小麦PPDK基因耐盐分子机制解析及耐盐优异变异筛选

There is mounting evidence that metabolic reprogramming is critical for the survival of organisms exposed to stressed environments. Microbes may escape the effects of stress by re-engineering metabolic networks mediated by pyruvate, orthophosphate dikinase (PPDK). Many types of stress induce PPDK protein in plants, while its molecular mechanism is not fully understood. The study of PPDK enhancing salt tlerance in wheat is still a blank. Our previous results clearly indicated that wheat TaPPDK is involved in responding to high salinity stress. Overexpression of TaPPDK gene in Arabidopsis thaliana and soybean hair roots enhances salinity tolerance. In this study, we will further clearify the salinity stress inffluence on the expression and activity of PPDK, by western blot and analyzing the posttranslation modification of wheat PPDK. To investigate the role of PPDK in salinity tolerance, we will use three approaches. First, we will analyze microarray data to investigate pathways within which PPDK may function during salt stress. Second, we will perform metabolic fingerprinting after feeding 13C-labelled pyruvate, the substrate of PPDK, to leaves and roots under salt stress to determine how it is metabolized. Third, we will generate A. thaliana in which the TaPPDK gene was over-expressed. By association analysis of TaPPDK allelic variations with salt tolerance in wheat accessions, we will identify the salt tolerant haplotype. The results and achievements will provide the gene associated with salt tolerance with great potential for wheat improvement and the theoretically basis for deciphering the salt tolerance mechanism of the candidate gene.

越来越多的证据证明生物在应对逆境胁迫过程中会通过重建代谢网络来维持生存。丙酮酸磷酸双激酶PPDK参与的代谢网络重建在细菌应对逆境过程中发挥重要作用。但PPDK在植物抵御逆境中发挥作用的分子机制尚不清楚，在小麦中的相关研究更是空白。前期工作已经证明小麦PPDK基因在拟南芥及大豆发状根中过表达显著提高转基因植物耐盐性。本项目拟在进一步明确盐胁迫条件下PPDK变化规律的基础上，通过盐胁迫下小麦转录组数据分析，筛选与TaPPDK协同变化的基因，预测PPDK参与的代谢途径；再通过13C标记PPDK反应底物丙酮酸的同位素示踪技术、转TaPPDK基因耐盐拟南芥生理生化参数分析，确定盐胁迫下PPDK参与的物质代谢过程，解析TaPPDK耐盐分子机制。并分析TaPPDK基因在小麦种质资源中的自然变异与资源耐盐性的关系，筛选TaPPDK基因耐盐优异等位变异，为小麦耐盐遗传育种提供重要的候选基因及功能基因信息。

我国土壤盐碱化日益严重，对我国的粮食安全造成了严重威胁。丙酮酸磷酸双激酶PPDK在植物抵御逆境胁迫中发挥重要作用，但其耐盐分子机制尚不清楚，在小麦中的相关研究更是空白。我们研究发现小麦TaPPDK基因受盐胁迫诱导表达，且在拟南芥及大豆发状根中过表达显著提高转基因植物耐盐性。通过13C标记PPDK反应底物丙酮酸的同位素示踪技术、盐胁迫下小麦转录组数据分析以及转TaPPDK基因耐盐拟南芥生理生化参数分析，发现盐胁迫下PPDK在产生转运氨基酸谷氨酰胺的途径中起作用，编码PPDK代谢途径中相关酶类的基因在盐胁迫下上调表达，与TaPPDK表达变化成正相关性，且TaPPDK过表达转基因拟南芥的氮含量高于对照而过氧化物含量低于对照，故推测谷氨酸及谷氨酰胺可能通过参与氧化胁迫及氮代谢过程应答植物高盐胁迫。另外，通过分析TaPPDK基因在小麦种质资源中的自然变异与资源耐盐性的关系，在TaPPDK基因启动子区筛选到1种耐盐优异等位变异，且在小麦育种进程中得到人工选择的筛选作用。研究结果将为小麦耐盐遗传育种提供重要的候选基因及功能基因信息。