CN112522311B - 用于adcy3基因编辑的crispr系统及其在构建肥胖症猪核移植供体细胞中应用 - Google Patents
用于adcy3基因编辑的crispr系统及其在构建肥胖症猪核移植供体细胞中应用 Download PDFInfo
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Abstract
本发明公开了用于ADCY3基因编辑的CRISPR系统及其在构建肥胖症猪核移植供体细胞中应用。一种用于猪ADCY3基因编辑的CRISPR/Cas9系统,包含Cas9表达载体和针对猪ADCY3基因的gRNA表达载体;所述的Cas9表达载体为质粒全序列如SEQ ID NO.2所示,所述的gRNA表达载体表达SEQ ID NO.22所示的gRNA,其靶点如SEQ ID NO.18所示。本发明针对猪ADCY3基因设计了四个gRNA,从中筛选高效gRNA后再进行预设靶点的敲除,可有效降低后期单克隆细胞鉴定筛选的工作量,并可直接用PCR产物测序来检测基因编辑效率。
Description
技术领域
本发明属于生物技术领域,具体涉及用于ADCY3基因编辑的CRISPR/Cas9系统及其应用。
背景技术
肥胖症(Obesity)是指体脂肪累积过多而对健康造成负面影响的身体状态,可能导致寿命减短及各种健康问题。肥胖是全世界主要的可预防死因,也是21世纪最重要的公共卫生问题之一。目前成人与儿童的肥胖盛行率都在上升,且女性较男性更常发生。2013年,包括美国医学会和美国心脏协会等数个医学会将肥胖定义为一种疾病,即为肥胖症。2015年,全球有6亿名成人(13%)和4200万名五岁以下的孩童有肥胖问题。世卫组织更是发出警告,指出超重和肥胖是全球引起死亡的第五大风险,全球每年“胖死”的人至少280万。
尽管肥胖通常受到遗传和环境的共同影响,但肥胖症实际上是可遗传且由多基因作用的,只是不同人的易感性存在差异。在极少数情况下,遗传性肥胖症是由致病突变直接干扰能量稳态或脂肪沉积,例如人类单基因肥胖病的发生。其中,ADCY3基因的突变已被证实是造成人类先天性肥胖症最重要的基因之一。因此,迫切需要开发出基于ADCY3突变导致的先天性肥胖症动物模型以尽快解开发病机制谜团并为进一步的治疗奠定基础。猪作为大动物,是人类长期以来主要的肉食供应动物,易于大规模繁殖饲养,而且在伦理道德及动物保护等方面的要求较低,且猪体型大小和生理功能与人类近似,是理想的人类疾病模型动物。
基因编辑是近年来不断取得重大发展的一种生物技术,其包括从基于同源重组的基因编辑到基于核酸酶的ZFN、TALEN、CRISPR/Cas9等基因编辑手段,其中CRISPR/Cas9技术是当前最先进的基因编辑技术。目前,基因编辑技术被越来越多地应用到动物模型的制作上。
发明内容
本发明的目的是针对现有技术的上述不足,提供一种用于ADCY3基因编辑的CRISPR/Cas9系统。
本发明的另一目的是提供用于ADCY3基因编辑的gRNA及其表达载体。
本发明的又一目的是提供所述的CRISPR/Cas9系统在构建ADCY3基因突变的猪重组细胞中的应用。
本发明的目的可通过以下技术方案实现:
一种用于猪ADCY3基因编辑的CRISPR/Cas9系统,包含Cas9表达载体和针对猪ADCY3基因的gRNA表达载体;所述的Cas9表达载体为质粒全序列如SEQ ID NO.2所示的pU6gRNA-eEF1a-mNLS-hSpCas9-EGFP-PURO载体。
为了增加Cas9质粒的基因编辑能力,我们在购自addgene(Plasmid#42230,fromZhang Feng lab)pX330-U6-Chimeric_BB-CBh-hSpCas9(简称PX330)载体的基础上进行改造得到pU6gRNA-eEF1a-mNLS-hSpCas9-EGFP-PURO(简称粒pKG-GE3)。PX330的图谱如图1,改造方式如下:
1)去除原载体gRNA骨架中多余无效的序列;
2)改造启动子:将原有启动子(chickenβ-actin启动子)改造为具更高表达活性的EF1a启动子,增加Cas9基因的蛋白表达能力;
3)增加核定位信号:在Cas9的N端及C端均增加核定位信号编码序列(NLS),增加Cas9的核定位能力;
4)增加双筛选标记:原载体无任何筛选标记,不利于阳性转化细胞的筛选和富集,在Cas9的C端,插入P2A-EGFP-T2A-PURO,赋予载体荧光和抗性筛选能力;
5)插入WPRE和3’LTR等调控基因表达的序列:在基因读码框最后插入WPRE、3’LTR等序列,可增强Cas9基因的蛋白翻译能力。
改造后载体pU6gRNA-eEF1a-mNLS-hSpCas9-EGFP-PURO(简称pKG-GE3)及改造位点如图2,质粒全序列如SEQ ID NO:2所示;pKG-GE3的主要元件有:
1)gRNA表达元件:U6 gRNA scaffold;
2)启动子:EF1a启动子和CMV增强子;
3)含多个NLS的Cas9基因:含N端和C端多核定位信号(NLS)的Cas9基因;
4)筛选标记基因:荧光和抗性双筛选标记元件P2A-EGFP-T2A-PURO;
5)增强翻译的元件:WPRE和3’LTR增强Cas9及筛选标记基因的翻译效率;
6)转录终止信号:bGH polyA signal;
7)载体骨架:包括Amp抗性元件和ori复制子等。
质粒pKG-GE3中,具有特异融合基因;所述特异融合基因编码特异融合蛋白;
所述特异融合蛋白自N端至C端依次包括如下元件:两个核定位信号(NLS)、Cas9蛋白、两个核定位信号、自剪切多肽P2A、荧光报告蛋白、自裂解多肽T2A、抗性筛选标记蛋白;
质粒pKG-GE3中,由EF1a启动子启动所述特异融合基因的表达;
质粒pKG-GE3中,所述特异融合基因下游具有WPRE序列元件、3’LTR序列元件和bGHpoly(A)signal序列元件。
质粒pKG-GE3中,依次具有如下元件:CMV增强子、EF1a启动子、所述特异融合基因、WPRE序列元件、3’LTR序列元件、bGH poly(A)signal序列元件。
所述特异融合蛋白中,Cas9蛋白上游的两个核定位信号为SV40核定位信号,Cas9蛋白下游的两个核定位信号为nucleoplasmin核定位信号。
所述特异融合蛋白中,荧光报告蛋白具体可为EGFP蛋白。
所述特异融合蛋白中,抗性筛选标记蛋白具体可为Puromycin蛋白。
自剪切多肽P2A的氨基酸序列为“ATNFSLLKQAGDVEENPGP”(发生自剪切的断裂位置为C端开始第一个氨基酸残基和第二个氨基酸残基之间)。
自裂解多肽T2A的氨基酸序列为“EGRGSLLTCGDVEENPGP”(发生自裂解的断裂位置为C端开始第一个氨基酸残基和第二个氨基酸残基之间)。
特异融合基因具体如SEQ ID NO:2中第911-6706位核苷酸所示。
CMV增强子如SEQ ID NO:2中第395-680位核苷酸所示。
EF1a启动子如SEQ ID NO:2中第682-890位核苷酸所示。
WPRE序列元件如SEQ ID NO:2第6722-7310位核苷酸所示。
3’LTR序列元件如SEQ ID NO:2中第7382-7615位核苷酸所示。
bGH poly(A)signal序列元件如SEQ ID NO:2中第7647-7871位核苷酸所示。
作为本发明的一种优选,针对猪ADCY3基因的gRNA表达载体的载体骨架为pKG-U6gRNA,质粒全序列如SEQ ID NO.3所示。
作为本发明的一种优选,所述的CRISPR/Cas9系统,该表达载体表达SEQ ID NO.22所示的gRNA,其靶点如SEQ ID NO.18所示。
作为本发明的一种优选,所述的针对猪ADCY3基因的gRNA表达载体由SEQ IDNO.26和SEQ ID NO.27所示的单链DNA退火而成的双链插入载体骨架pKG-U6gRNA的限制性内切酶BbsI位点所得。
作为本发明的进一步优选,所述的gRNA表达载体和Cas9表达载体的摩尔比为1~3:1,进一步优选3:1。
本发明所述的CRISPR/Cas9系统在构建ADCY3基因突变的猪重组细胞中的应用。
一种重组细胞,由本发明所述的用于猪ADCY3基因编辑的CRISPR/Cas9系统共转染猪原代成纤维细胞经验证后所得。
本发明所述的重组细胞在构建ADCY3基因敲除的克隆猪中的应用;优选在构建ADCY3基因敲除的先天性肥胖症克隆猪中的应用。
针对猪ADCY3基因的gRNA,序列如SEQ ID NO.22所示。
一种针对猪ADCY3基因的gRNA表达载体,该表达载体表达SEQ ID NO.22所示的gRNA;且该表达载体的载体骨架为pKG-U6gRNA,质粒全序列如SEQ ID NO.3所示;所述的gRNA表达载体优选由SEQ ID NO.26和SEQ ID NO.27所示的单链DNA退火而成的双链插入载体骨架pKG-U6gRNA的限制性内切酶BbsI位点所得。
与现有技术相比,本发明至少具有如下有益效果:
(1)本发明研究对象(猪)比其他动物(大小鼠、灵长类)具有更好的应用性。
大小鼠等啮齿类动物不论从体型、器官大小、生理、病理等方面都与人相差巨大,无法真实地模拟人类正常的生理、病理状态。研究表明,95%以上在大小鼠中验证有效的药物在人类临床试验中是无效的。就大动物而言,灵长类是与人亲缘关系最近的动物,但其体型小、性成熟晚(6-7岁开始交配),且为单胎动物,群体扩繁速度极慢,饲养成本也高。另外,灵长类动物克隆效率低、难度大、成本高。
而猪作为模型动物就没有上述缺点,猪是除灵长类外与人亲缘关系最近的动物,其体型、体重、器官大小等与人相近,在解剖学、生理学、营养代谢、疾病发病机制方面等方面与人类极为相似。同时,猪的性成熟早(4-6个月),繁殖力高,一胎多仔,在2-3年内即可形成一个较大群体。另外,猪的克隆技术非常成熟,克隆及饲养成本较灵长类低得多;而且猪作为人类长期以来的肉食性动物,用猪作为疾病模型动物在动物保护和伦理等方面的要求较低。
(2)本发明针对猪ADCY3基因设计了四个gRNA,从中筛选高效gRNA后再进行预设靶点的敲除,可有效降低后期鉴定筛选的工作量,并可直接用PCR产物测序来检测基因编辑效率。
(3)采用本发明改造的Cas9高效表达载体进行基因编辑,编辑效率比原载体提高100%以上。
(4)采用本发明改造的Cas9高效表达载体进行基因编辑,通过靶标基因PCR产物测序结果可分析出所获细胞的基因型[纯合突变(包括双等位基因相同变异的突变和双等位基因不同变异的突变)、杂合突变或野生型],获得纯合突变的概率为30%~50%,大大优于使用胚胎注射技术的模型制备方法(即受精卵注射基因编辑材料)中获得纯合突变的概率(低于5%)。
(5)利用本发明所得到的纯合突变单克隆细胞株进行体细胞核移植动物克隆可直接得到含靶标基因纯合突变的克隆猪,并且该纯合突变可稳定遗传。
在小鼠模型制作中采用的受精卵显微注射基因编辑材料后再进行胚胎移植的方法,因其直接获得纯合突变后代的概率非常低(低于5%),需要进行后代的杂交选育,这不太适用于妊娠期较长的大动物(如猪)模型制作。因此,本发明采用技术难度大、挑战性高的原代细胞体外编辑并筛选阳性编辑单克隆细胞的方法,后期再通过体细胞核移植动物克隆技术直接获得相应疾病模型猪,可大大缩短模型猪制作周期并节省人力、物力、财力。
本发明为通过基因编辑手段获得先天性肥胖症猪模型奠定了坚实的基础,将有助于研究并揭示由ADCY3突变引起的先天性肥胖症发病机制,进一步的可用于进行药物筛选、药效检测、疾病病理、基因治疗及细胞治疗等研究,能够为进一步的临床应用提供有效的实验数据,进而为成功治疗人类因ADCY3突变导致的先天性肥胖症提供有力的实验手段。本发明对于先天性肥胖症药物的研发及揭示该病的发病机制具有重大应用价值。
附图说明
图1为质粒pX330的结构示意图。
图2为质粒pKG-GE3的结构示意图。
图3为pU6gRNACas9载体的结构示意图。
图4为pU6gRNA-eEF1a Cas9载体的结构图谱。
图5为pU6gRNA-eEF1a Cas9+nNLS载体图谱。
图6为质粒pKG-U6gRNA的结构示意图。
图7为将20bp左右的DNA分子(用于转录形成gRNA的靶序列结合区)插入质粒pKG-U6gRNA的示意图。
图8为质粒配比优化时的测序结果。
图9为质粒pX330和质粒pKG-GE3的效果比较时的测序结果。
图10为实施例3中以18只猪的基因组DNA为模板进行PCR扩增后的电泳图。
图11为实施例3的步骤四中的测序峰图。
图12为实施例4中得到的单克隆细胞的靶基因PCR产物电泳图。
图13为编号ADCY3-6的单克隆细胞的正向测序与野生型比对的结果。
图14为编号为ADCY3-22的单克隆细胞的正向测序与野生型比对的结果。
图15为编号为ADCY3-17的单克隆细胞的正向测序与野生型比对的结果。
具体实施方式
下面结合具体实施方式对本发明进行进一步的详细描述,给出的实施例仅为了阐明本发明,而不是为了限制本发明的范围。以下提供的实施例可作为本技术领域普通技术人员进行进一步改进的指南,并不以任何方式构成对本发明的限制。
下述实施例中的实验方法,如无特殊说明,均为常规方法,按照本领域内的文献所描述的技术或条件或者按照产品说明书进行。下述实施例中所用的材料、试剂等,如无特殊说明,均可从商业途径得到。实施例中构建的重组质粒,均已进行测序验证。完全培养液(%为体积比):15%胎牛血清(Gibco)+83%DMEM培养基(Gibco)+1%Penicillin-Streptomycin(Gibco)+1%HEPES(Solarbio)。细胞培养条件:37℃,5%CO2、5%O2的恒温培养箱。
制备猪原代成纤维细胞的方法:①取猪耳组织0.5g,除毛,然后用75﹪酒精浸泡30-40s,然后用含5%(体积比)Penicillin-Streptomycin(Gibco)的PBS缓冲液洗涤5次,然后用PBS缓冲液洗涤一次;②用剪刀将组织剪碎,采用5mL 1%胶原酶溶液(Sigma),37℃消化1h,然后500g离心5min,弃上清;③将沉淀用1mL完全培养液重悬,然后铺入含10mL完全培养基并已用0.2%明胶(VWR)封盘的直径为10的细胞培养皿中,培养至细胞长满皿底60%左右;④完成步骤③后,采用胰蛋白酶消化并收集细胞,然后重悬于完全培养液。
实施例1、质粒的制备
1.1制备质粒pU6gRNA eEF1a-mNLS-hSpCas9-EGFP-PURO(简称质粒pKG-GE3)
原始质粒pX330-U6-Chimeric_BB-CBh-hSpCas9(简称质粒pX330),序列如SEQ IDNO:1所示。质粒pX330的结构示意图见图1。SEQ ID NO:1中,第440-725位核苷酸组成CMV增强子,第727-1208位核苷酸组成chickenβ-actin启动子,第1304-1324位核苷酸编码SV40核定位信号(NLS),第1325-5449位核苷酸编码Cas9蛋白,第5450-5497位核苷酸编码nucleoplasmin核定位信号(NLS)。
质粒pU6gRNA eEF1a-mNLS-hSpCas9-EGFP-PURO,简称质粒pKG-GE3,核苷酸如SEQID NO:2所示。构建方法如下:
(1)去除gRNA骨架中多余无效的序列
质粒pX330用BbsI、XbaI酶切,回收载体片段(约8313bp左右),利用多片段重组法合成插入片段175bp(SEQ ID NO:4),与回收后载体片段重组得到pU6gRNACas9载体(图3)。
(2)改造启动子及增强子
对构建好的pU6gRNACas9载体,用XbaI和AgeI内切酶去除启动子(chickenβ-actin启动子)及增强子序列(CMV增强子),回收线性载体序列约7650bp,并利用多片段重组法合成554bp的包含CMV增强子及EF1a启动子的序列(SEQ ID NO:5),与酶切后载体pU6gRNACas9重组得到pU6gRNA-eEF1a Cas9载体(图4)。
(3)Cas9基因N端增加NLS序列
将构建好的载体pU6gRNA-eEF1a Cas9使用AgeI、BglII酶切,回收7786bp载体序列,并将增加了NLS的序列补充到酶切位点,即利用多片段重组法合成447bp的包括2个核定位信号及部分切除的Cas9编码序列(SEQ ID NO:6),重组得到pU6gRNA-eEF1a Cas9+nNLS载体(图5)。
(4)Cas9基因C端加入NLS、P2A-EGFP-T2A-PURO、WPRE-3’LTR-bGH polyA signals
以上构建好的载体命名为pU6gRNA-eEF1a Cas9+nNLS,使用FseI、SbfI酶切,回收载体序列7781bp,利用多片段重组法合成2727bp的包括NLS-P2A-EGFP-T2A-PURO-WPRE-3’LTR-bGH polyA signals(SEQ ID NO:7)的序列,与载体片段重组得到载体pU6gRNA-eEF1a-mNLS-hSpCas9-EGFP-PURO,简称pKG-GE3,质粒图谱如图2,核苷酸序列(SEQ ID NO:2)。
