利用CRISPR-Cas9基因编辑技术获得水稻osarf8-1杂合突变体

Obtain of osarf8-1 Heterozygous Mutant in Rice by CRISPR/Cas9 Genome Editing Technique

为了研究生长素是如何参与调控植物的生长发育过程,我们利用CRISPR-Cas9基因编辑技术,设计水稻生长素响应因子OsARF8的两个靶点,对水稻中的该基因进行敲除。构建基因编辑载体pBIN-sg R-Cas-Os,通过农杆菌介导转化水稻品种‘9522’。我们从潮霉素抗性筛选出的28株转基因植株中鉴定得到针对OsARF8的一种杂合突变类型,共计6棵在外显子第174号碱基处缺失一个C碱基的杂合突变类型osarf8-1,该处碱基突变所对应的是第7个氨基酸的变化,导致亮氨酸(Leu)变成色氨酸(Trp),自此处开始随后的氨基酸序列发生移码突变,并且提前终止于第123个氨基酸,正好位于Os ARF蛋白的DBD功能域的最始端(DBD为120~222个氨基酸),说明DBD功能域在该突变体中完全不能表达,ARF8蛋白的功能将受到极大的影响。本研究成功利用CRISPR-Cas9基因编辑技术精确地敲除了水稻OsARF8基因,为CRISPR技术在水稻中的应用提供了一个可供参考的范例,同时也为进一步研究OsARF8基因功能提供了重要的参考依据。

In order to inves tigate how auxin is involved in the regulation of plant growth and development process, we used the CRISPR-Cas9 gene editing technique to design two targets of the rice auxin response factor OsARF8, which was used to knockout this gene in rice. Gene editing vectors p BIN-sg R-Cas-Os was constructed,and then rice variety‘9522＇was treated by Agrobacterium mediated transformation. We identified a heterozygous mutation type for OsARF8 from 28 transgenic plants screened from hygromycin resistance, and a total of 6 heterozygous mutations that lacked a C base at exon 174 were obtained. The mutation of the base of this site corresponded to the change of the 7 th amino acid, resulting in leucine（Leu） becoming tryptophan（Trp）. Since then, the subsequent amino acid sequence had shift mutation, and previously terminated at 123 rd amino acids,which was located at the very beginning of the DBD domain of Os ARF protein（DBD contained 120~222 amino acids）, indicating that the DBD domain had no expression in the mutant, and the function of the ARF8 protein would be greatly affected in this mutant. In this study, CRISPR-Cas9 gene editing technique was successfully used to knock out the OsARF8 gene of rice accurately, which provided a reference for the applicatin of CRISPR technique in rice. Also, it provided theoretical basis for further study on the function of OsARF8 in rice.