CN112575032A - 一种抗Bt毒素家蚕的选育方法 - Google Patents
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Abstract
本方案公开一种抗Bt毒素家蚕的选育方法，利用基因组编辑技术，对ABCC2基因进行敲除，构建出对Bt Cry1Ac毒素有抗性的家蚕敲除品系。对照品系Cry1Ac毒素的LC50浓度为37.5 mg/L,而具有Cry1Ac抗性的实验品系在375 mg/L的浓度下仍然能够正常生长发育。通过将家蚕敲除品系与生产实用品系菁松进行杂交，将敲除后的ABCC2基因导入菁松基因组中，并通过菁松回交和分子标记辅助育种，培育出既具备优良经济性状，又对Cry1Ac毒素有较高抗性的实用品系,该品系能有效防止Bt Cry1Ac毒素对蚕业生产造成的危害。
Description
技术领域
本发明涉及基因工程领域，运用了CRISPR/Cas9介导的基因组编辑技术再结合遗传育种手段，主要涉及一种抗Bt毒素家蚕的选育方法。
背景技术
家蚕是重要的经济昆虫，也是我国许多农民和企业收入的来源，为我国的经济发展做出了重大的贡献。细菌病是危害蚕业生产的四大传染病之一，家蚕食下苏云金芽胞杆菌(简称Bt)及其变种会引起细菌性中毒病又名卒倒病是常见的细菌病，具有致病力强和传染性强的特点。本病在夏秋蚕期多有发生，当蚕食下毒素较多时，本病急性发作，食桑突然停止，前半身抬起，呈现痉挛性抖动，很快倒伏死亡。当蚕食下毒素较少时，本病慢性发展，一般经2-3天，食欲渐减，发育迟缓，大多数即出现空胸、下痢、粪结等症状。病蚕尸体初死时体色正常，体躯强直紧张，胸部稍为伸长和膨大，约经10多个小时，从胸腹交界处的环节，开始变色并向首尾伸展，以至全身变为黑褐色，内容物腐烂液化。体皮易破，流出黑褐色的恶臭污液。
随着分子时代的到来，家蚕抗病育种的手段已从传统的常规性育种转向转基因育种。传统选育方法筛选抗性品种面临着周期长、定向性差以及提高抗性的同时难以兼顾经济性状等局限。2013年初发现的人工核酸内切酶CRISPR/Cas9系统是一种更为简单有效的基因组编辑工具。它以其高效的敲除效率、简单的操作、靶点选择的宽泛性、广泛的物种适应性及实验周期短等优势，所以正受到越来越多研究者的青睐。
在实际生产中，家蚕茧丝产量才是至关重要的,所以在选育抗病家蚕的同时要兼顾家蚕的茧丝量。利用CRISPR/Cas9介导的基因组编辑技术对家蚕抗病相关的关键基因进行基因组编辑，得到需要的敲除品系，然后与体质强健、茧质优良的实用生产品种杂交，再通过回交和分子标记辅助育种的方法，培育出既具备抗病又保留优良经济性状的家蚕实用品系。是防止蚕病对蚕业生产造成危害最安全经济、绿色环保的手段，也将会是一条更快捷、更稳定更广泛的抗病育种新途径。
发明内容
本发明利用CRISPR/Cas9介导的基因组编辑技术对家蚕体内负责转运Bt毒素的ABCC2关键基因进行敲除，再结合遗传育种的手段，从而选育出抗Bt毒素家蚕的方法，具体方案如下：运用基因组编辑技术结合遗传育种手段，选育出具备Bt Cry1Ac毒素抗性的实用家蚕品种
进一步改进在于，通过破坏家蚕体内负责转运Bt毒素的ABCC2基因使家蚕对BtCry1Ac毒素产生抗性。
进一步改进在于，在ABCC2基因上设计sgRNA靶点，通过CRISPR/Cas9技术构建敲除ABCC2基因的家蚕实验品系。
进一步改进在于，通过将抗Bt Cry1Ac毒素的家蚕敲除品系与实用生产品系菁松进行杂交，再通过菁松回交和分子标记辅助育种的方法，仅将抗性基因导入到菁松品系，从而培育出既具备Bt Cry1Ac 毒素抗性又保留优良经济性状的家蚕实用品种。
抗Bt毒素家蚕的选育方法，包括以下步骤：
步骤一、G1代：运用CRISPR/Cas9介导的基因组编辑技术，首先要设计靶点并验证，构建PXL-IE1-DsRed-U6-sgRNA载体，然后通过显微注射技术将构建好的质粒注射到Nistari家蚕的蚕卵体内，等蚕卵孵出后饲养到蛾期自交；
