CN111996215B - 一种全身性Plin1基因敲除动物模型构建及鉴定方法 - Google Patents
一种全身性Plin1基因敲除动物模型构建及鉴定方法 Download PDFInfo
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Abstract
本发明属生物技术领域，提供一种全身性Plin1基因敲除动物模型构建及其鉴定方法。构建方法步骤为：针对Plin1基因2号外显子前后序列设计sgRNA序列，构建Plin1基因敲除载体并进行体外转录获得sgRNA；将sgRNA与Cas9蛋白混合后显微注射入小鼠受精卵中，获得全身性Plin1基因敲除小鼠。本项目的有益效果在于筛选出了一对评分最高的sgRNA序列，保证了项目的成功率；实现了Plin1基因2号外显子的大片段敲除，提高了敲除效率；针对敲除区域设计3条引物，成功鉴定出了小鼠的各类基因型，避免了大片段敲除时2条引物可能无法区分基因型的情况。为研究PLIN1的生物学功能及肥胖相关代谢性疾病的发病机制提供便捷、可靠、经济的动物模型和良好的基础。
Description
技术领域
本发明属于生物技术领域，具体涉及一种全身性Plin1基因敲除动物模型构建及其鉴定方法。
背景技术
随着经济的快速发展和人民生活水平的提高，肥胖已经成为日益严重的全球性健康问题。近年数十年来，全球超重和肥胖的发病率增长明显，并在发达国家和发展中国家中均呈快速蔓延之势。普遍认为，近年来生活环境的改变与肥胖症的发生有着直接的关系。不健康的饮食习惯，久坐的生活方式，缺乏运动，睡眠习惯以及文化和人口背景的变化均可能诱发肥胖。但在一项国外最新的研究中发现，遗传因素在生活环境的改变下可能在肥胖的发生中占据着主要地位。随着人类全基因计划和全基因组关联研究的日渐深入，发现了数十种与肥胖发生相关的基因，针对这些基因开展系列研究有助于全面理解肥胖的发病机制并可能为肥胖防治的提供潜在靶点。
Plin1基因位于人类15号染色体的q26区域，其DNA长度大于15 kb，包含9个外显子和8个内含子，其多个单核酸多态性位点会增加肥胖的发生风险，被认为是肥胖相关的候选基因。在哺乳动物中，Plin1基因在白色脂肪组织中高度表达，单拷贝编码完全定位于脂滴表面的围脂滴蛋白（perilipin1, PLIN1），对脂滴的生物合成具有十分重要的作用。PLIN1可以增强甘油三酯（triglyceride, TG）的合成并促进单房脂滴（直径≥10um）的形成，进而提高脂肪组织储存脂肪的能力。同时，PLIN1还可以通过与甘油三酯脂肪酶（adiposetriglyceride Lipase, ATGL）和激素敏感性脂肪酶（hormone-sensitive lipase, HSL）相互作用，参与脂解的调节。最近还有研究表明，PLIN1可能也参与了脂肪组织中的炎症反应。目前，对于PLIN1的生物学功能虽尚未完全阐明，但已经发现其基因表达量下调与2型糖尿病、高血压、脂肪肝等多种代谢性疾病的发生密切相关，进一步研究Plin1基因的功能与作用对于阐明这些疾病的发病机制具有重要意义。
基因编辑技术是一种对靶基因进行定点改造的技术，在基因结构与功能研究、细胞的基因工程改造，动植物的新品种培育及疾病的基因治疗方面被广泛应用。近年来，得益于转基因技术的发展和疾病模型构建的需要，转基因动物成为基因编辑研究领域的一大热点。通过运用各种方法将外源基因导入动物的基因组内，外源基因片段即可实现对动物细胞染色体的的修饰，达到在转基因动物体内稳定遗传的目的。转基因动物模型的构建打破了自然界基因组序列的固有格局，使得人们可以更加深入的对基因的结构和功能进行研究，为基因及遗传信息的研究创造了更加广阔的平台。
CRISPR/Cas9技术是近年来新出现的一种基因编辑技术，与锌指核酸酶(ZFN)技术和类转录样效应因子核酸酶(TALEN)技术相比，因其操作简单、高效率和低成本的优势在基因编辑领域被广泛应用。
CRISPR/Cas9***元件包括Cas9蛋白和sgRNA，Cas9蛋白上具有两种核酸内切酶的活性，它能够与目的序列的引导序列sgRNA相结合，并被sgRNA引导到靶点位置进行切割，造成双链断裂（DSB），在DNA损伤修复机制的作用下引入了突变，实现基因敲除、修复或***（图1）。自CRISPR/Cas9技术诞生以来，相关研究人员已对其进行了多次改进，形成了现在仅需sgRNA和Cas9蛋白共同作用即可实现对目的序列的编辑的工程化***。该***中的sgRNA和Cas9蛋白可以通过不同载体作用于目的基因，无论是质粒、慢病毒或者直接转染sgRNA与Cas9蛋白的混合物，都可以很方便的实现基因编辑。
中国专利文献CN107043787A公开了一种基于CRISPR/Cas9获得MARF1定点突变小鼠模型的构建方法。中国专利文献CN104293831A公开了一种基于CRISPR/Cas9基因敲除技术建立高血压小鼠模型的方法。中国专利文献CN105950639A公开了一种金黄色葡萄球菌CRISPR/Cas9***的制备方法及其在构建基因修饰小鼠模型中的应用。中国专利文献CN106172238A公开了一种利用CRISPR/Cas9基因敲除技术建立miR-124基因敲除小鼠动物模型的方法和其应用。中国专利文献CN107475300 A公开了一种利用CRISPR/Cas9技术建立Ifit3-eKO1基因敲除小鼠动物模型的方法和应用。但关于全身性Plin1基因敲除小鼠动物模型的构建及鉴定方法目前还未见报道。
发明内容
本发明的目的是针对现有技术中的不足，提供了一种全身性Plin1基因敲除小鼠动物模型的构建及鉴定方法。
本发明的第二个目的是，提供了一对基于CRISPR/Cas9技术靶向Plin1基因2号外显子的sgRNA序列。
本发明的第三个目的是，提供上述sgRNA的应用。
为实现上述目的，本发明采取的技术方案是：一种全身性Plin1基因敲除小鼠动物模型的构建方法，所述方法基于CRISPR/Cas9基因敲除技术，包括以下步骤：
（1）针对PLIN1基因2号外显子前后序列设计1对sgRNA序列，通过构建PLIN1基因敲除载体及体外转录获得sgRNA；
（2）将sgRNA与Cas9蛋白混合组成的Cas9 sgRNA体系的小鼠受精卵显微注射，F0代小鼠出生及鉴定，F0代小鼠性成熟配繁，F1代小鼠鉴定，F1代阳性小鼠雌雄近交获得F2代小鼠，即为全身性Plin1基因敲除小鼠。
进一步地，sgRNA核苷酸序列如SEQ ID NO:1， SEQ ID NO:2所示。
所述sgRNA的筛选及获得方法为：
（1）针对小鼠Plin1基因2号外显子序列，在NCBI中查询小鼠Plin1基因的基本信息，应用在线工具http://crispr.mit.edu设计识别Plin1基因2号外显子前后的sgRNA序列对；
（2）根据脱靶位点、基因数和发生错配的可能性大小进行评估筛选，选择1对sgRNA序列，其核苷酸序列如SEQ ID NO:1，SEQ ID NO:2所示；
（3）选择CRISPR基因敲除质粒pX330作为载体，用限制性内切酶BBS切割pX330质粒BBS酶切位点使其线性化；
（4）向2条Plin1 sgRNA的5’端添加粘性末端后与线性化载体用T4 DNA连接酶进行连接，在pX330 gRNA scafflod位置加入sgRNA序列构建Plin1基因CRISPR/Cas9敲除载体Plin1-1，Plin1-2；构建好的质粒载体转化至DH5α菌群中，2 d后挑取单克隆菌落测序及限制性核酸内切酶ApaLI+NcoI双酶切法鉴定，载体序列信息如SEQ ID NO:3,4所示；
（5）按照sgRNA体外转录试剂盒即PC1380说明书设计正向引物SG1-F、SG2-F和通用下游引物SG-R，序列如SEQ ID NO:5,6,7所示，PCR扩增转录模板后进行sgRNA转录，转录完全并纯化后进行琼脂糖凝胶电泳实验检测sgRNA片段大小及完整性。
鉴定Plin1基因敲除小鼠动物模型的方法为：
A、待F0代小鼠出生三周后，提取小鼠尾部DNA，利用引物F，R进行PCR扩增琼脂糖凝胶电泳并测序鉴定基因型，引物序列如SEQ ID NO:8,9所示；
B、选取F0代阳性小鼠与野生型异性小鼠交配获的F1代小鼠，利用引物F及Wt/He-R进行PCR扩增琼脂糖凝胶电泳测序鉴定基因型，引物序列如SEQ ID NO:8,10所示；
C、选取F1代阳性小鼠雌雄近交获得F2代小鼠，按照F1代小鼠基因型鉴定方法获得纯合型小鼠，即为小鼠动物模型。
所述的sgRNA在建立基因缺陷小鼠中的应用。
