TaPLC1基因参与小麦耐热的功能和机理研究

Heat stress is one of the major factors limiting wheat yield and quality. To study the mechanism of wheat responses to heat stress, our previous results showed that TaPLC1 gene was related with wheat thermotolerance, and the inhibition of TaPLC1 gene's expression showed less termotolerance in wheat seedlings. By this project, we will explore the function and mechanism of TaPLC1 involved in heat tolerance. In order to further prove the function of the gene TaPLC1 in heat tolerance, wheat transformation lines with suppressing (RNA interference) or overexpression of the gene are obtained by transformation technology, respectively. To determine the action site of TaPLC1, a subcellular localization assay will be conducted by transiently expressing TaPLC1-GFP fusion protein in onion epidermal cells and Arabidopsis protoplasts. Various plasmid vectors with 5’-truncated TaPLC1 promoters fused with the reported gene GUS will be constructed and transformed into wheat callus, respectively. Activity of the gene TaPLC1 promoter under heat treatments will be analyzed. RNA-Seq will be used for gene expression analysis in response to heat stress treatments in the seedlings of wheat transformation lines and their wild type. Downstream target genes of the gene TaPLC1 will be screened out. Regulation of downstream target genes of TaPLC1 will be illustrated. Interaction proteins of TaPLC1 will be screened out using yeast two-hybrid and bimolecular fluorescence complementation. The results are very helpful for the theoretical study of heat tolerance and breeding of thermotolerance cultivars in wheat.

高温胁迫是严重影响小麦产量和品质的重要因素之一。为研究小麦对高温胁迫的响应机制，课题组前期研究发现小麦磷脂酶基因TaPLC1与高温胁迫相关，其抑制表达使小麦耐热性显著降低。本项目拟对其功能及调控机理进行解析。通过分析TaPLC1基因的过表达和RNAi沉默转基因小麦，进一步鉴定该基因的耐热性功能，通过研究TaPLC1基因在拟南芥原生质体和洋葱表皮细胞瞬时表达，以及5’系列缺失片段驱动GUS基因在小麦响应热胁迫瞬时表达，明确TaPLC1基因表达蛋白的细胞部位并初步揭示启动子响应热胁迫机制；通过小麦转基因后代和野生型的转录组测序和分析，筛选与耐热相关下游靶基因；用酵母双杂交和双分子荧光互补技术，鉴定互作蛋白，从而揭示TaPLC1基因参与小麦耐热的的调控机理，为小麦耐热机制研究和耐热品种培育提供参考。

本项目对TaPLC1参与耐热的功能进行解析，扩增了TaPLC1基因上游启动子序列，分析发现其包括多个热响应原件。构建了TaPLC1基因过表达及沉默载体，完成小麦遗传转化，并获得了相应转基因植株。通过对气孔观察，我们发现转基因植株可以通过快速调节气孔关闭达到耐热。并对野生型及沉默转基因植株热胁迫前后进行转录组测序分析。进行TaPLC1基因亚细胞定位，发现其定位在细胞膜上。构建酵母双杂交文库，通过筛选共筛选到36个互作蛋白，进一步通过体外验证及测序分析，发现TaPLC1可能通过与小麦二磷酸核酮糖羧化小亚基叶绿体和过氧化物酶作用来调节小麦的热响应机制，通过本研究的实施揭示了TaPLC1基因参与小麦耐热的的调控机理，为小麦耐热机制研究和耐热品种培育提供参考。。