CN106554969A - 基于抑菌杀菌的多靶点CRISPR/Cas9表达载体 - Google Patents

基于抑菌杀菌的多靶点CRISPR/Cas9表达载体 Download PDF

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CN106554969A
CN106554969A CN201611026707.5A CN201611026707A CN106554969A CN 106554969 A CN106554969 A CN 106554969A CN 201611026707 A CN201611026707 A CN 201611026707A CN 106554969 A CN106554969 A CN 106554969A
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王令
张涛
路宏朝
杜伟立
尹亚军
杨理凯
郭苗
刘欢
尹美晨
李蕊清
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Abstract

本发明公开了一种基于抑菌杀菌的多靶点CRISPR/Cas9表达载体,该基于抑菌杀菌的多靶点CRISPR/Cas9表达载体的基因序列为SEQ ID NO1;Cas9表达载体基因序列为SEQ ID NO2;构建方法利用导向RNA特异识别基因的DNA片段,然后介导Cas9蛋白切割DNA双链,产生双链切口,破坏DNA序列,从而破坏DNA编码的功能蛋白的原理构建多靶点CRISPR/Cas9表达载体。本发明通过与阴性对照相比较,CRISPR/Cas9作用于DNA回旋酶和二氢叶酸还原酶后,细菌的存活率只有10%,杀菌效果达到90%以上。

Description

基于抑菌杀菌的多靶点CRISPR/Cas9表达载体
技术领域
本发明属于生物技术领域,尤其涉及一种基于抑菌杀菌的多靶点CRISPR/Cas9表达载体。
背景技术
传统的抗生素通过抑制细菌分裂或代谢相关基因的表达,实现抑菌杀菌的目的。如青霉素类、头孢菌素类抗生素阻碍细菌细胞壁的合成;氨基苷类、大环内酯类抗生素抑制细菌蛋白质的合成;喹诺酮类抗生素抑制细菌DNA的复制;利福平抑制细菌RNA的合成;多粘霉素和新霉素等抗生素影响细胞膜的通透性;磺胺类抗生素影响细菌叶酸的合成。其中细菌DNA旋转酶为喹诺酮类药物作用靶位,二氢叶酸还原酶是磺胺类抗生素作用靶位。在抗生素长期的选择压力下,细菌代谢相关酶的编码基因发生突变,从而丧失抗生素的作用靶位,抵御抗生素的作用,从而产生耐药性,甚至导致超级细菌的诞生。此外,抗生素的滥用加快了耐药性病原菌的产生,从而极大的威胁着人类的健康和公共卫生安全。
综上所述,目前关于CRISPR/Cas9作用于DNA回旋酶和二氢叶酸的杀菌效果还没有有效的理论指导,以减少耐药性的产生,达到抑菌杀菌的效果。
发明内容
本发明的目的在于提供一种基于抑菌杀菌的多靶点CRISPR/Cas9表达载体,旨在解决关于CRISPR/Cas9作用于DNA回旋酶和二氢叶酸的杀菌效果还没有有效的理论指导,减少耐药性的产生,达到抑菌杀菌的效果的问题。
本发明是这样实现的,
一种基于抑菌杀菌的多靶点CRISPR/Cas9表达载体,该基于抑菌杀菌的多靶点CRISPR/Cas9表达载体的基因序列为SEQ ID NO1;Cas9表达载体基因序列为SEQ ID NO2。
本发明另一目的在于提供一种基于抑菌杀菌的多靶点CRISPR/Cas9表达载体的构建方法,该构建方法利用导向RNA特异识别基因的DNA片段,然后介导Cas9蛋白切割DNA双链,产生双链切口,破坏DNA序列,从而破坏DNA编码的功能蛋白的原理构建多靶点CRISPR/Cas9表达载体;
该构建方法具体包括多靶点CRISPR表达载体的构建,该多靶点CRISPR表达载体的构建的方法包括:
上游特异引物的寡聚核苷酸包含靶点序列,5’端含有Sal Ⅰ酶切位点,3’端为部分重复序列(direct repeat,DR);
下游通用引物的5’端携带Xho Ⅰ和BamH Ⅰ酶切位点,3’端重复序列的11个核苷酸与上游引物3’端碱基互补,上游特异引物和下游通用引物二者形成引物二聚体;
该引物二聚体在Klenow酶的作用下延伸,合成特异的靶序列DNA,该引物二聚体含有靶序列、重复序列、Sal Ⅰ、Xho Ⅰ和BamH Ⅰ酶切位点;
利用Sal Ⅰ和BamH Ⅰ双酶切靶序列DNA,克隆至经Xho Ⅰ和BamH Ⅰ双酶切的骨架载体pCRISPR载体中,获得单靶点载体pCRISPR-T1;
Sal Ⅰ和Xho Ⅰ为同尾酶,连接后Sal Ⅰ和Xho Ⅰ这两个酶切位点都消失,载体中先前的Xho Ⅰ酶切位点被破坏,同时在克隆的靶序列DNA中加入一个Xho Ⅰ酶切位点;连接之后的单靶点载体中仍然含有Xho Ⅰ和BamH Ⅰ多克隆位点;
随后,利用相同的克隆方法将靶序列2插入pCRISPR-T1载体的Xho Ⅰ和BamH Ⅰ位点,获得双靶点载体pCRISPR-T1-T2,同时该双靶点载体pCRISPR-T1-T2仍然携带Xho Ⅰ和BamH Ⅰ位点,作为插入第三个靶点的骨架载体,构建三靶点表达载体,依次加入更多靶点。
进一步,所述多靶点CRISPR/Cas9表达载体的构建方法还包括Cas9表达载体的构建,该Cas9表达载体的构建的方法包括:
利用特异引物Fcas和Rcas,以嗜热链球菌基因组为模板,扩增Cas9基因,正向和反向引物分别携带BamH Ⅰ和Xho Ⅰ酶切位点,将扩增的Cas9基因片段克隆至载体pET28a中,获得Cas9诱导表达载体pET28a-Cas9;
通过多靶点CRISPR表达载体的构建和Cas9表达载体的构建后形成双质粒的CRISPR/Cas9表达载体。
进一步,上游特异引物的寡聚核苷酸包含靶点序列包括以大肠杆菌DNA旋转酶A亚基引物、B亚基引物和二氢叶酸还原酶引物;所述A亚基引物、B亚基引物和二氢叶酸还原酶引物的DNA序列分别为SEQ ID NO3、SEQ ID NO4和SEQ ID NO5;
所述下游通用引物的DNA序列为SEQ ID NO6。
进一步,所述上游特异引物和下游通用引物二者形成引物二聚体中含靶序列的上游引物和通用引物UR的3’末端有11个碱基互补,二者在反应体系中配对形成二聚体,在Klenow酶的作用下延伸合成双链DNA;
具体包括:50μL反应体系中上下游引物各15μL,5μL缓冲液,1μL Klenow酶,2μLdNTP,12μL ddH2O,反应条件37℃保持30min;利用2%的琼脂糖凝胶电泳检测合成的双链DNA。
本发明另一目的在于提供一种利用上述的基于抑菌杀菌的多靶点CRISPR/Cas9表达载体在杀菌上的应用方法,该应用方法包括:
将0.5μg CRISPR表达载体和0.5μg Cas9表达载体与100μL大肠杆菌感受态细胞混合,转移至2mm电击杯,电击条件2.5kv,5ms,转化至大肠杆菌细胞;然后将转化混合体转移至1mL无抗生素LB培养基中,37℃孵育1h后平均涂布于两个LB固体平板;
两个LB平板都含有Amp和Kan抗生素,一个加入浓度2%的葡萄糖,另一个加入浓度1mM的IPTG;平板置于37℃培养箱,培养14小时~16小时后,置于成像系统仪拍照并统计克隆数;
表达载体pET28a-Cas9在IPTG诱导作用下表达Cas9蛋白,将转化混合体涂布于含葡萄糖的LB双抗平板,Cas9蛋白表达被抑制,质粒成功转化至大肠杆菌细胞,在平板上存活,检测质粒转化效率;
将另一等份转化混合体涂布于含IPTG的双抗平板上,形成特异性CRISPR/Cas9核酸酶,切割靶基因DNA旋转酶或二氢叶酸还原酶,使细菌不能存活;
统计同一转化混合体在两种不同平板上的克隆数,杀菌效率=含IPTG平板克隆数/含葡糖糖平板克隆数×100%。
本发明提供的基于抑菌杀菌的多靶点CRISPR/Cas9表达载体及其构建方法通过与阴性对照相比较,CRISPR/Cas9作用于DNA回旋酶和二氢叶酸还原酶后,细菌的存活率不足10%,杀菌效果达到90%以上。
