CN110551720A - 基于美洲大蠊Dsx基因设计的dsRNA、其制备方法、编码基因及应用 - Google Patents
基于美洲大蠊Dsx基因设计的dsRNA、其制备方法、编码基因及应用 Download PDFInfo
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Abstract
本发明公开了基于美洲大蠊Dsx基因设计的dsRNA、其制备方法、编码基因及应用,所述dsRNA为由如Seq ID No.1所示的核苷酸序列作为正义链及由与Seq ID No.1所示的核苷酸序列反向互补的核苷酸序列作为反义链组成的双链RNA。所述dsRNA可广泛应用于制备防治美洲大蠊、干扰美洲大蠊化学通讯或控制美洲大蠊雌性成虫生殖的产品或防控美洲大蠊及其他具有相同基因靶标序列的昆虫中。以昆虫性别决定基因Dsx为靶标,基于Dsx中的基因片段设计一种dsRNA,将该dsRNA导入到成年雌性美洲大蠊体内,抑制其卵子发生和卵巢成熟,从而控制雌性美洲大蠊的生殖,达到害虫防控的目的。
Description
技术领域
本发明涉及卫生害虫防治领域,具体涉及基于美洲大蠊Dsx基因设计的dsRNA、其制备方法、编码基因及应用。
背景技术
1965年,在果蝇中发现了一种使后代的雄性和雌性发育为中间性的隐性突变,这个突变体被称为doublesex(Dsx)[1]。研究表明,Dsx基因通过其转录本性别特异性的选择性剪接产生一种雄性和一种雌性特异性DSX蛋白,发挥其体细胞性分化中的双功能作用,从而调控下游与性别特异性特征相关的靶基因[2]。Dsx曾被认为是所有昆虫性别决定级联中的最终主调控基因,然而,后来的研究否定了这一说法,认为它是性别决定和性别分化之间的中枢联系[3]。Dsx同源基因在不同昆虫物种中具有不同数量,例如双翅目昆虫具有多个,膜翅目昆虫有两个,而在鳞翅目昆虫中只有一个。Dsx具有多种末端调控功能,包括调节翅型大小、拟态、性二型性、行为以及荷尔蒙分泌等,而生殖过程中,Dsx能调控卵黄生成,表明Dsx在生殖调控中具有重要作用[4]。在琥珀蚕(Antheraea assama)中,RNAi介导Dsx沉默完全抑制Vg基因的表达,表明Vg是Dsx的下游靶基因,并且能导致卵巢畸形,生殖力下降和卵的完全致死,揭示了Dsx作为末端调节基因在生殖中的重要性[5]。在赤拟谷盗中,Vg及其受体基因VgR(Vitellogenin receptors)被鉴定为Dsx的靶标基因,Dsx RNAi导致这两个基因的表达下调,卵子发生受到抑制,产卵率和卵的孵化率显著降低[6]。
美洲大蠊(Periplaneta americana)属蜚蠊目,蜚蠊科昆虫,俗称蟑螂,适应能力和繁殖力都很强,是世界公认的城市卫生害虫。传播致病生物,如细菌,原生动物和病毒;并引发过敏反应和哮喘[7]。随着全球气候变暖,城市化进程的加快,交通和贸易的快速发展,危害程度日趋严重,已成为宾馆、饭店、家庭、医院、学校、食品加工制售、餐饮等单位重要的卫生害虫。美洲大蠊属于不完全***昆虫,具有强大的繁殖力和环境适应力,能进行孤雌生殖,而且传播致病微生物,是危害严重的城市卫生害虫。目前化学防治仍是控制美洲大蠊种群数量的主要防治方式,有机磷类、氨基甲酸酯类和拟除虫菊酯类化学杀虫剂的长期滥用势必导致害虫抗药性的提高和环境的污染。因此,建立安全有效的蟑螂防控机制势在必行。
雌性昆虫的生殖对于物种繁殖具有重要作用,而且可以作为害虫防控靶标,具有巨大的应用潜力,因而受到广泛的关注。近年来,由于生物技术的快速发展,运用生物信息学从昆虫基因组中寻找有效的靶标基因,进而运用RNA干扰等分子生物学的手段研究该靶标基因对害虫的影响。该技术具有特异性高、特异性强且不会产生污染环境有害物质等优点,为害虫防控提供了新的方向。通过以Dsx为靶标,控制雌性美洲大蠊的繁殖,进而达到害虫防控的目的,将有望成为解决现有技术中美洲大蠊繁殖速度快且耐药性强等问题,然而,现有技术中尚未有相关报道。
参考文献:
[1]Burtis KC,Baker BS.Drosophila Doublesex Gene Controls SomaticSexual Differentiation by Producing Alternatively Spliced Mrnas EncodingRelated Sex-Specific Polypeptides.Cell[J].1989,56(6):997-1010.
