CN110551190A - 一种利用家蚕生产蜘蛛丝的方法 - Google Patents

一种利用家蚕生产蜘蛛丝的方法 Download PDF

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CN110551190A
CN110551190A CN201810562242.8A CN201810562242A CN110551190A CN 110551190 A CN110551190 A CN 110551190A CN 201810562242 A CN201810562242 A CN 201810562242A CN 110551190 A CN110551190 A CN 110551190A
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谭安江
许军
牛宝龙
计东风
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Abstract

本发明公开了一种利用家蚕生产蜘蛛丝蛋白的方法，包括如下步骤：1)在家蚕的丝素重链基因FibH上选择TALENs的两个靶点；2)分别构建用于表达TALENs的质粒；3)体外转录TALENs mRNA；4)构建包含蜘蛛丝基因MaSp1的donor载体；5)将TALENs mRNA和donor载体混合，对家蚕进行显微注射，注射后的蚕卵进行催青直到孵化，饲养出生产蜘蛛丝蛋白的家蚕个体。实验表明饲养孵化出来的G1代蚕卵中蜘蛛丝MaSp1基因替换了家蚕FibH基因，饲养的家蚕个体分泌出了蜘蛛丝，突破了传统转基因家蚕不能在家蚕丝腺中大量合成蜘蛛丝蛋白的限制。

Description

一种利用家蚕生产蜘蛛丝的方法
技术领域
本发明属于生物技术领域，涉及一种利用家蚕生产蜘蛛丝的方法，尤其涉及一种利用家蚕生产蜘蛛丝蛋白的方法。
背景技术
家蚕(Bombyx mori)是一种绢丝昆虫，属于鳞翅目昆虫，也是一种模式生物。经过数千年的驯化和改良，家蚕已经成为我国最为重要的经济昆虫并支撑着蚕丝行业。家蚕幼虫期大量摄食桑叶，在丝腺中合成并分泌蚕丝蛋白，幼虫末期经吐丝结茧形成蚕丝。蚕丝不仅可以作为纺织业的原材料，在化工、医学以及食品工业等领域也发挥着重要作用，是我国一些地区的重要支柱产业。我国蚕丝生产历史悠久，规模庞大，近年来，丝绸行业的总产值高达两千亿元。此外，由于具有劳动密集型的特点，蚕丝行业为我国大量的农业劳动力创造了工作机会，帮助国家解决了就业问题。随着我国家蚕基因组计划宣布家蚕基因组框架图的完成，标志着家蚕后基因组时代的到来，功能基因组学的主要工作包括：大量功能基因的克隆鉴定、重要生物学性状的分子调控机理，以及实现人为控制和调节这些基因和性状以满足人类的需求。
蜘蛛丝目前算得上是自然界中最结实、最具有延展性的材料。蜘蛛丝的理化性质与蚕丝相比，具有非常明显的优势。在力学强度方面，蜘蛛丝纤维与强度最高的碳纤维及高强合纤Aramid、Kelve等强度相接近，但它的韧性明显优于上述几种纤维。因此，蜘蛛丝纤维在国防、军事(防弹衣)、建筑等领域具有广阔应用前景。天然蜘蛛丝主要来源于结网，产量非常低，而且蜘蛛具有同类相食的个性，无法像家蚕一样高密度养殖。所以要从天然蜘蛛中取得蛛丝产量很有限。随着现代生物工程发展，用基因工程手段人工合成蜘蛛丝蛋白是一种新突破，有可能形成具有远大前景的蜘蛛丝产业。由于家蚕是吐丝昆虫，其作为表达蜘蛛丝蛋白的表达宿主是不二选择。
基因组编辑技术是进行功能基因组研究的重要工具，锌指核酸酶技术(ZFNs)、类转录激活因子核酸酶(Transcription activator-like effector nucleases,TALENs，转录激活子样效应因子核酸酶)技术以及CRISPR/Cas9技术是近年来发展起来的三种主流基因组编辑技术。这三种基因组编辑技术的原理都是通过在生物基因组特定位点制造DNA断裂损伤，从而激活机体自身的DNA损伤修复机制，在此过程中引发各种变异。目前，三种技术都在多种物种中成功应用。在昆虫中，基因组编辑技术首先在果蝇中得以开展。2002年，Bibikova等人使用ZFNs技术在基因组水平上成功地将果蝇的yellow基因敲除。2010年Takatsu等人使用ZFNs技术在家蚕基因组水平上敲除了BmBLOS2基因，突变个体变现出油蚕表型，并且这种变异成功遗传到下一代。2012年Ma等人使用TALENs技术敲除了家蚕BmBLOS2基因，突变个体表现出油蚕表型，同时还使用两组TALENs在两个位点切割，造成了靶标位点中间片段的缺失。Wang等人在家蚕中成功建立起CRISPR/Cas9基因组编辑技术平台以及基于TALEN的基因敲入技术。这些研究进展预示着家蚕具有生产蜘蛛丝蛋白的可能性。但是，迄今尚未有通过在家蚕中用蜘蛛丝基因替换丝素基因进行蜘蛛丝生产的报道。
发明内容
为了解决了这一瓶颈，扩展家蚕的应用领域，保持蚕业的可持续发展，创造了利用非转基因方式的利用家蚕生产蜘蛛丝蛋白的方法，利用该方法可以大量表达外源蛋白。
本发明基于TALEN的基因敲入技术实现蜘蛛Nephila clavipes(圆网蛛棒络新妇蛛)丝基因MaSp1在家蚕丝素重链基因座对FibH基因的替换，获得一种家蚕非转基因形式的丝素重链替换系统，可将家蚕的丝腺组织作为生物反应器用来大量生产蜘蛛丝蛋白。具体而言，本发明包含如下技术方案。
一种利用家蚕生产蜘蛛丝蛋白的方法，包括如下步骤：
1)在家蚕的丝素重链基因FibH上选择TALENs的两个靶点，第一靶点(即FibH1TALENs位点)位于FibH基因的二号外显子上，第一靶点包括左半位点FibH1-L即TALEN左臂和右半位点FibH1-R即TALEN右臂，其中FibH1-L与FibH1-R之间有12-20bp的第一间隔序列作为切割序列；第二靶点(即FibH2TALENs位点)位于FibH基因的3’非翻译区(即3’-UTR)之外，第二靶点包括左半位点FibH2-L即TALEN左臂和右半位点FibH2-R即TALEN右臂，其中FibH2-L与FibH2-R之间有12-20bp的第二间隔序列作为切割序列；