基于细菌耐药性问题,本发明通过CRISPR/Cas9直接切割细菌生长的重要基因,产生DNA双链切口,破坏重要功能基因,使细菌无法正常分裂和生长,达到抑菌杀菌的效果。
附图说明
图1是本发明实施例提供的基于抑菌杀菌的多靶点CRISPR表达载体的构建方法流程图。
具体实施方式
为了使本发明的目的、技术方案及优点更加清楚明白,以下结合实施例,对本发明进行进一步详细说明。应当理解,此处所描述的具体实施例仅仅用以解释本发明,并不用于限定本发明。
下面结合附图对本发明的应用原理作进一步描述。
本发明实施例提供的基于抑菌杀菌的多靶点CRISPR/Cas9表达载体,该基于抑菌杀菌的多靶点CRISPR/Cas9表达载体的基因序列为SEQ ID NO1;Cas9表达载体基因序列为SEQ ID NO2。
本发明实施例提供的基于抑菌杀菌的多靶点CRISPR/Cas9表达载体的构建方法,该构建方法利用导向RNA特异识别基因的DNA片段,然后介导Cas9蛋白切割DNA双链,产生双链切口,破坏DNA序列,从而破坏DNA编码的功能蛋白的原理构建多靶点CRISPR/Cas9表达载体;
如图1所示,本发明实施例提供的基于抑菌杀菌的多靶点CRISPR表达载体的构建的方法包括:
上游特异引物的寡聚核苷酸包含靶点序列,5’端含有Sal Ⅰ酶切位点,3’端为部分重复序列(direct repeat,DR);
下游通用引物的5’端携带Xho Ⅰ和BamH Ⅰ酶切位点,3’端重复序列的11个核苷酸与上游引物3’端碱基互补,上游特异引物和下游通用引物二者形成引物二聚体;
该引物二聚体在Klenow酶的作用下延伸,合成特异的靶序列DNA,该引物二聚体含有靶序列、重复序列、Sal Ⅰ、Xho Ⅰ和BamH Ⅰ酶切位点;
利用Sal Ⅰ和BamH Ⅰ双酶切靶序列DNA,克隆至经Xho Ⅰ和BamH Ⅰ双酶切的骨架载体pCRISPR载体中,获得单靶点载体pCRISPR-T1;
Sal Ⅰ和Xho Ⅰ为同尾酶,连接后Sal Ⅰ和Xho Ⅰ这两个酶切位点都消失,载体中先前的Xho Ⅰ酶切位点被破坏,同时在克隆的靶序列DNA中加入一个Xho Ⅰ酶切位点;连接之后的单靶点载体中仍然含有Xho Ⅰ和BamH Ⅰ多克隆位点;
随后,利用相同的克隆方法将靶序列2插入pCRISPR-T1载体的Xho Ⅰ和BamH Ⅰ位点,获得双靶点载体pCRISPR-T1-T2,同时该双靶点载体pCRISPR-T1-T2仍然携带Xho Ⅰ和BamH Ⅰ位点,作为插入第三个靶点的骨架载体,构建三靶点表达载体,依次加入更多靶点。
进一步,所述多靶点CRISPR/Cas9表达载体的构建方法还包括Cas9表达载体的构建,该Cas9表达载体的构建的方法包括:
利用特异引物Fcas和Rcas,以嗜热链球菌基因组为模板,扩增Cas9基因,正向和反向引物分别携带BamH Ⅰ和Xho Ⅰ酶切位点,将扩增的Cas9基因片段克隆至载体pET28a中,获得Cas9诱导表达载体pET28a-Cas9;
所述特异引物FcasDNA序列为SEQ ID NO7
gccATGACTAAGCCATACTCAAT,
所述特异引物RcasDNA序列为SEQ ID NO8
gccTTAACCCTCTCCTAGTTTG;
通过多靶点CRISPR表达载体的构建和Cas9表达载体的构建后形成双质粒的CRISPR/Cas9表达载体。
进一步,上游特异引物的寡聚核苷酸包含靶点序列包括以大肠杆菌DNA旋转酶A亚基引物、B亚基引物和二氢叶酸还原酶引物;所述A亚基引物、B亚基引物和二氢叶酸还原酶引物的DNA序列分别为SEQ ID NO3 gcaGTCGACCGGTCTATGACACGATCGTCCGCAGTTTTAGAGCTGTGTTG,
SEQ ID NO4
gcaGTCGACTCATAACAGTTCAGAACTATTACAGTTTTAGAGCTGTGTTG,
和SEQ ID NO5
gcaGTCGACCGGGTACGGACGATCGCGTAACGTGTTTTAGAGCTGTGTTG;
所述下游通用引物的DNA序列为SEQ ID NO6
gcaATCGTTTTGGAACCATTCGAAACAACACAGCTC。
进一步,所述上游特异引物和下游通用引物二者形成引物二聚体中含靶序列的上游引物和通用引物UR的3’末端有11个碱基互补,二者在反应体系中配对形成二聚体,在Klenow酶的作用下延伸合成双链DNA;
具体包括:50μL反应体系中上下游引物各15μL,5μL缓冲液,1μL Klenow酶,2μLdNTP,12μL ddH2O,反应条件37℃保持30 min;利用2%的琼脂糖凝胶电泳检测合成的双链DNA。
本发明实施例提供一种利用上述基于抑菌杀菌的多靶点CRISPR/Cas9表达载体在杀菌上的应用方法,该应用方法包括:
将0.5μg CRISPR表达载体和0.5μg Cas9表达载体与100μL大肠杆菌感受态细胞混合,转移至2mm电击杯,电击条件2.5 kv,5ms,转化至大肠杆菌细胞;然后将转化混合体转移至1mL无抗生素LB培养基中,37℃孵育1h后平均涂布于两个LB固体平板;
两个LB平板都含有Amp和Kan抗生素,一个加入浓度2%的葡萄糖,另一个加入浓度1mM的IPTG;平板置于37℃培养箱,培养14小时~16小时后,置于成像系统仪拍照并统计克隆数;
表达载体pET28a-Cas9在IPTG诱导作用下表达Cas9蛋白,将转化混合体涂布于含葡萄糖的LB双抗平板,Cas9蛋白表达被抑制,质粒成功转化至大肠杆菌细胞,在平板上存活,检测质粒转化效率;
将另一等份转化混合体涂布于含IPTG的双抗平板上,形成特异性CRISPR/Cas9核酸酶,切割靶基因DNA旋转酶或二氢叶酸还原酶,使细菌不能存活;
统计同一转化混合体在两种不同平板上的克隆数,杀菌效率=含IPTG平板克隆数/含葡糖糖平板克隆数×100%。
下面结合实施例对本发明的应用原理作进一步描述。
1)上游特异引物的寡聚核苷酸包含靶点序列,5’端含有Sal Ⅰ酶切位点,3’端为部分重复序列(direct repeat,DR)。下游通用引物的5’端携带Xho Ⅰ和BamH Ⅰ酶切位点,3’端重复序列的11个核苷酸与上游引物3’端碱基互补,二者可以形成引物二聚体。二聚体在Klenow酶的作用下延伸,合成特异的靶序列DNA,其含有靶序列、重复序列、Sal Ⅰ、Xho Ⅰ和BamH Ⅰ酶切位点。利用Sal Ⅰ和BamH Ⅰ双酶切靶序列DNA,克隆至经Xho Ⅰ和BamH Ⅰ双酶切的骨架载体pCRISPR载体中,获得单靶点载体pCRISPR-T1。由于Sal Ⅰ和Xho Ⅰ为同尾酶,连接后这两个酶切位点都消失,所以载体中先前的Xho Ⅰ酶切位点被破坏,同时本发明在克隆的靶序列DNA中加入了一个Xho Ⅰ位点,所以,连接之后的单靶点载体中仍然含有Xho Ⅰ和BamHⅠ多克隆位点(图1)。随后,利用相同的克隆方法将靶序列2插入pCRISPR-T1载体的Xho Ⅰ和BamH Ⅰ位点,获得双靶点载体pCRISPR-T1-T2,同时该载体仍然携带Xho Ⅰ和BamH Ⅰ位点,可以作为插入第三个靶点的骨架载体,构建三靶点表达载体,依次可以加入更多靶点。
2)靶点的选择和靶序列合成
以大肠杆菌DNA旋转酶A亚基(Gene ID:946614)、B亚基(Gene ID:948211)和二氢叶酸还原酶(Gene ID:13906554)3个编码基因为靶基因,根据NCBI公布的基因序列,遵循NGG的PAM原则寻找靶位点,在DNA旋转酶的A亚基和B亚基分别选择一个靶点A1,B1,在二氢叶酸还原酶基因序列中选择一个靶位点,简称为靶点R2,3个靶点序列的具体信息见表1。
表1靶点序列
注:下划线碱基为PAM序列。
根据所选择的靶位点,设计引物(表2),其中上游引物包含靶序列,重复序列和SalⅠ酶切位点,通用下游引物UR含重复序列,BamH Ⅰ和Xho Ⅰ酶切位点。
表2引物序列
注:双下划线和波浪线分别表示Xho Ⅰ和BamH Ⅰ酶切位点,单下划线为Sal Ⅰ酶切位点。
