CN111630165A - 通过抑制条件性必需基因进行反向选择 - Google Patents
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Abstract
本发明涉及一种通过抑制条件性必需基因进行反向选择的方法。
Description
序列表的引用
本申请含有处于计算机可读形式的序列表,将其通过引用并入本文。
技术领域
本发明涉及一种通过抑制条件性必需基因进行反向选择的方法。
背景技术
所谓的CRISPR-Cas9基因组编辑***已被广泛用作一种工具来修饰多种生物的基因组。CRISPR-Cas9***的力量在于其简单性,可在特异性目的基因中靶向和编辑单个碱基对。Cas9蛋白是双RNA指导的核酸内切酶,其核酸酶活性由所谓的指导RNA(gRNA)分子引导。通过改变gRNA的20bp序列窗口以匹配靶DNA序列来选择Cas9靶序列。当与gRNA分子复合时,Cas9蛋白然后将结合其靶DNA序列并使用两个催化结构域产生双-链断裂。Cas9可以进一步被工程化为在两个催化结构域中的任一个中含有单个氨基酸突变。在这种情况下,Cas9作为切割酶起作用,即具有单链切割活性。
除了其在基因组编辑中使用外,CRISPR-Cas9***还用于控制基因表达。这种应用,通常被称为CRISPR抑制或CRISPRi,允许基因的序列特异性抑制或激活。CRISPR干扰利用了一种缺乏核酸内切酶活性的无催化活性的(死)Cas9变体(称作Cas9d)。Cas9d-gRNA复合物保留了与靶DNA序列结合的能力,但不会在DNA链中引入任何断裂。通过改变gRNA序列,可以控制Cas9d-gRNA复合物的靶DNA序列,从而调节任何生物中几乎任何基因的表达,条件是存在功能性PAM序列。
在工业生物技术中,持续需要适合于开发经优化的生产宿主的强力且有效的选择***。鉴于CRISPR-Cas9技术的多功能性和精确性,据推测该***可用于反向选择目的。然而,迄今为止,利用CRISPR-Cas9技术进行直接选择的尝试是困难的。这对于细菌宿主细胞尤其如此,因为归因于已知来自真核生物的非同源末端连接(NHEJ)***对双链(DS)断裂的低效修复机制,导致许多原核生物对Cas9-gRNA复合物的核酸内切酶活性非常敏感(参见,例如,Su等人,Scientific Reports[科学报告]2016,6,37895;Altenbuchner,Applied andEnvironmental Microbiology[应用与环境微生物学]2016,82,第5421-5427页;Peters等人,Current Opinion in Microbiology[微生物学当前观点]2015,27,第121-126页;Aravind和Koonin,Genome Research[基因组研究]2001,11,第1365-1374页)。
此外,如果多于一个位点靶向DS断裂,则使用CRISPR-Cas9技术的直接选择将变得越来越困难。
许多研究者报道了通过同源重组(HR),将目的基因(GOI)成功整合到染色体上的gRNA靶点中,并且然后引入CRISPR-Cas9活性进行DS断裂以杀死保留原始gRNA靶序列的细胞。以此方式,就有可能有效地富集已经接受GOI的细胞。然而,HR和DS活性的这些事件的时机非常重要。CRISPR-Cas9复合物在产生DS断裂方面非常活跃,并且不应被表达,直至发生同源重组并去除gRNA靶点。
发明内容
本发明提供了在适用于细菌宿主细胞的选择***中,利用CRISPR-Cas9技术的多功能性和精确性的手段和方法。
因此,在第一方面,本发明涉及用于将至少一种目的多核苷酸***至宿主细胞基因组的方法,所述方法包括以下步骤:
a)提供一种宿主细胞,所述宿主细胞在其基因组中包含以下:
i.编码包含靶序列的选择性标记的多核苷酸,所述靶序列侧接针对II类Cas9蛋白的功能性PAM序列;
ii.编码gRNA的至少一种多核苷酸,所述gRNA与靶序列至少80%互补并且能够与所述靶序列杂交;以及
iii.编码II类Cas9蛋白的无效核酸酶变体的多核苷酸,所述II类Cas9蛋白能够与gRNA相互作用并与靶序列结合,从而抑制选择性标记的表达;
b)用至少一种目的多核苷酸转化所述宿主细胞,并且所述至少一种目的多核苷酸能够使所述编码gRNA的至少一种多核苷酸失活;
c)选择由选择性标记赋予的性状;以及
d)识别转化的宿主细胞,其中所述编码gRNA的至少一种多核苷酸已被所述至少一种目的多核苷酸灭活。
在第二方面,本发明涉及用于将至少两种不同的目的多核苷酸***至宿主细胞基因组的方法,所述方法包括以下步骤:
a)提供一种宿主细胞,所述宿主细胞在其基因组中包含以下:
i.编码至少两种不同的选择性标记的至少两种多核苷酸,每种选择性标记包含侧接针对II类Cas9蛋白的功能性PAM序列的不同靶序列;
ii.编码至少两个gRNA的至少两种多核苷酸,所述至少两个gRNA与至少两个不同的靶序列至少80%互补并且能够与所述至少两个不同的靶序列杂交;
iii.编码II类Cas9蛋白的无效核酸酶变体的多核苷酸,所述II类Cas9蛋白能够与至少两个gRNA相互作用并与至少两个不同的靶序列结合,从而抑制两种不同的选择性标记的表达;
b)用至少两种不同的目的多核苷酸转化所述宿主细胞,所述多核苷酸能够使所述编码至少两个gRNA的至少两种多核苷酸失活;和
c)选择由至少两种不同的选择性标记赋予的性状;以及
d)识别转化的宿主细胞,其中所述编码至少两个gRNA的至少两种多核苷酸已被所述至少两种不同的目的多核苷酸灭活。
附图说明
图1显示了PP3811-cas9d菌株中的bglC-cas9d基因座。
图2显示了PP3811-gDNA1菌株中的gnt-dsRED-gDNA(cat)基因座。
图3显示了PP3811-gDNA2菌株中的amyL-dsRED-gDNA(cat)基因座。
图4显示了PP3811-gDNA3菌株中的lacA2-dsRED-gDNA(cat)基因座。
图5显示了整合amyL后PP3811-amyL3中的gnt基因座。
图6显示了重新整合amyL后PP3811-amyL3中的amyL基因座。
图7显示了整合amyL后PP3811-amyL3中的lacA2基因座。
图8显示了PP3811-gDNA3菌株的示意图。
图9显示了pPPamyL-attP质粒。
图10显示了pSJ14072质粒。
定义
cDNA:术语“cDNA”意指可以通过从获得自真核或原核细胞的成熟的、剪接的mRNA分子进行反转录而制备的DNA分子。cDNA缺乏可以存在于对应基因组DNA中的内含子序列。初始的初级RNA转录物是mRNA的前体,其要通过一系列的步骤(包括剪接)进行加工,之后呈现为成熟的剪接的mRNA。
编码序列:术语“编码序列”意指直接指定多肽的氨基酸序列的多核苷酸。编码序列的边界通常由可读框确定,该可读框以起始密码子(例如ATG、GTG或TTG)开始并且以终止密码子(例如TAA、TAG或TGA)结束。编码序列可为基因组DNA、cDNA、合成DNA或其组合。
条件性必需基因:条件性必需基因或基因座可以作为选择性标记起作用。细菌条件性必需选择性标记的实例是来自枯草芽孢杆菌或地衣芽孢杆菌的dal基因,这些基因仅当在D-丙氨酸存在下培养细菌时是必需的;或是编码酶的基因,当细胞在半乳糖存在下生长时,这些酶参与UDP半乳糖从细菌细胞中的去除。此类基因的非限制性实例是编码UTP依赖性磷酸化酶(EC2.7.7.10)、UDP-葡萄糖依赖性尿苷基转移酶(EC 2.7.7.12)或UDP-半乳糖差向异构酶(EC 5.1.3.2)的来自枯草芽孢杆菌或地衣芽孢杆菌的那些。如果必需基因或基因座失活,将使得所得菌株具有缺陷(例如,不能代谢特定碳源),或生长需要(例如,变成氨基酸营养缺陷型,或对给定的压力变得敏感)。条件性必需基因的非限制性实例是编码D-丙氨酸消旋酶的基因、编码木糖异构酶的基因、葡萄糖酸操纵子的基因。优选地,所述条件性必需基因选自由以下组成的组:dal、lysA、araA、galE、antK、metC、xylA、gntP、gntK、glpD、glpF、glpK、glpP、lacA2、hisC、gapA、和aspB。
控制序列:术语“控制序列”意指表达编码本发明的成熟多肽的多核苷酸所必需的核酸序列。每个控制序列对于编码所述多肽的多核苷酸来说可以是天然的(即,来自相同基因)或外源的(即,来自不同基因),或相对于彼此是天然的或外源的。此类控制序列包括但不限于前导序列、多腺苷酸化序列、前肽序列、启动子、信号肽序列、以及转录终止子。最少,控制序列包括启动子、以及转录和翻译终止信号。出于引入有利于将控制序列与编码多肽的多核苷酸的编码区连接的特异性限制位点的目的,所述控制序列可以提供有多个接头。
表达:术语“表达”包括涉及多肽产生的任何步骤,包括但不限于:转录、转录后修饰、翻译、翻译后修饰、以及分泌。
表达载体:术语“表达载体”意指直链或环状DNA分子,其包含编码多肽的多核苷酸并且可操作地连接至提供用于其表达的控制序列。
宿主细胞:术语“宿主细胞”意指易于用包含本发明的多核苷酸的核酸构建体或表达载体进行转化、转染、转导等的任何细胞类型。术语“宿主细胞”涵盖由于复制期间出现的突变而与亲本细胞不完全相同的任何亲本细胞子代。
分离的:术语“分离的”意指处于自然界中不存在的形式或环境中的物质。分离的物质的非限制性实例包括(1)任何非天然存在的物质;(2)至少部分地从与它天然相关的一个或多个或所有天然存在的成分中去除的任何物质,包括但不限于:任何酶、变体、核酸、蛋白质、肽或辅因子;(3)相对于在自然界中发现的物质经过人为改变的任何物质;或(4)相对于与它天然相关的其他组分通过增加所述物质的量(例如,在宿主细胞中的重组生产;编码所述物质的基因的多个拷贝;以及使用比与编码所述物质的基因天然相关联的启动子更强的启动子)而改变的任何物质。分离的物质可以存在于发酵液样品中;例如宿主细胞可以经遗传修饰以表达本发明的多肽。来自所述宿主细胞的发酵液将包含分离的多肽。
核酸构建体:术语“核酸构建体”意指单链或双链的核酸分子,所述核酸分子是从天然存在的基因中分离的,或以原本不存在于自然界中的方式被修饰成包含核酸的区段,或者是合成的,所述核酸分子包含一个或多个控制序列。
可操作地连接:术语“可操作地连接”意指如下构型,在所述构型中,控制序列被放置在相对于多核苷酸的编码序列适当的位置处,使得所述控制序列引导所述编码序列的表达。
序列同一性:两个氨基酸序列之间或两个核苷酸序列之间的关联度通过参数“序列同一性”来描述。
出于本发明的目的,使用如在EMBOSS软件包(EMBOSS:欧洲分子生物学开放软件包(EMBOSS:The European Molecular Biology Open Software Suite),Rice等人,2000,Trends Genet.[遗传学趋势]16:276-277)(优选5.0.0版本或更新版本)的尼德尔程序中所实施的尼德曼-翁施算法(Needleman-Wunsch algorithm)(Needleman和Wunsch,1970,J.Mol.Biol.[分子生物学杂志]48:443-453)来确定两个氨基酸序列之间的序列同一性。所使用的参数是空位开放罚分10、空位延伸罚分0.5、以及EBLOSUM62(BLOSUM62的EMBOSS版本)取代矩阵。使用尼德尔标记的“最长同一性”的输出(使用非简化(-nobrief)选项获得)作为同一性百分比并且如下计算:
(相同的残基x 100)/(比对长度-比对中的空位总数)
出于本发明的目的,使用如在EMBOSS软件包(EMBOSS:欧洲分子生物学开放软件包(EMBOSS:The European Molecular Biology Open Software Suite),Rice等人,2000,同上)(优选5.0.0版本或更新版本)的尼德尔程序中所实施的尼德曼-翁施算法(Needleman和Wunsch,1970,同上)来确定两个脱氧核糖核苷酸序列之间的序列同一性。所使用的参数是空位开放罚分10、空位延伸罚分0.5、以及EDNAFULL(NCBI NUC4.4的EMBOSS版本)取代矩阵。使用尼德尔标记的“最长同一性”的输出(使用非简化(-nobrief)选项获得)作为同一性百分比并且如下计算:
(相同的脱氧核糖核苷酸x 100)/(比对长度-比对中的空位总数)
具体实施方式
本发明提供了在适用于细菌宿主细胞的选择***中,利用CRISPR-Cas9技术的多功能性和精确性的手段和方法。通过使用编码CRISPRi中的gRNA的DNA序列(表示为‘gDNA’)作为间接的反向选择性标记,本发明人已表明,可以通过选择不存在编码gRNA的gDNA将多个基因拷贝***至宿主细胞基因组中。
如本文实例中所示,合适的选择***可以基于抗生素抗性基因,如赋予对氯霉素抗性的cat基因。包含编码cat基因的多核苷酸以及编码II类Cas9蛋白的无效核酸酶变体的多核苷酸和编码针对cat基因的gRNA的多核苷酸的宿主细胞将因此仅在不存在氯霉素的情况下才能生长,因为Cas9d-gRNA复合物将抑制cat基因的表达。只要宿主细胞表达II类Cas9蛋白的无效核酸酶变体和gRNA,所述宿主细胞保持对氯霉素敏感。
在下一步中,用多核苷酸转化宿主细胞,这允许用目的基因替代gDNA。通过随后对氯霉素抗性的选择,由于gRNA不再表达,仅具有被目的基因替换的gDNA的细胞才能存活,这使得正确转化的宿主细胞对氯霉素具有抗性。
如本文所附实例中所示,本发明的方法特别适合于在宿主细胞染色体上的单独基因座上一个或多个特定表达盒的一步多***。本发明的方法提供了含有多个表达盒的宿主细胞,即多拷贝宿主细胞,其由于表达盒被***到染色体上的单独基因座上而高度稳定。此类细胞在工业生物技术中作为用于生产目的多肽的强力主力是高度可靠的。
因此,在第一方面,本发明涉及用于将至少一种目的多核苷酸***至宿主细胞基因组的方法,所述方法包括以下步骤:
a)提供一种宿主细胞,所述宿主细胞在其基因组中包含以下:
i.编码包含靶序列的选择性标记的多核苷酸,所述靶序列侧接针对II类Cas9蛋白的功能性PAM序列;
ii.编码gRNA的至少一种多核苷酸,所述gRNA与靶序列至少80%互补并且能够与所述靶序列杂交;以及
iii.编码II类Cas9蛋白的无效核酸酶变体的多核苷酸,所述II类Cas9蛋白能够与gRNA相互作用并与靶序列结合,从而抑制选择性标记的表达;
b)用至少一种目的多核苷酸转化所述宿主细胞,并且所述至少一种目的多核苷酸能够使所述编码gRNA的至少一种多核苷酸失活;
c)选择由选择性标记赋予的性状;以及
d)识别转化的宿主细胞,其中所述编码gRNA的至少一种多核苷酸已被所述至少一种目的多核苷酸灭活。
在第一方面的方法的步骤(a)中提供的宿主细胞包含编码gRNA的至少一种多核苷酸。优选地,编码gRNA的多核苷酸的数目是至少一个,如至少两个、至少三个、至少四个、至少五个、至少六个、至少七个、至少八个、至少九个、至少十个、至少15个、至少20个、至少25个或更多个。
在第一方面的方法的步骤(b)中用至少一种目的多核苷酸转化宿主细胞。优选地,目的多核苷酸的数目是至少一个,如至少两个、至少三个、至少四个、至少五个、至少六个、至少七个、至少八个、至少九个、至少十个、至少15个、至少20个、至少25个或更多个。
优选地,所述至少一种目的多核苷酸编码蛋白质;更优选地,所述目的多核苷酸编码选自由以下组成的组的酶:水解酶、异构酶、连接酶、裂解酶、氧化还原酶、或转移酶;更优选地是氨肽酶、淀粉酶、糖酶、羧肽酶、过氧化氢酶、纤维二糖水解酶、纤维素酶、几丁质酶、角质酶、环糊精糖基转移酶、脱氧核糖核酸酶、内切葡聚糖酶、酯酶、α-半乳糖苷酶、β-半乳糖苷酶、葡糖淀粉酶、α-葡糖苷酶、β-葡糖苷酶、转化酶、漆酶、脂肪酶、甘露糖苷酶、变聚糖酶、氧化酶、果胶分解酶、过氧化物酶、磷酸二酯酶、植酸酶、多酚氧化酶、蛋白水解酶、核糖核酸酶、转谷氨酰胺酶、木聚糖酶、和β-木糖苷酶。
优选地,选择性标记是赋予对氯霉素、四环素、氨苄青霉素、大观霉素、卡那霉素或新霉素的抗性的抗生素抗性基因;更优选地,选择性标记是赋予对氯霉素的抗性的抗生素抗性基因。
还优选地,选择性标记是选自由以下组成的组的抗生素抗性基因:cat、erm、tet、amp、spec、kana、和neo;更优选地,选择性标记是cat基因。
可替代地,并且还优选地,选择性标记是赋予对宿主细胞的营养缺陷型的基因。优选地,选择性标记是选自由以下组成的组的条件性必需基因:dal、lysA、araA、galE、antKmetC、xylA、gntP、glpD、glpF、glpK、glpP、lacA2、hisC、gapA、和aspB基因。更优选地,选择性标记是dal基因。
有许多众所周知的使基因失活的方法,例如,通过引入非正义突变或者移码突变来突变所述基因、或者通过使开放阅读框的部分或全部缺失、或者通过操作一个或多个控制序列。
相应地,在第一方面的优选的实施例中,所述编码gRNA的至少一种多核苷酸通过所述多核苷酸的部分或全部缺失而失活。
在第一方面的优选的实施例中,所述编码gRNA的至少一种多核苷酸已在宿主细胞基因组中被步骤(d)中的至少一种目的多核苷酸部分或完全替换,从而使所述编码gRNA的至少一种多核苷酸失活。
在第二方面,本发明涉及用于将至少两种不同的目的多核苷酸***至宿主细胞基因组的方法,所述方法包括以下步骤:
a)提供一种宿主细胞,所述宿主细胞在其基因组中包含以下:
i.编码至少两种不同的选择性标记的至少两种多核苷酸,每种选择性标记包含侧接针对II类Cas9蛋白的功能性PAM序列的不同靶序列;
ii.编码至少两个gRNA的至少两种多核苷酸,所述至少两个gRNA与至少两个不同的靶序列至少80%互补并且能够与所述至少两个不同的靶序列杂交;
iii.编码II类Cas9蛋白的无效核酸酶变体的多核苷酸,所述II类Cas9蛋白能够与至少两个gRNA相互作用并与至少两个不同的靶序列结合,从而抑制两种不同的选择性标记的表达;
b)用至少两种不同的目的多核苷酸转化所述宿主细胞,所述多核苷酸能够使所述编码至少两个gRNA的至少两种多核苷酸失活;和
c)选择由至少两种不同的选择性标记赋予的性状;以及
d)识别转化的宿主细胞,其中所述编码至少两个gRNA的至少两种多核苷酸已被所述至少两种不同的目的多核苷酸灭活。
在第二方面的方法的步骤(a)中提供的宿主细胞包含编码至少两种不同的选择性标记的至少两种多核苷酸和编码至少两个gRNA的至少两种多核苷酸。优选地,编码至少两种不同的选择性标记和至少两个gRNA的多核苷酸的数目独立地为至少两个,如至少三个、至少四个、至少五个、至少六个、至少七个、至少八个、至少九个、至少十个、至少15个、至少20个、至少25个或更多个。
在第二方面的方法的步骤(b)中用至少两种不同的目的多核苷酸转化宿主细胞。优选地,不同的目的多核苷酸的数目是至少两个,如至少三个、至少四个、至少五个、至少六个、至少七个、至少八个、至少九个、至少十个、至少15个、至少20个、至少25个或更多个。
优选地,所述至少两种不同的目的多核苷酸编码至少两种蛋白质;更优选地,所述至少两种不同的目的多核苷酸编码独立地选自由以下组成的组的酶:水解酶、异构酶、连接酶、裂解酶、氧化还原酶、或转移酶;更优选地是氨肽酶、淀粉酶、糖酶、羧肽酶、过氧化氢酶、纤维二糖水解酶、纤维素酶、几丁质酶、角质酶、环糊精糖基转移酶、脱氧核糖核酸酶、内切葡聚糖酶、酯酶、α-半乳糖苷酶、β-半乳糖苷酶、葡糖淀粉酶、α-葡糖苷酶、β-葡糖苷酶、转化酶、漆酶、脂肪酶、甘露糖苷酶、变聚糖酶、氧化酶、果胶分解酶、过氧化物酶、磷酸二酯酶、植酸酶、多酚氧化酶、蛋白水解酶、核糖核酸酶、转谷氨酰胺酶、木聚糖酶、和β-木糖苷酶。
在第二方面的优选的实施例中,所述至少两种不同的选择性标记独立地选自由以下组成的组:抗生素抗性基因和赋予对宿主细胞的营养缺陷型的基因;优选地,所述至少两种不同的选择性标记独立地选自由以下组成的组的基因:cat、erm、tet、amp、spec、kana、neo、dal、lysA、araA、galE、antKmetC、xylA、gntP、glpD、glpF、glpK、glpP、lacA2、hisC、gapA、和aspB。
在第二方面的优选的实施例中,所述编码至少两个gRNA的至少两种多核苷酸通过所述多核苷酸的部分或全部缺失而失活。
在第二方面的优选的实施例中,所述编码至少两个gRNA的至少两种多核苷酸已在宿主细胞基因组中被步骤(d)中的至少两种不同的目的多核苷酸部分或完全替换,从而使所述编码至少两个gRNA的至少两种多核苷酸失活。
多核苷酸
本发明还涉及本发明的多核苷酸,包括目的多核苷酸以及编码选择性标记、gRNA和II类Cas9蛋白的无效核酸酶变体的多核苷酸。在一个实施例中,此类多核苷酸已经被分离。
用于分离或克隆多核苷酸的技术是本领域已知的且包括从基因组DNA或cDNA或其组合进行分离。来自基因组DNA的多核苷酸的克隆可以例如通过使用众所周知的聚合酶链式反应(PCR)或用以对具有共有的结构特征的克隆的DNA片段进行检测的表达库抗体筛选来实现。参见例如,Innis等人,1990,PCR:A Guide to Methods and Application[PCR:方法和应用指南],Academic Press[学术出版社],纽约。可以使用其他核酸扩增程序例如连接酶链式反应(LCR)、连接激活转录(LAT)和基于多核苷酸的扩增(NASBA)。
核酸构建体
本发明还涉及核酸构建体,这些核酸构建体包含可操作地连接至一个或多个控制序列的本发明的多核苷酸,在与控制序列相容的条件下,所述一个或多个控制序列指导所述编码序列在适合的宿主细胞中的表达。
可用许多方式操作所述多核苷酸以提供用于它们的表达。取决于表达载体,在多核苷酸***载体之前对其进行操作可以是理想的或必需的。用于利用重组DNA方法修饰多核苷酸的技术是本领域熟知的。
所述控制序列可为启动子,即,被宿主细胞识别用于表达编码本发明的多肽的多核苷酸的多核苷酸。所述启动子包含介导多肽的表达的转录控制序列。所述启动子可以是在宿主细胞中显示出转录活性的任何多核苷酸,包括变体、截短型及杂合型启动子,并且可以从编码与所述宿主细胞同源或异源的细胞外或细胞内多肽的基因获得。
用于在细菌宿主细胞中指导本发明核酸构建体的转录的适合启动子的实例是从以下基因中获得的启动子:解淀粉芽孢杆菌α-淀粉酶基因(amyQ)、地衣芽孢杆菌α-淀粉酶基因(amyL)、地衣芽孢杆菌青霉素酶基因(penP)、嗜热脂肪芽孢杆菌产麦芽糖淀粉酶基因(amyM)、枯草芽孢杆菌果聚糖蔗糖酶基因(sacB)、枯草芽孢杆菌xylA和xylB基因、苏云金芽孢杆菌cryIIIA基因(Agaisse和Lereclus,1994,Molecular Microbiology[分子微生物学]13:97-107)、大肠杆菌lac操纵子、大肠杆菌trc启动子(Egon等人,1988,Gene[基因]69:301-315)、天蓝链霉菌琼脂水解酶基因(dagA)和原核β-内酰胺酶基因(Villa-Kamaroff等人,1978,Proc.Natl.Acad.Sci.USA[美国国家科学院院刊]75:3727-3731)以及tac启动子(DeBoer等人,1983,Proc.Natl.Acad.Sci.USA[美国国家科学院院刊]80:21-25)。其他启动子描述于Gilbert等人,1980,Scientific American[科学美国人]242:74-94的“Usefulproteins from recombinant bacteria[来自重组细菌的有用蛋白质]”;和在Sambrook等人,1989,同上。串联启动子的实例披露于WO 99/43835中。
用于在丝状真菌宿主细胞中指导本发明核酸构建体的转录的适合启动子的实例是从以下基因中获得的启动子:构巢曲霉乙酰胺酶、黑曲霉中性α-淀粉酶、黑曲霉酸稳定性α-淀粉酶、黑曲霉或泡盛曲霉葡糖淀粉酶(glaA)、米曲霉TAKA淀粉酶、米曲霉碱性蛋白酶、米曲霉丙糖磷酸异构酶、尖孢胰蛋白酶样蛋白酶(WO 96/00787)、镶片镰孢菌淀粉葡糖苷酶(WO 00/56900)、镶片镰孢菌Daria(WO 00/56900)、镶片镰孢菌Quinn(WO 00/56900)、米黑根毛霉脂肪酶、米黑根毛霉天冬氨酸蛋白酶、里氏木霉β-葡糖苷酶、里氏木霉纤维二糖水解酶I、里氏木霉纤维二糖水解酶II、里氏木霉内切葡聚糖酶I、里氏木霉内切葡聚糖酶II、里氏木霉内切葡聚糖酶III、里氏木霉内切葡聚糖酶V、里氏木霉木聚糖酶I、里氏木霉木聚糖酶II、里氏木霉木聚糖酶III、里氏木霉β-木糖苷酶,以及里氏木霉翻译延伸因子,以及NA2-tpi启动子(来自编码中性α-淀粉酶的曲霉属基因的修饰的启动子,其中已经用来自编码丙糖磷酸异构酶的曲霉属基因的未翻译的前导序列替换未翻译的前导序列;非限制性实例包括来自黑曲霉中性α-淀粉酶基因的修饰的启动子,其中已经用来自构巢曲霉或米曲霉丙糖磷酸异构酶基因的未翻译的前导序列替换未翻译的前导序列);及其变体、截短型、以及杂合型启动子。其他启动子在美国专利号6,011,147中描述。
在酵母宿主中,有用的启动子从以下的基因获得:酿酒酵母烯醇酶(ENO-1)、酿酒酵母半乳糖激酶(GAL1)、酿酒酵母醇脱氢酶/甘油醛-3磷酸脱氢酶(ADH1、ADH2/GAP)、酿酒酵母丙糖磷酸异构酶(TPI)、酿酒酵母金属硫蛋白(CUP1)以及酿酒酵母3磷酸甘油酸激酶。酵母宿主细胞的其他有用的启动子由Romanos等人,1992,Yeast[酵母]8:423-488描述。
控制序列也可以是由宿主细胞识别以终止转录的转录终止子。所述终止子可操作地连接至多核苷酸的3'-末端。在宿主细胞中有功能的任何终止子可用于本发明中。
细菌宿主细胞的优选终止子从以下的基因获得:克劳氏芽孢杆菌碱性蛋白酶(aprH)、地衣芽孢杆菌α-淀粉酶(amyL)、和大肠杆菌核糖体RNA(rrnB)。
用于丝状真菌宿主细胞的优选终止子从以下的基因获得:构巢曲霉乙酰胺酶、构巢曲霉邻氨基苯甲酸合酶、黑曲霉葡糖淀粉酶、黑曲霉α-葡糖苷酶、米曲霉TAKA淀粉酶、尖孢镰孢胰蛋白酶样蛋白酶、里氏木霉β-葡糖苷酶、里氏木霉纤维二糖水解酶I、里氏木霉纤维二糖水解酶II、里氏木霉内切葡聚糖酶I、里氏木霉内切葡聚糖酶II、里氏木霉内切葡聚糖酶III、里氏木霉内切葡聚糖酶V、里氏木霉木聚糖酶I、里氏木霉木聚糖酶II、里氏木霉木聚糖酶III、里氏木霉β-木糖苷酶以及里氏木霉翻译延伸因子。