2)分别构建用于表达类转录激活因子效应物核酸酶TALEN1-L、TALEN1-R、TALEN2-L和TALEN2-R的质粒，其中TALEN1-L、TALEN1-R、TALEN2-L和TALEN2-R分别用于切割FibH1-L、FibH1-R、FibH2-L和FibH2-R；
3)体外转录用于表达TALEN1-L、TALEN1-R、TALEN2-L和TALEN2-R的质粒，合成得到作为TALEN1-L、TALEN1-R、TALEN2-L和TALEN2-R翻译模板的TALENsmRNA；
4)构建包含蜘蛛丝基因MaSp1的donor载体，该donor载体的两端分别含有第一靶点两旁的同源序列和第二靶点两旁的同源序列，两端同源序列之间含有用于替换基因FibH的目的基因MaSp1；
5)将步骤3)中得到的mRNA和步骤4)中得到的donor载体混合，两者的终浓度各自控制在200-300ng/μl，对家蚕进行显微注射，注射后的蚕卵进行催青直到孵化，饲养孵化出来的蚁蚕，将当代(G0)的蚕蛾自交，获得G1代蚕卵，饲养出生产蜘蛛丝蛋白的家蚕个体。
上述靶点序列FibH1-L、FibH1-R、FibH2-L和FibH2-R的长度为14-18bp。
优选地，上述第一间隔序列的长度为14-18bp，第二间隔序列的长度为14-18bp。
在一种实施方式中，步骤2)中所述的用于表达TALEN1-L、TALEN1-R、TALEN2-L和TALEN2-R的质粒分别为pSW-epeas-FibH1-L、pSW-epeas-FibH1-R、pSW-epeas-FibH2-L、pSW-epeas-FibH2-R。
在一种实施方式中，针对两个靶点即FibH1TALENs位点和FibH2TALENs位点的TALENs骨架由北京维尚立德公司合成，合成后亚克隆入载体pSW-epeas-T中，分别构成pSW-epeas-FibH1-L、pSW-epeas-FibH1-R、pSW-epeas-FibH2-L、pSW-epeas-FibH2-R。其中TALENs骨架包含TALE蛋白的DNA结合结构域和Fok I核酸内切酶切割结构域。
优选地，步骤1)中所述FibH1-L序列是SEQ ID NO:1(5’-ACGTATCAAACAGT-3’)；FibH1-R序列是SEQ ID NO:2(5’-AAGGATATACGAGCG-3’)；FibH2-L序列是SEQ ID NO:3(5’-TCTTTGAGATATGGGTC-3’)；FibH2-R序列是SEQ ID NO:4(5’-GAGGGTACAAAGAATACA-3’)。这种情况下，第一靶点FibH1-L与FibH1-R之间有16bp的间隔序列作为切割序列；第二靶点FibH2-L与FibH2-R之间有14bp的间隔序列作为切割序列。第一靶点(即FibH1TALENs位点)与第二靶点(即FibH2TALENs位点)之间有15794bp的距离。
步骤3)中体外转录TALENs mRNA参照Ambion公司的mMESSAGE T7Kit试剂盒进行，具体方法是：将pSW-epeas-FibH1-L、pSW-epeas-FibH1-R、pSW-epeas-FibH2-L、pSW-epeas-FibH2-L四个质粒用NotI限制性内切酶(Fermentas公司)线性化，mRNA合成参照试剂盒的具体操作进行。合成好的mRNA放置于-80℃冰箱备用。
在一种实施方式中，步骤4)中所述基因MaSp1是序列SEQ ID NO:5。
优选地，上述步骤4)中所述TALEN1-L的氨基酸序列为SEQ ID NO:6；TALEN1-R的氨基酸序列为SEQ ID NO:7；TALEN2-L的氨基酸序列为SEQ ID NO:8；TALEN2-R的氨基酸序列为SEQ ID NO:9。
在一种优选的实施方式中，步骤4)中所述donor载体上还含有筛选标签，优选红色荧光蛋白(DsRed)。
本发明的基因替代方法是在TALENs诱导的基因组断裂之后，在基因发生同源修复的时候加入含有断裂位点两旁的同源序列的donor载体，donor载体的两段同源序列之间含有替换的目的基因MaSp1以及筛选标签。
可选地，上述步骤4)中所述donor载体的构建方法是：克隆出FibH1TALENs位点上游1356bp和FibH2TALENs位点下游1113bp的两个片段；将这两个片段依次连接到PJET-1.2载体上；将去除ATG起始系列的MaSp1基因连接在上述1356bp和1113bp的两个片段之间，并在MaSp1序列后加入家蚕FibH基因的3’-UTR序列，构成donor载体。
当donor载体上还含有筛选标签时，donor载体的构建方法可以是：克隆出FibH1TALENs位点上游1356bp和FibH2TALENs位点下游1113bp的两个片段；将这两个片段依次连接到PJET-1.2载体(Fermentas公司)上；将去除ATG起始系列的MaSp1基因连接在上述1356bp和1113bp的两个片段之间，并在MaSp1序列后加入家蚕FibH基因的3’-UTR序列；再将红色荧光蛋白筛选标签克隆(比如是IE1-DsRed2-SV40，来自Addgene公司的#26852)连接在3’-UTR序列之后，构成donor载体PJET-1.2-MaSp1-donor。
上述donor载体PJET-1.2-FibH-donor的质粒抽提与纯化可以用Qiagen公司的Plasmid Midi kit试剂盒，具体方法操作按照试剂盒说明书进行，纯化好的质粒放置于-20℃冰箱备用。
可选地，步骤5)中家蚕胚胎显微注射方法参照文献(Tan et al.,2013Proc.Natl.Acad.Sci.U.S.A.110,6766–6770.)描述的方法进行家蚕显微注射，注射后的蚕卵用无毒胶封口以防止污染，在25℃条件下，进行催青直到孵化，饲养孵化出来的蚁蚕，将当代(G0)的蚕蛾自交，获得G1代蚕卵，在荧光显微镜下挑出红色荧光个体饲养。
