拟南芥Athspr新基因的耐盐功能及其分子机理研究

High Salinity in soils has seriously affected the yields and quality of many crops in the world, and it is a significant and important project to isolate novel genes and to dissect their molecular mechanism resistant to high salinity. A novel Arabidopsis T-DNA insertion mutant that shows a high hypersensitivity to salt stress has been isolated in our lab. And its related gene, AtHSPR(heat shock protein-related in Arabidopsis thaliana), has also been cloned in our previous studies. Athspr gene encodes a new protein that share some similar conservative domains with ClpB/HSP100 according to the analysis of bioinformatics. In the present project, the differences in phenotypes, resistance to salt stress, cellular and subcellullar structures between wild type and Athspr mutant plants will be studied using conventional methods and laser scanning confocal microscopy as well as transmission and scanning electron microcopy; the spatiotemporal expression pattern of Athspr gene and its protein in wild type and the mutant will be investigated under normal and salt stress conditions by Real Time-PCR and immunoblotting; the relationship between over-expression of Athspr in transgenic Arabidopsis lines driven by the constitutive promoter or Athspr-specific promoter and the tolerance of salt stress will be studied here by Real Time-PCR. And the interaction between AtHSPR and HSP101 that is a member of ClpB/HSP100 or Na+/H+ exchanger in both wild type and Athspr mutant plants under normal or salt stress will also be investigated using methods of yeast two-hybrid, co-immunopricipitation and GST pull-down. In addition, the effects of Abscisic acid (ABA) and reactive oxygen on Athspr gene expression and its function will be studied through the treatments such as ABA and hydrogen peroxide; the levels of ABA in wild type and Athspr mutant will be measured by HPLC (High Performance Liquid Chromatography). Moreover, chaperone activity of AtHSPR and its ability of DNA-binding and DNA recombination will also be determined. The studies above will help us to dissect the function of Athspr gene and its molecular mechanism in tolerance to salt stress, which will provide important and valuable information for the plant how to resist against salt stress.

土壤盐渍化己严重影响到世界范围内许多重要作物的品质和产量，而发掘耐盐新基因并解析其耐盐分子机理是一个意义重大的研究课题。前期研究中，我们分离到一个对盐非常敏感的拟南芥T-DNA插入突变体，其关联基因Athspr编码一个与ClpB/HSP100同源且功能未知的新蛋白。本项目拟以Athspr突变体为材料，采用常规及多种分子生物学手段，结合盐、ABA和H2O2处理，从不同水平比较野生型与突变体的表型与耐盐差异；研究钠盐胁迫下ABA和ROS对Athspr的表达调控；解析Athspr基因及其蛋白的时空表达模式以及Athspr过表达对植株耐盐性的影响；分析AtHSPR的分子伴侣活性、DNA结合及同源重组能力；探究 AtHSPR与HSP101、SOS、HKT1、NHX1是否存在相互作用；最终阐明Athspr基因的耐盐功能及其分子机理，为更深层次揭示植物耐盐的分子机制提供重要而有意义的信息。

土壤盐渍化已经严重影响到世界范围内许多重要作物的品质和产量，而发掘耐盐新基因并解析其耐盐机理是一个意义重大的研究课题。本课题组在拟南芥C24生态型中分离到一个T-DNA插入突变体athspr（Arabidopsis thaliana heat shock protein-related），与野生型相比，该突变体器官体积减小、细胞体积缩减，对盐高度敏感。本项目通过多种常规及分子生物学方法，对AtHSPR基因参与植物耐受盐胁迫的生理功能和分子机制进行了系统的分析。主要研究成果如下：1）athspr突变体对NaCl极为敏感，50mM NaCl处理即可抑制其幼苗生长，150mM时则完全死亡，但在相同条件下野生型表现出相对较小的生长抑制；2）在盐胁迫条件下，athspr突变体中ROS积累和细胞膜损伤程度显著高于野生型，且对ABA的敏感性降低、ABA的积累减少、叶尖气孔指数增加，并且减慢了ABA诱导的气孔关闭，当外源施加ABA时可以部分恢复athspr突变体的盐敏性；3）AtHSPR基因编码一个细胞核定位的ATP水解酶蛋白，其特异地表达在拟南芥维管组织中，且AtHSPR基因的表达受到盐和ABA的诱导而增强；4）与野生型相比，在盐处理条件下ABA/胁迫响应、SOS系统和抗氧化系统相关基因的表达在athspr突变体中降低而在AtHSPR过表达系中升高，且AtHSPR过表达系维持了体内较高的抗氧化活性和K/Na离子平衡，因而增强了转基因拟南芥的盐耐受性，并通过减少叶片水分的散失和ABA介导的气孔开闭增强了转基因的抗旱性；5）转录组学分析显示：athspr突变体在盐处理条件下，涉及ROS积累、ABA信号转导、细胞死亡、胁迫响应和光合作用等相关基因的转录响应与野生型相比有较大差异，AtHSPR基因可能通过调控这些信号通路来参与拟南芥的盐胁迫应答；6）通过酵母双杂交系统筛选验证了包括与钾离子转运相关的KCO5和抗氧化相关的FeSOD1在内的11个与AtHSPR相互作用的蛋白，并在盐耐受性中发挥重要作用。. 综上所述，AtHSPR作为一个ABA介导的盐耐受性的正向调控子，通过清除过度积累的ROS、调控ABA诱导的气孔关闭和ABA信号转导、维持光合作用和体内Na/K离子平衡来增强植物的耐盐性。本研究不仅具有重要的科学意义，同时还为通过转基因提高农作物的盐旱耐受能力奠定了基础。