白菜近等基因系叶缘裂刻形成和发育的分子机理研究

Diversity in leaf shape is produced by alterations of the margin. It suggests that the development of leaf lobes, leaf initiation and compound leaves is involved in common mechanism. Leaf-lobed mutant play an important role in leaf developmental mechanisms. Lobed leaves increased heat dissipation per unit area and improved water balance under dry and high atmospheric conditions. A previous study showed that a new BrcLL1 gene was fine mapped on chromosome A10 to a 86-kb region with a mapping population of near-isogenic lines derived from a lobe-leafed mutant of 'Aikangqing' inbred lines. Genetic studies have shown that its leaf lobe gene (BrcLL1) is monogenic dominant. In the present study, A map-based cloning strategy was employed to isolate the BrcLL1 gene. Several approaches will be used to determine which of the candidate genes correspond to the target, such as qPCR, RNA in-situ hybridization, genetic transformation and so on. Non-heading Chinese cabbage NILs of smooth leaf and lobed leaf were used as materials, The vein-cleared treatment and SEM were carried out to analyze the histological characterization and the semi-quantitative RT-PCR and Northern blot were adopted to study the expressions of leaf lobe-related genes in SAM with young leaf. These results provide a foundation for understanding the leaf developmental mechanisms and for genetic improvement of the plant architecture potential of non-heading Chinese cabbage.

叶缘裂刻的改变在很大程度上决定叶形的多样性，其形态发生同叶的起始、复叶发育分享着相同的分子机制。叶缘裂刻突变株在探索叶发育调控机制中发挥着重要的作用，叶缘裂刻形态结构有助于散热和保水，是植物抵御高温伤害和控制水分胁迫的有力工具。本项目组利用白菜"矮抗青"叶缘裂刻变异株，选育出外型新颖、叶缘深裂近等基因系新种质（BrcLL1），遗传分析表明叶缘裂刻性状由单显性基因控制，前期将其精细定位于86kb区间，是一个叶缘裂刻功能未被报道的新基因。本项目采用图位克隆法鉴定候选基因；采用qPCR、RNA 原位杂交、十字花科种质资源BrcLL1 等位基因多样性分析、基因组遗传互补等技术验证BrcLL1 候选基因的功能。通过观测叶脉、叶缘细胞组织特征及分析叶缘发育关键基因表达变化，揭示叶裂形成的分子基础。该研究结果对植物叶发育调控机制、白菜类作物株型的设计和品种改良有重要的理论与实践意义。

植物的叶形多种多样，叶是植物光合产物的重要来源。揭示叶形变异的遗传学基础是提高农艺产值的关键。十字花科油菜、萝卜、白菜、羽衣甘蓝存在天然的叶缘裂刻变异体，项目前期对叶缘裂刻基因进行初步定位，发现A10连锁群上的Bra009510为可能的候选基因，为研究白菜农艺性状及叶发育奠定了基础。本研究发现Bra009510在拟南芥中同源基因LMI1（late meristem identity1）为白菜叶缘形成的基因，在白菜裂叶材料中存在2次等位复制，2个拷贝在基因组的内含子中有245bp插入/缺失，cDNA区域仅存在个别SNP，2个拷贝在拟南芥中表达均造成叶缘裂刻的产生，但裂叶中独有的拷贝形成裂刻更深。但不会造成株型变小的表型。本研究结果表明，园叶白菜在进化过程中仅保留了基因Bra009510-A，裂叶中同时存在Bra009510-A和Bra009510-B，基因的等位复制及结构上的变异，造成叶缘裂刻形状各异。尤其是裂叶中存在独特Bra009510-B拷贝，可以诱导产生丰富的叶形变异体，该基因响应环境和激素调控。应用转录组测序技术，发现裂叶相对圆叶存在564个差异显著的差异表达基因（different expressed genes，DEGs），其中302个基因表达量上调，262个基因表达量下调，对转录组数据分析获得了7个参与叶发育的候选基因，分别为TCP13（Bra001032）、DPA4（Bra009171）、LOM2（Bra014479）、GRF6(Bra015184)、 Cyp79B2(Bra017871)、Avp (Bra018135)和 LMI1（Bra009510），并对相关基因BrLOM2进行克隆与表达分析。该研究结果可以为育种者根据育种目标创制理想叶形的白菜基因型。