含靶序列的上游引物和通用引物UR的3’末端有11个碱基互补,二者在反应体系中配对形成二聚体,其扮演了引物和模板的双重角色,在Klenow酶的作用下延伸合成双链DNA。50μL反应体系中有上下游引物各15μL,5μL缓冲液,1μL Klenow酶,2μL dNTP,12μLddH2O,反应条件37℃保持30min。利用2%的琼脂糖凝胶电泳检测合成的双链DNA。
3)多靶点载体的构建
根据图1所示的表达载体构建策略,利用Sal Ⅰ和BamH Ⅰ双酶切A1靶序列DNA,同时以Xho Ⅰ和BamH Ⅰ双酶切骨架载体pCRISPR。纯化酶切后的DNA产物,在T4DNA连接酶的作用下将靶序列DNA克隆至骨架载体中,同时以只加入酶切的骨架载体为对照,连接产物转化至大肠杆菌Top10感受态细胞,比较实验组和对照组中单克隆数。选择实验组的2个单克隆,提取质粒并送至北京鼎盛生物有限公司,以引物M13F(-47)测序,检测靶点A1插入骨架载体。
Xho Ⅰ和BamH Ⅰ双酶切单靶点载体pCRISPR-A1,同时用Sal Ⅰ和BamH Ⅰ双酶切B1靶序列DNA。然后纯化酶切后的DNA片段和载体,加入连接体系,将B1靶DNA分别克隆至单靶点载体中,测序鉴定,分别获得含A1-B1的双靶点表达载体pCRISPR-A1-B1。
三靶点载体构建以获得的双靶点载体pCRISPR-A1-B1为骨架,Xho Ⅰ和BamH Ⅰ双酶切骨架载体,利用Sal Ⅰ和BamH Ⅰ双酶切R2靶序列DNA,然后克隆R2靶序列至pCRISPR-A1-B1载体中,获得含三靶点A1-B1-R2的表达载体pCRISPR-A1-B1-R2。
4)CRISPR表达载体构建的特殊之处
本发明在靶点DNA合成过程中引入Sal Ⅰ、Xho Ⅰ和BamH Ⅰ多克隆位点(图1)。同尾酶Sal Ⅰ和Xho Ⅰ酶切后产生相同的黏性末端,连接后两个酶切位点都被破坏。当Sal Ⅰ、BamH Ⅰ酶切的第一个靶序列和Xho Ⅰ、BamH Ⅰ酶切的骨架载体连接后,接口处无Sal Ⅰ和XhoⅠ酶切位点,但是在BamH Ⅰ位点前引入了Xho Ⅰ,从而使获得单靶点载体中仍然有Xho Ⅰ和BamH Ⅰ双酶切位点。以单靶点载体为骨架,利用相同的方法克隆第二个靶点至第一个靶点之后,形成双靶点载体,同时保留了Xho Ⅰ和BamH Ⅰ多克隆位点,方便后续靶点的插入。以此类推,此载体中可以无限制的加入多个靶点,实现一个载体表达多个靶点的gRNA。
5)pCas9表达载体构建
除了CRISPR表达载体外,还需要构建Cas9表达载体,形成双质粒的CRISPR/Cas9系统。利用特异引物Fcas和Rcas(表2),以嗜热链球菌基因组为模板,扩增Cas9基因,正向和反向引物分别携带BamH Ⅰ和Xho Ⅰ酶切位点,将扩增的Cas9基因片段克隆至载体pET28a中,获得Cas9诱导表达载体pET28a-Cas9。
6)杀菌实验
将0.5μg CRISPR表达载体和0.5μg Cas9表达载体与100μL大肠杆菌感受态细胞混合,转移至2mm电击杯,电击条件2.5kv,5ms,转化至大肠杆菌细胞。然后将转化混合体转移至1mL无抗生素LB培养基中,37℃孵育1h后平均涂布于两个LB固体平板。两个LB平板都含有Amp和Kan抗生素,其中一个加入葡萄糖(终浓度2%),另一个加入IPTG(终浓度1mM)。平板置于37℃培养箱,培养14-16小时后,置于成像系统仪拍照并统计克隆数。
表达载体pET28a-Cas9在IPTG诱导作用下表达Cas9蛋白,而高浓度的葡萄糖能抑制诱导启动子的表达。所以,本发明将转化产物涂布于含葡萄糖的LB双抗平板,Cas9蛋白表达被抑制,只要质粒成功转化至大肠杆菌细胞,则能在平板上存活,以此检测质粒转化效率。此外,将另一等份转化产物涂布于含IPTG的双抗平板上,形成特异性CRISPR/Cas9核酸酶,切割靶基因DNA旋转酶或二氢叶酸还原酶,导致细菌不能存活。统计同一转化产物在两种不同平板上的克隆数,杀菌效率=含IPTG平板克隆数/含葡糖糖平板克隆数×100%。
本发明构建了三靶点载体pCRISPR-A1-B1-R2,将三靶点表达载体和Cas9表达载体转化至大肠杆菌细胞内,统计细菌的相对成活率,以反映CRISPR/Cas9酶对细菌的破坏能力,同时以空表达载体转化的大肠杆菌作为阴性对照。
下面结合原理分析对本发明的基于抑菌杀菌的多靶点CRISPR/Cas9表达载体作进一步描述。
DNA回旋酶和二氢叶酸还原酶是细菌生长的重要功能基因,同时是抗菌药物的作用靶点,抑制或破坏这两种酶的活性,即能实现抑菌或杀菌的目的,但是靶基因的突变使细菌出现耐药性。CRISPR/Cas9技术一种高效的基因编辑技术,其利用导向RNA特异识别基因的DNA片段,然后介导Cas9蛋白切割DNA双链,产生双链切口,破坏DNA序列,从而破坏DNA编码的功能蛋白。本发明基于CRISPR/Cas9技术原理,以大肠杆菌的DNA旋转酶和二氢叶酸还原酶为靶基因构建针对目标基因的多靶点CRISPR/Cas9表达载体,特异性破坏DNA回旋酶和二氢叶酸还原酶基因,导致大肠杆菌不能正常生长,达到抑菌和杀菌的目的。
本发明提供的基于抑菌杀菌的多靶点CRISPR/Cas9表达载体及其构建方法通过与阴性对照相比较,CRISPR/Cas9作用于DNA回旋酶和二氢叶酸还原酶后,细菌的存活率不足10%,杀菌效果达到90%以上。
以上所述仅为本发明的较佳实施例而已,并不用以限制本发明,凡在本发明的精神和原则之内所作的任何修改、等同替换和改进等,均应包含在本发明的保护范围之内。
<110>申请人名称:陕西理工学院
<120>基于抑菌杀菌的多靶点CRISPRCas9表达载体
<160> 8
<210>1
<211>5139
<212> DNA
<213>人工序列
<400>DNA序列
TGAGTTATTTCAAGACTATCTTTTAGTATTTAGTAGTTTCTGTATGAAGTTGAATGGGAT
AATCATTTTGTTAGAGAGTAGATTATAAGGATTTGATAGAGGAGGAATTAAGTTGCTTGA
CATATGATTATTAAGAAATAATCTAATATGGTGACAGTCACATCTTGTCTAAAACGTTGA
TATATAAGGATTTTTAAGGTATAATAAATATAAAATTGGAATTATTTTGAAGCTGAAGTC
ATGCTGAGATTAATAGTGCGATTACGAAATCTGGTAGAAAAGATATCCTACGAGGCTCGA
CGTTTTAGAGCTGTGTTGTTTCGAATGGTTCCAAAACCTCGACCGGTCTATGACACGATC
GTCCGCAGTTTTAGAGCTGTGTTGTTTCGAATGGTTCCAAAACCTCGACTCATAACAGTT
CAGAACTATTACAGTTTTAGAGCTGTGTTGTTTCGAATGGTTCCAAAACCTCGACGACGC
ATATCGACGCAGAAGTGGAGTTTTAGAGCTGTGTTGTTTCGAATGGTTCCAAAACCTCGA
GGATGGATCCTTTTGTTATCACAATTTTCGGTTGACATCTCTTAGAACTCATCTTATCAT
AAAGGAGTCTAGTATTAAAATATGAGAAGGAACATGTTATACGAATATCCAGCGTCGACG
ATGGTACCCAATTCGCCCTATAGTGAGTCGTATTACGCGCGCTCACTGGCCGTCGTTTTA
CAACGTCGTGACTGGGAAAACCCTGGCGTTACCCAACTTAATCGCCTTGCAGCACATCCC
CCTTTCGCCAGCTGGCGTAATAGCGAAGAGGCCCGCACCGATCGCCCTTCCCAACAGTTG
CGCAGCCTGAATGGCGAATGGGACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTG
GTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCT
TTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGG
CTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACCTCGACCCCAAAAAACTTGATTAG
GGTGATGGTTCACGTAGTGGGCCATCGCCCTGATAGACGGTTTTTCGCCCTTTGACGTTG
GAGTCCACGTTCTTTAATAGTGGACTCTTGTTCCAAACTGGAACAACACTCAACCCTATC
TCGGTCTATTCTTTTGATTTATAAGGGATTTTGCCGATTTCGGCCTATTGGTTAAAAAAT
GAGCTGATTTAACAAAAATTTAACGCGAATTTTAACAAAATATTAACGCTTACAATTTAG
GTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATT
CAAATATGTATCCGCTCATGAGACAATAACCCTGATAAATGCTTCAATAATATTGAAAAA
GGAAGAGTATGAGTATTCAACATTTCCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTT
GCCTTCCTGTTTTTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGT
TGGGTGCACGAGTGGGTTACATCGAACTGGATCTCAACAGCGGTAAGATCCTTGAGAGTT
TTCGCCCCGAAGAACGTTTTCCAATGATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGG
TATTATCCCGTATTGACGCCGGGCAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGA
ATGACTTGGTTGAGTACTCACCAGTCACAGAAAAGCATCTTACGGATGGCATGACAGTAA
GAGAATTATGCAGTGCTGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGA
CAACGATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGATCATGTAA
CTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCATACCAAACGACGAGCGTGACA
CCACGATGCCTGTAGCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTTA
CTCTAGCTTCCCGGCAACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGGACCAC
TTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGC
GTGGGTCTCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGTATCGTAG
TTATCTACACGACGGGGAGTCAGGCAACTATGGATGAACGAAATAGACAGATCGCTGAGA
TAGGTGCCTCACTGATTAAGCATTGGTAACTGTCAGACCAAGTTTACTCATATATACTTT
AGATTGATTTAAAACTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATA
ATCTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAG
AAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAA
CAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTT
TTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTCCTTCTAGTGTAGC
CGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAA
TCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAA
GACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGC
CCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAA
GCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAA
CAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCG
GGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCC
TATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTG
CTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTG
AGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGG
AAGCGGAAGAGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAAT
GCAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGAGCGCAACGCAATTAATG
TGAGTTAGCTCACTCATTAGGCACCCCAGGCTTTACACTTTATGCTTCCGGCTCGTATGT
TGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGCTATGACCATGATTACG
CCAAGCGCGCAATTAACCCTCACTAAAGGGAACAAAAGCTGGAGCTCCTAGCTAGCCCTT
TGGATGATTCCAAGGGGCTCTTTGTGTATAAAAAAAACACCGAATCGGTGCCACCTTTTC
AAGTTGAGTACGGACTAAGCCTTATTTTAACTCGCTGTGTTGTTTCGAATGGTTTCAAAC
CACAATTATTATTATATAGTTTTATAACTAATATGTCAATTTACTATAGGGTAAAGTATA
AAAAATTTTATTCTTGATTACAAGATTTCAACGTCATACTATTTAATCAGTAATTCTATT
AGATGTTGAAATGTTGTTTAATAAGTGTATAATAATTTCTATAAATTTAGATTTTTCAAA
TAAGGAGAAATGTATGACTAAGCCATACTCAATTGGACTTGATATTGGAACGAATAGTGT
TGGATGGGCTGTAATAACTGATAATTACAAGGTTCCGTCTAAAAAAATGAAAGTCTTAGG
AAATACGAGTAAAAAGTATATCAAAAAGAACCTGTTAGGTGTATTACTCTTTGACTCTGG
AATCACAGCAGAAGGAAGAAGATTGAAGCGTACTGCAAGAAGACGTTATACTAGACGCCG