SEQ ID NO:2中,第395-680位核苷酸组成CMV增强子,第682-890位核苷酸组成EF1a启动子,第986-1006位核苷酸编码核定位信号(NLS),第1016-1036位核苷酸编码核定位信号(NLS),第1037-5161位核苷酸编码Cas9蛋白,第5162-5209位核苷酸编码核定位信号(NLS),第5219-5266位核苷酸编码核定位信号(NLS),第5276-5332位核苷酸编码自剪切多肽P2A(自剪切多肽P2A的氨基酸序列为“ATNFSLLKQAGDVEENPGP”,发生自剪切的断裂位置为C端开始第一个氨基酸残基和第二个氨基酸残基之间),第5333-6046位核苷酸编码EGFP蛋白,第6056-6109位核苷酸编码自裂解多肽T2A(自裂解多肽T2A的氨基酸序列为“EGRGSLLTCGDVEENPGP”,发生自裂解的断裂位置为C端开始第一个氨基酸残基和第二个氨基酸残基之间),第6110-6703位核苷酸编码Puromycin蛋白(简称Puro蛋白),第6722-7310位核苷酸组成WPRE序列元件,第7382-7615位核苷酸组成3’LTR序列元件,第7647-7871位核苷酸组成bGH poly(A)signal序列元件。SEQ ID NO:2中,第911-6706形成融合基因,表达融合蛋白。由于自剪切多肽P2A和自裂解多肽T2A的存在,融合蛋白自发形成如下三个蛋白:具有Cas9蛋白的蛋白、具有EGFP蛋白的蛋白和具有Puro蛋白的蛋白。
与质粒pX330相比,质粒pKG-GE3主要进行了如下改造:①去除残留的gRNA骨架序列(GTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTTT),降低干扰;②将原有chickenβ-actin启动子改造为具更高表达活性的EF1a启动子,增加Cas9基因的蛋白表达能力;③在Cas9基因的上游和下游均增加核定位信号编码基因(NLS),增加Cas9蛋白的核定位能力;④原质粒无任何真核细胞筛选标记,不利于阳性转化细胞的筛选和富集,依次在Cas9基因的下游插入P2A-EGFP-T2A-PURO编码基因,赋予载体荧光和真核细胞抗性筛选能力;⑤插入WPRE元件和3’LTR序列元件,增强Cas9基因的蛋白翻译能力。
1.2构建pKG-U6gRNA载体
来源pUC57载体,通过EcoRV酶切位点,连接pKG-U6gRNA插入序列(含U6启动子、BbsI酶切位点和sgRNA骨架序列的DNA片段,序列如SEQ ID NO:8所示),反向插入到pUC57载体,得到pKG-U6gRNA载体全序列(SEQ ID NO:3),SEQ ID NO:3中,第2280-2539位核苷酸组成hU6启动子,第2558-2637位核苷酸用于转录形成gRNA骨架。使用时,将20bp左右的DNA分子(用于转录形成gRNA的靶序列结合区)插入质粒pKG-U6gRNA,形成重组质粒,示意图见图4,在细胞中重组质粒转录得到gRNA。所构建的pKG-U6gRNA载体图谱如图6。
实施例2、质粒pX330和质粒pKG-GE3的效果比较
选择位于RAG1基因的高效gRNA靶点:
RAG1-gRNA4的靶点:5’-AGTTATGGCAGAACTCAGTG-3’(SEQ ID NO:9)。
用于扩增和检测包含靶点的片段的引物如下:
RAG1-nF126:5’-CCCCATCCAAAGTTTTTAAAGGA-3’(SEQ ID NO:10);
RAG1-nR525:5’-TGTGGCAGATGTCACAGTTTAGG-3’(SEQ ID NO:11)
初生从江香猪(雌性,血型AO),制备猪原代成纤维细胞。
一、制备重组质粒
取质粒pKG-U6gRNA,用限制性内切酶BbsI进行酶切,回收载体骨架(约3kb的线性大片段)。分别合成RAG1-4S和RAG1-4A,然后混合并进行退火,得到具有粘性末端的双链DNA分子。将具有粘性末端的双链DNA分子和载体骨架连接,得到质粒pKG-U6gRNA(RAG1-gRNA4)。
RAG1-4S:5’-caccgAGTTATGGCAGAACTCAGTG-3’(SEQ ID NO:12);
RAG1-4A:5’-aaacCACTGAGTTCTGCCATAACTc-3’(SEQ ID NO:13)。
RAG1-4S和RAG1-4A均为单链DNA分子。
二、质粒配比优化
第一组:将质粒pKG-U6gRNA(RAG1-gRNA4)和质粒pKG-GE3共转染猪原代成纤维细胞。配比:约20万个猪原代成纤维细胞:0.44μg质粒pKG-U6gRNA(RAG1-gRNA4):1.56μg质粒pKG-GE3。即质粒pKG-U6gRNA(RAG1-gRNA4)和质粒pKG-GE3的摩尔配比为:1:1。
第二组:将质粒pKG-U6gRNA(RAG1-gRNA4)和质粒pKG-GE3共转染猪原代成纤维细胞。配比:约20万个猪原代成纤维细胞:0.72μg质粒pKG-U6gRNA(RAG1-gRNA4):1.28μg质粒pKG-GE3。即质粒pKG-U6gRNA(RAG1-gRNA4)和质粒pKG-GE3的摩尔配比为:2:1。
第三组:将质粒pKG-U6gRNA(RAG1-gRNA4)和质粒pKG-GE3共转染猪原代成纤维细胞。配比:约20万个猪原代成纤维细胞:0.92μg质粒pKG-U6gRNA(RAG1-gRNA4):1.08μg质粒pKG-GE3。即质粒pKG-U6gRNA(RAG1-gRNA4)和质粒pKG-GE3的摩尔配比为:3:1。
第四组:将质粒pKG-U6gRNA(RAG1-gRNA4)转染致猪原代成纤维细胞。配比:约20万个猪原代成纤维细胞:1μg质粒pKG-U6gRNA(RAG1-gRNA4)。
共转染采用电击转染的方式,采用哺乳动物核转染试剂盒(Neon kit,Thermofisher)与Neon TM transfection system电转仪(参数设置为:1450V、10ms、3pulse)。
2、完成步骤1后,采用完全培养液培养16-18小时,然后更换新的完全培养液进行培养。培养总时间为48小时。
3、完成步骤2后,采用胰蛋白酶消化并收集细胞,提取基因组DNA,采用RAG1-nF126和RAG1-nR525组成的引物对进行PCR扩增,然后进行电泳。
电泳后回收目的条带并进行测序,测序结果见图8。
通过利用Synthego ICE工具分析测序峰图得出不同靶点的编辑效率。第一组至第三组的基因编辑效率依次为9%、53%、66%。第四组不发生基因编辑。结果表明,第三组编辑效率最高,确定单gRNA质粒与Cas9质粒最适用量为摩尔比3:1,质粒实际用量为0.92μg:1.08μg。
三、质粒pX330和质粒pKG-GE3的效果比较
1、共转染
RAG1-B组:将质粒pKG-U6gRNA(RAG1-gRNA4)转染猪原代成纤维细胞。配比:约20万个猪原代成纤维细胞:0.92μg质粒pKG-U6gRNA(RAG1-gRNA4)。
RAG1-330组:将质粒pKG-U6gRNA(RAG1-gRNA4)和质粒pX330共转染猪原代成纤维细胞。配比:约20万个猪原代成纤维细胞:0.92μg质粒pKG-U6gRNA(RAG1-gRNA4):1.08μg质粒pX330。
RAG1-KG组:将质粒pKG-U6gRNA(RAG1-gRNA4)和质粒pKG-GE3共转染猪原代成纤维细胞。配比:约20万个猪原代成纤维细胞:0.92μg质粒pKG-U6gRNA(RAG1-gRNA4):1.08μg质粒pKG-GE3。
共转染采用电击转染的方式,采用哺乳动物核转染试剂盒(Neon kit,Thermofisher)与Neon TM transfection system电转仪(参数设置为:1450V、10ms、3pulse)。
2、完成步骤1后,采用完全培养液培养16-18小时,然后更换新的完全培养液进行培养。培养总时间为48小时。
3、完成步骤2后,采用胰蛋白酶消化并收集细胞,提取基因组DNA,采用RAG1-nF126和RAG1-nR525组成的引物对进行PCR扩增,将产物进行测序。
通过利用Synthego ICE工具分析测序峰图得出不同靶点的编辑效率。RAG1-B组不发生基因编辑。RAG1-330组、RAG1-KG组的编辑效率依次为28%、68%。测序结果示例性峰图见图9。结果表明,与采用质粒pX330相比,采用质粒pKG-GE3使得基因编辑效率显著提高。
实施例3、用于ADCY3基因敲除的靶点筛选
猪ADCY3基因信息:编码adenylate cyclase 3蛋白;位于猪3号染色体;
GeneID为100514402,Sus scrofa。猪ADCY3基因编码的蛋白质如GENBANKACCESSION NO.XP_020943435.1(linear CON 12-JAN-2018)所示。基因组DNA中,猪ADCY3基因具有22个外显子,其中第3外显子如SEQ ID NO:14所示,其编码的蛋白质片段如SEQ IDNO:15所示。
一、ADCY3基因敲除预设靶点及邻近基因组序列保守性分析
18只初生从江香猪,其中雌性10只(分别命名1、2、3、4、5、6、7、8、9、10)、雄性8只(分别命名为A、B、C、D、E、F、G、H)。
分别以18只猪的基因组DNA为模板,采用引物对(引物对的靶序列包括猪ADCY3基因第12外显子)进行PCR扩增,然后进行电泳。回收PCR扩增产物并进行测序,将测序结果与公共数据库中的ADCY3基因序列进行比对分析。根据比对结果,设计用于检测突变的引物(引物本身避开可能的突变位点)。设计的用于检测突变的引物为:ADCY3-E3-F/ADCY3-E3-R。采用ADCY3-E3-F/ADCY3-E3-R组成的引物对对18只猪的基因组DNA进行PCR扩增后的电泳图见图7。
ADCY3-E3-F:5’-TTGGGGTTGAATCGTGGTCA-3’(SEQ ID NO:16);
ADCY3-E3-R:5’-AGGGAGCCAGCGAAAGATAAC-3’(SEQ ID NO:17)。
二、筛选靶点
通过筛选NGG(避开可能的突变位点)初步筛选到若干靶点,经过预实验进一步从中筛选到4个靶点。
4个靶点分别如下:
sgRNAADCY3-E3-g1靶点:5’-GGAGGTGAAGATGAACCTGG-3’(SEQ ID NO:18);
sgRNAADCY3-E3-g2靶点:5’-GCACCGCAAAGCCTTCCTGG-3’(SEQ ID NO:19);
sgRNAADCY3-E3-g3靶点:5’-GGGCCTCCAGGAAGGCTTTG-3’(SEQ ID NO:20);
sgRNAADCY3-E3-g4靶点:5’-GGAAGGCTTTGCGGTGCTTG-3’(SEQ ID NO:21)。
三、制备重组质粒
取质粒pKG-U6gRNA,用限制性内切酶BbsI进行酶切,回收载体骨架(约3kb的线性大片段)。
分别合成ADCY3-E3-gRNA1-S和ADCY3-E3-gRNA1-A,然后混合并进行退火,得到具有粘性末端的双链DNA分子。将具有粘性末端的双链DNA分子和载体骨架连接,得到质粒pKG-U6gRNA(ADCY3-E3-g1)。质粒pKG-U6gRNA(ADCY3-E3-g1)表达SEQ ID NO:22所示的sgRNAADCY3-E3-g1。
SEQ ID NO:22:
GGAGGUGAAGAUGAACCUGGguuuuagagcuagaaauagcaaguuaaaauaaggcuaguccguuaucaacuugaaaaaguggcaccgagucggugcuuuu
分别合成ADCY3-E3-gRNA2-S和ADCY3-E3-gRNA2-A,然后混合并进行退火,得到具有粘性末端的双链DNA分子。将具有粘性末端的双链DNA分子和载体骨架连接,得到质粒pKG-U6gRNA(ADCY3-E3-g2)。质粒pKG-U6gRNA(ADCY3-E3-g2)表达SEQ ID NO:23所示的sgRNAADCY3-E3-g2。
SEQ ID NO:23
GCACCGCAAAGCCUUCCUGGguuuuagagcuagaaauagcaaguuaaaauaaggcuaguccguuaucaacuugaaaaaguggcaccgagucggugcuuuu
分别合成ADCY3-E3-gRNA3-S和ADCY3-E3-gRNA3-A,然后混合并进行退火,得到具有粘性末端的双链DNA分子。将具有粘性末端的双链DNA分子和载体骨架连接,得到质粒pKG-U6gRNA(ADCY3-E3-g3)。质粒pKG-U6gRNA(ADCY3-E3-g3)表达SEQ ID NO:24所示的sgRNAADCY3-E3-g3。
SEQ ID NO:24:
GGGCCUCCAGGAAGGCUUUGguuuuagagcuagaaauagcaaguuaaaauaaggcuaguccguuaucaacuugaaaaaguggcaccgagucggugcuuuu
分别合成ADCY3-E3-gRNA4-S和ADCY3-E3-gRNA4-A,然后混合并进行退火,得到具有粘性末端的双链DNA分子。将具有粘性末端的双链DNA分子和载体骨架连接,得到质粒pKG-U6gRNA(ADCY3-E3-g4)。质粒pKG-U6gRNA(ADCY3-E3-g4)表达SEQ ID NO:25所示的sgRNAADCY3-E3-g1。
SEQ ID NO:25:
GGAAGGCUUUGCGGUGCUUGguuuuagagcuagaaauagcaaguuaaaauaaggcuaguccguuaucaacuugaaaaaguggcaccgagucggugcuuuu
ADCY3-E3-gRNA1-S:5’-caccGGAGGTGAAGATGAACCTGG-3’(SEQ ID NO:26);
ADCY3-E3-gRNA1-A:5’-aaacCCAGGTTCATCTTCACCTCC-3’(SEQ ID NO:27);
ADCY3-E3-gRNA2-S:5’-caccGCACCGCAAAGCCTTCCTGG-3’(SEQ ID NO:28);
ADCY3-E3-gRNA2-A:5’-aaacCCAGGAAGGCTTTGCGGTGC-3’(SEQ ID NO:29);
ADCY3-E3-gRNA3-S:5’-caccGGGCCTCCAGGAAGGCTTTG-3’(SEQ ID NO:30);
ADCY3-E3-gRNA3-A:5’-aaacCAAAGCCTTCCTGGAGGCCC-3’(SEQ ID NO:31);
ADCY3-E3-gRNA4-S:5’-caccGGAAGGCTTTGCGGTGCTTG-3’(SEQ ID NO:32);
ADCY3-E3-gRNA4-A:5’-aaacCAAGCACCGCAAAGCCTTCC-3’(SEQ ID NO:33)。
ADCY3-E3-gRNA1-S、ADCY3-E3-gRNA1-A、ADCY3-E3-gRNA2-S、ADCY3-E3-gRNA2-A、ADCY3-E3-gRNA3-S、ADCY3-E3-gRNA3-A、ADCY3-E3-gRNA4-S、ADCY3-E3-gRNA4-A均为单链DNA分子。
四、不同靶点的编辑效率比较
从初生从江香猪(雌性,血型AO)的耳组织制备猪原代成纤维细胞。
1、共转染
第一组:将质粒pKG-U6gRNA(ADCY3-E3-g1)和质粒pKG-GE3共转染猪原代成纤维细胞。配比:约20万个猪原代成纤维细胞:0.92μg质粒pKG-U6gRNA(ADCY3-E3-g1):1.08μg质粒pKG-GE3。
第二组:将质粒pKG-U6gRNA(ADCY3-E3-g2)和质粒pKG-GE3共转染猪原代成纤维细胞。配比:约20万个猪原代成纤维细胞:0.92μg质粒pKG-U6gRNA(ADCY3-E3-g2):1.08μg质粒pKG-GE3。
第三组:将质粒pKG-U6gRNA(ADCY3-E3-g3)和质粒pKG-GE3共转染猪原代成纤维细胞。配比:约20万个猪原代成纤维细胞:0.92μg质粒pKG-U6gRNA(ADCY3-E3-g3):1.08μg质粒pKG-GE3。
第四组:将质粒pKG-U6gRNA(ADCY3-E3-g4)和质粒pKG-GE3共转染猪原代成纤维细胞。配比:约20万个猪原代成纤维细胞:0.92μg质粒pKG-U6gRNA(ADCY3-E3-g4):1.08μg质粒pKG-GE3。
第五组:猪原代成纤维细胞,未进行任何转染操作。
共转染采用电击转染的方式,采用哺乳动物核转染试剂盒(Neon kit,Thermofisher)与Neon TM transfection system电转仪(参数设置为:1450V、10ms、3pulse)。
2、完成步骤1后,采用完全培养液培养16-18小时,然后更换新的完全培养液进行培养。培养总时间为48小时。
3、完成步骤2后,采用胰蛋白酶消化并收集细胞,然后裂解细胞并提取基因组DNA,采用ADCY3-E3-F和ADCY3-E3-R组成的引物对进行PCR扩增,然后进行电泳。回收目标片段并进行测序,测序峰图见图8。利用Synthego ICE工具分析测序峰图得出不同靶点的基因编辑效率。第一组至第四组的基因编辑效率依次为43%、17%、2%、3%。第五组不发生基因编辑。结果表明,第一组编辑效率最高,sgRNAADCY3-E3-g1的靶点为最优靶点。
