCGAAGACGGGACCGCCGGCGAGCAGCTGC
ACAAAGCCATGAAGCGCTACGCCCTGGTGCCCGGCACCATCGCCTTTACCGACGCACAT
ATCGAGGTGGACATTACCTACGCCGAGTACTTCGAGATGAGCGTTCGGCTGGCAGAAGC
TATGAAGCGCTATGGGCTGAATACAAACCATCGGATCGTGGTGTGCAGCGAGAATAGCT
TGCAGTTCTTCATGCCCGTGTTGGGTGCCCTGTTCATCGGTGTGGCTGTGGCCCCAGCTA
ACGACATCTACAACGAGCGCGAGCTGCTGAACAGCATGGGCATCAGCCAGCCCACCGT
CGTATTCGTGAGCAAGAAAGGGCTGCAAAAGATCCTCAACGTGCAAAAGAAGCTACCG
ATCATACAAAAGATCATCATCATGGATAGCAAGACCGACTACCAGGGCTTCCAAAGCATG
TACACCTTCGTGACTTCCCATTTGCCACCCGGCTTCAACGAGTACGACTTCGTGCCCGA
GAGCTTCGACCGGGACAAAACCATCGCCCTGATCATGAACAGTAGTGGCAGTACCGGAT
TGCCCAAGGGCGTAGCCCTACCGCACCGCACCGCTTGTGTCCGATTCAGTCATGCCCGC
GACCCCATCTTCGGCAACCAGATCATCCCCGACACCGCTATCCTCAGCGTGGTGCCATTT
CACCACGGCTTCGGCATGTTCACCACGCTGGGCTACTTGATCTGCGGCTTTCGGGTCGT
GCTCATGTACCGCTTCGAGGAGGAGCTATTCTTGCGCAGCTTGCAAGACTATAAGATTCA
ATCTGCCCTGCTGGTGCCCACACTATTTAGCTTCTTCGCTAAGAGCACTCTCATCGACAA
GTACGACCTAAGCAACTTGCACGAGATCGCCAGCGGCGGGGCGCCGCTCAGCAAGGAG
GTAGGTGAGGCCGTGGCCAAACGCTTCCACCTACCAGGCATCCGCCAGGGCTACGGCCT
GACAGAAACAACCAGCGCCATTCTGATCACCCCCGAAGGGGACGACAAGCCTGGCGCA
GTAGGCAAGGTGGTGCCCTTCTTCGAGGCTAAGGTGGTGGACTTGGACACCGGTAAGA
CACTGGGTGTGAACCAGCGCGGCGAGCTGTGCGTCCGTGGCCCCATGATCATGAGCGG
CTACGTTAACAACCCCGAGGCTACAAACGCTCTCATCGACAAGGACGGCTGGCTGCACA
GCGGCGACATCGCCTACTGGGACGAGGACGAGCACTTCTTCATCGTGGACCGGCTGAA
GAGCCTGATCAAATACAAGGGCTACCAGGTAGCCCCAGCCGAACTGGAGAGCATCCTGC
TGCAACACCCCAACATCTTCGACGCCGGGGTCGCCGGCCTGCCCGACGACGATGCCGG
CGAGCTGCCCGCCGCAGTCGTCGTGCTGGAACACGGTAAAACCATGACCGAGAAGGAG
ATCGTGGACTATGTGGCCAGCCAGGTTACAACCGCCAAGAAGCTGCGCGGTGGTGTTGT
GTTCGTGGACGAGGTGCCTAAAGGACTGACCGGCAAGTTGGACGCCCGCAAGATCCGC
GAGATTCTCATTAAGGCCAAGAAGGGCGGCAAGATCGCCGTGTAATAATTCTAGATACGT
ATTGCCATGTGTATGTGGGAGACGGTCGGGTCCAGATATTCGTATCTGTCGAGTAGAGTG
TGGGCTCCCACATACTCTGATGATCCTTCGGGATCATTCATGGCAAGTCGACCTAGCATA
ACCCCTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTGCTGAAAGATATCGATATCCA
ATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCA
ACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCC
TTCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTA
TCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTACACGACGG
GGAGTCAGGCAACTATGGATGAACGAAATAGACAGATCGCTGAGATAGGTGCCTCACTG
ATTAAGCATTGGTAACTGTCAGACCAAGTTTACTCATATATACTTTAGATTGATTTAAAAC
TTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAAT
CCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCTTAATAAGATGATCTTCTTG
AGATCGTTTTGGTCTGCGCGTAATCTCTTGCTCTGAAAACGAAAAAACCGCCTTGCAGG
GCGGTTTTTCGAAGGTTCTCTGAGCTACCAACTCTTTGAACCGAGGTAACTGGCTTGGA
GGAGCGCAGTCACCAAAACTTGTCCTTTCAGTTTAGCCTTAACCGGCGCATGACTTCAA
GACTAACTCCTCTAAATCAATTACCAGTGGCTGCTGCCAGTGGTGCTTTTGCATGTCTTT
CCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGACTGAACGGG
GGGTTCGTGCATACAGTCCAGCTTGGAGCGAACTGCCTACCCGGAACTGAGTGTCAGGC
GTGGAATGAGACAAACGCGGCCATAACAGCGGAATGACACCGGTAAACCGAAAGGCAG
GAACAGGAGAGCGCACGAGGGAGCCGCCAGGGGGAAACGCCTGGTATCTTTATAGTCC
TGTCGGGTTTCGCCACCACTGATTTGAGCGTCAGATTTCGTGATGCTTGTCAGGGGGGC
GGAGCCTATGGAAAAACGGCTTTGCCGCGGCCCTCTCACTTCCCTGTTAAGTATCTTCCT
GGCATCTTCCAGGAAATCTCCGCCCCGTTCGTAAGCCATTTCCGCTCGCCGCAGTCGAA
CGACCGAGCGTAGCGAGTCAGTGAGCGAGGAAGCGGAATATATCCTGTATCACATATTCT
GCTGACGCACCGGTGCAGCCTTTTTTCTCCTGCCACATGAAGCACTTCACTGACACCCT
CATCAGTGCCAACATAGTAAGCCAGTATACACTCCGCTAGCGCTGAGGTCTGCCTCGTG
AAGAAGGTGTTGCTGACTCATACCAGGCCTGAATCGCCCCATCATCCAGCCAGAAAGTG
AGGGAGCCACGGTTGATGAGAGCTTTGTTGTAGGTGGACCAGTTGGTGATTTTGAACTT
TTGCTTTGCCACGGAACGGTCTGCGTTGTCGGGAAGATGCGTGATCTGATCCTTCAACT
CAGCAAAAGTTCGATTTATTCAACAAAGCCACGTTGTGTCTCAAAATCTCTGATGTTACA
TTGCACAAGATAAAAATATATCATCATGAACAATAAAACTGTCTGCTTACATAAACAGTA
ATACAAGGGGTGTTATGAGCCATATTCAACGGGAAACGTCTTGCTCGAGGCCGCGATTA
AATTCCAACATGGATGCTGATTTATATGGGTATAAATGGGCTCGCGATAATGTCGGGCAAT
CAGGTGCGACAATCTATCGATTGTATGGGAAGCCCGATGCGCCAGAGTTGTTTCTGAAA
CATGGCAAAGGTAGCGTTGCCAATGATGTTACAGATGAGATGGTCAGACTAAACTGGCT
GACGGAATTTATGCCTCTTCCGACCATCAAGCATTTTATCCGTACTCCTGATGATGCATGG
TTACTCACCACTGCGATCCCCGGGAAAACAGCATTCCAGGTATTAGAAGAATATCCTGAT
TCAGGTGAAAATATTGTTGATGCGCTGGCAGTGTTCCTGCGCCGGTTGCATTCGATTCCT
GTTTGTAATTGTCCTTTTAACAGCGATCGCGTATTTCGTCTCGCTCAGGCGCAATCACGA
ATGAATAACGGTTTGGTTGATGCGAGTGATTTTGATGACGAGCGTAATGGCTGGCCTGTT
GAACAAGTCTGGAAAGAAATGCATAAGCTTTTGCCATTCTCACCGGATTCAGTCGTCAC
TCATGGTGATTTCTCACTTGATAACCTTATTTTTGACGAGGGGAAATTAATAGGTTGTATT
GATGTTGGACGAGTCGGAATCGCAGACCGATACCAGGATCTTGCCATCCTATGGAACTG
CCTCGGTGAGTTTTCTCCTTCATTACAGAAACGGCTTTTTCAAAAATATGGTATTGATAAT
CCTGATATGAATAAATTGCAGTTTCATTTGATGCTCGATGAGTTTTTCTAATCAGAATTGG
TTAATTGGTTGTAACACTGGCAGAGCATTACGCTGACTTGACGGGACGGCGGCTTTGTT
GAATAAATCGAACTTTTGCTGAGTTGAAGGATCAGATCACGCATCTTCCCGACAACGCA
GACCGTTCCGTGGCAAAGCAAAAGTTCAAAATCACCAACTGGTCCACCTACAACAAAG
CTCTCATCAACCGTGGCTCCCTCACTTTCTGGCTGGATGATGGGGCGATTCAGGCCTGGT
ATGAGTCAGCAACACCTTCTTCACGAGGCAGACCTCAGCGCTCAAAGATGCAGGGGTA
AAAGCTAACCGCATCTTTACCGACAAGGCATCCGGCAGTTCAACAGATCGGGAAGGGCT
GGATTTGCTGAGGATGAAGGTGGAGGAAGGTGATGTCATTCTGGTGAAGAAGCTCGAC
CGTCTTGGCCGCGACACCGCCGACATGATCCAACTGATAAAAGAGTTTGATGCTCAGGG
TGTAGCGGTTCGGTTTATTGACGACGGGATCAGTACCGACGGTGATATGGGGCAAATGG
TGGTCACCATCCTGTCGGCTGTGGCACAGGCTGAACGCCGGAGGATCAAGTCGGTCAA
GCCAAGCGCAACCAGCGGCACCGCCGCGAGCAACGTCGCAAGGGCGATCAGGGGACG
ATTTTTGCGAAGAATTTCCACGGTAAGAATCCAATCTCTCGAATTTAGGGTGAAAGAAG
CTTGGCATAGGGGTGTGCACGAACTCGGTGGAGGAAATTTCCGCGGGGCAAGGCTTCG
CGAAGCGGAGTCGCGGCAGTGGCTTTGAAGATCTTTGGGAGCAGTCCTTGTGCGCTTAC
GAGGTGAGCCGGTGGGGAACCGTTATCTGCCTATGGTGTGAGCCCCCCTAGAGAGCTTC
AAGAGCAATCAGCCCGACCTAGAAAGGAGGCCAAGAGAGAGACCCCTACGGGGGGAA
CCGTTTTCTGCCTACGAGATGGCACATTTACTGGGAAGCTTTACGGCGTCCTCGTGGAAG
TTCAATGCCCGCAGACTTAAGTGCTCTATTCACGGTCTGACGTGACACGCTAAATTCAGA
CATAGCTTCATTGATTGTCGGCCACGAGCCAGTCTCTCCCTCAACAGTCATAAACCAACC
TGCAATGGTCAAGCGATTTCCTTTAGCTTTCCTAGCTTGTCGTTGACTGGACTTAGCTAG
TTTTTCTCGCTGTGCTCGGGCGTACTCACTGTTTGGGTCTTTCCAGCGTTCTGCGGCCTT
TTTACCGCCACGTCTTCCCATAGTGGCCAGAGCTTTTCGCCCTCGGCTGCTCTGCGTCTC
TGTCTGACGAGCAGGGACGACTGGCTGGCCTTTAGCGACGTAGCCGCGCACACGTCGC
GCCATCGTCTGGCGGTCACGCATCGGCGGCAGATCAGGCTCACGGCCGTCTGCTCCGAC
CGCCTGAGCGACGGTGTAGGCACGCTCGTAGGCGTCGATGATCTTGGTGTCTTTTAGGC
GCTCACCAGCCGCTTTTAACTGGTATCCCACAGTCAAAGCGTGGCGAAAAGCCGTCTCA
TCACGGGCGGCACGCCCTGGAGCAGTCCAGAGGACACGGACGCCGTCGATCAGCTCTC
CAGACGCTTCAGCGGCGCTCGGCAGGCTTGCTTCAAGCGTGGCAAGTGCTTTTGCTTCC
GCAGTGGCTTTTCTTGCCGCTTCGATACGTGCCCGTCCGCTAGAAAACTCCTGCTCATAG
CGTTTTTTAGGTTTTTCTGTGCCTGAGATCATGCGAGCAACCTCCATAAGATCAGCTAGG
CGATCCACGCGATTGTGCTGGGCATGCCAGCGGTACGCGGTGGGATCGTCGGAGACGTG
CAGTGGCCACCGGCTCAGCCTATGTGAAAAAGCCTGGTCAGCGCCGAAAACGCGGGTC
ATTTCCTCGGTCGTTGCAGCCAGCAGGCGCATATTCGGGCTGCTCATGCCTGCTGCGGCA
TACACCGGATCAATGAGCCAGATGAGCTGGCATTTCCCGCTCAGTGGATTCACGCCGATC
CAAGCTGGCGCTTTTTCCAGGCGTGCCCAGCGCTCCAAAATCGCGTAGACCTCGGGGTT
TACGTGCTCGATTTTCCCGCCGGCCTGGTGGCTCGGCACATCAATGTCCAGGACAAGCA
CGGCTGCGTGCTGCGCGTGCGTCAGAGCAACATACTGGCACCGGGCAAGCGATTTTGA
ACCAACTCGGTATAACTTCGGCTGTGTTTCTCCCGTGTCCGGGTCTTTGATCCAAGCGCT
GGCGAAGTCGCGGGTCTTGCTGCCCTGGAAATTTTCTCTGCCCAGGTGAGCGAGGAATT
CGCGGCGGTCTTCGCTCGTCCAGCCACGTGATCGCAGCGCGAGCTCGGGATGGGTGTCG
AACAGATCAGCGGAAAATTTCCAGGCCGGTGTGTCAATGTCTCGTGAATCCGCTAGAGT
CATTTTTGAGCGCTTTCTCCCAGGTTTGGACTGGGGGTTAGCCGACGCCCTGTGAGTTAC
CGCTCACGGGGCGTTCAACATTTTTCAGGTATTCGTGCAGCTTATCGCTTCTTGCCGCCT
GTGCGCTTTTTCGACGCGCGACGCTGCTGCCGATTCGGTGCAGGTGGTGGCGGCGCTGA
CACGTCCTGGGCGGCCACGGCCACACGAAACGCGGCATTTACGATGTTTGTCATGCCTG
CGGGCACCGCGCCACGATCGCGGATAATTCTCGCTGCCGCTTCCAGCTCTGTGACGACC
ATGGCCAAAATTTCGCTCGGGGGACGCACTTCCAGCGCCATTTGCGACCTAGCCGCCTC
CAGCTCCTCGGCGTGGCGTTTGTTGGCGCGCTCGCGGCTGGCTGCGGCACGACACGCAT
CTGAGCAATATTTTGCGCGCCGTCCTCGCGGGTCAGGCCGGGGAGGAATCAGGCCACCG
CAGTAGGCGCAACTGATTCGATCCTCCACTACTGTGCGTCCTCCTGGCGCTGCCGAGCA
CGCAGCTCGTCAGCCAGCTCCTCAAGATCCGCCACGAGAGTTTCTAGGTCGCTCGCGGC
ACTGGCCCAGTCTCGTGATGCTGGCGCGTCCGTCGTATCGAGAGCTCGGAAAAATCCGA
TCACCGTTTTTAAATCGACGGCAGCATCGAGCGCGTCGGACTCCAGCGCGACATCAGAG
AGATCCATAGCTGATGATTCGGGCCAATTTTGGTACTTCGTCGTGAAGGTCATGACACCA
TTATAACGAACGTTCGTTAAAGTTTTTGGCGGAAAATCACGCGGCACGAAAATTTTCAC
GAAGCGGGACTTTGCGCAGCTCAGGGGTGCTAAAAATTTTGTATCGCACTTGATTTTTCC
GAAAGACAGATTATCTGCAAACGGTGTGTCGTATTTCTGGCTTGGTTTTTAAAAAATCTG
GAATCGAAAATTTGCGGGGCGACCGAGAAGTTTTTTACAAAAGGCAAAAACTTTTTCGG
GATCGACAGAAATAAAACGATCGAC
SEQ ID NO:112
5’UTR-egfp(由维多利亚绿锥虫修饰)：
GGAGACCCAAGCTGGCTAGCGTTTAAACTTAAGCTTCGAATTCTGCAGTCGACGGTACCGCGGGCCCGGGATCCACCGGTCGCCACCATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAGTAA
SEQ ID NO:82
5’UTR-luc2(萤火虫)：
GGAGACCCAAGCTGGCTAGCGTTTAAACTTAAGCTTGGCAATCCGGTACTGTTGGTAAAGCCACCATGGAAGATGCCAAAAACATTAAGAAGGGCCCAGCGCCATTCTACCCACTCGAAGACGGGACCGCCGGCGAGCAGCTGCACAAAGCCATGAAGCGCTACGCCCTGGTGCCCGGCACCATCGCCTTTACCGACGCACATATCGAGGTGGACATTACCTACGCCGAGTACTTCGAGATGAGCGTTCGGCTGGCAGAAGCTATGAAGCGCTATGGGCTGAATACAAACCATCGGATCGTGGTGTGCAGCGAGAATAGCTTGCAGTTCTTCATGCCCGTGTTGGGTGCCCTGTTCATCGGTGTGGCTGTGGCCCCAGCTAACGACATCTACAACGAGCGCGAGCTGCTGAACAGCATGGGCATCAGCCAGCCCACCGTCGTATTCGTGAGCAAGAAAGGGCTGCAAAAGATCCTCAACGTGCAAAAGAAGCTACCGATCATACAAAAGATCATCATCATGGATAGCAAGACCGACTACCAGGGCTTCCAAAGCATGTACACCTTCGTGACTTCCCATTTGCCACCCGGCTTCAACGAGTACGACTTCGTGCCCGAGAGCTTCGACCGGGACAAAACCATCGCCCTGATCATGAACAGTAGTGGCAGTACCGGATTGCCCAAGGGCGTAGCCCTACCGCACCGCACCGCTTGTGTCCGATTCAGTCATGCCCGCGACCCCATCTTCGGCAACCAGATCATCCCCGACACCGCTATCCTCAGCGTGGTGCCATTTCACCACGGCTTCGGCATGTTCACCACGCTGGGCTACTTGATCTGCGGCTTTCGGGTCGTGCTCATGTACCGCTTCGAGGAGGAGCTATTCTTGCGCAGCTTGCAAGACTATAAGATTCAATCTGCCCTGCTGGTGCCCACACTATTTAGCTTCTTCGCTAAGAGCACTCTCATCGACAAGTACGACCTAAGCAACTTGCACGAGATCGCCAGCGGCGGGGCGCCGCTCAGCAAGGAGGTAGGTGAGGCCGTGGCCAAACGCTTCCACCTACCAGGCATCCGCCAGGGCTACGGCCTGACAGAAACAACCAGCGCCATTCTGATCACCCCCGAAGGGGACGACAAGCCTGGCGCAGTAGGCAAGGTGGTGCCCTTCTTCGAGGCTAAGGTGGTGGACTTGGACACCGGTAAGACACTGGGTGTGAACCAGCGCGGCGAGCTGTGCGTCCGTGGCCCCATGATCATGAGCGGCTACGTTAACAACCCCGAGGCTACAAACGCTCTCATCGACAAGGACGGCTGGCTGCACAGCGGCGACATCGCCTACTGGGACGAGGACGAGCACTTCTTCATCGTGGACCGGCTGAAGAGCCTGATCAAATACAAGGGCTACCAGGTAGCCCCAGCCGAACTGGAGAGCATCCTGCTGCAACACCCCAACATCTTCGACGCCGGGGTCGCCGGCCTGCCCGACGACGATGCCGGCGAGCTGCCCGCCGCAGTCGTCGTGCTGGAACACGGTAAAACCATGACCGAGAAGGAGATCGTGGACTATGTGGCCAGCCAGGTTACAACCGCCAAGAAGCTGCGCGGTGGTGTTGTGTTCGTGGACGAGGTGCCTAAAGGACTGACCGGCAAGTTGGACGCCCGCAAGATCCGCGAGATTCTCATTAAGGCCAAGAAGGGCGGCAAGATCGCCGTGTAA
b)质粒pJC1-PT7-egfp-西兰花-TT7和pJC1-PT7-luc2-西兰花-TT7在谷氨酸棒状杆菌ATCC 13032(DE3)_Δcg2273中的转化
谷氨酸棒状杆菌ATCC 13032(DE3)_Δcg2273的构建在实施例5b中进行了描述。根据Tauch等人，2002(Tauch,A.,Kirchner,O.,B.,/>S.,Pühler,A.,andKalinowski,J.Efficient electrotransformation of Corynebacterium diphtheriaewith a Mini-Replicon Derived from the Corynebacterium glutamicum plasmid pGA1.Curr.Microbiol.2002；45,362-367)，制备谷氨酸棒状杆菌菌株ATCC 13032(DE3)_Δcg2273的感受态细胞，并通过电穿孔用载体pJC1-PT7-egfp-西兰花-TT7或用pJC1-PT7-luc2-西兰花-TT7转化。转化子的选择是在含有25μg/ml卡那霉素的BHI琼脂(1％)平板上进行的。由此获得的克隆被命名为谷氨酸棒状杆菌ATCC 13032(DE3)_Δcg2273_pJC1_PT7-egfp-西兰花-TT7和谷氨酸棒状杆菌ATCC 13032(DE3)_Δcg2273_pJC1_PT7-luc2-西兰花-TT7。
c)使用荧光活化细胞分选(FACS)的细胞培养和表型验证
将产生的谷氨酸棒状杆菌ATCC 13032(DE3)_Δcg2273_pJC1_PT7-egfp-西兰花-TT7和谷氨酸棒状杆菌ATCC 13032(DE3)_Δcg2273_pJC1_PT7-luc2-西兰花-TT7，在含有25μg/ml卡那霉素的BHI琼脂平板上划线，在30℃下培养过夜。将生长的细胞重悬于含有25μg/ml卡那霉素的CGIII培养基中，并在含2mL有抗生素的CGIII培育基的试管中将OD600调节至0.75。细胞在30℃和120rpm下孵育，培养6小时后用1.5mM IPTG诱导。之后，继续培养6小时。随后，使用最终浓度为500μM DFHBI的PBS将细胞稀释至OD600为0.6。使用实施例1d)中已经描述的AriaIII高速细胞分选器对细胞进行分析。与未染色的细胞相比，DFHBI染色的细胞显示出显著增加的荧光输出(参见图7)。
d)RNA的提取及RT-PCR分析
根据实施例1f)从1.38×109个细胞中分离RNA。为了验证得到的RNA片段，使用One-Taq一步RT-PCR试剂盒(New England Biolabs，Ipswich，MA，USA)进行逆转录酶PCR(RT-PCR)，其中引物egfp_for(SEQ ID NO:109)和西兰花_rev(SEQ ID NO:113)用于验证egfp-西兰花片段的内部，引物luc2_for(SEQ ID NO:114)和brococli_rev(SEQ ID NO:113)以验证luc2-西兰花片段的内部部分。最初，使用ProtoScriptII逆转录酶从RNA中产生cDNA，随后，使用上述引物通过OneTaq热启动DNA聚合酶扩增得到的cDNA片段。在制备的RNA样品中鉴定出831bp大小的谷氨酸棒状杆菌ATCC 13032(DE3)_Δcg2273_pJC1_PT7-egfp-西兰花-TT7(总转录长度为973nts)和1774bp大小的谷氨酸棒状杆菌ATCC 13032(DE3)_Δcg2273_pJC1_PT7-luc2-西兰花-TT7(总转录长度为1894nts)。作为阳性对照，使用上述引物通过OneTaq热启动DNA聚合酶扩增载体pJC1_PT7-egfp-西兰花-TT7和pJC1_PT7-luc2-西兰花-TT7。作为阴性对照，在不进行逆转录酶步骤的情况下，通过OneTaq热启动DNA聚合酶扩增RNA样品。琼脂糖凝胶上没有出现条带，表明制备的RNA样品不含原始载体DNA(参见图8)。
SEQ ID NO:109
egfp_for:
ATGGTGAGCAAGGGCGAGGA
SEQ ID NO:113
西兰花_rev:
TTGCCATGAATGATCCCGAAG
SEQ ID NO:114
luc2_for:
ATGGAAGATGCCAAAAACATTAAGAAG
该实验显示了迄今为止不显著的表型(RNA产生)与荧光输出的成功连接。在这个实验中，产生的RNA具有973或1894个核苷酸的长度，并从载体上转录。因此，根据实施例1中所示的程序，使用本发明可以优化长度为至少1894个核苷酸的RNA的生产。
序列表
<110> 森斯艾普有限公司
<120> RNA核酸适配体传感器
<130> P2E231038WPCN
<160> 122
<170> PatentIn version 3.5
<210> 1
<211> 29
<212> RNA
<213> 人工序列
<220>
<223> Aptamer
<400> 1
agguucgaag cuuuugcuug gacgaaccg 29
<210> 2
<211> 23
<212> RNA
<213> 人工序列
<220>
<223> Aptamer
<400> 2
gaagggacgg ugcggagagg aga 23
<210> 3
<211> 24
<212> RNA
<213> 人工序列
<220>
<223> Aptamer
<400> 3
gaaggagagg agaggaagag gaga 24
<210> 4
<211> 22
<212> RNA
<213> 人工序列
<220>
<223> Aptamer
<400> 4
ggaaggauug guauguggua ua 22
<210> 5
<211> 25
<212> RNA
<213> 人工序列
<220>
<223> Aptamer
<400> 5
cgagggagug gugaggauga ggcga 25
<210> 6
<211> 98
<212> RNA
<213> 人工序列
<220>
<223> Aptamer
<400> 6
gacgcaacug aaugaaaugg ugaaggacgg guccaggugu ggcugcuucg gcagugcagc 60
uuguugagua gagugugagc uccguaacua gucgcguc 98
<210> 7
<211> 95
<212> RNA
<213> 人工序列
<220>
<223> Aptamer
<400> 7
gauguaacug aaugaaaugg ugaaggacgg guccaguagg cugcuucggc agccuacuug 60
uugaguagag ugugagcucc guaacuaguu acauc 95
<210> 8
<211> 47
<212> RNA
<213> 人工序列
<220>
<223> Aptamer
<400> 8
agacggucgg guccagauau ucguaucugu cgaguagagu gugggcu 47
<210> 9
<211> 65
<212> RNA
<213> 人工序列
<220>
<223> Scaffold
<220>
<221> misc_feature
<223> tRNALys
<220>
<221> misc_feature
<222> (28)..(31)
<223> Insertion site for aptamer
<400> 9
gcccggauag cucagucggu agagcagnnn ncggguccag gguucaaguc ccuguucggg 60
cgcca 65
<210> 10
<211> 86
<212> RNA
<213> 人工序列
<220>
<223> Scaffold
<220>
<221> misc_feature
<222> (23)..(26)
<223> Insertion site
<400> 10
ugccuggcga ccauagcgau ugnnnncaau uagcgccgau gguagugugg gguuucccca 60
ugugagagua ggacaucgcc aggcau 86
<210> 11
<211> 62
<212> RNA
<213> 人工序列
<220>
<223> Scaffold
<220>
<221> misc_feature
<223> F29
<220>
<221> misc_feature
<222> (19)..(22)
<223> Insertion site for aptamer
<220>
<221> misc_feature
<222> (43)..(46)
<223> Insertion site for aptamer
<400> 11
uugucacgug uaugugggnn nncccacaua cuuuguugau ccnnnnggau caaucauggc 60
aa 62
<210> 12
<211> 62
<212> RNA
<213> 人工序列
<220>
<223> Scaffold
<220>
<221> misc_feature
<223> F30
<220>
<221> misc_feature
<222> (19)..(22)
<223> Insertion site for aptamer
<220>
<221> misc_feature
<222> (43)..(46)
<223> Insertion site for aptamer
<400> 12
uugccaugug uaugugggnn nncccacaua cucugaugau ccnnnnggau cauucauggc 60
aa 62
<210> 13
<211> 28
<212> RNA
<213> 人工序列
<220>
<223> Aptamer
<400> 13
cgaggaagga ggucugagga ggucacug 28
<210> 14
<211> 52
<212> RNA
<213> 人工序列
<220>
<223> Aptamer
<400> 14
agacggucgg guccaggugc acaaaugugg ccuguugagu agcguguggg cu 52
<210> 15
<211> 52
<212> RNA
<213> 人工序列
<220>
<223> Aptamer
<400> 15
agacggucgg guccaguccc aacgauguug gcuguugagu aguguguggg cu 52
<210> 16
<211> 29
<212> RNA
<213> 人工序列
<220>
<223> Aptamer
<400> 16
aaggacgggu ccguugagua gagugugag 29
<210> 17
<211> 90
<212> RNA
<213> 人工序列
<220>
<223> Scaffold and aptamer
<400> 17
gcccggauag cucagucggu agagcagagg uucgaagcuu uugcuuggac gaaccgcggg 60
uccaggguuc aagucccugu ucgggcgcca 90
<210> 18
<211> 84
<212> RNA
<213> 人工序列
<220>
<223> Scaffold + aptamer
<400> 18
gcccggauag cucagucggu agagcaggaa gggacggugc ggagaggaga cggguccagg 60
guucaagucc cuguucgggc gcca 84
<210> 19
<211> 85
<212> RNA
<213> 人工序列
<220>
<223> RNA scaffold + aptamar
<400> 19
gcccggauag cucagucggu agagcaggaa ggagaggaga ggaagaggag acggguccag 60
gguucaaguc ccuguucggg cgcca 85
<210> 20
<211> 83
<212> RNA
<213> 人工序列
<220>
<223> RNA scaffold + aptamer
<400> 20
gcccggauag cucagucggu agagcaggga aggauuggua ugugguauac ggguccaggg 60
uucaaguccc uguucgggcg cca 83
<210> 21
<211> 86
<212> RNA
<213> 人工序列
<220>
<223> RNA scaffold + aptamer
<400> 21
gcccggauag cucagucggu agagcagcga gggaguggug aggaugaggc gacgggucca 60
ggguucaagu cccuguucgg gcgcca 86
<210> 22
<211> 159
<212> RNA
<213> 人工序列
<220>
<223> RNA scaffold + aptamer
<400> 22
gcccggauag cucagucggu agagcaggac gcaacugaau gaaaugguga aggacggguc 60
cagguguggc ugcuucggca gugcagcuug uugaguagag ugugagcucc guaacuaguc 120
gcguccgggu ccaggguuca agucccuguu cgggcgcca 159
<210> 23
<211> 156
<212> RNA
<213> 人工序列
<220>
<223> RNA scaffold + aptamer
<400> 23
gcccggauag cucagucggu agagcaggau guaacugaau gaaaugguga aggacggguc 60
caguaggcug cuucggcagc cuacuuguug aguagagugu gagcuccgua acuaguuaca 120
uccgggucca ggguucaagu cccuguucgg gcgcca 156
<210> 24
<211> 108
<212> RNA
<213> 人工序列
<220>
<223> RNA scaffold + aptamer
<400> 24
gcccggauag cucagucggu agagcagaga cggucggguc cagauauucg uaucugucga 60
guagagugug ggcucggguc caggguucaa gucccuguuc gggcgcca 108
<210> 25
<211> 89
<212> RNA
<213> 人工序列
<220>
<223> RNA scaffold + aptamer
<400> 25
gcccggauag cucagucggu agagcagcga ggaaggaggu cugaggaggu cacugcgggu 60
ccaggguuca agucccuguu cgggcgcca 89
<210> 26
<211> 113
<212> RNA
<213> 人工序列
<220>
<223> RNA scaffold + aptamer
<400> 26
gcccggauag cucagucggu agagcagaga cggucggguc caggugcaca aauguggccu 60
guugaguagc gugugggcuc ggguccaggg uucaaguccc uguucgggcg cca 113
<210> 27
<211> 113
<212> RNA
<213> 人工序列
<220>
<223> RNA scaffold + aptamer
<400> 27
gcccggauag cucagucggu agagcagaga cggucggguc cagucccaac gauguuggcu 60
guugaguagu gugugggcuc ggguccaggg uucaaguccc uguucgggcg cca 113
<210> 28
<211> 90
<212> RNA
<213> 人工序列
<220>
<223> RNA scaffold + aptamer
<400> 28
gcccggauag cucagucggu agagcagaag gacggguccg uugaguagag ugugagcggg 60
uccaggguuc aagucccugu ucgggcgcca 90
<210> 29
<211> 111
<212> RNA
<213> 人工序列
<220>
<223> RNA scaffold + aptamer
<400> 29
ugccuggcga ccauagcgau ugagguucga agcuuuugcu uggacgaacc gcaauuagcg 60
ccgaugguag ugugggguuu ccccauguga gaguaggaca ucgccaggca u 111
<210> 30
<211> 105
<212> RNA
<213> 人工序列
<220>
<223> RNA scaffold + aptamer
<400> 30