TAATCGTATCCTTTATTTGCAGGAAATTTTTAGCACAGAGATGGCTACATTAGATGATGC
TTTCTTTCAAAGACTTGACGATTCGTTTTTAGTTCCTGATGATAAACGTGATAGTAAGTA
TCCGATATTTGGAAACTTAGTAGAAGAAAAAGCCTATCATGATGAATTTCCAACTATCTA
TCATTTAAGGAAATATTTAGCAGATAGTACTAAAAAAGCAGATTTGCGTCTAGTTTATCT
TGCATTGGCTCATATGATTAAATATAGAGGTCACTTCTTAATTGAAGGAGAGTTTAATTC
AAAAAATAATGATATTCAGAAGAATTTTCAAGACTTTTTGGACACTTATAATGCTATTTT
TGAATCGGATTTATCACTTGAGAATAGTAAACAACTTGAGGAAATTGTTAAAGATAAGAT
TAGTAAATTAGAAAAGAAAGATCGTATTTTAAAACTCTTCCCTGGGGAGAAGAATTCTAT
CAGTATGATGAACAATCGAATTTAACAATATTTGATAGTAAAATAAGGAGTATAAGGTCC
TCTGAATTACTTTTAATAACGGATATTTTAGGTTACGATATTAATACTTCTCAGGTATTA
AAGTTGCTTCACACAGATATTGTAAGCCAGTTGAATGATAAACCTGAAGTTAGATCAGAA
ATAGATTCTTTGGTATCATTAATTACTGATATTATAATGGCAGAGTGTATAGAAAATGAA
CTAGATATAGAGTATGATGAGATTACACTTTTAGAACTAATTAAAGCATTAGGTGTCAGA
ATCGAAACTAAGTCTTGTACGGTTTTTGAAAAAATATTTGAGATTTTACAGATTTTTAAA
TATTTAGTTAAAAAGAGAATTTTAGTATTCGTCAATAGCTTGTCGTATTTTTCTAAAGAT
GAAATTTATCAAATCTTGGAATATACAAAGTTATCACAAGCTGATGTATTATTTTTGGAA
CCTAGACAGATTGAAGGGATTCAACAATTTATTTTAGACAAGGATTATATTTTGATGCCC
TATAATAACTAGTAAATTAGTAATAAGTATAGATAGTCT
<210>2
<211>9502
<212> DNA
<213>人工序列
<400>DNA序列
ATCCGGATATAGTTCCTCCTTTCAGCAAAAAACCCCTCAAGACCCGTTTAGAGGCCCCAA
GGGGTTATGCTAGTTATTGCTCAGCGGTGGCAGCAGCCAACTCAGCTTCCTTTCGGGCTT
TGTTAGCAGCCGGATCTCAGTGGTGGTGGTGGTGGTGCTCGAGTTAACCCTCTCCTAGTT
TGGCAAGGTCTATTCGTGTTTCATAGAGGCCTGTAACAGATTGATGAATAAGTGTGGCAT
CTTTTAATAGGGATGATGGGGTATAGTCTCTATACCTTGGAATTTTAACACCTAAAAATT
CAAAATCAGCAGCACTTCCACGAGAGGTTAATTCAAATAGCCCCTTTCTTTCACTTCCGG
TAGGTCCTATAAAACTACTACAGAGTTCATCTATACTATGATTTTGCCAAGATTGAAAGG
CAGAGTTTAAGAGTTTACCATTCTTTTTAGCTCCAACATAATTCTCATTAAACTCAAGAA
TGTAGTAAAATAATTCTTCAAACTCTTTTTTATGGTTCTCAACATATTTTCTATGATTCT
CATTAATTGTGTTACTTATTCTCTTAGCATGATAAAGTAATTTCACAAACTTCTGTGAAA
GAAAAATCTGATTTCCTTTGTGAATCTCTCCCCTCTTATTATTCGTTGACAAAATACTAG
CCAACATACGACGTGAACCATCTGAAAGTTCAAATAAACTATATTTAGGTAGTTCAATAA
TTAACTCAATATCTTTATAACCTTTTTCAAGTAAAAAATTAAGTTTATCTTTTCTATAAT
TAATCCTATCTAAAATAGAAATACCTTGAAATTCTAGTACATTTGTTATTTTTTTCTTAG
CACCTTTTTCAATTGTCCCTTTAACAAGAACAGTAAAAGAATTAGAAATTCCAGCATACC
CCCCATACTTTTTGGGGTCAAGATACTCTTTAGCACCTACTAAATTTTCATTACTATTTG
GTTTTGGCTTTGAGGAAAGATTTGCATTAAACAATCCCTTTGGTTTTCCTCTATCCAATC
CGTGATTCTGTTCCTCAACTTTTTTCACAACATTTACTTGCGGATAAGAGAGTACTCTCC
TTACAGTTGCTAAATCAGATTCTTTATTCCAAACGGATTCGCCGGTCTCCTCATTTACCT
CAATAAGTGGTCTTTCAATAACTCTACCATCAGCTAAAGAAATAGATTTTTTAAAGATAT
TCATGATATTTGAATAGAAATATACCTTTTCTGTAGCGGACTTTCTTTCTCTAAAACTAT
TGTATTTTGGATAATCACCGTACACAAATTCTGGCTCTAGTTTAGGGTATTTCTTAAGTA
AAGCACTTGCTACAACGGCATTCAAGTAAGCATCATGAGCATGATGAAAATCATTGATTT
CACGAACTTTATAAAGTTCAAAATCCTTACGAAATTGAGAAACTAAGGTAGATTTCAAGG
TAATAATTTTTACTGTTCGTACCGCTCTATTATTTTCATCTTTTTTATTATTAAATTTCT
CATCAAGTAAACGAGCTACATGTTTTGTTATTTGACGTGTTTCAACCAACTGGCGTTGAA
TAAAACCAGCTTTGTCCTCAGGTGACAATCCTCCCCGTTCAGCTTTTGTCAGATTATCAA
ATTTTCGTTGAGAAATTAATTTTGATTTCAATAATTGATACCAAAATGTCTTTCTTTTTT
TGACAACCTCTAAACTTGGAACATCATCTGATTTACCACGGTTACTAGCAGATGAAACAA
GTACTTTATTGTCAATAGAATTATCTTTCAAAAAAGCTTGAGGAATAATATGATCAATAT
CATAATTACTTAATCTATCAATATCTAAATCATCTCCGGTATACATATCTTTTCCATTTT
GAAGATAGTATAAGTAAAGTCGATCATTTTGAAGTGCGTTATTGTCTATTTTAGAAAGTT
TTGCAGGAATATTTTCCTTAAGTATCTTACTACCTAACTCTTTGAGAGATTTTTCTAAAC
GTTTCAAGCGTTGTTGGGAATTAGACTTACCTTGATTGGTATATTGATTTTCACGAGCCA
TCTCAACAACAATTGACTCGGGTTTTCTTCCTCCCATTACTTTGACCAATTCATCTACAA
TTTTTATGCTTTGTAAAATACCTTTTTTAATCGCAGGACTACCTGGCAAAGACTTAACGA
CCTCTTTAATATTACCTTTATCTTCGTCACCAATAATTTGTGCTTTCTGTATCTTCTTTT