实施例4、制备ADCY3基因编辑单克隆细胞
从初生从江香猪(雌性,血型AO)的耳组织制备猪原代成纤维细胞。
1、共转染
将质粒pKG-U6gRNA(ADCY3-E3-g1)和质粒pKG-GE3共转染猪原代成纤维细胞。配比:约20万个猪原代成纤维细胞:0.92μg质粒pKG-U6gRNA(ADCY3-E3-g1):1.08μg质粒pKG-GE3。
共转染采用电击转染的方式,采用哺乳动物核转染试剂盒(Neon kit,Thermofisher)与Neon TM transfection system电转仪(参数设置为:1450V、10ms、3pulse)。
2、完成步骤1后,采用完全培养液培养16-18小时,然后更换新的完全培养液进行培养。培养总时间为48小时。
3、完成步骤2后,采用胰蛋白酶消化并收集细胞,用完全培养液洗涤,再用完全培养液重悬,然后分别挑取各个单细胞至96孔板的不同孔中(每个孔1个细胞,每个孔中装有100μl完全培养液),培养2周(每2-3天更换新的完全培养液)。
4、完成步骤3后,采用胰蛋白酶消化并收集细胞(每孔得到的细胞,约2/3接种到装有完全培养液的6孔板中,剩余的1/3收集在1.5mL离心管中用于后续的基因型测序检测)。
5、取步骤4的6孔板,培养至细胞长至80%汇合度,采用胰蛋白酶消化并收集细胞,使用细胞冻存液(90%完全培养基+10%DMSO,体积比)将细胞冻存。
6、取步骤4的离心管,取细胞,提取基因组DNA,采用ADCY3-E3-F和ADCY3-E3-R组成的引物对进行PCR扩增,然后进行电泳。将猪原代成纤维细胞作为野生型对照。电泳图见图9。图9中的泳道编号与表1中的细胞编号一致。
7、完成步骤6后,回收PCR扩增产物并测序。
猪原代成纤维细胞的测序结果只有一种,其基因型为野生型。如果某一单克隆细胞的测序结果有两种,一种与猪原代成纤维细胞的测序结果一致,另一种与猪原代成纤维细胞的测序结果相比发生了突变(突变包括一个或多个核苷酸的缺失、插入或替换),该单克隆细胞的基因型为杂合突变型;如果某一单克隆细胞的测序结果为两种,均与猪原代成纤维细胞的测序结果相比发生了突变(突变包括一个或多个核苷酸的缺失、插入或替换),该单克隆细胞的基因型为双等位基因不同变异的纯合突变型;如果某一单克隆细胞的测序结果为一种,且与猪原代成纤维细胞的测序结果相比发生了突变(突变包括一个或多个核苷酸的缺失、插入或替换),该单克隆细胞的基因型为双等位基因相同变异的纯合突变型;如果某一单克隆细胞的测序结果为一种,且与猪原代成纤维细胞的测序结果一致,该单克隆细胞的基因型为野生型。
结果见表1。编号为1、3、4、7、10、11、13、17、21、26、31、36、41、42的单克隆细胞的基因型为双等位基因不同变异的纯合突变型。编号为22、28、40的单克隆细胞的基因型为杂合突变型。编号为2、5、12、23、25的单克隆细胞均显示为复杂的套峰,因此未能获得有效的序列,不能确定基因型和具体形式,但可以判断发生了基因编辑。得到基因编辑单克隆细胞的比率为22/41。
示例性的测序比对结果见图10至图12。图10是编号为ADCY3-6的单克隆细胞的正向测序与野生型比对的结果,判定为野生型。图11是编号为ADCY3-22的单克隆细胞的正向测序与野生型比对的结果,判定为杂合突变型。图12是编号为ADCY3-17的单克隆细胞的正向测序与野生型比对的结果,判定为双等位基因不同变异的纯合突变型。
表1
以上对本发明进行了详述。对于本领域技术人员来说,在不脱离本发明的宗旨和范围,以及无需进行不必要的实验情况下,可在等同参数、浓度和条件下,在较宽范围内实施本发明。虽然本发明给出了特殊的实施例,应该理解为,可以对本发明作进一步的改进。总之,按本发明的原理,本申请欲包括任何变更、用途或对本发明的改进,包括脱离了本申请中已公开范围,而用本领域已知的常规技术进行的改变。按以下附带的权利要求的范围,可以进行一些基本特征的应用。
序列表
<110> 南京启真基因工程有限公司
<120> 用于ADCY3基因编辑的CRISPR 系统及其在构建肥胖症猪核移植供体细胞中应用
<160> 33
<170> SIPOSequenceListing 1.0
<210> 1
<211> 8484
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 1
gagggcctat ttcccatgat tccttcatat ttgcatatac gatacaaggc tgttagagag 60
ataattggaa ttaatttgac tgtaaacaca aagatattag tacaaaatac gtgacgtaga 120
aagtaataat ttcttgggta gtttgcagtt ttaaaattat gttttaaaat ggactatcat 180
atgcttaccg taacttgaaa gtatttcgat ttcttggctt tatatatctt gtggaaagga 240
cgaaacaccg ggtcttcgag aagacctgtt ttagagctag aaatagcaag ttaaaataag 300
gctagtccgt tatcaacttg aaaaagtggc accgagtcgg tgcttttttg ttttagagct 360
agaaatagca agttaaaata aggctagtcc gtttttagcg cgtgcgccaa ttctgcagac 420
aaatggctct agaggtaccc gttacataac ttacggtaaa tggcccgcct ggctgaccgc 480
ccaacgaccc ccgcccattg acgtcaatag taacgccaat agggactttc cattgacgtc 540
aatgggtgga gtatttacgg taaactgccc acttggcagt acatcaagtg tatcatatgc 600
caagtacgcc ccctattgac gtcaatgacg gtaaatggcc cgcctggcat tgtgcccagt 660
acatgacctt atgggacttt cctacttggc agtacatcta cgtattagtc atcgctatta 720
ccatggtcga ggtgagcccc acgttctgct tcactctccc catctccccc ccctccccac 780
ccccaatttt gtatttattt attttttaat tattttgtgc agcgatgggg gcgggggggg 840
ggggggggcg gggcgagggg cggggcgggg cgaggcggag aggtgcggcg gcagccaatc 900
agagcggcgc gctccgaaag tttcctttta tggcgaggcg gcggcggcgg cggccctata 960
aaaagcgaag cgcgcggcgg gcgggagtcg ctgcgcgctg ccttcgcccc gtgccccgct 1020
ccgccgccgc ctcgcgccgc ccgccccggc tctgactgac cgcgttactc ccacaggtga 1080
gcgggcggga cggcccttct cctccgggct gtaattagct gagcaagagg taagggttta 1140
agggatggtt ggttggtggg gtattaatgt ttaattacct ggagcacctg cctgaaatca 1200
ctttttttca ggttggaccg gtgccaccat ggactataag gaccacgacg gagactacaa 1260
ggatcatgat attgattaca aagacgatga cgataagatg gccccaaaga agaagcggaa 1320
ggtcggtatc cacggagtcc cagcagccga caagaagtac agcatcggcc tggacatcgg 1380
caccaactct gtgggctggg ccgtgatcac cgacgagtac aaggtgccca gcaagaaatt 1440
caaggtgctg ggcaacaccg accggcacag catcaagaag aacctgatcg gagccctgct 1500
gttcgacagc ggcgaaacag ccgaggccac ccggctgaag agaaccgcca gaagaagata 1560
caccagacgg aagaaccgga tctgctatct gcaagagatc ttcagcaacg agatggccaa 1620
ggtggacgac agcttcttcc acagactgga agagtccttc ctggtggaag aggataagaa 1680
gcacgagcgg caccccatct tcggcaacat cgtggacgag gtggcctacc acgagaagta 1740
ccccaccatc taccacctga gaaagaaact ggtggacagc accgacaagg ccgacctgcg 1800
gctgatctat ctggccctgg cccacatgat caagttccgg ggccacttcc tgatcgaggg 1860
cgacctgaac cccgacaaca gcgacgtgga caagctgttc atccagctgg tgcagaccta 1920
caaccagctg ttcgaggaaa accccatcaa cgccagcggc gtggacgcca aggccatcct 1980
gtctgccaga ctgagcaaga gcagacggct ggaaaatctg atcgcccagc tgcccggcga 2040
gaagaagaat ggcctgttcg gaaacctgat tgccctgagc ctgggcctga cccccaactt 2100
caagagcaac ttcgacctgg ccgaggatgc caaactgcag ctgagcaagg acacctacga 2160
cgacgacctg gacaacctgc tggcccagat cggcgaccag tacgccgacc tgtttctggc 2220
cgccaagaac ctgtccgacg ccatcctgct gagcgacatc ctgagagtga acaccgagat 2280
caccaaggcc cccctgagcg cctctatgat caagagatac gacgagcacc accaggacct 2340
gaccctgctg aaagctctcg tgcggcagca gctgcctgag aagtacaaag agattttctt 2400
cgaccagagc aagaacggct acgccggcta cattgacggc ggagccagcc aggaagagtt 2460
ctacaagttc atcaagccca tcctggaaaa gatggacggc accgaggaac tgctcgtgaa 2520
gctgaacaga gaggacctgc tgcggaagca gcggaccttc gacaacggca gcatccccca 2580
ccagatccac ctgggagagc tgcacgccat tctgcggcgg caggaagatt tttacccatt 2640
cctgaaggac aaccgggaaa agatcgagaa gatcctgacc ttccgcatcc cctactacgt 2700
gggccctctg gccaggggaa acagcagatt cgcctggatg accagaaaga gcgaggaaac 2760
catcaccccc tggaacttcg aggaagtggt ggacaagggc gcttccgccc agagcttcat 2820
cgagcggatg accaacttcg ataagaacct gcccaacgag aaggtgctgc ccaagcacag 2880
cctgctgtac gagtacttca ccgtgtataa cgagctgacc aaagtgaaat acgtgaccga 2940
gggaatgaga aagcccgcct tcctgagcgg cgagcagaaa aaggccatcg tggacctgct 3000
gttcaagacc aaccggaaag tgaccgtgaa gcagctgaaa gaggactact tcaagaaaat 3060
cgagtgcttc gactccgtgg aaatctccgg cgtggaagat cggttcaacg cctccctggg 3120
cacataccac gatctgctga aaattatcaa ggacaaggac ttcctggaca atgaggaaaa 3180
cgaggacatt ctggaagata tcgtgctgac cctgacactg tttgaggaca gagagatgat 3240
cgaggaacgg ctgaaaacct atgcccacct gttcgacgac aaagtgatga agcagctgaa 3300
gcggcggaga tacaccggct ggggcaggct gagccggaag ctgatcaacg gcatccggga 3360
caagcagtcc ggcaagacaa tcctggattt cctgaagtcc gacggcttcg ccaacagaaa 3420
cttcatgcag ctgatccacg acgacagcct gacctttaaa gaggacatcc agaaagccca 3480
ggtgtccggc cagggcgata gcctgcacga gcacattgcc aatctggccg gcagccccgc 3540
cattaagaag ggcatcctgc agacagtgaa ggtggtggac gagctcgtga aagtgatggg 3600
ccggcacaag cccgagaaca tcgtgatcga aatggccaga gagaaccaga ccacccagaa 3660
gggacagaag aacagccgcg agagaatgaa gcggatcgaa gagggcatca aagagctggg 3720
cagccagatc ctgaaagaac accccgtgga aaacacccag ctgcagaacg agaagctgta 3780
cctgtactac ctgcagaatg ggcgggatat gtacgtggac caggaactgg acatcaaccg 3840
gctgtccgac tacgatgtgg accatatcgt gcctcagagc tttctgaagg acgactccat 3900
cgacaacaag gtgctgacca gaagcgacaa gaaccggggc aagagcgaca acgtgccctc 3960
cgaagaggtc gtgaagaaga tgaagaacta ctggcggcag ctgctgaacg ccaagctgat 4020
tacccagaga aagttcgaca atctgaccaa ggccgagaga ggcggcctga gcgaactgga 4080
taaggccggc ttcatcaaga gacagctggt ggaaacccgg cagatcacaa agcacgtggc 4140
acagatcctg gactcccgga tgaacactaa gtacgacgag aatgacaagc tgatccggga 4200
agtgaaagtg atcaccctga agtccaagct ggtgtccgat ttccggaagg atttccagtt 4260
ttacaaagtg cgcgagatca acaactacca ccacgcccac gacgcctacc tgaacgccgt 4320
cgtgggaacc gccctgatca aaaagtaccc taagctggaa agcgagttcg tgtacggcga 4380
ctacaaggtg tacgacgtgc ggaagatgat cgccaagagc gagcaggaaa tcggcaaggc 4440
taccgccaag tacttcttct acagcaacat catgaacttt ttcaagaccg agattaccct 4500
