【作者】 李慧玉；

【导师】 姜静； 王玉成；

【作者基本信息】 东北林业大学 ， 林木遗传育种， 2007， 博士

【摘要】 柽柳(Taramix sp.)具有强的耐盐碱能力，是进行植物耐盐碱机理研究的理想材料。为研究盐胁迫下柽柳根部的耐盐机理，本研究以0.4 mol／L NaCl胁迫处理刚毛柽柳(Tamarixhispida)根部组织为试材，分别构建对照及胁迫24h、48h的3个cDNA文库。3个文库的初始滴度为1.0×10~6 pfu、1.4×10~6 pfu、1.2×10~6 pfu，插入片段平均长度分别为0.8 kb、0.9kb、0.85kb，平均重组率为98.2％。通过对3个cDNA文库克隆的随机测序共获得7726条有效的EST序列，其GenBank注册号为EG966290～EG974015，这些EST代表了4168条无重复的单一序列(uniquesequence)，其中包括1142条contigs和3026条singlets。其中4508条EST与与NCBI的Nr数据库中的已知蛋白序列相似，并将它们按功能分成11类，占比例最高的是蛋白质合成类(protein synthesis)基因，约21.4％，功能未知(Function unknown)和转运类(Transport facilitation)分别占15％和12.2％，代谢类(Metabolism)占11.2％、细胞防御(Cell rescue，defense)占7.3％、能量(Energy)占7.0％、细胞结构类(Cell structure)6.8％、转录(Transcription)占5.1％、蛋白质定位(Protein destination)4.8％、信号转导(Signal transduction)占4.1％、细胞生长／分裂(Cell growth，division)占3.7％。3个cDNA文库中共有860条EST与抗逆相关，其中，活性氧清除的基因(27％)、胁迫响应(16％)、细胞结构(16％)、蛋白质合成及降解基因(10％)，代谢和能量、信号转导及转运所占比例次之，转录调控和渗透调物质合成基因最少，各占3％。通过对3个文库的EST的分析及比较，发现共有106条基因在NaCl胁迫前后呈差异表达，发现34条上调表达基因，27条下调表达基因，45条基因出现暂时差异表达，其中H~+-ATP合成酶、ABA胁迫成熟蛋白、脂质转运蛋白、金属硫蛋白、类萌芽素蛋白、晚期胚胎富集蛋白类ERD15和LEA5等基因胁迫后表达丰度上升；而细胞壁结构蛋白富含脯氨酸蛋白及木糖葡聚糖型内转运糖基化酶等基因胁迫后表达量下降；蛋白质翻译延伸因子EF1α和翻译起始因子eIF 5a等基因胁迫后出现瞬时的上升或下降。采用实时荧光定量PCR技术，分别研究文库中获得的8种POD和7种水通道蛋白基因在NaCl胁迫前后柽柳根和叶组织中的表达差异。结果表明，盐胁迫后POD基因在柽柳根和叶组织细胞中的表达模式相同，在胁迫24h时出现瞬时抑制现象；水通道蛋白基因在盐胁迫在根、叶器官中有表达模式有明显的差异。盐胁迫后7种水通道蛋白基因在根部表达量上升；在叶部它们胁迫后表达量下降。从文库中克隆获得了25条抗逆相关基因的全长cDNA序列，这些基因这些基因分属于：细胞防御、离子平衡和转运、抗病、细胞壁成分、信号转导与调控、水分胁迫相关蛋白、蛋白质的合成与降解。从盐胁迫下柽柳众多基因的表达特征信息可推断，柽柳根部对盐胁迫响应的过程可能是一个复杂的多基因协同作用的过程，涉及活性氧清除、细胞壁组分、胁迫信号转导与基因的表达调控、水分和离子的转运、脱水保护等多种途径。从而为系统阐明柽柳抗盐分子机理奠定坚实的基础。对刚毛柽柳抗逆基因的克隆，可为基因工程育种提供优良的侯选基因。更多还原

