油菜叶色基因BnaC.HO1的功能分析及其突变导致叶色变异的机理

The mutation of leaf coloration is very obvious trait mutation and commonly ocurred. The genes caused leaf color variation affected the metabolic process of chlorophyll directly or indirectly. In our study, a chlorophyll-deficient mutant with yellow-green leaves of Brassica napus was obtained by treatment with EMS. The chlorophyll-deficient mutant had decreased total Chlorophyll content, contained less granal stacks and lacked granal membranes. By a map-based cloning approach with the candidate gene approach, the leaf coloration gene BnaC.HO1 was isolated. BnaC.HO1 gene was highly homogous to the HY1 that catalysed decomposition of heme b in phytochrome chromophore synthesis-the heme branch in tetrapyrrole metabolism of Arabidopsis thaliana. In further research, we will conduct several following investigations:1. The analysis of qPCR and Enzyme activity detection will be carried out to clarify the fuctions of the BnaC.HO1 gene and its homologous gene. 2. The cis-acting elements in the promoter region will be identified by the promoter truncated method. The transcription factors will be selected through the yeast one-hybrid technology and the functions of the transcription factors will be pointed out by transformation experiments. 3.The genes that interacted with the BnaC.HO1 gene will be screened out through the yeast two hybrid technology and the functions of these genes will be pointed out through the gene expression analysis、RNAi analysis in Brassica napus and mutants analysis in Arabidopsis thaliana. In addition, BnaC.HO1 gene, the transcription factors and the genes interacted with BnaC.HO1 will be integrated into the regulatory networks of the tetrapyrrole metabolism and chloroplast development via transcriptome sequencing analysis and genetic analysis. Finally, these results will bring to light the molecular mechanisms of the leaf color variation in chlorophyll-deficient mutant BnaC.ygl.

叶色突变体是植物中突变频率较高且易于鉴定的突变性状，造成叶色变化的基因直接或间接影响着叶绿素代谢过程，本项目的研究材料是经EMS诱变获得油菜黄化突变体BnaC.ygl，该突变体叶绿素合成减少，叶绿体发育不良，该黄化表型是BnaC.HO1基因的缺失造成。BnaC.HO1与拟南芥中血红素加氧酶（HO1）同源，HO1参与四吡咯代谢的血红素分支，主要催化血红素b分解。我们拟以该基因为研究对象进行以下工作：1.通过qPCR分析及酶活分析解析该基因及其同源基因的表达模式及功能；2.鉴定出BnaC.HO1基因启动子区一个起主要作用的顺式作用元件，通过酵母单杂技术获得与该顺式作用元件结合的转录因子并进行功能分析；3.通过酵母双杂技术筛选与其互作的蛋白，并对互作基因进行功能解析；4. 利用转录组等分析将该基因、转录因子及其互作基因整合到四吡咯代谢及叶绿发育的调控网络。最终揭示该突变体叶色变异机理

四吡咯在植物光合作用和呼吸作用等生命活动中不可或缺，血红素-光敏色素生色团和叶绿素的合成是四吡咯代谢途径中两个重要的分支。血红素加氧酶HO是血红素-光敏色素生色团分支的限速酶，主要催化血红素的分解打开吡咯环为光敏色素生色团的形成做准备。前期工作中我们通过对油菜黄化突变体ygl的遗传分析和图位克隆，成功分离了叶色基因BnaC07.YGL（BnaC07.HO1）。.为了探讨BnaC07.HO1的功能、调控网络和黄化的机理，我们通过遗传分析（互补及干涉分析）确定了BnaC07.HO1的缺失导致突变体ygl黄化。BnaC07.HO1在苗期叶片中的表达量最高，表明其在油菜发育早期发挥着重要的作用。原核诱导和纯化了融合蛋白His-BnaC07.HO1并酶活检测证实该重组蛋白具有催化血红素分解为BV IXa的活性。利用RNA-seq分析表明BnaC07.HO1的缺失导致突变体中四吡咯代谢路径特别是叶绿素合成相关基因的表达上调。突变体中四吡咯代谢中关键的中间产物ALA、 Proto IX、Mg-proto IX和Pchlide均显著下降。总结来说，在突变体ygl中BnaC07.HO1的缺失导致HO1的催化功能大大降低，使体内血红素积累过量反馈抑制了ALA合成，使整个四吡咯合成受到抑制，叶绿素合成减少导致黄化的表型。.同时利用酵母双杂我们筛选到与BnaC07.HO1互作的基因121-2，该基因的敲除导致转基因植株苗期严重失绿，同时叶绿体发育异常，说明121-2在叶绿体正常发育的过程起着非常重要的作用；早期我们发现突变体ygl苗期叶绿体发育也异常，因此我们推测BnaC07.HO1基因缺失可能抑制了121-2基因的功能，使叶绿体发育异常，不能正常合成叶绿素，导致ygl黄化的表型。.项目执行中我们还发现与Darmor-bzh参考基因比较，突变体及野生型T6中BnaA07.HO1启动子上插入一个非编码长链RNA，该插入在甘蓝型油菜中普遍存在并下调BnaA07.HO1基因的表达。遗传分析表明对于无lncRNA插入材料，突变体ygl的黄叶表型受到两对隐性基因控制。在突变体ygl中过表达BnaA07.HO1基因能使叶片颜色恢复正常，这些结果表明突变体ygl的叶片黄化归因于BnaC07.HO1的缺失和BnaA07.HO1的下调表达。这给我们对突变体ygl的黄化机制带来了一个新的认识。