[2]Hildreth PE.Doublesex,a Recessive Gene That Tranforms Both Malesand Females of Drosophila into Intersexes.Genetics[J].1965,51(4):659-678.
[3]Nagaraju J,Gopinath G,Sharma V,Shukla JN.Lepidopteran SexDetermination:A Cascade of Surprises.Sexual Development[J].2014,8(1-3):104-112.
[4]Shukla J,Nagaraju J.Two Female-Specific Dsx Proteins Are Encodedby the Sex-Specific Transcripts of Dsx,and Are Required for Female SexualDifferentiation in Two Wild Silkmoth Species,Antheraea Assama and AntheraeaMylitta(Lepidoptera,Saturniidae).Insect Biochemical and Molecular Biology[J].2010,40(9):672-682.
[5]Shukla JN,Palli SR.Doublesex Target Genes in the Red Flour Beetle,Tribolium Castaneum.Scientific Reports[J].2012,2(12):948.
[6]Verhulst EC,van de Zande L.Double Nexus-Doublesex Is theConnecting Element in Sex Determination.Briefings in functional genomics[J].2015,14(6):396-406.
[7]Gomez OE,Belles X.Microrna-Dependent Metamorphosis inHemimetabolan Insects.Proceedings of the National Academy of Sciences[J].2009,106(51):21678-21682.
发明内容
本发明的所要解决的第一个技术问题在于:提供一种能够用于防控昆虫的的dsRNA。
本发明的所要解决的第二个技术问题在于:提供上述dsRNA的制备方法。
本发明的所要解决的第三个技术问题在于:提供编码上述dsRNA的基因及含有该编码基因的表达载体、转基因细胞系或宿主菌。
本发明的所要解决的第四个技术问题在于:提供上述dsRNA的应用。
为解决上述第一个技术问题,本发明的技术方案为:基于美洲大蠊Dsx基因设计的dsRNA,所述dsRNA为由如Seq ID No.1所示的核苷酸序列作为正义链及由与Seq ID No.1所示的核苷酸序列反向互补的核苷酸序列作为反义链组成的双链RNA。
为解决上述第二个技术问题,本发明的技术方案为:上述dsRNA的制备方法,包括以下步骤:将核苷酸序列如Seq ID No.3所示的DNA片段克隆到载体中,基于克隆后的载体设计引物并进行PCR扩增后,将PCR扩增产物转录合成即得。
优选地,所述制备方法具体为:以引物1和引物2扩增用于设计靶向沉默Dsx dsRNA的DNA片段,将扩增得到的DNA片段克隆到pTOPO载体,命名为pTOPO-Dsx,然后以pTOPO-Dsx为模板,设计两端含有T7启动子的引物3和引物4,进行PCR扩增,转录合成所述引物3的核苷酸序列如Seq ID No.6所示,引物4的核苷酸序列如Seq ID No.7所示。
优选地,采用T7RiboMAX Express RNAi System转录合成即得。
为解决上述第三个技术问题,本发明的技术方案为:编码上述dsRNA的基因。
上述基因的制备方法,包括以下步骤:基于上述基因设计启动子引物对,进行PCR扩增即得。
含有上述基因的表达载体、表达盒、转基因细胞系或宿主菌。
为解决上述第四个技术问题,本发明的技术方案为:上述dsRNA在制备用于防治美洲大蠊或控制美洲大蠊雌性成虫生殖的产品中的应用。
上述dsRNA在防控美洲大蠊及其他具有相同基因靶标序列的昆虫生殖中的应用。
一种防控美洲大蠊的方法,包括以下步骤:将上述dsRNA导入到美洲大蠊雌虫体内。
进一步地,所述导入操作通过注射的方式;优选地,所述注射操作是从沿腹部第二体节注入。
进一步地,所述导入操作还可以通过饲喂的方式。
进一步地,导入操作是在羽化后的第2、4和6天分别注射dsRNA一次。