【Abstract】 Taramix hispida, a halophyte with high capacity of salt tolerance, is an excellent model plantin plant salt tolerance research. Three cDNA libraries were constructed from the root tissues of T.hispida of well-watered and exposed to NaC1 (0.4 M NaCl)for 24 and 48 h, respectively (named ascontrol, 24 and 48 h library, respectively). Average primary titer of the three libraries was 1.0×10~6pfu, 1.4×10~6 pfu, 1.2×10~6 pfu, respectively, with a rate of recombination about 98.％. PCRdetection revealed that their average inserts size was respectively 0.8, 0.9, and 0.85 kb.A total of 7 726 expressed sequence tags(ESTs) were obtained through randomly selectedsequencing process, with the GenBank accession numbers of EG966290-EG974015. The total of 7726 ESTs represented 4 168 non-redundant unique transcripts including 1 142 contigs and 3 026singletons. Total of 4 508 ESTs showed there existed significant similarityto the sequences with thesequences in NCBI Nr database, which were further classfied into 11 functional categories based ontheir putative functions, i.e., protein synthesis (21.4％), Function unknown (15％), Transportfacilitation (12.2％), Metabolism (11.2％), Cell rescue, defense (7.3％), Energy (7.0％), Cellstructure (6.8％), Transcription (5.1％), Protein destination (4.8％), Signal transduction (4.1％) andCell growth, division (3.7％). It should be emphasized that a remarkable variability of number of cellstructure genes was found among the three libraries. The percent of the cell structure genes in 24 hlibrary was three times of the control and 48 h library.Nearly 860 ESTs involved in salt tolerance were found from the 3 cDNA librarilies. Amongthem, scavenging reactive oxygen species was the most aboundant one, accounting for 27％, stressrelated protein and protein synthesis accounted for 16％respectively. Protein synthesis and degrationwas 10％, followed by metabolism and energy, signal transduction and transport. The smallest weretranscription and osmolyte biosynthesis genes, accounting for 3％respectively.EST analysis showed that total 106 unique genes showed different expression patterns afterexposure to NaCl; among them, 34 genes were up-regulated, 27 genes were down-regulated and theother 45 genes were transiently down or up-regulated. Among these differentially regualted genes,H~+-ATPsynthase, ABA stress ripening protein, lipid transfer protein, metallothionein, germin-likeprotein, ERD15 and LEA5 were up-regulated; however, the frequency of ESTs of structural cell wallprotein, including proline-rich protein and xyloglucan endotransglucosylase/hydrolase protein etcwere down-regulated, and EF1αand eIF 5a were transiently down or up-regulated.To explore the differences expression mode between roots and leaves of T. hispida exposed toNaCl, eight unique transcripts of POD and seven unique transcripts of aquaporin were selected forexpression analysis using real-time reverse transcription-polymerase chain reaction (RT-PCR).Theresults showed that gene expression pattems of eight POD genes were similar with each other in roots and leaves of T. hispida after exposing to NaCl for 0, 24 and 48 h, with repressed at 24 h andrecovered at 48 h. The different time course expression patterns of aquapofin appeared between inroots and leaves of T. hispida, seven aquaporin transcripts were up-regulated under NaCl stress inroots, however, they were down-regulated in leaves.Totally, 25 full-length genes were cloned from T hispida. These genes involved in the celldefense, anti-disease, ion homeostasis and transport, water stress related protein, cell structure,protein synthase and degration, and signal transduction.Based on the above, it could be concluded that salt-tolerance mechanism of T. hispida is acomplex system, involving with the cooperation of many pathways, such as scavenging reactiveoxygen, cell wall component, signal tranduction and transcription regulating, transporting of waterand ions, protection of dehydration damage. The studies could be used as a foundation in thefuture comprehensive reseaches of the salt resistance molecular mechanism of T. hispida. Thecloning of stress-tolerance genes may offer some attractive candidate genes for genetic engineeringof trees.更多还原