ugccuggcga ccauagcgau uggaagggac ggugcggaga ggagacaauu agcgccgaug 60
guaguguggg guuuccccau gugagaguag gacaucgcca ggcau 105
<210> 31
<211> 106
<212> RNA
<213> 人工序列
<220>
<223> RNA scaffold + aptamer
<400> 31
ugccuggcga ccauagcgau uggaaggaga ggagaggaag aggagacaau uagcgccgau 60
gguagugugg gguuucccca ugugagagua ggacaucgcc aggcau 106
<210> 32
<211> 104
<212> RNA
<213> 人工序列
<220>
<223> RNA scaffold + aptamer
<400> 32
ugccuggcga ccauagcgau ugggaaggau ugguaugugg uauacaauua gcgccgaugg 60
uagugugggg uuuccccaug ugagaguagg acaucgccag gcau 104
<210> 33
<211> 107
<212> RNA
<213> 人工序列
<220>
<223> RNA scaffold + aptamer
<400> 33
ugccuggcga ccauagcgau ugcgagggag uggugaggau gaggcgacaa uuagcgccga 60
ugguagugug ggguuucccc augugagagu aggacaucgc caggcau 107
<210> 34
<211> 180
<212> RNA
<213> 人工序列
<220>
<223> RNA scaffold + aptamer
<400> 34
ugccuggcga ccauagcgau uggacgcaac ugaaugaaau ggugaaggac ggguccaggu 60
guggcugcuu cggcagugca gcuuguugag uagaguguga gcuccguaac uagucgcguc 120
caauuagcgc cgaugguagu gugggguuuc cccaugugag aguaggacau cgccaggcau 180
<210> 35
<211> 177
<212> RNA
<213> 人工序列
<220>
<223> RNA scaffold + aptamer
<400> 35
ugccuggcga ccauagcgau uggauguaac ugaaugaaau ggugaaggac ggguccagua 60
ggcugcuucg gcagccuacu uguugaguag agugugagcu ccguaacuag uuacauccaa 120
uuagcgccga ugguagugug ggguuucccc augugagagu aggacaucgc caggcau 177
<210> 36
<211> 129
<212> RNA
<213> 人工序列
<220>
<223> RNA scaffold + aptamer
<400> 36
ugccuggcga ccauagcgau ugagacgguc ggguccagau auucguaucu gucgaguaga 60
gugugggcuc aauuagcgcc gaugguagug ugggguuucc ccaugugaga guaggacauc 120
gccaggcau 129
<210> 37
<211> 110
<212> RNA
<213> 人工序列
<220>
<223> RNA scaffold + aptamer
<400> 37
ugccuggcga ccauagcgau ugcgaggaag gaggucugag gaggucacug caauuagcgc 60
cgaugguagu gugggguuuc cccaugugag aguaggacau cgccaggcau 110
<210> 38
<211> 268
<212> RNA
<213> 人工序列
<220>
<223> RNA scaffold + aptamer
<400> 38
ugccuggcga ccauagcgau ugagacgguc ggguccaggu gcacaaaugu ggccuguuga 60
guagcgugug ggcucaauua gcgccgaugg uagugugggg uuuccccaug ugagaguagg 120
acaucgccag gcauugccug gcgaccauag cgauugagac ggucgggucc aggugcacaa 180
auguggccug uugaguagcg ugugggcuca auuagcgccg augguagugu gggguuuccc 240
caugugagag uaggacaucg ccaggcau 268
<210> 39
<211> 134
<212> RNA
<213> 人工序列
<220>
<223> RNA scaffold + aptamer
<400> 39
ugccuggcga ccauagcgau ugagacgguc ggguccaguc ccaacgaugu uggcuguuga 60
guagugugug ggcucaauua gcgccgaugg uagugugggg uuuccccaug ugagaguagg 120
acaucgccag gcau 134
<210> 40
<211> 111
<212> RNA
<213> 人工序列
<220>
<223> RNA scaffold + aptamer
<400> 40
ugccuggcga ccauagcgau ugaaggacgg guccguugag uagaguguga gcaauuagcg 60
ccgaugguag ugugggguuu ccccauguga gaguaggaca ucgccaggca u 111
<210> 41
<211> 87
<212> RNA
<213> 人工序列
<220>
<223> RNA scaffold + aptamer
<220>
<221> misc_feature
<222> (68)..(71)
<223> Insertion site for aptamer
<400> 41
uugucacgug uaugugggag guucgaagcu uuugcuugga cgaaccgccc acauacuuug 60
uugauccnnn nggaucaauc auggcaa 87
<210> 42
<211> 81
<212> RNA
<213> 人工序列
<220>
<223> RNA scaffold + aptamer
<220>
<221> misc_feature
<222> (62)..(65)
<223> Insertion site for aptamer
<400> 42
uugucacgug uaugugggga agggacggug cggagaggag acccacauac uuuguugauc 60
cnnnnggauc aaucauggca a 81
<210> 43
<211> 82
<212> RNA
<213> 人工序列
<220>
<223> RNA scaffold + aptamer
<220>
<221> misc_feature
<222> (63)..(63)
<223> n is a, c, g, or u
<220>
<221> misc_feature
<222> (64)..(67)
<223> Insertion site for aptamer
<400> 43
uugucacgug uaugugggga aggagaggag aggaagagga gacccacaua cuuuguugau 60
ccnnnnggau caaucauggc aa 82
<210> 44
<211> 80
<212> RNA
<213> 人工序列
<220>
<223> RNA scaffold + aptamer
<220>
<221> misc_feature
<222> (61)..(63)
<223> n is a, c, g, or u
<220>
<221> misc_feature
<222> (64)..(67)
<223> Insertion site for aptamer
<400> 44
uugucacgug uauguggggg aaggauuggu augugguaua cccacauacu uuguugaucc 60
nnnnggauca aucauggcaa 80
<210> 45
<211> 83
<212> RNA
<213> 人工序列
<220>
<223> RNA scaffold + aptamer
<220>
<221> misc_feature
<222> (64)..(67)
<223> Insertion site for aptamer
<400> 45
uugucacgug uaugugggcg agggaguggu gaggaugagg cgacccacau acuuuguuga 60
uccnnnngga ucaaucaugg caa 83
<210> 46
<211> 156
<212> RNA
<213> 人工序列
<220>
<223> RNA scaffold + aptamer
<220>
<221> misc_feature
<222> (137)..(140)
<223> Insertion site for aptamer
<400> 46
uugucacgug uaugugggga cgcaacugaa ugaaauggug aaggacgggu ccaggugugg 60
cugcuucggc agugcagcuu guugaguaga gugugagcuc cguaacuagu cgcgucccca 120
cauacuuugu ugauccnnnn ggaucaauca uggcaa 156
<210> 47
<211> 153
<212> RNA
<213> 人工序列
<220>
<223> RNA scaffold + aptamer
<220>
<221> misc_feature
<222> (134)..(137)
<223> Insertion site for aptamer
<400> 47
uugucacgug uaugugggga uguaacugaa ugaaauggug aaggacgggu ccaguaggcu 60
gcuucggcag ccuacuuguu gaguagagug ugagcuccgu aacuaguuac auccccacau 120
acuuuguuga uccnnnngga ucaaucaugg caa 153
<210> 48
<211> 105
<212> RNA
<213> 人工序列
<220>
<223> RNA scaffold + aptamer
<220>
<221> misc_feature
<222> (86)..(89)
<223> Insertion site
<400> 48
uugucacgug uaugugggag acggucgggu ccagauauuc guaucugucg aguagagugu 60
gggcucccac auacuuuguu gauccnnnng gaucaaucau ggcaa 105
<210> 49
<211> 86
<212> RNA
<213> 人工序列
<220>
<223> RNA scaffold + aptamer
<220>
<221> misc_feature
<222> (67)..(70)
<223> Insertion site
<400> 49
uugucacgug uaugugggcg aggaaggagg ucugaggagg ucacugccca cauacuuugu 60
ugauccnnnn ggaucaauca uggcaa 86
<210> 50
<211> 110
<212> RNA
<213> 人工序列
<220>
<223> RNA scaffold + aptamer
<220>
<221> misc_feature
<222> (91)..(94)
<223> Insertion site for aptamer
<400> 50
uugucacgug uaugugggag acggucgggu ccaggugcac aaauguggcc uguugaguag 60
cgugugggcu cccacauacu uuguugaucc nnnnggauca aucauggcaa 110
<210> 51
<211> 110
<212> RNA
<213> 人工序列
<220>
<223> RNA scaffold + aptamer
<220>
<221> misc_feature
<222> (91)..(94)
<223> Insertion site for aptamer
<400> 51
uugucacgug uaugugggag acggucgggu ccagucccaa cgauguuggc uguugaguag 60
ugugugggcu cccacauacu uuguugaucc nnnnggauca aucauggcaa 110
<210> 52
<211> 87
<212> RNA
<213> 人工序列
<220>
<223> RNA scaffold + aptamer
<220>
<221> misc_feature
<222> (68)..(71)
<223> Insertion site for aptamer
<400> 52
uugucacgug uaugugggaa ggacgggucc guugaguaga gugugagccc acauacuuug 60
uugauccnnn nggaucaauc auggcaa 87
<210> 53
<211> 87
<212> RNA
<213> 人工序列
<220>
<223> RNA scaffold + aptamer
<220>
<221> misc_feature
<222> (68)..(71)
<223> Insertion site for aptatmer
<400> 53
uugccaugug uaugugggag guucgaagcu uuugcuugga cgaaccgccc acauacucug 60
augauccnnn nggaucauuc auggcaa 87
<210> 54
<211> 81
<212> RNA
<213> 人工序列
<220>
<223> RNA scaffold + aptamer
<220>
<221> misc_feature
<222> (62)..(65)
<223> Insertion site for aptamer
<400> 54
uugccaugug uaugugggga agggacggug cggagaggag acccacauac ucugaugauc 60
cnnnnggauc auucauggca a 81
<210> 55
<211> 82
<212> RNA
<213> 人工序列
<220>
<223> RNA scaffold + aptamer
<220>
<221> misc_feature
<222> (63)..(63)
<223> n is a, c, g, or u
<220>
<221> misc_feature
<222> (64)..(67)
<223> Insertion site for aptamer
<400> 55
uugccaugug uaugugggga aggagaggag aggaagagga gacccacaua cucugaugau 60
ccnnnnggau cauucauggc aa 82
<210> 56
<211> 80
<212> RNA
<213> 人工序列
<220>
<223> RNA scaffold + aptamer
<220>
<221> misc_feature
<222> (61)..(63)
<223> n is a, c, g, or u
<220>
<221> misc_feature
<222> (64)..(67)
<223> Insertion site for aptamer
<400> 56
uugccaugug uauguggggg aaggauuggu augugguaua cccacauacu cugaugaucc 60
nnnnggauca uucauggcaa 80
<210> 57
<211> 83
<212> RNA
<213> 人工序列
<220>
<223> RNA scaffold + aptamer
<220>
<221> misc_feature
<223> F30::Mango4
<220>
<221> misc_feature
<222> (64)..(67)
<223> Insertion site for aptamer
<400> 57
uugccaugug uaugugggcg agggaguggu gaggaugagg cgacccacau acucugauga 60
uccnnnngga ucauucaugg caa 83
<210> 58
<211> 156
<212> RNA
<213> 人工序列
<220>
<223> RNA scaffold + aptamer
<220>
<221> misc_feature
<222> (137)..(140)
<223> Insertion site for aptamer
<400> 58
uugccaugug uaugugggga cgcaacugaa ugaaauggug aaggacgggu ccaggugugg 60
cugcuucggc agugcagcuu guugaguaga gugugagcuc cguaacuagu cgcgucccca 120
cauacucuga ugauccnnnn ggaucauuca uggcaa 156
<210> 59
<211> 153
<212> RNA
<213> 人工序列
<220>
<223> RNA scaffold + aptamer
<220>
<221> misc_feature
<223> F30::Spinach2
<220>
<221> misc_feature
<222> (134)..(137)
<223> Insertion site for aptamer
<400> 59
uugccaugug uaugugggga uguaacugaa ugaaauggug aaggacgggu ccaguaggcu 60
gcuucggcag ccuacuuguu gaguagagug ugagcuccgu aacuaguuac auccccacau 120
acucugauga uccnnnngga ucauucaugg caa 153
<210> 60
<211> 105
<212> RNA
<213> 人工序列
<220>
<223> RNA scaffold + aptamer
<220>
<221> misc_feature
<222> (86)..(89)
<223> Insertion site for aptamer
<400> 60
uugccaugug uaugugggag acggucgggu ccagauauuc guaucugucg aguagagugu 60
gggcucccac auacucugau gauccnnnng gaucauucau ggcaa 105
<210> 61
<211> 86
<212> RNA
<213> 人工序列
<220>
<223> RNA scaffold + aptamer
<220>
<221> misc_feature
<222> (67)..(70)
<223> Insertion site for aptamer
<400> 61
uugccaugug uaugugggcg aggaaggagg ucugaggagg ucacugccca cauacucuga 60
ugauccnnnn ggaucauuca uggcaa 86
<210> 62
<211> 110
<212> RNA
<213> 人工序列
<220>
<223> RNA scaffold + aptamer
<220>
<221> misc_feature
<222> (91)..(94)
<223> Insertion site for aptamer
<400> 62
uugccaugug uaugugggag acggucgggu ccaggugcac aaauguggcc uguugaguag 60
cgugugggcu cccacauacu cugaugaucc nnnnggauca uucauggcaa 110
<210> 63
<211> 110
<212> RNA
<213> 人工序列
<220>
<223> RNA scaffold + aptamer
<220>
<221> misc_feature
<222> (91)..(94)
<223> Insertion site for aptamer
<400> 63
uugccaugug uaugugggag acggucgggu ccagucccaa cgauguuggc uguugaguag 60
ugugugggcu cccacauacu cugaugaucc nnnnggauca uucauggcaa 110
<210> 64
<211> 87
<212> RNA
<213> 人工序列
<220>
<223> RNA scaffold + aptamer
<220>
<221> misc_feature
<222> (68)..(71)
<223> Insertion site for aptamer
<400> 64
uugccaugug uaugugggaa ggacgggucc guugaguaga gugugagccc acauacucug 60
augauccnnn nggaucauuc auggcaa 87
<210> 65
<211> 52
<212> RNA
<213> 人工序列
<220>
<223> Aptamer
<400> 65
agacggucgg guccaggugc acaaaugugg ccuguugagu agcguguggg cu 52
<210> 66
<211> 52
<212> RNA
<213> 人工序列
<220>
<223> Aptamer
<400> 66
agacggucgg guccaguccc aacgauguug gcuguugagu aguguguggg cu 52
<210> 67
<211> 61
<212> DNA
<213> 人工序列
<220>
<223> Pf1 promoter
<400> 67
ctcgagcggg acggtcgaac cagcttcaag cgaccggatg agtatgttac agtagatagc 60
g 61
<210> 68
<211> 105
<212> DNA
<213> 人工序列
<220>
<223> Target RNA
<400> 68
agcgggagac cgctcgacct tagttctcct gttgcggggg agttcatggg atccaggtac 60
ccagggcgag gcttatccat tgcactccgg atgtgctgac ccctg 105
<210> 69
<211> 105
<212> DNA
<213> 人工序列
<220>
<223> F30 with broccoli in insertion site 1 and TTCG in insertion site
<400> 69
ttgccatgtg tatgtgggag acggtcgggt ccagatattc gtatctgtcg agtagagtgt 60
gggctcccac atactctgat gatccttcgg gatcattcat ggcaa 105
<210> 70
<211> 69
<212> DNA
<213> 人工序列
<220>
<223> TF1 Terminator sequence
<400> 70
ctagcatagc ataaaataac gccccacctt cttaacggga ggtggggcgt tatttttacg 60
gggatcgct 69
<210> 71
<211> 354
<212> DNA
<213> 人工序列
<220>
<223> SEQ-ID No: 67-70 combined, with XbaI recognition site on 5'-end,
SalI recognition site on 3'-end
<400> 71
ctgtctctag actcgagcgg gacggtcgaa ccagcttcaa gcgaccggat gagtatgtta 60
cagtagatag cgagcgggag accgctcgac cttagttctc ctgttgcggg ggagttcatg 120
ggatccaggt acccagggcg aggcttatcc attgcactcc ggatgtgctg acccctgttg 180
ccatgtgtat gtgggagacg gtcgggtcca gatattcgta tctgtcgagt agagtgtggg 240
ctcccacata ctctgatgat ccttcgggat cattcatggc aagctagcat agcataaaat 300
aacgccccac cttcttaacg ggaggtgggg cgttattttt acggtcgacc tgtc 354
<210> 72
<211> 249
<212> DNA
<213> 人工序列
<220>
<223> SEQ-ID No: 67, 68 and 70, with XbaI recognition site on 5'-end,
SalI recognition site on 3'-end
<400> 72
ctgtctctag actcgagcgg gacggtcgaa ccagcttcaa gcgaccggat gagtatgtta 60
cagtagatag cgagcgggag accgctcgac cttagttctc ctgttgcggg ggagttcatg 120
ggatccaggt acccagggcg aggcttatcc attgcactcc ggatgtgctg acccctggct 180
agcatagcat aaaataacgc cccaccttct taacgggagg tggggcgtta tttttacggt 240
cgacctgtc 249
<210> 73
<211> 20
<212> DNA
<213> 人工序列
<220>
<223> pJC1_check_f
<400> 73
tgaagaccgt caaccaaagg 20
<210> 74
<211> 20
<212> DNA
<213> 人工序列
<220>
<223> pJC1_check_rev
<400> 74
tgccgggaag ctagagtaag 20
<210> 75
<211> 6440
<212> DNA
<213> 人工序列
<220>
<223> pJC1-PF1-U1A-F30::broccoli/UUCG-TF1
<400> 75
ccgagaagtt ttttacaaaa ggcaaaaact ttttcgggat cgacagaaat aaaacgatcg 60
acggtacgca acaaaaaagc gtcaggatcg ccgtagagcg attgaagacc gtcaaccaaa 120
ggggaagcct ccaatcgacg cgacgcgcgc tctacggcga tcctgacgca gatttttagc 180
tatctgtcgc agcgccctca gggacaagcc acccgcacaa cgtcgcgagg gcgatcagcg 240
acgccgcagg gggatcctct agactcgagc gggacggtcg aaccagcttc aagcgaccgg 300
atgagtatgt tacagtagat agcgagcggg agaccgctcg accttagttc tcctgttgcg 360
ggggagttca tgggatccag gtacccaggg cgaggcttat ccattgcact ccggatgtgc 420
tgacccctgt tgccatgtgt atgtgggaga cggtcgggtc cagatattcg tatctgtcga 480
gtagagtgtg ggctcccaca tactctgatg atccttcggg atcattcatg gcaagctagc 540
atagcataaa ataacgcccc accttcttaa cgggaggtgg ggcgttattt ttacggtcga 600
cctgcagcaa tggcaacaac gttgcgcaaa ctattaactg gcgaactact tactctagct 660
tcccggcaac aattaataga ctggatggag gcggataaag ttgcaggacc acttctgcgc 720
tcggcccttc cggctggctg gtttattgct gataaatctg gagccggtga gcgtgggtct 780
cgcggtatca ttgcagcact ggggccagat ggtaagccct cccgtatcgt agttatctac 840
acgacgggga gtcaggcaac tatggatgaa cgaaatagac agatcgctga gataggtgcc 900
tcactgatta agcattggta actgtcagac caagtttact catatatact ttagattgat 960
ttaaaacttc atttttaatt taaaaggatc taggtgaaga tcctttttga taatctcatg 1020
accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagacccctt aataagatga 1080
tcttcttgag atcgttttgg tctgcgcgta atctcttgct ctgaaaacga aaaaaccgcc 1140
ttgcagggcg gtttttcgaa ggttctctga gctaccaact ctttgaaccg aggtaactgg 1200
cttggaggag cgcagtcacc aaaacttgtc ctttcagttt agccttaacc ggcgcatgac 1260
ttcaagacta actcctctaa atcaattacc agtggctgct gccagtggtg cttttgcatg 1320
tctttccggg ttggactcaa gacgatagtt accggataag gcgcagcggt cggactgaac 1380
ggggggttcg tgcatacagt ccagcttgga gcgaactgcc tacccggaac tgagtgtcag 1440
gcgtggaatg agacaaacgc ggccataaca gcggaatgac accggtaaac cgaaaggcag 1500
gaacaggaga gcgcacgagg gagccgccag ggggaaacgc ctggtatctt tatagtcctg 1560
tcgggtttcg ccaccactga tttgagcgtc agatttcgtg atgcttgtca ggggggcgga 1620
gcctatggaa aaacggcttt gccgcggccc tctcacttcc ctgttaagta tcttcctggc 1680
atcttccagg aaatctccgc cccgttcgta agccatttcc gctcgccgca gtcgaacgac 1740
cgagcgtagc gagtcagtga gcgaggaagc ggaatatatc ctgtatcaca tattctgctg 1800
acgcaccggt gcagcctttt ttctcctgcc acatgaagca cttcactgac accctcatca 1860
gtgccaacat agtaagccag tatacactcc gctagcgctg aggtctgcct cgtgaagaag 1920
gtgttgctga ctcataccag gcctgaatcg ccccatcatc cagccagaaa gtgagggagc 1980
cacggttgat gagagctttg ttgtaggtgg accagttggt gattttgaac ttttgctttg 2040
ccacggaacg gtctgcgttg tcgggaagat gcgtgatctg atccttcaac tcagcaaaag 2100
ttcgatttat tcaacaaagc cacgttgtgt ctcaaaatct ctgatgttac attgcacaag 2160
ataaaaatat atcatcatga acaataaaac tgtctgctta cataaacagt aatacaaggg 2220
gtgttatgag ccatattcaa cgggaaacgt cttgctcgag gccgcgatta aattccaaca 2280
tggatgctga tttatatggg tataaatggg ctcgcgataa tgtcgggcaa tcaggtgcga 2340
caatctatcg attgtatggg aagcccgatg cgccagagtt gtttctgaaa catggcaaag 2400
gtagcgttgc caatgatgtt acagatgaga tggtcagact aaactggctg acggaattta 2460
tgcctcttcc gaccatcaag cattttatcc gtactcctga tgatgcatgg ttactcacca 2520
ctgcgatccc cgggaaaaca gcattccagg tattagaaga atatcctgat tcaggtgaaa 2580
atattgttga tgcgctggca gtgttcctgc gccggttgca ttcgattcct gtttgtaatt 2640
gtccttttaa cagcgatcgc gtatttcgtc tcgctcaggc gcaatcacga atgaataacg 2700
gtttggttga tgcgagtgat tttgatgacg agcgtaatgg ctggcctgtt gaacaagtct 2760
ggaaagaaat gcataagctt ttgccattct caccggattc agtcgtcact catggtgatt 2820
tctcacttga taaccttatt tttgacgagg ggaaattaat aggttgtatt gatgttggac 2880
gagtcggaat cgcagaccga taccaggatc ttgccatcct atggaactgc ctcggtgagt 2940
tttctccttc attacagaaa cggctttttc aaaaatatgg tattgataat cctgatatga 3000
ataaattgca gtttcatttg atgctcgatg agtttttcta atcagaattg gttaattggt 3060
tgtaacactg gcagagcatt acgctgactt gacgggacgg cggctttgtt gaataaatcg 3120
aacttttgct gagttgaagg atcagatcac gcatcttccc gacaacgcag accgttccgt 3180
ggcaaagcaa aagttcaaaa tcaccaactg gtccacctac aacaaagctc tcatcaaccg 3240
tggctccctc actttctggc tggatgatgg ggcgattcag gcctggtatg agtcagcaac 3300
accttcttca cgaggcagac ctcagcgctc aaagatgcag gggtaaaagc taaccgcatc 3360
tttaccgaca aggcatccgg cagttcaaca gatcgggaag ggctggattt gctgaggatg 3420
aaggtggagg aaggtgatgt cattctggtg aagaagctcg accgtcttgg ccgcgacacc 3480
gccgacatga tccaactgat aaaagagttt gatgctcagg gtgtagcggt tcggtttatt 3540
gacgacggga tcagtaccga cggtgatatg gggcaaatgg tggtcaccat cctgtcggct 3600
gtggcacagg ctgaacgccg gaggatcaag tcggtcaagc caagcgcaac cagcggcacc 3660
gccgcgagca acgtcgcaag ggcgatcagg ggacgatttt tgcgaagaat ttccacggta 3720
agaatccaat ctctcgaatt tagggtgaaa gaagcttggc ataggggtgt gcacgaactc 3780
ggtggaggaa atttccgcgg ggcaaggctt cgcgaagcgg agtcgcggca gtggctttga 3840
agatctttgg gagcagtcct tgtgcgctta cgaggtgagc cggtggggaa ccgttatctg 3900
cctatggtgt gagcccccct agagagcttc aagagcaatc agcccgacct agaaaggagg 3960
ccaagagaga gacccctacg gggggaaccg ttttctgcct acgagatggc acatttactg 4020
ggaagcttta cggcgtcctc gtggaagttc aatgcccgca gacttaagtg ctctattcac 4080
ggtctgacgt gacacgctaa attcagacat agcttcattg attgtcggcc acgagccagt 4140
ctctccctca acagtcataa accaacctgc aatggtcaag cgatttcctt tagctttcct 4200
agcttgtcgt tgactggact tagctagttt ttctcgctgt gctcgggcgt actcactgtt 4260
tgggtctttc cagcgttctg cggccttttt accgccacgt cttcccatag tggccagagc 4320
ttttcgccct cggctgctct gcgtctctgt ctgacgagca gggacgactg gctggccttt 4380
agcgacgtag ccgcgcacac gtcgcgccat cgtctggcgg tcacgcatcg gcggcagatc 4440
aggctcacgg ccgtctgctc cgaccgcctg agcgacggtg taggcacgct cgtaggcgtc 4500