酵母宿主细胞的优选终止子从以下的基因获得:酿酒酵母烯醇酶、酿酒酵母细胞色素C(CYC1)以及酿酒酵母甘油醛-3磷酸脱氢酶。酵母宿主细胞的其他有用的终止子由Romanos等人(1992,同上)描述。
控制序列还可以是启动子下游和基因的编码序列上游的mRNA稳定子区域,其增加所述基因的表达。
适合的mRNA稳定子区域的实例从以下获得:苏云金芽孢杆菌cryIIIA基因(WO 94/25612)和枯草芽孢杆菌SP82基因(Hue等人,1995,Journal of Bacteriology[细菌学杂志]177:3465-3471)。
控制序列也可以是前导序列,即对宿主细胞翻译很重要的mRNA的非翻译区域。所述前导序列可操作地连接至多核苷酸的5'-末端。可以使用在宿主细胞中有功能的任何前导序列。
用于丝状真菌宿主细胞的优选前导序列从米曲霉TAKA淀粉酶和构巢曲霉丙糖磷酸异构酶的基因获得。
酵母宿主细胞的适合的前导序列从以下的基因获得:酿酒酵母烯醇酶(ENO-1)、酿酒酵母3磷酸甘油酸激酶、酿酒酵母α-因子和酿酒酵母醇脱氢酶/甘油醛-3磷酸脱氢酶(ADH2/GAP)。
控制序列还可以是多腺苷酸化序列,一种可操作地连接至所述多核苷酸的3’-末端并且当转录时由宿主细胞识别为将多腺苷酸残基添加至所转录的mRNA的信号的序列。可以使用在宿主细胞中有功能的任何多腺苷酸化序列。
用于丝状真菌宿主细胞的优选多腺苷酸化序列从以下酶的基因获得:构巢曲霉邻氨基苯甲酸合酶、黑曲霉葡糖淀粉酶、黑曲霉α-葡糖苷酶、米曲霉TAKA淀粉酶以及尖孢镰孢胰蛋白酶样蛋白酶。
酵母宿主细胞的有用的多腺苷酸化序列由Guo和Sherman,1995,Mol.CellularBiol.[分子细胞生物学]15:5983-5990描述。
控制序列还可以是编码与多肽的N末端连接的信号肽并指导多肽进入细胞的分泌途径的信号肽编码区。多核苷酸的编码序列的5’端本身可以含有在翻译阅读框中天然与编码多肽的编码序列区段相连接的信号肽编码序列。可替代地,编码序列的5’端可以含有对于所述编码序列是外源的信号肽编码序列。在编码序列天然地不含信号肽编码序列的情况下,可能需要外源信号肽编码序列。可替代地,外源信号肽编码序列可以单纯地替换天然信号肽编码序列以便增强多肽的分泌。然而,可以使用指导已表达多肽进入宿主细胞的分泌途径的任何信号肽编码序列。
用于细菌宿主细胞的有效信号肽编码序列是从芽孢杆菌NCIB 11837产麦芽糖淀粉酶、地衣芽孢杆菌枯草杆菌蛋白酶、地衣芽孢杆菌β-内酰胺酶、嗜热脂肪芽孢杆菌α-淀粉酶、嗜热脂肪芽孢杆菌中性蛋白酶(nprT、nprS、nprM)和枯草芽孢杆菌prsA的基因获得的信号肽编码序列。另外的信号肽由Simonen和Palva,1993,Microbiological Reviews[微生物评论]57:109-137描述。
用于丝状真菌宿主细胞的有效的信号肽编码序列是从以下酶的基因获得的信号肽编码序列:黑曲霉中性淀粉酶、黑曲霉葡糖淀粉酶、米曲霉TAKA淀粉酶、特异腐质霉纤维素酶、特异腐质霉内切葡聚糖酶V、疏棉状腐质霉脂肪酶和米黑根毛霉天冬氨酸蛋白酶。
用于酵母宿主细胞的有用的信号肽从酿酒酵母α-因子和酿酒酵母转化酶的基因获得。其他的有用的信号肽编码序列由Romanos等人(1992,同上)描述。
控制序列还可以是编码位于多肽的N末端的前肽的前肽编码序列。所得的多肽被称为前体酶或多肽原(或在一些情况下被称为酶原(zymogen))。多肽原通常是无活性的并且可通过催化切割或自身催化切割来自多肽原的前肽而转化为活性多肽。前肽编码序列可以从以下的基因获得:枯草芽孢杆菌碱性蛋白酶(aprE)、枯草芽孢杆菌中性蛋白酶(nprT)、嗜热毁丝霉漆酶(WO 95/33836)、米黑根毛霉天冬氨酸蛋白酶和酿酒酵母α-因子。
在信号肽序列和前肽序列二者都存在的情况下,所述前肽序列位于紧邻多肽的N-末端且所述信号肽序列位于紧邻前肽序列的N-末端。
还可希望的是添加调节序列,所述调节序列调节宿主细胞生长相关的多核苷酸的表达。调节序列的实例是引起多核苷酸表达以响应于化学或物理刺激(包括调节化合物的存在)而开启或关闭的那些。原核***中的调节序列包括lac、tac、和trp操纵子***。在酵母中,可以使用ADH2***或GAL1***。在丝状真菌中,可以使用黑曲霉葡糖淀粉酶启动子、米曲霉TAKA α-淀粉酶启动子和米曲霉葡糖淀粉酶启动子、里氏木霉纤维二糖水解酶I启动子以及里氏木霉纤维二糖水解酶II启动子。调节序列的其他实例是允许基因扩增的那些序列。在真核***中,这些调节序列包括在甲氨蝶呤存在下扩增的二氢叶酸还原酶基因以及用重金属扩增的金属硫蛋白基因。在这些情况中,多核苷酸将与调节序列可操作地连接。
表达载体
本发明还涉及包含本发明的多核苷酸、启动子、以及转录和翻译终止信号的重组表达载体。各种核苷酸和控制序列可以连接在一起以产生重组表达载体,所述重组表达载体可以包括一个或多个合宜的限制位点以允许在此类位点处***或取代多核苷酸。可替代地,可以通过将多核苷酸或包含所述多核苷酸的核酸构建体***用于表达的适当载体中而表达所述多核苷酸。在产生表达载体时,编码序列如此位于载体中,使得编码序列与用于表达的适当控制序列可操作地连接。
重组表达载体可以是可以方便地经受重组DNA程序并且可以引起多核苷酸表达的任何载体(例如,质粒或病毒)。载体的选择将典型地取决于载体与待引入载体的宿主细胞的相容性。载体可以是线性或闭合环状质粒。
载体可以是自主复制载体,即作为染色体外实体存在的载体,其复制独立于染色体复制,例如质粒、染色体外元件、微染色体或人工染色体。载体可以含有用于确保自我复制的任何手段。可替代地,载体可以是这样的载体,当它引入宿主细胞中时整合入基因组中并与其中已整合了它的一个或多个染色体一起复制。此外,可以使用单独的载体或质粒或两个或更多个载体或质粒,其共同含有待引入宿主细胞基因组的总DNA,或可以使用转座子。
载体优选地含有允许方便地选择转化细胞、转染细胞、转导细胞等细胞的一个或多个选择性标记。选择性标记是一种基因,其产物提供了杀生物剂抗性或病毒抗性、对重金属抗性、对营养缺陷型的原养型等。
细菌选择性标记的实例是地衣芽孢杆菌或枯草芽孢杆菌dal基因、赋予氨基酸或其他代谢物营养缺陷型的标记、或赋予抗生素抗性(如氨苄青霉素、氯霉素、卡那霉素、新霉素、大观霉素、或四环素抗性)的标记。酵母宿主细胞的适合的标记包括但不限于:ADE2、HIS3、LEU2、LYS2、MET3、TRP1和URA3。用于在丝状真菌宿主细胞中使用的选择性标记包括但不限于adeA(磷酸核糖酰氨基咪唑-琥珀羧胺合酶)、adeB(磷酸核糖酰-氨基咪唑合酶)、amdS(乙酰胺酶)、argB(鸟氨酸氨甲酰基转移酶)、bar(草丁膦乙酰转移酶)、hph(潮霉素磷酸转移酶)、niaD(硝酸还原酶)、pyrG(乳清酸核苷-5'磷酸脱羧酶)、sC(硫酸腺苷基转移酶)以及trpC(邻氨基苯甲酸合酶)连同其等效物。优选的用于曲霉细胞中的是构巢曲霉或米曲霉amdS和pyrG基因以及吸水链霉菌(Streptomyces hygroscopicus)bar基因。优选的用于木霉属细胞的是adeA、adeB、amdS、hph以及pyrG基因。
选择性标记可以是如WO 2010/039889中所述的双选择性标记***。在一个方面,双选择性标记是hph-tk双选择性标记***。
载体优选地含有允许载体整合到宿主细胞的基因组中或载体在细胞中独立于基因组自主复制的一个或多个元件。
对于整合到宿主细胞基因组中,载体可以依靠多核苷酸序列或用于通过同源或非同源重组整合到基因组中的所述载体的任何其他元件。可替代地,所述载体可以含有用于指导通过同源重组而整合入宿主细胞基因组中的一个或多个染色体中的一个或多个精确位置处的另外的多核苷酸。为了增加在精确位置处整合的可能性,整合元件应当含有足够数目的核酸,例如100至10,000个碱基对、400至10,000个碱基对和800至10,000个碱基对,所述核酸与相应的靶序列具有高度序列同一性以增强同源重组的概率。整合元件可以是与宿主细胞基因组内的靶序列同源的任何序列。此外,整合元件可以是非编码或编码的多核苷酸。另一方面,载体可以通过非同源重组整合入宿主细胞的基因组中。
为了自主复制,载体还可以另外包含复制起点,所述复制起点使得载体在讨论中的宿主细胞中自主复制成为可能。复制起点可以是在细胞中发挥作用的介导自主复制的任何质粒复制子。术语“复制起点”或“质粒复制子”意指使质粒或载体能够在体内复制的多核苷酸。
细菌复制起点的实例是允许在大肠杆菌中复制的质粒pBR322、pUC19、pACYC177、和pACYC184的复制起点,以及允许在芽孢杆菌属中复制的质粒pUB110、pE194、pTA1060、和pAMβ1的复制起点。
用于酵母宿主细胞中的复制起点的实例是2微米复制起点、ARS1、ARS4、ARS1与CEN3的组合、及ARS4与CEN6的组合。
在丝状真菌细胞中有用的复制起点的实例是AMA1和ANS1(Gems等人,1991,Gene[基因]98:61-67;Cullen等人,1987,Nucleic Acids Res.[核酸研究]15:9163-9175;WO00/24883)。可以根据WO 00/24883中披露的方法完成AMA1基因的分离和包含所述基因的质粒或载体的构建。
可将本发明多核苷酸的多于一个拷贝***宿主细胞以增加多肽的产生。通过将序列的至少一个另外的拷贝整合到宿主细胞基因组中或通过包括一个与所述多核苷酸一起的可扩增的选择性标记基因可以获得所述多核苷酸的增加的拷贝数目,其中通过在适当的选择性试剂的存在下培养细胞可以选择含有选择性标记基因的经扩增的拷贝的细胞、以及由此所述多核苷酸的另外的拷贝。
用于连接以上所述的元件以构建本发明的重组表达载体的程序是本领域的普通技术人员熟知的(参见例如,Sambrook等人,1989,同上)。
宿主细胞
本发明还涉及重组宿主细胞,所述重组宿主细胞包含可操作地连接至一个或多个控制序列的本发明的多核苷酸,所述一个或多个控制序列指导本发明的多核苷酸的表达。将包含多核苷酸的构建体或载体引入到宿主细胞中,这样使得所述构建体或载体被维持作为染色体整合体或作为自主复制的染色体外载体,如早前所描述。术语“宿主细胞”涵盖由于复制期间出现的突变而与亲本细胞不完全相同的任何亲本细胞子代。
宿主细胞可以是任何有用的细胞,例如原核细胞或真核细胞。
原核宿主细胞可以是任何革兰氏阳性或革兰氏阴性细菌。***包括但不限于:芽孢杆菌属、梭菌属、肠球菌属、土芽孢杆菌属(Geobacillus)、乳杆菌属、乳球菌属、大洋芽孢杆菌属、葡萄球菌属、链球菌属和链霉菌属。革兰氏阴性细菌包括但不限于弯曲杆菌属、大肠杆菌、黄杆菌属、梭杆菌属、螺杆菌属、泥杆菌属、奈瑟氏菌属、假单胞菌属、沙门氏菌属、以及脲原体属。
细菌宿主细胞可以是任何芽孢杆菌属细胞,包括但不限于:嗜碱芽孢杆菌、高地芽孢杆菌、解淀粉芽孢杆菌、植物解淀粉芽孢杆菌亚种、短芽孢杆菌、环状芽孢杆菌、克劳氏芽孢杆菌、凝结芽孢杆菌、坚强芽孢杆菌、灿烂芽孢杆菌、迟缓芽孢杆菌、地衣芽孢杆菌、巨大芽孢杆菌、甲基营养型芽孢杆菌、短小芽孢杆菌、沙福芽孢杆菌、嗜热脂肪芽孢杆菌、枯草芽孢杆菌、和苏云金芽孢杆菌细胞。
细菌宿主细胞还可以是任何链球菌属细胞,包括但不限于类马链球菌、酿脓链球菌、***链球菌以及马链球菌兽疫亚种细胞。
细菌宿主细胞还可以是任何链霉菌属细胞,包括但不限于:不产色链霉菌、除虫链霉菌、天蓝链霉菌、灰色链霉菌以及浅青紫链霉菌细胞。
将DNA引入芽孢杆菌属细胞中可以通过以下来实现:原生质体转化(参见,例如,Chang和Cohen,1979,Mol.Gen.Genet.[分子遗传学与基因组学]168:111-115)、感受态细胞转化(参见例如,Young和Spizizen,1961,J.Bacteriol.[细菌学杂志]81:823-829;或Dubnau和Davidoff-Abelson,1971,J.Mol.Biol.[分子生物学杂志]56:209-221)、电穿孔(参见例如,Shigekawa和Dower,1988,Biotechniques[生物技术]6:742-751)或接合(参见例如,Koehler和Thorne,1987,J.Bacteriol.[细菌学杂志]169:5271-5278)。将DNA引入大肠杆菌细胞中可以通过以下方式来实现:原生质体转化(参见例如,Hanahan,1983,J.Mol.Biol.[分子生物学杂志]166:557-580)或电穿孔(参见例如,Dower等人,1988,Nucleic Acids Res.[核酸研究]16:6127-6145)。将DNA引入链霉菌属细胞中可以通过以下方式来实现:原生质体转化、电穿孔(参见例如,Gong等人,2004,Folia Microbiol.(Praha)[叶线形微生物学(布拉格)]49:399-405)、接合(参见例如,Mazodier等人,1989,J.Bacteriol.[细菌学杂志]171:3583-3585)、或转导(参见例如,Burke等人,2001,Proc.Natl.Acad.Sci.USA[美国国家科学院院刊]98:6289-6294)。将DNA引入假单孢菌属细胞中可以通过以下方式来实现:电穿孔(参见例如,Choi等人,2006,J.Microbiol.Methods[微生物学方法杂志]64:391-397)或接合(参见例如,Pinedo和Smets,2005,Appl.Environ.Microbiol.[应用与环境微生物学]71:51-57)。将DNA引入链球菌属细胞中可以通过以下方式来实现:天然感受态(natural competence)(参见例如,Perry和Kuramitsu,1981,Infect.Immun.[感染与免疫]32:1295-1297)、原生质体转化(参见例如,Catt和Jollick,1991,Microbios[微生物学]68:189-207)、电穿孔(参见例如,Buckley等人,1999,Appl.Environ.Microbiol.[应用与环境微生物学]65:3800-3804)、或接合(参见例如,Clewell,1981,Microbiol.Rev.[微生物学评论]45:409-436)。然而,可以使用本领域已知的将DNA引入宿主细胞中的任何方法。
宿主细胞还可以是真核生物,如哺乳动物、昆虫、植物或真菌细胞。
宿主细胞可以是真菌细胞。如本文所用的“真菌”包括子囊菌门(Ascomycota)、担子菌门(Basidiomycota)、壶菌门(Chytridiomycota)和接合菌门(Zygomycota)以及卵菌门(Oomycota)和所有有丝***孢子真菌(如由Hawksworth等人在以下文献中所定义:Ainsworth and Bisby’s Dictionary of The Fungi[安斯沃思和拜斯比真菌字典],第8版,1995,CAB International[国际应用生物科学中心],University Press[大学出版社],Cambridge,UK[英国剑桥])。
真菌宿主细胞可以为酵母细胞。如本文所用的“酵母”包括产子囊酵母(内孢霉目)、产担子酵母及属于半知菌纲(芽孢纲)的酵母。由于对酵母的分类将来可能变化,出于本发明的目的,酵母应当按照Biology and Activities of Yeast[酵母的生物学与活性](Skinner,Passmore和Davenport编辑,Soc.App.Bacteriol.Symposium Series No.9[应用细菌学学会专题论文集系列9],1980)中所描述的定义。
酵母宿主细胞可以是假丝酵母属(Candida)、汉逊酵母属(Hansenula)、克鲁弗酵母属(Kluyveromyces)、毕赤酵母属(Pichia)、酵母菌属(Saccharomyces)、裂殖酵母属(Schizosaccharomyces)或耶氏酵母属(Yarrowia)细胞,如乳酸克鲁维酵母(Kluyveromyces lactis)、卡尔酵母(Saccharomyces carlsbergensis)、酿酒酵母(Saccharomyces cerevisiae)、糖化酵母(Saccharomyces diastaticus)、道格拉氏酵母(Saccharomyces douglasii)、克鲁弗酵母(Saccharomyces kluyveri)、诺地酵母(Saccharomyces norbensis)、卵形酵母(Saccharomyces oviformis)或解脂耶氏酵母(Yarrowia lipolytica)细胞。
真菌宿主细胞可为丝状真菌细胞。“丝状真菌”包括真菌门(Eumycota)和卵菌门(Oomycota)的亚门的所有丝状形式(如由Hawksworth等人,1995(同上)所定义的)。丝状真菌通常的特征在于由几丁质、纤维素、葡聚糖、壳聚糖、甘露聚糖和其他复杂多糖构成的菌丝体壁。营养生长是通过菌丝延伸来进行的,而碳分解代谢是专性需氧的。相反,酵母(如酿酒酵母)的营养生长是通过单细胞菌体的出芽(budding)来进行的,而碳分解代谢可以是发酵性的。
丝状真菌宿主细胞可以是枝顶孢霉属、曲霉属、短梗霉属、烟管霉属(Bjerkandera)、拟腊菌属、金孢子菌属、鬼伞属、革盖菌属(Coriolus)、隐球菌属、线黑粉菌科(Filibasidium)、镰孢属、腐质霉属、梨孢菌属、毛霉属、毁丝霉属、新美鞭菌属、链孢菌属、拟青霉属、青霉属、平革菌属、射脉菌属(Phlebia)、瘤胃壶菌属、侧耳属(Pleurotus)、裂褶菌属、篮状菌属、嗜热子囊菌属、梭孢壳属、弯颈霉属、栓菌属(Trametes)或木霉属细胞。
例如,丝状真菌宿主细胞可以是泡盛曲霉、臭曲霉、烟曲霉、日本曲霉、构巢曲霉、黑曲霉、米曲霉、黑刺烟管菌(Bjerkandera adusta)、干拟蜡菌(Ceriporiopsisaneirina)、卡内基拟蜡菌(Ceriporiopsis caregiea)、浅黄拟蜡孔菌(Ceriporiopsisgilvescens)、潘诺希塔拟蜡菌(Ceriporiopsis pannocinta)、环带拟蜡菌(Ceriporiopsisrivulosa)、微红拟蜡菌(Ceriporiopsis subrufa)、虫拟蜡菌(Ceriporiopsissubvermispora)、狭边金孢子菌(Chrysosporium inops)、嗜角质金孢子菌、卢克诺文思金孢子菌(Chrysosporium lucknowense)、粪状金孢子菌(Chrysosporium merdarium)、租金孢子菌、女王杜香金孢子菌(Chrysosporium queenslandicum)、热带金孢子菌、褐薄金孢子菌(Chrysosporium zonatum)、灰盖鬼伞(Coprinus cinereus)、毛革盖菌(Coriolushirsutus)、杆孢状镰孢、谷类镰孢、库威镰孢、大刀镰孢、禾谷镰孢、禾赤镰孢、异孢镰孢、合欢木镰孢、尖孢镰孢、多枝镰孢、粉红镰孢、接骨木镰孢、肤色镰孢、拟分枝孢镰孢、硫色镰孢、圆镰孢、拟丝孢镰孢、镶片镰孢、特异腐质霉、柔毛腐质霉、米黑毛霉、嗜热毁丝霉、粗糙链孢菌、产紫青霉、黄孢平革菌(Phanerochaete chrysosporium)、射脉菌(Phlebiaradiata)、刺芹侧耳(Pleurotus eryngii)、土生梭孢壳霉、长域毛栓菌(Trametesvillosa)、变色栓菌(Trametes versicolor)、哈茨木霉、康宁木霉、长枝木霉、里氏木霉、或绿色木霉细胞。
可以将真菌细胞通过涉及原生质体形成、原生质体转化、以及细胞壁再生的方法以本身已知的方式转化。用于转化曲霉属和木霉属宿主细胞的适合程序描述于以下文献中:EP 238023,Yelton等人,1984,Proc.Natl.Acad.Sci.USA[美国国家科学院院刊]81:1470-1474以及Christensen等人,1988,Bio/Technology[生物/技术]6:1419-1422。用于转化镰孢属物种的适合方法由Malardier等人,1989,Gene[基因]78:147-156和WO 96/00787描述。可以使用由以下文献描述的程序转化酵母:Becker和Guarente,在Abelson,J.N.和Simon,M.I.编辑,Guide to Yeast Genetics and Molecular Biology[酵母遗传学与分子生物学指南],Methods in Enzymology[酶学方法],第194卷,第182-187页,AcademicPress,Inc.[学术出版社有限公司],纽约);Ito等人,1983,J.Bacteriol.[细菌学杂志]153:163;以及Hinnen等人,1978,Proc.Natl.Acad.Sci.USA[美国国家科学院院刊]75:1920。
II类Cas9蛋白的无效核酸酶变体
若干个II类Cas9类似物或同系物是已知的,并且随着近几年科学兴趣增加被更多发现;在Makarova等人的综述(20015,An updated evolutionary classification ofCRISPR–Cas systems[CRISPR–Cas***的更新的进化分类],Nature[自然]13:722-736)中提供。
酿脓链球菌的Cas9蛋白(SEQ ID NO:2)是一种标准II类Cas9蛋白并且迄今为止具有最佳特征。通过在位置10:D10A处取代单个氨基酸,天冬氨酸替代丙氨酸,开发了仅具有一个活性核酸酶结构域(与野生型蛋白中的两个活性结构域相反)的这种蛋白的变体。通过在位置840:H840A处取代单个氨基酸,组氨酸替代丙氨酸,开发了仅具有一个活性核酸酶结构域(与野生型蛋白中的两个活性结构域相反)的这种蛋白的另一种变体。双取代的(D10A、H840A)变体是无效核酸酶变体。预期其他II类Cas9酶可以被类似地修饰。
相应地,在优选的实施例中,II类Cas9蛋白的无效核酸酶变体在对应于酿脓链球菌Cas9氨基酸序列中的位置10的氨基酸位置处包含取代;优选地,所述II类Cas9蛋白的无效核酸酶变体在酿脓链球菌Cas9氨基酸序列中包含天冬氨酸取代丙氨酸,D10A。还优选地,所述II类Cas9蛋白的无效核酸酶变体在对应于酿脓链球菌Cas9氨基酸序列中的位置840的氨基酸位置处包含取代;优选地,所述II类Cas9蛋白的无效核酸酶变体在酿脓链球菌Cas9氨基酸序列中包含组氨酸取代丙氨酸,H840A。
预期其他核酸内切酶(如Cpf1)(Zetsche等人,Cell[细胞]2015,163,第759-771页)以及Inscripta公司(www.inscripta.com,前身为慕芮斯生物科技(Muse Bio))发布的MAD酶(如MAD7)的无效核酸酶变体也可用于如本文所述的反向选择目的。相应地,此类无效核酸酶变体在本发明的范围内。
指导RNA
CRISPR-Cas9基因组编辑中的gRNA构成了可重复编程的部分,使得***如此通用。在天然酿脓链球菌***中,gRNA实际上是两种RNA多核苷酸的复合物,第一crRNA含有约20个核苷酸,其确定了Cas9蛋白的特异性和与crRNA杂交以形成与Cas9相互作用的RNA复合物的tracr RNA(参见Jinek等人,2012,A programmable dual-RNA-guided DNAendonuclease in adaptive bacterial immunity[在自适应细菌免疫中的可编程双重-RNA-指导的DNA核酸内切酶],Science[科学]337:816-821)。术语crRNA和tracrRNA在本文中与术语tracr-配对RNA和tracr RNA可互换地使用。
由于CRISPR-Cas9***的发现,单一多核苷酸gRNA已经被开发并成功应用,与天然两部分gRNA复合物一样有效。
在优选的实施例中,所述单一gRNA或RNA复合物包含第一RNA,所述第一RNA包含与所述一个或多个基因组靶序列至少85%互补并且能够与所述一个或多个基因组靶序列杂交的20个或更多个核苷酸;优选地,所述20个或更多个核苷酸与所述一个或多个基因组靶序列至少90%、95%、97%、98%、99%或甚至100%互补并且能够与所述一个或多个基因组靶序列杂交。
在另一个优选的实施例中,宿主细胞包含单一gRNA,所述单一gRNA包含单个多核苷酸形式的第一和第二RNA,并且其中当彼此杂交时,tracr配对序列和tracr序列形成茎环结构。
为了使Cas9-gRNA复合物能够与给定的靶序列杂交,所述靶序列应侧接针对II类Cas9蛋白的功能性PAM序列。有关PAM序列的综述,参见,例如,Shah等人,2013,Protospacerrecognition motifs[原型间隔子识别基序],RNA Biol.[RNA生物学]10(5):891–899。
实例
材料与方法
用作缓冲液和底物的化学品是至少试剂级的商业产品。
使用标准教科书程序,采用商业热循环仪和来自商业供应商的Ready-To-Go PCRbeads,Phusion聚合酶或RED-TAQ聚合酶进行PCR扩增。
LB琼脂:参见EP 0 506 780。
LBPSG琼脂板含有LB琼脂,补充有磷酸盐(0.01M K3PO4)、葡萄糖(0.4%)和淀粉(0.5%);参见EP 0 805 867B1。
TY(液体肉汤培养基;参见WO 1994/14968,p16)。
寡核苷酸引物获得自DNA技术(奥尔胡斯,丹麦)。用可商购的试剂盒和试剂,使用标准教科书程序进行DNA操作(质粒和基因组DNA制备、限制性消化、纯化、连接、DNA测序)。
在一些情况下,使用来自通用电气医疗集团(GE Healthcare)的TempliPhi试剂盒,在等温滚环扩增反应中扩增连接混合物。
使用两步式程序(Yasbin等人,1975,J.Bacteriol.[细菌学杂志]121:296-304.)或一步式程序将DNA引入到枯草芽孢杆菌经提炼的天然感受态细胞中,其中将来自琼脂板的细胞材料再悬浮于Spizisen 1培养基(12ml)(WO 2014/052630)中,在37℃以200rpm振荡约4小时,将DNA添加至400微升等分试样中,并且在选择性琼脂板上铺板之前,将这些等分试样在所希望的温度下,以150rpm振荡另外1小时。
使用含有pLS20的经修饰的枯草芽孢杆菌供体菌株PP3724,基本上如之前所描述的(EP 2 029 732 B1),通过来自枯草芽孢杆菌的接合,将DNA引入到地衣芽孢杆菌中,其中甲基化酶基因M.bli1904II(US 20130177942)从在amyE基因座处的三联启动子表达,pBC16衍生的orfβ和枯草芽孢杆菌comS基因(和卡那霉素抗性基因)从在alr基因座处的三联启动子表达(使得需要D-丙氨酸菌株),并且枯草芽孢杆菌comS基因(和cat基因)从在pel基因座处的三联启动子表达。
