【作者】 李朔；

【导师】 夏光敏；

【作者基本信息】 山东大学 ， 遗传学， 2009， 博士

【摘要】 植物的耐盐性是由多基因控制的复杂性状,盐胁迫应答基因涉及代谢、防御反应、能量、离子平衡和物质转运等诸多方面。深入理解控制这个性状的分子机制,有助于农作物的基因改良从而减少盐害带来的损失。对盐胁迫下植物转录组整体水平上的研究对于耐盐机理的揭示具有重要意义。目前对模式植物拟南芥和水稻盐胁迫下转录组变化研究取得了许多进展,而对非模式植物如小麦的研究较少。山融3号是从普通小麦济南177（Triticum aestivum L.2n=42）与其耐盐近缘属长穗偃麦草（Thinopyrum ponticum 2n=70）不对称体细胞融合杂种中筛选出来的耐盐渐渗系新品种。遗传和生理生化分析表明山融3号基因组中含有渐渗的长穗偃麦草基因组小片段,其耐盐指数及各项耐盐生理指标均优于其亲本,且其耐盐性状由一个主效基因位点和多个微效基因位点共同控制。本论文设计了山融3号原位合成长oligo表达谱芯片,包含15000个unigene,将之用于研究盐胁迫和渗透势相当的PEG（polyethylen glycol）胁迫下山融3号及其亲本济南177根部转录组的变化情况,通过对芯片杂交结果的分析,系统性的阐明了山融3号耐逆可能涉及的途径,还对盐胁迫早期渗透胁迫与离子胁迫对转录组影响的关系进行了探讨;在此基础上,进一步着重研究了山融3号中克隆的4个盐胁迫响应基因,结合在拟南芥中过表达等多种手段,进行功能相关研究,指出了这些基因在山融3号中参与耐逆调控的可能方式。主要的研究内容及结果包括:1.山融3号表达谱芯片的设计山融3号的来源与遗传背景特殊,其中有别于普通小麦的转录子可能对耐盐性状有着重要贡献,为了将这些转录子囊括于芯片分析的范围之内,首先构建了山融3号和济南177根部盐胁迫下的SSH cDNA文库,将文库测序得到的1000个EST序列聚类成180个unigene、连同山融3号全长cDNA文库EST序列聚类而成的2539个unigene、并整合DFCI小麦数据库中的unigene资源,设计了代表山融3号15000个unigene的原位合成长oligo表达谱芯片。2.山融3号及其亲本济南177根部盐胁迫转录组变化差异的分析利用上述芯片研究了山融3号及其亲本济南177在盐胁迫下不同时间点根转录组之间的差异,发现了836个具有显著性差异的探针,对这些差异基因的差异模式和功能的分析表明:山融3号在金属离子、水分和营养元素的运输、抗氧化、类黄酮类合成、部分ABA途径基因、部分防御反应相关基因上具有表达优势,而表达显著下降的基因包括大量的光合作用相关基因以及部分防御反应基因;对调控类基因（如转录因子和激素合成与代谢相关基因）和转运蛋白基因响应模式的分析发现,山融3号中盐胁迫下上调的探针数目更多,且上调程度更高,而盐胁迫下调探针的下调程度则较济南177中低,暗示山融3号的胁迫适应性反应强度提高,进而提高了胁迫耐受性;相对与济南177,JA（jasmonic acid）合成关键基因和GA（gibberellin）合成关键基因在山融3号中分别组成型的大幅度表达量降低和升高,暗示了两个品种中不同的激素平衡情况;最后,总结了芯片中54个在两个品种间表达有差异且代表未知小麦EST的探针,这些EST序列均来源于山融3号自身,有可能是体细胞融合带来的外源基因或基因变异的产物。以上发现有助于阐述山融3号的耐盐机理,并提供了许多潜在的耐盐候选基因,同时证明了体细胞融合对转录组的重大影响,以数据说明了该技术应用于耐盐品种培育的价值。3.利用NaCl和PEG等渗处理研究盐胁迫早期响应机制为了研究小麦根部对盐胁迫早期响应机制,并区分渗透胁迫与离子胁迫的关系,利用渗透势相当的NaCl和PEG对山融3号和济南177进行等渗胁迫处理,基于芯片杂交数据,分析了根部胁迫早期的转录组变化,发现:盐胁迫早期响应以调控基因的上调和转运相关基因的下调为主;等渗处理下盐胁迫和单纯渗透胁迫引发的转录组变化有很大一致性,但PEG胁迫引起了更大范围的转录组变化,同时调控类基因（转录因子和激素合成与代谢相关基因）对PEG胁迫的响应程度更大;存在非渗透胁迫响应的盐胁迫响应探针,并且转运蛋白基因中在胁迫早期下调的探针在NaCl胁迫下比PEG胁迫下下调程度更大。这些发现首次在转录组水平证明盐胁迫早期以渗透胁迫的影响为主,而离子胁迫的影响也同时存在。所筛出的差异探针也为研究早期盐胁迫信号传导和离子胁迫特异性信号传导提供了候选基因。4.山融3号盐胁迫早期响应基因TaDi19A和TaDi19B的克隆与功能研究基于对山融3号和济南177盐胁迫根部SSH文库表达差异筛选的结果,由EST片段克隆到山融3号TaDi19A和TaDi19B基因,它们都属于Di19（DROUGHTINDUCED19）基因家族。其中:TaDi19A定位于小麦3B染色体长臂,主要在细胞核中起作用,在非胁迫条件下小麦的根和叶中都有基本表达,在NaCl、PEG、冷以及非生物胁迫相关激素ABA和乙烯的处理下迅速上调表达;该基因在拟南芥中的组成型表达造成转基因植株在种子萌发阶段对盐胁迫、渗透胁迫以及ABA处理超敏感;转基因植株根的伸长实验表明植株盐耐受性降低且对乙烯的敏感性降低;在H₂O₂处理下,转基因植株的开花期比对照大大提前。另外,转基因拟南芥中ABA信号传导途径基因ABI1、RAB18、ERD15和ABF3,以及SOS（salt overly sensitive）途径基因SOS2的转录水平发生改变。因此,TaDi19A可能是作为胁迫信号传导的调控因子,通过改变这些基因的转录来对植物非生物胁迫及相关激素的响应产生影响。对TaDi19B基因的序列特征、亚细胞定位、表达谱进行了研究,并建立了拟南芥组成型表达的转基因株系。发现它主要定位于细胞核,其表达受到盐、渗透胁迫和冷胁迫的诱导,并且在山融3号中的上调更明显,推测它可能对山融3号的高耐盐性有贡献,参与了盐等非生物胁迫的响应。