PePAL1和Pe4CL3在杏鲍菇采后两次木质化中的转录调控机制研究

Postharvest lignification is a common quality deterioration phenomenon occurred in horticultural products during storage and transportation. Studies on the formation and regulation mechanisms of postharvest lignification are of great significance for the preservation and quality of horticultural products. The results of our previous study showed that two lignifications easily occurs on postharvest Pleurotus eryngii, and the expression of PePAL1 and Pe4CL3 was specifically regulated in two lignifications, respectively, which suggests that the regulation of these genes may play a key role in the occurrence of two lignifications. So, the transcriptional regulation mechanisms of PePAL1 and Pe4CL3 in two postharvest lignifications are selected to be the main research contents in this project based on previous research results. The lignification specific transcription factors of PePAL1 and Pe4CL3 will be identified by the interaction analyses of core promoter and transcription factor in vitro and in vivo using electrophoretic mobility shift assay, chromatin immunoprecipitation assay and other technologies. The functions in two postharvest lignifications and the regulation mechanisms on target genes of lignification specific transcription factors will be elucidated by detecting their functions, recognition sites, key structural domains and interacting proteins using gene expression interference, yeast two-hybrid system, co-immunoprecipitation and other technologies. The results will provide new theoretical foundation for improving postharvest technology of Pleurotus eryngii in practice.

采后木质化是园艺产品贮运过程中常见的品质劣变现象，其发生及调控机理的研究对园艺产品的保鲜保质具有重要意义。我们前期的研究结果表明，杏鲍菇采后会发生两次木质化，PePAL1和Pe4CL3的表达分别在两次木质化时受到特异性的调控，而该调控可能对两次木质化的发生起着关键作用。为此，在原有工作基础上，本项目拟以PePAL1和Pe4CL3在两次木质化中的转录调控为主线，通过核心启动子与转录因子在体内、体外的互作分析，利用凝胶阻滞实验、染色质免疫共沉淀等方法鉴定PePAL1和Pe4CL3的木质化特异转录因子；通过识别位点、关键结构域和互作蛋白等研究，采用基因表达干涉、酵母双杂交、Co-IP等技术明确特异性转录因子在两次木质化中的功能及其对靶基因的调控机制。研究结果可为生产上改进杏鲍菇采后保鲜技术措施提供新的理论依据。

采后木质化发生及调控机理的研究对食用菌采后保鲜保质具有重要意义。PePAL1和Pe4CL3的表达在杏鲍菇采后两次木质化中的特异性调控可能对两次木质化的发生起着关键作用。本项目对PePAL1和Pe4CL3在杏鲍菇采后两次木质化中的转录调控机制进行了研究。1）构建了PePAL1和Pe4CL3的干扰和过表达载体的构建与菌丝遗传转化，从理化特征和基因表达层面分析PePAL1和Pe4CL3的表达水平改变对两次木质化的影响，验证了PePAL1和Pe4CL3与杏鲍菇采后两次木质化的关系；2）克隆得到PePAL1，Pe4CL1，PePAL2和Pe4CL3的启动子全长序列，鉴定了PePAL1和Pe4CL3的核心启动子；筛选得到4个两次木质化中特异性正调控PePAL1和Pe4CL3表达的转录因子PeMYB1/2/6/17，分析了所筛转录因子的转录活性及其与靶基因启动子在体内和体外的结合，并进行了亚细胞定位分析。通过上述工作鉴定了调控PePAL1和Pe4CL3的木质化特异转录因子；3）构建了特异性转录因子PeMYB1的过表达和干涉表达载体，获得了六个PeMYB1过表达转化子，分析了木质化特异性转录因子PeMYB1在杏鲍菇采后两次木质化中的功能；4）分析了特异性转录因子PeMYB1识别关键酶基因启动子的位点和转录因子的转录调控功能域；筛选得到15个分别可与转录因子PeMYB1和PeMYB2/6互作的蛋白，从逆境胁迫信号传导途径分析了这些蛋白与木质化特异转录因子PeMYB1和PeMYB2/6互作对靶基因PePAL1和Pe4CL3调控机制。本研究解析了杏鲍菇采后两次木质化关键基因转录调控的机制，研究结果可为生产上改进杏鲍菇采后保鲜技术措施提供新的理论依据。