枯草芽孢杆菌JA1343:JA1343是PL1801的孢子形成阴性衍生物(WO 2005/042750)。部分spollAC基因已经被删除,以获得孢子形成阴性表型。
实例中描述的所有构建都是从基因艺术-赛默飞世尔科技公司(GeneArt-ThermoFisher Scientific)订购的合成DNA片段组装而成。如实例中所述,片段通过序列重叠延伸(SOE)进行组装。
根据先前描述的方法(美国专利号5,843,720),通过染色体整合与切割将本专利中使用的温度敏感性质粒并入地衣芽孢杆菌的基因组中。含质粒的地衣芽孢杆菌转化子在50℃在具有红霉素的LBPG选择性培养基上生长,以迫使载体在相同的序列处整合到染色体上。基于其在50℃、在LBPG+红霉素选择性培养基上生长的能力来选择所希望的整合体。然后,使整合体于37℃无选择性地生长在LBPG培养基中,以允许切割整合的质粒。将细胞铺在LBPG板上并且针对红霉素-敏感性进行筛选。检查敏感性克隆是否正确整合了所需的构建体。
根据先前描述的方法(Pitcher等人,同上)或通过使用从凯杰公司(Qiagen)可商购的QIAamp DNA血液试剂盒,从以上若干红霉素敏感性分离株中制备基因组DNA。
菌株
PP3724:含有pLS20,其中甲基化酶基因M.bli1904II(US 20130177942)从在amyE基因座处的三联启动子表达,pBC16衍生的orfβ和枯草芽孢杆菌comS基因(和卡那霉素抗性基因)从在alr基因座处的三联启动子表达(使得需要D-丙氨酸菌株),并且枯草芽孢杆菌comS基因(和cat基因)从在pel基因座处的三联启动子表达。
JA1622:这种菌株是具有破坏的spoIIAC基因(sigF)的枯草芽孢杆菌168衍生物JA578,其描述于WO 2002/00907中。所述基因型是:amyE::repF(pE194)、spoIIAC。
SJ1904:这种菌株是描述于WO 2008/066931中的地衣芽孢杆菌菌株。编码碱性蛋白酶的基因(aprL)是失活的。
PP3811:地衣芽孢杆菌菌株SJ1904的衍生物,其中碱性蛋白酶基因aprL、金属蛋白酶mprL和spoIIAC基因是失活的。
PP3811-cas9d:这种菌株是地衣芽孢杆菌菌株PP3811,其中cas9d基因在bglC基因座处***。最终***物具有从WO 1993/010249中描述的PamyL启动子变体转录的cas9d基因。整合后染色体上的最终序列在图1和SEQ ID NO:3中描述。
PP3811-gDNA1:这种菌株是地衣芽孢杆菌菌株PP3811-cas9d,其中将dsRED基因和转录针对地衣芽孢杆菌中catL基因的gRNA(cat)的gDNA(cat)***至gnt基因座。在gDNA的更下游,定位了来自噬菌体TP901-1的attB位点(WO 2006/042548)。dsRED基因从WO 1999/043835中描述的三联启动子表达。整合后染色体上的最终序列在图2和SEQ ID NO:4中描述。
PP3811-gDNA2:这种菌株是地衣芽孢杆菌菌株PP3811-gDNA1,其中将dsRED基因和转录针对地衣芽孢杆菌中catL基因的gRNA(cat)的gDNA(cat)***至amyL基因座。在gDNA的更下游,定位了attB位点(参见上文)。整合后染色体上的最终序列在图3和SEQ ID NO:5中描述。
PP3811-gDNA3:这种菌株是地衣芽孢杆菌PP3811-gDNA2,其中将dsRED基因和转录针对地衣芽孢杆菌中catL基因的gRNA(cat)的gDNA(cat)***至lacA2基因座。在gDNA的更下游,定位了attB位点(参见上文)。整合后染色体上的最终序列在图4和SEQ ID NO:6中描述。
PP3811-amyL3:这是地衣芽孢杆菌菌株PP3811-gDNA3,其中dsRED基因和gDNA(cat)的三个拷贝被编码地衣芽孢杆菌α-淀粉酶的amyL基因的三个拷贝替换。图5-7和SEQID NO:7-9中描述了替换后染色体三个基因座的最终序列。
PP3724-pPPamyL-attP:这种菌株是含有质粒pPPamyL-attP的接合供体菌株PP3724。
质粒
pC194:从金黄色葡萄球菌中分离的质粒(Horinouchi和Weisblum,1982)。
pE194:从金黄色葡萄球菌中分离的质粒(Horinouchi和Weisblum,1982)。
pUB110:从金黄色葡萄球菌中分离的质粒(McKenzie等人,1986)
pPPamyL-attP:在实例6中为本发明构建的质粒。所述质粒通过合成序列组装制备,以产生载体,所述载体含有:(1)编码地衣芽孢杆菌α-淀粉酶的amyL基因,其前接用于整合的cry3A稳定子(2)WO 2006/042548中描述的来自TP901-1的attP和整合酶(int)。所述整合酶促进了地衣芽孢杆菌宿主质粒上attP位点和染色体上attB位点的整合。
序列
实例1.将cas9d染色体整合到地衣芽孢杆菌的bglC基因座中
将表达盒***bglC基因座处,其中编码Cas9(Cas9d)的无活性变体的cas9d基因从由WO 1993/010249中先前描述的amyL启动子(P4199)表达。用于整合的DNA作为合成DNA订购(基因艺术-赛默飞世尔科技公司),并克隆到如先前在WO 2006/042548中描述的整合载体中。在图1中显示了bglC基因座的最终图谱。所述基因座的核苷酸序列可在SEQ ID NO:3中找到。
用于PCR扩增的条件如下:使用Phusion热启动DNA聚合酶***(赛默科技公司(Thermo Scientific))通过PCR扩增对应的DNA片段。PCR扩增反应混合物含有1ul(约0,1ug)的模板DNA、2ul的正义引物(20pmol/ul)、2ul的反义引物(20pmol/ul)、10ul的5X PCR缓冲液(具有7.5mM MgCl2、8ul的dNTP混合物(各1,25mM)、37μl水和0.5ul(2U/ul)DNA聚合酶混合物)。使用热循环仪来扩增片段。根据制造商的说明书,使用QIAGENQIAquick凝胶提取试剂盒(凯杰公司(Qiagen,Inc.),巴伦西亚,加利福尼亚州),用1x TBE缓冲液从1.2%琼脂糖凝胶中纯化PCR产物。
将PCR产物用于随后的PCR反应中,以使用剪接重叠PCR(SOE)使用Phusion热启动DNA聚合酶***(赛默科技公司)如下产生单个质粒。PCR扩增反应混合物含有两种凝胶纯化的PCR产物和合成片段各50ng,并用热循环仪组装并扩增质粒。将所得的SOE产物直接用于转化枯草芽孢杆菌宿主JA1622以建立质粒。将质粒通过感受态转移到供体菌株PP3724中。
将菌株地衣芽孢杆菌菌株用上述质粒转化,并根据上文材料与方法部分中所述的程序整合和切割。通过该程序,染色体上bglC基因座被通过质粒递送的克隆的构建体替换。已用图1所示的构建体替换了最终菌株的bglC基因座,并且质粒在50℃的限制性温度下丢失。
最终构建体具有从染色体上的bglC基因座表达的cas9d基因。菌株命名为PP3811-cas9d。
实例2.将dsRED-gDNA(cat)染色体整合到地衣芽孢杆菌的gnt基因座中
将表达盒***gnt基因座处,其中编码红色荧光蛋白的dsRED标记基因从由WO2005/098016中先前描述的P3启动子表达。在dsRED标记基因的下游,gDNA序列从解淀粉芽孢杆菌的amyQ启动子表达。gDNA转录针对cat标记基因的gRNA。cat标记基因编码地衣芽孢杆菌的乙酰转移酶,所述乙酰转移酶赋予对氯霉素的抗性。已在WO 2007/138049中描述了将DNA染色体整合到地衣芽孢杆菌中。用于整合的DNA作为合成DNA订购(基因艺术-赛默飞世尔科技公司),通过SOE-PCR组装并克隆到如先前描述的基于pE194的温度敏感性整合载体中。在图1中显示了gnt基因座的最终图谱。所述基因座的核苷酸序列可在SEQ ID NO:4中找到。
PCR产物如实例1中所述制备并用于随后的PCR反应中,以使用剪接重叠PCR(SOE)使用Phusion热启动DNA聚合酶***(赛默科技公司)如下产生单个质粒。PCR扩增反应混合物含有两种凝胶纯化的PCR产物和合成片段各50ng,并用热循环仪组装并扩增整合质粒。将所得的SOE产物直接用于转化枯草芽孢杆菌宿主JA1622以建立整合质粒。将质粒转移到供体菌株PP3724中并用于接合。所述质粒用于根据实例1中所述的程序在地衣芽孢杆菌的gnt基因座处***dsRED基因和gDNA(cat)。最终菌株命名为PP3811-gDNA1。
实例3.将dsRED-gDNA(cat)染色体整合到地衣芽孢杆菌的amyL基因座中
在amyL基因座处***与实例2中所述的表达盒相同的表达盒。用于整合的DNA作为合成DNA订购(基因艺术-赛默飞世尔科技公司),通过SOE-PCR组装并克隆到如先前在WO2006/042548中描述的基于pE194的温度敏感性整合载体中。在图3中显示了amyL基因座的最终图谱。所述基因座的核苷酸序列可在SEQ ID NO:5中找到。
PCR产物如实例1中所述制备并用于随后的PCR反应中,以使用剪接重叠PCR(SOE)使用Phusion热启动DNA聚合酶***(赛默科技公司)如下产生单个质粒。PCR扩增反应混合物含有两种凝胶纯化的PCR产物和合成片段各50ng,并用热循环仪组装并扩增整合质粒。将所得的SOE产物直接用于转化枯草芽孢杆菌宿主JA1622以建立整合质粒。该质粒用于如以上在实例1中所述的在地衣芽孢杆菌的amyL基因座处***dsRED基因和gDNA(cat)。最终菌株命名为PP3811-gDNA2。
实例4.将dsRED-gDNA(cat)染色体整合到地衣芽孢杆菌的lacA2基因座中
在lacA2基因座处***与实例2和3中所述的表达盒几乎相同的表达盒。唯一的区别是dsRED基因的可替代的合成序列(dsREDsyn)。该基因变体仍编码相同的荧光蛋白。用于整合的DNA作为合成DNA订购(基因艺术-赛默飞世尔科技公司),并克隆到先前在WO 2006/042548中描述的整合载体中。在图4中显示了amyL基因座的最终图谱。所述基因座的核苷酸序列可在SEQ ID NO:6中找到。
PCR产物如实例1中所述制备并用于随后的PCR反应中,以使用剪接重叠PCR(SOE)使用Phusion热启动DNA聚合酶***(赛默科技公司)如下产生单个质粒。PCR扩增反应混合物含有两种凝胶纯化的PCR产物和合成片段各50ng,并用热循环仪组装并扩增整合质粒。将所得的SOE产物直接用于转化枯草芽孢杆菌宿主JA1622以建立整合质粒。该质粒用于如以上在实例1中所述的在地衣芽孢杆菌的lacA2基因座处***dsRED基因和gDNA(cat)。最终菌株命名为PP3811-gDNA3,其具有dsRED基因的三个拷贝和gDNA(catL)盒以及从bglC基因座表达的Cas9d(图8)。
实例5.质粒pPPamyL-attP的构建
质粒pPPamyL-attP由从基因艺术公司订购的DNA序列组装。在图9中描绘了完整的质粒和标注。所述质粒的核苷酸序列可在SEQ ID NO:10中找到。
用于PCR扩增的条件如实例1中所述。将纯化的PCR产物用于随后的PCR反应中,以使用剪接重叠PCR(SOE)使用Phusion热启动DNA聚合酶***(赛默科技公司)如下产生单个质粒。PCR扩增反应混合物含有六种凝胶纯化的PCR产物各50ng,并用热循环仪组装并扩增9550bp的质粒(图1)。将所得的SOE产物直接用于转化枯草芽孢杆菌宿主JA1622以建立质粒pPPamyL-attP。所述质粒在实例6中用于转化实例4中描述的宿主菌株PP64。
所述质粒编码来自地衣芽孢杆菌的淀粉酶基因amyL,其侧接在cry3A稳定子区域和attP噬菌体整合位点的上游。
amyL整合入染色体将发生在存在于宿主菌株PP3811-gDNA3中和质粒上的cry3A稳定子区域以及分别在染色体和质粒上的attB和attP位点之间。
实例6.选择淀粉酶基因amyL的三拷贝整合
将实例5中所述的质粒pPPamyL-attP转化到地衣芽孢杆菌菌株PP3811-gDNA3中,用于对三个不同基因座(gnt:dsRED-gDNA(cat)、amyL:dsRED-gDNA(cat)和lacA2:dsRED-gDNA(cat))中的amyL表达盒的分步整合进行选择。在该步骤中,gDNA(cat)和dsRED基因被amyL表达盒替换。通过在染色体上gDNA基因座上的侧翼区和引入的质粒之间的重组增强替换。上游是宿主菌株PP3811-gDNA3的染色体上和质粒pPPamyL-attP上存在的相同的cry3A稳定子区域。下游是分别在染色体和质粒上的attB和attP位点。
质粒转化PP3811-gDNA3后,将细胞在34℃下用1ug/ml红霉素在LBPG板上铺板三天,以允许在许可温度下在染色体和质粒之间发生扩增和重组事件。将菌落在200ul TY中洗涤,并且将50ul转移到5ml TY液体培养物中,并在34℃下以200rpm孵育24小时。在LBPG板上用6ug/ml氯霉素(cam)对培养物进行划线,以选择其中所有三个gDNA(cat)基因座都被amyL表达盒替换的菌株。
对来自cam板的十个不同菌落进行重新划线,并测试所有三个基因座中的amyL整合。所有十个菌落均在琼脂糖凝胶上显示出预期的条带。
所有三个基因座均被有效替换,并按预期***amyL表达盒。图5-7显示了替换后的所有基因座和用于验证***的引物。菌株命名为PP3811-3amyL
氯霉素抗性克隆均具有淀粉酶活性,这通过在补充有淀粉的LBPG板上进行铺板显示。所有菌落均在补充有淀粉的板上显示出大圈,这证实了淀粉酶的表达。
该实例表明,gDNA(cat)与Cas9d组合可以非常有效地用作一种工具,所述工具用于对地衣芽孢杆菌染色体上表达盒的至少三个拷贝的整合进行选择。
实例7.使用flp/FRT技术以选择DNA的三拷贝整合的宿主细胞构建。
地衣芽孢杆菌菌株PP3811-gDNA3(实例4)用作构建菌株的基础,其中可以通过flp/FRT技术(WO 2018/077796)对目的基因的三个拷贝的整合进行选择。
在实例6中,将amyL整合盒***到可用于菌株PP3811-gDNA3的三个不同基因座(即:gnt:dsRED-gDNA(cat)、amyL:dsRED-gDNA(cat)和lacA2:dsRED-gDNA(cat))中的每一个。
在本实例中,除选择CamR之外,还使用如用于amyL整合的相同的程序来将如下区段整合到PP3811-gDNA3中,所述区段由FRT-F、绿色荧光蛋白(GFP)编码区、转录针对cat标记基因的gRNA的gDNA序列(从解淀粉芽孢杆菌的amyQ启动子表达)和FRT-F3组成。用于整合的DNA作为合成DNA订购(基因艺术-赛默飞世尔科技公司),并且接受在保持为pSJ13864的载体中。将1.6kb BglII-MluI片段从pSJ13864切割并连接到pPP3676的7.4kb BglII-MluI片段,产生pSJ14072(图10)。所述1.6kb BglII-MluI片段具有SEQ ID NO:15中给出的DNA序列。
将pSJ14072引入接合供体菌株PP3724中,并将所得的转化子库用作供体与PP3811-gDNA3接合。如实例6中所述进行质粒和染色体之间的重组。将由所有三个基因座中所希望的整合产生的菌株分离为绿色荧光菌落,其相对于亲本生物的红色荧光菌落容易被看到。所有细胞仍然对氯霉素敏感,因为转录针对cat的gRNA的gDNA仍然存在于所有3个基因座处。这种宿主细胞被保持为SJ14111。
实例8.使用flp/FRT技术分离被选择以含有三个拷贝的淀粉酶基因amyL的菌株。
为了构建用于flp/FRT***的α-淀粉酶整合载体,将amyL编码区使用引物#B692和#B693从地衣芽孢杆菌扩增,并克隆到如先前描述(WO 2018/077796)的pSJ13654中,获得所希望的重组质粒(pSJ13835),将其引入枯草芽孢杆菌接合供体菌株和保持为SJ13837的转化子中。因此,整合载体携带由FRT-F、amyL和FRT-F3组成的区段。
将SJ13837用作供体与SJ14111接合。混合供体/受体培养物在固体培养基上生长后,用红霉素(2ug/ml)在培养基上进行影印,将板在33℃下孵育4天,随后将一条混合转移接合子(使用10ul的接种环)接种于含或不含氯霉素(6ug/ml)的液体培养基(TY)中,并在37℃下振荡孵育。第二天,不含氯霉素的培养物显示出强绿色荧光,而含氯霉素的培养物则不显示荧光。这与以下观点一致,即如果某种物质(在这种情况下是amyL)替换了染色体中的GFP+gDNA(cat),则获得氯霉素抗性。使用培养物接种新的培养物,这次是在含或不含10ug/ml氯霉素的情况下进行的,并且再次含氯霉素的培养物不显示荧光。铺板后,获得大量非荧光菌落。证实四个这样的菌落是氯霉素抗性和红霉素敏感的,并且当通过PCR扩增分析三个整合基因座中的每一个时,所有菌落均被证实含有侧翼为FRT的amyL构建体的***。
序列表
<110> 诺维信公司(Novozymes A/S)
<120> 通过抑制条件性必需基因进行反向选择
<130> 14386-WO-PCT
<160> 15
<170> PatentIn 3.5版
<210> 1
<211> 4107
<212> DNA
<213> 酿脓链球菌
<400> 1
atggacaaaa aatacagcat cggcctggat attggcacaa attcagttgg ctgggcagtt 60
atcacagacg aatataaagt tccgagcaaa aaatttaaag tcctgggcaa tacagatcgc 120
catagcatca aaaaaaacct gattggcgca ctgctgtttg attcaggcga aacagcagaa 180
gcaacaagac ttaaaagaac agcaagacgc agatatacaa gacgcaaaaa tcgcatttgc 240
tatctgcaag aaatctttag caacgaaatg gcgaaagtcg acgacagctt ttttcataga 300
ctggaagaat catttctggt cgaagaagat aaaaaacacg aacgccatcc gatttttggc 360
aacattgttg atgaagtcgc gtatcatgaa aaatacccga caatttatca tctgcgcaaa 420
aaactggttg acagcacaga taaagcagat cttcgcctga tttatctggc actggcacat 480
atgatcaaat ttagaggcca ttttctgatc gaaggcgatc tgaatccgga taattcagat 540
gtcgacaaac tgtttattca gctggtccag acatataacc agctgtttga agaaaatccg 600
attaatgcat caggcgttga tgcaaaagca attctgtcag caagactgtc aaaatcaaga 660
cgcctggaaa atctgattgc acaactgcct ggcgaaaaaa aaaatggact gtttggcaat 720
cttattgcac tgtcactggg cctgacaccg aactttaaat caaattttga tctggcggaa 780
gatgcgaaac tgcaactttc aaaagatacg tatgatgacg atctggataa tctgctggcg 840
caaattggcg atcaatatgc agatcttttt ctggcagcga aaaatctgtc agatgcaatt 900
ctgctgtcag atattctgcg cgtcaataca gaaattacaa aagcaccgct gagcgcgagc 960
atgattaaaa gatatgatga acatcatcag gacctgacac tgctgaaagc actggttaga 1020
caacaactgc cggaaaaata caaagaaatc ttttttgatc agagcaaaaa cggctatgca 1080
ggctatattg atggcggagc atcacaagaa gaattttaca aatttatcaa accgatcctg 1140
gaaaaaatgg atggaacaga agaactgctg gttaaactga atcgcgaaga tttactgaga 1200
aaacagcgca catttgataa tggctcaatt ccgcatcaaa ttcatctggg cgaactgcat 1260
gcgattctta gacgccaaga agatttttat ccgtttctga aagacaaccg ggaaaaaatt 1320
gaaaaaatcc tgacatttcg catcccgtat tatgtcggac cgctggcaag aggcaattca 1380
agatttgcat ggatgacacg caaaagcgaa gaaacaatta caccgtggaa ttttgaagaa 1440
gtcgttgata aaggcgcaag cgcacaatca tttattgaac gcatgacgaa ctttgacaaa 1500
aacctgccga atgaaaaagt cctgccgaaa cattcactgc tgtatgaata ctttacggtc 1560
tataatgaac tgacgaaagt caaatatgtc acagaaggca tgagaaaacc ggcatttctg 1620
tcaggcgaac agaaaaaagc gattgtcgat cttctgttta aaacgaaccg caaagtcaca 1680
gtgaaacagc tgaaagaaga ttactttaaa aaaatcgaat gctttgatag cgtcgaaatc 1740
tcaggcgtcg aagatagatt taatgcaagc ctgggcacat atcatgatct gctgaaaatc 1800
atcaaagata aagattttct ggataacgaa gaaaacgaag atatcctgga agatattgtg 1860
ctgacactga cgctttttga agatcgcgaa atgattgaag aacgcctgaa aacatatgcg 1920
cacctgtttg atgataaagt catgaaacaa cttaaacgca gacgctatac aggctggggc 1980
agactttcaa gaaaactgat taacggcatt cgcgataaac aaagcggcaa aacaatcctg 2040
gattttctga aatcagatgg ctttgcgaat cgcaatttta tgcagctgat tcatgatgac 2100
agcctgacgt ttaaagaaga tattcagaaa gcacaagttt caggccaagg cgattcactg 2160
catgaacata ttgcaaatct ggcaggctca ccggcaatca aaaaaggcat tctgcaaaca 2220
gttaaagtcg tcgatgaact ggttaaagtt atgggcagac ataaaccgga aaacatcgtt 2280
attgaaatgg cacgcgaaaa tcagacaaca caaaaaggac agaaaaattc acgcgaacgg 2340
atgaaaagaa ttgaagaagg cattaaagaa ctgggcagcc aaatcctgaa agaacatccg 2400
gttgaaaata cacagctgca gaacgaaaaa ctgtatctgt attatctgca gaatggacgc 2460
gatatgtatg tcgatcaaga actggatatt aatcgcctga gcgattatga tgtggatcat 2520
attgttccgc agagctttct taaagatgat agcatcgata acaaagtcct gacacgctca 2580
gataaaaaca gaggcaaatc agataatgtc ccgtcagaag aggttgtcaa aaaaatgaaa 2640
aactactggc gtcaactgct gaacgcgaaa cttattacac aacgcaaatt tgacaatctg 2700
acaaaagcag aaagaggcgg actgtcagaa cttgataaag cgggttttat caaaagacag 2760
ctggtcgaaa cacgccagat tacaaaacat gttgcgcaaa ttctggatag ccgcatgaac 2820
acaaaatatg acgaaaacga taaactgatc cgggaagtca aagtcattac gctgaaatca 2880
aaactggtca gcgattttcg caaagacttt cagttttaca aagtccgcga aatcaacaac 2940
taccatcatg cacatgatgc atatctgaat gcagttgtcg gcacagcgct tatcaaaaaa 3000
taccctaaac tggaaagcga atttgtctac ggcgactata aagtctatga tgtccgcaaa 3060
atgattgcga aaagcgaaca agaaattggc aaagcgacag cgaaatactt tttttacagc 3120
aacatcatga acttttttaa aacggaaatc acactggcga acggcgaaat tagaaaaaga 3180
ccgcttattg aaacgaacgg tgaaacaggc gaaattgttt gggataaagg cagagatttt 3240