本发明的有益效果体现在：本发明针对Plin1基因2号外显子的前后序列，通过脱靶位点、基因数和发生错配的可能性大小进行评估，筛选出了一对评分最高的sgRNA序列，保证了项目的成功率；同时，通过上述sgRNA作为打靶载体，实现了Plin1基因2号外显子的大片段敲除，很大程度上提高了敲除效率。另外，我们针对敲除区域设计了3条引物，成功鉴定出了小鼠的各类基因型，避免了大片段敲除时2条引物可能无法区分基因型的情况。本发明使用CRISPR/Cas9基因敲除技术，首次实现了小鼠Plin1基因2号外显子的大片段敲除，获得了全身性Plin1基因敲除的小鼠动物模型，项目的成功实施为研究PLIN1的生物学功能以及肥胖相关代谢性疾病的发病机制提供了便捷、可靠、经济的动物模型。
本发明使用CRISPR/Cas9基因敲除技术，首次实现了小鼠Plin1基因2号外显子的大片段敲除，并通过多条引物进行基因型鉴定，获得了全身性PLIN1基因敲除的小鼠动物模型。项目的成功实施为研究PLIN1的生物学功能及肥胖相关代谢性疾病的发病机制提供便捷、可靠、经济的动物模型和良好的基础。
附图说明
图1为基于CRISPR/Cas9技术建立全身性Plin1基因敲除小鼠模型的技术路线图；
图2为基于CRISPR/Cas9技术敲除Plin1基因2号外显子的策略示意图；
图3为pX330质粒载体图谱；
图4为Plin1基因敲除载体测序结果及酶切鉴定电泳图；图中：上图为载体测序结果图；下图为酶切鉴定电泳图：M：DNA marker；1：Plin1--1载体酶切片段；2：Plin1--1载体；3：Plin1--2载体酶切片段；4：Plin1--2载体；
图5为sgRNA体外转录的电泳图；图中：M：DNA marker；1-3：sgRNA- Plin1--1；4-6：sgRNA-Plin1-2；
图6为基于Plin1基因2号外显子敲除小鼠的鉴定策略图；
图7为F0代小鼠PCR鉴定的电泳图；图中：Water：空白对照；1-23：小鼠编号；WT：野生型小鼠；M：DNAmarker；
图8为F0代8号嵌合体前后测序结果比对及PCR鉴定的电泳图；图中：左图F0代8号嵌合体为Plin1基因PCR鉴定的电泳图；右图为F0代8号嵌合体Plin1基因前后测序结果比对；
图9为F1代阳性基因敲除小鼠的前后测序结果比对和电泳图；图中：上图为1,2,4,8,12,13,15号小鼠Plin1基因PCR鉴定的电泳图，下图为1,2,4,8,12,13,15号Plin1基因前后测序结果比对；
图10为F2代基因敲除小鼠的电泳图；图中：上图为利用引物F，R PCR鉴定1-17号小鼠基因型的电泳图，下图为利用引物F，Wt/He-R PCR鉴定1-17号小鼠基因型的电泳图，图中：白色箭头指示为纯合型小鼠基因型。
具体实施方式
下面对本发明的具体实施方式做出进一步的说明。以便于本领域技术人员理解本发明，但应该清楚，本发明不限于具体实施方式的范围，对本领域技术人员来说，只要各种变化在权利要求限定和确定的本发明的精神和范围内，这些变化是显而易见的，一切利用本发明构思的发明创造均在保护之列。
除另外定义，本文所用的所有科技术语一般为本发明所属领域普通技术人员所通常理解的含义。本文所用的命名和实验方法是公知的，并且在本领域中常规使用。使用标准技术进行的所有操作通常是根据仪器耗材生产商的产品说明书和常规技术要求以及本文中提供的参考资料进行的。
基于CRISPR/Cas9基因敲除技术建立全身性Plin1基因敲除小鼠模型的方法，其技术路线如图1所示，具体方法为：
一、在NCBI中查询小鼠Plin1基因的基本信息
（1）敲除基因名称（Ensembl/MGI/GenBank号）：Plin1（ENSMUSG00000030546/1890505/NM_001113471.1）。
（2）敲除基因网址链接（NCBI）：https://www.ncbi.nlm.nih.gov/gene/103968；
（3）敲除针对的exon：exon 2。
二．基于CRISPR/Cas9技术敲除Plin1基因的策略示意图，如图2所示，实现Plin1基因2号外显子的大片段敲除。
三．确认基因敲除位点上下游序列信息
应用在线工具(http://crispr.mit.edu)设计2对可以识别Plin1基因2号外显子前后序列的sgRNA序列，并根据脱靶位点、基因数和发生错配的可能性大小进行分析，选择一对评分最高的sgRNA并互补配对相应的反义链后合成，sgRNA序列如SEQ ID NO:1，SEQ IDNO:2所示。
四．构建sgRNA表达载体
选择CRISPR质粒pX330（图3）作为骨架载体。利用限制性内切酶BBS切割pX330质粒BBS酶切位点（第245、267位置）使其线性化，37℃孵育30 min后，1%的琼脂糖凝胶电泳，利用回收试剂盒回收线性化载体；向2条Plin1基因sgRNA的5’端添加粘性末端调整浓度至100 μM，在含有T4 PNK的体系中37℃孵育30 min，再经95℃孵育5 min后缓慢降至25℃，使sgRNA退火磷酸化；将退火磷酸化的sgRNA稀释至1 μM后与回收的线性化载体用T4 DNA连接酶进行连接，37℃孵育1 h后16℃过夜，构建Plin1基因CRISPR/Cas9敲除载体Plin1-1、Plin-2，载体信息如SEQ ID NO:3,4；
pX330载体线性化反应体系
sgRNA与pX330载体连接反应体系
五、构建好的sgRNA载体转化至DH5α大肠杆菌菌群中，常规培养2 d后挑取单克隆菌落提取质粒DNA测序并通过限制性核酸内切酶ApaLI+NcoI双酶切法鉴定载体，测序及酶切结果如图4所示，成功的在pX330 gRNA scafflod位置加入sgRNA序列，并获得5个预期的大小的片段（497、527、959、1246和5195 bp）。
六、sgRNA体外转录
根据sgRNA序列设计体外转录模板的正向引物SG1-F、SG2-F和通用下游引物SG-R，序列如SEQ ID NO:5,6,7所示，PCR扩增sgRNA转录模板及回收后，按照体外转录试剂盒PC1380（购自苏州泓迅生物科技股份有限公司）说明书进行sgRNA转录，转录完全并纯化后进行2%琼脂糖凝胶电泳实验检测sgRNA片段大小及完整性；如图5所示，sgRNA体外转录产物片段完整，大小介于100-200 bp之间，与预期结果相同。
七、超数***与受精卵的获取
8周龄左右C57BL/6J雌性小鼠腹腔注射5-10 UI 孕马血清***，46-48小时后注射5-10 UI 人绒毛膜***促使小鼠超数***，与雄性小鼠合笼交配，次日挑取见栓雌鼠，断头处死后用75%乙醇消毒腹部，打开腹腔后剪取双侧输卵管，移入预热至37℃，含有3%透明质酸酶的M2培养基中，在倒置显微镜下用精细镊子刺破输卵管膨大的壶腹部使受精卵流出，静置消化1-3分钟使受精卵散开后，再用预热的M2培养基清洗3次，将获取的受精卵每30-50枚移入另一含有新鲜M2培养基液滴的培养皿中并用矿物油覆盖，置于显微操作***的载物台上，共获得113枚受精卵。
八、结扎雄鼠与假孕母鼠的构建
8周龄左右C57BL/6J雄性小鼠腹腔注射0.15 ml 5%水合氯醛，待小鼠完全麻醉后，75%乙醇消毒小鼠两后肢中间位置，用眼科剪横切一个1 cm左右的开口，用钝的镊子夹住小鼠睾丸周围的脂肪垫将输精管、附睾、睾丸等拉出，轻轻提起输精管，用剪刀剪断（剪去一节），利用同样的操作切除另一侧的输精管。完成将睾丸等塞回小鼠体内并复位，缝合小鼠并将其转移至新的鼠笼中标号。待小鼠苏醒后，正常喂养2周使小鼠完全恢复，获得结扎雄鼠。将结扎雄鼠与正常8周龄雌鼠合笼，次日挑取见栓雌鼠为假孕母鼠。
九、受精卵的显微注射与胚胎移植
将步骤六中获得sgRNA与购买的Cas9蛋白混合并调整至适宜浓度，通过显微注射针注入1-2 pl至受精***中，所有受精卵注射完毕后，在倒置显微镜下挑取注射后存活的受精卵，移入预热至37℃的M16培养基中，置于细胞培养箱37℃、5% CO2条件下培养2 h，共有63枚受精卵存活。5%水合氯醛麻醉步骤八中获得的假孕母鼠，75%乙醇消毒后背部后沿着背中线从最后肋骨开始剪开一个1-2 cm的小口，用镊子找到脂肪垫后缓慢拉出卵巢、输卵管及子宫，在体式显微镜下用精细镊子撕开包被卵巢与输卵管的薄壁，暴露输卵管伞端，将培养后的受精卵用移卵针通过输卵管伞端吹入输卵管中，对侧输卵管相同操作后复位小鼠卵巢及脂肪垫等组织并缝合开口，完成胚胎移植，共移植4只假孕雌鼠。待假孕雌鼠苏醒，19-21 d后分娩的小鼠即为F0代小鼠。
十．基于敲除Plin1基因2号外显子小鼠基因型鉴定的策略示意图，如图6所示，设计了F，R和Wt/He-R 3条引物分别用于鉴定突变型和野生型小鼠。引物序列如SEQ ID NO：8,9,10所示。
十一．待F0代小鼠出生三周后，提取小鼠尾部DNA，利用引物F，R进行PCR扩增琼脂糖凝胶电泳鉴定基因型，电泳结果如图7所示，共出生24只小鼠，其中12只为阳性小鼠（4♂8♀）。敲除片段大小约为700 bp左右。