本发明首次利用TALENs的基因替代方法在家蚕丝素重链基因座上替换上蜘蛛丝基因MaSp1，通过在家蚕胚胎期显微注射靶向家蚕内源的FibH基因的两对TALENsmRNA和含有MaSp1基因的donor载体，成功地实现了蜘蛛丝MaSp1基因替换家蚕FibH基因的品系，可以在家蚕丝腺组织中大量生产蜘蛛丝蛋白，饲养的家蚕个体分泌出了蜘蛛丝，突破了传统转基因家蚕不能在家蚕丝腺中大量的合成蜘蛛丝蛋白的限制，创造了利用非转基因方式的利用家蚕生产蜘蛛丝蛋白的方法。
附图说明
图1为家蚕基因组上实现蜘蛛丝MaSp1基因替换家蚕FibH基因的构建示意图。其中引物F1(5’-ACGCCGATTGGCAAACAAAAACT-3’)和R2(5’-CGCAAGCCTATGTTTGTTCCTA-3’)位于家蚕基因组上，引物F2(5’-CCCCCTGAACCTGAAACATA-3’)和R3(5’-CGGCTGCACCAGCGCTAGAGA-3’)位于donor载体上；IE1-DsRed2-SV40筛选标签克隆被连接到丝素重链基因FibH上。
图2为Western blot检测茧中内源基因FibH和外源基因MaSp1表达的照片。Anti-Fib-H是内源蛋白检测结果，Anti-MaSp1是外源蛋白检测结果。Control是对照家蚕，MaSp1+/-是杂合体，MaSp1+/+是纯合体。
具体实施方式
以下结合具体实施例对本发明做进一步详细说明。应理解，以下实施例仅用于说明本发明而非用于限定本发明的范围。
本发明将家蚕胚胎显微注射技术、基因组编辑技术与分子生物操作相结合，成功构建了利用家蚕丝腺大量生产外源蜘蛛丝蛋白的高效表达系统。图1显示了家蚕基因组上实现蜘蛛丝MaSp1基因替换家蚕FibH基因的构建示意图。
本发明用蜘蛛丝素重链基因比如MaSp1替代家蚕丝素重链基因FibH，检测到外源基因蜘蛛丝丝素重链基因的表达，确认家蚕内源的丝素重链基因消失。获得基因替代的材料可以通过观察红色荧光挑选出有红色荧光蛋白表达的蚕卵即可，检测插入位点的正确性用PCR扩增和测序。检测外源基因MaSp1和内源FibH表达用qPCR和western blot方法。
本发明首次开发和利用了基于TALENs的基因替代方法，通过家蚕胚胎显微注射技术，突破了传统转基因家蚕不能在家蚕丝腺中大量合成蜘蛛丝蛋白的限制。我们首次利用基于TALENs的基因替代方式在家蚕丝素重链基因位坐上替换上Nephila clavipes丝基因，通过在家蚕胚胎期显微注射靶向家蚕内源的FibH基因的两对TALENs mRNA和含有MaSp1基因的donor载体，成功实现了蜘蛛丝MaSp1基因替换家蚕FibH基因的品系。
对于本发明，术语“载体”可以是指用于包含基因序列的表达盒或者质粒，这是本领域技术人员所熟知的。
在本文中，有时为了描述简便，会将蜘蛛丝MaSp1蛋白名称与其编码基因(DNA)名称混用，本领域技术人员应能理解它们在不同描述场合表示不同的物质。例如，对于MaSp1(基因)，用于描述在蚕茧中的成分类别时，指的是蛋白质；在作为一种基因描述时，指的是编码该MaSp1蛋白的基因MaSp1。类似地，有时会将家蚕FibH或者Fib-H蛋白名称与其编码基因(DNA)名称混用，本领域技术人员应能理解它们在不同描述场合表示不同的物质。
本文实施例中涉及到多种物质的添加量、含量及浓度，其中所述的百分含量，除特别说明外，皆指质量百分含量。
实施例
材料和方法
本文中的全基因合成、引物合成及测序皆由北京维尚立德公司和生工生物工程(上海)股份有限公司完成。
本文中的分子生物学实验包括质粒构建、酶切、感受态细胞制备、转化等主要参照《分子克隆实验指南》(第三版)，J.萨姆布鲁克，D.W.拉塞尔(美)编著，黄培堂等译，科学出版社，北京，2002)进行。
可根据相关试剂盒说明书操作。必要时可以通过简单试验确定具体实验条件比如PCR条件。
实施例1：TALENs骨架合成
TALENs骨架结构包括转录启动子序列T7(TAATACGACTCACTATAGGG)、靶向结合位点的TALENs单体结构、以及Fok I核酸内切酶切割结构域，各个模块合成由北京维尚立德公司完成。
实施例2：TALENs表达质粒构建
通过Golden gate方法将实施例1中合成的TALENs骨架基因克隆入载体pSW-epeas-T(北京维尚立德公司)中，分别构成pSW-epeas-FibH1-L、pSW-epeas-FibH1-R、pSW-epeas-FibH2-L、pSW-epeas-FibH2-R。
实施例3：体外转录TALENs mRNA
使用Ambion公司的mMESSAGE T7Kit试剂盒进行TALENs mRNA的体外转录。具体方法是：分别将实施例2中构建的四个质粒pSW-epeas-FibH1-L、pSW-epeas-FibH1-R、pSW-epeas-FibH2-L、pSW-epeas-FibH2-L用NotI限制性内切酶(Fermentas公司)线性化，mRNA合成参照试剂盒的具体操作进行。
合成好的TALENs mRNA放置于-80℃冰箱备用。
实施例4：Donor载体构建
4.1通过PCR从家蚕基因组克隆出FibH1TALENs位点上游1356bp和FibH2TALENs位点下游1113bp的两个片段，引物分别为(5’-3’)
Right-arm-F：AGGCGCGCCTATGGTAAAGAAAGGACACGAG；
Right-arm-R：ATTGTATTCTTTGTACCCTCATACCTCAAAGAACGACCCATATCTCAAAGACAAGCCTGTCCCTCAAGGATTTCTTCG；
Left-arm-F：ATGATATCTATTAACAATTGCTATTGCC；
Left-arm-R：TGACTGCAGCACTAGTGCTGAAAT。
使用KOD酶(上海东洋)进行扩增，PCR反应条件一般为：94℃变性5min，94℃变性30s，55℃复性30s，72℃延伸1min/1kb，扩增30个循环；72℃延伸保温10min，10℃保温。
4.2将所得到的两个片段依次通过同源重组连接到PJET-1.2载体(Fermentas公司)上。