gcaacagtta gaaaagttct gagcatgccg caagtcaaca tcgtgaaaaa aacagaagtt 3300
cagacaggcg gatttagcaa agaatcaatt cttccgaaac gcaactcaga caaactgatt 3360
gcgcgtaaaa aagactggga cccgaaaaaa tacggtggct ttgattcacc gacagttgca 3420
tattcagttc tggttgttgc gaaagtggaa aaaggcaaat ccaaaaaact taaaagcgtg 3480
aaagaacttc tgggcatcac aattatggaa cgctcgagct ttgaaaaaaa cccgatcgac 3540
tttctggaag ccaaaggcta taaagaagtg aaaaaagacc ttattatcaa actgccgaaa 3600
tacagcctgt ttgaactgga aaatggcaga aaacgcatgc tggcatcagc aggcgaactt 3660
cagaaaggca atgaactggc actgccgtca aaatatgtta actttctgta tctggcgagc 3720
cattacgaaa aacttaaagg ctcaccggaa gataacgaac agaaacaact gtttgtcgaa 3780
cagcataaac attacctgga cgaaatcatc gaacaaatca gcgaattttc aaaacgcgtt 3840
attctggcag atgcgaacct ggataaagtt cttagcgcat ataacaaaca ccgggataaa 3900
ccgattagag aacaagcgga aaatatcatt cacctgttta cactgacaaa tcttggcgca 3960
ccggcagcgt ttaaatactt tgatacaaca attgaccgca aacgctacac aagcacaaaa 4020
gaagttctgg acgcaacact gattcatcaa tcaattacag gcctttatga aacgcgcatt 4080
gatctgtcac aactgggagg cgattga 4107
<210> 2
<211> 1368
<212> PRT
<213> 酿脓链球菌
<400> 2
Met Asp Lys Lys Tyr Ser Ile Gly Leu Asp Ile Gly Thr Asn Ser Val
1 5 10 15
Gly Trp Ala Val Ile Thr Asp Glu Tyr Lys Val Pro Ser Lys Lys Phe
20 25 30
Lys Val Leu Gly Asn Thr Asp Arg His Ser Ile Lys Lys Asn Leu Ile
35 40 45
Gly Ala Leu Leu Phe Asp Ser Gly Glu Thr Ala Glu Ala Thr Arg Leu
50 55 60
Lys Arg Thr Ala Arg Arg Arg Tyr Thr Arg Arg Lys Asn Arg Ile Cys
65 70 75 80
Tyr Leu Gln Glu Ile Phe Ser Asn Glu Met Ala Lys Val Asp Asp Ser
85 90 95
Phe Phe His Arg Leu Glu Glu Ser Phe Leu Val Glu Glu Asp Lys Lys
100 105 110
His Glu Arg His Pro Ile Phe Gly Asn Ile Val Asp Glu Val Ala Tyr
115 120 125
His Glu Lys Tyr Pro Thr Ile Tyr His Leu Arg Lys Lys Leu Val Asp
130 135 140
Ser Thr Asp Lys Ala Asp Leu Arg Leu Ile Tyr Leu Ala Leu Ala His
145 150 155 160
Met Ile Lys Phe Arg Gly His Phe Leu Ile Glu Gly Asp Leu Asn Pro
165 170 175
Asp Asn Ser Asp Val Asp Lys Leu Phe Ile Gln Leu Val Gln Thr Tyr
180 185 190
Asn Gln Leu Phe Glu Glu Asn Pro Ile Asn Ala Ser Gly Val Asp Ala
195 200 205
Lys Ala Ile Leu Ser Ala Arg Leu Ser Lys Ser Arg Arg Leu Glu Asn
210 215 220
Leu Ile Ala Gln Leu Pro Gly Glu Lys Lys Asn Gly Leu Phe Gly Asn
225 230 235 240
Leu Ile Ala Leu Ser Leu Gly Leu Thr Pro Asn Phe Lys Ser Asn Phe
245 250 255
Asp Leu Ala Glu Asp Ala Lys Leu Gln Leu Ser Lys Asp Thr Tyr Asp
260 265 270
Asp Asp Leu Asp Asn Leu Leu Ala Gln Ile Gly Asp Gln Tyr Ala Asp
275 280 285
Leu Phe Leu Ala Ala Lys Asn Leu Ser Asp Ala Ile Leu Leu Ser Asp
290 295 300
Ile Leu Arg Val Asn Thr Glu Ile Thr Lys Ala Pro Leu Ser Ala Ser
305 310 315 320
Met Ile Lys Arg Tyr Asp Glu His His Gln Asp Leu Thr Leu Leu Lys
325 330 335
Ala Leu Val Arg Gln Gln Leu Pro Glu Lys Tyr Lys Glu Ile Phe Phe
340 345 350
Asp Gln Ser Lys Asn Gly Tyr Ala Gly Tyr Ile Asp Gly Gly Ala Ser
355 360 365
Gln Glu Glu Phe Tyr Lys Phe Ile Lys Pro Ile Leu Glu Lys Met Asp
370 375 380
Gly Thr Glu Glu Leu Leu Val Lys Leu Asn Arg Glu Asp Leu Leu Arg
385 390 395 400
Lys Gln Arg Thr Phe Asp Asn Gly Ser Ile Pro His Gln Ile His Leu
405 410 415
Gly Glu Leu His Ala Ile Leu Arg Arg Gln Glu Asp Phe Tyr Pro Phe
420 425 430
Leu Lys Asp Asn Arg Glu Lys Ile Glu Lys Ile Leu Thr Phe Arg Ile
435 440 445
Pro Tyr Tyr Val Gly Pro Leu Ala Arg Gly Asn Ser Arg Phe Ala Trp
450 455 460
Met Thr Arg Lys Ser Glu Glu Thr Ile Thr Pro Trp Asn Phe Glu Glu
465 470 475 480
Val Val Asp Lys Gly Ala Ser Ala Gln Ser Phe Ile Glu Arg Met Thr
485 490 495
Asn Phe Asp Lys Asn Leu Pro Asn Glu Lys Val Leu Pro Lys His Ser
500 505 510
Leu Leu Tyr Glu Tyr Phe Thr Val Tyr Asn Glu Leu Thr Lys Val Lys
515 520 525
Tyr Val Thr Glu Gly Met Arg Lys Pro Ala Phe Leu Ser Gly Glu Gln
530 535 540
Lys Lys Ala Ile Val Asp Leu Leu Phe Lys Thr Asn Arg Lys Val Thr
545 550 555 560
Val Lys Gln Leu Lys Glu Asp Tyr Phe Lys Lys Ile Glu Cys Phe Asp
565 570 575
Ser Val Glu Ile Ser Gly Val Glu Asp Arg Phe Asn Ala Ser Leu Gly
580 585 590
Thr Tyr His Asp Leu Leu Lys Ile Ile Lys Asp Lys Asp Phe Leu Asp
595 600 605
Asn Glu Glu Asn Glu Asp Ile Leu Glu Asp Ile Val Leu Thr Leu Thr
610 615 620
Leu Phe Glu Asp Arg Glu Met Ile Glu Glu Arg Leu Lys Thr Tyr Ala
625 630 635 640
His Leu Phe Asp Asp Lys Val Met Lys Gln Leu Lys Arg Arg Arg Tyr
645 650 655
Thr Gly Trp Gly Arg Leu Ser Arg Lys Leu Ile Asn Gly Ile Arg Asp
660 665 670
Lys Gln Ser Gly Lys Thr Ile Leu Asp Phe Leu Lys Ser Asp Gly Phe
675 680 685
Ala Asn Arg Asn Phe Met Gln Leu Ile His Asp Asp Ser Leu Thr Phe
690 695 700
Lys Glu Asp Ile Gln Lys Ala Gln Val Ser Gly Gln Gly Asp Ser Leu
705 710 715 720
His Glu His Ile Ala Asn Leu Ala Gly Ser Pro Ala Ile Lys Lys Gly
725 730 735
Ile Leu Gln Thr Val Lys Val Val Asp Glu Leu Val Lys Val Met Gly
740 745 750
Arg His Lys Pro Glu Asn Ile Val Ile Glu Met Ala Arg Glu Asn Gln
755 760 765
Thr Thr Gln Lys Gly Gln Lys Asn Ser Arg Glu Arg Met Lys Arg Ile
770 775 780
Glu Glu Gly Ile Lys Glu Leu Gly Ser Gln Ile Leu Lys Glu His Pro
785 790 795 800
Val Glu Asn Thr Gln Leu Gln Asn Glu Lys Leu Tyr Leu Tyr Tyr Leu
805 810 815
Gln Asn Gly Arg Asp Met Tyr Val Asp Gln Glu Leu Asp Ile Asn Arg
820 825 830
Leu Ser Asp Tyr Asp Val Asp His Ile Val Pro Gln Ser Phe Leu Lys
835 840 845
Asp Asp Ser Ile Asp Asn Lys Val Leu Thr Arg Ser Asp Lys Asn Arg
850 855 860
Gly Lys Ser Asp Asn Val Pro Ser Glu Glu Val Val Lys Lys Met Lys
865 870 875 880
Asn Tyr Trp Arg Gln Leu Leu Asn Ala Lys Leu Ile Thr Gln Arg Lys
885 890 895
Phe Asp Asn Leu Thr Lys Ala Glu Arg Gly Gly Leu Ser Glu Leu Asp
900 905 910
Lys Ala Gly Phe Ile Lys Arg Gln Leu Val Glu Thr Arg Gln Ile Thr
915 920 925
Lys His Val Ala Gln Ile Leu Asp Ser Arg Met Asn Thr Lys Tyr Asp
930 935 940
Glu Asn Asp Lys Leu Ile Arg Glu Val Lys Val Ile Thr Leu Lys Ser
945 950 955 960
Lys Leu Val Ser Asp Phe Arg Lys Asp Phe Gln Phe Tyr Lys Val Arg
965 970 975
Glu Ile Asn Asn Tyr His His Ala His Asp Ala Tyr Leu Asn Ala Val
980 985 990
Val Gly Thr Ala Leu Ile Lys Lys Tyr Pro Lys Leu Glu Ser Glu Phe
995 1000 1005
Val Tyr Gly Asp Tyr Lys Val Tyr Asp Val Arg Lys Met Ile Ala
1010 1015 1020
Lys Ser Glu Gln Glu Ile Gly Lys Ala Thr Ala Lys Tyr Phe Phe
1025 1030 1035
Tyr Ser Asn Ile Met Asn Phe Phe Lys Thr Glu Ile Thr Leu Ala
1040 1045 1050
Asn Gly Glu Ile Arg Lys Arg Pro Leu Ile Glu Thr Asn Gly Glu
1055 1060 1065
Thr Gly Glu Ile Val Trp Asp Lys Gly Arg Asp Phe Ala Thr Val
1070 1075 1080
Arg Lys Val Leu Ser Met Pro Gln Val Asn Ile Val Lys Lys Thr
1085 1090 1095
Glu Val Gln Thr Gly Gly Phe Ser Lys Glu Ser Ile Leu Pro Lys
1100 1105 1110
Arg Asn Ser Asp Lys Leu Ile Ala Arg Lys Lys Asp Trp Asp Pro
1115 1120 1125
Lys Lys Tyr Gly Gly Phe Asp Ser Pro Thr Val Ala Tyr Ser Val
1130 1135 1140
Leu Val Val Ala Lys Val Glu Lys Gly Lys Ser Lys Lys Leu Lys
1145 1150 1155
Ser Val Lys Glu Leu Leu Gly Ile Thr Ile Met Glu Arg Ser Ser
1160 1165 1170
Phe Glu Lys Asn Pro Ile Asp Phe Leu Glu Ala Lys Gly Tyr Lys
1175 1180 1185
Glu Val Lys Lys Asp Leu Ile Ile Lys Leu Pro Lys Tyr Ser Leu
1190 1195 1200
Phe Glu Leu Glu Asn Gly Arg Lys Arg Met Leu Ala Ser Ala Gly
1205 1210 1215
Glu Leu Gln Lys Gly Asn Glu Leu Ala Leu Pro Ser Lys Tyr Val
1220 1225 1230
Asn Phe Leu Tyr Leu Ala Ser His Tyr Glu Lys Leu Lys Gly Ser
1235 1240 1245
Pro Glu Asp Asn Glu Gln Lys Gln Leu Phe Val Glu Gln His Lys
1250 1255 1260
His Tyr Leu Asp Glu Ile Ile Glu Gln Ile Ser Glu Phe Ser Lys
1265 1270 1275
Arg Val Ile Leu Ala Asp Ala Asn Leu Asp Lys Val Leu Ser Ala
1280 1285 1290
Tyr Asn Lys His Arg Asp Lys Pro Ile Arg Glu Gln Ala Glu Asn
1295 1300 1305
Ile Ile His Leu Phe Thr Leu Thr Asn Leu Gly Ala Pro Ala Ala
1310 1315 1320
Phe Lys Tyr Phe Asp Thr Thr Ile Asp Arg Lys Arg Tyr Thr Ser
1325 1330 1335
Thr Lys Glu Val Leu Asp Ala Thr Leu Ile His Gln Ser Ile Thr
1340 1345 1350
Gly Leu Tyr Glu Thr Arg Ile Asp Leu Ser Gln Leu Gly Gly Asp
1355 1360 1365
<210> 3
<211> 5888
<212> DNA
<213> 人工序列
<220>
<223> bglC-cas9d基因座的序列
<400> 3
tgtttttgat aagatcacgg agtttatccg gaaaccgttc atgaagaaga agcagacgat 60
tgatgaacaa gggcatgtag aaacgaaaaa agtgccgaaa tcaaacttcg gctatttgct 120
gaattgctat tggtgcgcag ggatatggtg cgcgttgatc attgctgtcg gatatctgat 180
tgccccaaaa gcgatattcc cgttgatttt gattttgtcg gtcgccgggg ggcaggcgat 240
tcttgaaacg tttgtcggtg tcgccacaaa acttgtcggc tttttctccg atttaaagaa 300
gtaaaccatt ccaagcggat ggttttattt ttttgtcaat aaagtgatac aaacagcaga 360
gagaacgtgt cagttttatg aacttttcac agcgattttt cccggatgcg gcattttagg 420
cagagaggaa gcatctcatt gtaaagattt cagtttttaa aatttagaat tgagagaaaa 480
aggatgtgca aagtccccgg agctcggatc cactagtaac ggccgccagt gtgctggaat 540
tcgcccttgc ggccgctcgc tttccaatct gaaggtttca ttgtgggatg ttgatccgga 600
agattggaag tacaaaaata agcaaaagat tgtcaatcat gtcatgagcc atgcgggaga 660
cggaaaaatc gtcttaatgc acgatattta tgcaacgtcc gcagatgctg ctgaagagat 720
tattaaaaag ctgaaagcaa aaggctatca attggtaact gtatctcagc ttgaagaagt 780
gaagaagcag agaggctatt gaataaatga gtagaaagcg ccatatcggc gcttttcttt 840
tggaagaaaa tatagggaaa atggtacttg ttaaaaattc ggaatattta tacaatatca 900
tatgtatcac attgaaaggg gaggagaatc atggacaaaa aatacagcat cggcctggct 960
attggcacaa attcagttgg ctgggcagtt atcacagacg aatataaagt tccgagcaaa 1020
aaatttaaag tcctgggcaa tacagatcgc catagcatca aaaaaaacct gattggcgca 1080
ctgctgtttg attcaggcga aacagcagaa gcaacaagac ttaaaagaac agcaagacgc 1140
agatatacaa gacgcaaaaa tcgcatttgc tatctgcaag aaatctttag caacgaaatg 1200
gcgaaagtcg acgacagctt ttttcataga ctggaagaat catttctggt cgaagaagat 1260
aaaaaacacg aacgccatcc gatttttggc aacattgttg atgaagtcgc gtatcatgaa 1320
aaatacccga caatttatca tctgcgcaaa aaactggttg acagcacaga taaagcagat 1380
cttcgcctga tttatctggc actggcacat atgatcaaat ttagaggcca ttttctgatc 1440
gaaggcgatc tgaatccgga taattcagat gtcgacaaac tgtttattca gctggtccag 1500
acatataacc agctgtttga agaaaatccg attaatgcat caggcgttga tgcaaaagca 1560
attctgtcag caagactgtc aaaatcaaga cgcctggaaa atctgattgc acaactgcct 1620
ggcgaaaaaa aaaatggact gtttggcaat cttattgcac tgtcactggg cctgacaccg 1680
aactttaaat caaattttga tctggcggaa gatgcgaaac tgcaactttc aaaagatacg 1740
tatgatgacg atctggataa tctgctggcg caaattggcg atcaatatgc agatcttttt 1800
ctggcagcga aaaatctgtc agatgcaatt ctgctgtcag atattctgcg cgtcaataca 1860
gaaattacaa aagcaccgct gagcgcgagc atgattaaaa gatatgatga acatcatcag 1920
gacctgacac tgctgaaagc actggttaga caacaactgc cggaaaaata caaagaaatc 1980
ttttttgatc agagcaaaaa cggctatgca ggctatattg atggcggagc atcacaagaa 2040
gaattttaca aatttatcaa accgatcctg gaaaaaatgg atggaacaga agaactgctg 2100
gttaaactga atcgcgaaga tttactgaga aaacagcgca catttgataa tggctcaatt 2160
ccgcatcaaa ttcatctggg cgaactgcat gcgattctta gacgccaaga agatttttat 2220
ccgtttctga aagacaaccg ggaaaaaatt gaaaaaatcc tgacatttcg catcccgtat 2280
tatgtcggac cgctggcaag aggcaattca agatttgcat ggatgacacg caaaagcgaa 2340
gaaacaatta caccgtggaa ttttgaagaa gtcgttgata aaggcgcaag cgcacaatca 2400
tttattgaac gcatgacgaa ctttgacaaa aacctgccga atgaaaaagt cctgccgaaa 2460
cattcactgc tgtatgaata ctttacggtc tataatgaac tgacgaaagt caaatatgtc 2520
acagaaggca tgagaaaacc ggcatttctg tcaggcgaac agaaaaaagc gattgtcgat 2580
cttctgttta aaacgaaccg caaagtcaca gtgaaacagc tgaaagaaga ttactttaaa 2640
aaaatcgaat gctttgatag cgtcgaaatc tcaggcgtcg aagatagatt taatgcaagc 2700
ctgggcacat atcatgatct gctgaaaatc atcaaagata aagattttct ggataacgaa 2760
gaaaacgaag atatcctgga agatattgtg ctgacactga cgctttttga agatcgcgaa 2820
atgattgaag aacgcctgaa aacatatgcg cacctgtttg atgataaagt catgaaacaa 2880
cttaaacgca gacgctatac aggctggggc agactttcaa gaaaactgat taacggcatt 2940