十二．选取F0代8号进行电泳及测序鉴定，结果如图8所示，敲除了包含Plin1基因2号外显子序列在内的741bp；将F0代8号和11号雄性小鼠分别与野生型雌性小鼠交配获得F1代小鼠，提取小鼠尾部DNA，利用引物F，R和Wt/He-R进行PCR扩增琼脂糖凝胶电泳并及测序鉴定基因型，电泳及测序结果如图9所示；11号小鼠后代1,2,4号小鼠（均为♂）与8号小鼠后代8号（♂）,12,13,15号（均为♀）小鼠均为F1阳性杂合型小鼠；11号小鼠F1代Plin1基因敲除片段大小与8号小鼠及其F1代略有不同，缺失了747 bp。
十三、选取F1阳性杂合型小鼠8号与12,13,15号小鼠雌雄近交，待小鼠出生后即为F2代小鼠，提取小鼠尾部DNA，利用引物F，R和Wt/He-RPCR扩增琼脂糖凝胶电泳鉴定基因型，电泳结果如图10所示，共出生17只小鼠，其中12,13号（1♂1♀）均为纯合型小鼠；11,17号（2♀）均为野生型小鼠；1-10,14-16号（6♂7♀）小鼠均为杂合型小鼠，基本符合孟德尔遗传定律。
PCR反应体系
PCR反应条件
十四．F2代中基因型纯合型小鼠即为小鼠动物模型。
序列表
<110> 山西医科大学
<120> 一种全身性Plin1基因敲除动物模型构建及鉴定方法
<160> 10
<170> SIPOSequenceListing 1.0
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ttttgctcac atgtgagggc ctatttccca tgattccttc atatttgcat atacgataca 60
aggctgttag agagataatt ggaattaatt tgactgtaaa cacaaagata ttagtacaaa 120
atacgtgacg tagaaagtaa taatttcttg ggtagtttgc agttttaaaa ttatgtttta 180
aaatggacta tcatatgctt accgtaactt gaaagtattt cgatttcttg gctttatata 240
tcttgtggaa aggacgaaac accgttggag ggcgaatgtt acatgtttta gagctagaaa 300
tagcaagtta aaataaggct agtccgttat caacttgaaa aagtggcacc gagtcggtgc 360
tttttttcta gacgttacat aacttacggt aaatggcccg cctggctgac cgcccaacga 420
cccccgccca ttgacgtcaa tagtaacgcc aatagggact ttccattgac gtcaatgggt 480
ggagtattta cggtaaactg cccacttggc agtacatcaa gtgtatcata tgccaagtac 540
gccccctatt gacgtcaatg acggtaaatg gcccgcctgg cattgtgccc agtacatgac 600
cttatgggac tttcctactt ggcagtacat ctacgtatta gtcatcgcta ttaccatggt 660
cgaggtgagc cccacgttct gcttcactct ccccatctcc cccccctccc cacccccaat 720
tttgtattta tttatttttt aattattttg tgcagcgatg ggggcggggg gggggggggg 780
gcgcgcgcca ggcggggcgg ggcggggcga ggggcggggc ggggcgaggc ggagaggtgc 840
ggcggcagcc aatcagagcg gcgcgctccg aaagtttcct tttatggcga ggcggcggcg 900
gcggcggccc tataaaaagc gaagcgcgcg gcgggcggga gtcgctgcgc gctgccttcg 960
ccccgtgccc cgctccgccg ccgcctcgcg ccgcccgccc cggctctgac tgaccgcgtt 1020
actcccacag gtgagcgggc gggacggccc ttctcctccg ggctgtaatt agctgagcaa 1080
gaggtaaggg tttaagggat ggttggttgg tggggtatta atgtttaatt acctggagca 1140
cctgcctgaa atcacttttt ttcaggttgg accggtgcca ccatggacta taaggaccac 1200
gacggagact acaaggatca tgatattgat tacaaagacg atgacgataa gatggcccca 1260
aagaagaagc ggaaggtcgg tatccacgga gtcccagcag ccgacaagaa gtacagcatc 1320
ggcctggaca tcggcaccaa ctctgtgggc tgggccgtga tcaccgacga gtacaaggtg 1380
cccagcaaga aattcaaggt gctgggcaac accgaccggc acagcatcaa gaagaacctg 1440
atcggagccc tgctgttcga cagcggcgaa acagccgagg ccacccggct gaagagaacc 1500
gccagaagaa gatacaccag acggaagaac cggatctgct atctgcaaga gatcttcagc 1560
aacgagatgg ccaaggtgga cgacagcttc ttccacagac tggaagagtc cttcctggtg 1620
gaagaggata agaagcacga gcggcacccc atcttcggca acatcgtgga cgaggtggcc 1680
taccacgaga agtaccccac catctaccac ctgagaaaga aactggtgga cagcaccgac 1740
aaggccgacc tgcggctgat ctatctggcc ctggcccaca tgatcaagtt ccggggccac 1800
ttcctgatcg agggcgacct gaaccccgac aacagcgacg tggacaagct gttcatccag 1860
ctggtgcaga cctacaacca gctgttcgag gaaaacccca tcaacgccag cggcgtggac 1920
gccaaggcca tcctgtctgc cagactgagc aagagcagac ggctggaaaa tctgatcgcc 1980
cagctgcccg gcgagaagaa gaatggcctg ttcggaaacc tgattgccct gagcctgggc 2040
ctgaccccca acttcaagag caacttcgac ctggccgagg atgccaaact gcagctgagc 2100
aaggacacct acgacgacga cctggacaac ctgctggccc agatcggcga ccagtacgcc 2160
gacctgtttc tggccgccaa gaacctgtcc gacgccatcc tgctgagcga catcctgaga 2220
gtgaacaccg agatcaccaa ggcccccctg agcgcctcta tgatcaagag atacgacgag 2280
caccaccagg acctgaccct gctgaaagct ctcgtgcggc agcagctgcc tgagaagtac 2340
aaagagattt tcttcgacca gagcaagaac ggctacgccg gctacattga cggcggagcc 2400
agccaggaag agttctacaa gttcatcaag cccatcctgg aaaagatgga cggcaccgag 2460
gaactgctcg tgaagctgaa cagagaggac ctgctgcgga agcagcggac cttcgacaac 2520
ggcagcatcc cccaccagat ccacctggga gagctgcacg ccattctgcg gcggcaggaa 2580
gatttttacc cattcctgaa ggacaaccgg gaaaagatcg agaagatcct gaccttccgc 2640
atcccctact acgtgggccc tctggccagg ggaaacagca gattcgcctg gatgaccaga 2700