TAAAAGAAAGAGCATCATCGTGAATAAGTTGCATGAAATTACGGTTAGAAATACCATCAT
CAATTAAGTAATCAAGAATAGTATTACCAGATTTTTCATCTCGAATACCATTAATAAGCT
TAGCAGATAACTTACCCCAGCCAGTGTAATGTCTACGAGATAACTTTTTCAAAACGGATT
TATCGAATATATTCTCAAATTTTGAAAGACGTTGTTTTATCATCTCTCTATCTTCAAAAA
TTGTCAAAGTATGGATAATTTCTTCGATAATCGCTTCATTTGAACTATCATCCAAAAACT
CTTTATCATTAATAATATTTAAAAGATCGTGATAAGTAGATAAACTAGAATTAAACTGTT
TCTCTATGCCTTTTAATTCAATTCCATCATACCCATAAATTGCATGTAAATATTCAATAA
TATCCTTATCAGTAACTTTCCTTTTATCTTTAAAATAAAGTCTAACAATATCTTTCTTCT
GCTTACTATCTAAAAATTGATAATCTCTCATACTTTCGGCAATAAATCTAACTTTTGTTA
ATTCATTATATACATTAAAAGTTTCGTATAAGAGACTATGCTTTGGAAGTACCTTCTCTT
CTGGCAAATACAAATCAAAACTAGTCATTCGATTAATGAAGGCCTCTGCCGAAGATTCTT
TGTCAATAACGTCCTCAAAATTCCAAGGTGTAATTTTTTCATTTCGTTTTCTTATTGACC
AGGCAAAATCACTATTCCCTCTCGCAAGTGGACCTACATAATAAGGAATTCGGAAGGTTA
AAATCTTCTCGATTCTTTCTTTATTTTTAGCCAAGAAAGGATAAAATTTAGCTTGCTTAT
CAAGAATTGCTCTCATTTCTTGAAGATGAATCTGATATGGTATCGAACCATTGTCAAATG
TACGTTGCTTTCTCAAAAAATCTTCTCGATCAATTTTTTCAAGAAAATAATCCGCACCTT
CAAATTCAGCCAATAGTTTTTTTAGATATACGTAGAAATCTTCCTGATTTGTTTTTCCAT
CAATATAACCAGCATAACCATTTTTGGTGTCATCTTTAAATACTTCATTATACGTTTTTA
GTGAAATATTTCTTATATATTCCTTTAGTAACGCTAAATCTTCTTTGTGTTCATTATATC
GCTTTATCATAGCAGAAGAGAGAGGTGCTTCTGTCTCATTATCAGTTACAGTCAGAAAAC
CCGATAAAAGAATAGCATCATAAAGTTTCTTTGCTTTGAGAAAGACATCACTGTAATCAT
CTCCAATATAACCTAACAAAGTCTCTAAATCTTCATCATAGCTTTCTTTGGAAAAATGTA
AGGAGGCTTTTTCGTCTAAATTAAAACATTTCCTAAAATCAGCTTGATTTCCTACAATCA
ACTTTAGAAACTCTGAAAAAATCCCCGAATTCTTCTCCCCAGGGAAGAGTTTTAAAATAC
GATCTTTCTTTTCTAATTTACTAATCTTATCTTTAACAATTTCCTCAAGTTGTTTACTAT
TCTCAAGTGATAAATCCGATTCAAAAATAGCATTATAAGTGTCCAAAAAGTCTTGAAAAT
TCTTCTGAATATCATTATTTTTTGAATTAAACTCTCCTTCAATTAAGAAGTGACCTCTAT
ATTTAATCATATGAGCCAATGCAAGATAAACTAGACGCAAATCTGCTTTTTTAGTACTAT
CTGCTAAATATTTCCTTAAATGATAGATAGTTGGAAATTCATCATGATAGGCTTTTTCTT
CTACTAAGTTTCCAAATATCGGATACTTACTATCACGTTTATCATCAGGAACTAAAAACG
AATCGTCAAGTCTTTGAAAGAAAGCATCATCTAATGTAGCCATCTCTGTGCTAAAAATTT
CCTGCAAATAAAGGATACGATTACGGCGTCTAGTATAACGTCTTCTTGCAGTACGCTTCA
ATCTTCTTCCTTCTGCTGTGATTCCAGAGTCAAAGAGTAATACACCTAACAGGTTCTTTT
TGATATACTTTTTACTCGTATTTCCTAAGACTTTCATTTTTTTAGACGGAACCTTGTAAT
TATCAGTTATTACAGCCCATCCAACACTATTCGTTCCAATATCAAGTCCAATTGAGTATG
GCTTAGTCATGGATCCGCGACCCATTTGCTGTCCACCAGTCATGCTAGCCATATGGCTGC
CGCGCGGCACCAGGCCGCTGCTGTGATGATGATGATGATGGCTGCTGCCCATGGTATATC
TCCTTCTTAAAGTTAAACAAAATTATTTCTAGAGGGGAATTGTTATCCGCTCACAATTCC
CCTATAGTGAGTCGTATTAATTTCGCGGGATCGAGATCTCGATCCTCTACGCCGGACGCA
TCGTGGCCGGCATCACCGGCGCCACAGGTGCGGTTGCTGGCGCCTATATCGCCGACATCA
CCGATGGGGAAGATCGGGCTCGCCACTTCGGGCTCATGAGCGCTTGTTTCGGCGTGGGTA
TGGTGGCAGGCCCCGTGGCCGGGGGACTGTTGGGCGCCATCTCCTTGCATGCACCATTCC
TTGCGGCGGCGGTGCTCAACGGCCTCAACCTACTACTGGGCTGCTTCCTAATGCAGGAGT
CGCATAAGGGAGAGCGTCGAGATCCCGGACACCATCGAATGGCGCAAAACCTTTCGCGGT
ATGGCATGATAGCGCCCGGAAGAGAGTCAATTCAGGGTGGTGAATGTGAAACCAGTAACG
TTATACGATGTCGCAGAGTATGCCGGTGTCTCTTATCAGACCGTTTCCCGCGTGGTGAAC
CAGGCCAGCCACGTTTCTGCGAAAACGCGGGAAAAAGTGGAAGCGGCGATGGCGGAGCTG
AATTACATTCCCAACCGCGTGGCACAACAACTGGCGGGCAAACAGTCGTTGCTGATTGGC
GTTGCCACCTCCAGTCTGGCCCTGCACGCGCCGTCGCAAATTGTCGCGGCGATTAAATCT
CGCGCCGATCAACTGGGTGCCAGCGTGGTGGTGTCGATGGTAGAACGAAGCGGCGTCGAA
GCCTGTAAAGCGGCGGTGCACAATCTTCTCGCGCAACGCGTCAGTGGGCTGATCATTAAC
TATCCGCTGGATGACCAGGATGCCATTGCTGTGGAAGCTGCCTGCACTAATGTTCCGGCG
TTATTTCTTGATGTCTCTGACCAGACACCCATCAACAGTATTATTTTCTCCCATGAAGAC
GGTACGCGACTGGGCGTGGAGCATCTGGTCGCATTGGGTCACCAGCAAATCGCGCTGTTA
GCGGGCCCATTAAGTTCTGTCTCGGCGCGTCTGCGTCTGGCTGGCTGGCATAAATATCTC
ACTCGCAATCAAATTCAGCCGATAGCGGAACGGGAAGGCGACTGGAGTGCCATGTCCGGT