本发明的有益效果在于:本发明方案首次将Dsx基因作为美洲大蠊的防控靶标;以昆虫性别决定基因Dsx为靶标,基于Dsx中的基因片段设计一种dsRNA,将该dsRNA导入到成年雌性美洲大蠊体内,抑制其卵子发生和卵巢成熟,从而控制雌性美洲大蠊的生殖,达到害虫防控的目的;本发明方案dsRNA特异性高,靶向于特定昆虫的特异基因,不会产生污染环境的有害物质,应用本发明方案设计的RNAi,使得美洲大蠊雌性成虫的卵子发生和卵巢发育受到了显著抑制,对于其后代繁殖的产生造成了严重影响,为害虫防控提供了新方法。
附图说明
图1为本发明实施例2中注射方法与检测方法的操作流程图;
图2为本发明实施例2中美洲大蠊雌性成虫在羽化后脂肪体中Dsx和Vg mRNA水平的发育变化趋势图;
图3为本发明实施例2中DsxRNAi后表达量检测结果图;
图4为本发明实施例2中Dsx RNAi后卵巢形态的变化图;
图5为本发明实施例2中Dsx RNAi后GSI的变化量柱状图;
图6为本发明实施例2中Dsx RNAi后卵巢粒卵长度的变化量柱状图;
图7为本发明实施例2中Dsx RNAi和Vg RNAi后卵巢中Vg的变化的凝胶电泳图及蛋白条带的灰度量化图,其中,(a)Dsx RNAi后Vg含量的变化;(b)Vg RNAi后Vg含量的变化;(c)Dsx RNAi后蛋白条带的灰度量化;(d)Vg RNAi后蛋白条带的灰度量化;
图8为本发明实施例2中Dsx RNAi后滤泡细胞和Patency的形态变化及DsxRNAi后滤胞细胞核直径的变化对照图;其中,(a)Dsx RNAi后卵巢滤泡细胞和Patency的表型变化;(b)Dsx RNAi后单位面积滤泡细胞数量的变化;(c)Dsx RNAi后滤泡细胞核直径的变化。
其中,图中标识的“*”代表“显著性水平P<0.05”,“***”代表“显著性水平P<0.001”。
具体实施方式
为详细说明本发明的技术内容、构造特征、所实现目的及效果,以下结合实施方式并配合附图详予说明。
本发明实施例1为:一种基于美洲大蠊Dsx基因设计的dsRNA,该dsRNA的核苷酸的正义链序列如Seq ID No.1所示,反义链为与Seq ID No.1所示序列反向互补的核苷酸序列,Seq ID No.1所示的核苷酸序列具体如下:
CGCCCGCUGUAGGAACCACCGCCUCAAGAUAGGCCUCAAGGGCCACAAGCGCUACUGCAAGUACCGGUACUGCAACUGCGACAAGUGCUGUCUGACGGCG。
其制备过程包括以下步骤:
(1)引物设计
根据美洲大蠊基因组数据库中的Dsx基因序列(如SEQ ID No.2所示),以其保守性较高的区域片段(如SEQ ID No.3所示),用于设计为dsRNA。利用引物设计软件PrimerPremier 5设计干扰片段的引物,扩增片段的长度100bp。片段正向引物Dsx-FP:CGCCCGCTGTAGGAACCAC(如SEQ ID No.4所示),反向引物Dsx-RP:CGCCGTCAGACAGCACTTG(如SEQ ID No.5所示)。使用pSTBlue-1载体(dsCK)的一段92bp的非编码序列作为阴性对照dsRNA[7],正向引物CK-FP:GAAAGCTC,反向引物CK-RP:GAATACAGCGGCCGCGAG。
美洲大蠊Dsx基因序列(SEQ ID No.2)具体如下:
ATGTCTGAGAACGGTGGCGAGCCAGGGCAGGAAGGCGCCCGGCCGGACGTTGCGGCTACTAGCAGCAGTTCGCAGAGTCCTCGTACGCCCCCCAATTGCGCCCGCTGTAGGAACCACCGCCTCAAGATAGGCCTCAAGGGCCACAAGCGCTACTGCAAGTACCGGTACTGCAACTGCGACAAGTGCTGTCTGACGGCGGAGCGCCAGCGGGTCATGGCGCTGCAGACGGCGCTGCGACGCGCCCAGGCGCAGGACGAGGCCCGCCTGGCCGGCCAGGTCGGAGGCCTCAGCATCGAGAACGGCGTGCCCGTCACCCAAACGTCCGCCCCCGGGGAGCCACCCGCCCCCGGCACCAGCGCCGCCGCCATCGTGTCCCGTTCCATGGAAGGCAGCTGCGATTCGTCGTCCTCGTCGCCCTGCTCCACCGGCGGCAGAGCGCTGTCCTCCGTCACCAGCGCGGGCGGCGCCGCCCTACGCGCCAGGGTGAATCCCCCGCACACCAACTCCGCTACTCCAGTCGAATTCCAGCCGATGACAGCTGGAACTTCCCTGAAGTCTTTCCCATCACTTTCCTTCCAGGATTCTTCCCGCCACCACTCCGCGCATCTCGATAATACATTGGACACTATCAACGCCCAAGGAGAGAGTTCGGAAGTTTCCGGCGAATCTCTCCAGATGCTGCTGGAAATGTTTAGGTTTCCTCCAGTAGCGCTGCCTCTAATCTACGTTGTTCTACAAGTCTCGCAATCGGATGTCAATGTGGCATACAATCGCATCATACAGGGAAAAATGTCGATAATGTATATTATATATGCAATAAGAATATATCACAAATTTTATGTGAATATCTCTAAAGAAATAAGTTGCTTGTTGGAAAAGGATATCTTTCAAAGTACATCTGTATTTTCTGCAGAATTTCAAGTAATTCTGAGTGCACTGAATTCATATCAGTAG。