aagagcgagg aaaccatcac cccctggaac ttcgaggaag tggtggacaa gggcgcttcc 2760
gcccagagct tcatcgagcg gatgaccaac ttcgataaga acctgcccaa cgagaaggtg 2820
ctgcccaagc acagcctgct gtacgagtac ttcaccgtgt ataacgagct gaccaaagtg 2880
aaatacgtga ccgagggaat gagaaagccc gccttcctga gcggcgagca gaaaaaggcc 2940
atcgtggacc tgctgttcaa gaccaaccgg aaagtgaccg tgaagcagct gaaagaggac 3000
tacttcaaga aaatcgagtg cttcgactcc gtggaaatct ccggcgtgga agatcggttc 3060
aacgcctccc tgggcacata ccacgatctg ctgaaaatta tcaaggacaa ggacttcctg 3120
gacaatgagg aaaacgagga cattctggaa gatatcgtgc tgaccctgac actgtttgag 3180
gacagagaga tgatcgagga acggctgaaa acctatgccc acctgttcga cgacaaagtg 3240
atgaagcagc tgaagcggcg gagatacacc ggctggggca ggctgagccg gaagctgatc 3300
aacggcatcc gggacaagca gtccggcaag acaatcctgg atttcctgaa gtccgacggc 3360
ttcgccaaca gaaacttcat gcagctgatc cacgacgaca gcctgacctt taaagaggac 3420
atccagaaag cccaggtgtc cggccagggc gatagcctgc acgagcacat tgccaatctg 3480
gccggcagcc ccgccattaa gaagggcatc ctgcagacag tgaaggtggt ggacgagctc 3540
gtgaaagtga tgggccggca caagcccgag aacatcgtga tcgaaatggc cagagagaac 3600
cagaccaccc agaagggaca gaagaacagc cgcgagagaa tgaagcggat cgaagagggc 3660
atcaaagagc tgggcagcca gatcctgaaa gaacaccccg tggaaaacac ccagctgcag 3720
aacgagaagc tgtacctgta ctacctgcag aatgggcggg atatgtacgt ggaccaggaa 3780
ctggacatca accggctgtc cgactacgat gtggaccata tcgtgcctca gagctttctg 3840
aaggacgact ccatcgacaa caaggtgctg accagaagcg acaagaaccg gggcaagagc 3900
gacaacgtgc cctccgaaga ggtcgtgaag aagatgaaga actactggcg gcagctgctg 3960
aacgccaagc tgattaccca gagaaagttc gacaatctga ccaaggccga gagaggcggc 4020
ctgagcgaac tggataaggc cggcttcatc aagagacagc tggtggaaac ccggcagatc 4080
acaaagcacg tggcacagat cctggactcc cggatgaaca ctaagtacga cgagaatgac 4140
aagctgatcc gggaagtgaa agtgatcacc ctgaagtcca agctggtgtc cgatttccgg 4200
aaggatttcc agttttacaa agtgcgcgag atcaacaact accaccacgc ccacgacgcc 4260
tacctgaacg ccgtcgtggg aaccgccctg atcaaaaagt accctaagct ggaaagcgag 4320
ttcgtgtacg gcgactacaa ggtgtacgac gtgcggaaga tgatcgccaa gagcgagcag 4380
gaaatcggca aggctaccgc caagtacttc ttctacagca acatcatgaa ctttttcaag 4440
accgagatta ccctggccaa cggcgagatc cggaagcggc ctctgatcga gacaaacggc 4500
gaaaccgggg agatcgtgtg ggataagggc cgggattttg ccaccgtgcg gaaagtgctg 4560
agcatgcccc aagtgaatat cgtgaaaaag accgaggtgc agacaggcgg cttcagcaaa 4620
gagtctatcc tgcccaagag gaacagcgat aagctgatcg ccagaaagaa ggactgggac 4680
cctaagaagt acggcggctt cgacagcccc accgtggcct attctgtgct ggtggtggcc 4740
aaagtggaaa agggcaagtc caagaaactg aagagtgtga aagagctgct ggggatcacc 4800
atcatggaaa gaagcagctt cgagaagaat cccatcgact ttctggaagc caagggctac 4860
aaagaagtga aaaaggacct gatcatcaag ctgcctaagt actccctgtt cgagctggaa 4920
aacggccgga agagaatgct ggcctctgcc ggcgaactgc agaagggaaa cgaactggcc 4980
ctgccctcca aatatgtgaa cttcctgtac ctggccagcc actatgagaa gctgaagggc 5040
tcccccgagg ataatgagca gaaacagctg tttgtggaac agcacaagca ctacctggac 5100
gagatcatcg agcagatcag cgagttctcc aagagagtga tcctggccga cgctaatctg 5160
gacaaagtgc tgtccgccta caacaagcac cgggataagc ccatcagaga gcaggccgag 5220
aatatcatcc acctgtttac cctgaccaat ctgggagccc ctgccgcctt caagtacttt 5280
gacaccacca tcgaccggaa gaggtacacc agcaccaaag aggtgctgga cgccaccctg 5340
atccaccaga gcatcaccgg cctgtacgag acacggatcg acctgtctca gctgggaggc 5400
gacaaaaggc cggcggccac gaaaaaggcc ggccaggcaa aaaagaaaaa gtaagaattc 5460
ctagagctcg ctgatcagcc tcgactgtgc cttctagttg ccagccatct gttgtttgcc 5520
cctcccccgt gccttccttg accctggaag gtgccactcc cactgtcctt tcctaataaa 5580
atgaggaaat tgcatcgcat tgtctgagta ggtgtcattc tattctgggg ggtggggtgg 5640
ggcaggacag caagggggag gattgggaag agaatagcag gcatgctggg gagcggccgc 5700
aggaacccct agtgatggag ttggccactc cctctctgcg cgctcgctcg ctcactgagg 5760
ccgggcgacc aaaggtcgcc cgacgcccgg gctttgcccg ggcggcctca gtgagcgagc 5820
gagcgcgcag ctgcctgcag gggcgcctga tgcggtattt tctccttacg catctgtgcg 5880