ggccaacggc gagatccgga agcggcctct gatcgagaca aacggcgaaa ccggggagat 4560
cgtgtgggat aagggccggg attttgccac cgtgcggaaa gtgctgagca tgccccaagt 4620
gaatatcgtg aaaaagaccg aggtgcagac aggcggcttc agcaaagagt ctatcctgcc 4680
caagaggaac agcgataagc tgatcgccag aaagaaggac tgggacccta agaagtacgg 4740
cggcttcgac agccccaccg tggcctattc tgtgctggtg gtggccaaag tggaaaaggg 4800
caagtccaag aaactgaaga gtgtgaaaga gctgctgggg atcaccatca tggaaagaag 4860
cagcttcgag aagaatccca tcgactttct ggaagccaag ggctacaaag aagtgaaaaa 4920
ggacctgatc atcaagctgc ctaagtactc cctgttcgag ctggaaaacg gccggaagag 4980
aatgctggcc tctgccggcg aactgcagaa gggaaacgaa ctggccctgc cctccaaata 5040
tgtgaacttc ctgtacctgg ccagccacta tgagaagctg aagggctccc ccgaggataa 5100
tgagcagaaa cagctgtttg tggaacagca caagcactac ctggacgaga tcatcgagca 5160
gatcagcgag ttctccaaga gagtgatcct ggccgacgct aatctggaca aagtgctgtc 5220
cgcctacaac aagcaccggg ataagcccat cagagagcag gccgagaata tcatccacct 5280
gtttaccctg accaatctgg gagcccctgc cgccttcaag tactttgaca ccaccatcga 5340
ccggaagagg tacaccagca ccaaagaggt gctggacgcc accctgatcc accagagcat 5400
caccggcctg tacgagacac ggatcgacct gtctcagctg ggaggcgaca aaaggccggc 5460
ggccacgaaa aaggccggcc aggcaaaaaa gaaaaagtaa gaattcctag agctcgctga 5520
tcagcctcga ctgtgccttc tagttgccag ccatctgttg tttgcccctc ccccgtgcct 5580
tccttgaccc tggaaggtgc cactcccact gtcctttcct aataaaatga ggaaattgca 5640
tcgcattgtc tgagtaggtg tcattctatt ctggggggtg gggtggggca ggacagcaag 5700
ggggaggatt gggaagagaa tagcaggcat gctggggagc ggccgcagga acccctagtg 5760
atggagttgg ccactccctc tctgcgcgct cgctcgctca ctgaggccgg gcgaccaaag 5820
gtcgcccgac gcccgggctt tgcccgggcg gcctcagtga gcgagcgagc gcgcagctgc 5880
ctgcaggggc gcctgatgcg gtattttctc cttacgcatc tgtgcggtat ttcacaccgc 5940
atacgtcaaa gcaaccatag tacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg 6000
tggttacgcg cagcgtgacc gctacacttg ccagcgcctt agcgcccgct cctttcgctt 6060
tcttcccttc ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc 6120
tccctttagg gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgatttgg 6180
gtgatggttc acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg 6240
agtccacgtt ctttaatagt ggactcttgt tccaaactgg aacaacactc aactctatct 6300
cgggctattc ttttgattta taagggattt tgccgatttc ggtctattgg ttaaaaaatg 6360
agctgattta acaaaaattt aacgcgaatt ttaacaaaat attaacgttt acaattttat 6420
ggtgcactct cagtacaatc tgctctgatg ccgcatagtt aagccagccc cgacacccgc 6480
caacacccgc tgacgcgccc tgacgggctt gtctgctccc ggcatccgct tacagacaag 6540
ctgtgaccgt ctccgggagc tgcatgtgtc agaggttttc accgtcatca ccgaaacgcg 6600
cgagacgaaa gggcctcgtg atacgcctat ttttataggt taatgtcatg ataataatgg 6660
tttcttagac gtcaggtggc acttttcggg gaaatgtgcg cggaacccct atttgtttat 6720
ttttctaaat acattcaaat atgtatccgc tcatgagaca ataaccctga taaatgcttc 6780
aataatattg aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc cttattccct 6840
tttttgcggc attttgcctt cctgtttttg ctcacccaga aacgctggtg aaagtaaaag 6900
atgctgaaga tcagttgggt gcacgagtgg gttacatcga actggatctc aacagcggta 6960
agatccttga gagttttcgc cccgaagaac gttttccaat gatgagcact tttaaagttc 7020
tgctatgtgg cgcggtatta tcccgtattg acgccgggca agagcaactc ggtcgccgca 7080
tacactattc tcagaatgac ttggttgagt actcaccagt cacagaaaag catcttacgg 7140
atggcatgac agtaagagaa ttatgcagtg ctgccataac catgagtgat aacactgcgg 7200
ccaacttact tctgacaacg atcggaggac cgaaggagct aaccgctttt ttgcacaaca 7260
tgggggatca tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa gccataccaa 7320
acgacgagcg tgacaccacg atgcctgtag caatggcaac aacgttgcgc aaactattaa 7380
ctggcgaact acttactcta gcttcccggc aacaattaat agactggatg gaggcggata 7440
aagttgcagg accacttctg cgctcggccc ttccggctgg ctggtttatt gctgataaat 7500
ctggagccgg tgagcgtgga agccgcggta tcattgcagc actggggcca gatggtaagc 7560
cctcccgtat cgtagttatc tacacgacgg ggagtcaggc aactatggat gaacgaaata 7620
gacagatcgc tgagataggt gcctcactga ttaagcattg gtaactgtca gaccaagttt 7680
actcatatat actttagatt gatttaaaac ttcattttta atttaaaagg atctaggtga 7740
agatcctttt tgataatctc atgaccaaaa tcccttaacg tgagttttcg ttccactgag 7800
cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa 7860
tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag 7920
agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg 7980
ttcttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat 8040
acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta 8100
ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg 8160
gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc 8220
gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa 8280
gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc 8340
tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt 8400
caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct 8460
tttgctggcc ttttgctcac atgt 8484
<210> 2
<211> 10476
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 2
gagggcctat ttcccatgat tccttcatat ttgcatatac gatacaaggc tgttagagag 60
ataattggaa ttaatttgac tgtaaacaca aagatattag tacaaaatac gtgacgtaga 120
aagtaataat ttcttgggta gtttgcagtt ttaaaattat gttttaaaat ggactatcat 180
atgcttaccg taacttgaaa gtatttcgat ttcttggctt tatatatctt gtggaaagga 240
cgaaacaccg ggtcttcgag aagacctgtt ttagagctag aaatagcaag ttaaaataag 300
gctagtccgt tatcaacttg aaaaagtggc accgagtcgg tgcttttttc tagcgcgtgc 360
gccaattctg cagacaaatg gctctagagg tacccgttac ataacttacg gtaaatggcc 420
cgcctggctg accgcccaac gacccccgcc cattgacgtc aatagtaacg ccaataggga 480
ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc 540
aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct 600
ggcattgtgc ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat 660
tagtcatcgc tattaccatg ggggcagagc gcacatcgcc cacagtcccc gagaagttgg 720
ggggaggggt cggcaattga tccggtgcct agagaaggtg gcgcggggta aactgggaaa 780
gtgatgtcgt gtactggctc cgcctttttc ccgagggtgg gggagaaccg tatataagtg 840
cagtagtcgc cgtgaacgtt ctttttcgca acgggtttgc cgccagaaca caggttggac 900
cggtgccacc atggactata aggaccacga cggagactac aaggatcatg atattgatta 960
caaagacgat gacgataaga tggcccccaa aaagaaacga aaggtgggtg ggtccccaaa 1020
gaagaagcgg aaggtcggta tccacggagt cccagcagcc gacaagaagt acagcatcgg 1080
cctggacatc ggcaccaact ctgtgggctg ggccgtgatc accgacgagt acaaggtgcc 1140
cagcaagaaa ttcaaggtgc tgggcaacac cgaccggcac agcatcaaga agaacctgat 1200
cggagccctg ctgttcgaca gcggcgaaac agccgaggcc acccggctga agagaaccgc 1260
cagaagaaga tacaccagac ggaagaaccg gatctgctat ctgcaagaga tcttcagcaa 1320
cgagatggcc aaggtggacg acagcttctt ccacagactg gaagagtcct tcctggtgga 1380
agaggataag aagcacgagc ggcaccccat cttcggcaac atcgtggacg aggtggccta 1440
ccacgagaag taccccacca tctaccacct gagaaagaaa ctggtggaca gcaccgacaa 1500
ggccgacctg cggctgatct atctggccct ggcccacatg atcaagttcc ggggccactt 1560
cctgatcgag ggcgacctga accccgacaa cagcgacgtg gacaagctgt tcatccagct 1620
ggtgcagacc tacaaccagc tgttcgagga aaaccccatc aacgccagcg gcgtggacgc 1680
caaggccatc ctgtctgcca gactgagcaa gagcagacgg ctggaaaatc tgatcgccca 1740
gctgcccggc gagaagaaga atggcctgtt cggaaacctg attgccctga gcctgggcct 1800
gacccccaac ttcaagagca acttcgacct ggccgaggat gccaaactgc agctgagcaa 1860
ggacacctac gacgacgacc tggacaacct gctggcccag atcggcgacc agtacgccga 1920
cctgtttctg gccgccaaga acctgtccga cgccatcctg ctgagcgaca tcctgagagt 1980
gaacaccgag atcaccaagg cccccctgag cgcctctatg atcaagagat acgacgagca 2040
ccaccaggac ctgaccctgc tgaaagctct cgtgcggcag cagctgcctg agaagtacaa 2100
agagattttc ttcgaccaga gcaagaacgg ctacgccggc tacattgacg gcggagccag 2160
