大白菜自交不亲和类钙调蛋白BrCML关键基因筛选与功能鉴定

Self-incompatibility is widely existed in Brassica crops, including Chinese cabbage. The utilization of self-incompatible lines is a major approach for hybridization breeding in Chinese cabbage. Calmodulin-like proteins (CMLs) are the vital regulators in pollen germination and tube growth of self-incompatibility. Our previous studies have identified some BrCML genes in Chinese cabbage. However, the functions of BrCML genes during self-incompatibility are still unclear. (1) In this study, in order to exploring the characteristic of Chinese cabbage self-incompatibility, the pollen germination and tube growth after self-pollination will be observed, and the distribution of Ca2+ in stigma and style will be detected by using the self-incompatible and self-compatible Chinese cabbage. (2) The identification and expression analysis of critical BrCML genes during self-incompatibility will be performed by using RNA-Seq. Then, the dynamic relationship among the gene expressions, the occurrence of self-incompatibility, pollen germination and tube growth, and Ca2+ distributions will be analyzed. (3) Moreover, critical BrCML genes are over-expressed and transformed the Arabidopsis for validating their functions. Finally, the regulatory mechanisms of critical BrCML genes in determining the pollen germination and tube growth of self-incompatibility will be understood. These results will provide theoretical references for uncovering the molecular mechanism of sporophytic self-incompatibility in Chinese cabbage.

自交不亲和现象普遍存在大白菜等芸薹属作物中，用自交不亲和系制种是当前大白菜杂种优势利用的重要方式。类钙调蛋白CML是自交不亲和花粉萌发及花粉管生长的重要调控因子，本项目前期已鉴定大白菜类钙调蛋白BrCML家族基因，但其在自交不亲和过程的具体功能尚不清楚。本项目拟利用大白菜自交不亲和系与自交亲和系，（1）观察自花授粉后花粉萌发及花粉管生长情况，检测柱头与花柱中的钙离子分布，明确大白菜自交不亲和反应特征；（2）通过RNA-Seq测序，筛选大白菜自交不亲和过程关键BrCML基因，分析其表达特征，并明确关键BrCML基因动态表达与自交不亲和反应、花粉萌发及花粉管生长、钙离子分布的关系；（3）进一步通过过表达转化拟南芥植株，验证关键BrCML基因的功能，最终明晰大白菜关键BrCML基因对自交不亲和花粉萌发及花粉管生长的调控机制。研究结果将为阐明大白菜自交不亲和分子调控机理提供理论依据。

大白菜具有典型的孢子体自交不亲和现象。类钙调蛋白在花粉萌发和花粉管生长中起到重要调控作用，然而其在大白菜自交不亲和花粉萌发和花粉管生长中的作用机制仍不明确。本项目探讨了大白菜自交不亲和反应发生特征，以及关键基因BrCML23、BrCML24和BrCML49对花粉萌发及花粉管生长的调控功能，取得以下主要结果：（1）通过田间自交亲和指数统计、S单元型鉴定以及自花花粉萌发荧光观察，评估了9个大白菜自交系的自交不亲和性，其中91-125为强自交不亲和材料，14S23为强自交亲和材料；（2）透射电镜观察发现大白菜91-125自花授粉8h柱头乳突细胞中游离Ca2+水平升高，且主要分布在液泡膜、细胞壁和细胞间质中；（3）RNA-Seq测序筛选到大白菜自交不亲和过程中一系列差异表达基因，包括S位点基因和钙结合蛋白，其中BrCML23、BrCML24和BrCML49基因在自交不亲和过程显著下调表达；（4）基于大白菜基因组BRAD，克隆分离了BrCML23、BrCML24和BrCML49基因全长序列，亚细胞定位分析发现三个蛋白均定位在细胞核和细胞膜上；（5）大白菜BrCML49基因过表达遗传转化使拟南芥植株抽薹开花时间提前，并促进花粉和花粉管的萌发；（6）大白菜BrCML23和BrCML24基因过表达载体的成功构建和拟南芥的遗传转化，为后续基因功能研究打下基础。以上研究结果为探明大白菜自交不亲和遗传机理提供参考，并为进一步明确大白菜BrCML基因响应Ca2+信号调控自交不亲和花粉萌发与花粉管生长的分子机理提供重要依据。