gtatttcaca ccgcatacgt caaagcaacc atagtacgcg ccctgtagcg gcgcattaag 5940
cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg ccttagcgcc 6000
cgctcctttc gctttcttcc cttcctttct cgccacgttc gccggctttc cccgtcaagc 6060
tctaaatcgg gggctccctt tagggttccg atttagtgct ttacggcacc tcgaccccaa 6120
aaaacttgat ttgggtgatg gttcacgtag tgggccatcg ccctgataga cggtttttcg 6180
ccctttgacg ttggagtcca cgttctttaa tagtggactc ttgttccaaa ctggaacaac 6240
actcaactct atctcgggct attcttttga tttataaggg attttgccga tttcggtcta 6300
ttggttaaaa aatgagctga tttaacaaaa atttaacgcg aattttaaca aaatattaac 6360
gtttacaatt ttatggtgca ctctcagtac aatctgctct gatgccgcat agttaagcca 6420
gccccgacac ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc 6480
cgcttacaga caagctgtga ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc 6540
atcaccgaaa cgcgcgagac gaaagggcct cgtgatacgc ctatttttat aggttaatgt 6600
catgataata atggtttctt agacgtcagg tggcactttt cggggaaatg tgcgcggaac 6660
ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga gacaataacc 6720
ctgataaatg cttcaataat attgaaaaag gaagagtatg agtattcaac atttccgtgt 6780
cgcccttatt cccttttttg cggcattttg ccttcctgtt tttgctcacc cagaaacgct 6840
ggtgaaagta aaagatgctg aagatcagtt gggtgcacga gtgggttaca tcgaactgga 6900
tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc caatgatgag 6960
cacttttaaa gttctgctat gtggcgcggt attatcccgt attgacgccg ggcaagagca 7020
actcggtcgc cgcatacact attctcagaa tgacttggtt gagtactcac cagtcacaga 7080
aaagcatctt acggatggca tgacagtaag agaattatgc agtgctgcca taaccatgag 7140
tgataacact gcggccaact tacttctgac aacgatcgga ggaccgaagg agctaaccgc 7200
ttttttgcac aacatggggg atcatgtaac tcgccttgat cgttgggaac cggagctgaa 7260
tgaagccata ccaaacgacg agcgtgacac cacgatgcct gtagcaatgg caacaacgtt 7320
gcgcaaacta ttaactggcg aactacttac tctagcttcc cggcaacaat taatagactg 7380
gatggaggcg gataaagttg caggaccact tctgcgctcg gcccttccgg ctggctggtt 7440
tattgctgat aaatctggag ccggtgagcg tggaagccgc ggtatcattg cagcactggg 7500
gccagatggt aagccctccc gtatcgtagt tatctacacg acggggagtc aggcaactat 7560
ggatgaacga aatagacaga tcgctgagat aggtgcctca ctgattaagc attggtaact 7620
gtcagaccaa gtttactcat atatacttta gattgattta aaacttcatt tttaatttaa 7680
aaggatctag gtgaagatcc tttttgataa tctcatgacc aaaatccctt aacgtgagtt 7740
ttcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt 7800
ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg 7860
tttgccggat caagagctac caactctttt tccgaaggta actggcttca gcagagcgca 7920
gataccaaat actgttcttc tagtgtagcc gtagttaggc caccacttca agaactctgt 7980
agcaccgcct acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga 8040
taagtcgtgt cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc 8100
gggctgaacg gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact 8160
gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga 8220
caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg 8280
aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt 8340
tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt 8400
acggttcctg gccttttgct ggcc 8424
<210> 4
<211> 8424
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 4
ttttgctcac atgtgagggc ctatttccca tgattccttc atatttgcat atacgataca 60
aggctgttag agagataatt ggaattaatt tgactgtaaa cacaaagata ttagtacaaa 120
atacgtgacg tagaaagtaa taatttcttg ggtagtttgc agttttaaaa ttatgtttta 180
aaatggacta tcatatgctt accgtaactt gaaagtattt cgatttcttg gctttatata 240
tcttgtggaa aggacgaaac accggaactt caggcgtatg acccgtttta gagctagaaa 300
tagcaagtta aaataaggct agtccgttat caacttgaaa aagtggcacc gagtcggtgc 360
tttttttcta gacgttacat aacttacggt aaatggcccg cctggctgac cgcccaacga 420
cccccgccca ttgacgtcaa tagtaacgcc aatagggact ttccattgac gtcaatgggt 480
ggagtattta cggtaaactg cccacttggc agtacatcaa gtgtatcata tgccaagtac 540
gccccctatt gacgtcaatg acggtaaatg gcccgcctgg cattgtgccc agtacatgac 600