步骤二、G2代：G1代自交的蚕卵孵出后，筛选出红色荧光个体，即为携带可遗传的sgRNA的个体，根据CRISPR/Cas9二元杂交系统，红荧光个体与绿荧光Cas9转基因Nistari品系家蚕杂交，通过筛选双荧光个体家蚕从而构建ABCC2的基因敲除品系；
步骤三、G3代：G2代杂交的蚕卵孵出后，通过筛选双荧光个体家蚕，即为ABCC2基因敲除个体，2龄起蚕时添毒进行筛选，再通过检测敲除体和对照组基因组序列的差异，初步得到抗Bt Cry1Ac毒素的Nistari品系家蚕；
步骤四、G4代：G3代家蚕自交，对孵出的家蚕进行荧光检测、添毒和基因组序列鉴定，筛选出纯合的无荧光抗Bt Cry1Ac毒素Nistari品系家蚕，得到非转基因品系家蚕能排除sgRNA和Cas9转基因质粒的影响；
步骤五、G5-G9代：G4代中筛选出纯合的抗病家蚕与具有优良经济性状的菁松品种家蚕进行杂交，再与菁松回交4代并通过分子标记辅助育种，仅将抗性基因导入到菁松品系，而基因组的其它部分仍保留菁松的特征；
步骤六、G10代：G9代家蚕品种自交，进行抗Cry1Ac毒素能力和基因组序列的鉴定，筛选出对Cry1Ac毒素具有较高抗性的纯合实用家蚕品种，该品种既具备Bt Cry1Ac毒素抗性又保留优良经济性状。
相对于现有技术，本方案的有益效果在于：通过CRISPR/Cas9介导的基因组编辑技术再结合遗传育种手段，有选择性的对家蚕体内负责转运Bt Cry1Ac毒素的ABCC2关键基因进行敲除，得到抗Bt Cry1Ac毒素的Nistari品系家蚕，再与具有优良经济性状的菁松品种家蚕进行杂交，再通过与菁松回交和分子标记辅助育种的方法，培育出既具备Bt Cry1Ac毒素抗性又保留优良经济性状的家蚕实用品系，有效的防止Bt Cry1Ac毒素对蚕业生产造成的危害。
附图说明
图1是育种方法流程图；
图2 是 CRISPR/Cas9系统的PXL-IE1-DsRed-U6-sgRNA和Cas9质粒图谱；
图3是预测ABCC2基因的功能域示意图；
图4 是靶点在ABCC2基因外显子上的位置示意图；
图5中 A图为靶点序列扩增的琼脂糖凝胶电泳图，B图为重组质粒PXL-IE1-DsRed-U6-sgRNA的菌液PCR验证结果示意图，C图为重组质粒的测序验证结果示意图；
图6 是突变体和对照组野生型突变家蚕个体荧光检测图；
图7是敲除体（a）和对照组（b）2龄起蚕添食Bt Cry1Ac毒素48h后的表现型结构示意图；
图8 是敲除组Nistari和对照组Nistari基因组DNA序列差异示意图。
具体实施方式
为了使本技术领域的人员更好地理解本发明方案，下面将结合本发明实施例，对本发明实施例中的技术方案进行清楚、完整地描述。显然，所描述的实施例仅仅是本发明一部分实施例，而不是全部的实施例，说明书中给出了本发明的较佳实施例。本发明可以以许多不同的形式来实现，并不限于本文所描述的实施例，相反地，提供这些实施例的目的是使对本发明的公开内容的理解更加透彻全面。基于本发明中的实施例，本领域普通技术人员在没有作出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。
本发明提供了一种抗Bt毒素家蚕的选育方法，育种技术流程图如图1所示。该方法通过CRISPR/Cas9介导的基因组编辑技术，对家蚕体内负责转运Bt毒素的ABCC2关键基因进行敲除，再结合遗传育种手段，培育出既具备Bt毒素抗性又保留优良经济性状的家蚕实用品系。
实施例，我们通过对家蚕遗传和分子水平的研究发现家蚕体内ABCC2基因与BtCry1Ac毒素中的转运密切相关，破坏ABCC2基因能够使家蚕对Cry1Ac毒素产生抗性。且该抗性属于常染色体隐性遗传，实验选用的是Nistari家蚕品种和实用生产品系菁松家蚕，它们都对Bt Cry1Ac毒素敏感。Nistari家蚕品种是无滞育多化性品种不仅对Bt Cry1Ac毒素敏感，而且体质强健，在秋冬季很容易饲养成活，适合做为选育抗Bt Cry1Ac毒素的实验品种
