Chen Rongrong, Xu Qilong, Liu Yan, Zhang Jiaojiao, Ren Dongtao, Wang Guoying, Liu Yunjun
Institute of Crop Sciences, Chinese Academy of Agricultural Sciences, Beijing, China.
State Key Laboratory of Plant Physiology and Biochemistry, College of Biological Sciences, China Agricultural University, Beijing, China.
Front Plant Sci. 2018 Sep 7;9:1180. doi: 10.3389/fpls.2018.01180. eCollection 2018.
Male sterility (MS) provides a useful breeding tool to harness hybrid vigor for hybrid seed production. It is necessary to generate new male sterile mutant lines for the development of hybrid seed production technology. The CRISPR/Cas9 technology is well suited for targeting genomes to generate male sterile mutants. In this study, we artificially synthesized gene with biased codons of maize. A CRISPR/Cas9 vector targeting the gene of maize was constructed and transformed into maize using an -mediated method, and eight T independent transgenic lines were generated. Sequencing results showed that genes in these T transgenic lines were not mutated. However, we detected mutations in the gene in F and F progenies of the transgenic line H17. A potential off-target site sequence which had a single nucleotide that was different from the target was also mutated in the F progeny of the transgenic line H17. Mutation in the gene and the male sterile phenotype could be stably inherited by the next generation in a Mendelian fashion. Transgene-free male sterile plants were obtained by screening the F generation of male sterile plants, and the MS phenotype could be introduced into other elite inbred lines for hybrid production.
雄性不育（MS）为利用杂种优势进行杂交种子生产提供了一种有用的育种工具。为了开发杂交种子生产技术，有必要培育新的雄性不育突变系。CRISPR/Cas9技术非常适合靶向基因组以产生雄性不育突变体。在本研究中，我们人工合成了具有玉米偏好密码子的基因。构建了一个靶向玉米基因的CRISPR/Cas9载体，并使用介导法将其转化到玉米中，获得了8个T代独立转基因株系。测序结果表明，这些T代转基因株系中的基因未发生突变。然而，我们在转基因株系H17的F1和F2后代中检测到了该基因的突变。转基因株系H17的F2后代中，一个与靶位点有单个核苷酸差异的潜在脱靶位点序列也发生了突变。该基因的突变和雄性不育表型能够以孟德尔方式稳定地遗传给下一代。通过筛选雄性不育植株的F3代获得了无转基因的雄性不育植株，并且可以将MS表型导入其他优良自交系用于杂交生产。