美洲大蠊Dsx基因序列保守型较高区域片段序列具体如下:
CGCCCGCTGTAGGAACCACCGCCTCAAGATAGGCCTCAAGGGCCACAAGCGCTACTGCAAGTACCGGTACTGCAACTGCGACAAGTGCTGTCTGACGGCG
(2)构建载体与转化
将单一的PCR产物连接入pTOPO(艾德莱)载体中,制作成重组载体,命名为pTOPO-Dsx。将构建好的载体连接转化感受态细菌(DH5α),制作成重组菌株。筛选出阳性克隆,扩大培养后提取重组质粒。
(3)合成Dsx dsRNA(Promega T7RiboMAXTM Express RNAi System)
dsRNA模板的合成:在正反向引物的5’端加上T7RNA聚合酶启动子序列5′-TAATACGACTCACTATAGGN(17–22)-3’,分别为正向引物Dsx-FP:TAATACGACTCACTATAGGCGCCCGCTGTAGGAACCAC(如SEQ ID No.6所示),反向引物Dsx-RP:TAATACGACTCACTATAGGCGCCGTCAGACAGCACTTG(如SEQ ID No.7所示)。将质粒稀释至10ng左右以其作为模板,进行PCR扩增。胶回收PCR产物,作为dsRNA合成的模板,按T7RiboMAX Express RNAi System试剂盒说明书操作。
反应体系(20μl)具体组成如下:
(a)轻轻混匀,37℃保温30min;
(b)70℃保温10分钟,缓慢冷却到室温;
(c)1:200稀释RNase Solution.(1μl RNase Solution加入到199μl nucleasefree water);
(d)加1μl稀释后的RNase Solution和1μl RQ1RNase free DNase到20μl的反应体系内,37℃保温30分钟;
(e)加入0.1倍体积3M NaAc(醋酸钠),3倍体积异丙醇,混匀后冰上放置5分钟,4℃13000rpm/min离心10min,弃上清;
(f)除去上层液体,用500μl 75%乙醇(DEPC水配置)洗涤dsRNA沉淀,4℃13000rpm/min离心10min,弃上清;
(g)室温放置(约5-10min)晾干,根据产量,加入适量Nuclease-Free ddH2O溶解dsRNA;
(h)NanoDrop测定dsRNA浓度,电泳检测dsRNA质量;
(i)分装,稀释为相应浓度,放于-20℃保存,备用。
注意:如果有多管相同反应体系,在第(e)步后可以合并到一个1.5ml的离心管里,便于操作。
本发明实施例二为:上述dsRNA在美洲大蠊防治中的应用,包括以下步骤:如图1所示,取刚羽化为成虫的美洲大蠊,第2、4和6天注射Dsx dsRNA。第5天取脂肪体提取RNA用于实时荧光定量PCR检测,取卵巢提取蛋白进行SDS-PAGE凝胶电泳;第6天取卵巢进行DAPI和phalloidin染色后,置于共聚焦显微镜拍照;第7天去卵巢置于体视显微镜拍照。
具体操作如下:
(1)dsRNA的注射及表型观察统计
用二氧化碳将羽化后第2天健康的雌性美洲大蠊充分麻醉,用微量注射器向美洲大蠊腹部第二体节注射dsRNA,每只蟑螂注射3μg(3μl,1μg/μl)Dsx dsRNA。CK-dsRNA为对照。每隔两天注射一次,共注射三次。其中,每个处理注射20只。第5天取脂肪体,每3只蟑螂的组织合并为一个样品,放入液氮中速冻,然后-80℃保存备用,用于总RNA和蛋白提取;第6天,取卵巢用于细胞染色;观察第7天,取卵巢,用于表型观察,拍照。实验重复3次。
(2)RNA干扰后基因表达量检测