参照图2，本实例采用CRISPR/Cas9介导的基因组编辑技术进行品种选育，需要先得到PXL-IE1-DsRed-U6-sgRNA质粒，然后显微注射得到sgRNA转基因Nistari家蚕，带有红荧光。Cas9转基因Nistari家蚕带有绿荧光无需构建由中国农业科学院蚕业研究所提供。
首先在NCBI网站(https://www.ncbi.nlm.nih.gov/)查找家蚕ABCC2基因的ORF序列和基因组序列，在SMART网站(http://smart.embl.de/)对该基因进行功能域分析，参照图3。根据ABCC2基因的功能域，用Cas9 Target Finder 网站对该功能域内的外显子上设计靶点，筛选出特异性高的靶点，参照图4。在靶点基因组序列两边设计引物，PCR扩增序列连接PMD-19T进行验证,防止靶点序列已突变，导致敲除脱靶。
构建sgRNA转基因载体，采用同源重组的方法，将靶点连接到PXL-IE1-DsRed-U6载体。实验方法是以PXL-IE1-DsRed-U6空载体为模板，设计引物扩增出137bp的含靶点的片段，为sgRNA1，参照图5。图5中的C图为重组质粒的测序验证结果示意图，白框的为靶点序列，用NheI快切酶酶切空载体 PXL-IE1-DsRed-U6，用同源重组试剂盒将sgRNA1与酶切后的PXL载体同源重组，获得质粒。再通过菌液PCR和测序进一步确认。确认后对构建好的PXL-IE1-DsRed-U6-sgRNA质粒进行中抽。
运用显微注射的方法，将Nistari 品系蚕卵用37%的甲醛蒸气消毒5-10min后，用微量注射仪进行注射。注射物质为PXL-IE1-DsRed-U6-sgRNA转基因质粒和helper辅助质粒的混合物（1:1），质粒的浓度至少为400 ng/µL，注射体积3-5nL左右。家蚕胚胎注射后立即用“502”胶封住，放置于无菌操作台中，待胶晾干放在无菌透明盒中密封，置于25 ℃条件下催青。将孵化的Nistari蚕卵养成虫，为G1代。
G2代：因为G1代刚注射质粒，孵出的一龄蚕即使质粒导入成功，红色荧光也不强，无法筛选，所以G1代饲养至蛾期自交，产出的蚕卵孵出蚕蚁后筛选出带红荧光的家蚕个体，即为阳性个体。
G3代：根据二元杂交系统，G2代饲养至蛾期的家蚕与绿荧光Cas9转基因Nistari家蚕杂交，蚕卵孵出后通过筛选双荧光个体家蚕，构建ABCC2的基因敲除品系详见图6。图6 为突变家蚕个体荧光检测图，左右图为同一组蚕，只是在不同荧光检测下的情况，左侧图显示红绿荧光的是突变体，右侧图黑色的为对照组野生型。
验证基因组编辑后家蚕对Bt Cry1Ac毒素的反应。Bt Cry1Ac毒素对正常Nistari家蚕的LC50为37.5 mg/L,选择对照组和突变个体家蚕各200头， 2龄起蚕添食浓度为375mg/L的Bt Cry1Ac毒蛋白溶液，观察突变体和对照组在添毒后48h的情况，如图7，敲除体（a）和对照组（b）2龄起蚕添食Bt Cry1Ac毒素48h后的表现，图中所放的实验家蚕数量为实际的10%。结果显示，敏感Nistari体内ABCC2基因组被编辑后，纯合的突变体Nistari对BtCry1Ac产生了抗性，而对照组全部死亡。
为了确定CRISPR/Cas9介导的基因组编辑技术是否对ABCC2基因成功敲除，对突变体的敲除靶点基因组序列进行验证。突变体Nistari家蚕和对照组Nistari家蚕各随机选择3头，提取基因组DNA，设计引物PCR克隆扩增并测序。与对照组的比对结果显示，突变体Nistari靶点基因组发生了不同程度的缺失或突变，有的产生了148bp的缺失，有的只产生了11bp的缺失，且短的缺失与突变区域发生在基因的外显子部分，参照图8，图8.1中WTG是对照组Nistari，1、2为敲除体Nistari；标黑的序列是靶点序列；图8.2中WTG为对照组Nistari，3为敲除体Nistari，框里面的为靶点序列。这一结果显示CRISPR/Cas9介导的基因组编辑可以诱导该基因的核苷酸序列突变并且具有高效的敲除效率，干扰了ABCC2基因的正常功能，使敲除体家蚕Nistari对Bt Cry1Ac毒素产生了极强的抗性。具有双荧光且对BtCry1Ac毒素产生抗性的为G3代。
