黑麦草叶片衰老过程中LpSGR调控叶绿素降解的上游通路探析

Leaf senescence is one of the most important factors for forage yield and quality. Chlorophyll (chl) degradation is the visible marker for leaf senescence that causes yellowing leaves, protein runoff, and degraded forage quality during forage crop production and harvesting. In order to investigate the molecular regulatory mechanism underlying chl breakdown during leaf senescence in forage grasses, the applicants have cloned and functionally characterized the key gene LpSGR that involved in the chl breakdown pathway from perennial ryegrass, and found that LpSGR is tightly regulated at the transcriptional level during leave senescence and its promoter region has a conserved region that contributes to the specific gene expression in senescent leaves. Based on these preliminary data, we propose to further our study on the transcriptional regulation of LpSGR by: ①screening the full length cDNA library of ryegrass to fish out the upstream regulators of LpSGR; ②analyzing the functional domains of the upstream transcriptional factors and their binding DNA cis-elements; ③overexpressing or down-regulating the candidate genes to understand their biological functions; ④further screening the interacting proteins with the candidate regulators and analyzing the transcriptomic and genomic datasets to understand the upstream pathways that the SGR-regulated chl breakdown involves in during leaf senescence. The results from this project will provide a solid theoretical groundwork for forage improvement in the future.

叶片早衰是影响牧草产量和品质的重要因素。叶绿素降解是叶片衰老的重要标志，在饲草生产和收获过程中，叶绿素降解导致叶片枯黄，蛋白质降解加速，影响饲草产量和品质。为了深入研究禾本科牧草叶片衰老过程中叶绿素降解的分子调控机制，申请人以多年生黑麦草为材料，克隆了叶绿素降解的核心调控基因LpSGR，发现LpSGR在转录水平上受衰老信号调控，该基因启动子区域存在响应衰老信号的保守功能区。在此基础上，本研究拟进一步筛选、分析LpSGR的转录调控上游因子及信号转导通路，研究内容包括：①筛选黑麦草全长cDNA文库，捕获LpSGR的上游调控因子；②分析上游转录因子的功能域及其特异识别的关键DNA顺式元件；③过量或抑制表达调控因子编码基因，解析其生物学功能；④进一步筛选候选因子的互作蛋白，结合转录组和基因组分析，探析叶片衰老过程中LpSGR调控叶绿素降解的上游调控通路。研究结果将为饲草品种改良提供理论基础。

叶片早衰是影响牧草产量和品质的重要因素。叶绿素降解是叶片衰老的重要标志，在饲草生产和收获过程中，叶绿素降解导致叶片枯黄，蛋白质降解加速，影响饲草产量和品质。为了深入研究禾本科牧草叶片衰老过程中叶绿素降解的分子调控机制，我们以多年生黑麦草为材料，通过对不同截断活性进行检测，发现叶绿素降解基因LpSGR启动子中存在抑制该基因在功能叶片中表达的启动子区域（-467bp到-390 bp）；通过酵母单杂交技术，筛选到作用于LpSGR启动子的LpNAL、LpABI3、LpERF1、LpABF3等转录因子。LpNAL定位于细胞核且为转录抑制子，通过ChIP-qPCR、EMSA和LpSGR启动子驱动Luciferase报告基因等三种实验进一步证实LpNAL直接结合LpSGR启动子并抑制该基因的转录表达。过量或抑制LpNAL的表达能够延缓或促进黑麦草叶片的衰老。通过酵母双杂交筛库筛选到了一些与LpNAL互作的候选蛋白，其中包括LpORE1、LpGlb、LpMIEL1、LpNRPB3和LpPR5。其中LpORE1是乙烯信号通路的关键基因，LpNAL的表达也受到乙烯的诱导。因此推测LpNAL是乙烯诱导叶片衰老过程中的限速因子之一。此外，我们还对其他叶绿素降解途径基因及相关转录调控机理开展了研究。相关研究结果分析了叶绿素降解基因上游调控因子的生物学功能及参与的信号转导途径，为多年生牧草和草坪草分子育种提供了理论基础和基因资源。