5.山融3号盐胁迫早期响应基因TaERD15A和TaERD15B初步的功能研究同样基于SSH文库表达差异筛选的结果,从山融3号中克隆了TaFRD15A和TaERD15B基因,对它们的序列分析表明,这类基因编码的蛋白在各个物种中差异较大,保守性较差,其拟南芥同源基因ERD15是ABA途径负调控因子。对它们的基因结构、亚细胞定位、表达谱进行了分析,并分别获得了拟南芥组成型表达的转基因株系。其中,TaERD15A位于小麦1A染色体短臂,蛋白在细胞内主要定位于细胞核,并且在盐胁迫和PEG造成的渗透胁迫下上调表达,且在济南177中的表达量更高,同时,它也受到低温胁迫的诱导,但不受ABA诱导。该基因在拟南芥中的组成型表达造成转基因植株的盐耐受性降低。这说明它可能是胁迫耐受性的负调控因子,并且通过非ABA依赖的途径起作用,而盐胁迫下该基因在山融3号中更低的表达量可能是山融3号更高盐耐受性的原因之一。TaERD15B的亚细胞定位与TaERD15A类似,并同样受PEG和冷胁迫诱导,但是不受NaCl胁迫诱导,而在PEG处理带来的渗透胁迫下,该基因在济南177中的表达量高于山融3号,推测它可能主要参与渗透胁迫和冷胁迫的响应过程。更多还原

【Abstract】 As salt-stress tolerance characters in plants are controlled by quantitative trait loci,a comprehensive resolution of salt-tolerance mechanisms has been complicated.Microarrays have become powerful tools for high throughput screening of salt-stress responsive genes. In model plants,such as Arabidopsis and rice,tens of thousands of gene expression patterns in response to salt stress have been monitored.But for wheat,the information was still limited.A new somatic hybrid introgression line Shanrong No.3（SR3） has been generated in our lab from hybridization of common wheat Jinan 177（JN177） with Thinopyrum ponticum, a salt and drought tolerant grass.Cytological and molecular analysis showed that some nuclear and non-nuclear DNAs and even functional genes of donor T.ponticum were introgressed into this line.SR3 had a significantly higher yield than its parent JN177 in salt-alkali soil of Shandong,China.It has passed Shandong provincial regional yield trial for new salt-enduring wheat cultivar（Lu-Nong-Shen-Zi No.[2004]030）.The results of SSRs analysis suggest that salt tolerance of SR3 is controlled by a major salt tolerance gene and some microgenes.To investigate the salinity tolerance mechanism of SR3,we have developed a new long oligo-DNA microarray based on the EST sequences either from SR3 or from public wheat EST database,which harbors 15,000 unigenes.Using it,the transcriptomes of salt and osmotic stress responses were fully analyzed in SR3 and JN177. Additionally,four stress responsive genes were cloned from SR3 and functionally analyzed.The main research contents and results achieved in this work were summarized as follows.1.Probe design of oligo-DNA microarrays for SR3For the special genetic background of SR3,it may have transcripts other than in common wheat which may be related to the salt tolerance of SR3.To harbor these transcripts in the microarray,we generated a SSH cDNA library between SR3 and