4.3将去除ATG起始系列的MaSp1基因通过酶切连接连接在上一步骤所得的载体上，位于上述的1356bp和1113bp的两个片段之间。
4.4将IE1-DsRed2-SV40(本实验室保存质粒PXL-BacII-IE1-DsRed2-SV40)筛选标签克隆连接在3’-UTR序列之后，构成donor载体PJET-1.2-MaSp1-donor。测序正确后，进行质粒大量抽提。
4.5质粒抽提与纯化用Qiagen公司的Plasmid Midi kit试剂盒，具体方法操作按照试剂盒说明书进行，纯化好的PJET-1.2-FibH-donor质粒放置于-20℃冰箱备用。
实施例5：家蚕胚胎显微注射
参照文献Tan et al.,2013Proc.Natl.Acad.Sci.U.S.A.110,6766–6770.描述的方法进行家蚕显微注射。具体操作包括：
将实施例3中得到的mRNA和实施例4中得到的donor载体混合，两者的终浓度各自控制在200-300ng/μl，对家蚕进行显微注射。
注射后的蚕卵用无毒胶封口以防止污染，在25℃条件下，进行催青直到孵化，饲养孵化出来的蚁蚕，将当代(G0)的蚕蛾自交，获得G1代蚕卵，在荧光显微镜下挑出红色荧光个体饲养。
实施例6：蚕茧中内源基因FibH和外源基因MaSp1表达的比较
6.1对实施例5中获得的红色荧光家蚕个体(包括MaSp1+/-杂合体和MaSp1+/+纯合体)、未进行基因改造的阴性对照(control)家蚕个体分泌产生的蚕茧进行蛋白质提取。
将蚕丝用剪刀剪碎，蛋白提取在8M尿素溶液中裂解12小时，离心获得上清液。
6.2聚丙烯酰胺凝胶电泳
将上述等量蛋白和蛋白上样液混合后进行SDS聚丙烯酰胺凝胶电泳，在110V恒压下两个小时。
6.3Western blot
将上述获得的SDS聚丙烯酰胺凝胶在10mA的恒流条件下转膜一小时。然后进行免疫印记分析。
图2显示了蚕卵基因组的免疫印记照片。从图中可以看出，在对照家蚕蚕茧中FibH蛋白高表达，杂合体蚕茧中FibH蛋白表达下降，纯合体蚕茧中不含FibH蛋白；在对照家蚕蚕茧中不含MaSp1蛋白，杂合体蚕茧中含一定MaSp1蛋白，纯合体蚕茧中MaSp1蛋白高表达，表明本发明得到的纯合体是蜘蛛丝MaSp1基因替换家蚕FibH基因的品系。
上述实验表明，本发明可以在家蚕中成功实现蜘蛛丝MaSp1基因替换家蚕FibH基因，并表达蜘蛛丝蛋白，因此这种非转基因的家蚕养殖方式对于蜘蛛丝生产具有重要意义。应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本发明的保护范围。
序列表
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<120> 一种利用家蚕生产蜘蛛丝的方法
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Ala Gln Val Val Ala Ile Ala Ser Asn Asn Gly Gly Lys Gln Ala Leu
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Ala Gln Val Val Ala Ile Ala Ser Asn Gly Gly Gly Lys Gln Ala Leu
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Ala Leu Glu Ser Ile Val Ala Gln Leu Ser Arg Pro Asp Pro Ala Leu
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Ala Ala Leu Thr Asn Asp His Leu Val Ala Leu Ala Cys Leu Gly Gly
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Asn Cys Asn Gly Ala Val Leu Ser Val Glu Glu Leu Leu Ile Gly Gly
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Met Ala Pro Lys Lys Lys Arg Lys Val Asp Tyr Lys Asp His Asp Gly
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Asp Tyr Lys Asp His Asp Ile Asp Tyr Lys Asp Asp Asp Asp Lys Gly
20 25 30
Thr Val Asp Leu Arg Thr Leu Gly Tyr Ser Gln Gln Gln Gln Glu Lys
35 40 45
Ile Lys Pro Lys Val Arg Ser Thr Val Ala Gln His His Glu Ala Leu
50 55 60
Val Gly His Gly Phe Thr His Ala His Ile Val Ala Leu Ser Gln His
65 70 75 80
Pro Ala Ala Leu Gly Thr Val Ala Val Thr Tyr Gln His Ile Ile Thr
85 90 95
Ala Leu Pro Glu Ala Thr His Glu Asp Ile Val Gly Val Gly Lys Gln
100 105 110
Trp Ser Gly Ala Arg Ala Leu Glu Ala Leu Leu Thr Asp Ala Gly Glu
115 120 125
Leu Arg Gly Pro Pro Leu Gln Leu Asp Thr Gly Gln Leu Val Lys Ile
130 135 140
Ala Lys Arg Gly Gly Val Thr Ala Met Glu Ala Val His Ala Ser Arg