cgcgataaac aaagcggcaa aacaatcctg gattttctga aatcagatgg ctttgcgaat 3000
cgcaatttta tgcagctgat tcatgatgac agcctgacgt ttaaagaaga tattcagaaa 3060
gcacaagttt caggccaagg cgattcactg catgaacata ttgcaaatct ggcaggctca 3120
ccggcaatca aaaaaggcat tctgcaaaca gttaaagtcg tcgatgaact ggttaaagtt 3180
atgggcagac ataaaccgga aaacatcgtt attgaaatgg cacgcgaaaa tcagacaaca 3240
caaaaaggac agaaaaattc acgcgaacgg atgaaaagaa ttgaagaagg cattaaagaa 3300
ctgggcagcc aaatcctgaa agaacatccg gttgaaaata cacagctgca gaacgaaaaa 3360
ctgtatctgt attatctgca gaatggacgc gatatgtatg tcgatcaaga actggatatt 3420
aatcgcctga gcgattatga tgtggatgct attgttccgc agagctttct taaagatgat 3480
agcatcgata acaaagtcct gacacgctca gataaaaaca gaggcaaatc agataatgtc 3540
ccgtcagaag aggttgtcaa aaaaatgaaa aactactggc gtcaactgct gaacgcgaaa 3600
cttattacac aacgcaaatt tgacaatctg acaaaagcag aaagaggcgg actgtcagaa 3660
cttgataaag cgggttttat caaaagacag ctggtcgaaa cacgccagat tacaaaacat 3720
gttgcgcaaa ttctggatag ccgcatgaac acaaaatatg acgaaaacga taaactgatc 3780
cgggaagtca aagtcattac gctgaaatca aaactggtca gcgattttcg caaagacttt 3840
cagttttaca aagtccgcga aatcaacaac taccatcatg cacatgatgc atatctgaat 3900
gcagttgtcg gcacagcgct tatcaaaaaa taccctaaac tggaaagcga atttgtctac 3960
ggcgactata aagtctatga tgtccgcaaa atgattgcga aaagcgaaca agaaattggc 4020
aaagcgacag cgaaatactt tttttacagc aacatcatga acttttttaa aacggaaatc 4080
acactggcga acggcgaaat tagaaaaaga ccgcttattg aaacgaacgg tgaaacaggc 4140
gaaattgttt gggataaagg cagagatttt gcaacagtta gaaaagttct gagcatgccg 4200
caagtcaaca tcgtgaaaaa aacagaagtt cagacaggcg gatttagcaa agaatcaatt 4260
cttccgaaac gcaactcaga caaactgatt gcgcgtaaaa aagactggga cccgaaaaaa 4320
tacggtggct ttgattcacc gacagttgca tattcagttc tggttgttgc gaaagtggaa 4380
aaaggcaaat ccaaaaaact taaaagcgtg aaagaacttc tgggcatcac aattatggaa 4440
cgctcgagct ttgaaaaaaa cccgatcgac tttctggaag ccaaaggcta taaagaagtg 4500
aaaaaagacc ttattatcaa actgccgaaa tacagcctgt ttgaactgga aaatggcaga 4560
aaacgcatgc tggcatcagc aggcgaactt cagaaaggca atgaactggc actgccgtca 4620
aaatatgtta actttctgta tctggcgagc cattacgaaa aacttaaagg ctcaccggaa 4680
gataacgaac agaaacaact gtttgtcgaa cagcataaac attacctgga cgaaatcatc 4740
gaacaaatca gcgaattttc aaaacgcgtt attctggcag atgcgaacct ggataaagtt 4800
cttagcgcat ataacaaaca ccgggataaa ccgattagag aacaagcgga aaatatcatt 4860
cacctgttta cactgacaaa tcttggcgca ccggcagcgt ttaaatactt tgatacaaca 4920
attgaccgca aacgctacac aagcacaaaa gaagttctgg acgcaacact gattcatcaa 4980
tcaattacag gcctttatga aacgcgcatt gatctgtcac aactgggagg cgattgaatt 5040
gacactaaag ggatccagaa gcggcaacac gctaatcaat aaaaaaacgc tgtgcggtta 5100
aagggcacag cgtttttttg tgtatgaatc gaaaaagaga acagatcgca ggtctcaaaa 5160
atcgagcgta aagggctgat ccgcggccgc gtcgactaga agagcagaga ggacggattt 5220
cctgaaggaa atccgttttt ttattttgcc cgtcttataa atttcgttgt ccaactcgct 5280
taattgcgag tttttatttc gtttatttca atcaaggtaa atgctagcgg ccgcgtcgac 5340
tagaagagca gagaggacgg atttcctgaa ggaaatccgt ttttttattt tgcccgtctt 5400
ataaatttcg ttgccatggg atccgcggcc gcgctgcagc caacacgata gcagtacaat 5460
acagagcggg ggacaacaat gtaaacggca accaaatccg ccctcagctc aacattaaaa 5520
acaacagcaa aaaaaccgtc tctttaaatc gaatcaccgt ccgctactgg tataaaacga 5580
atcgcaaagg aaaaaatttt gactgcgact atgcccaaat cggctgcagc aaaatcacgc 5640
acaaattcgt ccaattaaaa aaagcggtaa acggagcaga cacgtatctt gaagtagggt 5700
ttaaaaatgg tacattggcg ccgggtgcaa gtacaggtga aatccagatc cgtcttcaca 5760
atgacggctg gagcaattat gcccaaagcg gcgactattc atttttaaat tcaaacacgt 5820
ttaaaaatac gaaaaaaatc acgttgtatg agaacggaaa gctgatttgg ggcactgaac 5880
ctaaataa 5888
<210> 4
<211> 3133
<212> DNA
<213> 人工序列
<220>
<223> PP3811-gDNA3的gnt基因座的序列:gnt-dsRED-gDNA(cat)
<400> 4
agcgaagcct tgtgcatagg cgcagatttt gcccatatat aatgcctgtc tgacgcggtc 60
gatccacacg ttttgatcca ggcgccgttc ttctgttgca ggtccggcca atactttttc 120
cgcagctgtc cgttcgtctt ttaatgatga caggtaacgg gcaaacaggg attccgtgat 180
aattgatgat ggaatgccgt tgtcgacggc ctgcaggctc gtccatttgc ccgtgccttt 240
ttggccggtt ttgtcgagga tgacgtcgat gagtggagcg cccgtcttct catccttttt 300
ccgcaggatc tccgccgtga tttcgattaa atagctgttc agctctcctt gattccacgt 360
gtcgaaaatg tcagcgattt catctatcgg caaaagaagc ttttctctta aaaacgtata 420
tgcttcggcg atgagctgca tgtctgcgta ttcgatgccg ttgtgcacca ttttgacaaa 480
atgacccgcg cctttcggac ggccgctcgc tttccaatct gaaggtttca ttgtgggatg 540
ttgatccgga agattggaag tacaaaaata agcaaaagat tgtcaatcat gtcatgagcc 600
atgcgggaga cggaaaaatc gtcttaatgc acgatattta tgcaacgtcc gcagatgctg 660
ctgaagagat tattaaaaag ctgaaagcaa aaggctatca attggtaact gtatctcagc 720
ttgaagaagt gaagaagcag agaggctatt gaataaatga gtagaaagcg ccatatcggc 780
gcttttcttt tggaagaaaa tatagggaaa atggtacttg ttaaaaattc ggaatattta 840
tacaatatca tatgtatcac attgaaagga ggggcctgct gtccagactg tccgctgtgt 900
aaaaaaaagg aataaagggg ggttgacatt attttactga tatgtataat ataatttgta 960
taagaaaatg gaggggccct cgaaacgtaa gatgaaacct tagataaaag tgcttttttt 1020
gttgcaattg aagaattatt aatgttaagc ttaattaaag ataatatctt tgaattgtaa 1080
cgcccctcaa aagtaagaac tacaaaaaaa gaatacgtta tatagaaata tgtttgaacc 1140
ttcttcagat tacaaatata ttcggacgga ctctacctca aatgcttatc taactataga 1200
atgacataca agcacaacct tgaaaatttg aaaatataac taccaatgaa cttgttcatg 1260
tgaattatcg ctgtatttaa ttttctcaat tcaatatata atatgccaat acattgttac 1320
aagtagaaat taagacaccc ttgatagcct tactatacct aacatgatgt agtattaaat 1380
gaatatgtaa atatatttat gataagaagc gacttattta taatcattac atatttttct 1440
attggaatga ttaagattcc aatagaatag tgtataaatt atttatcttg aaaggaggga 1500
tgcctaaaaa cgaagaacat taaaaacata tatttgcacc gtctaatgga tttatgaaaa 1560
atcattttat cagtttgaaa attatgtatt atggagctct ataaaaatga ggagggaacc 1620
gaatggcttc aactgaagac gtaatcaaag agttcatgcg cttcaaagtg cgaatggaag 1680
gaagtgtaaa cgggcatgag tttgaaattg aaggtgaagg tgaaggaagg ccttatgaag 1740
gaacgcaaac tgcaaaactt aaagtgacaa aaggaggacc gctgccgttt gcttgggaca 1800
tcttaagtcc gcagtttcag tatgggtcaa aagtttatgt aaagcatcct gctgacattc 1860
ctgattacaa aaagttaagt tttcctgaag gattcaagtg ggagcgcgta atgaactttg 1920
aagatggagg tgtcgtaact gtaacgcaag attcaagtct gcaagacggt tgcttcattt 1980
acaaagtaaa gttcattggc gtgaactttc caagtgatgg tcctgtaatg cagaaaaaga 2040
caatgggttg ggagccgtca actgagaggc tttatccgcg tgatggtgtc ttgaaaggtg 2100
aaattcacaa agccttaaag ttgaaagatg gagggcatta tcttgttgag ttcaagagca 2160
tttacatggc gaaaaagcct gtgcagcttc ctggctacta ctatgttgat tcaaaacttg 2220
acataactag tcacaacgaa gactacacaa ttgttgagca gtatgagcga actgaaggaa 2280
ggcatcatct ttttctttaa tgctgtccag actgtccgct gtgtaaaaaa aaggaataaa 2340
ggggggttga cattatttta ctgatatgta taatataatt tgtataagaa aatgcttcat 2400
gtaatggtca aaatgtttta gagctagaaa tagcaagtta aaataaggct agtccgttat 2460
caacttgaaa aagtggcacc gagtcggtgc tttctgataa ttgccaacac aattaacatc 2520
tcaatcaagg taaatgctag cggccgcgtc gactagaaga gcagagagga cggatttcct 2580
gaaggaaatc cgttttttta ttttgcccgt cttataaatt tcgttgagat cttttataca 2640
aataggctta acaataaagt aaatcctaat ccggccaccg cgataattgt ttcaagcagt 2700
gtccaggtgg cgaatgtttc tttcatgctc aggccgaaat actctttgaa catccagaag 2760
cccgcgtcgt tgacatggga agcgattaca cttccggccc ctgttgcaag cacaaccagt 2820
gcaagattga catcgctttg tccgagcatc ggaagaacga gtccggtcgt gcttaatgca 2880
gcaactgtcg cggaacctaa agagatgcgc agaatcgcgg cgatgaccca ggcgagcaag 2940
atcggcgaca tggccgttcc tttgaataat tcagctacat agtcgcctac tccgccgttg 3000
atcaagactt gtttgaatgc gccgccgccc ccgatgatca agagcatcat tccgatttga 3060
gtaatggcgg ttgaacagga atccatcact tgtttgatcg ggatctttct ggcgataccc 3120
atcgtataaa tcg 3133
<210> 5
<211> 2813
<212> DNA
<213> 人工序列
<220>
<223> PP3811-gDNA3中amyL基因座的序列:amyL-dsRED-gDNA(cat)
<400> 5
tacagaagca tgaagggcat gcgaccttct ttgtgcttgg aagcagagcg caatattatc 60
ccgaaacgat aaaacggatg ctgaaggaag gaaacgaagt cggcaaccat tcctgggacc 120
atccgttatt gacaaggctg tcaaacgaaa aagcgtatca ggagattaac gacacgcaag 180
aaatgatcga aaaaatcagc ggacacctgc ctgtacactt gcgtcctcca tacggcggga 240
tcaatgattc cgtccgctcg ctttccaatc tgaaggtttc attgtgggat gttgatccgg 300
aagattggaa gtacaaaaat aagcaaaaga ttgtcaatca tgtcatgagc catgcgggag 360
acggaaaaat cgtcttaatg cacgatattt atgcaacgtc cgcagatgct gctgaagaga 420
ttattaaaaa gctgaaagca aaaggctatc aattggtaac tgtatctcag cttgaagaag 480
tgaagaagca gagaggctat tgaataaatg agtagaaagc gccatatcgg cgcttttctt 540
ttggaagaaa atatagggaa aatggtactt gttaaaaatt cggaatattt atacaatatc 600
atatgtatca cattgaaagg aggggcctgc tgtccagact gtccgctgtg taaaaaaaag 660
gaataaaggg gggttgacat tattttactg atatgtataa tataatttgt ataagaaaat 720
ggaggggccc tcgaaacgta agatgaaacc ttagataaaa gtgctttttt tgttgcaatt 780
gaagaattat taatgttaag cttaattaaa gataatatct ttgaattgta acgcccctca 840
aaagtaagaa ctacaaaaaa agaatacgtt atatagaaat atgtttgaac cttcttcaga 900
ttacaaatat attcggacgg actctacctc aaatgcttat ctaactatag aatgacatac 960
aagcacaacc ttgaaaattt gaaaatataa ctaccaatga acttgttcat gtgaattatc 1020
gctgtattta attttctcaa ttcaatatat aatatgccaa tacattgtta caagtagaaa 1080
ttaagacacc cttgatagcc ttactatacc taacatgatg tagtattaaa tgaatatgta 1140
aatatattta tgataagaag cgacttattt ataatcatta catatttttc tattggaatg 1200
attaagattc caatagaata gtgtataaat tatttatctt gaaaggaggg atgcctaaaa 1260
acgaagaaca ttaaaaacat atatttgcac cgtctaatgg atttatgaaa aatcatttta 1320
tcagtttgaa aattatgtat tatggagctc ttataaaaat gaggagggaa ccgaatggct 1380
tcaactgaag acgtaatcaa agagttcatg cgcttcaaag tgcgaatgga aggaagtgta 1440
aacgggcatg agtttgaaat tgaaggtgaa ggtgaaggaa ggccttatga aggaacgcaa 1500
actgcaaaac ttaaagtgac aaaaggagga ccgctgccgt ttgcttggga catcttaagt 1560
ccgcagtttc agtatgggtc aaaagtttat gtaaagcatc ctgctgacat tcctgattac 1620
aaaaagttaa gttttcctga aggattcaag tgggagcgcg taatgaactt tgaagatgga 1680
ggtgtcgtaa ctgtaacgca agattcaagt ctgcaagacg gttgcttcat ttacaaagta 1740
aagttcattg gcgtgaactt tccaagtgat ggtcctgtaa tgcagaaaaa gacaatgggt 1800
tgggagccgt caactgagag gctttatccg cgtgatggtg tcttgaaagg tgaaattcac 1860
aaagccttaa agttgaaaga tggagggcat tatcttgttg agttcaagag catttacatg 1920
gcgaaaaagc ctgtgcagct tcctggctac tactatgttg attcaaaact tgacataact 1980
agtcacaacg aagactacac aattgttgag cagtatgagc gaactgaagg aaggcatcat 2040
ctttttcttt aatgctgtcc agactgtccg ctgtgtaaaa aaaaggaata aaggggggtt 2100
gacattattt tactgatatg tataatataa tttgtataag aaaatgcttc atgtaatggt 2160
caaaatgttt tagagctaga aatagcaagt taaaataagg ctagtccgtt atcaacttga 2220
aaaagtggca ccgagtcggt gctttactga taattgccaa cacaattaac atctcaatca 2280
aggtaaatgc tagcgcggcc gcgtcgacag gcctctttga ttacatttta taattaattt 2340
taacaaagtg tcatcagccc tcaggaagga cttgctgaca gtttgaatcg cataggtaag 2400
gcggggatga aatggcaacg ttatctgatg tagcaaagaa agcaaatgtg tcgaaaatga 2460
cggtatcgcg ggtgatcaat catcctgaga ctgtgacgga tgaattgaaa aagcttgttc 2520
attccgcaat gaaggagctc aattatatac cgaactatgc agcaagagcg ctcgttcaaa 2580
acagaacaca ggtcgtcaag ctgctcatac tggaagaaat ggatacaaca gaaccttatt 2640
atatgaatct gttaacggga atcagccgcg agctggaccg tcatcattat gctttgcagc 2700
ttgtcacaag gaaatctctc aatatcggcc agtgcgacgg cattattgcg acggggttga 2760
gaaaagccga ttttgaaggg ctcatcaagg tttttgaaaa gcctgtcgtt gta 2813
<210> 6
<211> 3045
<212> DNA
<213> 人工序列
<220>
<223> PP3811-gDNA3中lacA2基因座的序列:lacA2-dsRED-gDNA(cat)
<400> 6
tgttgattgg ctttggcctc cagcttttta taaatggatt caccgaagct ggttaagtag 60
atatagtggt tgcggctgtc ctcctcgctt ctctttttat agaccatatt ttctttttca 120
aaccgcttca ggatccggct gacatagccc cggtccaggc cgagcgtatc ttgaatcagt 180
ttggctgtac aatcggccgt attgtgaatt tcaaataata tccgggtttc cgtcaatgaa 240
aaagggctgt cataaatatg ttcattcaga aaaccgagca catttgtata gaatcgattg 300
aactttctga attttaaagt gatagaatga ttgatttctg tcatctcaaa acctctctcc 360
ctgtaaatcg ttgctttaat caattataat aaaatagttg atttagtcaa gtgtatggaa 420
