【作者】 李田；

【导师】 毕玉平；

【作者基本信息】 山东师范大学 ， 植物学， 2014， 博士

【摘要】 林业在国民经济发展、维护生态平衡、提供高产优质林木方面具有重要作用，而高盐等非生物胁迫因素则严重制约着林木的生长发育及产量。植物耐盐性状通常受多个基因的调控，其应答基因涉及植物生理代谢、离子运输以及物质合成等各个方面。阐明植物的耐盐调控分子机制，对于通过基因工程手段来增强树种耐盐性具有重要的理论指导意义。绒毛白蜡是我国重要的林木种质资源之一，以较强的耐盐、耐旱等特性著称，是优良的盐碱地造林绿化和城市园林绿化树种。目前对于盐胁迫下模式植物的转录组变化等分子生物信息研究相对深入，而对木本植物这方面的研究相对较少。本研究以耐盐树种绒毛白蜡为材料，通过高通量测序对不同时间段盐胁迫下的绒毛白蜡幼苗进行表达谱分析，从中获得了大量与盐诱导相关的基因，通过qRT-PCR验证了8个基因在盐胁迫下的表达模式；并在此基础上，克隆了2个MYB转录因子基因及其启动子序列进行功能研究。结果将为深入了解绒毛白蜡耐盐分子机制以及绒毛白蜡树种遗传改良奠定理论基础。主要研究内容及结果如下：1.绒毛白蜡盐胁迫下的表达谱分析（1）以高盐胁迫下的绒毛白蜡幼苗为材料，采用数字基因表达谱技术对本实验中的6份样品进行序列测定，总共进行了55,525,943次读数。经测序分析，在三组比较中分别得到差异表达的基因数为72、1345、1379个。其中有4个差异表达基因为3个差异文库所共有，每个差异文库特异性表达的基因数目分别为50、317和353个。（2）GO显著性富集分析结果发现：NaCl胁迫0.5h后显著上调的GO terms主要集中于氧化胁迫响应、逆境胁迫响应、硝酸盐反应、端粒酶活性调节以及端粒酶正向调控方面。NaCl胁迫24h后，显著上调的GO terms数量明显增多，除包括上述功能外，还包括脱水干旱响应、应激反应、非生物胁迫刺激响应、ABA信号通路的调控等。Pathway显著性富集分析共筛选出20条可能与盐胁迫有关的代谢途径，如植物激素信号转导途径、淀粉和蔗糖代谢途径、植物病原体互作途径、苯丙素生物合成途径、细胞凋亡途径、类胡萝卜素生物合成途径以及戊糖和葡萄糖醛酸酯转换途径等。（3）通过分析和比较NaCl胁迫不同时间点绒毛白蜡的基因表达谱，筛选得到许多盐胁迫响应基因。这些基因的功能主要涉及渗透调节与离子平衡、激素信号转导、活性氧清除、转录调控等方面。推测这些基因很可能参与了绒毛白蜡的耐盐胁迫过程。（4）为验证测序结果的准确性，选择了8个差异表达基因，利用实时定量qRT-PCR方法验证了上述基因在NaCl胁迫下的基因表达模式，发现在NaCl胁迫0.5h和24h后这些基因表达趋势与表达谱测序结果一致。2.绒毛白蜡MYB转录因子基因的克隆及表达分析（1）通过RACE手段结合表达谱测序结果，首次克隆到2个绒毛白蜡MYB基因的cDNA全长，命名为FvMYB1和FvMYB2。其中，FvMYB1基因cDNA全长为1203bp，编码301个氨基酸，分子量约为33.38kDa，基因组扩增表明该基因无内含子；FvMYB2基因cDNA全长为1201bp，编码311个氨基酸，分子量约为35.00kDa，基因组序列全长为1476bp，含有3个外显子和2个内含子。（2）基因组织表达模式分析结果表明：两者在被检测组织中均有表达，其中FvMYB1基因在茎中表达量最高，在根中表达量最低；FvMYB2基因的表达量在各组织中差异不大。对不同非生物胁迫下的基因表达模式研究表明：在NaCl、PEG和ABA处理下，FvMYB1和FvMYB2基因表达模式存在异同。其中2个基因的表达量最高值分别在各处理12h和6h时出现，但FvMYB1基因只对NaCl和PEG响应，而FvMYB2基因可响应上述三种胁迫。暗示后者可能在植物应对非生物胁迫过程中发挥着更重要的作用。（3）亚细胞定位结果表明，FvMYB1和FvMYB2基因编码的蛋白均主要定位于细胞核中。酵母转录激活实验表明，两者均有转录激活活性，能够激活下游报告基因的表达，其中FvMYB2半乳糖苷酶活性高于FvMYB1。（4）构建了HIS-FvMYB1和HIS-FvMYB2融合蛋白表达载体并在大肠杆菌BL21中诱导表达，通过SDS-PAGE以及Western杂交分别验证了2个融合蛋白的表达。从而为后续进行转基因植物中目的蛋白的Western杂交提供相应的抗体制备蛋白基础。（5）构建了CaMV35S启动子驱动的FvMYB1、FvMYB2基因的植物过表达载体，利用农杆菌介导法转化烟草，对Kan筛选获得的转基因植株进行PCR和Southern杂交检测。并对正常生长条件下转基因植株中胁迫相关基因的表达进行了分析，结果表明NtP5CS、NtLEA5、NtMnSOD的表达量均高于对照。进一步对转基因植株进行盐胁迫处理后的表型及生理生化分析，结果表明，转基因烟草的盐害症状轻于野生型烟草；且盐胁迫条件下，转基因植株在SOD、POD、CAT保护酶活性、脯氨酸含量方面均不同程度的高于野生型，MDA含量低于对照；而叶绿素含量仅FvMYB2转基因烟草高于对照。实验结果表明FvMYB1和FvMYB2基因具有增强植物耐盐性的功能。3.绒毛白蜡FvMYB1和FvMYB2启动子克隆及分析（1）为进一步鉴定该基因的功能，对FvMYB1和FvMYB2基因的启动子进行克隆及其表达研究。利用染色体步移法（Walking）从绒毛白蜡基因组中克隆到这两个基因起始密码子上游各自l140bp和1600bp的启动子序列。通过Plant CARE分析FvMYB1和FvMYB2启动子中含有的顺式作用元件，表明两者除具有TATA-box、CAAT-box等典型的真核生物顺式调控元件外，还含有大量的光反应相关元件：G-box、GATA-motif、I-box、Box4、GAG-motif、C-box等；胁迫响应元件：盐诱导元件GT1GMSCAM4、ABA响应元件ABRE、MBS干旱诱导元件、水杨酸反应元件TCA、热诱导元件HSE、低温响应元件LTR等。（2）为进一步研究各顺式元件的作用，将FvMYB1和FvMYB2启动子序列的不同5’端缺失片段替换植物表达载体pCAMBIA3301的35S启动子，构建了以GUS为报告基因的缺失表达载体，并通过农杆菌转化获得包含不同目的片段的转基因烟草。GUS组织化学染色结果表明：FvMYB1基因3个缺失片段长度的启动子（242bp、523bp和792bp）具有启动功能；FvMYB2基因中858bp和1600bp长度的启动子可使转基因烟草GUS染色呈蓝色。且其中4个启动子缺失片段在盐胁迫后GUS染色加深，表明FvMYB1和FvMYB2启动子均在一定程度上受盐胁迫诱导。更多还原

【Abstract】 Forestry plays a decisive role in the national economic development, maintaining ecologicalbalance and providing the high yield and good quality trees. However, high salt stress and otherabiotic stress factors severely restrict the growth and yield of trees. The salt tolerance traits ofplants are usually controlled by multiple genes, and their response genes involved in many sides,such as plant physiological metabolism, ion transport and material synthesis, etc.. Clarifying themolecular mechanism of plant salt tolerance has important theoretical guarding significance toenhance the salt resistance of tree species by genetic engineering. Fraxinus velutina Torr., themain greening tree species for saline-alkali land and urban, is one of the important species in theforest, also with strong resistance to salt, drought and other good features. Now the deepresearches