2021年4月22日 星期四
基于CRISPR/Cas9技术的水稻抗稻瘟病基因Pita突变体的创制
Creation of mutant of blast resistance gene Pita in Oryza sativa based on CRISPR/Cas9 technology
2020年 第29卷 第2期 页码[1-7]    下载全文[3.4MB]  
摘要

以粳稻品种‘日本晴’(Oryza sativa subsp. japonica ‘Nipponbare’)为转基因受体,利用CRISPR/Cas9技术对抗稻瘟病基因Pita的第6至第25位碱基进行定点编辑,对突变体进行鉴定,筛选出不含T-DNA区的纯合突变体,并对该基因纯合突变体的稻瘟病抗性及6个稻瘟病病程相关基因的表达水平进行分析。结果表明:在获得的8株T0代阳性转基因植株中,有7株为突变体植株,包括1株杂合体和6株纯合体,突变率和纯合突变率分别为87.5%和85.7%,突变类型包括碱基置换、碱基插入和碱基缺失。通过PCR检测获得42株T1代T-DNA阴性植株,全部为纯合突变体。纯合突变体的稻瘟病抗性分析结果表明:接种稻瘟病菌小种CH199(Magnaporthe oryzae strain CH199)后,Pita基因编辑材料V1-38-5的T2代植株叶片病斑面积明显大于野生型‘日本晴’,平均病级为4.1±0.2,极显著(P<0.01)高于野生型‘日本晴’(3.0±0.2)。此外,与野生型‘日本晴’相比,V1-38-5的T2代植株PR2和PR1b基因的相对表达量在接种稻瘟病菌小种CH199 12 h时较低,其PR2、PR1b、PR3和E2F基因的相对表达量在接种稻瘟病菌小种CH199 24 h时也较低。研究结果显示:利用CRISPR/Cas9技术对水稻Pita基因进行定点编辑能够获得稳定遗传且更易感病的纯合突变体材料,这些材料可用于水稻抗性品种选育和品质改良,在一定程度上加速水稻定向分子育种的进程。

Abstract

Taking Oryza sativa subsp. japonica ‘Nipponbare’ as transgenic acceptor, the fixed-point editing of the 6th to 25th bases of blast resistance gene Pita was performed by CRISPR/Cas9 technology, and the mutants were identified, the homozygous mutants without T-DNA region were screened out, and the blast resistance and the expression levels of six blast pathogenesis-related genes in homozygous mutants of this gene were analyzed. The results show that among eight T0 positive transgenic plants obtained, there are seven mutant plants, including one heterozygote and six homozygotes, and the mutation rate and homozygous mutation rate are 87.5% and 85.7%, respectively, and the mutation types include base substitution, base insertion, and base deletion. Forty-two T-DNA negative plants of T1 generation are obtained by using PCR detection, and all of them are homozygous mutants. Analysis result of blast resistance of homozygous mutants shows that after inoculating Magnaporthe oryzae strain CH199, leaf spot area of T2 generation plants of Pita gene editing material V1-38-5 is evidently larger than that of wild type of ‘Nipponbare’, and the average disease grade is 4.1±0.2, which is extremely significantly (P<0.01) higher than that of wild type of ‘Nipponbare’ (3.0±0.2). Besides, compared with wild type of ‘Nipponbare’, relative expression levels of PR2 and PR1b genes in T2 generation of V1-38-5 are relatively low when inoculating M. oryzae strain CH199 for 12 h, and those of PR2, PR1b, PR3, and E2F genes are also relatively low when inoculating M. oryzae strain CH199 for 24 h. It is suggested that the fixed-point editing Pita gene in O. sativa by CRISPR/Cas9 technology can obtain homozygous mutant materials with stable inheritance and more susceptible to diseases, these materials can be used for resistant variety breeding and quality improvement of O. sativa, and accelerate the process of directional molecular breeding of O. sativa to a certain extent.

关键词水稻; 抗稻瘟病基因Pita; CRISPR/Cas9技术; 基因编辑; 纯合突变体
Key wordsOryza sativa Linn.; blast resistance gene Pita; CRISPR/Cas9 technology; gene editing; homozygous mutant
作者吴凡, 王月, 陈闽, 刘佳, 杭悦宇, 孙小芹
所在单位江苏省中国科学院植物研究所(南京中山植物园), 江苏 南京 210014
点击量659
下载次数8
基金项目国家自然科学基金资助项目(31470448); 江苏省中国科学院植物研究所博士人才科研启动基金项目(011900610)