绒毛白蜡非ABA依赖型FvSnRK2.1基因对盐胁迫的响应机制研究

Lula-3 is a new variety of Fraxinus velutina Torr. Through high-throughput sequencing technologies we selected a large number of genes related to salt induced including three genes encoded SnRK2 Kinases. Among them FvSnRK2.1 belongs to GroupⅠand the expression level could not be induced by ABA stress. Compared with molecular cloning and characterization relatively little is known about the activation mechanism of GroupⅠmembers and their targets. The structure and function of FvSnRK2.1 was preliminary forecasted by bioinformatics analysis. The induced expression and tissue-specific expression profiles of FvSnRK2.1 were detected by real-time fluorescent quantitative PCR. Subcellular location was detected by monitoring the transient expression of FvSnRK2.1+ GFP fusion protein. The over-expression vector and Virus-induced gene silencing (VIGS) vector were constructed for Arabidopsis genetic transformation and Fraxinus velutina inoculated. The salt tolerance of transgenic plants was analyzed. Proteins that interacted with FvSnRK2.1 were screened by yeast two-hybrid system. The expression profile of the downstream genes of FvSnRK2.1 were investigated by RNA-seq and the signal pathway components were identified under different kind of salt stress. This research provides theoretical foundation for deep understanding the molecular mechanism of trees salt tolerance.

鲁蜡3号是绒毛白蜡耐盐新品种，高通量转录组分析筛选出三个编码SnRK2蛋白激酶的耐盐相关基因，其中FvSnRK2.1属于GroupⅠ，其表达不受ABA诱导，目前对此类型基因的研究多集中于功能分析，对其作用机理研究很少。本项目拟通过生物信息学分析初步预测FvSnRK2.1的结构与功能；利用荧光定量PCR检测基因的组织表达特异性和诱导表达模式；构建目的基因与GFP的融合表达载体进行亚细胞定位；构建FvSnRK2.1过表达载体和VIGS载体，分别进行拟南芥和绒毛白蜡的转化，分析过表达植株和功能缺失突变体的耐盐相关指标；采用酵母双杂技术分析其互作蛋白；利用转录组测序技术分析FvSnRK2.1下游基因的表达变化，探讨该基因可能参与的信号通路，同时比较不同盐胁迫下目的基因所在信号通路的差异，明确FvSnRK2.1耐盐作用机理，为培育耐盐绒毛白蜡品种提供新基因，也为揭示林木耐盐分子机制提供理论依据。

绒毛白蜡是我国北方重要的造林树种，具有较强的耐盐能力，目前有关绒毛白蜡耐盐相关基因研究较少。SnRK2是一个相对较小的植物专一性蛋白激酶家族，属Ser/Thr 类蛋白激酶，是植物响应干旱盐碱等导致的渗透性胁迫的主要调控原件。通过该项目的实施，我们从绒毛白蜡中克隆到一个FvSnRK2.1基因，进行了生物信息学分析、表达特异性分析及亚细胞定位；构建了过表达载体，完成了转基因拟南芥的筛选、鉴定，胁迫处理下表型、生理生化指标、基因表达及转录组分析；构建了绒毛白蜡cDNA文库，进行了互作蛋白的筛选。研究结果表明：FvSnRK2.1属于GroupⅠ，为ABA非依赖型SnRK2,定位于细胞核和细胞膜上；非生物胁迫下，该基因的表达受到不同程度的诱导；NaCl胁迫下MDA含量、抗氧化酶活性、气孔开度、干鲜重及表型变化均表明FvSnRK2.1增强了拟南芥的耐盐性；高通量转录组分析结果表明，FvSnRK2.1基因与氧化还原、胁迫防御、信号转导等基因在盐胁迫下相互作用从而达到增强耐盐的目的。这些结果揭示了FvSnRK2.1在盐胁迫中起着非常重要的作用，为培育耐盐绒毛白蜡品种提供新基因，也为揭示林木耐盐分子机制提供理依据。