根据美洲大蠊基因组数据库(Li et al.,2018)中的基因序列,利用引物设计软件Primer5设计荧光定量PCR引物,扩增片段的长度约80-150bp。荧光定量PCR检测所用到引物Dsx-FP:AGGAACCACCGCCTCAAGATAG(如SEQ ID No.8所示),Dsx-RP:CTCCGCCGTCAGACAGCACT(如SEQ ID No.9所示);Actin-FP:CATCCTGCGTTTGGATCTGG(如SEQ ID No.10所示),Actin-RP:TTTCTCGTTCGGCAGTGGTG(如SEQ ID No.11所示)。使用HieffTM qPCRGreenMaster Mix(Low Rox Plus)试剂,按照其说明书操作,进行荧光定量PCR检测。两组数据之间进行比较,利用管家基因β-actin对目的基因相对表达量进行计算,再使用t检验比较两组数据之间是否存在显著差异,得出目的基因在某处理或者某时间点的相对表达量。每个样品重复三次,每个处理进行三次生物学重复,取各组数据的平均值和标准误,绘制图表,如图2所示。取第一个生殖周期期间美洲大蠊雌虫的脂肪体检测Dsx的表达量变化,发现DsxmRNA的表达量(图2,左轴)在羽化后逐渐上升,到第5天达到峰值,后下降。Vg mRNA的表达量(图2,右轴)与Dsx具有类似的趋势,表明它们存在内在联系。
RNAi后,检测脂肪体中Dsx和Vg的表达量如图3所示,Dsx的表达量下调了94.96%,此时Vg的表达量显著下调71.76%,即干扰Dsx后Vg的表达也受到了抑制,表明Dsx是Vg的上游,促进Vg的表达。
(3)卵巢表型观察与生殖腺指数GSI(gonadosomatic index)测定
取出美洲大蠊,称量体重。冰上麻醉后,用昆虫针固定在蜡盘上,置于OlympusSZ61解剖显微镜下,剪开腹部,将卵巢从组织中剥离,取出放入培养皿中,加入蟑螂生理盐水,使卵巢漂浮于溶液中。继续在显微镜下解剖卵巢,取出卵巢外周肌肉组织后,置于尼康DS-Ri2照相机下拍照,记录卵巢发育情况。在显微镜下,用解剖镊子将卵巢剥离成单个的卵小管,观察卵小管的形态,用尼康DS-Ri2照相机拍照记录,使用NIS-Elements BR 4.50.00软件测量卵巢首粒卵的长度。
用镊子将上述解剖的卵巢从蟑螂生理盐水中取出,放置于滤纸上把表面水分吸干,再使用电子天平称量卵巢重量,并记录。GSI计算按照如下公式:
GSI=[卵巢重量/(体重-卵巢重量)]×100%
Dsx RNAi后卵巢发育受到抑制(如图4所示),与对照相比,生殖腺指数GSI分别显著减小了73.75%(如图5所示),首粒卵长度分别显著减小了39.15%(如图6所示)。以上结果表明Dsx RNAi后卵巢成熟受到一定程度地抑制,说明Dsx对卵子发生具有一定的促进作用。
(4)SDS-PAGE凝胶电泳
取美洲大蠊卵巢于碧云天RIPA裂解液中研磨(提前加入PMSF,终浓度1mM),于4℃,12000g下离心30min,取上清用0.22μm滤膜过滤。使用翊圣生物BCA试剂盒测定蛋白浓度,浓度调节至2μg/μl。在蛋白样品中加入Loading buffer,沸水浴5分钟;每个胶孔上样量10μl;电泳时先以80V电压30min,待样品进入分离胶后调至130V继续电泳90min;取下凝胶,放于加有考马斯亮蓝R250染色液的染色皿中,置于摇床上,设置转速45r/min,染色30min后倒掉染液,清水冲洗3遍;加入脱色液,放置于摇床上,条带清晰可见后倒掉脱色液;置于扫描仪中拍照。
对羽化后第2天的雌性美洲大蠊注射Dsx dsRNA进行RNA干扰,第4天再次干扰,第5天取卵巢并提取蛋白,进行SDS-PAGE凝胶电泳及考马斯亮蓝染色。SDS-PAGE凝胶电泳结果中发现,对Dsx干扰后,卵巢中Vg(100kD)的含量显著下降(如图7a)。如图7c所示,与对照相比,Dsx干扰后Vg减少了79.63%。Vg RNA干扰在第2天,48h后干扰一次,共干扰三次,第7天取卵巢,提取蛋白后进行SDS-PAGE电泳(图7b)。结果发现对Vg干扰后,卵黄发生受阻,卵巢中始终不能积累Vg(图7d)。以上结果说明Dsx可以通过促进Vg表达,促进卵巢中Vg的累积。
(5)滤泡细胞和Patency小孔数量及其直径统计
取出处理后第6天的蟑螂,麻醉后将卵巢解剖出来,放入0.5mL离心管中,加入400μl固定液,25℃摇床上固定40min;
(2)吸出固定液,加入400μl PBT清洗,倒掉废液,重复清洗4次;
(3)加入400μl PBT,25℃下置于摇床精洗1小时;
(4)吸出PBT,重新加入PBT 400μl,再加1μl DAPI/phalloidin(1:10000),用锡纸包裹离心管,避光于摇床上染色30min;
(5)再用PBT清洗4次,每次400μl;
(6)加入PBT 400μl,25℃下置于摇床精洗1小时;
(7)进行二次解剖,将组织取出置于载玻片上,吸干液体,滴一定量50%甘油,剖出所需组织,封片,在共聚焦显微镜下观察并拍照。
随机选取在共聚焦显微镜40×物镜视野下,以3.3倍放大倍数拍摄的卵巢滤泡细胞照片。统计照片视野中滤泡细胞和Patency小孔的数量;分别测量细胞核和Patency小孔的直径,每个细胞核和Patency小孔分别测量最长直径和最短直径,并以其平均值为该细胞的实际直径。每个处理随机选择3张照片测量。