in transcriptome and other molecular biology data under salt stress are mainly focus onthe model plants, but the studies of woody plants are very few.In this study, we analyzed the gene expression profile of Fraxinus velutina Torr. under differentperiods of salt stress through high-throughput sequencing technologies and selected a largenumber of genes related to salt induced. Besides these, expression differences of eight genesunder salt stress were verified by qRT-PCR. On this basis, we cloned and analyzed the functionsof two MYB transcription factor genes and their promoters, pointed out the possible regulationapproaches of the two genes involved in plant salt resistance. The results will provide insight intothe salt-tolerant molecular mechanism and lay a theoretical foundation for genetic improvementof Fraxinus velutina Torr.. The main results of this study were as follows:1．Gene expression profile analysis of Fraxinus velutina Torr. under salt stress(1) In this study, digital gene-expression (DGE) technologies were used to investigate theglobal gene expression profiles of Fraxinus velutina Torr. seedlings under salt stress. All the amounts of the data obtained from the six groups of Fraxinus velutina Torr. were around fifty-sixmillion reads after processing. By sequencing analysis,72,1345and1379differentially expressedgenes were detected, respectively. Among them,4differentially co-expressed genes were incommon,50,317and353were special expression genes in each library.(2) GO analysis indicated that, after0.5h NaCl stress, the significant increased GO termsmainly included oxidation stress response, adversity stress response, nitrate response, telomeraseactivity regulation and positive regulation of telomerase activity. And the number of GO termsincreased significantly after24h NaCl stress, which included dehydration drought response,stress response, response of stress stimulation and ABA signaling regulation, etc.. In addition,20pathways maybe related to salt stress were identified by KEEG analysis, which mainly includedplant hormone signal transduction, starch and sucrose metabolism, plant-pathogen interaction,phenylpropanoid biosynthesis pathway, apoptosis pathway, carotenoid biosynthetic pathway andpentose and glucuronate interconversions pathway, etc..(3) By gene expression profile analysis, a large number of salt stress response genes wereobtained and the main functions of these genes involved in osmotic regulation and ion balance,hormone signal transduction, reactive oxygen removal, transcription regulation and so on. Theresult showed that these genes are likely to participate in the salt-resistance process of Fraxinusvelutina Torr..(4) In order to test the accuracy of the sequencing results, eight differentially expressed geneswere selected to analyze the expression patterns using qRT-PCR. The results were consistent wellwith those obtained from expression profile sequencing.2. Cloning and expression analysis of MYB genes from Fraxinus velutina Torr.(1) Two cDNAs encoding MYB genes were cloned from Fraxinus velutina Torr. by RACEand designed as FvMYB1and FvMYB2. Sequence analysis of FvMYB1showed that it was1203bp long and encoded301amino acids, with a calculated molecular mass of33.38kDa, and thegene had no introns. FvMYB2encoding311amino acids was1201bp in full length, with a calculated molecular mass of35.00kDa, and the genomic DNA contained three exons and twointrons.