ccaggaagag ttctacaagt tcatcaagcc catcctggaa aagatggacg gcaccgagga 2220
actgctcgtg aagctgaaca gagaggacct gctgcggaag cagcggacct tcgacaacgg 2280
cagcatcccc caccagatcc acctgggaga gctgcacgcc attctgcggc ggcaggaaga 2340
tttttaccca ttcctgaagg acaaccggga aaagatcgag aagatcctga ccttccgcat 2400
cccctactac gtgggccctc tggccagggg aaacagcaga ttcgcctgga tgaccagaaa 2460
gagcgaggaa accatcaccc cctggaactt cgaggaagtg gtggacaagg gcgcttccgc 2520
ccagagcttc atcgagcgga tgaccaactt cgataagaac ctgcccaacg agaaggtgct 2580
gcccaagcac agcctgctgt acgagtactt caccgtgtat aacgagctga ccaaagtgaa 2640
atacgtgacc gagggaatga gaaagcccgc cttcctgagc ggcgagcaga aaaaggccat 2700
cgtggacctg ctgttcaaga ccaaccggaa agtgaccgtg aagcagctga aagaggacta 2760
cttcaagaaa atcgagtgct tcgactccgt ggaaatctcc ggcgtggaag atcggttcaa 2820
cgcctccctg ggcacatacc acgatctgct gaaaattatc aaggacaagg acttcctgga 2880
caatgaggaa aacgaggaca ttctggaaga tatcgtgctg accctgacac tgtttgagga 2940
cagagagatg atcgaggaac ggctgaaaac ctatgcccac ctgttcgacg acaaagtgat 3000
gaagcagctg aagcggcgga gatacaccgg ctggggcagg ctgagccgga agctgatcaa 3060
cggcatccgg gacaagcagt ccggcaagac aatcctggat ttcctgaagt ccgacggctt 3120
cgccaacaga aacttcatgc agctgatcca cgacgacagc ctgaccttta aagaggacat 3180
ccagaaagcc caggtgtccg gccagggcga tagcctgcac gagcacattg ccaatctggc 3240
cggcagcccc gccattaaga agggcatcct gcagacagtg aaggtggtgg acgagctcgt 3300
gaaagtgatg ggccggcaca agcccgagaa catcgtgatc gaaatggcca gagagaacca 3360
gaccacccag aagggacaga agaacagccg cgagagaatg aagcggatcg aagagggcat 3420
caaagagctg ggcagccaga tcctgaaaga acaccccgtg gaaaacaccc agctgcagaa 3480
cgagaagctg tacctgtact acctgcagaa tgggcgggat atgtacgtgg accaggaact 3540
ggacatcaac cggctgtccg actacgatgt ggaccatatc gtgcctcaga gctttctgaa 3600
ggacgactcc atcgacaaca aggtgctgac cagaagcgac aagaaccggg gcaagagcga 3660
caacgtgccc tccgaagagg tcgtgaagaa gatgaagaac tactggcggc agctgctgaa 3720
cgccaagctg attacccaga gaaagttcga caatctgacc aaggccgaga gaggcggcct 3780
gagcgaactg gataaggccg gcttcatcaa gagacagctg gtggaaaccc ggcagatcac 3840
aaagcacgtg gcacagatcc tggactcccg gatgaacact aagtacgacg agaatgacaa 3900
gctgatccgg gaagtgaaag tgatcaccct gaagtccaag ctggtgtccg atttccggaa 3960
ggatttccag ttttacaaag tgcgcgagat caacaactac caccacgccc acgacgccta 4020
cctgaacgcc gtcgtgggaa ccgccctgat caaaaagtac cctaagctgg aaagcgagtt 4080
cgtgtacggc gactacaagg tgtacgacgt gcggaagatg atcgccaaga gcgagcagga 4140
aatcggcaag gctaccgcca agtacttctt ctacagcaac atcatgaact ttttcaagac 4200
cgagattacc ctggccaacg gcgagatccg gaagcggcct ctgatcgaga caaacggcga 4260
aaccggggag atcgtgtggg ataagggccg ggattttgcc accgtgcgga aagtgctgag 4320
catgccccaa gtgaatatcg tgaaaaagac cgaggtgcag acaggcggct tcagcaaaga 4380
gtctatcctg cccaagagga acagcgataa gctgatcgcc agaaagaagg actgggaccc 4440
taagaagtac ggcggcttcg acagccccac cgtggcctat tctgtgctgg tggtggccaa 4500
agtggaaaag ggcaagtcca agaaactgaa gagtgtgaaa gagctgctgg ggatcaccat 4560
catggaaaga agcagcttcg agaagaatcc catcgacttt ctggaagcca agggctacaa 4620
agaagtgaaa aaggacctga tcatcaagct gcctaagtac tccctgttcg agctggaaaa 4680
cggccggaag agaatgctgg cctctgccgg cgaactgcag aagggaaacg aactggccct 4740
gccctccaaa tatgtgaact tcctgtacct ggccagccac tatgagaagc tgaagggctc 4800
ccccgaggat aatgagcaga aacagctgtt tgtggaacag cacaagcact acctggacga 4860
gatcatcgag cagatcagcg agttctccaa gagagtgatc ctggccgacg ctaatctgga 4920
caaagtgctg tccgcctaca acaagcaccg ggataagccc atcagagagc aggccgagaa 4980
tatcatccac ctgtttaccc tgaccaatct gggagcccct gccgccttca agtactttga 5040
caccaccatc gaccggaaga ggtacaccag caccaaagag gtgctggacg ccaccctgat 5100
ccaccagagc atcaccggcc tgtacgagac acggatcgac ctgtctcagc tgggaggcga 5160
caaaaggccg gcggccacga aaaaggccgg ccaggcaaaa aagaaaaagg gcggctccaa 5220
gcggcctgcc gcgacgaaga aagcgggaca ggccaagaaa aagaaaggat ccggcgcaac 5280
aaacttctct ctgctgaaac aagccggaga tgtcgaagag aatcctggac cggtgagcaa 5340
gggcgaggag ctgttcaccg gggtggtgcc catcctggtc gagctggacg gcgacgtaaa 5400
cggccacaag ttcagcgtgt ccggcgaggg cgagggcgat gccacctacg gcaagctgac 5460
cctgaagttc atctgcacca ccggcaagct gcccgtgccc tggcccaccc tcgtgaccac 5520
cctgacctac ggcgtgcagt gcttcagccg ctaccccgac cacatgaagc agcacgactt 5580
cttcaagtcc gccatgcccg aaggctacgt ccaggagcgc accatcttct tcaaggacga 5640
cggcaactac aagacccgcg ccgaggtgaa gttcgagggc gacaccctgg tgaaccgcat 5700
cgagctgaag ggcatcgact tcaaggagga cggcaacatc ctggggcaca agctggagta 5760
caactacaac agccacaacg tctatatcat ggccgacaag cagaagaacg gcatcaaggt 5820
gaacttcaag atccgccaca acatcgagga cggcagcgtg cagctcgccg accactacca 5880
gcagaacacc cccatcggcg acggccccgt gctgctgccc gacaaccact acctgagcac 5940
ccagtccgcc ctgagcaaag accccaacga gaagcgcgat cacatggtcc tgctggagtt 6000
cgtgaccgcc gccgggatca ctctcggcat ggacgagctg tacaagggct ccggcgaggg 6060
caggggaagt cttctaacat gcggggacgt ggaggaaaat cccggcccaa ccgagtacaa 6120
gcccacggtg cgcctcgcca cccgcgacga cgtccccagg gccgtacgca ccctcgccgc 6180
cgcgttcgcc gactaccccg ccacgcgcca caccgtcgat ccggaccgcc acatcgagcg 6240
ggtcaccgag ctgcaagaac tcttcctcac gcgcgtcggg ctcgacatcg gcaaggtgtg 6300
ggtcgcggac gacggcgccg cggtggcggt ctggaccacg ccggagagcg tcgaagcggg 6360
ggcggtgttc gccgagatcg gcccgcgcat ggccgagttg agcggttccc ggctggccgc 6420
gcagcaacag atggaaggcc tcctggcgcc gcaccggccc aaggagcccg cgtggttcct 6480
ggccaccgtc ggagtctcgc ccgaccacca gggcaagggt ctgggcagcg ccgtcgtgct 6540
ccccggagtg gaggcggccg agcgcgccgg ggtgcccgcc ttcctggaga cctccgcgcc 6600
ccgcaacctc cccttctacg agcggctcgg cttcaccgtc accgccgacg tcgaggtgcc 6660
cgaaggaccg cgcacctggt gcatgacccg caagcccggt gcctgaacgc gttaagtcga 6720
caatcaacct ctggattaca aaatttgtga aagattgact ggtattctta actatgttgc 6780
tccttttacg ctatgtggat acgctgcttt aatgcctttg tatcatgcta ttgcttcccg 6840
tatggctttc attttctcct ccttgtataa atcctggttg ctgtctcttt atgaggagtt 6900
gtggcccgtt gtcaggcaac gtggcgtggt gtgcactgtg tttgctgacg caacccccac 6960
tggttggggc attgccacca cctgtcagct cctttccggg actttcgctt tccccctccc 7020
tattgccacg gcggaactca tcgccgcctg ccttgcccgc tgctggacag gggctcggct 7080
gttgggcact gacaattccg tggtgttgtc ggggaaatca tcgtcctttc cttggctgct 7140
cgcctgtgtt gccacctgga ttctgcgcgg gacgtccttc tgctacgtcc cttcggccct 7200
caatccagcg gaccttcctt cccgcggcct gctgccggct ctgcggcctc ttccgcgtct 7260
tcgccttcgc cctcagacga gtcggatctc cctttgggcc gcctccccgc gtcgacttta 7320
agaccaatga cttacaaggc agctgtagat cttagccact ttttaaaaga aaagggggga 7380
ctggaagggc taattcactc ccaacgaaga caagatctgc tttttgcttg tactgggtct 7440
ctctggttag accagatctg agcctgggag ctctctggct aactagggaa cccactgctt 7500
aagcctcaat aaagcttgcc ttgagtgctt caagtagtgt gtgcccgtct gttgtgtgac 7560
tctggtaact agagatccct cagacccttt tagtcagtgt ggaaaatctc tagcagggcc 7620
cgtttaaacc cgctgatcag cctcgactgt gccttctagt tgccagccat ctgttgtttg 7680
cccctccccc gtgccttcct tgaccctgga aggtgccact cccactgtcc tttcctaata 7740
aaatgaggaa attgcatcgc attgtctgag taggtgtcat tctattctgg ggggtggggt 7800
ggggcaggac agcaaggggg aggattggga agacaatagc aggcatgctg gggatgcggt 7860
gggctctatg gcctgcaggg gcgcctgatg cggtattttc tccttacgca tctgtgcggt 7920
atttcacacc gcatacgtca aagcaaccat agtacgcgcc ctgtagcggc gcattaagcg 7980
cggcgggtgt ggtggttacg cgcagcgtga ccgctacact tgccagcgcc ttagcgcccg 8040
ctcctttcgc tttcttccct tcctttctcg ccacgttcgc cggctttccc cgtcaagctc 8100
taaatcgggg gctcccttta gggttccgat ttagtgcttt acggcacctc gaccccaaaa 8160
aacttgattt gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc 8220
ctttgacgtt ggagtccacg ttctttaata gtggactctt gttccaaact ggaacaacac 8280
tcaactctat ctcgggctat tcttttgatt tataagggat tttgccgatt tcggtctatt 8340
ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa ttttaacaaa atattaacgt 8400
ttacaatttt atggtgcact ctcagtacaa tctgctctga tgccgcatag ttaagccagc 8460
cccgacaccc gccaacaccc gctgacgcgc cctgacgggc ttgtctgctc ccggcatccg 8520
cttacagaca agctgtgacc gtctccggga gctgcatgtg tcagaggttt tcaccgtcat 8580
caccgaaacg cgcgagacga aagggcctcg tgatacgcct atttttatag gttaatgtca 8640
tgataataat ggtttcttag acgtcaggtg gcacttttcg gggaaatgtg cgcggaaccc 8700
ctatttgttt atttttctaa atacattcaa atatgtatcc gctcatgaga caataaccct 8760
gataaatgct tcaataatat tgaaaaagga agagtatgag tattcaacat ttccgtgtcg 8820
cccttattcc cttttttgcg gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg 8880
tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt gggttacatc gaactggatc 8940
tcaacagcgg taagatcctt gagagttttc gccccgaaga acgttttcca atgatgagca 9000
cttttaaagt tctgctatgt ggcgcggtat tatcccgtat tgacgccggg caagagcaac 9060
tcggtcgccg catacactat tctcagaatg acttggttga gtactcacca gtcacagaaa 9120
agcatcttac ggatggcatg acagtaagag aattatgcag tgctgccata accatgagtg 9180
ataacactgc ggccaactta cttctgacaa cgatcggagg accgaaggag ctaaccgctt 9240
ttttgcacaa catgggggat catgtaactc gccttgatcg ttgggaaccg gagctgaatg 9300
aagccatacc aaacgacgag cgtgacacca cgatgcctgt agcaatggca acaacgttgc 9360
gcaaactatt aactggcgaa ctacttactc tagcttcccg gcaacaatta atagactgga 9420
tggaggcgga taaagttgca ggaccacttc tgcgctcggc ccttccggct ggctggttta 9480
ttgctgataa atctggagcc ggtgagcgtg gaagccgcgg tatcattgca gcactggggc 9540
cagatggtaa gccctcccgt atcgtagtta tctacacgac ggggagtcag gcaactatgg 9600
atgaacgaaa tagacagatc gctgagatag gtgcctcact gattaagcat tggtaactgt 9660
cagaccaagt ttactcatat atactttaga ttgatttaaa acttcatttt taatttaaaa 9720
ggatctaggt gaagatcctt tttgataatc tcatgaccaa aatcccttaa cgtgagtttt 9780
cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt 9840
ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt 9900
tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga 9960
taccaaatac tgttcttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag 10020
caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata 10080
agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg 10140
gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga 10200
gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca 10260
ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa 10320
acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt 10380
tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac 10440
ggttcctggc cttttgctgg ccttttgctc acatgt 10476
<210> 3
<211> 3120
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 3
gacgaaaggg cctcgtgata cgcctatttt tataggttaa tgtcatgata ataatggttt 60
cttagacgtc aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt 120
tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat 180
aatattgaaa aaggaagagt atgagtattc aacatttccg tgtcgccctt attccctttt 240
ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg 300
ctgaagatca gttgggtgca cgagtgggtt acatcgaact ggatctcaac agcggtaaga 360
tccttgagag ttttcgcccc gaagaacgtt ttccaatgat gagcactttt aaagttctgc 420
tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga gcaactcggt cgccgcatac 480
actattctca gaatgacttg gttgagtact caccagtcac agaaaagcat cttacggatg 540
gcatgacagt aagagaatta tgcagtgctg ccataaccat gagtgataac actgcggcca 600
acttacttct gacaacgatc ggaggaccga aggagctaac cgcttttttg cacaacatgg 660
gggatcatgt aactcgcctt gatcgttggg aaccggagct gaatgaagcc ataccaaacg 720
acgagcgtga caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg 780
gcgaactact tactctagct tcccggcaac aattaataga ctggatggag gcggataaag 840
ttgcaggacc acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatctg 900
gagccggtga gcgtgggtct cgcggtatca ttgcagcact ggggccagat ggtaagccct 960
cccgtatcgt agttatctac acgacgggga gtcaggcaac tatggatgaa cgaaatagac 1020
agatcgctga gataggtgcc tcactgatta agcattggta actgtcagac caagtttact 1080
catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga 1140
tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt 1200
cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct 1260
gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc 1320
taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgttc 1380
ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc 1440
tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg 1500
ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt 1560
cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg 1620
agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg 1680
gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt 1740
atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag 1800
gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt 1860
gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta 1920
ttaccgcctt tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgagt 1980
cagtgagcga ggaagcggaa gagcgcccaa tacgcaaacc gcctctcccc gcgcgttggc 2040
cgattcatta atgcagctgg cacgacaggt ttcccgactg gaaagcgggc agtgagcgca 2100
acgcaattaa tgtgagttag ctcactcatt aggcacccca ggctttacac tttatgcttc 2160
cggctcgtat gttgtgtgga attgtgagcg gataacaatt tcacacagga aacagctatg 2220
accatgatta cgccaagctt gcatgcaggc ctctgcagtc gacgggcccg ggatccgatg 2280
ataaacatgt gagggcctat ttcccatgat tccttcatat ttgcatatac gatacaaggc 2340
tgttagagag ataattggaa ttaatttgac tgtaaacaca aagatattag tacaaaatac 2400
gtgacgtaga aagtaataat ttcttgggta gtttgcagtt ttaaaattat gttttaaaat 2460
ggactatcat atgcttaccg taacttgaaa gtatttcgat ttcttggctt tatatatctt 2520
gtggaaagga cgaaacaccg ggtcttcgag aagacctgtt ttagagctag aaatagcaag 2580
ttaaaataag gctagtccgt tatcaacttg aaaaagtggc accgagtcgg tgcttttttc 2640
tagcgcgtgc gccaattctg cagacaaatg gctctagagg tacccataga tctagatgca 2700
ttcgcgaggt accgagctcg aattcactgg ccgtcgtttt acaacgtcgt gactgggaaa 2760
accctggcgt tacccaactt aatcgccttg cagcacatcc ccctttcgcc agctggcgta 2820
atagcgaaga ggcccgcacc gatcgccctt cccaacagtt gcgcagcctg aatggcgaat 2880
ggcgcctgat gcggtatttt ctccttacgc atctgtgcgg tatttcacac cgcatatggt 2940
gcactctcag tacaatctgc tctgatgccg catagttaag ccagccccga cacccgccaa 3000
cacccgctga cgcgccctga cgggcttgtc tgctcccggc atccgcttac agacaagctg 3060
tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga 3120
<210> 4
<211> 175
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 4
tgtggaaagg acgaaacacc gggtcttcga gaagacctgt tttagagcta gaaatagcaa 60
gttaaaataa ggctagtccg ttatcaactt gaaaaagtgg caccgagtcg gtgctttttt 120
ctagcgcgtg cgccaattct gcagacaaat ggctctagag gtacccgtta cataa 175
<210> 5
<211> 554
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 5
tctgcagaca aatggctcta gaggtacccg ttacataact tacggtaaat ggcccgcctg 60
gctgaccgcc caacgacccc cgcccattga cgtcaatagt aacgccaata gggactttcc 120
attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt 180
atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt 240
gtgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca 300
tcgctattac catgggggca gagcgcacat cgcccacagt ccccgagaag ttggggggag 360
gggtcggcaa ttgatccggt gcctagagaa ggtggcgcgg ggtaaactgg gaaagtgatg 420
tcgtgtactg gctccgcctt tttcccgagg gtgggggaga accgtatata agtgcagtag 480
tcgccgtgaa cgttcttttt cgcaacgggt ttgccgccag aacacaggtt ggaccggtgc 540
caccatggac tata 554
<210> 6
<211> 447
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 6
ccagaacaca ggttggaccg gtgccaccat ggactataag gaccacgacg gagactacaa 60
