CRISPR/Cas9敲除恢复基因Rf6降低其近等基因系的育性

Fertility of Near Isogenic Lines Reduced by Knockout Restorer Gene Rf6 Using CRISPR/Cas9

利用CRISPR/Cas9系统的特异性及定位编辑的特性对近等基因恢复系L-Rf6中的Rf6进行基因编辑,以验证恢复基因敲除后该近等基因系的育性是否降低。本研究利用CRISPR/Cas9系统对水稻恢复基因Rf6的序列构建了pC1300-Cas9-Rf6载体,通过农杆菌介导转化近等基因恢复系L-Rf6 (具有红莲型不育系的细胞质和保持系的细胞核背景),得到转基因阳性植株,完成转基因植株中的目的基因的序列分析和育性统计。结果表明,T0-Cas9-L-Rf6转基因的编辑率为79.17%。T0-Cas9-L-Rf6育性统计显示,T0-Cas9-L-Rf6的植株的花粉育性和小穗结实率均明显降低。本研究为进一步探究恢复基因对不育基因的作用机理提供了实验材料和新的研究途径。

To verify whether the fertility of the nea r-isogenic line will reduced after the restorer gene Rf6 is knocked out in the near isogenic line L-Rf6, based on the specificity and the characteristics of location-editing of CRISPR/CAS9 system. The editing of Rf6 were performed in the restorer line L-Rf6. In this study, according to the sequence of restore gene Rf6, the pC1300-Cas9-Rf6 vectors were constructed using the CRISPR/CAS9 system to sequence Rf6 of rice restorer gene. The positive transgenic plants were obtained by Agrobacterium mediated transformation of near-isogenic restorer line L-Rf6(with cytoplasm of red-lotus sterile line and nuclear background of maintainer line). The the sequence of the target gene and the fertility in the transgenic plants were analyzed. The results showed that the editing rate of T0-Cas9-L-Rf6 transgenic plants was 79.17%. T0-Cas9-L-Rf6 fertility statistics showed that the pollen fertility and the spikelet fertility rate of T0-Cas9-L-Rf6 plants decreased obviously.This study could provide the experimental materials and the new research approaches for further exploring the mechanism of restoring genes to sterility genes.