gatgatcttg gtgtctttta ggcgctcacc agccgctttt aactggtatc ccacagtcaa 4560
agcgtggcga aaagccgtct catcacgggc ggcacgccct ggagcagtcc agaggacacg 4620
gacgccgtcg atcagctctc cagacgcttc agcggcgctc ggcaggcttg cttcaagcgt 4680
ggcaagtgct tttgcttccg cagtggcttt tcttgccgct tcgatacgtg cccgtccgct 4740
agaaaactcc tgctcatagc gttttttagg tttttctgtg cctgagatca tgcgagcaac 4800
ctccataaga tcagctaggc gatccacgcg attgtgctgg gcatgccagc ggtacgcggt 4860
gggatcgtcg gagacgtgca gtggccaccg gctcagccta tgtgaaaaag cctggtcagc 4920
gccgaaaacg cgggtcattt cctcggtcgt tgcagccagc aggcgcatat tcgggctgct 4980
catgcctgct gcggcataca ccggatcaat gagccagatg agctggcatt tcccgctcag 5040
tggattcacg ccgatccaag ctggcgcttt ttccaggcgt gcccagcgct ccaaaatcgc 5100
gtagacctcg gggtttacgt gctcgatttt cccgccggcc tggtggctcg gcacatcaat 5160
gtccaggaca agcacggctg cgtgctgcgc gtgcgtcaga gcaacatact ggcaccgggc 5220
aagcgatttt gaaccaactc ggtataactt cggctgtgtt tctcccgtgt ccgggtcttt 5280
gatccaagcg ctggcgaagt cgcgggtctt gctgccctgg aaattttctc tgcccaggtg 5340
agcgaggaat tcgcggcggt cttcgctcgt ccagccacgt gatcgcagcg cgagctcggg 5400
atgggtgtcg aacagatcag cggaaaattt ccaggccggt gtgtcaatgt ctcgtgaatc 5460
cgctagagtc atttttgagc gctttctccc aggtttggac tgggggttag ccgacgccct 5520
gtgagttacc gctcacgggg cgttcaacat ttttcaggta ttcgtgcagc ttatcgcttc 5580
ttgccgcctg tgcgcttttt cgacgcgcga cgctgctgcc gattcggtgc aggtggtggc 5640
ggcgctgaca cgtcctgggc ggccacggcc acacgaaacg cggcatttac gatgtttgtc 5700
atgcctgcgg gcaccgcgcc acgatcgcgg ataattctcg ctgccgcttc cagctctgtg 5760
acgaccatgg ccaaaatttc gctcggggga cgcacttcca gcgccatttg cgacctagcc 5820
gcctccagct cctcggcgtg gcgtttgttg gcgcgctcgc ggctggctgc ggcacgacac 5880
gcatctgagc aatattttgc gcgccgtcct cgcgggtcag gccggggagg aatcaggcca 5940
ccgcagtagg cgcaactgat tcgatcctcc actactgtgc gtcctcctgg cgctgccgag 6000
cacgcagctc gtcagccagc tcctcaagat ccgccacgag agtttctagg tcgctcgcgg 6060
cactggccca gtctcgtgat gctggcgcgt ccgtcgtatc gagagctcgg aaaaatccga 6120
tcaccgtttt taaatcgacg gcagcatcga gcgcgtcgga ctccagcgcg acatcagaga 6180
gatccatagc tgatgattcg ggccaatttt ggtacttcgt cgtgaaggtc atgacaccat 6240
tataacgaac gttcgttaaa gtttttggcg gaaaatcacg cggcacgaaa attttcacga 6300
agcgggactt tgcgcagctc aggggtgcta aaaattttgt atcgcacttg atttttccga 6360
aagacagatt atctgcaaac ggtgtgtcgt atttctggct tggtttttaa aaaatctgga 6420
atcgaaaatt tgcggggcga 6440
<210> 76
<211> 6395
<212> DNA
<213> 人工序列
<220>
<223> pJC1-PF1-U1A-TF1
<400> 76
ccgagaagtt ttttacaaaa ggcaaaaact ttttcgggat cgacagaaat aaaacgatcg 60
acggtacgca acaaaaaagc gtcaggatcg ccgtagagcg attgaagacc gtcaaccaaa 120
ggggaagcct ccaatcgacg cgacgcgcgc tctacggcga tcctgacgca gatttttagc 180
tatctgtcgc agcgccctca gggacaagcc acccgcacaa cgtcgcgagg gcgatcagcg 240
acgccgcagg gggatcctct agactcgagc gggacggtcg aaccagcttc aagcgaccgg 300
atgagtatgt tacagtagat agcgagcggg agaccgctcg accttagttc tcctgttgcg 360
ggggagttca tgggatccag gtacccaggg cgaggcttat ccattgcact ccggatgtgc 420
tgacccctgg ctagcatagc ataaaataac gccccacctt cttaacggga ggtggggcgt 480
tatttttacg gggatcgctt cttccgcttc ctcgctcact gactcgctgc gctcggtcgt 540
tcggctgcgg gtcgacctgc agcaatggca acaacgttgc gcaaactatt aactggcgaa 600
ctacttactc tagcttcccg gcaacaatta atagactgga tggaggcgga taaagttgca 660
ggaccacttc tgcgctcggc ccttccggct ggctggttta ttgctgataa atctggagcc 720
ggtgagcgtg ggtctcgcgg tatcattgca gcactggggc cagatggtaa gccctcccgt 780
atcgtagtta tctacacgac ggggagtcag gcaactatgg atgaacgaaa tagacagatc 840
gctgagatag gtgcctcact gattaagcat tggtaactgt cagaccaagt ttactcatat 900
atactttaga ttgatttaaa acttcatttt taatttaaaa ggatctaggt gaagatcctt 960
tttgataatc tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac 1020
cccttaataa gatgatcttc ttgagatcgt tttggtctgc gcgtaatctc ttgctctgaa 1080
aacgaaaaaa ccgccttgca gggcggtttt tcgaaggttc tctgagctac caactctttg 1140
aaccgaggta actggcttgg aggagcgcag tcaccaaaac ttgtcctttc agtttagcct 1200
taaccggcgc atgacttcaa gactaactcc tctaaatcaa ttaccagtgg ctgctgccag 1260
tggtgctttt gcatgtcttt ccgggttgga ctcaagacga tagttaccgg ataaggcgca 1320
gcggtcggac tgaacggggg gttcgtgcat acagtccagc ttggagcgaa ctgcctaccc 1380
ggaactgagt gtcaggcgtg gaatgagaca aacgcggcca taacagcgga atgacaccgg 1440
taaaccgaaa ggcaggaaca ggagagcgca cgagggagcc gccaggggga aacgcctggt 1500
atctttatag tcctgtcggg tttcgccacc actgatttga gcgtcagatt tcgtgatgct 1560
tgtcaggggg gcggagccta tggaaaaacg gctttgccgc ggccctctca cttccctgtt 1620
aagtatcttc ctggcatctt ccaggaaatc tccgccccgt tcgtaagcca tttccgctcg 1680
ccgcagtcga acgaccgagc gtagcgagtc agtgagcgag gaagcggaat atatcctgta 1740
tcacatattc tgctgacgca ccggtgcagc cttttttctc ctgccacatg aagcacttca 1800
ctgacaccct catcagtgcc aacatagtaa gccagtatac actccgctag cgctgaggtc 1860
tgcctcgtga agaaggtgtt gctgactcat accaggcctg aatcgcccca tcatccagcc 1920
agaaagtgag ggagccacgg ttgatgagag ctttgttgta ggtggaccag ttggtgattt 1980
tgaacttttg ctttgccacg gaacggtctg cgttgtcggg aagatgcgtg atctgatcct 2040
tcaactcagc aaaagttcga tttattcaac aaagccacgt tgtgtctcaa aatctctgat 2100
gttacattgc acaagataaa aatatatcat catgaacaat aaaactgtct gcttacataa 2160
acagtaatac aaggggtgtt atgagccata ttcaacggga aacgtcttgc tcgaggccgc 2220
gattaaattc caacatggat gctgatttat atgggtataa atgggctcgc gataatgtcg 2280
ggcaatcagg tgcgacaatc tatcgattgt atgggaagcc cgatgcgcca gagttgtttc 2340
tgaaacatgg caaaggtagc gttgccaatg atgttacaga tgagatggtc agactaaact 2400
ggctgacgga atttatgcct cttccgacca tcaagcattt tatccgtact cctgatgatg 2460
catggttact caccactgcg atccccggga aaacagcatt ccaggtatta gaagaatatc 2520
ctgattcagg tgaaaatatt gttgatgcgc tggcagtgtt cctgcgccgg ttgcattcga 2580
ttcctgtttg taattgtcct tttaacagcg atcgcgtatt tcgtctcgct caggcgcaat 2640
cacgaatgaa taacggtttg gttgatgcga gtgattttga tgacgagcgt aatggctggc 2700
ctgttgaaca agtctggaaa gaaatgcata agcttttgcc attctcaccg gattcagtcg 2760
tcactcatgg tgatttctca cttgataacc ttatttttga cgaggggaaa ttaataggtt 2820
gtattgatgt tggacgagtc ggaatcgcag accgatacca ggatcttgcc atcctatgga 2880
actgcctcgg tgagttttct ccttcattac agaaacggct ttttcaaaaa tatggtattg 2940
ataatcctga tatgaataaa ttgcagtttc atttgatgct cgatgagttt ttctaatcag 3000
aattggttaa ttggttgtaa cactggcaga gcattacgct gacttgacgg gacggcggct 3060
ttgttgaata aatcgaactt ttgctgagtt gaaggatcag atcacgcatc ttcccgacaa 3120
cgcagaccgt tccgtggcaa agcaaaagtt caaaatcacc aactggtcca cctacaacaa 3180
agctctcatc aaccgtggct ccctcacttt ctggctggat gatggggcga ttcaggcctg 3240
gtatgagtca gcaacacctt cttcacgagg cagacctcag cgctcaaaga tgcaggggta 3300
aaagctaacc gcatctttac cgacaaggca tccggcagtt caacagatcg ggaagggctg 3360
gatttgctga ggatgaaggt ggaggaaggt gatgtcattc tggtgaagaa gctcgaccgt 3420
cttggccgcg acaccgccga catgatccaa ctgataaaag agtttgatgc tcagggtgta 3480
gcggttcggt ttattgacga cgggatcagt accgacggtg atatggggca aatggtggtc 3540
accatcctgt cggctgtggc acaggctgaa cgccggagga tcaagtcggt caagccaagc 3600
gcaaccagcg gcaccgccgc gagcaacgtc gcaagggcga tcaggggacg atttttgcga 3660
agaatttcca cggtaagaat ccaatctctc gaatttaggg tgaaagaagc ttggcatagg 3720
ggtgtgcacg aactcggtgg aggaaatttc cgcggggcaa ggcttcgcga agcggagtcg 3780
cggcagtggc tttgaagatc tttgggagca gtccttgtgc gcttacgagg tgagccggtg 3840
gggaaccgtt atctgcctat ggtgtgagcc cccctagaga gcttcaagag caatcagccc 3900
gacctagaaa ggaggccaag agagagaccc ctacgggggg aaccgttttc tgcctacgag 3960
atggcacatt tactgggaag ctttacggcg tcctcgtgga agttcaatgc ccgcagactt 4020
aagtgctcta ttcacggtct gacgtgacac gctaaattca gacatagctt cattgattgt 4080
cggccacgag ccagtctctc cctcaacagt cataaaccaa cctgcaatgg tcaagcgatt 4140
tcctttagct ttcctagctt gtcgttgact ggacttagct agtttttctc gctgtgctcg 4200
ggcgtactca ctgtttgggt ctttccagcg ttctgcggcc tttttaccgc cacgtcttcc 4260
catagtggcc agagcttttc gccctcggct gctctgcgtc tctgtctgac gagcagggac 4320
gactggctgg cctttagcga cgtagccgcg cacacgtcgc gccatcgtct ggcggtcacg 4380
catcggcggc agatcaggct cacggccgtc tgctccgacc gcctgagcga cggtgtaggc 4440
acgctcgtag gcgtcgatga tcttggtgtc ttttaggcgc tcaccagccg cttttaactg 4500
gtatcccaca gtcaaagcgt ggcgaaaagc cgtctcatca cgggcggcac gccctggagc 4560
agtccagagg acacggacgc cgtcgatcag ctctccagac gcttcagcgg cgctcggcag 4620
gcttgcttca agcgtggcaa gtgcttttgc ttccgcagtg gcttttcttg ccgcttcgat 4680
acgtgcccgt ccgctagaaa actcctgctc atagcgtttt ttaggttttt ctgtgcctga 4740
gatcatgcga gcaacctcca taagatcagc taggcgatcc acgcgattgt gctgggcatg 4800
ccagcggtac gcggtgggat cgtcggagac gtgcagtggc caccggctca gcctatgtga 4860
aaaagcctgg tcagcgccga aaacgcgggt catttcctcg gtcgttgcag ccagcaggcg 4920
catattcggg ctgctcatgc ctgctgcggc atacaccgga tcaatgagcc agatgagctg 4980
gcatttcccg ctcagtggat tcacgccgat ccaagctggc gctttttcca ggcgtgccca 5040
gcgctccaaa atcgcgtaga cctcggggtt tacgtgctcg attttcccgc cggcctggtg 5100
gctcggcaca tcaatgtcca ggacaagcac ggctgcgtgc tgcgcgtgcg tcagagcaac 5160
atactggcac cgggcaagcg attttgaacc aactcggtat aacttcggct gtgtttctcc 5220
cgtgtccggg tctttgatcc aagcgctggc gaagtcgcgg gtcttgctgc cctggaaatt 5280
ttctctgccc aggtgagcga ggaattcgcg gcggtcttcg ctcgtccagc cacgtgatcg 5340
cagcgcgagc tcgggatggg tgtcgaacag atcagcggaa aatttccagg ccggtgtgtc 5400
aatgtctcgt gaatccgcta gagtcatttt tgagcgcttt ctcccaggtt tggactgggg 5460
gttagccgac gccctgtgag ttaccgctca cggggcgttc aacatttttc aggtattcgt 5520
gcagcttatc gcttcttgcc gcctgtgcgc tttttcgacg cgcgacgctg ctgccgattc 5580
ggtgcaggtg gtggcggcgc tgacacgtcc tgggcggcca cggccacacg aaacgcggca 5640
tttacgatgt ttgtcatgcc tgcgggcacc gcgccacgat cgcggataat tctcgctgcc 5700
gcttccagct ctgtgacgac catggccaaa atttcgctcg ggggacgcac ttccagcgcc 5760
atttgcgacc tagccgcctc cagctcctcg gcgtggcgtt tgttggcgcg ctcgcggctg 5820
gctgcggcac gacacgcatc tgagcaatat tttgcgcgcc gtcctcgcgg gtcaggccgg 5880
ggaggaatca ggccaccgca gtaggcgcaa ctgattcgat cctccactac tgtgcgtcct 5940
cctggcgctg ccgagcacgc agctcgtcag ccagctcctc aagatccgcc acgagagttt 6000
ctaggtcgct cgcggcactg gcccagtctc gtgatgctgg cgcgtccgtc gtatcgagag 6060
ctcggaaaaa tccgatcacc gtttttaaat cgacggcagc atcgagcgcg tcggactcca 6120
gcgcgacatc agagagatcc atagctgatg attcgggcca attttggtac ttcgtcgtga 6180
aggtcatgac accattataa cgaacgttcg ttaaagtttt tggcggaaaa tcacgcggca 6240
cgaaaatttt cacgaagcgg gactttgcgc agctcagggg tgctaaaaat tttgtatcgc 6300
acttgatttt tccgaaagac agattatctg caaacggtgt gtcgtatttc tggcttggtt 6360
tttaaaaaat ctggaatcga aaatttgcgg ggcga 6395
<210> 77
<211> 148
<212> RNA
<213> 人工序列
<220>
<223> Scaffold with two aptamers
<400> 77
uugccaugug uaugugggag acggucgggu ccagauauuc guaucugucg aguagagugu 60
gggcucccac auacucugau gauccagacg gucgggucca gauauucgua ucugucgagu 120
agaguguggg cuggaucauu cauggcaa 148
<210> 78
<211> 124
<212> RNA
<213> 人工序列
<220>
<223> Scaffold with two aptamers
<400> 78
uugccaugug uaugugggag acggucgggu ccagauauuc guaucugucg aguagagugu 60
gggcucccac auacucugau gauccgaagg gacggugcgg agaggagagg aucauucaug 120
gcaa 124
<210> 79
<211> 1191
<212> DNA
<213> 人工序列
<220>
<223> SEQ ID NO: 81, 82, 83 and 69 combined
<400> 79
cgcacaagcg gcggagcatg tggattaatt cgatgcaacg cgaagaacct tacctgggct 60
tgacatggac cggatcggcg tagagatacg ttttcccttg tggtcggttc acaggtggtg 120
catggttgtc gtcagctcgt gtcgtgagat gttgggttaa gtcccgcaac gagcgcaacc 180
cttgtcttat gttgccagca cattgtggtg ggtactcatg agagactgcc ggggttaact 240
cggaggaagg tggggatgac gtcaaatcat catgcccctt atgtccaggg cttcacacat 300
gctacaatgg tcggtacagc gagttgccac accgtgaggt ggagctaatc tcttaaagcc 360
ggcctcagtt cggattgggg tctgcaactc gaccccatga agtcggagtc gctagtaatc 420
gcagatcagc aacgctgcgg tgaatacgtt cccgggcctt gtacacaccg cccgtcacgt 480
catgaaagtt ggtaacaccc gaagccagtg gcccaacctt ttagggggga gctgtcgaag 540
gtgggatcgg cgattgggac gaagtcgtaa caaggtagcc gtaccggaag gtgcggctgg 600
atctagattg ccatgtgtat gtgggagacg gtcgggtcca gatattcgta tctgtcgagt 660
agagtgtggg ctcccacata ctctgatgat ccttcgggat cattcatggc aacctccttt 720
ctaaggagct ttattaaccc acatcagact gtgtctggtt ggtgggttgt tggtgttgga 780
acccgtatgt ggttgccatc aacatatttt taatcgggtg gagatgaccc ctcgggtgac 840
aacaacacag caaacagtgc tgtgattaat aggtggcatg ctgttgggtg tctggaatga 900
catcgcaagc atcacctttt ggtggtgtgt gtgggttgtt tctaacatcg agcatcgtca 960
acacgggtag agaatgttgt gttctttggt tgtggtgggg gtggtgtgtt gtgtgagaac 1020
tgtatagtgg acgcgagcat ctttattttt ttgttttttg ttgtgtgata ccgaacgcgc 1080
ccgcactttg tgtgtgggtt atagtatttt gtttgttgtt ttgtagggca cacggtggat 1140
gccttggcat atcaagccga tgaaggacgt gagaggctgc gttatgcctc g 1191
<210> 80
<211> 6867
<212> DNA
<213> 人工序列
<220>
<223> pk19msb_16s rrna-broccoli
<400> 80
cgcacaagcg gcggagcatg tggattaatt cgatgcaacg cgaagaacct tacctgggct 60
tgacatggac cggatcggcg tagagatacg ttttcccttg tggtcggttc acaggtggtg 120
catggttgtc gtcagctcgt gtcgtgagat gttgggttaa gtcccgcaac gagcgcaacc 180
cttgtcttat gttgccagca cattgtggtg ggtactcatg agagactgcc ggggttaact 240
cggaggaagg tggggatgac gtcaaatcat catgcccctt atgtccaggg cttcacacat 300
gctacaatgg tcggtacagc gagttgccac accgtgaggt ggagctaatc tcttaaagcc 360
ggcctcagtt cggattgggg tctgcaactc gaccccatga agtcggagtc gctagtaatc 420
gcagatcagc aacgctgcgg tgaatacgtt cccgggcctt gtacacaccg cccgtcacgt 480
catgaaagtt ggtaacaccc gaagccagtg gcccaacctt ttagggggga gctgtcgaag 540
gtgggatcgg cgattgggac gaagtcgtaa caaggtagcc gtaccggaag gtgcggctgg 600
atctagattg ccatgtgtat gtgggagacg gtcgggtcca gatattcgta tctgtcgagt 660
agagtgtggg ctcccacata ctctgatgat ccttcgggat cattcatggc aacctccttt 720
ctaaggagct ttattaaccc acatcagact gtgtctggtt ggtgggttgt tggtgttgga 780
acccgtatgt ggttgccatc aacatatttt taatcgggtg gagatgaccc ctcgggtgac 840
aacaacacag caaacagtgc tgtgattaat aggtggcatg ctgttgggtg tctggaatga 900
catcgcaagc atcacctttt ggtggtgtgt gtgggttgtt tctaacatcg agcatcgtca 960
acacgggtag agaatgttgt gttctttggt tgtggtgggg gtggtgtgtt gtgtgagaac 1020
tgtatagtgg acgcgagcat ctttattttt ttgttttttg ttgtgtgata ccgaacgcgc 1080
ccgcactttg tgtgtgggtt atagtatttt gtttgttgtt ttgtagggca cacggtggat 1140
gccttggcat atcaagccga tgaaggacgt gagaggctgc gttatgcctc gaagcttggc 1200
gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa 1260
catacgagcc ggaagcataa agtgtaaagc ctggggtgcc taatgagtga gctaactcac 1320
attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca 1380
ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgct cttccgcttc 1440
ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc 1500
aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc 1560
aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag 1620
gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc 1680
gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt 1740
tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct 1800
ttctcaatgc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg 1860
ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct 1920
tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat 1980
tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg 2040
ctacactaga aggacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa 2100
aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt 2160
ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc 2220
tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt 2280
atcaaaaagg atcttcacct agatcctttt ggggtgggcg aagaactcca gcatgagatc 2340
cccgcgctgg aggatcatcc agccctgata gaaacagaag ccactggagc acctcaaaaa 2400
caccatcata cactaaatca gtaagttggc agcatcaccc gacgcacttt gcgccgaata 2460
aatacctgtg acggaagatc acttcgcaga ataaataaat cctggtgtcc ctgttgatac 2520
cgggaagccc tgggccaact tttggcgaaa atgagacgtt gatcggcacg taagaggttc 2580
caactttcac cataatgaaa taagatcact accgggcgta ttttttgagt tatcgagatt 2640
ttcaggagct gatagaaaca gaagccactg gagcacctca aaaacaccat catacactaa 2700
atcagtaagt tggcagcatc acccgacgca ctttgcgccg aataaatacc tgtgacggaa 2760
gatcacttcg cagaataaat aaatcctggt gtccctgttg ataccgggaa gccctgggcc 2820
aacttttggc gaaaatgaga cgttgatcgg cacgtaagag gttccaactt tcaccataat 2880
gaaataagat cactaccggg cgtatttttt gagttatcga gattttcagg agctctttgg 2940
catcgtctct cgcctgtccc ctcagttcag taatttcctg catttgcctg tttccagtcg 3000
gtagatattc cacaaaacag cagggaagca gcgcttttcc gctgcataac cctgcttcgg 3060
ggtcattata gcgatttttt cggtatatcc atcctttttc gcacgatata caggattttg 3120
ccaaagggtt cgtgtagact ttccttggtg tatccaacgg cgtcagcggg gcaggatagg 3180
tgaagtaggc ccacccgcga gcgggtgttc cttcttcact gtcccttatt cgcacctggc 3240
ggtgctcaac gggaatcctg ctctgcgagg ctggccggct accgccggcg taacagatga 3300
gggcaagcgg atggctgatg aaaccaagcc aaccaggaag ggcagcccac ctatcaaggt 3360
gtactgcctt ccagacgaac gaagagcgat tgaggaaaag gcggcggcgg ccggcatgag 3420
cctgtcggcc tacctgctgg ccgtcggcca gggctacaaa atcacgggcg tcgtggacta 3480
tgagcacgtc cgcgagggcg tcccggaaaa cgattccgaa gcccaacctt tcatagaagg 3540
cggcggtgga atcgaaatct cgtgatggca ggttgggcgt cgcttggtcg gtcatttcgc 3600
tcggtaccca tcggcatttt cttttgcgtt tttatttgtt aactgttaat tgtccttgtt 3660
caaggatgct gtctttgaca acagatgttt tcttgccttt gatgttcagc aggaagctcg 3720
gcgcaaacgt tgattgtttg tctgcgtaga atcctctgtt tgtcatatag cttgtaatca 3780
cgacattgtt tcctttcgct tgaggtacag cgaagtgtga gtaagtaaag gttacatcgt 3840
taggatcaag atccattttt aacacaaggc cagttttgtt cagcggcttg tatgggccag 3900
ttaaagaatt agaaacataa ccaagcatgt aaatatcgtt agacgtaatg ccgtcaatcg 3960
tcatttttga tccgcgggag tcagtgaaca ggtaccattt gccgttcatt ttaaagacgt 4020
tcgcgcgttc aatttcatct gttactgtgt tagatgcaat cagcggtttc atcacttttt 4080
tcagtgtgta atcatcgttt agctcaatca taccgagagc gccgtttgct aactcagccg 4140
tgcgtttttt atcgctttgc agaagttttt gactttcttg acggaagaat gatgtgcttt 4200
tgccatagta tgctttgtta aataaagatt cttcgccttg gtagccatct tcagttccag 4260
tgtttgcttc aaatactaag tatttgtggc ctttatcttc tacgtagtga ggatctctca 4320
gcgtatggtt gtcgcctgag ctgtagttgc cttcatcgat gaactgctgt acattttgat 4380
acgtttttcc gtcaccgtca aagattgatt tataatcctc tacaccgttg atgttcaaag 4440
agctgtctga tgctgatacg ttaacttgtg cagttgtcag tgtttgtttg ccgtaatgtt 4500
