利用CRISPR/Cas9基因编辑技术创制水稻光敏色素PHYB基因突变体

Generation of the Mutations for PHYB in Rice Using CRISPR/Cas9 Approach

光敏色素是植物感受外界环境变化的最重要光受体之一,为研究光敏色素在水稻生长发育中的作用,本研究利用CRISPR/Cas9技术原理,设计水稻光敏色素基因PHYB的靶点,并将目的靶位点连接转化至载体pBUN411,成功构制了PHYB的CRISPR/Cas9表达载体,转化粳稻品种日本晴。由酶切及测序分析显示,转基因阳性植株中有6株靶位点序列发生改变,获得了碱基插入“A”或“T”和碱基缺失1bp或2bp的4种突变基因型,且在T0代就获得了纯合突变体。同时,本研究对T1代水稻phyB突变体进行红光照射处理,发现2叶1心期phyB突变体红光照射5d后,第3节间长度相对于野生型极显著增长。本研究利用CRISPR/Cas9系统成功对水稻PHYB基因进行了定点编辑,为进一步研究PHYB在水稻中的功能提供了参考依据。

Phytochrome is one of the mos t import ant photoreceptors for plants, as it has the ability to sense multiple parameters of ambient. To study the role of phytochromes in rice growth and development, the target of rice phytochrome gene PHYB was designed using CRISPR/Cas9 technology. Then the target site was ligated into vector pBUN411 for constructing. The CRISPR/Cas9 expression vector of PHYB successfully. The recombinant plasmids were transferred into japonica cultivar Nipponbare. Using enzyme digestion and sequencing analysis, six mutants were identified from the transgenic positive plants, including 4 mutation types with insert "A" or "T" and bases deletion 1 bp or 2 bp. The homozygous mutants could be obtained from the T0 generation. Moreover, we treated the rice phyB mutants of T1 generation in the 2-leaf 1 heart stage with red light irradiation for 5 days, and found that the length of the third internode was significantly increased compared with the wild type. In this study, the rice PHYB gene was knocked out successfully by CRISPR/Cas9 system, which laid an important foundation for further study on the function of PHYB in rice.