山黧豆CASase基因DNA全长序列的扩增及CRISPR/Cas9敲除载体的构建  

Amplification of CASase gene from Lathyrus sativus and construction of its knock-out vector via CRISPR/Cas9 system

作　　者：陶英杰[1] 刘凤娟[1] 毕春晓 任雪冰 曲瑞红 李科友[1] 徐全乐[1] TAO Yingjie;LIU Fengjuan;BI Chunxiao;REN QU Ruihong;LI Keyou;XU Quanle(College of Life Sciences,Northwest AS-F University,Yangling,Shaan.ri Xuebing,712100,China)

机构地区：[1]西北农林科技大学生命科学学院,陕西杨凌712100

出　　处：《西北农林科技大学学报（自然科学版）》2018年第8期23-28,38,共7页Journal of Northwest A&F University(Natural Science Edition)

基　　金：国家自然科学基金项目(31401910);中国博士后科学基金面上项目(2016M590975);陕西省博士后科研项目(2016BSHEDZZ119);中央高校基本科研业务费资助项目(2014YB040)

摘　　要：【目的】克隆山黧豆β-腈基丙氨酸合成酶基因(CASase)DNA全长序列,构建CRISPR/Cas9敲除载体,为筛选"低毒、高硫"山黧豆品系奠定基础。【方法】以萌发4d山黧豆幼苗根部为材料,提取其基因组DNA,利用PCR克隆CASase基因全长;经序列同源性、内含子、外显子、sgRNA靶位点和脱靶分析后,在CASasecDNA上选取5个sgRNA靶位点进行寡核苷酸链设计和体外活性检测;从5个sgRNA靶位点中取活性较高的4个sgRNA,利用酶切连接法构建基因敲除载体pPLHACas9-sgRNA20r、pPLHACas9-sgRNA68r、pPLHACas9-sgRNA225f和pPLHACas9-sgRNA256f。【结果】获得了CASase基因3 143bp的DNA全长序列,该序列编码β-腈基丙氨酸合成酶;在CASase cDNA中选取5个sgRNA靶位点在体外均能有效剪切靶序列;菌落PCR检测结果显示,4个CRISPR/Cas9基因敲除载体构建成功。【结论】克隆了山黧豆CASase基因DNA全长,成功构建了该基因上4个sgRNA靶位点的CRISPR/Cas9敲除载体。[Objective] This project cloned full-length DNA sequence of CASase gene from Lathyrus sa- tivus and construct its recombinant knock-out vector via the CRISPR/Cas9 system to provide basis for screening novel lines with low-ODAP and high sulfur-containing amino acids. [Method] The full-length DNA sequence of CASase gene was cloned from radicle DNA of L. sativus via PCR. After alignment, exons and off-target analysis, five target sites on CASase cDNA sequence were selected to design double-stranded Oligo DNA and were inserted into pPLatU6-sgRNA, which was then recombined into the expression vector to get pPLHACas9-sgRNA20r, pPLHACas9 sgRNA68r, pPLHACas9-sgRNA225f and pPLHACas9- sgRNA256f. [Result] The full length DNA sequence of CASase gene was obtained with 3 143 bp length and encoded β-cyanoalanine synthase （CASase）. All the five selected sgRNA worked with target sequence. Colony PCR and sequencing showed that four knockout vectors were conducted successfully. [Conclusion]The full-length DNA sequence of CASase gene was cloned and four knock-out vectors were conducted suc- cessfully via the CRISPR/Cas9 system.

关 键 词：山黧豆 CASase基因 基因克隆 载体构建 CRISPR/Cas9系统 

分 类 号：Q943.2[生物学—植物学] S529[农业科学—作物学]