大白菜叶色深绿突变体dg的候选基因鉴定及功能分析

Chinese cabbage (Brassica rapa ssp. pekinensis) is an important vegetable crops and its vegetative leaves are consumed as edible organs. The leaves performed dark green color has the great application value which can promote photosynthesis and effectively increase the crop yield and quality. However, few researches about dark green leaves functional genes, and the molecular mechanism of dark green leaves is unclear. In this research, the first single-base dark green leaf color mutant and wild type were used, the preliminarily analysis showed the dark green leaves character was controlled by a pair of recessive gene and mutant gene BrFC2 was mapped by MutMap, and then the separation ratio of mutant loci was analyzed in F2 population, the single plant with extreme traits will be sequenced to confirm the mutant gene; Using RNA-Seq and iTRAQ, the regulatory network of dark green leaves related genes will be revealed at transcription and translation level; Using the candidate gene overexpression vector and the genetic transformation system, the candidate gene function of dark green leaves will be verified; The detection of Heme and the analysis of candidate protein catalytic efficiency in vivo and in vitro will reveal the mechanism of BrFC2 regulating dark green leaf color. This research is the first finding that the single-base mutation of dark green leaf color mutant gene BrFC2 can affect leaf color deepening, it is the first to study the regulation of dark green leaf color by BrFC2 gene in Chinese cabbage, and has great significance to elucidate the new regulatory mechanism of chlorophyll synthesis; the dark green leaf color mutant has important application value in improving crop photosynthetic efficiency and quality.

大白菜以叶片为食用器官，其叶色深绿可促进光合作用，有效提高产量和品质。但发掘大白菜深绿叶色功能基因的研究鲜见报道，其分子调控机制尚不明确。本项目以首个大白菜单碱基突变叶色深绿突变体及其野生型为材料，在明确叶色深绿性状由候选突变基因BrFC2控制的基础上，分析突变位点在F2群体中的分离规律，结合极端性状单株测序验证候选基因突变位点；利用RNA-Seq和iTRAQ技术，在转录和翻译水平揭示叶色深绿相关基因的表达调控网络；利用候选基因过表达载体和大白菜遗传转化体系，验证叶色深绿候选基因功能；检测血红素含量，分析目标蛋白体内和体外催化效率，揭示BrFC2基因调控叶色深绿的机制。本研究是首次发现叶色深绿候选基因BrFC2点突变可以影响叶色加深，率先在大白菜中开展BrFC2基因对叶色深绿的调控研究，对阐释叶绿素合成调控新机制具有重大意义；该突变体在作物高光合效率品质改良方面具有重要的应用价值。

提高叶绿素含量可作为提高作物光合效率、增加作物产量的有效手段。然而，是否存在可增强叶绿素合成功能的功能获得型基因，是植物叶绿素研究的未知领域。本项目获得高光合效率、叶绿素含量增加、深绿色叶片大白菜EMS突变体dg，其铁螯合酶2基因（BrFC2）叶绿素a/b结合保守结构域发生精氨酸到赖氨酸的点突变(dBrFC2)。遗传转化结果表明，过表达dBrFC2在大白菜野生型中导致叶片颜色深绿，过表达BrFC2在大白菜野生型中表型不变。我们发现dBrFC2与原叶绿素氧化还原酶B1和B2 (BrPORB1和BrPORB2)互作，dBrFC2单氨基酸突变可加强与底物原叶绿素酸酯(Protochlorophyllide, Pchlide)亲和力，促进BrPORB催化反应，生成更多产物叶绿素酸酯（Chlide）。本项目提出了BrFC2-BrPORB模型共同介导叶绿素与血红素合成的新通路，丰富了叶绿素合成途径新认识，为作物品质精准育种提供基础。