ggatcatgat attgattaca aagacgatga cgataagatg gcccccaaaa agaaacgaaa 120
ggtgggtggg tccccaaaga agaagcggaa ggtcggtatc cacggagtcc cagcagccga 180
caagaagtac agcatcggcc tggacatcgg caccaactct gtgggctggg ccgtgatcac 240
cgacgagtac aaggtgccca gcaagaaatt caaggtgctg ggcaacaccg accggcacag 300
catcaagaag aacctgatcg gagccctgct gttcgacagc ggcgaaacag ccgaggccac 360
ccggctgaag agaaccgcca gaagaagata caccagacgg aagaaccgga tctgctatct 420
gcaagagatc ttcagcaacg agatggc 447
<210> 7
<211> 2727
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 7
cggcggccac gaaaaaggcc ggccaggcaa aaaagaaaaa gggcggctcc aagcggcctg 60
ccgcgacgaa gaaagcggga caggccaaga aaaagaaagg atccggcgca acaaacttct 120
ctctgctgaa acaagccgga gatgtcgaag agaatcctgg accggtgagc aagggcgagg 180
agctgttcac cggggtggtg cccatcctgg tcgagctgga cggcgacgta aacggccaca 240
agttcagcgt gtccggcgag ggcgagggcg atgccaccta cggcaagctg accctgaagt 300
tcatctgcac caccggcaag ctgcccgtgc cctggcccac cctcgtgacc accctgacct 360
acggcgtgca gtgcttcagc cgctaccccg accacatgaa gcagcacgac ttcttcaagt 420
ccgccatgcc cgaaggctac gtccaggagc gcaccatctt cttcaaggac gacggcaact 480
acaagacccg cgccgaggtg aagttcgagg gcgacaccct ggtgaaccgc atcgagctga 540
agggcatcga cttcaaggag gacggcaaca tcctggggca caagctggag tacaactaca 600
acagccacaa cgtctatatc atggccgaca agcagaagaa cggcatcaag gtgaacttca 660
agatccgcca caacatcgag gacggcagcg tgcagctcgc cgaccactac cagcagaaca 720
cccccatcgg cgacggcccc gtgctgctgc ccgacaacca ctacctgagc acccagtccg 780
ccctgagcaa agaccccaac gagaagcgcg atcacatggt cctgctggag ttcgtgaccg 840
ccgccgggat cactctcggc atggacgagc tgtacaaggg ctccggcgag ggcaggggaa 900
gtcttctaac atgcggggac gtggaggaaa atcccggccc aaccgagtac aagcccacgg 960
tgcgcctcgc cacccgcgac gacgtcccca gggccgtacg caccctcgcc gccgcgttcg 1020
ccgactaccc cgccacgcgc cacaccgtcg atccggaccg ccacatcgag cgggtcaccg 1080
agctgcaaga actcttcctc acgcgcgtcg ggctcgacat cggcaaggtg tgggtcgcgg 1140
acgacggcgc cgcggtggcg gtctggacca cgccggagag cgtcgaagcg ggggcggtgt 1200
tcgccgagat cggcccgcgc atggccgagt tgagcggttc ccggctggcc gcgcagcaac 1260
agatggaagg cctcctggcg ccgcaccggc ccaaggagcc cgcgtggttc ctggccaccg 1320
tcggagtctc gcccgaccac cagggcaagg gtctgggcag cgccgtcgtg ctccccggag 1380
tggaggcggc cgagcgcgcc ggggtgcccg ccttcctgga gacctccgcg ccccgcaacc 1440
tccccttcta cgagcggctc ggcttcaccg tcaccgccga cgtcgaggtg cccgaaggac 1500
cgcgcacctg gtgcatgacc cgcaagcccg gtgcctgaac gcgttaagtc gacaatcaac 1560
ctctggatta caaaatttgt gaaagattga ctggtattct taactatgtt gctcctttta 1620
cgctatgtgg atacgctgct ttaatgcctt tgtatcatgc tattgcttcc cgtatggctt 1680
tcattttctc ctccttgtat aaatcctggt tgctgtctct ttatgaggag ttgtggcccg 1740
ttgtcaggca acgtggcgtg gtgtgcactg tgtttgctga cgcaaccccc actggttggg 1800
gcattgccac cacctgtcag ctcctttccg ggactttcgc tttccccctc cctattgcca 1860
cggcggaact catcgccgcc tgccttgccc gctgctggac aggggctcgg ctgttgggca 1920
ctgacaattc cgtggtgttg tcggggaaat catcgtcctt tccttggctg ctcgcctgtg 1980
ttgccacctg gattctgcgc gggacgtcct tctgctacgt cccttcggcc ctcaatccag 2040
cggaccttcc ttcccgcggc ctgctgccgg ctctgcggcc tcttccgcgt cttcgccttc 2100
gccctcagac gagtcggatc tccctttggg ccgcctcccc gcgtcgactt taagaccaat 2160
gacttacaag gcagctgtag atcttagcca ctttttaaaa gaaaaggggg gactggaagg 2220
gctaattcac tcccaacgaa gacaagatct gctttttgct tgtactgggt ctctctggtt 2280
agaccagatc tgagcctggg agctctctgg ctaactaggg aacccactgc ttaagcctca 2340
ataaagcttg ccttgagtgc ttcaagtagt gtgtgcccgt ctgttgtgtg actctggtaa 2400
ctagagatcc ctcagaccct tttagtcagt gtggaaaatc tctagcaggg cccgtttaaa 2460
cccgctgatc agcctcgact gtgccttcta gttgccagcc atctgttgtt tgcccctccc 2520
ccgtgccttc cttgaccctg gaaggtgcca ctcccactgt cctttcctaa taaaatgagg 2580
aaattgcatc gcattgtctg agtaggtgtc attctattct ggggggtggg gtggggcagg 2640
acagcaaggg ggaggattgg gaagacaata gcaggcatgc tggggatgcg gtgggctcta 2700
tggcctgcag gggcgcctga tgcggta 2727
<210> 8
<211> 410
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 8
gataaacatg tgagggccta tttcccatga ttccttcata tttgcatata cgatacaagg 60
ctgttagaga gataattgga attaatttga ctgtaaacac aaagatatta gtacaaaata 120
cgtgacgtag aaagtaataa tttcttgggt agtttgcagt tttaaaatta tgttttaaaa 180
tggactatca tatgcttacc gtaacttgaa agtatttcga tttcttggct ttatatatct 240
tgtggaaagg acgaaacacc gggtcttcga gaagacctgt tttagagcta gaaatagcaa 300
gttaaaataa ggctagtccg ttatcaactt gaaaaagtgg caccgagtcg gtgctttttt 360
ctagcgcgtg cgccaattct gcagacaaat ggctctagag gtacccatag 410
<210> 9
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 9
agttatggca gaactcagtg 20
<210> 10
<211> 23
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 10
ccccatccaa agtttttaaa gga 23
<210> 11
<211> 23
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 11
tgtggcagat gtcacagttt agg 23
<210> 12
<211> 25
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 12
caccgagtta tggcagaact cagtg 25
<210> 13
<211> 25
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 13
aaaccactga gttctgccat aactc 25
<210> 14
<211> 1150
<212> DNA
<213> 猪(Sus scrofa)
<400> 14
ccctgtcata actgagtcag cctaggacct cttctggtct cctgtgacat cccattgcac 60
ctgcagctgt taggactgta cacgcctggc ccctggacgt gggcttagtt cacgttcctc 120
cccaaccagt cagcacagcg cccgcgggca tctgagaagt cagggctcca cagtggtggt 180
tgcacaggtg agcgagtgac aggatgagcg gctgagggac tgttcaggcc tcgcagctgg 240
ccggcatcag ccagctttgc tactgtggga aggggaacag cagtccccgg ccagccgtcc 300
aggtgtcacc catccatggc agtggggcag gctctactcc tgggacctcc atacccctcg 360
ggggtcagaa actgtgctgg agacgtccct ggagcctggg caacttgggg ttgaatcgtg 420
gtcactgctg gcccaccacc tccatctcgc tcccgaagac atgccttgac cctttgtcct 480
cttctccccc ttgggtacag atcctggcca acgtcttcct ctacctgtgt gccatcgtcg 540
tgggcatcat gtcctactac atggcggacc gcaagcaccg caaagccttc ctggaggccc 600
gccagtcgct ggaggtgaag atgaacctgg aggagcagag ccagcagcag gtgaggctct 660
ttgggggtgg cctgggggac aatgccagcc cggggtccag gcgcaaggcc tgttggaagg 720
agtgagccca ggtgctgcgg gggccccatg gggctcggag ttctcattct ttttctccgg 780
aggcatccct ctgtgttatc tttcgctggc tccctctgcc actcctggct gtgcaagggc 840
acatgctcac tgcgcatcta ctgggtgctc gcttggggct gctctccctg cggcagtcag 900
gggtcagcac ccacatcccc tggtctcgga ggtccttctg ggagaagtct gtccgcctct 960
cagcctcctc catgggtgct gttccctttg ccccgagggc actctcccca ccgccttctc 1020
ctgcgctgtc atttatgaag atgcaaagcc agcccccacc ccagggcccc cactctttgc 1080
ctcttccatc gaggggaggg aggcaggcgc ctgctgtccc tgctctttgt gtcctggccc 1140
tcctccagcc 1150
<210> 15
<211> 1145
<212> PRT
<213> 猪(Sus scrofa)
<400> 15
Met Pro Arg Thr Gln Gly Phe Ser Asp Pro Glu Tyr Ser Ala Glu Tyr
1 5 10 15
Ser Ala Glu Tyr Ser Val Ser Leu Pro Ser Asp Pro Glu Arg Gly Val
20 25 30
Gly Arg Thr His Glu Ile Ser Val Arg Asn Ser Gly Ser Cys Leu Cys
35 40 45
Leu Pro Arg Phe Met Arg Leu Thr Phe Val Pro Glu Ser Leu Glu Asn
50 55 60
Leu Tyr Gln Thr Tyr Phe Lys Arg Gln Arg His Glu Thr Leu Leu Val
65 70 75 80
Leu Val Val Phe Ala Ala Leu Phe Asp Cys Tyr Val Val Val Met Cys
85 90 95
Ala Val Val Phe Ser Ser Asp Lys Leu Ala Pro Leu Ala Val Ala Gly
100 105 110
Val Gly Leu Val Leu Asp Leu Ile Leu Phe Val Leu Cys Arg Lys Gly
115 120 125
Leu Leu Pro Ser Arg Val Thr Arg Lys Gly Val Pro Tyr Leu Leu Trp
130 135 140
Leu Leu Ile Thr Ala Gln Val Leu Ser Tyr Leu Gly Leu Asn Phe Ser
145 150 155 160
Gly Ala His Ala Ala Ser Asp Thr Val Gly Trp Gln Ala Phe Phe Val
165 170 175
Phe Ser Phe Phe Ile Thr Leu Pro Leu Ser Leu Ser Pro Ile Val Leu
180 185 190
Ile Ser Val Leu Ser Cys Val Val His Thr Leu Val Leu Gly Val Thr
195 200 205
Val Ala Gln Gln Gln Gln Asp Gly Leu Arg Gly Met Gln Leu Leu Arg