Dsx RNA干扰抑制卵巢滤泡细胞的发育(如图8a所示),与对照组相比,单位面积Patency小孔数量和滤泡细胞数量分别显著增加了60.44%和78%,然而Patency小孔数量与滤泡细胞数量之间的比值指数没有差异(如图8b所示);Dsx RNA干扰后滤泡细胞发育受到抑制,与对照相比,Patency小孔直径和滤泡细胞核直径分别显著减少了53.41%和28.41%,Patency小孔直径和滤泡细胞核直径的比值显著减小了34.83%(如图8c所示)。结果表明Dsx能够促进滤泡细胞的发育和Patency的形成。
综上所述,通过靶向沉默Dsx基因,即可控制雌虫生殖,达到防控的目的。
以上所述仅为本发明的实施例,并非因此限制本发明的专利范围,凡是利用本发明说明书及附图内容所作的等效结构或等效流程变换,或直接或间接运用在其他相关的技术领域,均同理包括在本发明的专利保护范围内。
序列表
<110> 梅州市华师昆虫发育生物学与应用技术重点实验室广梅园研发中心
华南师范大学
<120> 基于美洲大蠊Dsx基因设计的dsRNA、其制备方法、编码基因及应用
<160> 11
<170> SIPOSequenceListing 1.0
<210> 1
<211> 100
<212> RNA
<213> 人工序列(Artificial Sequence)
<400> 1
cgcccgcugu aggaaccacc gccucaagau aggccucaag ggccacaagc gcuacugcaa 60
guaccgguac ugcaacugcg acaagugcug ucugacggcg 100
<210> 2
<211> 954
<212> DNA
<213> Periplaneta americana
<400> 2
atgtctgaga acggtggcga gccagggcag gaaggcgccc ggccggacgt tgcggctact 60
agcagcagtt cgcagagtcc tcgtacgccc cccaattgcg cccgctgtag gaaccaccgc 120
ctcaagatag gcctcaaggg ccacaagcgc tactgcaagt accggtactg caactgcgac 180
aagtgctgtc tgacggcgga gcgccagcgg gtcatggcgc tgcagacggc gctgcgacgc 240
gcccaggcgc aggacgaggc ccgcctggcc ggccaggtcg gaggcctcag catcgagaac 300
ggcgtgcccg tcacccaaac gtccgccccc ggggagccac ccgcccccgg caccagcgcc 360
gccgccatcg tgtcccgttc catggaaggc agctgcgatt cgtcgtcctc gtcgccctgc 420
tccaccggcg gcagagcgct gtcctccgtc accagcgcgg gcggcgccgc cctacgcgcc 480
agggtgaatc ccccgcacac caactccgct actccagtcg aattccagcc gatgacagct 540
ggaacttccc tgaagtcttt cccatcactt tccttccagg attcttcccg ccaccactcc 600
gcgcatctcg ataatacatt ggacactatc aacgcccaag gagagagttc ggaagtttcc 660
ggcgaatctc tccagatgct gctggaaatg tttaggtttc ctccagtagc gctgcctcta 720
atctacgttg ttctacaagt ctcgcaatcg gatgtcaatg tggcatacaa tcgcatcata 780
cagggaaaaa tgtcgataat gtatattata tatgcaataa gaatatatca caaattttat 840
gtgaatatct ctaaagaaat aagttgcttg ttggaaaagg atatctttca aagtacatct 900
gtattttctg cagaatttca agtaattctg agtgcactga attcatatca gtag 954
<210> 3
<211> 100
<212> DNA
<213> Periplaneta americana
<400> 3
cgcccgctgt aggaaccacc gcctcaagat aggcctcaag ggccacaagc gctactgcaa 60
gtaccggtac tgcaactgcg acaagtgctg tctgacggcg 100
<210> 4
<211> 19