145 150 155 160
Asn Ala Leu Thr Gly Ala Pro Leu Asn Leu Thr Pro Asp Gln Val Val
165 170 175
Ala Ile Ala Ser His Asp Gly Gly Lys Gln Ala Leu Glu Thr Val Gln
180 185 190
Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Ala Gln
195 200 205
Val Val Ala Ile Ala Ser Asn Gly Gly Gly Lys Gln Ala Leu Glu Thr
210 215 220
Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro
225 230 235 240
Ala Gln Val Val Ala Ile Ala Ser Asn Gly Gly Gly Lys Gln Ala Leu
245 250 255
Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu
260 265 270
Thr Pro Ala Gln Val Val Ala Ile Ala Ser Asn Gly Gly Gly Lys Gln
275 280 285
Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His
290 295 300
Gly Leu Thr Pro Ala Gln Val Val Ala Ile Ala Ser Asn Asn Gly Gly
305 310 315 320
Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln
325 330 335
Ala His Gly Leu Thr Pro Asp Gln Val Val Ala Ile Ala Ser Asn Ile
340 345 350
Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu
355 360 365
Cys Gln Ala His Gly Leu Thr Pro Asp Gln Val Val Ala Ile Ala Ser
370 375 380
Asn Asn Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro
385 390 395 400
Val Leu Cys Gln Ala His Gly Leu Thr Pro Asp Gln Val Val Ala Ile
405 410 415
Ala Ser Asn Ile Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu
420 425 430
Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Asp Gln Val Val
435 440 445
Ala Ile Ala Ser Asn Gly Gly Gly Lys Gln Ala Leu Glu Thr Val Gln
450 455 460
Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Asp Gln
465 470 475 480
Val Val Ala Ile Ala Ser Asn Ile Gly Gly Lys Gln Ala Leu Glu Thr
485 490 495
Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro
500 505 510
Asp Gln Val Val Ala Ile Ala Ser Asn Gly Gly Gly Lys Gln Ala Leu
515 520 525
Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu
530 535 540
Thr Pro Asp Gln Val Val Ala Ile Ala Ser Asn Asn Gly Gly Lys Gln
545 550 555 560
Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His
565 570 575
Gly Leu Thr Pro Asp Gln Val Val Ala Ile Ala Ser Asn Asn Gly Gly
580 585 590
Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln
595 600 605
Ala His Gly Leu Thr Pro Asp Gln Val Val Ala Ile Ala Ser Asn Asn
610 615 620
Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu
625 630 635 640
Cys Gln Ala His Gly Leu Thr Pro Asp Gln Val Val Ala Ile Ala Ser
645 650 655
Asn Gly Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro
660 665 670
Val Leu Cys Gln Ala His Gly Leu Thr Pro Ala Gln Val Val Ala Ile
675 680 685
Ala Ser His Asp Gly Gly Arg Pro Ala Leu Glu Ser Ile Val Ala Gln
690 695 700
Leu Ser Arg Pro Asp Pro Ala Leu Ala Ala Leu Thr Asn Asp His Leu