TTTCAACAAACCATGCAAATGCTGAATGAGGGCATCGTTCCCACTGCGATGCTGGTTGCC
AACGATCAGATGGCGCTGGGCGCAATGCGCGCCATTACCGAGTCCGGGCTGCGCGTTGGT
GCGGATATCTCGGTAGTGGGATACGACGATACCGAAGACAGCTCATGTTATATCCCGCCG
TTAACCACCATCAAACAGGATTTTCGCCTGCTGGGGCAAACCAGCGTGGACCGCTTGCTG
CAACTCTCTCAGGGCCAGGCGGTGAAGGGCAATCAGCTGTTGCCCGTCTCACTGGTGAAA
AGAAAAACCACCCTGGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCA
TTAATGCAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGAGCGCAACGCAAT
TAATGTAAGTTAGCTCACTCATTAGGCACCGGGATCTCGACCGATGCCCTTGAGAGCCTT
CAACCCAGTCAGCTCCTTCCGGTGGGCGCGGGGCATGACTATCGTCGCCGCACTTATGAC
TGTCTTCTTTATCATGCAACTCGTAGGACAGGTGCCGGCAGCGCTCTGGGTCATTTTCGG
CGAGGACCGCTTTCGCTGGAGCGCGACGATGATCGGCCTGTCGCTTGCGGTATTCGGAAT
CTTGCACGCCCTCGCTCAAGCCTTCGTCACTGGTCCCGCCACCAAACGTTTCGGCGAGAA
GCAGGCCATTATCGCCGGCATGGCGGCCCCACGGGTGCGCATGATCGTGCTCCTGTCGTT
GAGGACCCGGCTAGGCTGGCGGGGTTGCCTTACTGGTTAGCAGAATGAATCACCGATACG
CGAGCGAACGTGAAGCGACTGCTGCTGCAAAACGTCTGCGACCTGAGCAACAACATGAAT
GGTCTTCGGTTTCCGTGTTTCGTAAAGTCTGGAAACGCGGAAGTCAGCGCCCTGCACCAT
TATGTTCCGGATCTGCATCGCAGGATGCTGCTGGCTACCCTGTGGAACACCTACATCTGT
ATTAACGAAGCGCTGGCATTGACCCTGAGTGATTTTTCTCTGGTCCCGCCGCATCCATAC
CGCCAGTTGTTTACCCTCACAACGTTCCAGTAACCGGGCATGTTCATCATCAGTAACCCG
TATCGTGAGCATCCTCTCTCGTTTCATCGGTATCATTACCCCCATGAACAGAAATCCCCC
TTACACGGAGGCATCAGTGACCAAACAGGAAAAAACCGCCCTTAACATGGCCCGCTTTAT
CAGAAGCCAGACATTAACGCTTCTGGAGAAACTCAACGAGCTGGACGCGGATGAACAGGC
AGACATCTGTGAATCGCTTCACGACCACGCTGATGAGCTTTACCGCAGCTGCCTCGCGCG
TTTCGGTGATGACGGTGAAAACCTCTGACACATGCAGCTCCCGGAGACGGTCACAGCTTG
TCTGTAAGCGGATGCCGGGAGCAGACAAGCCCGTCAGGGCGCGTCAGCGGGTGTTGGCGG
GTGTCGGGGCGCAGCCATGACCCAGTCACGTAGCGATAGCGGAGTGTATACTGGCTTAAC
TATGCGGCATCAGAGCAGATTGTACTGAGAGTGCACCATATATGCGGTGTGAAATACCGC
ACAGATGCGTAAGGAGAAAATACCGCATCAGGCGCTCTTCCGCTTCCTCGCTCACTGACT
CGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATAC
GGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAA
AGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTG
ACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAA
GATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGC
TTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCAC
GCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAAC
CCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGG
TAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGT
ATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGGA
CAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCT
CTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGA
TTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACG
CTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAACAATAAAACTGTCTGCT
TACATAAACAGTAATACAAGGGGTGTTATGAGCCATATTCAACGGGAAACGTCTTGCTCT
AGGCCGCGATTAAATTCCAACATGGATGCTGATTTATATGGGTATAAATGGGCTCGCGAT
AATGTCGGGCAATCAGGTGCGACAATCTATCGATTGTATGGGAAGCCCGATGCGCCAGAG
TTGTTTCTGAAACATGGCAAAGGTAGCGTTGCCAATGATGTTACAGATGAGATGGTCAGA
CTAAACTGGCTGACGGAATTTATGCCTCTTCCGACCATCAAGCATTTTATCCGTACTCCT
GATGATGCATGGTTACTCACCACTGCGATCCCCGGGAAAACAGCATTCCAGGTATTAGAA
GAATATCCTGATTCAGGTGAAAATATTGTTGATGCGCTGGCAGTGTTCCTGCGCCGGTTG
CATTCGATTCCTGTTTGTAATTGTCCTTTTAACAGCGATCGCGTATTTCGTCTCGCTCAG
GCGCAATCACGAATGAATAACGGTTTGGTTGATGCGAGTGATTTTGATGACGAGCGTAAT
GGCTGGCCTGTTGAACAAGTCTGGAAAGAAATGCATAAACTTTTGCCATTCTCACCGGAT
TCAGTCGTCACTCATGGTGATTTCTCACTTGATAACCTTATTTTTGACGAGGGGAAATTA
ATAGGTTGTATTGATGTTGGACGAGTCGGAATCGCAGACCGATACCAGGATCTTGCCATC
CTATGGAACTGCCTCGGTGAGTTTTCTCCTTCATTACAGAAACGGCTTTTTCAAAAATAT