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 4
cgcccgctgt aggaaccac 19
<210> 5
<211> 19
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 5
cgccgtcaga cagcacttg 19
<210> 6
<211> 38
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 6
taatacgact cactataggc gcccgctgta ggaaccac 38
<210> 7
<211> 38
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 7
taatacgact cactataggc gccgtcagac agcacttg 38
<210> 8
<211> 22
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 8
aggaaccacc gcctcaagat ag 22
<210> 9
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 9
ctccgccgtc agacagcact 20
<210> 10
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 10
catcctgcgt ttggatctgg 20
<210> 11
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 11
tttctcgttc ggcagtggtg 20
Claims (10)
1.基于美洲大蠊Dsx基因设计的dsRNA,其特征在于:所述dsRNA为由如Seq ID No.1所示的核苷酸序列作为正义链及由与Seq ID No.1所示的核苷酸序列反向互补的核苷酸序列作为反义链组成的双链RNA。
2.如权利要求1所述的dsRNA的制备方法,其特征在于:包括以下步骤:将核苷酸序列如Seq ID No.3所示的DNA片段克隆到载体中,基于克隆后的载体设计引物并进行PCR扩增后,将PCR扩增产物转录合成即得。
3.编码如权利要求1所述的dsRNA的基因。
4.如权利要求3所述的基因的制备方法,其特征在于:包括以下步骤:基于上述基因设计启动子引物对,进行PCR扩增即得。
5.含有如权利要求3所述的基因的表达载体、表达盒、转基因细胞系或宿主菌。
6.如权利要求1所述的dsRNA在制备用于防治美洲大蠊或控制美洲大蠊雌性成虫生殖的产品中的应用。
7.如权利要求1所述的dsRNA在防控美洲大蠊及其他具有相同基因靶标序列的昆虫生殖中的应用。
8.一种防控美洲大蠊的方法,其特征在于:包括以下步骤:将如权利要求1所述的dsRNA导入到美洲大蠊雌虫体内。
9.根据权利要求8所述的防控美洲大蠊的方法,其特征在于:所述导入操作通过注射的方式;优选地,所述注射操作是从沿腹部第二体节注入。
10.根据权利要求8所述的防控美洲大蠊的方法,其特征在于:导入操作是在羽化后的第2、4和6天分别注射dsRNA一次。
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Cited By (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN112458087A (zh) * | 2020-11-06 | 2021-03-09 | 华南师范大学 | 基于美洲大蠊HDAC1基因设计的dsRNA、其制备方法、编码基因及其应用 |
CN112522278A (zh) * | 2020-12-29 | 2021-03-19 | 华南师范大学 | 基于美洲大蠊嗅觉受体基因OR3X设计的dsRNA、编码基因及其制备方法与应用 |
CN112695033A (zh) * | 2020-12-29 | 2021-04-23 | 华南师范大学 | 基于美洲大蠊雄性附腺生殖相关基因SP28设计的siRNA及其制备方法与应用 |
CN112725345A (zh) * | 2020-12-29 | 2021-04-30 | 华南师范大学 | 基于美洲大蠊性信息素受体基因OR5M设计的dsRNA、编码基因及其制备方法与应用 |
CN116250548A (zh) * | 2022-08-30 | 2023-06-13 | 华南师范大学 | 抑咽侧体神经肽在防治美洲大蠊中的应用 |
Citations (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN101421408A (zh) * | 2006-02-10 | 2009-04-29 | 奥西泰克有限公司 | 昆虫中利用可变剪接的基因表达*** |
