CRISPR/Cas9编辑芥菜型多心皮油菜BjROP10基因的研究

油菜多心皮性状可显著增加油菜的每角果粒数，在油菜高产育种中有潜在的应用价值。前期研究发现，芥菜型多心皮油菜J163-4可形成稳定的四心皮性状，且小G蛋白基因BjROP10可能参与调控其多心皮的形成，但其分子机制尚不明确。本研究利用CRISPR/Cas9技术，构建了BjROP10的双靶点载体，并遗传转化芥菜型多心皮油菜J163-4。在T₀代，通过筛选鉴定获得了20株阳性苗，其中13株心皮数目发生改变。通过测序分析了7株心皮数发生变化的转基因阳性植株的BjROP10序列突变情况，结果显示其目标基因序列发生变异，导致无法编码形成正常的BjROP10蛋白。本研究对进一步解析BjROP10基因响应CLV信号调控油菜心皮数发育的功能提供了材料基础，也为研究油菜多心皮性状发育的分子机制奠定理论基础。

Preliminary study of gene editing of BjROP10 by CRISPR/Cas9 in Brassica juncea L.

The multi-carpel trait of rapeseed can significantly increase the number of seeds per silique of rapeseed, which has a potential application value for high-yield breeding. Previous studies have found that the multilocualr Brassica juncea, J163-4, can form a stable four-carpel trait, and the small G protein gene BjROP10 might be involved in regulating the formation of the multiloculus, but the molecular mechanism is still unclear. In this study, using CRISPR/Cas9 technology, a dual-target vector of BjROP10 was constructed and genetically transformed into J163-4. In the T₀ generation, 20 transgenetic positive seedlings were obtained, 13 of which showed changes of the number of carpels. The mutation sites of the target gene in 7 transgenic positive plants which showed the carpel number variation were analyzed by sequencing technology. The gene sequences of BjROP10 in the 7 transgenic positive plants were mutated, resulting in the inability of encoding a normal BjROP10 protein. This study provides a material basis for further analysis of the function of BjROP10 gene in regulating the development of carpel number of rapeseed in response to CLV signals, and provides experimental basis and experimental materials for studying the molecular mechanism of the development of multilocualr trait in rapeseed too.