cttatgggac tttcctactt ggcagtacat ctacgtatta gtcatcgcta ttaccatggt 660
cgaggtgagc cccacgttct gcttcactct ccccatctcc cccccctccc cacccccaat 720
tttgtattta tttatttttt aattattttg tgcagcgatg ggggcggggg gggggggggg 780
gcgcgcgcca ggcggggcgg ggcggggcga ggggcggggc ggggcgaggc ggagaggtgc 840
ggcggcagcc aatcagagcg gcgcgctccg aaagtttcct tttatggcga ggcggcggcg 900
gcggcggccc tataaaaagc gaagcgcgcg gcgggcggga gtcgctgcgc gctgccttcg 960
ccccgtgccc cgctccgccg ccgcctcgcg ccgcccgccc cggctctgac tgaccgcgtt 1020
actcccacag gtgagcgggc gggacggccc ttctcctccg ggctgtaatt agctgagcaa 1080
gaggtaaggg tttaagggat ggttggttgg tggggtatta atgtttaatt acctggagca 1140
cctgcctgaa atcacttttt ttcaggttgg accggtgcca ccatggacta taaggaccac 1200
gacggagact acaaggatca tgatattgat tacaaagacg atgacgataa gatggcccca 1260
aagaagaagc ggaaggtcgg tatccacgga gtcccagcag ccgacaagaa gtacagcatc 1320
ggcctggaca tcggcaccaa ctctgtgggc tgggccgtga tcaccgacga gtacaaggtg 1380
cccagcaaga aattcaaggt gctgggcaac accgaccggc acagcatcaa gaagaacctg 1440
atcggagccc tgctgttcga cagcggcgaa acagccgagg ccacccggct gaagagaacc 1500
gccagaagaa gatacaccag acggaagaac cggatctgct atctgcaaga gatcttcagc 1560
aacgagatgg ccaaggtgga cgacagcttc ttccacagac tggaagagtc cttcctggtg 1620
gaagaggata agaagcacga gcggcacccc atcttcggca acatcgtgga cgaggtggcc 1680
taccacgaga agtaccccac catctaccac ctgagaaaga aactggtgga cagcaccgac 1740
aaggccgacc tgcggctgat ctatctggcc ctggcccaca tgatcaagtt ccggggccac 1800
ttcctgatcg agggcgacct gaaccccgac aacagcgacg tggacaagct gttcatccag 1860
ctggtgcaga cctacaacca gctgttcgag gaaaacccca tcaacgccag cggcgtggac 1920
gccaaggcca tcctgtctgc cagactgagc aagagcagac ggctggaaaa tctgatcgcc 1980
cagctgcccg gcgagaagaa gaatggcctg ttcggaaacc tgattgccct gagcctgggc 2040
ctgaccccca acttcaagag caacttcgac ctggccgagg atgccaaact gcagctgagc 2100
aaggacacct acgacgacga cctggacaac ctgctggccc agatcggcga ccagtacgcc 2160
gacctgtttc tggccgccaa gaacctgtcc gacgccatcc tgctgagcga catcctgaga 2220
gtgaacaccg agatcaccaa ggcccccctg agcgcctcta tgatcaagag atacgacgag 2280
caccaccagg acctgaccct gctgaaagct ctcgtgcggc agcagctgcc tgagaagtac 2340
aaagagattt tcttcgacca gagcaagaac ggctacgccg gctacattga cggcggagcc 2400
agccaggaag agttctacaa gttcatcaag cccatcctgg aaaagatgga cggcaccgag 2460
gaactgctcg tgaagctgaa cagagaggac ctgctgcgga agcagcggac cttcgacaac 2520
ggcagcatcc cccaccagat ccacctggga gagctgcacg ccattctgcg gcggcaggaa 2580
gatttttacc cattcctgaa ggacaaccgg gaaaagatcg agaagatcct gaccttccgc 2640
atcccctact acgtgggccc tctggccagg ggaaacagca gattcgcctg gatgaccaga 2700
aagagcgagg aaaccatcac cccctggaac ttcgaggaag tggtggacaa gggcgcttcc 2760
gcccagagct tcatcgagcg gatgaccaac ttcgataaga acctgcccaa cgagaaggtg 2820
ctgcccaagc acagcctgct gtacgagtac ttcaccgtgt ataacgagct gaccaaagtg 2880
aaatacgtga ccgagggaat gagaaagccc gccttcctga gcggcgagca gaaaaaggcc 2940
atcgtggacc tgctgttcaa gaccaaccgg aaagtgaccg tgaagcagct gaaagaggac 3000
tacttcaaga aaatcgagtg cttcgactcc gtggaaatct ccggcgtgga agatcggttc 3060
aacgcctccc tgggcacata ccacgatctg ctgaaaatta tcaaggacaa ggacttcctg 3120
gacaatgagg aaaacgagga cattctggaa gatatcgtgc tgaccctgac actgtttgag 3180
gacagagaga tgatcgagga acggctgaaa acctatgccc acctgttcga cgacaaagtg 3240
atgaagcagc tgaagcggcg gagatacacc ggctggggca ggctgagccg gaagctgatc 3300
aacggcatcc gggacaagca gtccggcaag acaatcctgg atttcctgaa gtccgacggc 3360
ttcgccaaca gaaacttcat gcagctgatc cacgacgaca gcctgacctt taaagaggac 3420
atccagaaag cccaggtgtc cggccagggc gatagcctgc acgagcacat tgccaatctg 3480
gccggcagcc ccgccattaa gaagggcatc ctgcagacag tgaaggtggt ggacgagctc 3540
gtgaaagtga tgggccggca caagcccgag aacatcgtga tcgaaatggc cagagagaac 3600
cagaccaccc agaagggaca gaagaacagc cgcgagagaa tgaagcggat cgaagagggc 3660
atcaaagagc tgggcagcca gatcctgaaa gaacaccccg tggaaaacac ccagctgcag 3720
aacgagaagc tgtacctgta ctacctgcag aatgggcggg atatgtacgt ggaccaggaa 3780
