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

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.

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。

油菜CRABS CLAW (BnCRC) 转录因子可能发挥了重要作用在调控雌性不育上，定量PCR结果表明BnCRC在油菜营养器官（根、茎和叶）中不表达或低表达，在油菜花器官各组织中只在萼片中较高表达，其它花器官和角果中不表达或低表达；利用PCR和Southern Blot 技术开展了阳性验证和单拷贝筛选，结果获得13棵阳性转基因植株，其中单拷贝插入的转基因植株有6个。定量PCR分析表明BnCRC阳性植株中的平均表达量相对于对照下调2倍。制备了BnCRC抗体，其效价在512 K左右，纯度在85%以上。BnCRC基因RNA干涉敲除的油菜转基因植株花柱明显短于野生型，子房扁平，且明显宽于野生型。同时还伴随花药变短雄性不育及果柄变短等其它花器官变异性状；结合酵母双杂交互作蛋白的筛选结果、转录组与差异蛋白组的联合分析结果，BnCRC调控的主要途径为甲硫氨酸，糖代谢等，初步确定S-adenosylmethionine synthase 3、fructose-bisphosphate aldolase 1及mitochondrial outer membrane protein porin 1为BnCRC调控的主要候选基因。这些结果对于油菜杂种优势利用和杂交制种体系的改良具有重要应用价值。迄今已发表本项目标注（31571710）论文5篇，其中SCI论文1篇，国际会议摘要和海报展示一篇，另有一篇SCI论文在投，后续还将有论文发表。目前已经获得专利一项，申报专利一项。本项目构建的干扰表达载体及其中间载体已经提供给江苏大学、贵州油料所多家兄弟单位应用。本项目研究成果还参与获得了2017年湖北省科学技术进步奖一等奖（本人证书编号2017J- 241-1-038-001-R03）。