油菜CRABS CLAW (BnCRC) 转录因子调控油菜雌性不育的机理研究

甘蓝型油菜(Brassica napus L.)雌性不育突变体FS-M1从宁油10号品种中发现。通过抑制差减杂交（SSH）和基因芯片技术从该突变体中筛选到油菜CRABS CLAW (BnCRC)转录因子。克隆了BnCRC基因的全长，通过遗传转化获得BnCRC基因RNA干涉的转基因油菜植株，转基因植株具有和突变体FS-M1相似表型。为了进一步阐明BnCRC调控油菜雌性不育的机理，拟通过原位杂交分析BnCRC在油菜花发育过程中的时空表达特性；通过表型鉴定和细胞学等手段分析BnCRC的生物学功能；并利用酵母双杂交、高通量测序和蛋白质双向电泳技术，鉴定与BnCRC相互作用和受BnCRC调节的下游基因，利用定量PCR和原位杂交分析所鉴定的下游基因和互作蛋白在油菜花发育过程中的时空表达特性，并对这些基因进行转基因验证，进一步阐明BnCRC导致油菜雌性不育的调控机制及信号网络。

The Brassica napus female sterile mutant FS-M1 was isolated from spontaneous mutation of rapeseed culitvar ‘Ningyou10’. By suppression subtractive hybridization (SSH) and microarray technology, a Brassica napus CRABS CLAW (BnCRC) transcription factors was found to play an important role in the mutant traits. We have cloned the full-length cDNA and DNA of the BnCRC gene from Brassica napus. The BnCRC gene RNA interference knocked transgenic Brassica napus plants showed the same female sterile phenotype to the female sterile mutant FS-M1.To further elucidate the regulation mechanism of BnCRC on female sterile in Brassica napus, we plan to carry out following researches: (1) Temporal expression of BnCRC during the development of Brassica napus. will be analyzed by using in situ hybridization. (2) Biological function of BnCRC will be analyzed by means of phenotypic identification and cytological analysis. (3) Interactive proteins of BnCRC and downstream genes regulated by BnCRC will be identified by using the yeast two-hybrid, high-throughput sequencing and two-dimensional electrophoresis of protein techniques. (4) Temporal expression of interactive proteins of BnCRC and downstream genes regulated by BnCRC during the development of Brassica napus will be analyzed by using Real-time PCR and in situ hybridizationandtransgenic rape plants of the interactive proteins of BnCRC and downstream genes regulated by BnCRC will be developed to further clarify mechanisms and signaling networks of BnCRC result in female sterile in Brassica napus in view of systems biology.