利用CRISPR/Cas9基因编辑系统创制新型水稻温敏雄性核不育系

Generation of a New Thermo-sensitive Genic Male Sterile Line by Using CRISPR/Cas9 Gene Editing System

为迅速获得优良的温敏雄性核不育(TGMS)系,本研究利用CRISPR/Cas9基因编辑系统对一份优良的水稻品系GXU41的TMS5基因上2个靶点进行编辑,对编辑植株进行靶点突变检测,筛选无外源DNA的编辑TGMS系,并进行育性转换、农艺性状和米质分析评价。结果共获得12株T_(0)代转基因阳性植株,其中靶点1和靶点2的纯合突变率分别为50%和58.4%,一个或两个靶点为纯合突变占83.3%,双纯合突变率达到25%,在T_(1)代中筛选到无外源DNA的TGMS系。与野生型相比,编辑获得的TGMS系GXU41-5S随着温度升高小穗育性和花粉育性逐渐降低,在24℃时均表现完全不育;由于不育效应,GXU41-5S的株高增长和有效穗数增加显著,且GXU41-5S全部米质指标达到优质一等大米的标准要求。本研究证明了CRISPR/Cas9基因编辑系统是快速获得TGMS系有效途径,所创制的TGMS系对选育两系杂交水稻品种具有重要应用价值。

In order to quickly obtain excellent temperature-sensitive male sterile(TGMS) lines. In this study, the CRISPR/Cas9 gene editing system was used to edit two targets on the TMS5 gene of an excellent rice line GXU41,to detect target mutations in the edited plants, to screen the edited TGMS lines without exogenous DNA, and to breed them. Sex conversion, agronomic traits and rice quality were analysis and evaluation. A total of 12 T_(0)-generation transgenic positive plants were obtained in the results. The homozygous mutation rates of target 1 and target 2 were 50% and 58.4%, respectively. One or two target sites were homozygous mutations, accounting for83.3%, and the double homozygous mutation rate reached 25%. TGMS lines without exogenous DNA were selected in the T1generation. Compared with the wild type, the edited TGMS line GXU41-5S gradually reduced spikelet fertility and pollen fertility with increasing temperature, and showed complete sterility at 24 ℃;due to the sterility effect, the high growth rate and effective ear number of GXU41-5S strain increased significantly, and all the rice quality indicators of GXU41-5S reached the standard requirements of high-quality first-class rice. This study proved that the CRISPR/Cas9 gene editing system is an effective way to quickly obtain TGMS lines, and the created TGMS lines have important application value for breeding two-line hybrid rice varieties.