ctggacatca accggctgtc cgactacgat gtggaccata tcgtgcctca gagctttctg 3840
aaggacgact ccatcgacaa caaggtgctg accagaagcg acaagaaccg gggcaagagc 3900
gacaacgtgc cctccgaaga ggtcgtgaag aagatgaaga actactggcg gcagctgctg 3960
aacgccaagc tgattaccca gagaaagttc gacaatctga ccaaggccga gagaggcggc 4020
ctgagcgaac tggataaggc cggcttcatc aagagacagc tggtggaaac ccggcagatc 4080
acaaagcacg tggcacagat cctggactcc cggatgaaca ctaagtacga cgagaatgac 4140
aagctgatcc gggaagtgaa agtgatcacc ctgaagtcca agctggtgtc cgatttccgg 4200
aaggatttcc agttttacaa agtgcgcgag atcaacaact accaccacgc ccacgacgcc 4260
tacctgaacg ccgtcgtggg aaccgccctg atcaaaaagt accctaagct ggaaagcgag 4320
ttcgtgtacg gcgactacaa ggtgtacgac gtgcggaaga tgatcgccaa gagcgagcag 4380
gaaatcggca aggctaccgc caagtacttc ttctacagca acatcatgaa ctttttcaag 4440
accgagatta ccctggccaa cggcgagatc cggaagcggc ctctgatcga gacaaacggc 4500
gaaaccgggg agatcgtgtg ggataagggc cgggattttg ccaccgtgcg gaaagtgctg 4560
agcatgcccc aagtgaatat cgtgaaaaag accgaggtgc agacaggcgg cttcagcaaa 4620
gagtctatcc tgcccaagag gaacagcgat aagctgatcg ccagaaagaa ggactgggac 4680
cctaagaagt acggcggctt cgacagcccc accgtggcct attctgtgct ggtggtggcc 4740
aaagtggaaa agggcaagtc caagaaactg aagagtgtga aagagctgct ggggatcacc 4800
atcatggaaa gaagcagctt cgagaagaat cccatcgact ttctggaagc caagggctac 4860
aaagaagtga aaaaggacct gatcatcaag ctgcctaagt actccctgtt cgagctggaa 4920
aacggccgga agagaatgct ggcctctgcc ggcgaactgc agaagggaaa cgaactggcc 4980
ctgccctcca aatatgtgaa cttcctgtac ctggccagcc actatgagaa gctgaagggc 5040
tcccccgagg ataatgagca gaaacagctg tttgtggaac agcacaagca ctacctggac 5100
gagatcatcg agcagatcag cgagttctcc aagagagtga tcctggccga cgctaatctg 5160
gacaaagtgc tgtccgccta caacaagcac cgggataagc ccatcagaga gcaggccgag 5220
aatatcatcc acctgtttac cctgaccaat ctgggagccc ctgccgcctt caagtacttt 5280
gacaccacca tcgaccggaa gaggtacacc agcaccaaag aggtgctgga cgccaccctg 5340
atccaccaga gcatcaccgg cctgtacgag acacggatcg acctgtctca gctgggaggc 5400
gacaaaaggc cggcggccac gaaaaaggcc ggccaggcaa aaaagaaaaa gtaagaattc 5460
ctagagctcg ctgatcagcc tcgactgtgc cttctagttg ccagccatct gttgtttgcc 5520
cctcccccgt gccttccttg accctggaag gtgccactcc cactgtcctt tcctaataaa 5580
atgaggaaat tgcatcgcat tgtctgagta ggtgtcattc tattctgggg ggtggggtgg 5640
ggcaggacag caagggggag gattgggaag agaatagcag gcatgctggg gagcggccgc 5700
aggaacccct agtgatggag ttggccactc cctctctgcg cgctcgctcg ctcactgagg 5760
ccgggcgacc aaaggtcgcc cgacgcccgg gctttgcccg ggcggcctca gtgagcgagc 5820
gagcgcgcag ctgcctgcag gggcgcctga tgcggtattt tctccttacg catctgtgcg 5880
gtatttcaca ccgcatacgt caaagcaacc atagtacgcg ccctgtagcg gcgcattaag 5940
cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg ccttagcgcc 6000
cgctcctttc gctttcttcc cttcctttct cgccacgttc gccggctttc cccgtcaagc 6060
tctaaatcgg gggctccctt tagggttccg atttagtgct ttacggcacc tcgaccccaa 6120
aaaacttgat ttgggtgatg gttcacgtag tgggccatcg ccctgataga cggtttttcg 6180
ccctttgacg ttggagtcca cgttctttaa tagtggactc ttgttccaaa ctggaacaac 6240
actcaactct atctcgggct attcttttga tttataaggg attttgccga tttcggtcta 6300
ttggttaaaa aatgagctga tttaacaaaa atttaacgcg aattttaaca aaatattaac 6360
gtttacaatt ttatggtgca ctctcagtac aatctgctct gatgccgcat agttaagcca 6420
gccccgacac ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc 6480
cgcttacaga caagctgtga ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc 6540
atcaccgaaa cgcgcgagac gaaagggcct cgtgatacgc ctatttttat aggttaatgt 6600
catgataata atggtttctt agacgtcagg tggcactttt cggggaaatg tgcgcggaac 6660
ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga gacaataacc 6720
ctgataaatg cttcaataat attgaaaaag gaagagtatg agtattcaac atttccgtgt 6780
cgcccttatt cccttttttg cggcattttg ccttcctgtt tttgctcacc cagaaacgct 6840
ggtgaaagta aaagatgctg aagatcagtt gggtgcacga gtgggttaca tcgaactgga 6900
tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc caatgatgag 6960