atgaagttaa aaatgttaat gatagattat attttacaaa taaagaaaga taaattcaat 480
catacaggaa aattcatcca gcggccgctc gctttccaat ctgaaggttt cattgtggga 540
tgttgatccg gaagattgga agtacaaaaa taagcaaaag attgtcaatc atgtcatgag 600
ccatgcggga gacggaaaaa tcgtcttaat gcacgatatt tatgcaacgt ccgcagatgc 660
tgctgaagag attattaaaa agctgaaagc aaaaggctat caattggtaa ctgtatctca 720
gcttgaagaa gtgaagaagc agagaggcta ttgaataaat gagtagaaag cgccatatcg 780
gcgcttttct tttggaagaa aatataggga aaatggtact tgttaaaaat tcggaatatt 840
tatacaatat catatgtatc acattgaaag gaggggcctg ctgtccagac tgtccgctgt 900
gtaaaaaaaa ggaataaagg ggggttgaca ttattttact gatatgtata atataatttg 960
tataagaaaa tggaggggcc ctcgaaacgt aagatgaaac cttagataaa agtgcttttt 1020
ttgttgcaat tgaagaatta ttaatgttaa gcttaattaa agataatatc tttgaattgt 1080
aacgcccctc aaaagtaaga actacaaaaa aagaatacgt tatatagaaa tatgtttgaa 1140
ccttcttcag attacaaata tattcggacg gactctacct caaatgctta tctaactata 1200
gaatgacata caagcacaac cttgaaaatt tgaaaatata actaccaatg aacttgttca 1260
tgtgaattat cgctgtattt aattttctca attcaatata taatatgcca atacattgtt 1320
acaagtagaa attaagacac ccttgatagc cttactatac ctaacatgat gtagtattaa 1380
atgaatatgt aaatatattt atgataagaa gcgacttatt tataatcatt acatattttt 1440
ctattggaat gattaagatt ccaatagaat agtgtataaa ttatttatct tgaaaggagg 1500
gatggctaaa aacgaagaac attaaaaaca tatatttgca ccgtctaatg gatttatgaa 1560
aaatcatttt atcagtttga aaattatgta ttatggagct ctataaaaat gaggagggaa 1620
ccgaatggca tctacagaag atgtgatcaa ggaattcatg cggtttaagg tgagaatgga 1680
aggaagcgtg aacggacatg aatttgaaat cgagggggaa ggcgaaggca gaccctatga 1740
aggtacacag acagcaaagc tgaaggtgac aaagggtgga ccgctgcctt ttgcctggga 1800
catcctgagc ccacagtttc aatatgggag taaggtgtac gtgaagcatc cggctgacat 1860
cccggactat aagaagctgt ccttcccaga gggctttaag tgggaaagag tcatgaattt 1920
cgaagatggc ggtgtggtga cagtgacgca agatagctcc ctgcaagatg gatgctttat 1980
ctacaaggtg aagttcatcg gagtgaattt cccttcggat ggaccggtga tgcaaaagaa 2040
gacaatggga tgggaaccta gtacagaaag gctgtatccg agagatggag tgctgaaggg 2100
agaaatccac aaggcgctga agctgaagga tggcggacac tatctggtgg agtttaagag 2160
catctatatg gccaagaagc cagtgcaact gcctgggtac tactatgtgg actcgaagct 2220
ggatatcact tcacataacg aagactacac aatcgtggaa caatatgaac ggacggaagg 2280
aaggcatcac ctgtttctgt aatgctgtcc agactgtccg ctgtgtaaaa aaaaggaata 2340
aaggggggtt gacattattt tactgatatg tataatataa tttgtataag aaaatgcttc 2400
atgtaatggt caaaatgttt tagagctaga aatagcaagt taaaataagg ctagtccgtt 2460
atcaacttga aaaagtggca ccgagtcggt gctttctgat aattgccaac acaattaaca 2520
tctcaatcaa ggtaaatgct agcatcgatt acaacccgga tcaatggctt aaatatccgg 2580
acgtattaaa agaagatatc cgcctgatga aactgtcccg ctgcaatgtg atgtctgtcg 2640
gcattttctc ctgggtttcg ctcgagcctg aagaaggaag atttacattt gactggctcg 2700
atcaggttct tgatactttc aaggaaaacg gaatttatgc gtttttggct acaccgagcg 2760
gtgccagacc ggcttggatg tccaaaaagt atccagaggt gctgagaacg gagcgcaaca 2820
gggtcagaaa ccttcacgga aagcggcaca atcactgcta tacgtcgcct gtctaccgcc 2880
ggaaaacggc gatcataaac ggaaagctcg cggagcgcta tgcgcatcac ccggccgtca 2940
tcggctggca catttctaat gaatacggcg gagaatgcca ttgtgaactt tgccaagaca 3000
agttcagaga gtggctgctg gcgaaataca aaacgctgga ccgcc 3045
<210> 7
<211> 3915
<212> DNA
<213> 人工序列
<220>
<223> 整合amyL后PP3811-amyL3中gnt基因座的序列
<400> 7
agcgaagcct tgtgcatagg cgcagatttt gcccatatat aatgcctgtc tgacgcggtc 60
gatccacacg ttttgatcca ggcgccgttc ttctgttgca ggtccggcca atactttttc 120
cgcagctgtc cgttcgtctt ttaatgatga caggtaacgg gcaaacaggg attccgtgat 180
aattgatgat ggaatgccgt tgtcgacggc ctgcaggctc gtccatttgc ccgtgccttt 240
ttggccggtt ttgtcgagga tgacgtcgat gagtggagcg cccgtcttct catccttttt 300
ccgcaggatc tccgccgtga tttcgattaa atagctgttc agctctcctt gattccacgt 360
gtcgaaaatg tcagcgattt catctatcgg caaaagaagc ttttctctta aaaacgtata 420
tgcttcggcg atgagctgca tgtctgcgta ttcgatgccg ttgtgcacca ttttgacaaa 480
atgacccgcg cctttcggac ggccgctcgc tttccaatct gaaggtttca ttgtgggatg 540
ttgatccgga agattggaag tacaaaaata agcaaaagat tgtcaatcat gtcatgagcc 600
atgcgggaga cggaaaaatc gtcttaatgc acgatattta tgcaacgtcc gcagatgctg 660
ctgaagagat tattaaaaag ctgaaagcaa aaggctatca attggtaact gtatctcagc 720
ttgaagaagt gaagaagcag agaggctatt gaataaatga gtagaaagcg ccatatcggc 780
gcttttcttt tggaagaaaa tatagggaaa atggtacttg ttaaaaattc ggaatattta 840
tacaatatca tatgtatcac attgaaagga ggggcctgct gtccagactg tccgctgtgt 900
aaaaaaaagg aataaagggg ggttgacatt attttactga tatgtataat ataatttgta 960
taagaaaatg gaggggccct cgaaacgtaa gatgaaacct tagataaaag tgcttttttt 1020
gttgcaattg aagaattatt aatgttaagc ttaattaaag ataatatctt tgaattgtaa 1080
cgcccctcaa aagtaagaac tacaaaaaaa gaatacgtta tatagaaata tgtttgaacc 1140
ttcttcagat tacaaatata ttcggacgga ctctacctca aatgcttatc taactataga 1200
atgacataca agcacaacct tgaaaatttg aaaatataac taccaatgaa cttgttcatg 1260
tgaattatcg ctgtatttaa ttttctcaat tcaatatata atatgccaat acattgttac 1320
aagtagaaat taagacaccc ttgatagcct tactatacct aacatgatgt agtattaaat 1380
gaatatgtaa atatatttat gataagaagc gacttattta taatcattac atatttttct 1440
attggaatga ttaagattcc aatagaatag tgtataaatt atttatcttg aaaggaggga 1500
tgcctaaaaa cgaagaacat taaaaacata tatttgcacc gtctaatgga tttatgaaaa 1560
atcattttat cagtttgaaa attatgtatt atggccacat tgaaagggga ggagaatcat 1620
gaaacaacaa aaacggcttt acgcccgatt gctgacgctg ttatttgcgc tcatcttctt 1680
gctgcctcat tctgcagcag cggcggcaaa tcttaatggg acgctgatgc agtattttga 1740
atggtacatg cccaatgacg gccaacattg gaggcgtttg caaaacgact cggcatattt 1800
ggctgaacac ggtattactg ccgtctggat tcccccggca tataagggaa cgagccaagc 1860
ggatgtgggc tacggtgctt acgaccttta tgatttaggg gagtttcatc aaaaagggac 1920
ggttcggaca aagtacggca caaaaggaga gctgcaatct gcgatcaaaa gtcttcattc 1980
ccgcgacatt aacgtttacg gggatgtggt catcaaccac aaaggcggcg ctgatgcgac 2040
cgaagatgta accgcggttg aagtcgatcc cgctgaccgc aaccgcgtaa tttcaggaga 2100
acacctaatt aaagcctgga cacattttca ttttccgggg cgcggcagca catacagcga 2160
ttttaaatgg cattggtacc attttgacgg aaccgattgg gacgagtccc gaaagctgaa 2220
ccgcatctat aagtttcaag gaaaggcttg ggattgggaa gtttccaatg aaaacggcaa 2280
ctatgattat ttgatgtatg ccgacatcga ttatgaccat cctgatgtcg cagcagaaat 2340
taagagatgg ggcacttggt atgccaatga actgcaattg gacggtttcc gtcttgatgc 2400
tgtcaaacac attaaatttt cttttttgcg ggattgggtt aatcatgtca gggaaaaaac 2460
ggggaaggaa atgtttacgg tagctgaata ttggcagaat gacttgggcg cgctggaaaa 2520
ctatttgaac aaaacaaatt ttaatcattc agtgtttgac gtgccgcttc attatcagtt 2580
ccatgctgca tcgacacagg gaggcggcta tgatatgagg aaattgctga acggtacggt 2640
cgtttccaag catccgttga aatcggttac atttgtcgat aaccatgata cacagccggg 2700
gcaatcgctt gagtcgactg tccaaacatg gtttaagccg cttgcttacg cttttattct 2760
cacaagggaa tctggatacc ctcaggtttt ctacggggat atgtacggga cgaaaggaga 2820
ctcccagcgc gaaattcctg ccttgaaaca caaaattgaa ccgatcttaa aagcgagaaa 2880
acagtatgcg tacggagcac agcatgatta tttcgaccac catgacattg tcggctggac 2940
aagggaaggc gacagctcgg ttgcaaattc aggtttggcg gcattaataa cagacggacc 3000
cggtggggca aagcgaatgt atgtcggccg gcaaaacgcc ggtgagacat ggcatgacat 3060
taccggaaac cgttcggagc cggttgtcat caattcggaa ggctggggag agtttcacgt 3120
aaacggcggg tcggtttcaa tttatgttca aagatagacg cgtagggccc gcggctagcg 3180
gccgcgtcga ctagaagagc agagaggacg gatttcctga aggaaatccg tttttttatt 3240
ttgcccgtct tataaatttc gttgtccaac tcgcttaatt gcgagttttt atttcgttta 3300
tttcaatcaa ggtaaatgct agcggccgcg tcgactagaa gagcagagag gacggatttc 3360
ctgaaggaaa tccgtttttt tattttgccc gtcttataaa tttcgttgag atcttttata 3420
caaataggct taacaataaa gtaaatccta atccggccac cgcgataatt gtttcaagca 3480
gtgtccaggt ggcgaatgtt tctttcatgc tcaggccgaa atactctttg aacatccaga 3540
agcccgcgtc gttgacatgg gaagcgatta cacttccggc ccctgttgca agcacaacca 3600
gtgcaagatt gacatcgctt tgtccgagca tcggaagaac gagtccggtc gtgcttaatg 3660
cagcaactgt cgcggaacct aaagagatgc gcagaatcgc ggcgatgacc caggcgagca 3720
agatcggcga catggccgtt cctttgaata attcagctac atagtcgcct actccgccgt 3780
tgatcaagac ttgtttgaat gcgccgccgc ccccgatgat caagagcatc attccgattt 3840
gagtaatggc ggttgaacag gaatccatca cttgtttgat cgggatcttt ctggcgatac 3900
ccatcgtata aatcg 3915
<210> 8
<211> 3594
<212> DNA
<213> 人工序列
<220>
<223> 重新整合amyL后PP3811-amyL3中amyL基因座的序列
<400> 8
tacagaagca tgaagggcat gcgaccttct ttgtgcttgg aagcagagcg caatattatc 60
ccgaaacgat aaaacggatg ctgaaggaag gaaacgaagt cggcaaccat tcctgggacc 120
atccgttatt gacaaggctg tcaaacgaaa aagcgtatca ggagattaac gacacgcaag 180
aaatgatcga aaaaatcagc ggacacctgc ctgtacactt gcgtcctcca tacggcggga 240
tcaatgattc cgtccgctcg ctttccaatc tgaaggtttc attgtgggat gttgatccgg 300
aagattggaa gtacaaaaat aagcaaaaga ttgtcaatca tgtcatgagc catgcgggag 360
acggaaaaat cgtcttaatg cacgatattt atgcaacgtc cgcagatgct gctgaagaga 420
ttattaaaaa gctgaaagca aaaggctatc aattggtaac tgtatctcag cttgaagaag 480
tgaagaagca gagaggctat tgaataaatg agtagaaagc gccatatcgg cgcttttctt 540
ttggaagaaa atatagggaa aatggtactt gttaaaaatt cggaatattt atacaatatc 600
atatgtatca cattgaaagg aggggcctgc tgtccagact gtccgctgtg taaaaaaaag 660
gaataaaggg gggttgacat tattttactg atatgtataa tataatttgt ataagaaaat 720
ggaggggccc tcgaaacgta agatgaaacc ttagataaaa gtgctttttt tgttgcaatt 780
gaagaattat taatgttaag cttaattaaa gataatatct ttgaattgta acgcccctca 840
aaagtaagaa ctacaaaaaa agaatacgtt atatagaaat atgtttgaac cttcttcaga 900
ttacaaatat attcggacgg actctacctc aaatgcttat ctaactatag aatgacatac 960
aagcacaacc ttgaaaattt gaaaatataa ctaccaatga acttgttcat gtgaattatc 1020
gctgtattta attttctcaa ttcaatatat aatatgccaa tacattgtta caagtagaaa 1080
ttaagacacc cttgatagcc ttactatacc taacatgatg tagtattaaa tgaatatgta 1140
aatatattta tgataagaag cgacttattt ataatcatta catatttttc tattggaatg 1200
attaagattc caatagaata gtgtataaat tatttatctt gaaaggaggg atgcctaaaa 1260
acgaagaaca ttaaaaacat atatttgcac cgtctaatgg atttatgaaa aatcatttta 1320
tcagtttgaa aattatgtat tatggccaca ttgaaagggg aggagaatca tgaaacaaca 1380
aaaacggctt tacgcccgat tgctgacgct gttatttgcg ctcatcttct tgctgcctca 1440
ttctgcagca gcggcggcaa atcttaatgg gacgctgatg cagtattttg aatggtacat 1500
gcccaatgac ggccaacatt ggaggcgttt gcaaaacgac tcggcatatt tggctgaaca 1560
cggtattact gccgtctgga ttcccccggc atataaggga acgagccaag cggatgtggg 1620
ctacggtgct tacgaccttt atgatttagg ggagtttcat caaaaaggga cggttcggac 1680
aaagtacggc acaaaaggag agctgcaatc tgcgatcaaa agtcttcatt cccgcgacat 1740
taacgtttac ggggatgtgg tcatcaacca caaaggcggc gctgatgcga ccgaagatgt 1800
aaccgcggtt gaagtcgatc ccgctgaccg caaccgcgta atttcaggag aacacctaat 1860
taaagcctgg acacattttc attttccggg gcgcggcagc acatacagcg attttaaatg 1920
gcattggtac cattttgacg gaaccgattg ggacgagtcc cgaaagctga accgcatcta 1980
taagtttcaa ggaaaggctt gggattggga agtttccaat gaaaacggca actatgatta 2040
tttgatgtat gccgacatcg attatgacca tcctgatgtc gcagcagaaa ttaagagatg 2100
gggcacttgg tatgccaatg aactgcaatt ggacggtttc cgtcttgatg ctgtcaaaca 2160
cattaaattt tcttttttgc gggattgggt taatcatgtc agggaaaaaa cggggaagga 2220
aatgtttacg gtagctgaat attggcagaa tgacttgggc gcgctggaaa actatttgaa 2280
caaaacaaat tttaatcatt cagtgtttga cgtgccgctt cattatcagt tccatgctgc 2340
atcgacacag ggaggcggct atgatatgag gaaattgctg aacggtacgg tcgtttccaa 2400
gcatccgttg aaatcggtta catttgtcga taaccatgat acacagccgg ggcaatcgct 2460
tgagtcgact gtccaaacat ggtttaagcc gcttgcttac gcttttattc tcacaaggga 2520
atctggatac cctcaggttt tctacgggga tatgtacggg acgaaaggag actcccagcg 2580
cgaaattcct gccttgaaac acaaaattga accgatctta aaagcgagaa aacagtatgc 2640
gtacggagca cagcatgatt atttcgacca ccatgacatt gtcggctgga caagggaagg 2700
cgacagctcg gttgcaaatt caggtttggc ggcattaata acagacggac ccggtggggc 2760
aaagcgaatg tatgtcggcc ggcaaaacgc cggtgagaca tggcatgaca ttaccggaaa 2820
ccgttcggag ccggttgtca tcaattcgga aggctgggga gagtttcacg taaacggcgg 2880