taccggagaa atcagtgtag aataaacgga tttttccgtc agatgtaaat gtggctgaac 4560
ctgaccattc ttgtgtttgg tcttttagga tagaatcatt tgcatcgaat ttgtcgctgt 4620
ctttaaagac gcggccagcg tttttccagc tgtcaataga agtttcgccg actttttgat 4680
agaacatgta aatcgatgtg tcatccgcat ttttaggatc tccggctaat gcaaagacga 4740
tgtggtagcc gtgatagttt gcgacagtgc cgtcagcgtt ttgtaatggc cagctgtccc 4800
aaacgtccag gccttttgca gaagagatat ttttaattgt ggacgaatca aattcagaaa 4860
cttgatattt ttcatttttt tgctgttcag ggatttgcag catatcatgg cgtgtaatat 4920
gggaaatgcc gtatgtttcc ttatatggct tttggttcgt ttctttcgca aacgcttgag 4980
ttgcgcctcc tgccagcagt gcggtagtaa aggttaatac tgttgcttgt tttgcaaact 5040
ttttgatgtt catcgttcat gtctcctttt ttatgtactg tgttagcggt ctgcttcttc 5100
cagccctcct gtttgaagat ggcaagttag ttacgcacaa taaaaaaaga cctaaaatat 5160
gtaaggggtg acgccaaagt atacactttg ccctttacac attttaggtc ttgcctgctt 5220
tatcagtaac aaacccgcgc gatttacttt tcgacctcat tctattagac tctcgtttgg 5280
attgcaactg gtctattttc ctcttttgtt tgatagaaaa tcataaaagg atttgcagac 5340
tacgggccta aagaactaaa aaatctatct gtttcttttc attctctgta ttttttatag 5400
tttctgttgc atgggcataa agttgccttt ttaatcacaa ttcagaaaat atcataatat 5460
ctcatttcac taaataatag tgaacggcag gtatatgtga tgggttaaaa aggatcgatc 5520
ctctagcgaa ccccagagtc ccgctcagaa gaactcgtca agaaggcgat agaaggcgat 5580
gcgctgcgaa tcgggagcgg cgataccgta aagcacgagg aagcggtcag cccattcgcc 5640
gccaagctct tcagcaatat cacgggtagc caacgctatg tcctgatagc ggtccgccac 5700
acccagccgg ccacagtcga tgaatccaga aaagcggcca ttttccacca tgatattcgg 5760
caagcaggca tcgccatggg tcacgacgag atcctcgccg tcgggcatcc gcgccttgag 5820
cctggcgaac agttcggctg gcgcgagccc ctgatgctct tcgtccagat catcctgatc 5880
gacaagaccg gcttccatcc gagtacgtgc tcgctcgatg cgatgtttcg cttggtggtc 5940
gaatgggcag gtagccggat caagcgtatg cagccgccgc attgcatcag ccatgatgga 6000
tactttctcg gcaggagcaa ggtgagatga caggagatcc tgccccggca cttcgcccaa 6060
tagcagccag tcccttcccg cttcagtgac aacgtcgagc acagctgcgc aaggaacgcc 6120
cgtcgtggcc agccacgata gccgcgctgc ctcgtcttgg agttcattca gggcaccgga 6180
caggtcggtc ttgacaaaaa gaaccgggcg cccctgcgct gacagccgga acacggcggc 6240
atcagagcag ccgattgtct gttgtgccca gtcatagccg aatagcctct ccacccaagc 6300
ggccggagaa cctgcgtgca atccatcttg ttcaatcatg cgaaacgatc ctcatcctgt 6360
ctcttgatca gatcttgatc ccctgcgcca tcagatcctt ggcggcaaga aagccatcca 6420
gtttactttg cagggcttcc caaccttacc agagggcgcc ccagctggca attccggttc 6480
gcttgctgtc cataaaaccg cccagtctag ctatcgccat gtaagcccac tgcaagctac 6540
ctgctttctc tttgcgcttg cgttttccct tgtccagata gcccagtagc tgacattcat 6600
ccggggtcag caccgtttct gcggactggc tttctacgtg ttccgcttcc tttagcagcc 6660
cttgcgccct gagtgcttgc ggcagcgtga agctagctta tcgcgccatt cgccattcag 6720
gctgcgcaac tgttgggaag ggcgatcggt gcgggcctct tcgctattac gccagctggc 6780
gaaaggggga tgtgctgcaa ggcgattaag ttgggtaacg ccagggtttt cccagtcacg 6840
acgttgtaaa acgacggcca gtgaatt 6867
<210> 81
<211> 601
<212> DNA
<213> 人工序列
<220>
<223> Target integration site (upstream)
<400> 81
cgcacaagcg gcggagcatg tggattaatt cgatgcaacg cgaagaacct tacctgggct 60
tgacatggac cggatcggcg tagagatacg ttttcccttg tggtcggttc acaggtggtg 120
catggttgtc gtcagctcgt gtcgtgagat gttgggttaa gtcccgcaac gagcgcaacc 180
cttgtcttat gttgccagca cattgtggtg ggtactcatg agagactgcc ggggttaact 240
cggaggaagg tggggatgac gtcaaatcat catgcccctt atgtccaggg cttcacacat 300
gctacaatgg tcggtacagc gagttgccac accgtgaggt ggagctaatc tcttaaagcc 360
ggcctcagtt cggattgggg tctgcaactc gaccccatga agtcggagtc gctagtaatc 420
gcagatcagc aacgctgcgg tgaatacgtt cccgggcctt gtacacaccg cccgtcacgt 480
catgaaagtt ggtaacaccc gaagccagtg gcccaacctt ttagggggga gctgtcgaag 540
gtgggatcgg cgattgggac gaagtcgtaa caaggtagcc gtaccggaag gtgcggctgg 600
a 601
<210> 82
<211> 1718
<212> DNA
<213> 人工序列
<220>
<223> 5'UTR-luc2 (Photinus pyralis)
<400> 82
ggagacccaa gctggctagc gtttaaactt aagcttggca atccggtact gttggtaaag 60
ccaccatgga agatgccaaa aacattaaga agggcccagc gccattctac ccactcgaag 120
acgggaccgc cggcgagcag ctgcacaaag ccatgaagcg ctacgccctg gtgcccggca 180
ccatcgcctt taccgacgca catatcgagg tggacattac ctacgccgag tacttcgaga 240
tgagcgttcg gctggcagaa gctatgaagc gctatgggct gaatacaaac catcggatcg 300
tggtgtgcag cgagaatagc ttgcagttct tcatgcccgt gttgggtgcc ctgttcatcg 360
gtgtggctgt ggccccagct aacgacatct acaacgagcg cgagctgctg aacagcatgg 420
gcatcagcca gcccaccgtc gtattcgtga gcaagaaagg gctgcaaaag atcctcaacg 480
tgcaaaagaa gctaccgatc atacaaaaga tcatcatcat ggatagcaag accgactacc 540
agggcttcca aagcatgtac accttcgtga cttcccattt gccacccggc ttcaacgagt 600
acgacttcgt gcccgagagc ttcgaccggg acaaaaccat cgccctgatc atgaacagta 660
gtggcagtac cggattgccc aagggcgtag ccctaccgca ccgcaccgct tgtgtccgat 720
tcagtcatgc ccgcgacccc atcttcggca accagatcat ccccgacacc gctatcctca 780
gcgtggtgcc atttcaccac ggcttcggca tgttcaccac gctgggctac ttgatctgcg 840
gctttcgggt cgtgctcatg taccgcttcg aggaggagct attcttgcgc agcttgcaag 900
actataagat tcaatctgcc ctgctggtgc ccacactatt tagcttcttc gctaagagca 960
ctctcatcga caagtacgac ctaagcaact tgcacgagat cgccagcggc ggggcgccgc 1020
tcagcaagga ggtaggtgag gccgtggcca aacgcttcca cctaccaggc atccgccagg 1080
gctacggcct gacagaaaca accagcgcca ttctgatcac ccccgaaggg gacgacaagc 1140
ctggcgcagt aggcaaggtg gtgcccttct tcgaggctaa ggtggtggac ttggacaccg 1200
gtaagacact gggtgtgaac cagcgcggcg agctgtgcgt ccgtggcccc atgatcatga 1260
gcggctacgt taacaacccc gaggctacaa acgctctcat cgacaaggac ggctggctgc 1320
acagcggcga catcgcctac tgggacgagg acgagcactt cttcatcgtg gaccggctga 1380
agagcctgat caaatacaag ggctaccagg tagccccagc cgaactggag agcatcctgc 1440
tgcaacaccc caacatcttc gacgccgggg tcgccggcct gcccgacgac gatgccggcg 1500
agctgcccgc cgcagtcgtc gtgctggaac acggtaaaac catgaccgag aaggagatcg 1560
tggactatgt ggccagccag gttacaaccg ccaagaagct gcgcggtggt gttgtgttcg 1620
tggacgaggt gcctaaagga ctgaccggca agttggacgc ccgcaagatc cgcgagattc 1680
tcattaaggc caagaagggc ggcaagatcg ccgtgtaa 1718
<210> 83
<211> 479
<212> DNA
<213> 人工序列
<220>
<223> Target integration site (downstream)
<400> 83
cctcctttct aaggagcttt attaacccac atcagactgt gtctggttgg tgggttgttg 60
gtgttggaac ccgtatgtgg ttgccatcaa catattttta atcgggtgga gatgacccct 120
cgggtgacaa caacacagca aacagtgctg tgattaatag gtggcatgct gttgggtgtc 180
tggaatgaca tcgcaagcat caccttttgg tggtgtgtgt gggttgtttc taacatcgag 240
catcgtcaac acgggtagag aatgttgtgt tctttggttg tggtgggggt ggtgtgttgt 300
gtgagaactg tatagtggac gcgagcatct ttattttttt gttttttgtt gtgtgatacc 360
gaacgcgccc gcactttgtg tgtgggttat agtattttgt ttgttgtttt gtagggcaca 420
cggtggatgc cttggcatat caagccgatg aaggacgtga gaggctgcgt tatgcctcg 479
<210> 84
<211> 20
<212> DNA
<213> 人工序列
<220>
<223> 16S rRNA-broccoli_for
<400> 84
gtcttccacg acttctgtgc 20
<210> 85
<211> 20
<212> DNA
<213> 人工序列
<220>
<223> 16S rRNA-broccoli_rev
<400> 85
gtgtaaacct ccacaccagc 20
<210> 86
<211> 19
<212> DNA
<213> 人工序列
<220>
<223> rrnA_rev
<400> 86
cttgacctgg gaagttgcg 19
<210> 87
<211> 20
<212> DNA
<213> 人工序列
<220>
<223> rrnB_rev
<400> 87
gcaagattgc ttgctaccac 20
<210> 88
<211> 20
<212> DNA
<213> 人工序列
<220>
<223> rrnC_rev
<400> 88
agaaactcgg agcgaccatc 20
<210> 89
<211> 20
<212> DNA
<213> 人工序列
<220>
<223> rrnD_rev
<400> 89
cgtcacacat cgctctacag 20
<210> 90
<211> 21
<212> DNA
<213> 人工序列
<220>
<223> rrnE_rev
<400> 90
gaagccttcc catcaagcat c 21
<210> 91
<211> 20
<212> DNA
<213> 人工序列
<220>
<223> rrnF_rev
<400> 91
cacatcaagg tgacacggag 20
<210> 92
<211> 25
<212> DNA
<213> 人工序列
<220>
<223> qPCR_broc_fw
<400> 92
tcatgaaagt tggtaacacc cgaag 25
<210> 93
<211> 24
<212> DNA
<213> 人工序列
<220>
<223> qPCR_broc_rev
<400> 93
ttgccatgaa tgatcccgaa ggat 24
<210> 94
<211> 288
<212> DNA
<213> 人工序列
<220>
<223> SEQ ID No. 68, 69, 96 and 97 combined, with EcoRI recognition
site on 5'-end, HindIII recognition site on 3'-end
<400> 94
gaattctaat acgactcact atagagcggg agaccgctcg accttagttc tcctgttgcg 60
ggggagttca tgggatccag gtacccaggg cgaggcttat ccattgcact ccggatgtgc 120
tgacccctgt tgccatgtgt atgtgggaga cggtcgggtc cagatattcg tatctgtcga 180
gtagagtgtg ggctcccaca tactctgatg atccttcggg atcattcatg gcaactagca 240
taaccccttg gggcctctaa acgggtcttg aggggttttt tgaagctt 288
<210> 95
<211> 2917
<212> DNA
<213> 人工序列
<220>
<223> pUC18-PT7-U1A-F30::broccoli/UUCG-TT7
<400> 95
aattctaata cgactcacta tagagcggga gaccgctcga ccttagttct cctgttgcgg 60
gggagttcat gggatccagg tacccagggc gaggcttatc cattgcactc cggatgtgct 120
gacccctgtt gccatgtgta tgtgggagac ggtcgggtcc agatattcgt atctgtcgag 180
tagagtgtgg gctcccacat actctgatga tccttcggga tcattcatgg caactagcat 240
aaccccttgg ggcctctaaa cgggtcttga ggggtttttt gaagcttggc actggccgtc 300
gttttacaac gtcgtgactg ggaaaaccct ggcgttaccc aacttaatcg ccttgcagca 360
catccccctt tcgccagctg gcgtaatagc gaagaggccc gcaccgatcg cccttcccaa 420
cagttgcgca gcctgaatgg cgaatggcgc ctgatgcggt attttctcct tacgcatctg 480
tgcggtattt cacaccgcat atggtgcact ctcagtacaa tctgctctga tgccgcatag 540
ttaagccagc cccgacaccc gccaacaccc gctgacgcgc cctgacgggc ttgtctgctc 600
ccggcatccg cttacagaca agctgtgacc gtctccggga gctgcatgtg tcagaggttt 660
tcaccgtcat caccgaaacg cgcgagacga aagggcctcg tgatacgcct atttttatag 720
gttaatgtca tgataataat ggtttcttag acgtcaggtg gcacttttcg gggaaatgtg 780
cgcggaaccc ctatttgttt atttttctaa atacattcaa atatgtatcc gctcatgaga 840
caataaccct gataaatgct tcaataatat tgaaaaagga agagtatgag tattcaacat 900
ttccgtgtcg cccttattcc cttttttgcg gcattttgcc ttcctgtttt tgctcaccca 960
gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt gggttacatc 1020
gaactggatc tcaacagcgg taagatcctt gagagttttc gccccgaaga acgttttcca 1080
atgatgagca cttttaaagt tctgctatgt ggcgcggtat tatcccgtat tgacgccggg 1140
caagagcaac tcggtcgccg catacactat tctcagaatg acttggttga gtactcacca 1200
gtcacagaaa agcatcttac ggatggcatg acagtaagag aattatgcag tgctgccata 1260
accatgagtg ataacactgc ggccaactta cttctgacaa cgatcggagg accgaaggag 1320
ctaaccgctt ttttgcacaa catgggggat catgtaactc gccttgatcg ttgggaaccg 1380
gagctgaatg aagccatacc aaacgacgag cgtgacacca cgatgcctgt agcaatggca 1440
acaacgttgc gcaaactatt aactggcgaa ctacttactc tagcttcccg gcaacaatta 1500
atagactgga tggaggcgga taaagttgca ggaccacttc tgcgctcggc ccttccggct 1560
ggctggttta ttgctgataa atctggagcc ggtgagcgtg ggtctcgcgg tatcattgca 1620
gcactggggc cagatggtaa gccctcccgt atcgtagtta tctacacgac ggggagtcag 1680
gcaactatgg atgaacgaaa tagacagatc gctgagatag gtgcctcact gattaagcat 1740
tggtaactgt cagaccaagt ttactcatat atactttaga ttgatttaaa acttcatttt 1800
taatttaaaa ggatctaggt gaagatcctt tttgataatc tcatgaccaa aatcccttaa 1860
cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga 1920
gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg 1980
gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc 2040
agagcgcaga taccaaatac tgtccttcta gtgtagccgt agttaggcca ccacttcaag 2100
aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc 2160
agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg 2220
cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac 2280
accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga 2340
aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt 2400
ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag 2460
cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg 2520
gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgttctt tcctgcgtta 2580
tcccctgatt ctgtggataa ccgtattacc gcctttgagt gagctgatac cgctcgccgc 2640
agccgaacga ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg cccaatacgc 2700
aaaccgcctc tccccgcgcg ttggccgatt cattaatgca gctggcacga caggtttccc 2760
gactggaaag cgggcagtga gcgcaacgca attaatgtga gttagctcac tcattaggca 2820
ccccaggctt tacactttat gcttccggct cgtatgttgt gtggaattgt gagcggataa 2880
caatttcaca caggaaacag ctatgaccat gattacg 2917
<210> 96
<211> 18
<212> DNA
<213> 人工序列
<220>
<223> PT7
<400> 96
taatacgact cactatag 18
<210> 97
<211> 48
<212> DNA
<213> 人工序列
<220>
<223> TT7
<400> 97
ctagcataac cccttggggc ctctaaacgg gtcttgaggg gttttttg 48
<210> 98
<211> 19
<212> DNA
<213> 人工序列
<220>
<223> pUC18_check_f
<400> 98
ttccggctcg tatgttgtg 19
<210> 99
<211> 21
<212> DNA
<213> 人工序列
<220>
<223> pUC18_check_rev
<400> 99
aggcgattaa gttgggtaac g 21
<210> 100
<211> 330
<212> DNA
<213> 人工序列
<220>
<223> SEQ ID No. 67, 68, 101, 70 combined with XbaI recognition site on
5'-end, SalI recognition site on 3'-end
<400> 100
ctgtctctag actcgagcgg gacggtcgaa ccagcttcaa gcgaccggat gagtatgtta 60
cagtagatag cgagcgggag accgctcgac cttagttctc ctgttgcggg ggagttcatg 120
ggatccaggt acccagggcg aggcttatcc attgcactcc ggatgtgctg acccctggga 180
gctcttgcca tgtgtatgtg ggggaaggat tggtatgtgg tatacccaca tactctgatg 240
atccttcggg atcattcatg gcaagctagc atagcataaa ataacgcccc accttcttaa 300
cgggaggtgg ggcgttattt ttacgtcgac 330
<210> 101
<211> 80
<212> DNA
<213> 人工序列
<220>
<223> F30 with Mango3 in its insertion site
<400> 101
ttgccatgtg tatgtggggg aaggattggt atgtggtata cccacatact ctgatgatcc 60
ttcgggatca ttcatggcaa 80
<210> 102
<211> 6421
<212> DNA
<213> 人工序列
<220>
<223> pJC1-PF1-U1A-F30::mangoIII-TF1
<400> 102
ctagactcga gcgggacggt cgaaccagct tcaagcgacc ggatgagtat gttacagtag 60
atagcgagcg ggagaccgct cgaccttagt tctcctgttg cgggggagtt catgggatcc 120
aggtacccag ggcgaggctt atccattgca ctccggatgt gctgacccct gggagctctt 180
gccatgtgta tgtgggggaa ggattggtat gtggtatacc cacatactct gatgatcctt 240
cgggatcatt catggcaagc tagcatagca taaaataacg ccccaccttc ttaacgggag 300
gtggggcgtt atttttacgt cgacctgcag caatggcaac aacgttgcgc aaactattaa 360
ctggcgaact acttactcta gcttcccggc aacaattaat agactggatg gaggcggata 420
aagttgcagg accacttctg cgctcggccc ttccggctgg ctggtttatt gctgataaat 480
ctggagccgg tgagcgtggg tctcgcggta tcattgcagc actggggcca gatggtaagc 540
cctcccgtat cgtagttatc tacacgacgg ggagtcaggc aactatggat gaacgaaata 600
gacagatcgc tgagataggt gcctcactga ttaagcattg gtaactgtca gaccaagttt 660
actcatatat actttagatt gatttaaaac ttcattttta atttaaaagg atctaggtga 720
agatcctttt tgataatctc atgaccaaaa tcccttaacg tgagttttcg ttccactgag 780
cgtcagaccc cttaataaga tgatcttctt gagatcgttt tggtctgcgc gtaatctctt 840
gctctgaaaa cgaaaaaacc gccttgcagg gcggtttttc gaaggttctc tgagctacca 900
actctttgaa ccgaggtaac tggcttggag gagcgcagtc accaaaactt gtcctttcag 960
tttagcctta accggcgcat gacttcaaga ctaactcctc taaatcaatt accagtggct 1020
gctgccagtg gtgcttttgc atgtctttcc gggttggact caagacgata gttaccggat 1080
aaggcgcagc ggtcggactg aacggggggt tcgtgcatac agtccagctt ggagcgaact 1140
gcctacccgg aactgagtgt caggcgtgga atgagacaaa cgcggccata acagcggaat 1200
gacaccggta aaccgaaagg caggaacagg agagcgcacg agggagccgc cagggggaaa 1260
cgcctggtat ctttatagtc ctgtcgggtt tcgccaccac tgatttgagc gtcagatttc 1320
gtgatgcttg tcaggggggc ggagcctatg gaaaaacggc tttgccgcgg ccctctcact 1380
tccctgttaa gtatcttcct ggcatcttcc aggaaatctc cgccccgttc gtaagccatt 1440
tccgctcgcc gcagtcgaac gaccgagcgt agcgagtcag tgagcgagga agcggaatat 1500
atcctgtatc acatattctg ctgacgcacc ggtgcagcct tttttctcct gccacatgaa 1560
gcacttcact gacaccctca tcagtgccaa catagtaagc cagtatacac tccgctagcg 1620
ctgaggtctg cctcgtgaag aaggtgttgc tgactcatac caggcctgaa tcgccccatc 1680
atccagccag aaagtgaggg agccacggtt gatgagagct ttgttgtagg tggaccagtt 1740
ggtgattttg aacttttgct ttgccacgga acggtctgcg ttgtcgggaa gatgcgtgat 1800
ctgatccttc aactcagcaa aagttcgatt tattcaacaa agccacgttg tgtctcaaaa 1860
tctctgatgt tacattgcac aagataaaaa tatatcatca tgaacaataa aactgtctgc 1920
ttacataaac agtaatacaa ggggtgttat gagccatatt caacgggaaa cgtcttgctc 1980
gaggccgcga ttaaattcca acatggatgc tgatttatat gggtataaat gggctcgcga 2040
taatgtcggg caatcaggtg cgacaatcta tcgattgtat gggaagcccg atgcgccaga 2100
gttgtttctg aaacatggca aaggtagcgt tgccaatgat gttacagatg agatggtcag 2160
actaaactgg ctgacggaat ttatgcctct tccgaccatc aagcatttta tccgtactcc 2220
tgatgatgca tggttactca ccactgcgat ccccgggaaa acagcattcc aggtattaga 2280
agaatatcct gattcaggtg aaaatattgt tgatgcgctg gcagtgttcc tgcgccggtt 2340
gcattcgatt cctgtttgta attgtccttt taacagcgat cgcgtatttc gtctcgctca 2400
ggcgcaatca cgaatgaata acggtttggt tgatgcgagt gattttgatg acgagcgtaa 2460
tggctggcct gttgaacaag tctggaaaga aatgcataag cttttgccat tctcaccgga 2520
ttcagtcgtc actcatggtg atttctcact tgataacctt atttttgacg aggggaaatt 2580
aataggttgt attgatgttg gacgagtcgg aatcgcagac cgataccagg atcttgccat 2640
cctatggaac tgcctcggtg agttttctcc ttcattacag aaacggcttt ttcaaaaata 2700
tggtattgat aatcctgata tgaataaatt gcagtttcat ttgatgctcg atgagttttt 2760
ctaatcagaa ttggttaatt ggttgtaaca ctggcagagc attacgctga cttgacggga 2820
cggcggcttt gttgaataaa tcgaactttt gctgagttga aggatcagat cacgcatctt 2880
cccgacaacg cagaccgttc cgtggcaaag caaaagttca aaatcaccaa ctggtccacc 2940
tacaacaaag ctctcatcaa ccgtggctcc ctcactttct ggctggatga tggggcgatt 3000
caggcctggt atgagtcagc aacaccttct tcacgaggca gacctcagcg ctcaaagatg 3060
caggggtaaa agctaaccgc atctttaccg acaaggcatc cggcagttca acagatcggg 3120
aagggctgga tttgctgagg atgaaggtgg aggaaggtga tgtcattctg gtgaagaagc 3180
tcgaccgtct tggccgcgac accgccgaca tgatccaact gataaaagag tttgatgctc 3240
agggtgtagc ggttcggttt attgacgacg ggatcagtac cgacggtgat atggggcaaa 3300
tggtggtcac catcctgtcg gctgtggcac aggctgaacg ccggaggatc aagtcggtca 3360
agccaagcgc aaccagcggc accgccgcga gcaacgtcgc aagggcgatc aggggacgat 3420
ttttgcgaag aatttccacg gtaagaatcc aatctctcga atttagggtg aaagaagctt 3480
ggcatagggg tgtgcacgaa ctcggtggag gaaatttccg cggggcaagg cttcgcgaag 3540
cggagtcgcg gcagtggctt tgaagatctt tgggagcagt ccttgtgcgc ttacgaggtg 3600
agccggtggg gaaccgttat ctgcctatgg tgtgagcccc cctagagagc ttcaagagca 3660
atcagcccga cctagaaagg aggccaagag agagacccct acggggggaa ccgttttctg 3720
cctacgagat ggcacattta ctgggaagct ttacggcgtc ctcgtggaag ttcaatgccc 3780
gcagacttaa gtgctctatt cacggtctga cgtgacacgc taaattcaga catagcttca 3840
ttgattgtcg gccacgagcc agtctctccc tcaacagtca taaaccaacc tgcaatggtc 3900
aagcgatttc ctttagcttt cctagcttgt cgttgactgg acttagctag tttttctcgc 3960