cacttttaaa gttctgctat gtggcgcggt attatcccgt attgacgccg ggcaagagca 7020
actcggtcgc cgcatacact attctcagaa tgacttggtt gagtactcac cagtcacaga 7080
aaagcatctt acggatggca tgacagtaag agaattatgc agtgctgcca taaccatgag 7140
tgataacact gcggccaact tacttctgac aacgatcgga ggaccgaagg agctaaccgc 7200
ttttttgcac aacatggggg atcatgtaac tcgccttgat cgttgggaac cggagctgaa 7260
tgaagccata ccaaacgacg agcgtgacac cacgatgcct gtagcaatgg caacaacgtt 7320
gcgcaaacta ttaactggcg aactacttac tctagcttcc cggcaacaat taatagactg 7380
gatggaggcg gataaagttg caggaccact tctgcgctcg gcccttccgg ctggctggtt 7440
tattgctgat aaatctggag ccggtgagcg tggaagccgc ggtatcattg cagcactggg 7500
gccagatggt aagccctccc gtatcgtagt tatctacacg acggggagtc aggcaactat 7560
ggatgaacga aatagacaga tcgctgagat aggtgcctca ctgattaagc attggtaact 7620
gtcagaccaa gtttactcat atatacttta gattgattta aaacttcatt tttaatttaa 7680
aaggatctag gtgaagatcc tttttgataa tctcatgacc aaaatccctt aacgtgagtt 7740
ttcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt 7800
ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg 7860
tttgccggat caagagctac caactctttt tccgaaggta actggcttca gcagagcgca 7920
gataccaaat actgttcttc tagtgtagcc gtagttaggc caccacttca agaactctgt 7980
agcaccgcct acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga 8040
taagtcgtgt cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc 8100
gggctgaacg gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact 8160
gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga 8220
caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg 8280
aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt 8340
tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt 8400
acggttcctg gccttttgct ggcc 8424
<210> 5
<211> 60
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 5
ttaatacgac tcactatagg gttggagggc gaatgttaca tgttttagag ctagaaatag 60
<210> 6
<211> 60
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 6
ttaatacgac tcactatagg ggaacttcag gcgtatgacc cgttttagag ctagaaatag 60
<210> 7
<211> 31
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 7
aaaaaaagca ccgactcggt gccacttttt c 31
<210> 8
<211> 25
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 8
ctgagagaag gcttaacctt gctgg 25
<210> 9
<211> 26
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 9
agctttccat cctgcaagtg agtcag 26
<210> 10
<211> 25
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 10
aggtggcaag gacagagaca gtgag 25
Claims (3)
1.一种全身性Plin1基因敲除小鼠动物模型的构建方法，其特征在于：所述方法基于CRISPR/Cas9基因敲除技术，包括以下步骤：
（1）针对Plin1基因2号外显子前后序列设计1对sgRNA序列，通过构建Plin1基因敲除载体及体外转录获得sgRNA；
（2）将sgRNA与Cas9蛋白混合组成的Cas9 sgRNA体系的小鼠受精卵显微注射，F0代小鼠出生及鉴定，F0代小鼠性成熟配繁，F1代小鼠鉴定，F1代阳性小鼠雌雄近交获得F2代小鼠，即为全身性Plin1基因敲除小鼠；
所述sgRNA核苷酸序列如SEQ ID NO:1、SEQ ID NO:2所示；
所述sgRNA的筛选及获得方法为：
（1）针对小鼠Plin1基因2号外显子序列，在NCBI中查询小鼠Plin1基因的基本信息，应用在线工具http://crispr.mit.edu设计识别Plin1基因2号外显子前后的sgRNA序列对；
（2）根据脱靶位点、基因数和发生错配的可能性大小进行评估筛选，选择1对sgRNA序列，其核苷酸序列如SEQ ID NO:1，SEQ ID NO:2所示；
（3）选择CRISPR基因敲除质粒pX330作为载体，用限制性内切酶BBS切割pX330质粒BBS酶切位点即第245、267位置，使其线性化；
（4）向2条Plin1 sgRNA的5’端添加粘性末端后与线性化载体用T4 DNA连接酶进行连接，在pX330 gRNA scafflod位置加入sgRNA序列构建Plin1基因CRISPR/Cas9敲除载体Plin1-1，Plin1-2；构建好的质粒载体转化至DH5α菌群中，2 d后挑取单克隆菌落测序及限制性核酸内切酶ApaLI+NcoI双酶切法鉴定，载体序列信息如SEQ ID NO:3,4所示；
（5）按照sgRNA体外转录试剂盒即PC1380说明书设计正向引物SG1-F、SG2-F和通用下游引物SG-R，序列如SEQ ID NO：5，SEQ ID NO：6，SEQ ID NO：7所示，PCR扩增转录模板后进行sgRNA转录，转录完全并纯化后进行琼脂糖凝胶电泳实验检测sgRNA片段大小及完整性。
2.根据权利要求1所述的一种全身性Plin1基因敲除小鼠动物模型的构建方法，其特征在于：所述的方法还包括鉴定Plin1基因敲除小鼠动物模型。
3.根据权利要求2所述的一种全身性Plin1基因敲除小鼠动物模型的构建方法，其特征在于：所述鉴定Plin1基因敲除小鼠动物模型的方法为：
A、待F0代小鼠出生三周后，提取小鼠尾部DNA，利用引物F，R进行PCR扩增，琼脂糖凝胶电泳，测序鉴定基因型，引物序列如SEQ ID NO：8，SEQ ID NO：9所示；
B、选取F0代阳性小鼠与野生型异性小鼠交配获的F1代小鼠，利用引物F、Wt/He-R进行PCR扩增，琼脂糖凝胶电泳，测序鉴定基因型，引物序列如SEQ ID NO：8，SEQ ID NO：10所示；
C、选取F1代阳性小鼠雌雄近交获得F2代小鼠，按照F0代小鼠基因型鉴定方法获得纯合型小鼠，即为小鼠动物模型。
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