WO2015040574A1 (en) * | 2013-09-20 | 2015-03-26 | University Of Manitoba | Biological control of insects |
CN108823212A (zh) * | 2018-07-16 | 2018-11-16 | 华南师范大学 | 美洲大蠊断肢再生相关Wingless信号通路基因及其dsRNA的应用 |
CN108893472A (zh) * | 2018-07-16 | 2018-11-27 | 华南师范大学 | 一种dsRNA及其在防治德国小蠊上的应用 |
CN109207601A (zh) * | 2018-08-06 | 2019-01-15 | 华南师范大学 | 美洲大蠊断肢再生相关Hedgehog信号通路基因及其dsRNA的应用 |
-
2019
- 2019-08-13 CN CN201910744841.6A patent/CN110551720B/zh not_active Expired - Fee Related
Patent Citations (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN101421408A (zh) * | 2006-02-10 | 2009-04-29 | 奥西泰克有限公司 | 昆虫中利用可变剪接的基因表达*** |
WO2015040574A1 (en) * | 2013-09-20 | 2015-03-26 | University Of Manitoba | Biological control of insects |
CN106102456A (zh) * | 2013-09-20 | 2016-11-09 | 马尼托巴大学 | 昆虫的生物防治 |
CN108823212A (zh) * | 2018-07-16 | 2018-11-16 | 华南师范大学 | 美洲大蠊断肢再生相关Wingless信号通路基因及其dsRNA的应用 |
CN108893472A (zh) * | 2018-07-16 | 2018-11-27 | 华南师范大学 | 一种dsRNA及其在防治德国小蠊上的应用 |
CN109207601A (zh) * | 2018-08-06 | 2019-01-15 | 华南师范大学 | 美洲大蠊断肢再生相关Hedgehog信号通路基因及其dsRNA的应用 |
Non-Patent Citations (2)
Title |
---|
KESHAVA MYSORE ET AL.: "siRNA-Mediated Silencing of doublesex during Female Development of the Dengue Vector Mosquito Aedes aegypti", 《PLOS NEGLECTED TROPICAL DISEASE》 * |
SHIU-LINGCHEN: "Female-specific doublesex dsRNA interrupts yolk protein gene expression and reproductive ability in oriental fruit fly, Bactrocera dorsalis (Hendel)", 《INSECT BIOCHEMISTRY AND MOLECULAR BIOLOGY》 * |
Cited By (8)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN112458087A (zh) * | 2020-11-06 | 2021-03-09 | 华南师范大学 | 基于美洲大蠊HDAC1基因设计的dsRNA、其制备方法、编码基因及其应用 |
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CN112695033B (zh) * | 2020-12-29 | 2021-08-24 | 华南师范大学 | 基于美洲大蠊雄性附腺生殖相关基因SP28设计的siRNA及其制备方法与应用 |
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CN112725345B (zh) * | 2020-12-29 | 2021-09-28 | 华南师范大学 | 基于美洲大蠊性信息素受体基因OR5M设计的dsRNA、编码基因及其制备方法与应用 |
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