705 710 715 720
Val Ala Leu Ala Cys Leu Gly Gly Arg Pro Ala Met Asp Ala Val Lys
725 730 735
Lys Gly Leu Pro His Ala Pro Glu Leu Ile Arg Arg Val Asn Arg Arg
740 745 750
Ile Gly Glu Arg Thr Ser His Arg Val Ala Gly Ser Gln Leu Val Lys
755 760 765
Ser Glu Leu Glu Glu Lys Lys Ser Glu Leu Arg His Lys Leu Lys Tyr
770 775 780
Val Pro His Glu Tyr Ile Glu Leu Ile Glu Ile Ala Arg Asn Pro Thr
785 790 795 800
Gln Asp Arg Ile Leu Glu Met Lys Val Met Glu Phe Phe Met Lys Val
805 810 815
Tyr Gly Tyr Arg Gly Glu His Leu Gly Gly Ser Arg Lys Pro Asp Gly
820 825 830
Ala Ile Tyr Thr Val Gly Ser Pro Ile Asp Tyr Gly Val Ile Val Asp
835 840 845
Thr Lys Ala Tyr Ser Gly Gly Tyr Asn Leu Pro Ile Gly Gln Ala Asp
850 855 860
Ala Met Gln Ser Tyr Val Glu Glu Asn Gln Thr Arg Asn Lys His Ile
865 870 875 880
Asn Pro Asn Glu Trp Trp Lys Val Tyr Pro Ser Ser Val Thr Glu Phe
885 890 895
Lys Phe Leu Phe Val Ser Gly His Phe Lys Gly Asn Tyr Lys Ala Gln
900 905 910
Leu Thr Arg Leu Asn His Ile Thr Asn Cys Asn Gly Ala Val Leu Ser
915 920 925
Val Glu Glu Leu Leu Ile Gly Gly Glu Met Ile Lys Ala Gly Thr Leu
930 935 940
Thr Leu Glu Glu Val Arg Arg Lys Phe Asn Asn Gly Glu Ile Asn Phe
945 950 955 960
<210> 9
<211> 994
<212> PRT
<213> 人工序列()
<400> 9
Met Ala Pro Lys Lys Lys Arg Lys Val Asp Tyr Lys Asp His Asp Gly
1 5 10 15
Asp Tyr Lys Asp His Asp Ile Asp Tyr Lys Asp Asp Asp Asp Lys Gly
20 25 30
Thr Val Asp Leu Arg Thr Leu Gly Tyr Ser Gln Gln Gln Gln Glu Lys
35 40 45
Ile Lys Pro Lys Val Arg Ser Thr Val Ala Gln His His Glu Ala Leu
50 55 60
Val Gly His Gly Phe Thr His Ala His Ile Val Ala Leu Ser Gln His
65 70 75 80
Pro Ala Ala Leu Gly Thr Val Ala Val Thr Tyr Gln His Ile Ile Thr
85 90 95
Ala Leu Pro Glu Ala Thr His Glu Asp Ile Val Gly Val Gly Lys Gln
100 105 110
Trp Ser Gly Ala Arg Ala Leu Glu Ala Leu Leu Thr Asp Ala Gly Glu
115 120 125
Leu Arg Gly Pro Pro Leu Gln Leu Asp Thr Gly Gln Leu Val Lys Ile
130 135 140
Ala Lys Arg Gly Gly Val Thr Ala Met Glu Ala Val His Ala Ser Arg
145 150 155 160
Asn Ala Leu Thr Gly Ala Pro Leu Asn Leu Thr Pro Asp Gln Val Val
165 170 175
Ala Ile Ala Ser Asn Asn Gly Gly Lys Gln Ala Leu Glu Thr Val Gln
180 185 190
Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Asp Gln
195 200 205
Val Val Ala Ile Ala Ser Asn Gly Gly Gly Lys Gln Ala Leu Glu Thr
210 215 220
Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro
225 230 235 240
Ala Gln Val Val Ala Ile Ala Ser Asn Ile Gly Gly Lys Gln Ala Leu
245 250 255
Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu
260 265 270
Thr Pro Asp Gln Val Val Ala Ile Ala Ser Asn Gly Gly Gly Lys Gln
275 280 285
Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His
290 295 300
Gly Leu Thr Pro Ala Gln Val Val Ala Ile Ala Ser Asn Gly Gly Gly
305 310 315 320
Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln
325 330 335
Ala His Gly Leu Thr Pro Asp Gln Val Val Ala Ile Ala Ser His Asp
340 345 350
Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu
355 360 365
Cys Gln Ala His Gly Leu Thr Pro Ala Gln Val Val Ala Ile Ala Ser
370 375 380
Asn Gly Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro
385 390 395 400
Val Leu Cys Gln Ala His Gly Leu Thr Pro Asp Gln Val Val Ala Ile
405 410 415
Ala Ser Asn Gly Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu
420 425 430
Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Ala Gln Val Val
435 440 445
Ala Ile Ala Ser Asn Gly Gly Gly Lys Gln Ala Leu Glu Thr Val Gln
450 455 460
Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Asp Gln
465 470 475 480
Val Val Ala Ile Ala Ser Asn Asn Gly Gly Lys Gln Ala Leu Glu Thr
485 490 495
Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro
500 505 510
Asp Gln Val Val Ala Ile Ala Ser Asn Gly Gly Gly Lys Gln Ala Leu
515 520 525
Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu
530 535 540
Thr Pro Asp Gln Val Val Ala Ile Ala Ser Asn Ile Gly Gly Lys Gln
545 550 555 560
Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His
565 570 575
Gly Leu Thr Pro Asp Gln Val Val Ala Ile Ala Ser His Asp Gly Gly
580 585 590
Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln
595 600 605
Ala His Gly Leu Thr Pro Asp Gln Val Val Ala Ile Ala Ser His Asp
610 615 620
Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu
625 630 635 640
Cys Gln Ala His Gly Leu Thr Pro Ala Gln Val Val Ala Ile Ala Ser
645 650 655
His Asp Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro
660 665 670
Val Leu Cys Gln Ala His Gly Leu Thr Pro Asp Gln Val Val Ala Ile
675 680 685
Ala Ser Asn Gly Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu
690 695 700
Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Ala Gln Val Val
705 710 715 720
Ala Ile Ala Ser His Asp Gly Gly Arg Pro Ala Leu Glu Ser Ile Val
725 730 735
Ala Gln Leu Ser Arg Pro Asp Pro Ala Leu Ala Ala Leu Thr Asn Asp
740 745 750
His Leu Val Ala Leu Ala Cys Leu Gly Gly Arg Pro Ala Met Asp Ala
755 760 765
Val Lys Lys Gly Leu Pro His Ala Pro Glu Leu Ile Arg Arg Val Asn
770 775 780
Arg Arg Ile Gly Glu Arg Thr Ser His Arg Val Ala Gly Ser Gln Leu
785 790 795 800
Val Lys Ser Glu Leu Glu Glu Lys Lys Ser Glu Leu Arg His Lys Leu
805 810 815
Lys Tyr Val Pro His Glu Tyr Ile Glu Leu Ile Glu Ile Ala Arg Asn
820 825 830
Pro Thr Gln Asp Arg Ile Leu Glu Met Lys Val Met Glu Phe Phe Met
