辣椒温度敏感雄性不育是一种具有潜在利用价值的不育类型，但其育性转换的机理尚不清楚。项目前期以温敏型辣椒雄性不育系TCMS9704S等为试材，开展了育性转换过程的转录组测序，基因表达分析及转基因实验，项目组发现：CaHsfA2和CaHsfA6b异常转录与TCMS9704S育性转换具有重要相关性，CaHsfA6b 超表达能导致转基因植株高度雄性不育。本项目拟采用酵母双杂交技术研究CaHsfA2、CaHsfA6b互作调控功能；运用ChIP-Seq方法筛选、分离和鉴定CaHsfA2和CaHsfA6b在温敏雄性不育系育性转换过程中的调控靶标；进一步利用RT-PCR、Northern blot、Western blot等技术研究CaHsfA2和CaHsfA6b对雄性配子发育信号途径的调控。深入开展本项目研究，将为揭示CaHsfA2和CaHsfA6b转录调控辣椒温敏雄性不育系育性转换机制提供重要线索。

Thermo-sensitive male sterility has a potential value in hybrid breeding of pepper. However,the mechanism of fertility transition is not clear.Early in the project,through transcriptome profiling of TCMS9704S in pepper and transgenic experiments,We found that it has important correlation effects CaHsfA2and CaHsfA6b abnormal transcription on TCMS9704S fertility transition,Our obseration by transgene proved that overexpression of CaHsfA6b can results in male sterile in transgenic plants. This project will use yeast two-hybrid and other techniques to investigate the interaction function of CaHsfA2and CaHsfA6b,Furthermore, we will use ChIP-Seq technology to investigate the targets of CaHsfA2and CaHsfA6b in fertility transition of TCMS9704S. Technologies like RT-PCR,Northern blot,and Western blot are employded to understand the mechanism of CaHsfA2and CaHsfA6b on regulating related genes of male gamete development. A deep research on this project will provide an important view to understand the transcriptional regulation mechanism of CaHsfA2and CaHsfA6b in fertility transition of TCMS in pepper.

根据项目研究目标对辣椒热激转录因子CaHsfA6b进行了克隆及功能研究。研究结果表明CaHsfA6b在温敏雄性不育系9704S花蕾组织中具有明显高表达特征。同时发现CaHsfA6b表达水平显著受高温、ABA和NaCl等胁迫诱导。转基因研究表明 CaHsfA6b过表达烟草株系花粉活力受到显著性抑制，而拟南芥转化株系对高温、ABA和NaCl等逆境具有负向调控功能。利用酵母双杂交试验发现CaHsfA6b蛋白和细胞色素f存在互作关系，同时利用双荧光素酶检测和凝胶迁移实验发现CaHsfA6b蛋白可与CaBAG5启动子区热激元件结合并激活基因转录。辣椒CaHsfA6b可能通过结合细胞色素f调控CaBAG5介导的发育负调控途径，该途径可能对辣椒温敏不育系花粉育性变化具有重要作用。