【作者】 龙瑞才；

【导师】 张泽； 杨青川；

【作者基本信息】 重庆大学 ， 植物学， 2013， 博士

【摘要】 盐胁迫是全世界普遍存在的一种非生物胁迫，也是导致作物减产的主要环境因素之一。在盐渍化土壤中植物生长和生理功能受盐胁迫环境因素的影响。目前为止，已在植物中发现多个参与盐胁迫逆境调控的基因，这些基因对增强植物的耐盐性具有重要意义。紫花苜蓿是一种重要的豆科牧草植物，蒺藜苜蓿是一种重要的豆科模式植物，为了研究紫花苜蓿“中苜一号”（Medicago sativa cv. Zhongmu-1）和蒺藜苜蓿“Jamalong A-17”（Medicago truncatula）在盐胁迫下应激反应机理，本研究使用差异蛋白质双向电泳技术对300mM NaCl胁迫处理0h和10h后的中苜一号和Jamalong A-17根中的蛋白进行差异蛋白质组学分析。从中苜一号根蛋白双向电泳凝胶中鉴定出超过1000个非冗余蛋白点，经分析后发现其中40个蛋白点在盐胁迫后表达丰度下降，53个蛋白点在盐胁迫后表达丰度升高；在JamalongA-17根中发现8个蛋白点在盐胁迫后表达丰度下降，22个蛋白点在盐胁迫后表达丰度升高。使用质谱分析技术对这些差异蛋白点进行鉴定，最终从中苜一号和Jamalong A-17根蛋白中分别成功鉴定出60和26个蛋白点。蛋白功能分析结果表明从中苜一号和Jamalong A-17根中鉴定出的许多差异蛋白参与植物逆境应激调控过程，这些蛋白的功能主要包括离子转运、酶催化、DNA和RNA绑定等。植物具备在转录水平和转录后水平进行逆境应激调控的机制。21-24nt的小分子RNA特别是miRNA是近年来发现的一种重要的转录后调控分子。为研究小RNA在苜蓿盐胁迫下的调控作用，本研究构建了紫花苜蓿“中苜一号”和蒺藜苜蓿“Jamalong A-17”根在盐胁迫0h和10h后的小RNA文库，并使用高通量测序技术对这些小RNA进行测序分析。从紫花苜蓿文库中测序获得659个已知miRNA，在蒺藜苜蓿文库中测序获得677个已知miRNA，这些miRNA属于250个已知miRNA家族。另外还在紫花苜蓿和蒺藜苜蓿文库中分别预测出189和218个新miRNA。对这些文库中miRNA的读数进行分析，结果表明中苜一号和JamalongA-17根中多数miRNA在盐胁迫前后表达量没有显著变化，但也有许多miRNA的表达量在盐胁迫前后差异显著。分析表明这些差异表达miRNA的靶标基因功能分布较广，其中许多靶标基因可能参与盐胁迫应激调控作用，这为植物特别是苜蓿的转录后水平耐盐调控机理提供了新的证据和研究方向。为进一步研究苜蓿的耐盐机理，本研究从紫花苜蓿中一个盐诱导基因的EST序列基础上克隆得到一个与酵母PMP3（plasma membrane protein3）和拟南芥AtRCI2A同源的基因MsRCI2A。在蒺藜苜蓿基因组数据库中进行序列比对分析得到五个与MsRCI2A同源的基因（MtRCI2A-E），这些基因都编码疏水性较强的小分子蛋白，都含有两个跨膜区域。亚细胞定位分析结果表明MsRCI2A和MtRCI2A-E编码蛋白基本都定位于细胞膜上。转录表达水平分析结果表明MsRCI2A和MtRCI2A-D在盐胁迫下表达量明显升高，MtRCI2E的表达量无明显变化。MsRCI2A和MtRCI2A-C能使酵母PMP3基因缺失突变体的功能得到恢复。转基因研究结果表明在拟南芥中过量表达MsRCI2A后能够使其耐盐性得到明显增强。目前已在植物中发现多种参与逆境应激反应的甘氨酸富集蛋白（glycine richprotein，GRP），但其中许多甘氨酸富集蛋白的具体作用机理还未知。本研究从紫花苜蓿中分离克隆到一个受盐诱导的甘氨酸富集蛋白（MsGRP），对其编码蛋白序列分析结果表明MsGRP中不含有RRM结构域（常见的甘氨酸富集型RNA绑定蛋白所特有的结构域）。亚细胞定位分析表明MsGRP定位于细胞膜附近。转录表达分析结果显示MsGRP受盐、ABA和模拟干旱胁迫诱导。转基因研究结果表明过量表达MsGRP基因的拟南芥在种子萌发期和幼苗期都表现出比野生型拟南芥对盐和ABA更敏感。这些结果表明MsGRP可能通过ABA信号途径参与苜蓿的盐胁迫应激调控，且可能在盐胁迫等非生物胁迫下起到负调控作用。更多还原