G4代：G3代家蚕自交，对孵出的家蚕进行荧光、添毒和基因组序列检测，筛选出纯合的无荧光抗Bt毒素Nistari品系家蚕。该品系家蚕是非转基因的，排除sgRNA和Cas9转基因对家蚕食品安全的影响；
G5-G9代：G4代中筛选出纯合抗病家蚕与具有优良经济性状的菁松品种家蚕进行杂交，再与菁松回交四代并通过分子标记辅助育种，仅将抗性基因导入到菁松品系，而基因组的其它部分仍保留菁松的特征；
G10代：G9代家蚕品种自交，进行抗Cry1Ac毒素能力和基因组序列的鉴定，筛选出既具备Bt Cry1Ac毒素抗性又保留优良经济性状的家蚕实用品系。为了确定ABCC2基因的敲除后不会影响实用品种家蚕经济性状，选择野生型菁松家蚕为对照组，在相同且适宜的条件下饲养，对它们从5龄起蚕到熟蚕每天的体重以及化蛹后的蛹重及茧丝量进行调查。确保选育出既对Bt Cry1Ac毒素具备较高抗性又保留优良经济性状的实用家蚕品种。
综上所述，本发明利用CRISPR/Cas9介导的基因组编辑技术再结合遗传育种手段，对家蚕进行连续10代的选择育种，通过特定的筛选方法，选育出既具备Bt Cry1Ac毒素抗性又保留优良经济性状的家蚕实用品系，有效的防止Bt Cry1Ac毒素对蚕业生产造成的危害，符合实际农业生产的需要。
Claims (5)
1.一种抗Bt毒素家蚕的选育方法，其特征在于，运用基因组编辑技术结合遗传育种手段，选育出具备Bt Cry1Ac毒素抗性的实用家蚕品种。
2.如权利要求1所述的一种抗Bt毒素家蚕的选育方法，其特征在于，通过破坏家蚕体内负责转运Bt毒素的ABCC2基因使家蚕对Bt Cry1Ac毒素产生抗性。
3.如权利要求2所述的一种抗Bt毒素家蚕的选育方法，其特征在于，在ABCC2基因上设计sgRNA靶点，通过CRISPR/Cas9技术构建敲除ABCC2基因的家蚕敲除品系。
4.如权利要求3所述的一种抗Bt毒素家蚕的选育方法，其特征在于，通过将抗BtCry1Ac毒素的家蚕敲除品系与实用生产品系菁松进行杂交，再通过菁松回交和分子标记辅助育种的方法，仅将抗性基因导入到菁松品系，从而培育出家蚕实用品系。
5.如权利要求4所述的一种抗Bt毒素家蚕的选育方法，其特征在于，包括以下步骤：
步骤一、G1代：运用CRISPR/Cas9介导的基因组编辑技术，首先根据ABCC2基因的ORF序列设计靶点并验证，构建PXL-IE1-DsRed-U6-sgRNA载体，然后通过显微注射技术将构建好的质粒注射到Nistari家蚕的蚕卵体内，等蚕卵孵出后饲养到蛾期自交；
步骤二、G2代：G1代自交的蚕卵孵出后，筛选出红色荧光个体，即为携带可遗传的sgRNA的个体，根据CRISPR/Cas9二元杂交系统，红荧光个体与绿荧光Cas9转基因Nistari品系家蚕杂交，构建ABCC2的基因敲除品系；
步骤三、G3代：G2代杂交的蚕卵孵出后，通过筛选双荧光个体家蚕，即为ABCC2基因敲除个体，2龄起蚕时添毒进行筛选，再通过检测敲除体和对照组基因组序列的差异，初步得到抗Bt Cry1Ac毒素的Nistari品系家蚕；
步骤四、G4代：G3代家蚕自交，对孵出的家蚕进行荧光检测、添毒和基因组序列鉴定，筛选出纯合的无荧光抗Bt Cry1Ac毒素Nistari品系家蚕，得到非转基因品系家蚕能排除sgRNA和Cas9转基因质粒的影响；
步骤五、G5-G9代：G4代中筛选出纯合的抗病家蚕与具有优良经济性状的菁松品种家蚕进行杂交，再与菁松回交4代并通过分子标记辅助育种，仅将抗性基因导入到菁松品系，而基因组的其它部分仍保留菁松的特征；
步骤六、G10代：G9代家蚕品种自交，进行抗Cry1Ac毒素能力和基因组序列的鉴定，筛选出对Cry1Ac毒素具有较高抗性的纯合实用家蚕品种。
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