白菜花粉特异表达基因BGAL11功能和特异表达机制研究

Brassica includes several kinds of vegatables such as Brassica campestris,Brassica oleracea and Brassica juncea.All of them have high production effect,and have very important role in agricultural production in our country.Most of the crops in Brassica are cross-pollination,and have strong heterosis,using male sterile line is the ideal method to hybrid seed production..Pollen development abnormalities are the main reason of male sterile.Plant pollen development and its regulation is a very complex process which involves in the expression of many genes.So isolation and identification the pollen development related genes and pollen-specific expression promoters has important theoretical and practical significance for understanding the molecular mechanism of pollen development and creating a new sterile lines..This paper focus on the relationship between pollen-specific expression gene BcBGAL11 and pollen development in Brassica campestris, and the molecular mechanism of BcBGAL11 expressing specificly in fertile plants.T-DNA insertion mutants of its homologous gene in Arabidopsis,functional restoration plants and deletion mutants of BcBGAL in Brassica campestris are used for systematically studying the role of BcBGAL11 during pollen development.Promoter sequences of BcBGAL11 were isolated from fertile and sterile plants,and flower dip method is used to transform the promoters to Arabidopsis get evidence for molecular mechanism of BcBGAL11 expression-specific in pollen.

芸薹属是十字花科植物中最为重要的属，包括白菜类、甘蓝类和芥菜类等，其变种类型众多，生产效益高，在我国农业生产中占有极其重要的地位。本属作物多为异花授粉，杂种优势强，利用雄性不育系是其较理想的杂交制种途径。花粉发育的异常是导致雄性不育的主要原因，植物花粉发育及其调控是一个复杂的过程，涉及大量基因的表达和不同启动子的调控，特别是花粉特异性启动子在此过程中起着重要的调控作用，因此分离和鉴定花粉发育相关基因及花粉特异表达的启动子对于明确花粉发育的分子机制和创建新的雄性不育系具有重要的理论和实际意义。本研究围绕着花粉特异表达的BGAL11与花粉发育的关系及其特异表达的分子机制展开。通过观察其拟南芥同源基因的T-DNA插入突变体、功能互补植株和白菜中该基因缺失突变体来系统研究该基因在花粉发育过程中的作用；从核不育两用系中分离获得启动子序列，浸花法稳定转化至拟南芥中，为明确特异表达分子机制提供必要证据。

白菜类蔬菜是十字花科芸苔属作物，其变种类型多，生产效益高，在我国农业生产中占有十分重要的地位。白菜类等芸苔属作物多为异花授粉，具有很强的杂种优势，利用雄性不育系是其较理想的杂交制种途径。花粉发育所需基因表达异常是导致雄性不育的主要原因之一，本研究首先采用扫描电子显微镜观察白菜BGAL11的拟南芥同源基因AtBGAL11的突变体花粉形态发现突变体中花粉粒在四分体之后不能正常分开，Western杂交和转录组分析发现这是由突变体中BGAL活性显著降低和参与果胶等花粉壁成分多糖合成与降解途径中基因的表达明显降低等引起的。进而对由CRISPR敲除技术创建的白菜BGAL11的纯合T3代敲除突变体bcbgal11进行表型鉴定，与野生型95%的花粉萌发率相比，基因敲除突变体的花粉萌发率仅有约76%，并且突变体中花粉萌发管变短；亚历山大染色发现野生型花粉94%以上花粉均能染成红色，而突变体中只有约80%花粉粒能被染成红色，表明突变体中花粉活性降低了；采用扫描电子显微镜对野生型和突变体中的花粉微观结构进行分析发现，突变体中畸形花粉粒所占比率增加约11%，以上结果表明白菜BGAL11是花粉正常发育的必需基因之一。通过将可育株和不育株中BGAL11的启动子序列分别转化烟草和拟南芥发现不育株启动子驱动的GUS信号明显弱于可育株启动子驱动的GUS信号，表明白菜BGAL11在不育株和可育株的表达量的差异来源于启动子的差异。综上所述，本研究发现一个因启动子序列不同引起表达量明显有差异的BGAL编码基因白菜BGAL11并且其是花粉发育发育必需基因之一，为分子辅助育种和创建新的雄性不育系奠定理论基础。