一个新的甘蓝型油菜下卷叶突变基因的图位克隆与功能分析

Leaf, as the basic photosynthetic and vegetative plant organs, may directly decide the material production of plants. Leaf development closely related to the establishment of plant architecture and the formation of crop yield. The research on the mechanisms of leaf rolling in allotetraploid Brassica napus has not yet been reported. The novel down-curved leaf mutants M823, an EMS mutant of Brassica napus from our laboratory will be researched. Unlike the genetic mechanism reported for rolled-leaf traits in various plants, the down-curved leaf trait in M823 of Brassica napus has been demonstrated to be controlled by a single dominant gene. Morphological, cytological, physiological and biochemical analyses have been finished in our preliminary works. Based on these works, present research will finely map the mutated down-curved gene in M823 with the F2 population and using the 60k SNP chip as an effective approach for the positional cloning due to the enriched markers. We will obtain the candidate genes within the linked marker intervals for the mutation and clone the target gene, and then analysis the gene function. This work that exploring the key gene controlling down-curved leaf trait in Brassica napus will facilitate our understanding of the molecular mechanism underlying the leaf rolling of plants.

叶是植物的基本营养器官，决定植物体内干物质的积累。叶的发育与植物株型的建立和作物产量的形成密切相关。甘蓝型油菜为异源四倍体，是我国重要的油料作物，目前还未发现与油菜卷叶发生机制相关的研究报导。本实验室通过EMS诱变获得了一个新的甘蓝型油菜下卷叶突变体M823，与国内外已报道的卷叶基因的遗传模式不同，M823的下卷叶性状是由显性单基因控制的。本项目将在M823突变体形态学、细胞学和生理生化等方面鉴定的基础上，构建F2作图群体，利用高密度的60k SNP芯片对卷叶基因进行精细定位，筛选连锁标记区间内的候选基因，并克隆目标基因，对候选基因进行功能验证，以探寻M823中控制下卷叶突变的关键基因，从而为阐释卷叶发生的分子机理奠定基础。

叶是植物进行光合作用的主要场所，叶的发育与植物株型的建立和作物产量的形成密切相关。卷叶作为常见的表型在拟南芥和水稻中已有广泛的研究报道，然而目前在异源四倍体甘蓝型油菜中还未发现与卷叶发生机制相关的研究报导。本项目以EMS诱变获得的一个新的甘蓝型油菜下卷叶突变体为研究材料，命名为Bndcl1。遗传学分析表明，Bndcl1突变体下卷叶性状由显性单基因控制。利用构建的高密度SNP遗传图谱，将下卷叶显性位点定位至油菜C05染色体SNP标记M41893 和 M24380之间，区间大小为1.08Mb。进一步通过精细定位将该定位区间缩小至102kb，包含注释基因4个。我们克隆了定位区间的4个基因并比较了野生型和突变体的基因序列，表达水平差异和两个材料的蛋白质组差异，据此确定并克隆了下卷叶突变候选基因。QTL定位结果表明，BnDCL1位点是一个多效性位点，能同时降低株高，改变植株株型和叶形，导致突变体株型紧凑。基于iTRAQ的定量蛋白质组学研究表明，Bndcl1突变体和野生型中差异积累蛋白主要富集于代谢前体和能量的产生、光合作用、RNA修饰和核糖体合成等生物过程，并鉴定到与叶近远轴极性建成、BR信号途径以及非生物逆境胁迫相关的重要差异蛋白。项目研究结果为后续突变基因功能验证和植物卷叶发生分子机制研究奠定了坚实的基础，为甘蓝型油菜理想株型育种和机械化生产提供了宝贵材料。