tgtgctcggg cgtactcact gtttgggtct ttccagcgtt ctgcggcctt tttaccgcca 4020
cgtcttccca tagtggccag agcttttcgc cctcggctgc tctgcgtctc tgtctgacga 4080
gcagggacga ctggctggcc tttagcgacg tagccgcgca cacgtcgcgc catcgtctgg 4140
cggtcacgca tcggcggcag atcaggctca cggccgtctg ctccgaccgc ctgagcgacg 4200
gtgtaggcac gctcgtaggc gtcgatgatc ttggtgtctt ttaggcgctc accagccgct 4260
tttaactggt atcccacagt caaagcgtgg cgaaaagccg tctcatcacg ggcggcacgc 4320
cctggagcag tccagaggac acggacgccg tcgatcagct ctccagacgc ttcagcggcg 4380
ctcggcaggc ttgcttcaag cgtggcaagt gcttttgctt ccgcagtggc ttttcttgcc 4440
gcttcgatac gtgcccgtcc gctagaaaac tcctgctcat agcgtttttt aggtttttct 4500
gtgcctgaga tcatgcgagc aacctccata agatcagcta ggcgatccac gcgattgtgc 4560
tgggcatgcc agcggtacgc ggtgggatcg tcggagacgt gcagtggcca ccggctcagc 4620
ctatgtgaaa aagcctggtc agcgccgaaa acgcgggtca tttcctcggt cgttgcagcc 4680
agcaggcgca tattcgggct gctcatgcct gctgcggcat acaccggatc aatgagccag 4740
atgagctggc atttcccgct cagtggattc acgccgatcc aagctggcgc tttttccagg 4800
cgtgcccagc gctccaaaat cgcgtagacc tcggggttta cgtgctcgat tttcccgccg 4860
gcctggtggc tcggcacatc aatgtccagg acaagcacgg ctgcgtgctg cgcgtgcgtc 4920
agagcaacat actggcaccg ggcaagcgat tttgaaccaa ctcggtataa cttcggctgt 4980
gtttctcccg tgtccgggtc tttgatccaa gcgctggcga agtcgcgggt cttgctgccc 5040
tggaaatttt ctctgcccag gtgagcgagg aattcgcggc ggtcttcgct cgtccagcca 5100
cgtgatcgca gcgcgagctc gggatgggtg tcgaacagat cagcggaaaa tttccaggcc 5160
ggtgtgtcaa tgtctcgtga atccgctaga gtcatttttg agcgctttct cccaggtttg 5220
gactgggggt tagccgacgc cctgtgagtt accgctcacg gggcgttcaa catttttcag 5280
gtattcgtgc agcttatcgc ttcttgccgc ctgtgcgctt tttcgacgcg cgacgctgct 5340
gccgattcgg tgcaggtggt ggcggcgctg acacgtcctg ggcggccacg gccacacgaa 5400
acgcggcatt tacgatgttt gtcatgcctg cgggcaccgc gccacgatcg cggataattc 5460
tcgctgccgc ttccagctct gtgacgacca tggccaaaat ttcgctcggg ggacgcactt 5520
ccagcgccat ttgcgaccta gccgcctcca gctcctcggc gtggcgtttg ttggcgcgct 5580
cgcggctggc tgcggcacga cacgcatctg agcaatattt tgcgcgccgt cctcgcgggt 5640
caggccgggg aggaatcagg ccaccgcagt aggcgcaact gattcgatcc tccactactg 5700
tgcgtcctcc tggcgctgcc gagcacgcag ctcgtcagcc agctcctcaa gatccgccac 5760
gagagtttct aggtcgctcg cggcactggc ccagtctcgt gatgctggcg cgtccgtcgt 5820
atcgagagct cggaaaaatc cgatcaccgt ttttaaatcg acggcagcat cgagcgcgtc 5880
ggactccagc gcgacatcag agagatccat agctgatgat tcgggccaat tttggtactt 5940
cgtcgtgaag gtcatgacac cattataacg aacgttcgtt aaagtttttg gcggaaaatc 6000
acgcggcacg aaaattttca cgaagcggga ctttgcgcag ctcaggggtg ctaaaaattt 6060
tgtatcgcac ttgatttttc cgaaagacag attatctgca aacggtgtgt cgtatttctg 6120
gcttggtttt taaaaaatct ggaatcgaaa atttgcgggg cgaccgagaa gttttttaca 6180
aaaggcaaaa actttttcgg gatcgacaga aataaaacga tcgacggtac gcaacaaaaa 6240
agcgtcagga tcgccgtaga gcgattgaag accgtcaacc aaaggggaag cctccaatcg 6300
acgcgacgcg cgctctacgg cgatcctgac gcagattttt agctatctgt cgcagcgccc 6360
tcagggacaa gccacccgca caacgtcgcg agggcgatca gcgacgccgc agggggatcc 6420
t 6421
<210> 103
<211> 336
<212> DNA
<213> 人工序列
<220>
<223> SEQ ID No. 67, 68, cc2, 70 combined with XbaI recognition site on
5'-end, SalI recognition site on 3'-end
<400> 103
ctgtctctag actcgagcgg gacggtcgaa ccagcttcaa gcgaccggat gagtatgtta 60
cagtagatag cgagcgggag accgctcgac cttagttctc ctgttgcggg ggagttcatg 120
ggatccaggt acccagggcg aggcttatcc attgcactcc ggatgtgctg acccctggga 180
gctcttgcca tgtgtatgtg ggcgaggaag gaggtctgag gaggtcactg cccacatact 240
ctgatgatcc ttcgggatca ttcatggcaa gctagcatag cataaaataa cgccccacct 300
tcttaacggg aggtggggcg ttatttttac gtcgac 336
<210> 104
<211> 86
<212> DNA
<213> 人工序列
<220>
<223> F30 with corn in its insertion site1 and "TTCG" in insertion
site2
<400> 104
ttgccatgtg tatgtgggcg aggaaggagg tctgaggagg tcactgccca catactctga 60
tgatccttcg ggatcattca tggcaa 86
<210> 105
<211> 6427
<212> DNA
<213> 人工序列
<220>
<223> pJC1-PF1-U1A-F30::corn-TF1
<400> 105
ctagactcga gcgggacggt cgaaccagct tcaagcgacc ggatgagtat gttacagtag 60
atagcgagcg ggagaccgct cgaccttagt tctcctgttg cgggggagtt catgggatcc 120
aggtacccag ggcgaggctt atccattgca ctccggatgt gctgacccct gggagctctt 180
gccatgtgta tgtgggcgag gaaggaggtc tgaggaggtc actgcccaca tactctgatg 240
atccttcggg atcattcatg gcaagctagc atagcataaa ataacgcccc accttcttaa 300
cgggaggtgg ggcgttattt ttacgtcgac ctgcagcaat ggcaacaacg ttgcgcaaac 360
tattaactgg cgaactactt actctagctt cccggcaaca attaatagac tggatggagg 420
cggataaagt tgcaggacca cttctgcgct cggcccttcc ggctggctgg tttattgctg 480
ataaatctgg agccggtgag cgtgggtctc gcggtatcat tgcagcactg gggccagatg 540
gtaagccctc ccgtatcgta gttatctaca cgacggggag tcaggcaact atggatgaac 600
gaaatagaca gatcgctgag ataggtgcct cactgattaa gcattggtaa ctgtcagacc 660
aagtttactc atatatactt tagattgatt taaaacttca tttttaattt aaaaggatct 720
aggtgaagat cctttttgat aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc 780
actgagcgtc agacccctta ataagatgat cttcttgaga tcgttttggt ctgcgcgtaa 840
tctcttgctc tgaaaacgaa aaaaccgcct tgcagggcgg tttttcgaag gttctctgag 900
ctaccaactc tttgaaccga ggtaactggc ttggaggagc gcagtcacca aaacttgtcc 960
tttcagttta gccttaaccg gcgcatgact tcaagactaa ctcctctaaa tcaattacca 1020
gtggctgctg ccagtggtgc ttttgcatgt ctttccgggt tggactcaag acgatagtta 1080
ccggataagg cgcagcggtc ggactgaacg gggggttcgt gcatacagtc cagcttggag 1140
cgaactgcct acccggaact gagtgtcagg cgtggaatga gacaaacgcg gccataacag 1200
cggaatgaca ccggtaaacc gaaaggcagg aacaggagag cgcacgaggg agccgccagg 1260
gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc caccactgat ttgagcgtca 1320
gatttcgtga tgcttgtcag gggggcggag cctatggaaa aacggctttg ccgcggccct 1380
ctcacttccc tgttaagtat cttcctggca tcttccagga aatctccgcc ccgttcgtaa 1440
gccatttccg ctcgccgcag tcgaacgacc gagcgtagcg agtcagtgag cgaggaagcg 1500
gaatatatcc tgtatcacat attctgctga cgcaccggtg cagccttttt tctcctgcca 1560
catgaagcac ttcactgaca ccctcatcag tgccaacata gtaagccagt atacactccg 1620
ctagcgctga ggtctgcctc gtgaagaagg tgttgctgac tcataccagg cctgaatcgc 1680
cccatcatcc agccagaaag tgagggagcc acggttgatg agagctttgt tgtaggtgga 1740
ccagttggtg attttgaact tttgctttgc cacggaacgg tctgcgttgt cgggaagatg 1800
cgtgatctga tccttcaact cagcaaaagt tcgatttatt caacaaagcc acgttgtgtc 1860
tcaaaatctc tgatgttaca ttgcacaaga taaaaatata tcatcatgaa caataaaact 1920
gtctgcttac ataaacagta atacaagggg tgttatgagc catattcaac gggaaacgtc 1980
ttgctcgagg ccgcgattaa attccaacat ggatgctgat ttatatgggt ataaatgggc 2040
tcgcgataat gtcgggcaat caggtgcgac aatctatcga ttgtatggga agcccgatgc 2100
gccagagttg tttctgaaac atggcaaagg tagcgttgcc aatgatgtta cagatgagat 2160
ggtcagacta aactggctga cggaatttat gcctcttccg accatcaagc attttatccg 2220
tactcctgat gatgcatggt tactcaccac tgcgatcccc gggaaaacag cattccaggt 2280
attagaagaa tatcctgatt caggtgaaaa tattgttgat gcgctggcag tgttcctgcg 2340
ccggttgcat tcgattcctg tttgtaattg tccttttaac agcgatcgcg tatttcgtct 2400
cgctcaggcg caatcacgaa tgaataacgg tttggttgat gcgagtgatt ttgatgacga 2460
gcgtaatggc tggcctgttg aacaagtctg gaaagaaatg cataagcttt tgccattctc 2520
accggattca gtcgtcactc atggtgattt ctcacttgat aaccttattt ttgacgaggg 2580
gaaattaata ggttgtattg atgttggacg agtcggaatc gcagaccgat accaggatct 2640
tgccatccta tggaactgcc tcggtgagtt ttctccttca ttacagaaac ggctttttca 2700
aaaatatggt attgataatc ctgatatgaa taaattgcag tttcatttga tgctcgatga 2760
gtttttctaa tcagaattgg ttaattggtt gtaacactgg cagagcatta cgctgacttg 2820
acgggacggc ggctttgttg aataaatcga acttttgctg agttgaagga tcagatcacg 2880
catcttcccg acaacgcaga ccgttccgtg gcaaagcaaa agttcaaaat caccaactgg 2940
tccacctaca acaaagctct catcaaccgt ggctccctca ctttctggct ggatgatggg 3000
gcgattcagg cctggtatga gtcagcaaca ccttcttcac gaggcagacc tcagcgctca 3060
aagatgcagg ggtaaaagct aaccgcatct ttaccgacaa ggcatccggc agttcaacag 3120
atcgggaagg gctggatttg ctgaggatga aggtggagga aggtgatgtc attctggtga 3180
agaagctcga ccgtcttggc cgcgacaccg ccgacatgat ccaactgata aaagagtttg 3240
atgctcaggg tgtagcggtt cggtttattg acgacgggat cagtaccgac ggtgatatgg 3300
ggcaaatggt ggtcaccatc ctgtcggctg tggcacaggc tgaacgccgg aggatcaagt 3360
cggtcaagcc aagcgcaacc agcggcaccg ccgcgagcaa cgtcgcaagg gcgatcaggg 3420
gacgattttt gcgaagaatt tccacggtaa gaatccaatc tctcgaattt agggtgaaag 3480
aagcttggca taggggtgtg cacgaactcg gtggaggaaa tttccgcggg gcaaggcttc 3540
gcgaagcgga gtcgcggcag tggctttgaa gatctttggg agcagtcctt gtgcgcttac 3600
gaggtgagcc ggtggggaac cgttatctgc ctatggtgtg agccccccta gagagcttca 3660
agagcaatca gcccgaccta gaaaggaggc caagagagag acccctacgg ggggaaccgt 3720
tttctgccta cgagatggca catttactgg gaagctttac ggcgtcctcg tggaagttca 3780
atgcccgcag acttaagtgc tctattcacg gtctgacgtg acacgctaaa ttcagacata 3840
gcttcattga ttgtcggcca cgagccagtc tctccctcaa cagtcataaa ccaacctgca 3900
atggtcaagc gatttccttt agctttccta gcttgtcgtt gactggactt agctagtttt 3960
tctcgctgtg ctcgggcgta ctcactgttt gggtctttcc agcgttctgc ggccttttta 4020
ccgccacgtc ttcccatagt ggccagagct tttcgccctc ggctgctctg cgtctctgtc 4080
tgacgagcag ggacgactgg ctggccttta gcgacgtagc cgcgcacacg tcgcgccatc 4140
gtctggcggt cacgcatcgg cggcagatca ggctcacggc cgtctgctcc gaccgcctga 4200
gcgacggtgt aggcacgctc gtaggcgtcg atgatcttgg tgtcttttag gcgctcacca 4260
gccgctttta actggtatcc cacagtcaaa gcgtggcgaa aagccgtctc atcacgggcg 4320
gcacgccctg gagcagtcca gaggacacgg acgccgtcga tcagctctcc agacgcttca 4380
gcggcgctcg gcaggcttgc ttcaagcgtg gcaagtgctt ttgcttccgc agtggctttt 4440
cttgccgctt cgatacgtgc ccgtccgcta gaaaactcct gctcatagcg ttttttaggt 4500
ttttctgtgc ctgagatcat gcgagcaacc tccataagat cagctaggcg atccacgcga 4560
ttgtgctggg catgccagcg gtacgcggtg ggatcgtcgg agacgtgcag tggccaccgg 4620
ctcagcctat gtgaaaaagc ctggtcagcg ccgaaaacgc gggtcatttc ctcggtcgtt 4680
gcagccagca ggcgcatatt cgggctgctc atgcctgctg cggcatacac cggatcaatg 4740
agccagatga gctggcattt cccgctcagt ggattcacgc cgatccaagc tggcgctttt 4800
tccaggcgtg cccagcgctc caaaatcgcg tagacctcgg ggtttacgtg ctcgattttc 4860
ccgccggcct ggtggctcgg cacatcaatg tccaggacaa gcacggctgc gtgctgcgcg 4920
tgcgtcagag caacatactg gcaccgggca agcgattttg aaccaactcg gtataacttc 4980
ggctgtgttt ctcccgtgtc cgggtctttg atccaagcgc tggcgaagtc gcgggtcttg 5040
ctgccctgga aattttctct gcccaggtga gcgaggaatt cgcggcggtc ttcgctcgtc 5100
cagccacgtg atcgcagcgc gagctcggga tgggtgtcga acagatcagc ggaaaatttc 5160
caggccggtg tgtcaatgtc tcgtgaatcc gctagagtca tttttgagcg ctttctccca 5220
ggtttggact gggggttagc cgacgccctg tgagttaccg ctcacggggc gttcaacatt 5280
tttcaggtat tcgtgcagct tatcgcttct tgccgcctgt gcgctttttc gacgcgcgac 5340
gctgctgccg attcggtgca ggtggtggcg gcgctgacac gtcctgggcg gccacggcca 5400
cacgaaacgc ggcatttacg atgtttgtca tgcctgcggg caccgcgcca cgatcgcgga 5460
taattctcgc tgccgcttcc agctctgtga cgaccatggc caaaatttcg ctcgggggac 5520
gcacttccag cgccatttgc gacctagccg cctccagctc ctcggcgtgg cgtttgttgg 5580
cgcgctcgcg gctggctgcg gcacgacacg catctgagca atattttgcg cgccgtcctc 5640
gcgggtcagg ccggggagga atcaggccac cgcagtaggc gcaactgatt cgatcctcca 5700
ctactgtgcg tcctcctggc gctgccgagc acgcagctcg tcagccagct cctcaagatc 5760
cgccacgaga gtttctaggt cgctcgcggc actggcccag tctcgtgatg ctggcgcgtc 5820
cgtcgtatcg agagctcgga aaaatccgat caccgttttt aaatcgacgg cagcatcgag 5880
cgcgtcggac tccagcgcga catcagagag atccatagct gatgattcgg gccaattttg 5940
gtacttcgtc gtgaaggtca tgacaccatt ataacgaacg ttcgttaaag tttttggcgg 6000
aaaatcacgc ggcacgaaaa ttttcacgaa gcgggacttt gcgcagctca ggggtgctaa 6060
aaattttgta tcgcacttga tttttccgaa agacagatta tctgcaaacg gtgtgtcgta 6120
tttctggctt ggtttttaaa aaatctggaa tcgaaaattt gcggggcgac cgagaagttt 6180
tttacaaaag gcaaaaactt tttcgggatc gacagaaata aaacgatcga cggtacgcaa 6240
caaaaaagcg tcaggatcgc cgtagagcga ttgaagaccg tcaaccaaag gggaagcctc 6300
caatcgacgc gacgcgcgct ctacggcgat cctgacgcag atttttagct atctgtcgca 6360
gcgccctcag ggacaagcca cccgcacaac gtcgcgaggg cgatcagcga cgccgcaggg 6420
ggatcct 6427
<210> 106
<211> 1115
<212> DNA
<213> 人工序列
<220>
<223> dsRNA_PT7-alpha-tubulin-F30::broccoli
<400> 106
taatacgact cactataggg cgaatggaga acatcgcaca ggacttcggt aaaaagtgcc 60
gattgggctt cgccatctac ccggctccgc aggtttccac tgccgttgtc gaaccataca 120
actcggtttt gacgacacat gccaccctcg aacacgctga ctgcgtattc atgatggata 180
atgaggcgat ctatcagatc tgtcgtcgga atcttggagt tgaacgaccg gcgtatcaaa 240
atctcaatcg actgattagc caggccgttt cggcgataac cgcttctcta cgtttttccg 300
gagcgttgaa tgttgacctc aacgaatttc agacgaatct cgtcccctac ccgcgaatcc 360
atttcccgct cgtcacttat gctccgatta tttcggctga gaaggctcat cacgagcaac 420
ataacgtact ggaatacgta ttgccatgtg tatgtgggag acggtcgggt ccagatattc 480
gtatctgtcg agtagagtgt gggctcccac atactctgat gatccttcgg gatcattcat 540
ggcaagtcga cctagcataa ccccttgggg cctctaaacg ggtcttgagg ggttttttgc 600
tgaaagtatc atccacatat gagtactcta atacgactca ctatagggcg attccagtac 660
gttatgttgc tcgtgatgag ccttctcagc cgaaataatc ggagcataag tgacgagcgg 720
gaaatggatt cgcgggtagg ggacgagatt cgtctgaaat tcgttgaggt caacattcaa 780
cgctccggaa aaacgtagag aagcggttat cgccgaaacg gcctggctaa tcagtcgatt 840
gagattttga tacgccggtc gttcaactcc aagattccga cgacagatct gatagatcgc 900
ctcattatcc atcatgaata cgcagtcagc gtgttcgagg gtggcatgtg tcgtcaaaac 960
cgagttgtat ggttcgacaa cggcagtgga aacctgcgga gccgggtaga tggcgaagcc 1020
caatcggcac tttttaccga agtcctgtgc gatgttctcc atatagcata aaataacgcc 1080
ccaccttctt aacgggaggt ggggcgttat tttta 1115
<210> 107
<211> 6108
<212> DNA
<213> 人工序列
<220>
<223> pJC1
<400> 107
ccgagaagtt ttttacaaaa ggcaaaaact ttttcgggat cgacagaaat aaaacgatcg 60
acggtacgca acaaaaaagc gtcaggatcg ccgtagagcg attgaagacc gtcaaccaaa 120
ggggaagcct ccaatcgacg cgacgcgcgc tctacggcga tcctgacgca gatttttagc 180
tatctgtcgc agcgccctca gggacaagcc acccgcacaa cgtcgcgagg gcgatcagcg 240
acgccgcagg gggatcctct agagtcgacc tgcagcaatg gcaacaacgt tgcgcaaact 300
attaactggc gaactactta ctctagcttc ccggcaacaa ttaatagact ggatggaggc 360
ggataaagtt gcaggaccac ttctgcgctc ggcccttccg gctggctggt ttattgctga 420
taaatctgga gccggtgagc gtgggtctcg cggtatcatt gcagcactgg ggccagatgg 480
taagccctcc cgtatcgtag ttatctacac gacggggagt caggcaacta tggatgaacg 540
aaatagacag atcgctgaga taggtgcctc actgattaag cattggtaac tgtcagacca 600
agtttactca tatatacttt agattgattt aaaacttcat ttttaattta aaaggatcta 660
ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca 720
ctgagcgtca gaccccttaa taagatgatc ttcttgagat cgttttggtc tgcgcgtaat 780
ctcttgctct gaaaacgaaa aaaccgcctt gcagggcggt ttttcgaagg ttctctgagc 840
taccaactct ttgaaccgag gtaactggct tggaggagcg cagtcaccaa aacttgtcct 900
ttcagtttag ccttaaccgg cgcatgactt caagactaac tcctctaaat caattaccag 960
tggctgctgc cagtggtgct tttgcatgtc tttccgggtt ggactcaaga cgatagttac 1020
cggataaggc gcagcggtcg gactgaacgg ggggttcgtg catacagtcc agcttggagc 1080
gaactgccta cccggaactg agtgtcaggc gtggaatgag acaaacgcgg ccataacagc 1140
ggaatgacac cggtaaaccg aaaggcagga acaggagagc gcacgaggga gccgccaggg 1200
ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc accactgatt tgagcgtcag 1260
atttcgtgat gcttgtcagg ggggcggagc ctatggaaaa acggctttgc cgcggccctc 1320
tcacttccct gttaagtatc ttcctggcat cttccaggaa atctccgccc cgttcgtaag 1380
ccatttccgc tcgccgcagt cgaacgaccg agcgtagcga gtcagtgagc gaggaagcgg 1440
aatatatcct gtatcacata ttctgctgac gcaccggtgc agcctttttt ctcctgccac 1500
atgaagcact tcactgacac cctcatcagt gccaacatag taagccagta tacactccgc 1560
tagcgctgag gtctgcctcg tgaagaaggt gttgctgact cataccaggc ctgaatcgcc 1620
ccatcatcca gccagaaagt gagggagcca cggttgatga gagctttgtt gtaggtggac 1680
cagttggtga ttttgaactt ttgctttgcc acggaacggt ctgcgttgtc gggaagatgc 1740
gtgatctgat ccttcaactc agcaaaagtt cgatttattc aacaaagcca cgttgtgtct 1800
caaaatctct gatgttacat tgcacaagat aaaaatatat catcatgaac aataaaactg 1860
tctgcttaca taaacagtaa tacaaggggt gttatgagcc atattcaacg ggaaacgtct 1920
tgctcgaggc cgcgattaaa ttccaacatg gatgctgatt tatatgggta taaatgggct 1980
cgcgataatg tcgggcaatc aggtgcgaca atctatcgat tgtatgggaa gcccgatgcg 2040
ccagagttgt ttctgaaaca tggcaaaggt agcgttgcca atgatgttac agatgagatg 2100
gtcagactaa actggctgac ggaatttatg cctcttccga ccatcaagca ttttatccgt 2160
actcctgatg atgcatggtt actcaccact gcgatccccg ggaaaacagc attccaggta 2220
ttagaagaat atcctgattc aggtgaaaat attgttgatg cgctggcagt gttcctgcgc 2280
cggttgcatt cgattcctgt ttgtaattgt ccttttaaca gcgatcgcgt atttcgtctc 2340
gctcaggcgc aatcacgaat gaataacggt ttggttgatg cgagtgattt tgatgacgag 2400
cgtaatggct ggcctgttga acaagtctgg aaagaaatgc ataagctttt gccattctca 2460
ccggattcag tcgtcactca tggtgatttc tcacttgata accttatttt tgacgagggg 2520
aaattaatag gttgtattga tgttggacga gtcggaatcg cagaccgata ccaggatctt 2580
gccatcctat ggaactgcct cggtgagttt tctccttcat tacagaaacg gctttttcaa 2640
aaatatggta ttgataatcc tgatatgaat aaattgcagt ttcatttgat gctcgatgag 2700
tttttctaat cagaattggt taattggttg taacactggc agagcattac gctgacttga 2760
cgggacggcg gctttgttga ataaatcgaa cttttgctga gttgaaggat cagatcacgc 2820
atcttcccga caacgcagac cgttccgtgg caaagcaaaa gttcaaaatc accaactggt 2880
ccacctacaa caaagctctc atcaaccgtg gctccctcac tttctggctg gatgatgggg 2940
cgattcaggc ctggtatgag tcagcaacac cttcttcacg aggcagacct cagcgctcaa 3000
agatgcaggg gtaaaagcta accgcatctt taccgacaag gcatccggca gttcaacaga 3060
tcgggaaggg ctggatttgc tgaggatgaa ggtggaggaa ggtgatgtca ttctggtgaa 3120
gaagctcgac cgtcttggcc gcgacaccgc cgacatgatc caactgataa aagagtttga 3180
tgctcagggt gtagcggttc ggtttattga cgacgggatc agtaccgacg gtgatatggg 3240
gcaaatggtg gtcaccatcc tgtcggctgt ggcacaggct gaacgccgga ggatcaagtc 3300
ggtcaagcca agcgcaacca gcggcaccgc cgcgagcaac gtcgcaaggg cgatcagggg 3360
acgatttttg cgaagaattt ccacggtaag aatccaatct ctcgaattta gggtgaaaga 3420
agcttggcat aggggtgtgc acgaactcgg tggaggaaat ttccgcgggg caaggcttcg 3480
cgaagcggag tcgcggcagt ggctttgaag atctttggga gcagtccttg tgcgcttacg 3540
aggtgagccg gtggggaacc gttatctgcc tatggtgtga gcccccctag agagcttcaa 3600
gagcaatcag cccgacctag aaaggaggcc aagagagaga cccctacggg gggaaccgtt 3660