835 840 845
Lys Val Tyr Gly Tyr Arg Gly Glu His Leu Gly Gly Ser Arg Lys Pro
850 855 860
Asp Gly Ala Ile Tyr Thr Val Gly Ser Pro Ile Asp Tyr Gly Val Ile
865 870 875 880
Val Asp Thr Lys Ala Tyr Ser Gly Gly Tyr Asn Leu Pro Ile Gly Gln
885 890 895
Ala Arg Glu Met Gln Arg Tyr Val Glu Glu Asn Gln Thr Arg Asn Lys
900 905 910
His Ile Asn Pro Asn Glu Trp Trp Lys Val Tyr Pro Ser Ser Val Thr
915 920 925
Glu Phe Lys Phe Leu Phe Val Ser Gly His Phe Lys Gly Asn Tyr Lys
930 935 940
Ala Gln Leu Thr Arg Leu Asn His Ile Thr Asn Cys Asn Gly Ala Val
945 950 955 960
Leu Ser Val Glu Glu Leu Leu Ile Gly Gly Glu Met Ile Lys Ala Gly
965 970 975
Thr Leu Thr Leu Glu Glu Val Arg Arg Lys Phe Asn Asn Gly Glu Ile
980 985 990
Asn Phe

Claims (10)

1.一种利用家蚕生产蜘蛛丝蛋白的方法，包括如下步骤：
1)在家蚕的丝素重链基因FibH上选择TALENs的两个靶点，第一靶点位于FibH基因的二号外显子上，第一靶点包括左半位点FibH1-L即TALEN左臂和右半位点FibH1-R即TALEN右臂，其中FibH1-L与FibH1-R之间有12-20bp的第一间隔序列作为切割序列；第二靶点位于FibH基因的3’非翻译区之外，第二靶点包括左半位点FibH2-L即TALEN左臂和右半位点FibH2-R即TALEN右臂，其中FibH2-L与FibH2-R之间有12-20bp的第二间隔序列作为切割序列；
2)分别构建用于表达类转录激活因子效应物核酸酶TALEN1-L、TALEN1-R、TALEN2-L和TALEN2-R的质粒，其中TALEN1-L、TALEN1-R、TALEN2-L和TALEN2-R分别用于切割FibH1-L、FibH1-R、FibH2-L和FibH2-R；
3)体外转录用于表达TALEN1-L、TALEN1-R、TALEN2-L和TALEN2-R的质粒，合成得到作为TALEN1-L、TALEN1-R、TALEN2-L和TALEN2-R翻译模板的TALENs mRNA；
4)构建包含蜘蛛丝基因MaSp1的donor载体，该donor载体的两端分别含有第一靶点两旁的同源序列和第二靶点两旁的同源序列，两端同源序列之间含有用于替换基因FibH的目的基因MaSp1；
5)将步骤3)中得到的mRNA和步骤4)中得到的donor载体混合，两者的终浓度各自控制在200-300ng/μl，对家蚕进行显微注射，注射后的蚕卵进行催青直到孵化，饲养孵化出来的蚁蚕，将当代G0的蚕蛾自交，获得G1代蚕卵，饲养出生产蜘蛛丝蛋白的家蚕个体。
2.如权利要求1所述的方法，其特征在于，所述第一间隔序列的长度为14-18bp，所述第二间隔序列的长度为14-18bp。
3.如权利要求1所述的方法，其特征在于，用于表达TALEN1-L、TALEN1-R、TALEN2-L和TALEN2-R的质粒分别为pSW-epeas-FibH1-L、pSW-epeas-FibH1-R、pSW-epeas-FibH2-L、pSW-epeas-FibH2-R。
4.如权利要求1所述的方法，其特征在于，步骤1)中所述FibH1-L序列是SEQ ID NO:1；FibH1-R序列是SEQ ID NO:2；FibH2-L序列是SEQ ID NO:3；FibH2-R序列是SEQ ID NO:4。
5.如权利要求1所述的方法，其特征在于，步骤4)中所述基因MaSp1是序列SEQ ID NO:5。
6.如权利要求4所述的方法，其特征在于，步骤4)中所述TALEN1-L的氨基酸序列为SEQID NO:6；TALEN1-R的氨基酸序列为SEQ ID NO:7；TALEN2-L的氨基酸序列为SEQ ID NO:8；TALEN2-R的氨基酸序列为SEQ ID NO:9。
7.如权利要求1所述的方法，其特征在于，步骤4)中所述donor载体上还含有筛选标签。
8.如权利要求7所述的方法，其特征在于，筛选标签是红色荧光蛋白。
9.如权利要求4所述的方法，其特征在于，步骤4)中所述donor载体的构建方法是：克隆出FibH1 TALENs位点上游1356bp和FibH2 TALENs位点下游1113bp的两个片段；将这两个片段依次连接到PJET-1.2载体上；将去除ATG起始系列的MaSp1基因连接在上述1356bp和1113bp的两个片段之间，并在MaSp1序列后加入家蚕FibH基因的3’-UTR序列，构成donor载体。
10.如权利要求4所述的方法，其特征在于，步骤4)中所述donor载体的构建方法是：克隆出FibH1 TALENs位点上游1356bp和FibH2 TALENs位点下游1113bp的两个片段；将这两个片段依次连接到PJET-1.2载体上；将去除ATG起始系列的MaSp1基因连接在上述1356bp和1113bp的两个片段之间，并在MaSp1序列后加入家蚕FibH基因的3’-UTR序列；再将红色荧光蛋白筛选标签克隆连接在3’-UTR序列之后，构成donor载体PJET-1.2-MaSp1-donor。
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