代谢组学和转录组学关联解析德钦紫花苜蓿根部应答高温胁迫的调控机制

The wild resources of Medicago sativa Linn. ‘Deqin’ (hereinafter referred to as the ‘Deqin’), with strong drought and heat tolerance, distributed in dry-hot valley zone of Yangzi River, Yunnan province, China. Our previous research result shows that the special thermotolerance of ‘Deqin’ was closely related to its four strong antioxidant isozymes (SOD2, POD4, POD7 and APX3) activities and six up-regulated heat induced molecular chaperones (Hsp70, Hsp18.2, Le-Hsp17.6, DnaJ, DnaK and NEFs) , which were regulated by ABA, CTK and IAA. Compared with leaves, roots play critical roles in plant survival in environments with high soil temperature. However, there is no report about the regulation mechanisms of ‘Deqin’ roots in response to heat stress. Pot plants filled with loamy soil of ‘Deqin’ which are exposed to high temperature will be used as testing material in this project. The researchers will analyze the physiological change, ion fluxes and ATPase activity of plasma membrane in ‘Deqin’ roots. In addition, we will analyze the accumulation of metabolites in ‘Deqin’ roots and screen out 3 to 5 metabolite markers. And then we analyze the transcriptome of ‘Deqin’ roots, screen out 1 to 3 heat-resistance key gene (s) and combine with the result of physiology analysis and metabolites accumulation. Finally we validate these key genes on heat resistant in ‘Deqin’ roots by prokaryotic expression and qRT-PCR. All the results will give us a further understand about the regulation mechanism of ‘Deqin’ in response to heat stress, and lay the foundation for heat-resistance alfalfa varieties by molecular breeding.

德钦紫花苜蓿野生资源成片分布于云南省金沙江干热河谷地带，具有较强的耐干热性。本研究团队前期研究表明，在高温胁迫下，德钦紫花苜蓿叶片特异性耐热与其叶片四个抗氧化酶同工酶（SOD2、POD4、POD7和APX3）活性增强和6个逆境蛋白（Hsp70、Hsp18.2、Le-Hsp17.6、DnaJ、DnaK和NEFs）上调有关，受ABA、CTK和IAA共同调控。与叶片相比，根部在土温高的环境中对植物的存活与否起关键作用，但高温胁迫下德钦紫花苜蓿根部应答调控机制未见报道。本项目以德钦紫花苜蓿盆栽苗为材料，在不同的热激处理下，分析其根部质膜ATPase活性等生理变化和代谢产物积累特征，筛选3-5个标志物；进行根部转录组分析，筛选1-3个根部抗热关键基因；采用原核表达和qRT-PCR方法进行抗热特异基因的验证。研究结果将深入解析德钦紫花苜蓿根部应答高温胁迫的调控机制，为紫花苜蓿抗热分子育种奠定基础。

德钦紫花苜蓿地上部分具有较强的耐热性，但与叶片相比，根部在土温高的环境中对植物的存活与否起关键作用。本研究首先筛选出30℃根际高温进行后续试验。根际高温影响紫花苜蓿的形态和生理，表现为叶片失水反卷，叶缘焦枯，下部叶片变黄，根系减少。叶绿素含量总体下降，相对电导率和H2O2含量总体上升，可溶性糖含量先上升后下降。筛选出紫花苜蓿根系应答根际高温关键基因HSP90、HSP83、sHSPs、SOD、POD、CAT共6个，并进行SDS-PAGE验证。筛选出9个代谢标志物：槲皮素、山奈酚和木犀草素等苯丙烷类和聚酮类；柠檬酸、L-苹果酸、L-谷氨酰胺和L-赖氨酸等有机酸及其衍生物；核黄素等有机杂环化合物；琥珀酸等有机化合物。通过qRT-PCR验证代谢标志物的合成关键酶基因：苹果酸脱氢酶MDH1、苹果酸合成酶aceB、柠檬酸合成酶CS、顺乌头酸酶ACO、谷氨酸合成酶GLT、1-吡咯啉-5-羧酸脱氢酶P5CDH、类黄酮3’-羟化酶CYP75B1、NAD依赖型谷氨酸脱氢酶GLUD1和NADP依赖型谷氨酸脱氢酶gdhA共9个。筛选出与耐热相关性最高的ALA6和耐热相关性较高的ALA2的两个基因，比较它们应答瞬时高温、阶梯高温、低温+阶梯高温以及低温+高温共4种不同方式高温胁迫的特点。在蒺藜苜蓿(Medicago truncatula) A17上克隆ALA6成功，并测序和分析。相关后续的验证和蛋白表达工作正在进一步的开展和完善之中。项目的成果为下一步开展德钦紫花苜蓿耐热品种改良奠定了基础。项目资助共发表论文3篇，待发表4篇。授权专利1项，待授权1项。开展国内外交流共9人次，大会特邀报告１次。培养硕士生4名，其中3名已经取得硕士学位。项目投入经费39万元，支出26.39万元，各项支出基本与预算相符。剩余经费12.61万元，剩余经费计划用于本项目研究后续支出。