ttctgcctac gagatggcac atttactggg aagctttacg gcgtcctcgt ggaagttcaa 3720
tgcccgcaga cttaagtgct ctattcacgg tctgacgtga cacgctaaat tcagacatag 3780
cttcattgat tgtcggccac gagccagtct ctccctcaac agtcataaac caacctgcaa 3840
tggtcaagcg atttccttta gctttcctag cttgtcgttg actggactta gctagttttt 3900
ctcgctgtgc tcgggcgtac tcactgtttg ggtctttcca gcgttctgcg gcctttttac 3960
cgccacgtct tcccatagtg gccagagctt ttcgccctcg gctgctctgc gtctctgtct 4020
gacgagcagg gacgactggc tggcctttag cgacgtagcc gcgcacacgt cgcgccatcg 4080
tctggcggtc acgcatcggc ggcagatcag gctcacggcc gtctgctccg accgcctgag 4140
cgacggtgta ggcacgctcg taggcgtcga tgatcttggt gtcttttagg cgctcaccag 4200
ccgcttttaa ctggtatccc acagtcaaag cgtggcgaaa agccgtctca tcacgggcgg 4260
cacgccctgg agcagtccag aggacacgga cgccgtcgat cagctctcca gacgcttcag 4320
cggcgctcgg caggcttgct tcaagcgtgg caagtgcttt tgcttccgca gtggcttttc 4380
ttgccgcttc gatacgtgcc cgtccgctag aaaactcctg ctcatagcgt tttttaggtt 4440
tttctgtgcc tgagatcatg cgagcaacct ccataagatc agctaggcga tccacgcgat 4500
tgtgctgggc atgccagcgg tacgcggtgg gatcgtcgga gacgtgcagt ggccaccggc 4560
tcagcctatg tgaaaaagcc tggtcagcgc cgaaaacgcg ggtcatttcc tcggtcgttg 4620
cagccagcag gcgcatattc gggctgctca tgcctgctgc ggcatacacc ggatcaatga 4680
gccagatgag ctggcatttc ccgctcagtg gattcacgcc gatccaagct ggcgcttttt 4740
ccaggcgtgc ccagcgctcc aaaatcgcgt agacctcggg gtttacgtgc tcgattttcc 4800
cgccggcctg gtggctcggc acatcaatgt ccaggacaag cacggctgcg tgctgcgcgt 4860
gcgtcagagc aacatactgg caccgggcaa gcgattttga accaactcgg tataacttcg 4920
gctgtgtttc tcccgtgtcc gggtctttga tccaagcgct ggcgaagtcg cgggtcttgc 4980
tgccctggaa attttctctg cccaggtgag cgaggaattc gcggcggtct tcgctcgtcc 5040
agccacgtga tcgcagcgcg agctcgggat gggtgtcgaa cagatcagcg gaaaatttcc 5100
aggccggtgt gtcaatgtct cgtgaatccg ctagagtcat ttttgagcgc tttctcccag 5160
gtttggactg ggggttagcc gacgccctgt gagttaccgc tcacggggcg ttcaacattt 5220
ttcaggtatt cgtgcagctt atcgcttctt gccgcctgtg cgctttttcg acgcgcgacg 5280
ctgctgccga ttcggtgcag gtggtggcgg cgctgacacg tcctgggcgg ccacggccac 5340
acgaaacgcg gcatttacga tgtttgtcat gcctgcgggc accgcgccac gatcgcggat 5400
aattctcgct gccgcttcca gctctgtgac gaccatggcc aaaatttcgc tcgggggacg 5460
cacttccagc gccatttgcg acctagccgc ctccagctcc tcggcgtggc gtttgttggc 5520
gcgctcgcgg ctggctgcgg cacgacacgc atctgagcaa tattttgcgc gccgtcctcg 5580
cgggtcaggc cggggaggaa tcaggccacc gcagtaggcg caactgattc gatcctccac 5640
tactgtgcgt cctcctggcg ctgccgagca cgcagctcgt cagccagctc ctcaagatcc 5700
gccacgagag tttctaggtc gctcgcggca ctggcccagt ctcgtgatgc tggcgcgtcc 5760
gtcgtatcga gagctcggaa aaatccgatc accgttttta aatcgacggc agcatcgagc 5820
gcgtcggact ccagcgcgac atcagagaga tccatagctg atgattcggg ccaattttgg 5880
tacttcgtcg tgaaggtcat gacaccatta taacgaacgt tcgttaaagt ttttggcgga 5940
aaatcacgcg gcacgaaaat tttcacgaag cgggactttg cgcagctcag gggtgctaaa 6000
aattttgtat cgcacttgat ttttccgaaa gacagattat ctgcaaacgg tgtgtcgtat 6060
ttctggcttg gtttttaaaa aatctggaat cgaaaatttg cggggcga 6108
<210> 108
<211> 7236
<212> DNA
<213> 人工序列
<220>
<223> pJC1_dsRNA_PT7-alpha-tubulin-F30::broccoli
<400> 108
aacgtcttgc tcgaggccgc gattaaattc caacatggat gctgatttat atgggtataa 60
atgggctcgc gataatgtcg ggcaatcagg tgcgacaatc tatcgattgt atgggaagcc 120
cgatgcgcca gagttgtttc tgaaacatgg caaaggtagc gttgccaatg atgttacaga 180
tgagatggtc agactaaact ggctgacgga atttatgcct cttccgacca tcaagcattt 240
tatccgtact cctgatgatg catggttact caccactgcg atccccggga aaacagcatt 300
ccaggtatta gaagaatatc ctgattcagg tgaaaatatt gttgatgcgc tggcagtgtt 360
cctgcgccgg ttgcattcga ttcctgtttg taattgtcct tttaacagcg atcgcgtatt 420
tcgtctcgct caggcgcaat cacgaatgaa taacggtttg gttgatgcga gtgattttga 480
tgacgagcgt aatggctggc ctgttgaaca agtctggaaa gaaatgcata agcttttgcc 540
attctcaccg gattcagtcg tcactcatgg tgatttctca cttgataacc ttatttttga 600
cgaggggaaa ttaataggtt gtattgatgt tggacgagtc ggaatcgcag accgatacca 660
ggatcttgcc atcctatgga actgcctcgg tgagttttct ccttcattac agaaacggct 720
ttttcaaaaa tatggtattg ataatcctga tatgaataaa ttgcagtttc atttgatgct 780
cgatgagttt ttctaatcag aattggttaa ttggttgtaa cactggcaga gcattacgct 840
gacttgacgg gacggcggct ttgttgaata aatcgaactt ttgctgagtt gaaggatcag 900
atcacgcatc ttcccgacaa cgcagaccgt tccgtggcaa agcaaaagtt caaaatcacc 960
aactggtcca cctacaacaa agctctcatc aaccgtggct ccctcacttt ctggctggat 1020
gatggggcga ttcaggcctg gtatgagtca gcaacacctt cttcacgagg cagacctcag 1080
cgctcaaaga tgcaggggta aaagctaacc gcatctttac cgacaaggca tccggcagtt 1140
caacagatcg ggaagggctg gatttgctga ggatgaaggt ggaggaaggt gatgtcattc 1200
tggtgaagaa gctcgaccgt cttggccgcg acaccgccga catgatccaa ctgataaaag 1260
agtttgatgc tcagggtgta gcggttcggt ttattgacga cgggatcagt accgacggtg 1320
atatggggca aatggtggtc accatcctgt cggctgtggc acaggctgaa cgccggagga 1380
tcaagtcggt caagccaagc gcaaccagcg gcaccgccgc gagcaacgtc gcaagggcga 1440
tcaggggacg atttttgcga agaatttcca cggtaagaat ccaatctctc gaatttaggg 1500
tgaaagaagc ttggcatagg ggtgtgcacg aactcggtgg aggaaatttc cgcggggcaa 1560
ggcttcgcga agcggagtcg cggcagtggc tttgaagatc tttgggagca gtccttgtgc 1620
gcttacgagg tgagccggtg gggaaccgtt atctgcctat ggtgtgagcc cccctagaga 1680
gcttcaagag caatcagccc gacctagaaa ggaggccaag agagagaccc ctacgggggg 1740
aaccgttttc tgcctacgag atggcacatt tactgggaag ctttacggcg tcctcgtgga 1800
agttcaatgc ccgcagactt aagtgctcta ttcacggtct gacgtgacac gctaaattca 1860
gacatagctt cattgattgt cggccacgag ccagtctctc cctcaacagt cataaaccaa 1920
cctgcaatgg tcaagcgatt tcctttagct ttcctagctt gtcgttgact ggacttagct 1980
agtttttctc gctgtgctcg ggcgtactca ctgtttgggt ctttccagcg ttctgcggcc 2040
tttttaccgc cacgtcttcc catagtggcc agagcttttc gccctcggct gctctgcgtc 2100
tctgtctgac gagcagggac gactggctgg cctttagcga cgtagccgcg cacacgtcgc 2160
gccatcgtct ggcggtcacg catcggcggc agatcaggct cacggccgtc tgctccgacc 2220
gcctgagcga cggtgtaggc acgctcgtag gcgtcgatga tcttggtgtc ttttaggcgc 2280
tcaccagccg cttttaactg gtatcccaca gtcaaagcgt ggcgaaaagc cgtctcatca 2340
cgggcggcac gccctggagc agtccagagg acacggacgc cgtcgatcag ctctccagac 2400
gcttcagcgg cgctcggcag gcttgcttca agcgtggcaa gtgcttttgc ttccgcagtg 2460
gcttttcttg ccgcttcgat acgtgcccgt ccgctagaaa actcctgctc atagcgtttt 2520
ttaggttttt ctgtgcctga gatcatgcga gcaacctcca taagatcagc taggcgatcc 2580
acgcgattgt gctgggcatg ccagcggtac gcggtgggat cgtcggagac gtgcagtggc 2640
caccggctca gcctatgtga aaaagcctgg tcagcgccga aaacgcgggt catttcctcg 2700
gtcgttgcag ccagcaggcg catattcggg ctgctcatgc ctgctgcggc atacaccgga 2760
tcaatgagcc agatgagctg gcatttcccg ctcagtggat tcacgccgat ccaagctggc 2820
gctttttcca ggcgtgccca gcgctccaaa atcgcgtaga cctcggggtt tacgtgctcg 2880
attttcccgc cggcctggtg gctcggcaca tcaatgtcca ggacaagcac ggctgcgtgc 2940
tgcgcgtgcg tcagagcaac atactggcac cgggcaagcg attttgaacc aactcggtat 3000
aacttcggct gtgtttctcc cgtgtccggg tctttgatcc aagcgctggc gaagtcgcgg 3060
gtcttgctgc cctggaaatt ttctctgccc aggtgagcga ggaattcgcg gcggtcttcg 3120
ctcgtccagc cacgtgatcg cagcgcgagc tcgggatggg tgtcgaacag atcagcggaa 3180
aatttccagg ccggtgtgtc aatgtctcgt gaatccgcta gagtcatttt tgagcgcttt 3240
ctcccaggtt tggactgggg gttagccgac gccctgtgag ttaccgctca cggggcgttc 3300
aacatttttc aggtattcgt gcagcttatc gcttcttgcc gcctgtgcgc tttttcgacg 3360
cgcgacgctg ctgccgattc ggtgcaggtg gtggcggcgc tgacacgtcc tgggcggcca 3420
cggccacacg aaacgcggca tttacgatgt ttgtcatgcc tgcgggcacc gcgccacgat 3480
cgcggataat tctcgctgcc gcttccagct ctgtgacgac catggccaaa atttcgctcg 3540
ggggacgcac ttccagcgcc atttgcgacc tagccgcctc cagctcctcg gcgtggcgtt 3600
tgttggcgcg ctcgcggctg gctgcggcac gacacgcatc tgagcaatat tttgcgcgcc 3660
gtcctcgcgg gtcaggccgg ggaggaatca ggccaccgca gtaggcgcaa ctgattcgat 3720
cctccactac tgtgcgtcct cctggcgctg ccgagcacgc agctcgtcag ccagctcctc 3780
aagatccgcc acgagagttt ctaggtcgct cgcggcactg gcccagtctc gtgatgctgg 3840
cgcgtccgtc gtatcgagag ctcggaaaaa tccgatcacc gtttttaaat cgacggcagc 3900
atcgagcgcg tcggactcca gcgcgacatc agagagatcc atagctgatg attcgggcca 3960
attttggtac ttcgtcgtga aggtcatgac accattataa cgaacgttcg ttaaagtttt 4020
tggcggaaaa tcacgcggca cgaaaatttt cacgaagcgg gactttgcgc agctcagggg 4080
tgctaaaaat tttgtatcgc acttgatttt tccgaaagac agattatctg caaacggtgt 4140
gtcgtatttc tggcttggtt tttaaaaaat ctggaatcga aaatttgcgg ggcgaccgag 4200
aagtttttta caaaaggcaa aaactttttc gggatcgaca gaaataaaac gatcgacggt 4260
acgcaacaaa aaagcgtcag gatcgccgta gagcgattga agaccgtcaa ccaaagggga 4320
agcctccaat cgacgcgacg cgcgctctac ggcgatcctg acgcagattt ttagctatct 4380
gtcgcagcgc cctcagggac aagccacccg cacaacgtcg cgagggcgat cagcgacgcc 4440
gcagggggat ccggatcctc tagactaata cgactcacta tagggcgaat ggagaacatc 4500
gcacaggact tcggtaaaaa gtgccgattg ggcttcgcca tctacccggc tccgcaggtt 4560
tccactgccg ttgtcgaacc atacaactcg gttttgacga cacatgccac cctcgaacac 4620
gctgactgcg tattcatgat ggataatgag gcgatctatc agatctgtcg tcggaatctt 4680
ggagttgaac gaccggcgta tcaaaatctc aatcgactga ttagccaggc cgtttcggcg 4740
ataaccgctt ctctacgttt ttccggagcg ttgaatgttg acctcaacga atttcagacg 4800
aatctcgtcc cctacccgcg aatccatttc ccgctcgtca cttatgctcc gattatttcg 4860
gctgagaagg ctcatcacga gcaacataac gtactggaat acgtattgcc atgtgtatgt 4920
gggagacggt cgggtccaga tattcgtatc tgtcgagtag agtgtgggct cccacatact 4980
ctgatgatcc ttcgggatca ttcatggcaa gtcgacctag cataacccct tggggcctct 5040
aaacgggtct tgaggggttt tttgctgaaa gtatcatcca catatgagta ctctaatacg 5100
actcactata gggcgattcc agtacgttat gttgctcgtg atgagccttc tcagccgaaa 5160
taatcggagc ataagtgacg agcgggaaat ggattcgcgg gtaggggacg agattcgtct 5220
gaaattcgtt gaggtcaaca ttcaacgctc cggaaaaacg tagagaagcg gttatcgccg 5280
aaacggcctg gctaatcagt cgattgagat tttgatacgc cggtcgttca actccaagat 5340
tccgacgaca gatctgatag atcgcctcat tatccatcat gaatacgcag tcagcgtgtt 5400
cgagggtggc atgtgtcgtc aaaaccgagt tgtatggttc gacaacggca gtggaaacct 5460
gcggagccgg gtagatggcg aagcccaatc ggcacttttt accgaagtcc tgtgcgatgt 5520
tctccatata gcataaaata acgccccacc ttcttaacgg gaggtggggc gttattttta 5580
ggtaccgata tcgatatcca atggcaacaa cgttgcgcaa actattaact ggcgaactac 5640
ttactctagc ttcccggcaa caattaatag actggatgga ggcggataaa gttgcaggac 5700
cacttctgcg ctcggccctt ccggctggct ggtttattgc tgataaatct ggagccggtg 5760
agcgtgggtc tcgcggtatc attgcagcac tggggccaga tggtaagccc tcccgtatcg 5820
tagttatcta cacgacgggg agtcaggcaa ctatggatga acgaaataga cagatcgctg 5880
agataggtgc ctcactgatt aagcattggt aactgtcaga ccaagtttac tcatatatac 5940
tttagattga tttaaaactt catttttaat ttaaaaggat ctaggtgaag atcctttttg 6000
ataatctcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagacccct 6060
taataagatg atcttcttga gatcgttttg gtctgcgcgt aatctcttgc tctgaaaacg 6120
aaaaaaccgc cttgcagggc ggtttttcga aggttctctg agctaccaac tctttgaacc 6180
gaggtaactg gcttggagga gcgcagtcac caaaacttgt cctttcagtt tagccttaac 6240
cggcgcatga cttcaagact aactcctcta aatcaattac cagtggctgc tgccagtggt 6300
gcttttgcat gtctttccgg gttggactca agacgatagt taccggataa ggcgcagcgg 6360
tcggactgaa cggggggttc gtgcatacag tccagcttgg agcgaactgc ctacccggaa 6420
ctgagtgtca ggcgtggaat gagacaaacg cggccataac agcggaatga caccggtaaa 6480
ccgaaaggca ggaacaggag agcgcacgag ggagccgcca gggggaaacg cctggtatct 6540
ttatagtcct gtcgggtttc gccaccactg atttgagcgt cagatttcgt gatgcttgtc 6600
aggggggcgg agcctatgga aaaacggctt tgccgcggcc ctctcacttc cctgttaagt 6660
atcttcctgg catcttccag gaaatctccg ccccgttcgt aagccatttc cgctcgccgc 6720
agtcgaacga ccgagcgtag cgagtcagtg agcgaggaag cggaatatat cctgtatcac 6780
atattctgct gacgcaccgg tgcagccttt tttctcctgc cacatgaagc acttcactga 6840
caccctcatc agtgccaaca tagtaagcca gtatacactc cgctagcgct gaggtctgcc 6900
tcgtgaagaa ggtgttgctg actcatacca ggcctgaatc gccccatcat ccagccagaa 6960
agtgagggag ccacggttga tgagagcttt gttgtaggtg gaccagttgg tgattttgaa 7020
cttttgcttt gccacggaac ggtctgcgtt gtcgggaaga tgcgtgatct gatccttcaa 7080
ctcagcaaaa gttcgattta ttcaacaaag ccacgttgtg tctcaaaatc tctgatgtta 7140
cattgcacaa gataaaaata tatcatcatg aacaataaaa ctgtctgctt acataaacag 7200
taatacaagg ggtgttatga gccatattca acggga 7236
<210> 109
<211> 20
<212> DNA
<213> 人工序列
<220>
<223> egfp_for
<400> 109
atggtgagca agggcgagga 20
<210> 110
<211> 411
<212> DNA
<213> 人工序列
<220>
<223> alpha-tubulin RNA from Varroa destructor
<400> 110
atggagaaca tcgcacagga cttcggtaaa aagtgccgat tgggcttcgc catctacccg 60
gctccgcagg tttccactgc cgttgtcgaa ccatacaact cggttttgac gacacatgcc 120
accctcgaac acgctgactg cgtattcatg atggataatg aggcgatcta tcagatctgt 180
cgtcggaatc ttggagttga acgaccggcg tatcaaaatc tcaatcgact gattagccag 240
gccgtttcgg cgataaccgc ttctctacgt ttttccggag cgttgaatgt tgacctcaac 300
gaatttcaga cgaatctcgt cccctacccg cgaatccatt tcccgctcgt cacttatgct 360
ccgattattt cggctgagaa ggctcatcac gagcaacata acgtactgga a 411
<210> 111
<211> 411
<212> DNA
<213> 人工序列
<220>
<223> alpha-tubulin antisense RNA
<400> 111
ttccagtacg ttatgttgct cgtgatgagc cttctcagcc gaaataatcg gagcataagt 60
gacgagcggg aaatggattc gcgggtaggg gacgagattc gtctgaaatt cgttgaggtc 120
aacattcaac gctccggaaa aacgtagaga agcggttatc gccgaaacgg cctggctaat 180
cagtcgattg agattttgat acgccggtcg ttcaactcca agattccgac gacagatctg 240
atagatcgcc tcattatcca tcatgaatac gcagtcagcg tgttcgaggg tggcatgtgt 300
cgtcaaaacc gagttgtatg gttcgacaac ggcagtggaa acctgcggag ccgggtagat 360
ggcgaagccc aatcggcact ttttaccgaa gtcctgtgcg atgttctcca t 411
<210> 112
<211> 807
<212> DNA
<213> 人工序列
<220>
<223> 5'UTR-egfp (modified from Aequorea victoria)
<400> 112
ggagacccaa gctggctagc gtttaaactt aagcttcgaa ttctgcagtc gacggtaccg 60
cgggcccggg atccaccggt cgccaccatg gtgagcaagg gcgaggagct gttcaccggg 120
gtggtgccca tcctggtcga gctggacggc gacgtaaacg gccacaagtt cagcgtgtcc 180
ggcgagggcg agggcgatgc cacctacggc aagctgaccc tgaagttcat ctgcaccacc 240
ggcaagctgc ccgtgccctg gcccaccctc gtgaccaccc tgacctacgg cgtgcagtgc 300
ttcagccgct accccgacca catgaagcag cacgacttct tcaagtccgc catgcccgaa 360
ggctacgtcc aggagcgcac catcttcttc aaggacgacg gcaactacaa gacccgcgcc 420
gaggtgaagt tcgagggcga caccctggtg aaccgcatcg agctgaaggg catcgacttc 480
aaggaggacg gcaacatcct ggggcacaag ctggagtaca actacaacag ccacaacgtc 540
tatatcatgg ccgacaagca gaagaacggc atcaaggtga acttcaagat ccgccacaac 600
atcgaggacg gcagcgtgca gctcgccgac cactaccagc agaacacccc catcggcgac 660
ggccccgtgc tgctgcccga caaccactac ctgagcaccc agtccgccct gagcaaagac 720
cccaacgaga agcgcgatca catggtcctg ctggagttcg tgaccgccgc cgggatcact 780
ctcggcatgg acgagctgta caagtaa 807
<210> 113
<211> 21
<212> DNA
<213> 人工序列
<220>
<223> broccoli_rev
<400> 113
ttgccatgaa tgatcccgaa g 21
<210> 114
<211> 27
<212> DNA
<213> 人工序列
<220>
<223> luc2_for
<400> 114
atggaagatg ccaaaaacat taagaag 27
<210> 115
<211> 11662
<212> DNA
<213> 人工序列
<220>
<223> pK18mobsacB-DE3
<220>
<221> misc_feature
<222> (2314)..(2314)
<223> n is a, c, g, or t
<400> 115
ctagcttcac gctgccgcaa gcactcaggg cgcaagggct gctaaaggaa gcggaacacg 60
tagaaagcca gtccgcagaa acggtgctga ccccggatga atgtcagcta ctgggctatc 120
tggacaaggg aaaacgcaag cgcaaagaga aagcaggtag cttgcagtgg gcttacatgg 180
cgatagctag actgggcggt tttatggaca gcaagcgaac cggaattgcc agctggggcg 240
ccctctggta aggttgggaa gccctgcaaa gtaaactgga tggctttctt gccgccaagg 300
atctgatggc gcaggggatc aagatctgat caagagacag gatgaggatc gtttcgcatg 360
attgaacaag atggattgca cgcaggttct ccggccgctt gggtggagag gctattcggc 420
tatgactggg cacaacagac aatcggctgc tctgatgccg ccgtgttccg gctgtcagcg 480
caggggcgcc cggttctttt tgtcaagacc gacctgtccg gtgccctgaa tgaactccaa 540
gacgaggcag cgcggctatc gtggctggcc acgacgggcg ttccttgcgc agctgtgctc 600
gacgttgtca ctgaagcggg aagggactgg ctgctattgg gcgaagtgcc ggggcaggat 660
ctcctgtcat ctcaccttgc tcctgccgag aaagtatcca tcatggctga tgcaatgcgg 720
cggctgcata cgcttgatcc ggctacctgc ccattcgacc accaagcgaa acatcgcatc 780
gagcgagcac gtactcggat ggaagccggt cttgtcgatc aggatgatct ggacgaagag 840
catcaggggc tcgcgccagc cgaactgttc gccaggctca aggcgcggat gcccgacggc 900
gaggatctcg tcgtgaccca tggcgatgcc tgcttgccga atatcatggt ggaaaatggc 960
cgcttttctg gattcatcga ctgtggccgg ctgggtgtgg cggaccgcta tcaggacata 1020
gcgttggcta cccgtgatat tgctgaagag cttggcggcg aatgggctga ccgcttcctc 1080
gtgctttacg gtatcgccgc tcccgattcg cagcgcatcg ccttctatcg ccttcttgac 1140
gagttcttct gagcgggact ctggggttcg ctagaggatc gatccttttt aacccatcac 1200
atatacctgc cgttcactat tatttagtga aatgagatat tatgatattt tctgaattgt 1260
gattaaaaag gcaactttat gcccatgcaa cagaaactat aaaaaataca gagaatgaaa 1320
agaaacagat agatttttta gttctttagg cccgtagtct gcaaatcctt ttatgatttt 1380
ctatcaaaca aaagaggaaa atagaccagt tgcaatccaa acgagagtct aatagaatga 1440
ggtcgaaaag taaatcgcgc gggtttgtta ctgataaagc aggcaagacc taaaatgtgt 1500
aaagggcaaa gtgtatactt tggcgtcacc ccttacatat tttaggtctt tttttattgt 1560
gcgtaactaa cttgccatct tcaaacagga gggctggaag aagcagaccg ctaacacagt 1620
acataaaaaa ggagacatga acgatgaaca tcaaaaagtt tgcaaaacaa gcaacagtat 1680
taacctttac taccgcactg ctggcaggag gcgcaactca agcgtttgcg aaagaaacga 1740
accaaaagcc atataaggaa acatacggca tttcccatat tacacgccat gatatgctgc 1800
aaatccctga acagcaaaaa aatgaaaaat atcaagtttc tgaatttgat tcgtccacaa 1860
ttaaaaatat ctcttctgca aaaggcctgg acgtttggga cagctggcca ttacaaaacg 1920
ctgacggcac tgtcgcaaac tatcacggct accacatcgt ctttgcatta gccggagatc 1980
ctaaaaatgc ggatgacaca tcgatttaca tgttctatca aaaagtcggc gaaacttcta 2040
ttgacagctg gaaaaacgct ggccgcgtct ttaaagacag cgacaaattc gatgcaaatg 2100
attctatcct aaaagaccaa acacaagaat ggtcaggttc agccacattt acatctgacg 2160
gaaaaatccg tttattctac actgatttct ccggtaaaca ttacggcaaa caaacactga 2220
caactgcaca agttaacgta tcagcatcag acagctcttt gaacatcaac ggtgtagagg 2280
attataaatc aatctttgac ggtgacggaa aaancgtatc aaaatgtaca gcagttcatc 2340
gatgaaggca actacagctc aggcgacaac catacgctga gagatcctca ctacgtagaa 2400
gataaaggcc acaaatactt agtatttgaa gcaaacactg gaactgaaga tggctaccaa 2460
ggcgaagaat ctttatttaa caaagcatac tatggcaaaa gcacatcatt cttccgtcaa 2520
gaaagtcaaa aacttctgca aagcgataaa aaacgcacgg ctgagttagc aaacggcgct 2580
