基于CRISPR/Cas9系统的水稻光敏色素互作因子OsPIL15基因编辑  

CRISPR/Cas9 System-Based Editing of Phytochrome-Interacting Factor OsPIL15

作　　者：季新[1] 李飞[1] 晏云[1] 孙红正[1] 张静[1] 李俊周[1] 彭廷[1] 杜彦修[1] 赵全志[1] JI Xin LI Fei YAN Yun SUN HongZheng ZHANG Jing LI JunZhou PENG Ting DU YanXiu ZHAO QuanZhi(Agronomy College of Henan .4gricultural University/Collaborative Innovation Center of Henan Grain Crops/ Henan Key Laboratory of Rice Biology, Zhengzhou 45000)

机构地区：[1]河南农业大学农学院/河南粮食作物协同刨新中心/河南省水稻生物学重点实验室,郑州450002

出　　处：《中国农业科学》2017年第15期2861-2871,共11页Scientia Agricultura Sinica

基　　金：河南省重大科技专项(141100110600);河南省高等学校重点科研项目(16A210026)

摘　　要：【目的】光作为一种环境信号,可影响植物的基因表达、酶活性和形态建成。光敏色素互作因子在光信号传导过程中起着重要作用。本研究旨在构建水稻光敏色素互作因子OsPIL15的CRISPR/Cas9表达载体,创制OsPIL15突变体,挖掘水稻功能基因,丰富和完善水稻光信号调控分子机制。【方法】依据CRISPR/Cas9技术原理,设计OsPIL15突变靶点。将所设计靶序列在水稻基因组中进行比对,排除非特异性靶位点,同时使该靶序列含有常用酶切位点,方便后期突变体鉴定。化学合成靶位点寡核苷酸序列并与载体pBUN411连接构建CRISPR/Cas9表达载体,利用农杆菌介导法导入粳稻品种日本晴,以除草剂抗性标记筛选获得阳性转基因植株。利用酶切法判断T0代转基因植株是否发生突变,结合测序结果分析突变单株的突变基因型。将靶点序列在水稻全基因组中进行比对分析,选择5个与靶序列同源性较高且错配在4 bp以内的位点作为潜在脱靶位点进行脱靶效应评估,分析所设计靶序列特异性。【结果】所构建表达载体成功实现了对OsPIL15的定向编辑,酶切显示在选取的25株T0代转基因植株中获得15株突变体,其中包括5株纯合突变体、6株双等位突变体和4株杂合突变体,共10种不同突变基因型和11个突变株系。突变类型以单碱基插入或缺失为主,同时也得到2种56和66 bp较大片段缺失株系。对部分纯合突变、双等位突变和杂合突变体的T1代植株进行分析,结果表明,T0代产生的突变基因型绝大部分能稳定遗传给下一代。T0代纯合突变体后代为纯合突变单株,仅在株系14纯合突变体后代中检测到1株未突变单株;T0代双等位突变体后代可得到2种纯合突变型和1种双等位突变型;T0代杂合突变体后代则可得到纯合、杂合及未突变3种类型。对T0代未突变植株的后继世代酶切分析显示,62株T1代转基因植株均未发生突变,表�【Objective】As an important environmental signal,light can regulate gene expression,affect the activity of enzymes and plant morphogenesis.Phytochrome interacting factors play an important role in the signal transduction of light.Therefore,constructing the expression vector of CRISPR/Cas9 containing rice phytochrome-interacting factors OsPIL15 and creating the ospil15 mutants have important significance to exploit functional genes and enrich light signal regulation of the molecular mechanism of rice.【Method】According to the principle of CRISPR/Cas9 technology,the sg RNA was designed.To exclude non-specific target sites,the sg RNA was analyzed by sequence alignment in the rice genome database.At the same time,the target sequence contained the common restriction site to identify mutants.The oligonucleotides of sg RNA were chemically synthesized and inserted into linearized plasmid p BUN411 to construct the expression vector.Transgenic rice plants harboring sg RNA：Cas9 were obtained by A.tumefaciens-mediated stable transformation and the positive transgenic plants were screened by herbicide resistance.The PCR products of T0 transgenic plants were digested by restriction enzyme to judge whether they were mutants.Then,the mutated genotypes of these mutants were analyzed by DNA sequencing.After searching rice genome using sg RNA sequence,five highly identical sites with less 4 mismatching bases were selected to assess off-target efficiency and specificity of sg RNA.【Result】The recombinant vector succeeded in oriented editing of OsPIL15.The restriction enzyme analysis results indicated that 15 mutants from the 25 randomly selected T0 transgenic lines were obtained.They included 5 homozygous mutants,6 biallelic mutants and 4 heterozygous mutants,and a total of 10 different genotypes and 11 mutant lines.The mutant types were mainly insertions or deletions of single base,besides,two mutant types of large deletions with 56 and 66 bp were obtained.Analysis of T1 transgenic plants from some T0 mutants indicated t

关 键 词：水稻 CRISPR/Cas9 基因编辑 OsPIL15 脱靶效应 

分 类 号：Q943.2[生物学—植物学] S511[农业科学—作物学]