GGTATTGATAATCCTGATATGAATAAATTGCAGTTTCATTTGATGCTCGATGAGTTTTTC
TAAGAATTAATTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGG
GGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGAAATTGTAAACGTTAATATTTTGTT
AAAATTCGCGTTAAATTTTTGTTAAATCAGCTCATTTTTTAACCAATAGGCCGAAATCGG
CAAAATCCCTTATAAATCAAAAGAATAGACCGAGATAGGGTTGAGTGTTGTTCCAGTTTG
GAACAAGAGTCCACTATTAAAGAACGTGGACTCCAACGTCAAAGGGCGAAAAACCGTCTA
TCAGGGCGATGGCCCACTACGTGAACCATCACCCTAATCAAGTTTTTTGGGGTCGAGGTG
CCGTAAAGCACTAAATCGGAACCCTAAAGGGAGCCCCCGATTTAGAGCTTGACGGGGAAA
GCCGGCGAACGTGGCGAGAAAGGAAGGGAAGAAAGCGAAAGGAGCGGGCGCTAGGGCGCT
GGCAAGTGTAGCGGTCACGCTGCGCGTAACCACCACACCCGCCGCGCTTAATGCGCCGCT
ACAGGGCGCGTCCCATTCGCCA
<210>3
<211>50
<212> DNA
<213>人工序列
<400>DNA序列
gcaGTCGACCGGTCTATGACACGATCGTCCGCA GTTTTAGAGCTGTGTTG
<210>4
<211>50
<212>DNA
<213>人工序列
<400>DNA序列
gcaGTCGACTCATAACAGTTCAGAACTATTACAGTTTTAGAGCTGTGTTG
<210>5
<211>50
<212> DNA
<213>人工序列
<400>DNA序列
gcaGTCGACCGGGTACGGACGATCGCGTAACGTGTTTTAGAGCTGTGTTG
<210>6
<211>48
<212> DNA
<213>人工序列
<400>DNA序列
gcaGGATCCATCCTCGAGGTTTTGGAACCATTCGAAACAACACAGCTC
<210>7
<211>29
<212> DNA
<213>人工序列
<400>DNA序列
gcc GGATCC ATGACTAAGCCATACTCAAT
<210>8
<211>28
<212> DNA
<213>人工序列
<400>DNA序列
gcc CTCGAG TTAACCCTCTCCTAGTTTG。

Claims (6)

1.一种基于抑菌杀菌的多靶点CRISPR/Cas9表达载体,其特征在于,该基于抑菌杀菌的多靶点CRISPR/Cas9表达载体的基因序列为SEQ ID NO1;Cas9表达载体基因序列为SEQ IDNO2。
2.一种如权利要求1所述基于抑菌杀菌的多靶点CRISPR/Cas9表达载体的构建方法,其特征在于,该基于抑菌杀菌的多靶点CRISPR/Cas9表达载体的构建方法利用导向RNA特异识别基因的DNA片段,然后介导Cas9蛋白切割DNA双链,产生双链切口,破坏DNA序列,从而破坏DNA编码的功能蛋白的原理构建多靶点CRISPR/Cas9表达载体;
该基于抑菌杀菌的多靶点CRISPR/Cas9表达载体的构建方法具体包括多靶点CRISPR表达载体的构建,该多靶点CRISPR表达载体的构建的方法包括:
上游特异引物的寡聚核苷酸包含靶点序列,5’端含有Sal Ⅰ酶切位点,3’端为部分重复序列(direct repeat,DR);
下游通用引物的5’端携带Xho Ⅰ和BamH Ⅰ酶切位点,3’端重复序列的11个核苷酸与上游引物3’端碱基互补,上游特异引物和下游通用引物二者形成引物二聚体;
该引物二聚体在Klenow酶的作用下延伸,合成特异的靶序列DNA,该引物二聚体含有靶序列、重复序列、Sal Ⅰ、Xho Ⅰ和BamHⅠ酶切位点;
利用Sal Ⅰ和BamH Ⅰ双酶切靶序列DNA,克隆至经Xho Ⅰ和BamH Ⅰ双酶切的骨架载体pCRISPR载体中,获得单靶点载体pCRISPR-T1;
Sal Ⅰ和Xho Ⅰ为同尾酶,连接后Sal Ⅰ和Xho Ⅰ这两个酶切位点都消失,载体中先前的Xho Ⅰ酶切位点被破坏,同时在克隆的靶序列DNA中加入一个Xho Ⅰ酶切位点;连接之后的单靶点载体中仍然含有Xho Ⅰ和BamH Ⅰ多克隆位点;
随后,利用相同的克隆方法将靶序列2插入pCRISPR-T1载体的Xho Ⅰ和BamH Ⅰ位点,获得双靶点载体pCRISPR-T1-T2,同时该双靶点载体pCRISPR-T1-T2仍然携带Xho Ⅰ和BamH Ⅰ位点,作为插入第三个靶点的骨架载体,构建三靶点表达载体,依次加入更多靶点。
3.如权利要求2所述的基于抑菌杀菌的多靶点CRISPR/Cas9表达载体的构建方法,其特征在于,所述基于抑菌杀菌的多靶点CRISPR/Cas9表达载体的构建方法还包括Cas9表达载体的构建,该Cas9表达载体的构建的方法包括:
利用特异引物Fcas和Rcas,以嗜热链球菌基因组为模板,扩增Cas9基因,正向和反向引物分别携带BamHⅠ和XhoⅠ酶切位点,将扩增的Cas9基因片段克隆至载体pET28a中,获得Cas9诱导表达载体pET28a-Cas9;
通过多靶点CRISPR表达载体的构建和Cas9表达载体的构建后形成双质粒的CRISPR/Cas9表达载体。
4.如权利要求2所述的基于抑菌杀菌的多靶点CRISPR/Cas9表达载体的构建方法,其特征在于,上游特异引物的寡聚核苷酸包含靶点序列包括以大肠杆菌DNA旋转酶A亚基引物、B亚基引物和二氢叶酸还原酶引物;所述A亚基引物、B亚基引物和二氢叶酸还原酶引物的DNA序列分别为SEQ ID NO3、SEQ ID NO4和SEQ ID NO5;
所述下游通用引物的DNA序列为SEQ ID NO6。
5.如权利要求2所述的基于抑菌杀菌的多靶点CRISPR/Cas9表达载体的构建方法,其特征在于,所述上游特异引物和下游通用引物二者形成引物二聚体中含靶序列的上游引物和通用引物UR的3’末端有11个碱基互补,二者在反应体系中配对形成二聚体,在Klenow酶的作用下延伸合成双链DNA;
具体包括:50μL反应体系中上下游引物各15μL,5μL缓冲液,1μL Klenow酶,2μL dNTP,12μL ddH2O,反应条件37℃保持30min;利用2%的琼脂糖凝胶电泳检测合成的双链DNA。
6.一种利用权利要求1所述的基于抑菌杀菌的多靶点CRISPR/Cas9表达载体在杀菌上的应用方法,其特征在于,该应用方法包括:
将0.5μg CRISPR表达载体和0.5μg Cas9表达载体与100μL大肠杆菌感受态细胞混合,转移至2mm电击杯,电击条件2.5kv,5ms,转化至大肠杆菌细胞;然后将转化混合体转移至1mL无抗生素LB培养基中,37℃孵育1h后平均涂布于两个LB固体平板;
两个LB平板都含有Amp和Kan抗生素,一个加入浓度2%的葡萄糖,另一个加入浓度1mM的IPTG;平板置于37℃培养箱,培养14小时~16小时后,置于成像系统仪拍照并统计克隆数;
表达载体pET28a-Cas9在IPTG诱导作用下表达Cas9蛋白,将转化混合体涂布于含葡萄糖的LB双抗平板,Cas9蛋白表达被抑制,质粒成功转化至大肠杆菌细胞,在平板上存活,检测质粒转化效率;
将另一等份转化混合体涂布于含IPTG的双抗平板上,形成特异性CRISPR/Cas9核酸酶,切割靶基因DNA旋转酶或二氢叶酸还原酶,使细菌不能存活;
统计同一转化混合体在两种不同平板上的克隆数,杀菌效率=含IPTG平板克隆数/含葡糖糖平板克隆数×100%。
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