油茶雄性不育株花药瓣化败育的细胞机理研究

Camellia oleifera is an important woody edible oil tree species in China, and it plays an important role in promoting the accurate elimination of poverty in mountainous areas. In the early stage of the research, it was found that a mutant of C. oleifera ‘Hua shuo’ (designated HB-21) contains significant variation and petalization of its anthers that resulted in severe male sterility for the first time . Many of these valuable male-sterile C. oleifera trees are now asexually propagated and preserved. In preliminarily studies, we found that the anthers ofthese male-sterile C. oleifera flowers had severe petalization, could not producefunctional pollen at maturity, and were clearly different from normal anthers. The preliminary stage of this project intends to use as a foundation the large amountof research on normal, fertile C. oleifera anthers and use tissue sections (including paraffin, semithin, and ultrathin sections), optical microscopy, electron microscopy, histochemical localization, and other procedures to conduct a comparative study onthe differences and properties of each stage of the development of male-sterile and normal C. oleifera anthers. In addition, correlation analysis of these observations with the physiological and metabolic substances (Ca2+, polysaccharides, lipids, and proteins) produced during the same time will be performed in order to elucidate the causes of petalization and abortion in male-sterile anthers and thus revealing the cellular mechanism of male sterility in C. oleifera. This project will not only lay the foundation for the study of molecular biology of male sterility in C. oleifera, but is also highly significant for research on C. oleifera breeding of new male-sterile varieties, cross breeding, and other relevant topics.

油茶是我国重要的木本食用油料树种，在当前促进山区精准脱贫中具有重要战略地位。申请人前期在调研中首次发现，油茶产区主栽的国审品种‘华硕’中存在花药显著变异、瓣化现象严重的雄性不育突变株“HB-21”，立即无性扩繁并保存了该珍贵的雄性不育材料。申请人初步研究发现该雄性不育油茶花药严重瓣化，成熟时不能产生功能花粉，明显不同于正常花药。本项目拟在前期对油茶可育花药持续开展大量研究的基础上，应用组织切片（石蜡、半薄和超薄切片）、光学显微技术、电子显微技术和组织化学定位等手段，比较研究油茶雄性不育和可育花药发育过程中各阶段的变化特点和规律，并将这些规律与同期细胞中的生理代谢物质（钙、多糖、脂类和蛋白质）关联分析，以期阐明雄性不育花药发生瓣化败育的时期、部位和原因，从而揭示雄性不育的细胞学机理。本项目不仅可为解析油茶雄性不育的分子机制奠定前期基础，而且在雄性不育品种选育、杂交育种等研究中也具有重要意义。

油茶是我国重要的木本食用油料树种，生殖发育问题一直以来受到广泛关注。项目组前期在国审油茶良种‘华硕’中发现了花药瓣化败育的雄性不育株（“HB-21”），由于该不育株花药不能或很少产生花粉，导致接触异花花粉的几率增大，结实率也增大，在一定程度上避免了自交不亲和性发生，对油茶产量提升及雄性不育性研究具有重要作用。本项目采用组织切片、光学显微技术、电子显微技术对该雄性不育株进行了细胞学系统研究并开展了分子生物学初步研究，阐明了花药瓣化败育的细胞学机理。主要结果：（1）油茶不育株花药瓣化败育主要有两种类型，分别是花药完全瓣化不产生花粉的全瓣化类型和花药上部瓣化而下部具有正常花药结构并可以产生花粉的半瓣化类型。可育花药可产生3641粒花粉，经TTC和离体萌发检测活力分别达到85.54%、51.14%，而半瓣化花药产生903粒花粉，相对活力分别为3.24%、11.20%，全瓣化花药无花粉产生；（2）油茶不育株发生败育的关键时期在7月中旬，此时雄蕊刚分化出花丝和花药。花药瓣化的主要原因是在油茶花药发育中的第2阶段--孢原细胞发生异常，以花芽子房为中心作垂周分裂抑制了平周分裂，使花药扁平化不形成花粉囊。半瓣化败育类型的花药，在发育的第6-7阶段出现绒毡层降解异常，在第10阶段绒毡层提前降解，这些异常导致在第13阶段产生的花粉经苏木精染色呈现不均一的颜色状态，这种现象反映出花粉的内含物缺失；（3）运用SMRT和RNA-seq技术，对瓣化发育的关键基因进行鉴定。在转录组中发现了大量的选择性剪接事件，通过对瓣化与正常材料雄蕊间MADS及所有差异表达基因的共表达网络分析，预测了4个MADS转录因子基因CoSEP3.1、CoAGL6、CoSEP3.2和CoAP3是负责花药瓣化发育的关键基因。本项目研究结果对下一步开展油茶雄性不育品种定向培育、解析山茶科山茶属植物花药瓣化分子机制具有重要意义。