利用CRISPR/Cas9技术敲除水稻Pi21基因的效率分析

利用CRISPR/Cas9技术,针对Pi21基因的两个靶位点(靶位1和靶位2),构建基因敲除载体,转化水稻品种南粳9108。经PCR鉴定,获得了28株T0代阳性转基因植株。对靶位点酶切检测发现,靶位1突变效率为78.57%,靶位2突变效率为92.86%;靶位1和靶位2同时突变的效率为78.57%,突变效率较高。通过对靶位点进行测序,发现靶位点突变类型较多,包括碱基缺失、碱基插入、碱基缺失后插入其他碱基和大片段DNA缺失等类型。对突变株系进行氨基酸预测,发现大部分株系都存在移码突变现象而使基因突变彻底,少数株系表现为部分氨基酸的缺失或变异。成功敲除了水稻Pi21基因,并对突变效率和类型进行了分析,为进一步验证Pi21基因功能、培育广谱抗稻瘟病的水稻新品系奠定了基础。

Knock out Efficiency Analysis of Pi21 Gene Using CRISPR/Cas9 in Rice

A CRISPR/Cas9 vector, which contains two targets (Target 1 and Target 2) of Pi21gene, was constructed and transformed to rice variety Nanjing 9108. Twenty eight T0 transgenic lines were obtained and confirmed by T DNA specific PCR. Restriction digestion analysis revealed 78.57% mutant ratio in Target 1 site, 92.86% mutant ratio in Target 2 site, 78.57% ratio of lines containing both Target 1 and Target 2 mutant in transgenic lines. Through sequencing analysis, we found the mutant types of targets, such as deletion of bases, insertion of bases, insertion behinds deletion of bases, and long fragment deletion of DNA, etc. Moreover, except amino acid deletion, most transgenic lines displayed frameshift mutation,which caused function loss of Pi21. In this way, a series of Pi21 knock out rice lines were obtained, and further, could be used for functional analysis of Pi21 and rice blast broad spectrum resistant breeding.