210 215 220
Glu Ile Leu Ala Asn Val Phe Leu Tyr Leu Cys Ala Ile Val Val Gly
225 230 235 240
Ile Met Ser Tyr Tyr Met Ala Asp Arg Lys His Arg Lys Ala Phe Leu
245 250 255
Glu Ala Arg Gln Ser Leu Glu Val Lys Met Asn Leu Glu Glu Gln Ser
260 265 270
Gln Gln Gln Glu Asn Leu Met Leu Ser Ile Leu Pro Lys His Val Ala
275 280 285
Asp Glu Met Leu Lys Asp Met Lys Lys Asp Glu Ser Gln Lys Asp Gln
290 295 300
Gln Gln Phe Asn Thr Met Tyr Met Tyr Arg His Glu Asn Val Ser Ile
305 310 315 320
Leu Phe Ala Asp Ile Val Gly Phe Thr Gln Leu Ser Ser Ala Cys Ser
325 330 335
Ala Gln Glu Leu Val Lys Leu Leu Asn Glu Leu Phe Ala Arg Phe Asp
340 345 350
Lys Leu Ala Ala Lys Tyr His Gln Leu Arg Ile Lys Ile Leu Gly Asp
355 360 365
Cys Tyr Tyr Cys Ile Cys Gly Leu Pro Asp Tyr Arg Glu Asp His Ala
370 375 380
Val Cys Ser Ile Leu Met Gly Leu Ala Met Val Glu Ala Ile Ser Tyr
385 390 395 400
Val Arg Glu Lys Thr Lys Thr Gly Val Asp Met Arg Val Gly Val His
405 410 415
Thr Gly Thr Val Leu Gly Gly Val Leu Gly Gln Lys Arg Trp Gln Tyr
420 425 430
Asp Val Trp Ser Thr Asp Val Thr Val Ala Asn Lys Met Glu Ala Gly
435 440 445
Gly Ile Pro Gly Arg Val His Ile Ser Gln Ser Thr Met Asp Cys Leu
450 455 460
Lys Gly Glu Phe Asp Val Glu Pro Gly Asp Gly Gly Ser Arg Cys Asp
465 470 475 480
Tyr Leu Asp Glu Lys Gly Ile Glu Thr Tyr Leu Ile Ile Ala Ser Arg
485 490 495
Pro Glu Val Lys Lys Thr Ala Ala Gln Asn Gly Leu Asn Gly Ser Ala
500 505 510
Leu Pro Asn Gly Ala Leu Pro Ser Ser Lys Pro Ser Ser Pro Ala Leu
515 520 525
Ile Glu Thr Lys Glu Pro Asn Gly Ser Val His Thr Ser Gly Ser Thr
530 535 540
Ser Glu Glu Ala Glu Glu Gln Asp Ala Gln Ala Asp Asn Pro Ser Phe
545 550 555 560
Pro Asn Pro Arg Arg Arg Leu Arg Leu Gln Asp Leu Ala Asp Arg Val
565 570 575
Val Asp Ala Ser Glu Asp Glu His Glu Leu Asn Gln Leu Leu Asn Glu
580 585 590
Ala Leu Leu Glu Arg Glu Ser Ala Gln Val Val Lys Lys Arg Asn Thr
595 600 605
Phe Leu Leu Ser Met Arg Phe Met Asp Pro Glu Met Glu Thr Arg Tyr
610 615 620
Ser Val Glu Lys Glu Lys Gln Ser Gly Ala Ala Phe Ser Cys Ser Cys
625 630 635 640
Val Val Leu Leu Cys Thr Ala Leu Val Glu Ala Leu Ile Asp Pro Trp
645 650 655
Leu Met Thr Asn Tyr Val Thr Phe Val Val Gly Glu Val Leu Leu Leu
660 665 670
Ile Leu Thr Ile Cys Ser Leu Ala Ala Ile Phe Pro Arg Ala Phe Pro
675 680 685
Lys Lys Leu Val Ala Phe Ser Thr Trp Ile Asp Arg Thr Arg Trp Ala
690 695 700
Arg Asn Thr Trp Ala Met Leu Ala Ile Phe Val Leu Val Met Ala Asn
705 710 715 720
Val Val Asp Met Leu Ser Cys Leu Gln Ser Asp Ala Gly Pro Gly Asn
725 730 735
Ser Thr Ala Gly Ala Arg Leu Glu Asp Gly Cys Val Glu Asn Pro Lys
740 745 750
Tyr Tyr Ser Tyr Val Ala Val Leu Ser Leu Ile Ala Thr Ile Met Leu
755 760 765
Val Gln Val Ser His Met Val Lys Leu Thr Leu Met Leu Leu Ile Ala
770 775 780
Gly Ala Val Gly Thr Ile Asn Ile Tyr Ala Trp Arg Pro Ile Phe Asp
785 790 795 800
Glu Tyr Asp Arg Arg Arg Phe Gln Glu His Asp Phe Pro Met Val Ala
805 810 815
Leu Glu Lys Met Gln Val Phe Ser Ser Pro Gly Leu Asn Gly Thr Asp
820 825 830
Arg Pro Pro Leu Val Pro Ser Lys Tyr Ser Met Thr Ala Met Val Phe
835 840 845
Val Met Met Leu Ser Phe Tyr Tyr Phe Ser Arg His Val Glu Lys Leu
850 855 860
Ala Arg Thr Leu Phe Leu Trp Lys Ile Glu Val His Asp Gln Lys Glu
865 870 875 880
Arg Val Tyr Glu Met Arg Arg Trp Asn Glu Ala Leu Val Thr Asn Met
885 890 895
Leu Pro Glu His Val Ala Arg His Phe Leu Gly Ser Lys Lys Arg Asp
900 905 910
Glu Glu Leu Tyr Ser Gln Ser Tyr Asp Glu Ile Gly Val Met Phe Ala
915 920 925
Ser Leu Pro Asn Phe Ala Asp Phe Tyr Thr Glu Glu Ser Ile Asn Asn
930 935 940
Gly Gly Ile Glu Cys Leu Arg Phe Leu Asn Glu Ile Ile Ser Asp Phe
945 950 955 960
Asp Ser Leu Leu Asp Asn Pro Lys Phe Arg Val Ile Thr Lys Ile Lys
965 970 975
Thr Ile Gly Ser Thr Tyr Met Ala Ala Ser Gly Val Thr Pro Asp Val
980 985 990
Asn Thr Asn Gly Phe Thr Ser Ser Ala Lys Glu Glu Lys Ser Asp Arg
995 1000 1005
Glu Arg Trp Gln His Leu Ala Asp Leu Ala Asp Phe Ala Leu Ala Met
1010 1015 1020
Lys Asp Thr Leu Thr Asn Ile Asn Asn Gln Ser Phe Asn Asn Phe Met
1025 1030 1035 1040
Leu Arg Ile Gly Met Asn Lys Gly Gly Val Leu Ala Gly Val Ile Gly
1045 1050 1055
Ala Arg Lys Pro His Tyr Asp Ile Trp Gly Asn Thr Val Asn Val Ala
1060 1065 1070
Ser Arg Met Glu Ser Thr Gly Val Met Gly Asn Ile Gln Val Val Glu
1075 1080 1085
Glu Thr Gln Leu Ile Leu Arg Gln Tyr Ala Ser Arg Cys Val Arg Arg
1090 1095 1100
Gly Pro Ile Phe Val Lys Gly Lys Gly Glu Leu Leu Thr Phe Phe Leu
1105 1110 1115 1120
Lys Gly Arg Asp Lys Pro Ala Ala Phe Pro Asn Gly Ala Ser Val Thr
1125 1130 1135
Leu Pro His Gln Val Val Asp Ser Ser
1140 1145
<210> 16
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 16
ttggggttga atcgtggtca 20
<210> 17
<211> 21
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 17
agggagccag cgaaagataa c 21
<210> 18
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 18
ggaggtgaag atgaacctgg 20
<210> 19
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 19
gcaccgcaaa gccttcctgg 20
<210> 20
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 20
gggcctccag gaaggctttg 20
<210> 21
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 21
ggaaggcttt gcggtgcttg 20
<210> 22
<211> 100
<212> RNA
<213> 人工序列(Artificial Sequence)
<400> 22
ggaggugaag augaaccugg guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60
cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100
<210> 23
<211> 100
<212> RNA
<213> 人工序列(Artificial Sequence)
<400> 23
gcaccgcaaa gccuuccugg guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60
cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100
<210> 24
<211> 100
<212> RNA
<213> 人工序列(Artificial Sequence)
<400> 24
gggccuccag gaaggcuuug guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60
cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100
<210> 25
<211> 100
<212> RNA
<213> 人工序列(Artificial Sequence)
<400> 25
ggaaggcuuu gcggugcuug guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60
cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100
<210> 26
<211> 24
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 26
caccggaggt gaagatgaac ctgg 24
<210> 27
<211> 24
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 27
aaacccaggt tcatcttcac ctcc 24
<210> 28
<211> 24
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 28
caccgcaccg caaagccttc ctgg 24
<210> 29
<211> 24
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 29
aaacccagga aggctttgcg gtgc 24
<210> 30
<211> 24
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 30
caccgggcct ccaggaaggc tttg 24
<210> 31
<211> 24
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 31
aaaccaaagc cttcctggag gccc 24
<210> 32
<211> 24
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 32
caccggaagg ctttgcggtg cttg 24
<210> 33
<211> 24
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 33
aaaccaagca ccgcaaagcc ttcc 24
Claims (4)
1.一种用于猪ADCY3基因编辑的CRISPR/Cas9系统,其特征在于包含Cas9表达载体和针对猪ADCY3基因的gRNA表达载体;所述的Cas9表达载体为质粒全序列如SEQ ID NO.2所示的pU6gRNA-eEF1a-mNLS-hSpCas9-EGFP-PURO载体;所述的针对猪ADCY3基因的gRNA表达载体由SEQ ID NO.26和SEQ ID NO.27所示的单链DNA退火而成的双链插入载体骨架pKG-U6gRNA的限制性内切酶BbsI位点所得;该表达载体表达SEQ ID NO.22所示的gRNA,其靶点如SEQID NO.18所示;载体骨架为pKG-U6gRNA质粒全序列如SEQ ID NO.3所示;所述的gRNA表达载体和Cas9表达载体的摩尔比为3:1。
2.权利要求1所述的CRISPR/Cas9系统在构建猪ADCY3基因敲除的猪重组细胞中的应用。
3.一种敲除猪ADCY3基因的猪重组细胞,其特征在于由权利要求1所述的CRISPR/Cas9系统共转染猪原代成纤维细胞经验证后所得。
4.权利要求3所述的重组细胞在构建ADCY3基因敲除的克隆猪中的应用。
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