【Abstract】 Salt stress is one of the most significant abiotic stresses to cause crop failureworldwide. So far, many salt stress induced genes and improving salt tolerance geneshave been characterized in many plants. In saline lands, plant root growth and functionare determined by the action of environmental salt stress through specific genes thatadapt root development to the restrictive condition. Medicago sativa and Medicagotruncatula are two important legumes for one is a primary forage, the other is a modellegume. To understand better NaCl stress responses in Medicago. sativa (Zhongmu-1)and Medicago. truncatula (Jamalong A-17) roots, a comparative proteomic analysis ofroots that had been exposed to300mM NaCl for0h and10h was conducted. Changesin the abundance of protein species within roots were examined using two-dimensionalelectrophoresis. Among the>1000protein spots reproducibly detected on M. sativaroots gel, the abundance of40protein spots decreased and53increased, at10h timepoints, in response to NaCl treatment. Among the>1000protein spots reproduciblydetected on M. truncatula roots gel, the abundance of8protein spots decreased and22increased, at10h time points, in response to NaCl treatment. Through tandem massspectrometry, identity was assigned to60and26of the differentially abundant spots inM. sativa (Zhongmu-1) and M. truncatula (Jamalong A-17) roots. The proteinsidentified included many previously characterized stress-responsive proteins and someprocesses including iron transport, catalysis, DNA or RNA binding and so on.In plant, many functions are regulated in transcriptional and posttranscriptionallevels. Small21-to24-nucleotide RNAs, notably the microRNA (miRNA), areemerging as a posttranscriptional regulation mechanism. Four small RNA librariesconstructed from roots of Zhongmu-1(M. sativa, salt tolerance) and Jamalong A-17(M.truncatula, salt sensitive). High-throughput sequencing was used to sequence the smallRNAs in these libraries.659and677conserved miRNA belong to250miRNA familieswere identified in Zhongmu-1and Jamalong A-17, as well as189and218novelcandidate miRNAs in were identified in Zhongmu-1and Jamalong A-17. Statisticalanalysis on sequencing reads abundance revealed specific miRNA showing contrastingexpression patterns in Zhongmu-1and Jamalong A-17roots.The differentiallyexpressed conserved and novel miRNAs may target a large variety of mRNAs, some of which may play key roles in salt stress regulation. The spatial salt stress regulation ofmiRNAs may determine specialization of regulatory RNA networks in plant.In this study one new salt induced M. sativa (alfalfa) gene (MsRCI2A) was clonedand characterized based on its EST, which shows high homology to yeast PMP3gene(plasma membrane protein3) and Arabidopsis AtRCI2A. Sequence comparisons revealthat five genes (MtRCI2A-E) show high homology to MsRCI2A in M. truncatulagenome. MsRCI2A and MtRCI2A-E all encode small, highly hydrophobic proteinscontaining two putative transmembrane domains, and mostly localize in the plasmamembrane. Transcription analysis result suggests that MsRCI2A and MtRCI2A-D genesare highly induced by salt stress. Expression of MsRCI2A and MtRCI2A-C in yeastmutants lacking the PMP3gene can functionally complement the membranehyperpolarization and salt sensitivity phenotypes resulting from PMP3deletion. Toinvestigate whether the overexpression of RCI2-ralated gene causes an enhancedsalt-tolerant phenotype, MsRCI2A is overexpressed in Arabidopsis thaliana. The resultreveals that the salt tolerance of MsRCI2A-overexpressing transgenic plants is improvedcomparing to the wild type under high salinity treatment.Many glycine-rich proteins have been implicated in plant responses toenvironmental stresses, but the function and importance of some GRPs in stressresponses are largely unknown. In this study a novel salt induced glycine-rich proteingene (MsGRP) was isolated from alfalfa. Compared with some glycine-rich RNAbinding proteins, MsGRP contains no RRM motif and localizes in the cell membrane orcell wall according to the subcellular localization result. MsGRP mRNA is induced bysalt, ABA and drought stresses in alfalfa seedlings, and its overexpression driven by aconstitutive cauliflower mosaic virus-35S promoter in Arabidopsis plants conferssalinity and ABA sensitivity, as compared with WT plants. MsGRP retards seedgermination and seedling growth of transgenic Arabidopsis plants under salt and ABAtreatments, implying that MsGRP may affect germination and growth through an ABAdependent regulation pathway. These results provide indirect evidence indicating thatMsGRP plays important roles in seed germination and seedling growth of alfalfa undersome abiotic stress conditions.更多还原