(2) The expression patterns of FvMYB1and FvMYB2in different organs of Fraxinus velutinaTorr. were analyzed by semi-quantitative RT-PCR. The results showed that both of them werewidely distributed in all the tested tissues. It was also found that the expression level ofFvMYB1was highest in stem and lowest in root while FvMYB2gene expression showed noobvious differences in all the tested tissues. Further, the gene expression patterns of MYB genesunder abiotic stresses (salt, drought and ABA) were also analyzed. Results indicated that therewere some similarities and differences between them. For example, the highest amountexpression time of FvMYB1and FvMYB2was around12h or6h. However, FvMYB2couldresponse to all the stresses above while FvMYB1could not response to ABA. The resultssuggested that FvMYB2may play a more important role than FvMYB1in the process of plantresponse to abiotic stress.(3) The subcellular localization of FvMYB1and FvMYB2showed that both were mainlylocalized in nucleus and the results were consisted with the studies of other transcription factors.In addition, the transcriptional activities of FvMYB1and FvMYB2were analyzed using yeastone-hybrid system. Results indicated that both of them could activate the expression ofdownstream report genes and β-Galaetosidase activity assay showed that FvMYB2has highertranscriptional activity than FvMYB1.(4) The prokaryotic expression vectors of HIS-FvMYB1and HIS-FvMYB2were constructedand expressed in E.coli BL21. Further SDS-PAGE and western blot were carried out to test thesuccess expression of FvMYB1and FvMYB2fusion protein, respectively. This result would lay afoundation for preparing effective antibodies used for western blot of transgenic plants.(5) To further investigate the functions of FvMYB1and FvMYB2genes, twoplant over-expression vectors pROKII-35S-FvMYB1and pROKII-35S-FvMYB2wereconstructed and introduced into tobacco genome respectively by Agrobacterium-mediated transformation. The transgenic tobaccos after Kan selection were obtained and examined by PCRand Southern blot. In normal conditions, some stress-related genes, such as NtP5CS, NtLEA5andNtMnSOD, expressed more in the transgenic tobaccos than the wild plants. Resistance analysisshowed that the salt injury symptoms of strains over-expressing FvMYB1or FvMYB2gene werelighter than the wild strains, and the concentration of SOD, POD, CAT and free proline of thetransgenic plants were higher than control, and the concentration of MDA was reduced aftertreated with NaCl for24h. In addition, the chlorophyll content of FvMYB2transgenic strains wasincreased in comparison with the control. These results indicated the roles of FvMYB1andFvMYB2in salt stress regulation.3. FvMYB1and FvMYB2promoter cloning and analysis of Fraxinus velutina Torr.(1) For further identified the function of FvMYB1and FvMYB2, two promoters of l140bp and1600bp were cloned from Fraxinus velutina Torr. genomic DNA based on genomic walkingprocedure. By Plant Care software analysis of the DNA sequences showed that many basic motifswere found in the FvMYB1and FvMYB2promoters, besides, there were some light-response andstress-response motifs including GT1GMSCAM4, ABRE, MBS, TCA, HSE, LTR, etc..(2) To investigate the characterizations of the FvMYB1and FvMYB2promoters, differentpromoter fragments were used to replace the35s promoter of pCAMBIA3301to construct plantdeletion expression vectors. The results of GUS histochemical staining of transgenic plants withdifferent deletion vectors showed that three of FvMYB1promoter sequences could drive the GUSgene expression, which were242bp,523bp and792bp in length. The transgenic plants withFvMYB2promoters of858bp and1600bp length showed blue color after GUS staining. Besides,the GUS stainings of all of the four fragments after salt induced were raised. Therefore, thepromoters of FvMYB1and FvMYB2could be induced by salt stress to some extent.更多还原