ctcggtatga ttgagctaaa cgatgattac acactgaaaa aagtgatgaa accgctgatt 2640
gcatctaaca cagtaacaga tgaaattgaa cgcgcgaacg tctttaaaat gaacggcaaa 2700
tggtacctgt tcactgactc ccgcggatca aaaatgacga ttgacggcat tacgtctaac 2760
gatatttaca tgcttggtta tgtttctaat tctttaactg gcccatacaa gccgctgaac 2820
aaaactggcc ttgtgttaaa aatggatctt gatcctaacg atgtaacctt tacttactca 2880
cacttcgctg tacctcaagc gaaaggaaac aatgtcgtga ttacaagcta tatgacaaac 2940
agaggattct acgcagacaa acaatcaacg tttgcgccga gcttcctgct gaacatcaaa 3000
ggcaagaaaa catctgttgt caaagacagc atccttgaac aaggacaatt aacagttaac 3060
aaataaaaac gcaaaagaaa atgccgatgg gtaccgagcg aaatgaccga ccaagcgacg 3120
cccaacctgc catcacgaga tttcgattcc accgccgcct tctatgaaag gttgggcttc 3180
ggaatcgttt tccgggacgc cctcgcggac gtgctcatag tccacgacgc ccgtgatttt 3240
gtagccctgg ccgacggcca gcaggtaggc cgacaggctc atgccggccg ccgccgcctt 3300
ttcctcaatc gctcttcgtt cgtctggaag gcagtacacc ttgataggtg ggctgccctt 3360
cctggttggc ttggtttcat cagccatccg cttgccctca tctgttacgc cggcggtagc 3420
cggccagcct cgcagagcag gattcccgtt gagcaccgcc aggtgcgaat aagggacagt 3480
gaagaaggaa cacccgctcg cgggtgggcc tacttcacct atcctgcccc gctgacgccg 3540
ttggatacac caaggaaagt ctacacgaac cctttggcaa aatcctgtat atcgtgcgaa 3600
aaaggatgga tataccgaaa aaatcgctat aatgaccccg aagcagggtt atgcagcgga 3660
aaagcgctgc ttccctgctg ttttgtggaa tatctaccga ctggaaacag gcaaatgcag 3720
gaaattactg aactgagggg acaggcgaga gacgatgcca aagagctcct gaaaatctcg 3780
ataactcaaa aaatacgccc ggtagtgatc ttatttcatt atggtgaaag ttggaacctc 3840
ttacgtgccg atcaacgtct cattttcgcc aaaagttggc ccagggcttc ccggtatcaa 3900
cagggacacc aggatttatt tattctgcga agtgatcttc cgtcacaggt atttattcgg 3960
cgcaaagtgc gtcgggtgat gctgccaact tactgattta gtgtatgatg gtgtttttga 4020
ggtgctccag tggcttctgt ttctatcagc tcctgaaaat ctcgataact caaaaaatac 4080
gcccggtagt gatcttattt cattatggtg aaagttggaa cctcttacgt gccgatcaac 4140
gtctcatttt cgccaaaagt tggcccaggg cttcccggta tcaacaggga caccaggatt 4200
tatttattct gcgaagtgat cttccgtcac aggtatttat tcggcgcaaa gtgcgtcggg 4260
tgatgctgcc aacttactga tttagtgtat gatggtgttt ttgaggtgct ccagtggctt 4320
ctgtttctat cagggctgga tgatcctcca gcgcggggat ctcatgctgg agttcttcgc 4380
ccaccccaaa aggatctagg tgaagatcct ttttgataat ctcatgacca aaatccctta 4440
acgtgagttt tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg 4500
agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc 4560
ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag 4620
cagagcgcag ataccaaata ctgtccttct agtgtagccg tagttaggcc accacttcaa 4680
gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc 4740
cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc 4800
gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta 4860
caccgaactg agatacctac agcgtgagca ttgagaaagc gccacgcttc ccgaagggag 4920
aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct 4980
tccaggggga aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga 5040
gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc 5100
ggccttttta cggttcctgg ccttttgctg gccttttgct cacatgttct ttcctgcgtt 5160
atcccctgat tctgtggata accgtattac cgcctttgag tgagctgata ccgctcgccg 5220
cagccgaacg accgagcgca gcgagtcagt gagcgaggaa gcggaagagc gcccaatacg 5280
caaaccgcct ctccccgcgc gttggccgat tcattaatgc agctggcacg acaggtttcc 5340
cgactggaaa gcgggcagtg agcgcaacgc aattaatgtg agttagctca ctcattaggc 5400
accccaggct ttacacttta tgcttccggc tcgtatgttg tgtggaattg tgagcggata 5460
acaatttcac acaggaaaca gctatgacca tgattacgaa ttggctccgt tgttcacgtc 5520
tactacgacg gcgacgagaa cgacaaggaa accttcctca tcggtacccg tgctggcgct 5580
tccgagaacc cagatcttga gacctactct gagcagtccc cactcggcgc tgcaattctc 5640
ggagctcagg aaggcgacac ccgtcagtac accgctccaa atggttccgt tatctccgta 5700
actgttgttt ctgcagaacc atacaactca gcaaaagccg cgacactccg cggcaaaaac 5760
taaccaagga tttaaaagtc ttcaaaatga caactctttc acgtaagttc ttcgtttctg 5820
ctaccacagc cctggcggca gtcgcactgg ttgcgtgttc ccctaatgag attgattctg 5880
aactgaaggt gccaacggca actggcgttt ctttaccttc gaagaacgtt tccgcgacct 5940
caactgctac tacagatgag gatgcgcctg gctacattga ttgcgtagcc gcaccaactc 6000
agcaacctgc tgaaatctca ctaaactgtg caatggatat tgatcggctc acggatattt 6060
cttggagcga atgggatact gattccgcaa ctggaaccgg tacccgcatc gtaaccgctg 6120
caaatggtca agagaccgaa accgaagata ttgaggtgaa gctttccttc cccaccgagt 6180
cttcccaagg cctagtgttc actcaggtca ccgtcgatgg acaggttctc ttcctctaat 6240
cctccataat tagagagcgt aaggccccta cttcctgttt taggaaatag gggccttttg 6300
ttgtcttctc ctggaggcta tttaagaagt ttaaattgtg tccatgagtt cgctcgagaa 6360
ctgcgcaact cgtgaaaggt aggcggatcc agatcccgga caccatcgaa tggcgcaaaa 6420
cctttcgcgg tatggcatga tagcgcccgg aagagagtca attcagggtg gtgaatgtga 6480
aaccagtaac gttatacgat gtcgcagagt atgccggtgt ctcttatcag accgtttccc 6540
gcgtggtgaa ccaggccagc cacgtttctg cgaaaacgcg ggaaaaagtg gaagcggcga 6600
tggcggagct gaattacatt cccaaccgcg tggcacaaca actggcgggc aaacagtcgt 6660
tgctgattgg cgttgccacc tccagtctgg ccctgcacgc gccgtcgcaa attgtcgcgg 6720
cgattaaatc tcgcgccgat caactgggtg ccagcgtggt ggtgtcgatg gtagaacgaa 6780
gcggcgtcga agcctgtaaa gcggcggtgc acaatcttct cgcgcaacgc gtcagtgggc 6840
tgatcattaa ctatccgctg gatgaccagg atgccattgc tgtggaagct gcctgcacta 6900
atgttccggc gttatttctt gatgtctctg accagacacc catcaacagt attattttct 6960
cccatgaaga cggtacgcga ctgggcgtgg agcatctggt cgcattgggt caccagcaaa 7020
tcgcgctgtt agcgggccca ttaagttctg tctcggcgcg tctgcgtctg gctggctggc 7080
ataaatatct cactcgcaat caaattcagc cgatagcgga acgggaaggc gactggagtg 7140
ccatgtccgg ttttcaacaa accatgcaaa tgctgaatga gggcatcgtt cccactgcga 7200
tgctggttgc caacgatcag atggcgctgg gcgcaatgcg cgccattacc gagtccgggc 7260
tgcgcgttgg tgcggatatc tcggtagtgg gatacgacga taccgaagac agctcatgtt 7320
atatcccgcc gttaaccacc atcaaacagg attttcgcct gctggggcaa accagcgtgg 7380
accgcttgct gcaactctct cagggccagg cggtgaaggg caatcagctg ttgcccgtct 7440
cactggtgaa aagaaaaacc accctggcgc ccaatacgca aaccgcctct ccccgcgcgt 7500
tggccgattc attaatgcag ctggcacgac aggtttcccg actggaaagc gggcagtgag 7560
cgcaacgcaa ttaatgtaag ttagctcact cattaggcac cccaggcttt acactttatg 7620
cttccggctc gtataatgtg tggaattgtg agcggataac aatttcacac aggaaacagc 7680
tatgaccatg attacggatt cactggccgt cgttttacaa cgtcgtgact gggaaaaccc 7740
tggcgttacc caacttaatc gccttgcagc acatccccct ttcgccagct ggcgtaatag 7800
cgaagaggcc cgcaccgatc gcccttccca acagttgcgc agcctgaatg gcgaatggcg 7860
ctttgcctgg tttccggcac cagaagcggt gccggaaagc tggctggagt gcgatcttcc 7920
tgaggccgat actgtcgtcg tcccctcaaa ctggcagatg cacggttacg atgcgcccat 7980
ctacaccaac gtgacctatc ccattacggt caatccgccg tttgttccca cggagaatcc 8040
gacgggttgt tactcgctca catttaatgt tgatgaaagc tggctacagg aaggccagac 8100
gcgaattatt tttgatggcg tcgggatctg atccggattt actaactgga agaggcacta 8160
aatgaacacg attaacatcg ctaagaacga cttctctgac atcgaactgg ctgctatccc 8220
gttcaacact ctggctgacc attacggtga gcgtttagct cgcgaacagt tggcccttga 8280
gcatgagtct tacgagatgg gtgaagcacg cttccgcaag atgtttgagc gtcaacttaa 8340
agctggtgag gttgcggata acgctgccgc caagcctctc atcactaccc tactccctaa 8400
gatgattgca cgcatcaacg actggtttga ggaagtgaaa gctaagcgcg gcaagcgccc 8460
gacagccttc cagttcctgc aagaaatcaa gccggaagcc gtagcgtaca tcaccattaa 8520
gaccactctg gcttgcctaa ccagtgctga caatacaacc gttcaggctg tagcaagcgc 8580
aatcggtcgg gccattgagg acgaggctcg cttcggtcgt atccgtgacc ttgaagctaa 8640
gcacttcaag aaaaacgttg aggaacaact caacaagcgc gtagggcacg tctacaagaa 8700
agcatttatg caagttgtcg aggctgacat gctctctaag ggtctactcg gtggcgaggc 8760
gtggtcttcg tggcataagg aagactctat tcatgtagga gtacgctgca tcgagatgct 8820
cattgagtca accggaatgg ttagcttaca ccgccaaaat gctggcgtag taggtcaaga 8880
ctctgagact atcgaactcg cacctgaata cgctgaggct atcgcaaccc gtgcaggtgc 8940
gctggctggc atctctccga tgttccaacc ttgcgtagtt cctcctaagc cgtggactgg 9000
cattactggt ggtggctatt gggctaacgg tcgtcgtcct ctggcgctgg tgcgtactca 9060
cagtaagaaa gcactgatgc gctacgaaga cgtttacatg cctgaggtgt acaaagcgat 9120
taacattgcg caaaacaccg catggaaaat caacaagaaa gtcctagcgg tcgccaacgt 9180
aatcaccaag tggaagcatt gtccggtcga ggacatccct gcgattgagc gtgaagaact 9240
cccgatgaaa ccggaagaca tcgacatgaa tcctgaggct ctcaccgcgt ggaaacgtgc 9300
tgccgctgct gtgtaccgca aggacaaggc tcgcaagtct cgccgtatca gccttgagtt 9360
catgcttgag caagccaata agtttgctaa ccataaggcc atctggttcc cttacaacat 9420
ggactggcgc ggtcgtgttt acgctgtgtc aatgttcaac ccgcaaggta acgatatgac 9480
caaaggactg cttacgctgg cgaaaggtaa accaatcggt aaggaaggtt actactggct 9540
gaaaatccac ggtgcaaact gtgcgggtgt cgataaggtt ccgttccctg agcgcatcaa 9600
gttcattgag gaaaaccacg agaacatcat ggcttgcgct aagtctccac tggagaacac 9660
ttggtgggct gagcaagatt ctccgttctg cttccttgcg ttctgctttg agtacgctgg 9720
ggtacagcac cacggcctga gctataactg ctcccttccg ctggcgtttg acgggtcttg 9780
ctctggcatc cagcacttct ccgcgatgct ccgagatgag gtaggtggtc gcgcggttaa 9840
cttgcttcct agtgaaaccg ttcaggacat ctacgggatt gttgctaaga aagtcaacga 9900
gattctacaa gcagacgcaa tcaatgggac cgataacgaa gtagttaccg tgaccgatga 9960
gaacactggt gaaatctctg agaaagtcaa gctgggcact aaggcactgg ctggtcaatg 10020
gctggcttac ggtgttactc gcagtgtgac taagcgttca gtcatgacgc tggcttacgg 10080
gtccaaagag ttcggcttcc gtcaacaagt gctggaagat accattcagc cagctattga 10140
ttccggcaag ggtctgatgt tcactcagcc gaatcaggct gctggataca tggctaagct 10200
gatttgggaa tctgtgagcg tgacggtggt agctgcggtt gaagcaatga actggcttaa 10260
gtctgctgct aagctgctgg ctgctgaggt caaagataag aagactggag agattcttcg 10320
caagcgttgc gctgtgcatt gggtaactcc tgatggtttc cctgtgtggc aggaatacaa 10380
gaagcctatt cagacgcgct tgaacctgat gttcctcggt cagttccgct tacagcctac 10440
cattaacacc aacaaagata gcgagattga tgcacacaaa caggagtctg gtatcgctcc 10500
taactttgta cacagccaag acggtagcca ccttcgtaag actgtagtgt gggcacacga 10560
gaagtacgga atcgaatctt ttgcactgat tcacgactcc ttcggtacca ttccggctga 10620
cgctgcgaac ctgttcaaag cagtgcgcga aactatggtt gacacatatg agtcttgtga 10680
tgtactggct gatttctacg accagttcgc tgaccagttg cacgagtctc aattggacaa 10740
aatgccagca cttccggcta aaggtaactt gaacctccgt gacatcttag agtcggactt 10800
cgcgttcgcg taacgaattc gcgtatggca atgacagttt gagacggcca caggcgattc 10860
tgagaagcca ttttctttgg gcgccgtggc agtttttatt gggtcccacc gccgaactgc 10920
atattcgaac caaggagcct caaaaatcga gctcgctttg gtctcaaacg cacatttatc 10980
gcgcgttgaa gtgtgcgttt gagaccaaag agccctccac aacgcacgtc tttggtttgg 11040
atatgacagg tgcccaagaa ctcaccccgc cccatgctca cagagccccc atcagaagcc 11100
aaaagacccc ttccctgccc aagaagaaca ggatgaaggg gtcttgtgct gcgtaaacta 11160
gcggttttgg aagtagctaa gcagacgtag gatttcggtg tagagccaga ccaaggtcac 11220
tgcaagacca agcgcaacgc cccatgccat cttggaaggt gcaccttcgc ggacgaggcg 11280
gtcagctgca tcgaagtcgg agaggaagct gaatgctgcc aggccgatgc agaagaggga 11340
gaagataatc gcgatgattc caccgtcacg cagtgggctt gcgccaccag tgaacagtgc 11400
ccatacaacg ttgcccagga caagaaccag gacgccaacc atcatgccgg tgaggatgcg 11460
gttgaactta ggagtgacct tgatagcgcc agtcttgtat acaaacagca tgccaatgaa 11520
tacaccgatg gtgccaagga ctgcctggcc aatgaggcca cctgcgttgg cgttaccaac 11580
tgtgaagccg gacagcagaa gggaaattcc gccgacgaag aggccttcga atactgcgta 11640
aatcaaagtg actgccgcag at 11662
<210> 116
<211> 20
<212> DNA
<213> 人工序列
<220>
<223> DE3_for
<400> 116
tttggcgtgt ggttggttag 20
<210> 117
<211> 20
<212> DNA
<213> 人工序列
<220>
<223> DE3_rev
<400> 117
tctctgagct gctggccaac 20
<210> 118
<211> 990
<212> DNA
<213> 人工序列
<220>
<223> PT7-egfp-broccoli-TT7 (SEQ ID No. 96, 112, 69 and 97 combined)
<400> 118
taatacgact cactataggg agacccaagc tggctagcgt ttaaacttaa gcttcgaatt 60
ctgcagtcga cggtaccgcg ggcccgggat ccaccggtcg ccaccatggt gagcaagggc 120
gaggagctgt tcaccggggt ggtgcccatc ctggtcgagc tggacggcga cgtaaacggc 180
cacaagttca gcgtgtccgg cgagggcgag ggcgatgcca cctacggcaa gctgaccctg 240
aagttcatct gcaccaccgg caagctgccc gtgccctggc ccaccctcgt gaccaccctg 300
acctacggcg tgcagtgctt cagccgctac cccgaccaca tgaagcagca cgacttcttc 360
aagtccgcca tgcccgaagg ctacgtccag gagcgcacca tcttcttcaa ggacgacggc 420
aactacaaga cccgcgccga ggtgaagttc gagggcgaca ccctggtgaa ccgcatcgag 480
ctgaagggca tcgacttcaa ggaggacggc aacatcctgg ggcacaagct ggagtacaac 540
tacaacagcc acaacgtcta tatcatggcc gacaagcaga agaacggcat caaggtgaac 600
ttcaagatcc gccacaacat cgaggacggc agcgtgcagc tcgccgacca ctaccagcag 660
aacaccccca tcggcgacgg ccccgtgctg ctgcccgaca accactacct gagcacccag 720
tccgccctga gcaaagaccc caacgagaag cgcgatcaca tggtcctgct ggagttcgtg 780
accgccgccg ggatcactct cggcatggac gagctgtaca agtaatacgt attgccatgt 840
gtatgtggga gacggtcggg tccagatatt cgtatctgtc gagtagagtg tgggctccca 900
catactctga tgatccttcg ggatcattca tggcaagtcg acctagcata accccttggg 960
gcctctaaac gggtcttgag gggttttttg 990
<210> 119
<211> 1911
<212> DNA
<213> 人工序列
<220>
<223> PT7-luc2-broccoli-TT7 (SEQ ID No. 96, 82, 69 and 97 combined)
<400> 119
taatacgact cactataggg agacccaagc tggctagcgt ttaaacttaa gcttggcaat 60
ccggtactgt tggtaaagcc accatggaag atgccaaaaa cattaagaag ggcccagcgc 120
cattctaccc actcgaagac gggaccgccg gcgagcagct gcacaaagcc atgaagcgct 180
acgccctggt gcccggcacc atcgccttta ccgacgcaca tatcgaggtg gacattacct 240
acgccgagta cttcgagatg agcgttcggc tggcagaagc tatgaagcgc tatgggctga 300
atacaaacca tcggatcgtg gtgtgcagcg agaatagctt gcagttcttc atgcccgtgt 360
tgggtgccct gttcatcggt gtggctgtgg ccccagctaa cgacatctac aacgagcgcg 420
agctgctgaa cagcatgggc atcagccagc ccaccgtcgt attcgtgagc aagaaagggc 480
tgcaaaagat cctcaacgtg caaaagaagc taccgatcat acaaaagatc atcatcatgg 540
atagcaagac cgactaccag ggcttccaaa gcatgtacac cttcgtgact tcccatttgc 600
cacccggctt caacgagtac gacttcgtgc ccgagagctt cgaccgggac aaaaccatcg 660
ccctgatcat gaacagtagt ggcagtaccg gattgcccaa gggcgtagcc ctaccgcacc 720
gcaccgcttg tgtccgattc agtcatgccc gcgaccccat cttcggcaac cagatcatcc 780
ccgacaccgc tatcctcagc gtggtgccat ttcaccacgg cttcggcatg ttcaccacgc 840
tgggctactt gatctgcggc tttcgggtcg tgctcatgta ccgcttcgag gaggagctat 900
tcttgcgcag cttgcaagac tataagattc aatctgccct gctggtgccc acactattta 960
gcttcttcgc taagagcact ctcatcgaca agtacgacct aagcaacttg cacgagatcg 1020
ccagcggcgg ggcgccgctc agcaaggagg taggtgaggc cgtggccaaa cgcttccacc 1080
taccaggcat ccgccagggc tacggcctga cagaaacaac cagcgccatt ctgatcaccc 1140
ccgaagggga cgacaagcct ggcgcagtag gcaaggtggt gcccttcttc gaggctaagg 1200
tggtggactt ggacaccggt aagacactgg gtgtgaacca gcgcggcgag ctgtgcgtcc 1260
gtggccccat gatcatgagc ggctacgtta acaaccccga ggctacaaac gctctcatcg 1320
acaaggacgg ctggctgcac agcggcgaca tcgcctactg ggacgaggac gagcacttct 1380
tcatcgtgga ccggctgaag agcctgatca aatacaaggg ctaccaggta gccccagccg 1440
aactggagag catcctgctg caacacccca acatcttcga cgccggggtc gccggcctgc 1500
ccgacgacga tgccggcgag ctgcccgccg cagtcgtcgt gctggaacac ggtaaaacca 1560
tgaccgagaa ggagatcgtg gactatgtgg ccagccaggt tacaaccgcc aagaagctgc 1620
gcggtggtgt tgtgttcgtg gacgaggtgc ctaaaggact gaccggcaag ttggacgccc 1680
gcaagatccg cgagattctc attaaggcca agaagggcgg caagatcgcc gtgtaataat 1740
tctagatacg tattgccatg tgtatgtggg agacggtcgg gtccagatat tcgtatctgt 1800
cgagtagagt gtgggctccc acatactctg atgatccttc gggatcattc atggcaagtc 1860
gacctagcat aaccccttgg ggcctctaaa cgggtcttga ggggtttttt g 1911
<210> 120
<211> 7102
<212> DNA
<213> 人工序列
<220>
<223> pJC1-PT7-egfp-broccoli-TT7
<400> 120
ccgagaagtt ttttacaaaa ggcaaaaact ttttcgggat cgacagaaat aaaacgatcg 60
acggtacgca acaaaaaagc gtcaggatcg ccgtagagcg attgaagacc gtcaaccaaa 120
ggggaagcct ccaatcgacg cgacgcgcgc tctacggcga tcctgacgca gatttttagc 180
tatctgtcgc agcgccctca gggacaagcc acccgcacaa cgtcgcgagg gcgatcagcg 240
acgccgcagg gtagtactca gctgtaatac gactcactat agggagaccc aagctggcta 300
gcgtttaaac ttaagcttcg aattctgcag tcgacggtac cgcgggcccg ggatccaccg 360
gtcgccacca tggtgagcaa gggcgaggag ctgttcaccg gggtggtgcc catcctggtc 420
gagctggacg gcgacgtaaa cggccacaag ttcagcgtgt ccggcgaggg cgagggcgat 480
gccacctacg gcaagctgac cctgaagttc atctgcacca ccggcaagct gcccgtgccc 540
tggcccaccc tcgtgaccac cctgacctac ggcgtgcagt gcttcagccg ctaccccgac 600
cacatgaagc agcacgactt cttcaagtcc gccatgcccg aaggctacgt ccaggagcgc 660
accatcttct tcaaggacga cggcaactac aagacccgcg ccgaggtgaa gttcgagggc 720
gacaccctgg tgaaccgcat cgagctgaag ggcatcgact tcaaggagga cggcaacatc 780
ctggggcaca agctggagta caactacaac agccacaacg tctatatcat ggccgacaag 840
cagaagaacg gcatcaaggt gaacttcaag atccgccaca acatcgagga cggcagcgtg 900
cagctcgccg accactacca gcagaacacc cccatcggcg acggccccgt gctgctgccc 960
gacaaccact acctgagcac ccagtccgcc ctgagcaaag accccaacga gaagcgcgat 1020
cacatggtcc tgctggagtt cgtgaccgcc gccgggatca ctctcggcat ggacgagctg 1080
tacaagtaat acgtattgcc atgtgtatgt gggagacggt cgggtccaga tattcgtatc 1140
tgtcgagtag agtgtgggct cccacatact ctgatgatcc ttcgggatca ttcatggcaa 1200
gtcgacctag cataacccct tggggcctct aaacgggtct tgaggggttt tttgctgaaa 1260
gatatcatcc aatggcaaca acgttgcgca aactattaac tggcgaacta cttactctag 1320
cttcccggca acaattaata gactggatgg aggcggataa agttgcagga ccacttctgc 1380
gctcggccct tccggctggc tggtttattg ctgataaatc tggagccggt gagcgtgggt 1440
ctcgcggtat cattgcagca ctggggccag atggtaagcc ctcccgtatc gtagttatct 1500
acacgacggg gagtcaggca actatggatg aacgaaatag acagatcgct gagataggtg 1560
cctcactgat taagcattgg taactgtcag accaagttta ctcatatata ctttagattg 1620
atttaaaact tcatttttaa tttaaaagga tctaggtgaa gatccttttt gataatctca 1680
tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc ttaataagat 1740
gatcttcttg agatcgtttt ggtctgcgcg taatctcttg ctctgaaaac gaaaaaaccg 1800
ccttgcaggg cggtttttcg aaggttctct gagctaccaa ctctttgaac cgaggtaact 1860
ggcttggagg agcgcagtca ccaaaacttg tcctttcagt ttagccttaa ccggcgcatg 1920
acttcaagac taactcctct aaatcaatta ccagtggctg ctgccagtgg tgcttttgca 1980
tgtctttccg ggttggactc aagacgatag ttaccggata aggcgcagcg gtcggactga 2040
acggggggtt cgtgcataca gtccagcttg gagcgaactg cctacccgga actgagtgtc 2100
aggcgtggaa tgagacaaac gcggccataa cagcggaatg acaccggtaa accgaaaggc 2160
aggaacagga gagcgcacga gggagccgcc agggggaaac gcctggtatc tttatagtcc 2220
