BnCIPK9调控油菜种子含油量的分子机制

Previous studies have demonstrated that the gene CIPK9 is related to plant stress resistances, almost nothing is reported about the regulation of seed oil accumulation by CIPK9. Our previous study had showed that BnCIPK9 is a negative regulator for the seed oil content by more than 5 percentage points in Brassica napus, but we do not know how BnCIPK9 control the seed oil. In this research project, we plan to conduct several following investigations. To analyze the copy number of BnCIPK9, and to investigate the function of BnCIPK9 through the genetic complementation test and CRISPR technology based on the resequencing data of 218 varieties in Brassica napus, and to ascertain the functional copy of BnCIPK9. With the use of candidate gene association analysis, we will detect the variation of BnCIPK9 in a natural population of 218 varieties, and discover the excellent alleles and functional SNPs of BnCIPK9. The genes that interacted with the BnCIPK9 will be screened out through the yeast two hybrid technology and RNA-seq of those materials developed from Westar. And finally, we will gain comprehensively insight into the mechanism of BnCIPK9 regulating seed oil accumulation in rapeseed. The results of this research will broaden the research ideas on seed oil accumulation of rapeseed/plant, which will provide new references and evidences for the biosynthesis and regulation of vegetable oils, make the theory of synthesis and accumulation mechanism of SNF1 homologous gene SnRKs more scientific, and be very helpful in improving rapeseed/plant high-oil breeding programs.

CIPK9是业已证明了的植物抗逆相关基因，其对种子含油量的影响未见报道。我们的前期研究发现，BnCIPK9负调控油菜种子含油量超过5个百分点，但其作用机制仍不清楚。本申请拟开展研究内容包括，分析油菜中BnCIPK9的拷贝数，利用CRISPR及互补实验等方法验证BnCIPK9的功能，确定其功能拷贝；利用候选基因关联分析法挖掘BnCIPK9的优异等位基因并寻找功能性SNP位点；利用酵母双杂筛选与BnCIPK9互作的蛋白，通过源于Westar的不同油菜材料的不同发育时期角果转录组测序筛选差异表达基因，解析BnCIPK9参与的代谢路径，最终揭示BnCIPK9在油菜种子含油量调控中的作用机理。本项目的开展将拓宽油菜种子含油量调控的研究思路，既为植物油脂生物合成及调控提供新的参考和证据，使酵母SNF1同源基因SnRKs在植物油脂合成与积累机制的理论更加科学完善，亦为油菜乃至植物高油改良提供新途径。

植物中相关研究表明，CIPK9可参与种子含油量调控。为了对BnaCIPK9在油菜种子油脂调控中发挥的功能进行解析本项目主要开展了以下研究：甘蓝型油菜SnRK基因家族与含油量相关候选基因关联分析以及SnRK家族基因特性和功能系统分析；BnaCIPK9基因沉默和bnacipk9功能缺失甘蓝型油菜突变体材料创建；BnaCIPK9调控种子油脂合成代谢生理路径功能分析；BnaCIPK9同源拷贝功能研究；油脂合成积累关键时期种子转录组和脂质组分析；BnaCIPK9互作蛋白筛选与蛋白互作验证研究。本项目取得的主要研究成果如下：.1）含油量性状的SnRK家族候选基因关联分析发现，2个基因（BnaA07g12290D和BnaA10g22850D）与含油量显著关联。.2）基因组数据库基因BLAST分析显示甘蓝型油菜中BnaCIPK9存在四个同源拷贝，遗传分析研究表明，BnaCIPK9同源拷贝均具有调控种子油脂的功能，但在甘蓝型油菜种子中只有BnaA10.CIPK9和BnaC05.CIPK9发挥油脂调控功能。.3）种子油脂分析研究表明，BnaCIPK9基因突变种子含油量提高，脂肪酸C18:1组分增加，C18:2和C18:3组分减少。.4）功能缺失突变体bnacipk9植株能够正常完成植物生长发育周期，叶片和角果生长量增强，植株衰老延迟。激素和糖类物质水平以及相关代谢和信号转导路径分析表明，BnaCIPK9通过调控Glucose信号与ABA信号转导路径参与种子油脂合成实现植物细胞糖稳态调节。.5）甘蓝型油菜种子转录组与脂质组分析表明，bnacipk9功能缺失突变体种子中糖酵解，丙酮酸代谢和能量代谢路径生理活性更加旺盛，且de novo脂肪酸合成提升，游离脂肪酸含量水平提升。.6）基于转录组差异表达基因分析结果结合酵母双杂交，LUC和BiFiC实验表明NBKX与BnaCIPK9存在互作。