gtcggtttca atttatgttc aaagatagac gcgtagggcc cgcggctagc ggccgcgtcg 2940
actagaagag cagagaggac ggatttcctg aaggaaatcc gtttttttat tttgcccgtc 3000
ttataaattt cgttgtccaa ctcgcttaat tgcgagtttt tatttcgttt atttcaatca 3060
aggtaaatgg ctagcgcggc cgcgtcgaca ggcctctttg attacatttt ataattaatt 3120
ttaacaaagt gtcatcagcc ctcaggaagg acttgctgac agtttgaatc gcataggtaa 3180
ggcggggatg aaatggcaac gttatctgat gtagcaaaga aagcaaatgt gtcgaaaatg 3240
acggtatcgc gggtgatcaa tcatcctgag actgtgacgg atgaattgaa aaagcttgtt 3300
cattccgcaa tgaaggagct caattatata ccgaactatg cagcaagagc gctcgttcaa 3360
aacagaacac aggtcgtcaa gctgctcata ctggaagaaa tggatacaac agaaccttat 3420
tatatgaatc tgttaacggg aatcagccgc gagctggacc gtcatcatta tgctttgcag 3480
cttgtcacaa ggaaatctct caatatcggc cagtgcgacg gcattattgc gacggggttg 3540
agaaaagccg attttgaagg gctcatcaag gtttttgaaa agcctgtcgt tgta 3594
<210> 9
<211> 3852
<212> DNA
<213> 人工序列
<220>
<223> 整合amyL后PP3811-amyL3中lacA2基因座的序列
<400> 9
tgttgattgg ctttggcctc cagcttttta taaatggatt caccgaagct ggttaagtag 60
atatagtggt tgcggctgtc ctcctcgctt ctctttttat agaccatatt ttctttttca 120
aaccgcttca ggatccggct gacatagccc cggtccaggc cgagcgtatc ttgaatcagt 180
ttggctgtac aatcggccgt attgtgaatt tcaaataata tccgggtttc cgtcaatgaa 240
aaagggctgt cataaatatg ttcattcaga aaaccgagca catttgtata gaatcgattg 300
aactttctga attttaaagt gatagaatga ttgatttctg tcatctcaaa acctctctcc 360
ctgtaaatcg ttgctttaat caattataat aaaatagttg atttagtcaa gtgtatggaa 420
atgaagttaa aaatgttaat gatagattat attttacaaa taaagaaaga taaattcaat 480
catacaggaa aattcatcca gcggccgctc gctttccaat ctgaaggttt cattgtggga 540
tgttgatccg gaagattgga agtacaaaaa taagcaaaag attgtcaatc atgtcatgag 600
ccatgcggga gacggaaaaa tcgtcttaat gcacgatatt tatgcaacgt ccgcagatgc 660
tgctgaagag attattaaaa agctgaaagc aaaaggctat caattggtaa ctgtatctca 720
gcttgaagaa gtgaagaagc agagaggcta ttgaataaat gagtagaaag cgccatatcg 780
gcgcttttct tttggaagaa aatataggga aaatggtact tgttaaaaat tcggaatatt 840
tatacaatat catatgtatc acattgaaag gaggggcctg ctgtccagac tgtccgctgt 900
gtaaaaaaaa ggaataaagg ggggttgaca ttattttact gatatgtata atataatttg 960
tataagaaaa tggaggggcc ctcgaaacgt aagatgaaac cttagataaa agtgcttttt 1020
ttgttgcaat tgaagaatta ttaatgttaa gcttaattaa agataatatc tttgaattgt 1080
aacgcccctc aaaagtaaga actacaaaaa aagaatacgt tatatagaaa tatgtttgaa 1140
ccttcttcag attacaaata tattcggacg gactctacct caaatgctta tctaactata 1200
gaatgacata caagcacaac cttgaaaatt tgaaaatata actaccaatg aacttgttca 1260
tgtgaattat cgctgtattt aattttctca attcaatata taatatgcca atacattgtt 1320
acaagtagaa attaagacac ccttgatagc cttactatac ctaacatgat gtagtattaa 1380
atgaatatgt aaatatattt atgataagaa gcgacttatt tataatcatt acatattttt 1440
ctattggaat gattaagatt ccaatagaat agtgtataaa ttatttatct tgaaaggagg 1500
gatgcctaaa aacgaagaac attaaaaaca tatatttgca ccgtctaatg gatagaaagg 1560
aggtgatcca gccgcacctt atgaaaaatc attttatcag tttgaaaatt atgtattatg 1620
gccacattga aaggggagga gaatcatgaa acaacaaaaa cggctttacg cccgattgct 1680
gacgctgtta tttgcgctca tcttcttgct gcctcattct gcagcagcgg cggcaaatct 1740
taatgggacg ctgatgcagt attttgaatg gtacatgccc aatgacggcc aacattggag 1800
gcgtttgcaa aacgactcgg catatttggc tgaacacggt attactgccg tctggattcc 1860
cccggcatat aagggaacga gccaagcgga tgtgggctac ggtgcttacg acctttatga 1920
tttaggggag tttcatcaaa aagggacggt tcggacaaag tacggcacaa aaggagagct 1980
gcaatctgcg atcaaaagtc ttcattcccg cgacattaac gtttacgggg atgtggtcat 2040
caaccacaaa ggcggcgctg atgcgaccga agatgtaacc gcggttgaag tcgatcccgc 2100
tgaccgcaac cgcgtaattt caggagaaca cctaattaaa gcctggacac attttcattt 2160
tccggggcgc ggcagcacat acagcgattt taaatggcat tggtaccatt ttgacggaac 2220
cgattgggac gagtcccgaa agctgaaccg catctataag tttcaaggaa aggcttggga 2280
ttgggaagtt tccaatgaaa acggcaacta tgattatttg atgtatgccg acatcgatta 2340
tgaccatcct gatgtcgcag cagaaattaa gagatggggc acttggtatg ccaatgaact 2400
gcaattggac ggtttccgtc ttgatgctgt caaacacatt aaattttctt ttttgcggga 2460
ttgggttaat catgtcaggg aaaaaacggg gaaggaaatg tttacggtag ctgaatattg 2520
gcagaatgac ttgggcgcgc tggaaaacta tttgaacaaa acaaatttta atcattcagt 2580
gtttgacgtg ccgcttcatt atcagttcca tgctgcatcg acacagggag gcggctatga 2640
tatgaggaaa ttgctgaacg gtacggtcgt ttccaagcat ccgttgaaat cggttacatt 2700
tgtcgataac catgatacac agccggggca atcgcttgag tcgactgtcc aaacatggtt 2760
taagccgctt gcttacgctt ttattctcac aagggaatct ggataccctc aggttttcta 2820
cggggatatg tacgggacga aaggagactc ccagcgcgaa attcctgcct tgaaacacaa 2880
aattgaaccg atcttaaaag cgagaaaaca gtatgcgtac ggagcacagc atgattattt 2940
cgaccaccat gacattgtcg gctggacaag ggaaggcgac agctcggttg caaattcagg 3000
tttggcggca ttaataacag acggacccgg tggggcaaag cgaatgtatg tcggccggca 3060
aaacgccggt gagacatggc atgacattac cggaaaccgt tcggagccgg ttgtcatcaa 3120
ttcggaaggc tggggagagt ttcacgtaaa cggcgggtcg gtttcaattt atgttcaaag 3180
atagacgcgt agggcccgcg gctagcggcc gcgtcgacta gaagagcaga gaggacggat 3240
ttcctgaagg aaatccgttt ttttattttg cccgtcttat aaatttcgtt gtccaactcg 3300
cttaattgcg agtttttatt tcgtttattt caatcaaggt aaatgctagc atcgattaca 3360
acccggatca atggcttaaa tatccggacg tattaaaaga agatatccgc ctgatgaaac 3420
tgtcccgctg caatgtgatg tctgtcggca ttttctcctg ggtttcgctc gagcctgaag 3480
aaggaagatt tacatttgac tggctcgatc aggttcttga tactttcaag gaaaacggaa 3540
tttatgcgtt tttggctaca ccgagcggtg ccagaccggc ttggatgtcc aaaaagtatc 3600
cagaggtgct gagaacggag cgcaacaggg tcagaaacct tcacggaaag cggcacaatc 3660
actgctatac gtcgcctgtc taccgccgga aaacggcgat cataaacgga aagctcgcgg 3720
agcgctatgc gcatcacccg gccgtcatcg gctggcacat ttctaatgaa tacggcggag 3780
aatgccattg tgaactttgc caagacaagt tcagagagtg gctgctggcg aaatacaaaa 3840
cgctggaccg cc 3852
<210> 10
<211> 9550
<212> DNA
<213> 人工序列
<220>
<223> 质粒pPPamyL-attP的序列
<400> 10
gagctcgtta ttaatctgtt cagcaatcgg gcgcgattgc tgaataaaag atacgagaga 60
cctctcttgt atctttttta ttttgagtgg ttttgtccgt tacactagaa aaccgaaaga 120
caataaaaat tttattcttg ctgagtctgg ctttcggtaa gctagacaaa acggacaaaa 180
taaaaattgg caagggttta aaggtggaga ttttttgagt gatcttctca aaaaatacta 240
cctgtccctt gctgattttt aaacgagcac gagagcaaaa cccccctttg ctgaggtggc 300
agagggcagg tttttttgtt tcttttttct cgtaaaaaaa agaaaggtct taaaggtttt 360
atggttttgg tcggcactgc cgacagcctc gcagagcaca cactttatga atataaagta 420
tagtgtgtta tactttactt ggaagtggtt gccggaaaga gcgaaaatgc ctcacatttg 480
tgccacctaa aaaggagcga tttacatatg agttatgcag tttgtagaat gcaaaaagtg 540
aaatcagctg gactaaaagg cagagctcgg taccagatct aaagataata tctttgaatt 600
gtaacccccc tcaaaagtaa gaactacaaa aaaagaatac gttatataga aatatgtttg 660
aaccttcttc agattacaaa tatattcgga cggactctac ctcaaatgct tatctaacta 720
tagaatgaca tacaagcaca accttgaaaa tttgaaaata taactaccaa tgaacttgtt 780
catgtgaatt atcgctgtat ttaattttct caattcaata tataatatgc caatacattg 840
ttacaagtag aaattaagac acccttgata gccttactat acctaacatg atgtagtatt 900
aaatgaatat gtaaatatat ttatgataag aagcgactta tttataatca ttacatattt 960
ttctattgga atgattaaga ttccaataga atagtgtata aattatttat cttgaaagga 1020
gggatgccta aaaacgaaga acattaaaaa catatatttg caccgtctaa tggatagaaa 1080
ggaggtgatc cagccgcacc ttatgaaaaa tcattttatc agtttgaaaa ttatgtatta 1140
tggccacatt gaaaggggag gagaatcatg aaacaacaaa aacggcttta cgcccgattg 1200
ctgacgctgt tatttgcgct catcttcttg ctgcctcatt ctgcagcagc ggcggcaaat 1260
cttaatggga cgctgatgca gtattttgaa tggtacatgc ccaatgacgg ccaacattgg 1320
aggcgtttgc aaaacgactc ggcatatttg gctgaacacg gtattactgc cgtctggatt 1380
cccccggcat ataagggaac gagccaagcg gatgtgggct acggtgctta cgacctttat 1440
gatttagggg agtttcatca aaaagggacg gttcggacaa agtacggcac aaaaggagag 1500
ctgcaatctg cgatcaaaag tcttcattcc cgcgacatta acgtttacgg ggatgtggtc 1560
atcaaccaca aaggcggcgc tgatgcgacc gaagatgtaa ccgcggttga agtcgatccc 1620
gctgaccgca accgcgtaat ttcaggagaa cacctaatta aagcctggac acattttcat 1680
tttccggggc gcggcagcac atacagcgat tttaaatggc attggtacca ttttgacgga 1740
accgattggg acgagtcccg aaagctgaac cgcatctata agtttcaagg aaaggcttgg 1800
gattgggaag tttccaatga aaacggcaac tatgattatt tgatgtatgc cgacatcgat 1860
tatgaccatc ctgatgtcgc agcagaaatt aagagatggg gcacttggta tgccaatgaa 1920
ctgcaattgg acggtttccg tcttgatgct gtcaaacaca ttaaattttc ttttttgcgg 1980
gattgggtta atcatgtcag ggaaaaaacg gggaaggaaa tgtttacggt agctgaatat 2040
tggcagaatg acttgggcgc gctggaaaac tatttgaaca aaacaaattt taatcattca 2100
gtgtttgacg tgccgcttca ttatcagttc catgctgcat cgacacaggg aggcggctat 2160
gatatgagga aattgctgaa cggtacggtc gtttccaagc atccgttgaa atcggttaca 2220
tttgtcgata accatgatac acagccgggg caatcgcttg agtcgactgt ccaaacatgg 2280
tttaagccgc ttgcttacgc ttttattctc acaagggaat ctggataccc tcaggttttc 2340
tacggggata tgtacgggac gaaaggagac tcccagcgcg aaattcctgc cttgaaacac 2400
aaaattgaac cgatcttaaa agcgagaaaa cagtatgcgt acggagcaca gcatgattat 2460
ttcgaccacc atgacattgt cggctggaca agggaaggcg acagctcggt tgcaaattca 2520
ggtttggcgg cattaataac agacggaccc ggtggggcaa agcgaatgta tgtcggccgg 2580
caaaacgccg gtgagacatg gcatgacatt accggaaacc gttcggagcc ggttgtcatc 2640
aattcggaag gctggggaga gtttcacgta aacggcgggt cggtttcaat ttatgttcaa 2700
agatagacgc gtagggcccg cggctagcgg ccgcgtcgac tagaagagca gagaggacgg 2760
atttcctgaa ggaaatccgt ttttttattt tgcccgtctt ataaatttcg ttgtccaact 2820
cgcttaattg cgagttttta tttcgtttat ttcaattaag gtaactaaag atcctctaga 2880
gtcgattatg tcttttgcgc agtcggctta aaccagtttt cgctggtgcg aaaaaagagt 2940
gtcttgtgac acctaaattc aaaatctatc ggtcagattt ataccgattt gattttatat 3000
attcttgaat aacatacgcc gagttatcac ataaaagcgg gaaccaatca tcaaatttaa 3060
acttcattgc ataatccatt aaactcttaa attctacgat tccttgttca tcaataaact 3120
caatcatttc tttaattaat ttatatctat ctgttgttgt tttctttaat aattcatcaa 3180
catctacacc gccataaact atcatatctt ctttttgata tttaaattta ttaggatcgt 3240
ccatgtgaag catatatctc acaagacctt tcacacttcc tgcaatctgc ggaatagtcg 3300
cattcaattc ttctgtaatt atttttatct gttcataaga tttattaccc tcatacatca 3360
ctagaatatg ataatgctct tttttcatcc taccttctgt atcagtatcc ctatcatgta 3420
atggagacac tacaaattga atgtgtaact cttttaaata ctctaaccac tcggcttttg 3480
ctgattctgg atataaaaca aatgtccaat tacgtcctct tgaatttttc ttgttttcag 3540
tttcttttat tacattttcg ctcatgatat aataacggtg ctaatacact taacaaaatt 3600
tagtcataga taggcagcat gccagtgctg tctatctttt tttgtttaaa atgcaccgta 3660
ttcctccttt gcatattttt ttattagaat accggttgca tctgatttgc taatattata 3720
tttttctttg attctattta atatctcatt ttcttctgtt gtaagtctta aagtaacagc 3780
aacttttttc tcttcttttc tatctacaac catcactgta cctcccaaca tctgtttttt 3840
tcactttaac ataaaaaaca accttttaac attaaaaacc caatatttat ttatttgttt 3900
ggacaatgga caatggacac ctagggggga ggtcgtagta cccccctatg ttttctcccc 3960
taaataaccc caaaaatcta agaaaaaaag acctcaaaaa ggtctttaat taacatctca 4020
aatttcgcat ttattccaat ttcctttttg cgtgtgatgc gctgcgtcca ttaaaaatcc 4080
tagagctttg aaaccgaaag ttaatagctg tcgctactac tttcgcttac gctctaagta 4140
tattttaagg actgtcacac gcaaaaagtt ttctcggcat aaaagtacct ctacatctct 4200
aaatcgtctg tacgctgttt ctcacgcttt ctatcgacct tctggacatt atcctgtaca 4260
acatccataa actgtcccac acgctcaaat ttggaatcat taaagaattt ctctttaagc 4320
ctattaaacc ctttctcaaa cccagggaaa ttcgccctcg cagcacgata taaagtcact 4380
gtactagctt gaaatttctc tgatacattc aactgctcat tcaaactatc attctctcgc 4440
tttaatttat taacctcttt acttttttcg tgatacccct ctttccatgt attcactact 4500
tctttcaaac tctctctacg tttttttaat tcttgatttt ctgtgtaata gtctgtgctc 4560
ttaatatttt cgtaatcatc aacaatccgt tctgcagaag agattgtttc ttgcaggcgt 4620
tcaaattcat cagcagttaa tatctttcta ccagtctctt cacgtccaga gaacaaacct 4680
gtacgctcat tttcataatc aaagggtttc gtagacctca tatgctctat tccactctgt 4740
aactgcttat ttgccttctg taactcatcc ttaacttctt gcagttcctg tttatgaaat 4800
acagtatctt tcttgtactg atccatcgct ttatgttctc gttctgtaac ctctttggac 4860