tgtcgggttt cgccaccact gatttgagcg tcagatttcg tgatgcttgt caggggggcg 2280
gagcctatgg aaaaacggct ttgccgcggc cctctcactt ccctgttaag tatcttcctg 2340
gcatcttcca ggaaatctcc gccccgttcg taagccattt ccgctcgccg cagtcgaacg 2400
accgagcgta gcgagtcagt gagcgaggaa gcggaatata tcctgtatca catattctgc 2460
tgacgcaccg gtgcagcctt ttttctcctg ccacatgaag cacttcactg acaccctcat 2520
cagtgccaac atagtaagcc agtatacact ccgctagcgc tgaggtctgc ctcgtgaaga 2580
aggtgttgct gactcatacc aggcctgaat cgccccatca tccagccaga aagtgaggga 2640
gccacggttg atgagagctt tgttgtaggt ggaccagttg gtgattttga acttttgctt 2700
tgccacggaa cggtctgcgt tgtcgggaag atgcgtgatc tgatccttca actcagcaaa 2760
agttcgattt attcaacaaa gccacgttgt gtctcaaaat ctctgatgtt acattgcaca 2820
agataaaaat atatcatcat gaacaataaa actgtctgct tacataaaca gtaatacaag 2880
gggtgttatg agccatattc aacgggaaac gtcttgctcg aggccgcgat taaattccaa 2940
catggatgct gatttatatg ggtataaatg ggctcgcgat aatgtcgggc aatcaggtgc 3000
gacaatctat cgattgtatg ggaagcccga tgcgccagag ttgtttctga aacatggcaa 3060
aggtagcgtt gccaatgatg ttacagatga gatggtcaga ctaaactggc tgacggaatt 3120
tatgcctctt ccgaccatca agcattttat ccgtactcct gatgatgcat ggttactcac 3180
cactgcgatc cccgggaaaa cagcattcca ggtattagaa gaatatcctg attcaggtga 3240
aaatattgtt gatgcgctgg cagtgttcct gcgccggttg cattcgattc ctgtttgtaa 3300
ttgtcctttt aacagcgatc gcgtatttcg tctcgctcag gcgcaatcac gaatgaataa 3360
cggtttggtt gatgcgagtg attttgatga cgagcgtaat ggctggcctg ttgaacaagt 3420
ctggaaagaa atgcataagc ttttgccatt ctcaccggat tcagtcgtca ctcatggtga 3480
tttctcactt gataacctta tttttgacga ggggaaatta ataggttgta ttgatgttgg 3540
acgagtcgga atcgcagacc gataccagga tcttgccatc ctatggaact gcctcggtga 3600
gttttctcct tcattacaga aacggctttt tcaaaaatat ggtattgata atcctgatat 3660
gaataaattg cagtttcatt tgatgctcga tgagtttttc taatcagaat tggttaattg 3720
gttgtaacac tggcagagca ttacgctgac ttgacgggac ggcggctttg ttgaataaat 3780
cgaacttttg ctgagttgaa ggatcagatc acgcatcttc ccgacaacgc agaccgttcc 3840
gtggcaaagc aaaagttcaa aatcaccaac tggtccacct acaacaaagc tctcatcaac 3900
cgtggctccc tcactttctg gctggatgat ggggcgattc aggcctggta tgagtcagca 3960
acaccttctt cacgaggcag acctcagcgc tcaaagatgc aggggtaaaa gctaaccgca 4020
tctttaccga caaggcatcc ggcagttcaa cagatcggga agggctggat ttgctgagga 4080
tgaaggtgga ggaaggtgat gtcattctgg tgaagaagct cgaccgtctt ggccgcgaca 4140
ccgccgacat gatccaactg ataaaagagt ttgatgctca gggtgtagcg gttcggttta 4200
ttgacgacgg gatcagtacc gacggtgata tggggcaaat ggtggtcacc atcctgtcgg 4260
ctgtggcaca ggctgaacgc cggaggatca agtcggtcaa gccaagcgca accagcggca 4320
ccgccgcgag caacgtcgca agggcgatca ggggacgatt tttgcgaaga atttccacgg 4380
taagaatcca atctctcgaa tttagggtga aagaagcttg gcataggggt gtgcacgaac 4440
tcggtggagg aaatttccgc ggggcaaggc ttcgcgaagc ggagtcgcgg cagtggcttt 4500
gaagatcttt gggagcagtc cttgtgcgct tacgaggtga gccggtgggg aaccgttatc 4560
tgcctatggt gtgagccccc ctagagagct tcaagagcaa tcagcccgac ctagaaagga 4620
ggccaagaga gagaccccta cggggggaac cgttttctgc ctacgagatg gcacatttac 4680
tgggaagctt tacggcgtcc tcgtggaagt tcaatgcccg cagacttaag tgctctattc 4740
acggtctgac gtgacacgct aaattcagac atagcttcat tgattgtcgg ccacgagcca 4800
gtctctccct caacagtcat aaaccaacct gcaatggtca agcgatttcc tttagctttc 4860
ctagcttgtc gttgactgga cttagctagt ttttctcgct gtgctcgggc gtactcactg 4920
tttgggtctt tccagcgttc tgcggccttt ttaccgccac gtcttcccat agtggccaga 4980
gcttttcgcc ctcggctgct ctgcgtctct gtctgacgag cagggacgac tggctggcct 5040
ttagcgacgt agccgcgcac acgtcgcgcc atcgtctggc ggtcacgcat cggcggcaga 5100
tcaggctcac ggccgtctgc tccgaccgcc tgagcgacgg tgtaggcacg ctcgtaggcg 5160
tcgatgatct tggtgtcttt taggcgctca ccagccgctt ttaactggta tcccacagtc 5220
aaagcgtggc gaaaagccgt ctcatcacgg gcggcacgcc ctggagcagt ccagaggaca 5280
cggacgccgt cgatcagctc tccagacgct tcagcggcgc tcggcaggct tgcttcaagc 5340
gtggcaagtg cttttgcttc cgcagtggct tttcttgccg cttcgatacg tgcccgtccg 5400
ctagaaaact cctgctcata gcgtttttta ggtttttctg tgcctgagat catgcgagca 5460
acctccataa gatcagctag gcgatccacg cgattgtgct gggcatgcca gcggtacgcg 5520
gtgggatcgt cggagacgtg cagtggccac cggctcagcc tatgtgaaaa agcctggtca 5580
gcgccgaaaa cgcgggtcat ttcctcggtc gttgcagcca gcaggcgcat attcgggctg 5640
ctcatgcctg ctgcggcata caccggatca atgagccaga tgagctggca tttcccgctc 5700
agtggattca cgccgatcca agctggcgct ttttccaggc gtgcccagcg ctccaaaatc 5760
gcgtagacct cggggtttac gtgctcgatt ttcccgccgg cctggtggct cggcacatca 5820
atgtccagga caagcacggc tgcgtgctgc gcgtgcgtca gagcaacata ctggcaccgg 5880
gcaagcgatt ttgaaccaac tcggtataac ttcggctgtg tttctcccgt gtccgggtct 5940
ttgatccaag cgctggcgaa gtcgcgggtc ttgctgccct ggaaattttc tctgcccagg 6000
tgagcgagga attcgcggcg gtcttcgctc gtccagccac gtgatcgcag cgcgagctcg 6060
ggatgggtgt cgaacagatc agcggaaaat ttccaggccg gtgtgtcaat gtctcgtgaa 6120
tccgctagag tcatttttga gcgctttctc ccaggtttgg actgggggtt agccgacgcc 6180
ctgtgagtta ccgctcacgg ggcgttcaac atttttcagg tattcgtgca gcttatcgct 6240
tcttgccgcc tgtgcgcttt ttcgacgcgc gacgctgctg ccgattcggt gcaggtggtg 6300
gcggcgctga cacgtcctgg gcggccacgg ccacacgaaa cgcggcattt acgatgtttg 6360
tcatgcctgc gggcaccgcg ccacgatcgc ggataattct cgctgccgct tccagctctg 6420
tgacgaccat ggccaaaatt tcgctcgggg gacgcacttc cagcgccatt tgcgacctag 6480
ccgcctccag ctcctcggcg tggcgtttgt tggcgcgctc gcggctggct gcggcacgac 6540
acgcatctga gcaatatttt gcgcgccgtc ctcgcgggtc aggccgggga ggaatcaggc 6600
caccgcagta ggcgcaactg attcgatcct ccactactgt gcgtcctcct ggcgctgccg 6660
agcacgcagc tcgtcagcca gctcctcaag atccgccacg agagtttcta ggtcgctcgc 6720
ggcactggcc cagtctcgtg atgctggcgc gtccgtcgta tcgagagctc ggaaaaatcc 6780
gatcaccgtt tttaaatcga cggcagcatc gagcgcgtcg gactccagcg cgacatcaga 6840
gagatccata gctgatgatt cgggccaatt ttggtacttc gtcgtgaagg tcatgacacc 6900
attataacga acgttcgtta aagtttttgg cggaaaatca cgcggcacga aaattttcac 6960
gaagcgggac tttgcgcagc tcaggggtgc taaaaatttt gtatcgcact tgatttttcc 7020
gaaagacaga ttatctgcaa acggtgtgtc gtatttctgg cttggttttt aaaaaatctg 7080
gaatcgaaaa tttgcggggc ga 7102
<210> 121
<211> 8070
<212> DNA
<213> 人工序列
<220>
<223> pJC1-PT7-luc2-broccoli-TT7
<400> 121
ggtacgcaac aaaaaagcgt caggatcgcc gtagagcgat tgaagaccgt caaccaaagg 60
ggaagcctcc aatcgacgcg acgcgcgctc tacggcgatc ctgacgcaga tttttagcta 120
tctgtcgcag cgccctcagg gacaagccac ccgcacaacg tcgcgagggc gatcagcgac 180
gccgcagggg gatccagtac tcagctggct aactagagaa cccactgctt actggcttat 240
cgaaattaat acgactcact atagggagac ccaagctggc tagcgtttaa acttaagctt 300
ggcaatccgg tactgttggt aaagccacca tggaagatgc caaaaacatt aagaagggcc 360
cagcgccatt ctacccactc gaagacggga ccgccggcga gcagctgcac aaagccatga 420
agcgctacgc cctggtgccc ggcaccatcg cctttaccga cgcacatatc gaggtggaca 480
ttacctacgc cgagtacttc gagatgagcg ttcggctggc agaagctatg aagcgctatg 540
ggctgaatac aaaccatcgg atcgtggtgt gcagcgagaa tagcttgcag ttcttcatgc 600
ccgtgttggg tgccctgttc atcggtgtgg ctgtggcccc agctaacgac atctacaacg 660
agcgcgagct gctgaacagc atgggcatca gccagcccac cgtcgtattc gtgagcaaga 720
aagggctgca aaagatcctc aacgtgcaaa agaagctacc gatcatacaa aagatcatca 780
tcatggatag caagaccgac taccagggct tccaaagcat gtacaccttc gtgacttccc 840
atttgccacc cggcttcaac gagtacgact tcgtgcccga gagcttcgac cgggacaaaa 900
ccatcgccct gatcatgaac agtagtggca gtaccggatt gcccaagggc gtagccctac 960
cgcaccgcac cgcttgtgtc cgattcagtc atgcccgcga ccccatcttc ggcaaccaga 1020
tcatccccga caccgctatc ctcagcgtgg tgccatttca ccacggcttc ggcatgttca 1080
ccacgctggg ctacttgatc tgcggctttc gggtcgtgct catgtaccgc ttcgaggagg 1140
agctattctt gcgcagcttg caagactata agattcaatc tgccctgctg gtgcccacac 1200
tatttagctt cttcgctaag agcactctca tcgacaagta cgacctaagc aacttgcacg 1260
agatcgccag cggcggggcg ccgctcagca aggaggtagg tgaggccgtg gccaaacgct 1320
tccacctacc aggcatccgc cagggctacg gcctgacaga aacaaccagc gccattctga 1380
tcacccccga aggggacgac aagcctggcg cagtaggcaa ggtggtgccc ttcttcgagg 1440
ctaaggtggt ggacttggac accggtaaga cactgggtgt gaaccagcgc ggcgagctgt 1500
gcgtccgtgg ccccatgatc atgagcggct acgttaacaa ccccgaggct acaaacgctc 1560
tcatcgacaa ggacggctgg ctgcacagcg gcgacatcgc ctactgggac gaggacgagc 1620
acttcttcat cgtggaccgg ctgaagagcc tgatcaaata caagggctac caggtagccc 1680
cagccgaact ggagagcatc ctgctgcaac accccaacat cttcgacgcc ggggtcgccg 1740
gcctgcccga cgacgatgcc ggcgagctgc ccgccgcagt cgtcgtgctg gaacacggta 1800
aaaccatgac cgagaaggag atcgtggact atgtggccag ccaggttaca accgccaaga 1860
agctgcgcgg tggtgttgtg ttcgtggacg aggtgcctaa aggactgacc ggcaagttgg 1920
acgcccgcaa gatccgcgag attctcatta aggccaagaa gggcggcaag atcgccgtgt 1980
aataattcta gatacgtatt gccatgtgta tgtgggagac ggtcgggtcc agatattcgt 2040
atctgtcgag tagagtgtgg gctcccacat actctgatga tccttcggga tcattcatgg 2100
caagtcgacc tagcataacc ccttggggcc tctaaacggg tcttgagggg ttttttgctg 2160
aaagatatcg atatccaatg gcaacaacgt tgcgcaaact attaactggc gaactactta 2220
ctctagcttc ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac 2280
ttctgcgctc ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc 2340
gtgggtctcg cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag 2400
ttatctacac gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga 2460
taggtgcctc actgattaag cattggtaac tgtcagacca agtttactca tatatacttt 2520
agattgattt aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata 2580
atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccttaa 2640
taagatgatc ttcttgagat cgttttggtc tgcgcgtaat ctcttgctct gaaaacgaaa 2700
aaaccgcctt gcagggcggt ttttcgaagg ttctctgagc taccaactct ttgaaccgag 2760
gtaactggct tggaggagcg cagtcaccaa aacttgtcct ttcagtttag ccttaaccgg 2820
cgcatgactt caagactaac tcctctaaat caattaccag tggctgctgc cagtggtgct 2880
tttgcatgtc tttccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg 2940
gactgaacgg ggggttcgtg catacagtcc agcttggagc gaactgccta cccggaactg 3000
agtgtcaggc gtggaatgag acaaacgcgg ccataacagc ggaatgacac cggtaaaccg 3060
aaaggcagga acaggagagc gcacgaggga gccgccaggg ggaaacgcct ggtatcttta 3120
tagtcctgtc gggtttcgcc accactgatt tgagcgtcag atttcgtgat gcttgtcagg 3180
ggggcggagc ctatggaaaa acggctttgc cgcggccctc tcacttccct gttaagtatc 3240
ttcctggcat cttccaggaa atctccgccc cgttcgtaag ccatttccgc tcgccgcagt 3300
cgaacgaccg agcgtagcga gtcagtgagc gaggaagcgg aatatatcct gtatcacata 3360
ttctgctgac gcaccggtgc agcctttttt ctcctgccac atgaagcact tcactgacac 3420
cctcatcagt gccaacatag taagccagta tacactccgc tagcgctgag gtctgcctcg 3480
tgaagaaggt gttgctgact cataccaggc ctgaatcgcc ccatcatcca gccagaaagt 3540
gagggagcca cggttgatga gagctttgtt gtaggtggac cagttggtga ttttgaactt 3600
ttgctttgcc acggaacggt ctgcgttgtc gggaagatgc gtgatctgat ccttcaactc 3660
agcaaaagtt cgatttattc aacaaagcca cgttgtgtct caaaatctct gatgttacat 3720
tgcacaagat aaaaatatat catcatgaac aataaaactg tctgcttaca taaacagtaa 3780
tacaaggggt gttatgagcc atattcaacg ggaaacgtct tgctcgaggc cgcgattaaa 3840
ttccaacatg gatgctgatt tatatgggta taaatgggct cgcgataatg tcgggcaatc 3900
aggtgcgaca atctatcgat tgtatgggaa gcccgatgcg ccagagttgt ttctgaaaca 3960
tggcaaaggt agcgttgcca atgatgttac agatgagatg gtcagactaa actggctgac 4020
ggaatttatg cctcttccga ccatcaagca ttttatccgt actcctgatg atgcatggtt 4080
actcaccact gcgatccccg ggaaaacagc attccaggta ttagaagaat atcctgattc 4140
aggtgaaaat attgttgatg cgctggcagt gttcctgcgc cggttgcatt cgattcctgt 4200
ttgtaattgt ccttttaaca gcgatcgcgt atttcgtctc gctcaggcgc aatcacgaat 4260
gaataacggt ttggttgatg cgagtgattt tgatgacgag cgtaatggct ggcctgttga 4320
acaagtctgg aaagaaatgc ataagctttt gccattctca ccggattcag tcgtcactca 4380
tggtgatttc tcacttgata accttatttt tgacgagggg aaattaatag gttgtattga 4440
tgttggacga gtcggaatcg cagaccgata ccaggatctt gccatcctat ggaactgcct 4500
cggtgagttt tctccttcat tacagaaacg gctttttcaa aaatatggta ttgataatcc 4560
tgatatgaat aaattgcagt ttcatttgat gctcgatgag tttttctaat cagaattggt 4620
taattggttg taacactggc agagcattac gctgacttga cgggacggcg gctttgttga 4680
ataaatcgaa cttttgctga gttgaaggat cagatcacgc atcttcccga caacgcagac 4740
cgttccgtgg caaagcaaaa gttcaaaatc accaactggt ccacctacaa caaagctctc 4800
atcaaccgtg gctccctcac tttctggctg gatgatgggg cgattcaggc ctggtatgag 4860
tcagcaacac cttcttcacg aggcagacct cagcgctcaa agatgcaggg gtaaaagcta 4920
accgcatctt taccgacaag gcatccggca gttcaacaga tcgggaaggg ctggatttgc 4980
tgaggatgaa ggtggaggaa ggtgatgtca ttctggtgaa gaagctcgac cgtcttggcc 5040
gcgacaccgc cgacatgatc caactgataa aagagtttga tgctcagggt gtagcggttc 5100
ggtttattga cgacgggatc agtaccgacg gtgatatggg gcaaatggtg gtcaccatcc 5160
tgtcggctgt ggcacaggct gaacgccgga ggatcaagtc ggtcaagcca agcgcaacca 5220
gcggcaccgc cgcgagcaac gtcgcaaggg cgatcagggg acgatttttg cgaagaattt 5280
ccacggtaag aatccaatct ctcgaattta gggtgaaaga agcttggcat aggggtgtgc 5340
acgaactcgg tggaggaaat ttccgcgggg caaggcttcg cgaagcggag tcgcggcagt 5400
ggctttgaag atctttggga gcagtccttg tgcgcttacg aggtgagccg gtggggaacc 5460
gttatctgcc tatggtgtga gcccccctag agagcttcaa gagcaatcag cccgacctag 5520
aaaggaggcc aagagagaga cccctacggg gggaaccgtt ttctgcctac gagatggcac 5580
atttactggg aagctttacg gcgtcctcgt ggaagttcaa tgcccgcaga cttaagtgct 5640
ctattcacgg tctgacgtga cacgctaaat tcagacatag cttcattgat tgtcggccac 5700
gagccagtct ctccctcaac agtcataaac caacctgcaa tggtcaagcg atttccttta 5760
gctttcctag cttgtcgttg actggactta gctagttttt ctcgctgtgc tcgggcgtac 5820
tcactgtttg ggtctttcca gcgttctgcg gcctttttac cgccacgtct tcccatagtg 5880
gccagagctt ttcgccctcg gctgctctgc gtctctgtct gacgagcagg gacgactggc 5940
tggcctttag cgacgtagcc gcgcacacgt cgcgccatcg tctggcggtc acgcatcggc 6000
ggcagatcag gctcacggcc gtctgctccg accgcctgag cgacggtgta ggcacgctcg 6060
taggcgtcga tgatcttggt gtcttttagg cgctcaccag ccgcttttaa ctggtatccc 6120
acagtcaaag cgtggcgaaa agccgtctca tcacgggcgg cacgccctgg agcagtccag 6180
aggacacgga cgccgtcgat cagctctcca gacgcttcag cggcgctcgg caggcttgct 6240
tcaagcgtgg caagtgcttt tgcttccgca gtggcttttc ttgccgcttc gatacgtgcc 6300
cgtccgctag aaaactcctg ctcatagcgt tttttaggtt tttctgtgcc tgagatcatg 6360
cgagcaacct ccataagatc agctaggcga tccacgcgat tgtgctgggc atgccagcgg 6420
tacgcggtgg gatcgtcgga gacgtgcagt ggccaccggc tcagcctatg tgaaaaagcc 6480
tggtcagcgc cgaaaacgcg ggtcatttcc tcggtcgttg cagccagcag gcgcatattc 6540
gggctgctca tgcctgctgc ggcatacacc ggatcaatga gccagatgag ctggcatttc 6600
ccgctcagtg gattcacgcc gatccaagct ggcgcttttt ccaggcgtgc ccagcgctcc 6660
aaaatcgcgt agacctcggg gtttacgtgc tcgattttcc cgccggcctg gtggctcggc 6720
acatcaatgt ccaggacaag cacggctgcg tgctgcgcgt gcgtcagagc aacatactgg 6780
caccgggcaa gcgattttga accaactcgg tataacttcg gctgtgtttc tcccgtgtcc 6840
gggtctttga tccaagcgct ggcgaagtcg cgggtcttgc tgccctggaa attttctctg 6900
cccaggtgag cgaggaattc gcggcggtct tcgctcgtcc agccacgtga tcgcagcgcg 6960
agctcgggat gggtgtcgaa cagatcagcg gaaaatttcc aggccggtgt gtcaatgtct 7020
cgtgaatccg ctagagtcat ttttgagcgc tttctcccag gtttggactg ggggttagcc 7080
gacgccctgt gagttaccgc tcacggggcg ttcaacattt ttcaggtatt cgtgcagctt 7140
atcgcttctt gccgcctgtg cgctttttcg acgcgcgacg ctgctgccga ttcggtgcag 7200
gtggtggcgg cgctgacacg tcctgggcgg ccacggccac acgaaacgcg gcatttacga 7260
tgtttgtcat gcctgcgggc accgcgccac gatcgcggat aattctcgct gccgcttcca 7320
gctctgtgac gaccatggcc aaaatttcgc tcgggggacg cacttccagc gccatttgcg 7380
acctagccgc ctccagctcc tcggcgtggc gtttgttggc gcgctcgcgg ctggctgcgg 7440
cacgacacgc atctgagcaa tattttgcgc gccgtcctcg cgggtcaggc cggggaggaa 7500
tcaggccacc gcagtaggcg caactgattc gatcctccac tactgtgcgt cctcctggcg 7560
ctgccgagca cgcagctcgt cagccagctc ctcaagatcc gccacgagag tttctaggtc 7620
gctcgcggca ctggcccagt ctcgtgatgc tggcgcgtcc gtcgtatcga gagctcggaa 7680
aaatccgatc accgttttta aatcgacggc agcatcgagc gcgtcggact ccagcgcgac 7740
atcagagaga tccatagctg atgattcggg ccaattttgg tacttcgtcg tgaaggtcat 7800
gacaccatta taacgaacgt tcgttaaagt ttttggcgga aaatcacgcg gcacgaaaat 7860
tttcacgaag cgggactttg cgcagctcag gggtgctaaa aattttgtat cgcacttgat 7920
ttttccgaaa gacagattat ctgcaaacgg tgtgtcgtat ttctggcttg gtttttaaaa 7980
aatctggaat cgaaaatttg cggggcgacc gagaagtttt ttacaaaagg caaaaacttt 8040
ttcgggatcg acagaaataa aacgatcgac 8070
<210> 122
<211> 6
<212> DNA
<213> 人工序列
<220>
<223> Restriction site for verification of positive integration
<400> 122
tctaga 6

Claims (15)

1.一种用于优化细胞中感兴趣的异源RNA序列的产生的方法，包括以下步骤：
a)将能够表达感兴趣的异源RNA的载体引入多个细胞中，其中所述的RNA用RNA标签标记，所述的RNA标签包含
-能够稳定荧光团的核酸适配体，以及
-能够稳定所述的核酸适配体的支架；
b)在允许感兴趣的异源RNA表达的条件下，在培养基中培养细胞；
c)将所述的荧光团加入所述的培养基中；
d)鉴定那些显示出最高荧光强度并因此显示出感兴趣的RNA的高表达的细胞。
2.一种生产感兴趣的异源RNA的方法，包括以下步骤：
a)将能够表达感兴趣的异源RNA的载体引入多个细胞，其中所述的RNA用RNA标签标记，所述的RNA标签包含能够稳定荧光团的核酸适配体和能够稳定所述的核酸适配体的支架；
b)在允许感兴趣的异源RNA表达的条件下，在培养基中培养细胞；
c)将所述的荧光团加入所述的培养基中；
d)鉴定和分离那些显示出最高荧光强度并因此显示出感兴趣的RNA的高表达的细胞；
e)从在步骤d)中分离的细胞中去除步骤a)的第一载体；
f)将能够在没有RNA标签的情况下表达感兴趣的异源RNA的第二载体引入步骤e)获得的细胞中；
g)通过培养在步骤f)获得的细胞来产生感兴趣的RNA。
3.一种用于比较不同细胞对感兴趣的异源RNA序列的生产能力的方法，包括以下步骤：
a)将能够表达感兴趣的异源RNA的载体引入多个细胞中，其中所述的RNA用RNA标签标记，所述的RNA标签包含
-能够稳定荧光团的核酸适配体，以及
-能够稳定所述的核酸适配体的支架；
b)在允许感兴趣的RNA表达的条件下，在培养基中培养细胞；
c)将所述的荧光团加入所述的培养基中；
d)比较所述的多个细胞之间的荧光强度。
4.根据权利要求1至3中任一项所述的方法，其特征在于，所述的细胞是微生物细胞，特别是***细胞。
5.根据权利要求1至4中任一项所述的方法，其特征在于，在步骤d)中，使用流式细胞术鉴定显示最高荧光强度的细胞。
6.根据权利要求1至5中任一项所述的方法，其特征在于，所述的细胞由于培养条件，特别是温度、压力和/或培养基、染色体遗传改变或载体中的遗传改变而表现出不同水平的异源RNA的表达。
7.根据权利要求1至6中任一项所述的方法，其特征在于，所述的核酸适配体包含如SEQID NO:1至8、13至16、65或66的序列。
8.根据权利要求1至7中任一项所述的方法，其特征在于，能够稳定核酸适配体的RNA支架包含如SEQ ID NO:9至12的序列。
9.根据权利要求1至8中任一项所述的方法，其特征在于，所述的RNA标签包含如SEQ IDNO:17至64、69、77或78的序列。
10.一种携带能够表达感兴趣的异源RNA的载体的细胞，其特征在于，所述的RNA用RNA标签标记，所述的RNA标签包含
-能够稳定荧光团的核酸适配体，以及
-能够稳定所述的核酸适配体的RNA支架。
11.根据权利要求10所述的细胞，其特征在于，所述的细胞是来自棒状杆菌属的***细胞，特别是谷氨酸棒状杆菌。
12.根据权利要求10或11所述的细胞，其特征在于，所述的核酸适配体包含如SEQ IDNO:1至8、13至16、65或66的序列。
13.根据权利要求10至12中任一项所述的细胞，其特征在于，所述的核酸适配体能够稳定选自2-HBI、DFHBI、DFHBI-1T、DFHBI-2T、DMABI或DMHBI、TO1、TO3或Hoechst1C的任何荧光团。
14.根据权利要求10至13中任一项所述的细胞，其特征在于，能够稳定所述的核酸适配体的RNA支架包含如SEQ ID NO:9至12的序列。
15.根据权利要求10至14中任一项所述的细胞，其特征在于，所述的RNA标签包含如SEQID NO:17至64、69、77或78的序列。
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