gtgcctcttt caagttcata acctttctca ttcacatact cattaaatct atcttgtaat 4920
tgagtaaagt ctttcttgtt gcctaactgt tcttttgcag acaatctccc gtcctctgtt 4980
aaagggacaa aaccaaagtg catatgtggg actctttcat ccagatggac agtcgcatac 5040
agcatatttt ccttaccgta ttcattttct agaaactcca agctatcttt aaaaaatcgt 5100
tctatttctt ctccgcttaa atcatcaaag aaatctttat cacttgtaac cagtccgtcc 5160
acatgtcgaa ttgcatctga ccgaatttta cgtttccctg aataattctc atcaatcgtt 5220
tcatcaattt tatctttata ctttatattt tgtgcgttaa tcaaatcata atttttatat 5280
gtttcctcat gatttatgtc tttattatta tagtttttat tctctctttg attatgtctt 5340
tgtatcccgt ttgtattact tgatccttta actctggcaa ccctcaaaat tgaatgagac 5400
atgctacacc tccggataat aaatatatat aaacgtatat agatttcata aagtctaaca 5460
cactagactt atttacttcg taattaagtc gttaaaccgt gtgctctacg accaaaacta 5520
taaaaccttt aagaactttc tttttttaca agaaaaaaga aattagataa atctctcata 5580
tcttttattc aataatcgca tccgattgca gtataaattt aacgatcact catcatgttc 5640
atatttatca gagctcgtgc tataattata ctaattttat aaggaggaaa aaatatgggc 5700
atttttagta tttttgtaat cagcacagtt cattatcaac caaacaaaaa ataagtggtt 5760
ataatgaatc gttaataagc aaaattcata taaccaaatt aaagagggtt ataatgaacg 5820
agaaaaatat aaaacacagt caaaacttta ttacttcaaa acataatata gataaaataa 5880
tgacaaatat aagattaaat gaacatgata atatctttga aatcggctca ggaaaaggcc 5940
attttaccct tgaattagta aagaggtgta atttcgtaac tgccattgaa atagaccata 6000
aattatgcaa aactacagaa aataaacttg ttgatcacga taatttccaa gttttaaaca 6060
aggatatatt gcagtttaaa tttcctaaaa accaatccta taaaatatat ggtaatatac 6120
cttataacat aagtacggat ataatacgca aaattgtttt tgatagtata gctaatgaga 6180
tttatttaat cgtggaatac gggtttgcta aaagattatt aaatacaaaa cgctcattgg 6240
cattactttt aatggcagaa gttgatattt ctatattaag tatggttcca agagaatatt 6300
ttcatcctaa acctaaagtg aatagctcac ttatcagatt aagtagaaaa aaatcaagaa 6360
tatcacacaa agataaacaa aagtataatt atttcgttat gaaatgggtt aacaaagaat 6420
acaagaaaat atttacaaaa aatcaattta acaattcctt aaaacatgca ggaattgacg 6480
atttaaacaa tattagcttt gaacaattct tatctctttt caatagctat aaattattta 6540
ataagtaagt taagggatgc ataaactgca tcccttaact tgtttttcgt gtgcctattt 6600
tttgtgaatc gacctgcagg catgcaagct taagcgagtt ggaatttaaa tatgatatct 6660
acattatcag cagtaacatc aacctttgat acaaggttgt tgacgatttt ctttttatta 6720
tcatatgata gttcattaat cggaattgag cccaactgag ttttaactaa ctcaaaaaca 6780
tcagtagagt cattaaattt attttcgcta atcttagctt taagcagctt tttctcagcc 6840
tgaagggaat cagtacgatc tttcaactca tccatagtga taaaatcatt taggtacaaa 6900
tcagagttct tttgtatttt tttatcgatc tgtgaaattt gctttttaaa tgacgaagta 6960
tcaagaatag gttggttgtt gccattgata attttcaata aggagtcatt attttcttga 7020
aatccaatca ggttgtcaat aacagtattt tctaaattac ttaaatcata agttcctgaa 7080
tcacactttt tattgtcatt atatactgta attccttttg tttttcgagg aaatctattt 7140
gcacagtgat atttcatagt gcggcttcca tcttttcttt tgtggccaag aacaattttt 7200
aaaggtgctc cacagtaacc gcaccttgcc atccctgaca gcatatattt agcttggaaa 7260
ggtctagggt tgttatttct ttcataagtc tgctgttgtc tttcttctag ctctttttga 7320
acttttaaat aagtctcata agggataatt ggtttgtgca taccttcaaa taggctgtcc 7380
ttaaatttga tataaccaca gtaaactgga ttatcaagtg tttgtcttag ggtacgataa 7440
gaccacggta tatctttacc gatgtgtcca gattcattga gtttatctct taattttgta 7500
agtgatattc ctgataaata atcagtgaat atttgttcaa ctattgtagc ttgtaaagga 7560
acaatttcta atatacctgt ctttctgttg tggtaatacc caaaagctgt cttagtccac 7620
atcatagact taccagattt cgctcgccct agtttaccca tagtcatgcg ttcttttata 7680
ttctctcttt caaactcatt aattgcagaa agaatagtga gaaacaagct acccatagca 7740
gaagaagtat caatactttc attaagcgag ataaagtcta ttttattttt tgtgaacaca 7800
tccttaacaa gataaagagt atctcttaca ctacgtgaaa ggcggtctag cttatataca 7860
agaactgtat caaaagcttt attctcgata tcgttgatta atctttgcat tgctgggcgt 7920
tcaagtttgg cccctgaaaa accagcatca gtataagtat cagatacttg ccaccccatt 7980
gcttcagcat attttgttaa acggtcaatt tgctcatcaa ttgagaaccc ttcctctgct 8040
tggttagtag tggatactcg tgtatagatt gctactttct tagtcatgag atttccccct 8100
taaaaataaa ttcattcaaa tacagatgca ttttatttca tatagtaagt acatcaccta 8160
ttagtttgtt gtttaaacaa actaacttat tttcatctta tataacctcg tcagtatttt 8220
caatattttt tttagttttt tatgaacaca ttagatttaa taaagggaag attcgctatg 8280
tactatgttg atacttaatt taaagattaa acaaatggag tggatgaagt ggatatcgct 8340
gatcaaacct ttgtcaaaaa agtaaatcaa aagttattat taaaagaaat ccttaaaaat 8400
tcacctattt caagagcaaa attatctgaa atgactggat taaataaatc aactgtctca 8460
tcacaggtaa acacgttaat gaaagaaagt atggtatttg aaataggtca aggacaatca 8520
agtggcggaa gaagacctgt catgcttgtt tttaataaaa aggcaggata ctccgttgga 8580
atagatgttg gtgtggatta tattaatggc attttaacag accttgaagg aacaatcgtt 8640
cttgatcaat accgccattt ggaatccaat tctccagaaa taacgaaaga cattttgatt 8700
gatatgattc atcactttat tacgcaaatg ccccaatctc cgtacgggtt tattggtata 8760
ggtatttgcg tgcctggact cattgataaa gatcaaaaaa ttgttttcac tccgaactcc 8820
aactggagag atattgactt aaaatcttcg atacaagaga agtacaatgt gtctgttttt 8880
attgaaaatg aggcaaatgc tggcgcatat ggagaaaaac tatttggagc tgcaaaaaat 8940
cacgataaca ttatttacgt aagtatcagc acaggaatag ggatcggtgt tattatcaac 9000
aatcatttat atagaggagt aagcggcttc tctggagaaa tgggacatat gacaatagac 9060
tttaatggtc ctaaatgcag ttgcggaaac cgaggatgct gggaattgta tgcttcagag 9120
aaggctttat taaaatctct tcagaccaaa gagaaaaaac tgtcctatca agatatcata 9180
aacctcgccc atctgaatga tatcggaacc ttaaatgcat tacaaaattt tggattctat 9240
ttaggaatag gccttaccaa tattctaaat actttcaacc cacaagccgt aattttaaga 9300
aatagcataa ttgaatcgca tcctatggtt ttaaattcaa tgagaagtga agtatcatca 9360
agggtttatt cccaattagg caatagctat gaattattgc catcttcctt aggacagaat 9420
gcaccggcat taggaatgtc ctccattgtg attgatcatt ttctggacat gattacaatg 9480
taatttttta tggaatggac agctcatctt taaagatgag tttttttatt ctaggagtat 9540
ttctgaattc 9550
<210> 11
<211> 22
<212> DNA
<213> 人工序列
<220>
<223> 引物1
<400> 11
gtttcacgta aacggcgggt cg 22
<210> 12
<211> 23
<212> DNA
<213> 人工序列
<220>
<223> 引物2
<400> 12
gatgggtatc gccagaaaga tcc 23
<210> 13
<211> 23
<212> DNA
<213> 人工序列
<220>
<223> 引物3
<400> 13
caacgacagg cttttcaaaa acc 23
<210> 14
<211> 23
<212> DNA
<213> 人工序列
<220>
<223> 引物4
<400> 14
cggtccagcg ttttgtattt cgc 23
<210> 15
<211> 1612
<212> DNA
<213> 人工序列
<220>
<223> BgIII-MluI片段的序列
<400> 15
agatctgtaa agataatatc tttgaattgt aacccccctc aaaagtaaga actacaaaaa 60
aagaatacgt tatatagaaa tatgtttgaa ccttcttcag attacaaata tattcggacg 120
gactctacct caaatgctta tctaactata gaatgacata caagcacaac cttgaaaatt 180
tgaaaatata actaccaatg aacttgttca tgtgaattat cgctgtattt aattttctca 240
attcaatata taatatgcca atacattgtt acaagtagaa attaagacac ccttgatagc 300
cttactatac ctaacatgat gtagtattaa atgaatatgt aaatatattt atgataagaa 360
gcgacttatt tataatcatt acatattttt ctattggaat gattaagatt ccaatagaat 420
agtgtataaa ttatttatct tgaaaggagg gatgcctaaa aacgaagaac attaaaaaca 480
tatatttgca ccgtctaatg gatagaaagg aggtgatcca gccgcacctt atgaaaaatc 540
attttatcag tttgaaaatt atgtattatg tggccagaag ttcctattcc gaagttccta 600
ttctctagaa agtataggaa cttcttataa aaatgaggag ggaaccgaat gagtaaagga 660
gaagaacttt tcactggagt tgtcccaatt cttgttgaat tagatggcga tgttaatggg 720
caaaaattct ctgttagtgg agagggtgaa ggtgatgcaa catacggaaa acttaccctt 780
aaatttattt gcactactgg gaagctacct gttccatggc caacgcttgt cactactctc 840
acttatggtg ttcaatgctt ttctagatac ccagatcata tgaaacagca tgactttttc 900
aagagtgcca tgcccgaagg ttatgtacag gaaagaacta tattttacaa agatgacggg 960
aactacaaga cacgtgctga agtcaagttt gaaggtgata cccttgttaa tagaatcgag 1020
ttaaaaggta ttgattttaa agaagatgga aacattcttg gacacaaaat ggaatacaat 1080
tataactcac ataatgtata catcatggca gacaaaccaa agaatggcat caaagttaac 1140
ttcaaaatta gacacaacat taaagatgga agcgttcaat tagcagacca ttatcaacaa 1200
aatactccaa ttggcgatgg ccctgtcctt ttaccagaca accattacct gtccacgcaa 1260
tctgcccttt ccaaagatcc caacgaaaag agagatcaca tgatccttct tgagtttgta 1320
acagctgctg ggattacaca tggcatggat gaactataca aataatgctg tccagactgt 1380
ccgctgtgta aaaaaaagga ataaaggggg gttgacatta ttttactgat atgtataata 1440
taatttgtat aagaaaatgc ttcatgtaat ggtcaaaatg ttttagagct agaaatagca 1500
agttaaaata aggctagtcc gttatcaact tgaaaaagtg gcaccgagtc ggtgctttga 1560
agttcctatt ccgaagttcc tattcttcaa atagtatagg aacttcacgc gt 1612
Claims (11)
1.一种用于将至少一种目的多核苷酸***至宿主细胞基因组的方法,所述方法包括以下步骤:
a)提供一种宿主细胞,所述宿主细胞在其基因组中包含以下:
i.编码包含靶序列的选择性标记的多核苷酸,所述靶序列侧接针对II类Cas9蛋白的功能性PAM序列;
ii.编码gRNA的至少一种多核苷酸,所述gRNA与靶序列至少80%互补并且能够与所述靶序列杂交;以及
iii.编码II类Cas9蛋白的无效核酸酶变体的多核苷酸,所述II类Cas9蛋白能够与gRNA相互作用并与靶序列结合,从而抑制选择性标记的表达;
b)用至少一种目的多核苷酸转化所述宿主细胞,并且所述至少一种目的多核苷酸能够使所述编码gRNA的至少一种多核苷酸失活;
c)选择由选择性标记赋予的性状;以及
d)识别转化的宿主细胞,其中所述编码gRNA的至少一种多核苷酸已被所述至少一种目的多核苷酸灭活。
2.一种用于将至少两种不同的目的多核苷酸***至宿主细胞基因组的方法,所述方法包括以下步骤:
a)提供一种宿主细胞,所述宿主细胞在其基因组中包含以下:
i.编码至少两种不同的选择性标记的至少两种多核苷酸,每种选择性标记包含侧接针对II类Cas9蛋白的功能性PAM序列的不同靶序列;
ii.编码至少两个gRNA的至少两种多核苷酸,所述至少两个gRNA与至少两个不同的靶序列至少80%互补并且能够与所述至少两个不同的靶序列杂交;
iii.编码II类Cas9蛋白的无效核酸酶变体的多核苷酸,所述II类Cas9蛋白能够与至少两个gRNA相互作用并与至少两个不同的靶序列结合,从而抑制两种不同的选择性标记的表达;
b)用至少两种不同的目的多核苷酸转化所述宿主细胞,所述多核苷酸能够使所述编码至少两个gRNA的至少两种多核苷酸失活;和
c)选择由至少两种不同的选择性标记赋予的性状;以及
d)识别转化的宿主细胞,其中所述编码至少两个gRNA的至少两种多核苷酸已被所述至少两种不同的目的多核苷酸灭活。
3.根据前述权利要求中任一项所述的方法,其中所述至少一种目的多核苷酸或所述至少两种目的多核苷酸编码蛋白质;优选地,所述至少一种目的多核苷酸或所述至少两种目的多核苷酸编码酶,所述酶独立地选自由以下组成的组:水解酶、异构酶、连接酶、裂解酶、氧化还原酶、或转移酶;更优选地是氨肽酶、淀粉酶、糖酶、羧肽酶、过氧化氢酶、纤维二糖水解酶、纤维素酶、几丁质酶、角质酶、环糊精糖基转移酶、脱氧核糖核酸酶、内切葡聚糖酶、酯酶、α-半乳糖苷酶、β-半乳糖苷酶、葡糖淀粉酶、α-葡糖苷酶、β-葡糖苷酶、转化酶、漆酶、脂肪酶、甘露糖苷酶、变聚糖酶、氧化酶、果胶分解酶、过氧化物酶、磷酸二酯酶、植酸酶、多酚氧化酶、蛋白水解酶、核糖核酸酶、转谷氨酰胺酶、木聚糖酶、和β-木糖苷酶。
4.根据前述权利要求中任一项所述的方法,其中所述宿主细胞是革兰氏阳性宿主细胞;优选地,所述宿主细胞选自由以下组成的组:芽孢杆菌属、链霉菌属、链球菌属、和乳杆菌属宿主细胞;更优选地,所述宿主细胞选自由以下组成的组:嗜碱芽孢杆菌、高地芽孢杆菌、解淀粉芽孢杆菌、植物解淀粉芽孢杆菌亚种、短芽孢杆菌、环状芽孢杆菌、克劳氏芽孢杆菌、凝结芽孢杆菌、坚硬芽孢杆菌、灿烂芽孢杆菌、迟缓芽孢杆菌、地衣芽孢杆菌、巨大芽孢杆菌、甲基营养型芽孢杆菌、短小芽孢杆菌、沙福芽孢杆菌、嗜热脂肪芽孢杆菌、枯草芽孢杆菌、以及苏云金芽孢杆菌细胞;最优选地,所述原核宿主细胞是地衣芽孢杆菌宿主细胞。
5.根据权利要求1-3中任一项所述的方法,其中所述宿主细胞是选自由以下组成的组的真菌宿主细胞:枝顶孢霉属、曲霉属、短梗霉属、烟管霉属(Bjerkandera)、拟腊菌属、金孢子菌属、鬼伞属、革盖菌属(Coriolus)、隐球菌属、线黑粉菌科(Filibasidium)、镰孢属、腐质霉属、梨孢菌属、毛霉属、毁丝霉属、新美鞭菌属、链孢菌属、拟青霉属、青霉属、平革菌属、射脉菌属(Phlebia)、瘤胃壶菌属、侧耳属(Pleurotus)、裂褶菌属、篮状菌属、嗜热子囊菌属、梭孢壳属、弯颈霉属、栓菌属(Trametes)、和木霉属细胞;优选地,所述真菌宿主细胞选自由以下组成的组:泡盛曲霉、臭曲霉、烟曲霉、日本曲霉、构巢曲霉、黑曲霉、米曲霉、黑刺烟管菌、干拟蜡菌、卡内基拟蜡菌、浅黄拟蜡孔菌、潘诺希塔拟蜡菌、环带拟蜡菌、微红拟蜡菌、虫拟蜡菌、狭边金孢子菌、嗜角质金孢子菌、卢克诺文思金孢子菌、粪状金孢子菌、租金孢子菌、女王杜香金孢子菌、热带金孢子菌、褐薄金孢子菌、灰盖鬼伞、毛革盖菌、杆孢状镰孢、谷类镰孢、库威镰孢、大刀镰孢、禾谷镰孢、禾赤镰孢、异孢镰孢、合欢木镰孢、尖孢镰孢、多枝镰孢、粉红镰孢、接骨木镰孢、肤色镰孢、拟分枝孢镰孢、硫色镰孢、圆镰孢、拟丝孢镰孢、镶片镰孢、特异腐质霉、柔毛腐质霉、米黑毛霉、嗜热毁丝霉、粗糙链孢菌、产紫青霉、黄孢平革菌、射脉菌、刺芹侧耳、土生梭孢壳霉、长域毛栓菌、变色栓菌、哈茨木霉、康宁木霉、长枝木霉、里氏木霉、和绿色木霉细胞。
6.根据权利要求1-3中任一项所述的方法,其中所述宿主细胞是选自由以下组成的组的酵母宿主细胞:假丝酵母属、汉逊酵母属、克鲁维酵母菌属、毕赤酵母属、酵母菌属、裂殖酵母属、和耶氏酵母属细胞;优选地,所述宿主细胞选自由以下组成的组:乳酸克鲁维酵母、巴斯德毕赤酵母、卡尔酵母、酿酒酵母、糖化酵母、道格拉氏酵母、克鲁弗酵母、诺地酵母、卵形酵母、和解脂耶氏酵母细胞。
7.根据前述权利要求中任一项所述的方法,其中所述选择性标记或所述至少两种不同的选择性标记独立地是阳性选择性标记、阴性选择标记、双向标记、或条件性必需基因。
8.根据前述权利要求中任一项所述的方法,其中所述选择性标记或所述至少两种不同的选择性标记独立地选自由以下组成的组的基因:cat、erm、tet、amp、spec、kana、neo、dal、lysA、araA、galE、antK、metC、xylA、gntP、glpD、glpF、glpK、glpP、lacA2、hisC、gapA、和aspB。
9.根据前述权利要求中任一项所述的方法,其中所述gRNA或所述至少两个gRNA包含第一RNA,所述第一RNA包含与所述编码一个或多个选择性标记的一个或多个多核苷酸至少85%互补并且能够与所述编码一个或多个选择性标记的一个或多个多核苷酸杂交的20个或更多个核苷酸;优选地,所述20个或更多个核苷酸与所述编码一个或多个选择性标记的一个或多个多核苷酸至少90%、95%、97%、98%、99%或甚至100%互补并且能够与所述编码一个或多个选择性标记的一个或多个多核苷酸杂交。
10.根据前述权利要求中任一项所述的方法,其中所述II类Cas9蛋白的无效核酸酶变体包含对应于SEQ ID NO:2的位置10和位置840的氨基酸的改变;更优选地,所述变体包含天冬氨酸取代丙氨酸,D10A和组氨酸取代丙氨酸,H840A。
11.根据前述权利要求中任一项所述的方法,其中所述编码gRNA的至少一种多核苷酸或所述编码至少两个gRNA的至少两种多核苷酸已在宿主细胞基因组中被至少一种目的多核苷酸或至少两种不同的目的多核苷酸部分或完全替换,从而使所述编码gRNA的至少一种多核苷酸或所述编码至少两个gRNA的至少两种多核苷酸失活。
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LT3728583T (lt) | 2023-06-12 |
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WO2019121192A1 (en) | 2019-06-27 |
US20210017544A1 (en) | 2021-01-21 |
CN111630165B (zh) | 2024-07-16 |
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DK3728583T3 (da) | 2023-05-30 |
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