JN177 under salt treatment.One thousand sequences from the library were clustered into 180 unigenes.We also get 2,539 unigenes from a complete cDNA library of SR3 which was constructed before.Based on these sequences,together with another 12,281 unigenes from DFCI Wheat Gene Index,probes were designed and synthesized in situ on glass slides by Agilent Technologies.2.Transcriptomes comparison between SR3 and JN177 under salt stressUsing the microarray,root transcriptomes were compared between SR3 and JN177 under time course salt treatment,and 836 probes were identified to be expressed with significant difference.By analyzing the expression patterns and potential functions of the probes,we found that:Genes related to transport（of metal ions,water and nutrient）,antioxidant production, flavonoid biosynthesis,some ABA pathway components and defense,were expressed more in SR3 than in JN177.While genes involved in photosynthesis and defense were expressed less.Based on the responsive patterns of regulatory genes（including transcription factor genes and genes involved in hormone biosynthesis and metabolism） and transporter genes under salt stress,it was found that more of these genes were up-regulated in SR3 than in JN177,and the up-regulation was also greater in SR3.However the down-regulation was more moderate in SR3.This may indicate that the adaptational response is stronger in SR3. Expression of the key genes involved in JA and GA biosynthesis were greatly suppressed and over-expressed respectively in SR3 than in JN177.This suggested the difference of the hormone balance between the two lines.Finally,54 differently expressed probes which represented new ESTs were highlighted. They were all from SR3 cDNA libraries and might be exogenous or mutant genes which resulted from the somatic hybridization.These findings can not only help to reveal the salinity tolerance mechanism of SR3 and provided lots of candidate salt-tolerance related genes,but also confirmed the great impression on transcriptome by somatic hybridization and further indicated the worth of applying this technique on salt tolerant crop breeding.3.Transcriptome analysis under NaCl and PEG treatments with equal osmotic potential revealed the mechanism of early salt responsesTo investigate the mechanism of early salt responses in wheat roots,especially the relationship between osmotic and ionic stress during salt stress,we treated SR3 and JN177 with NaCl and PEG which conferred equal osmotic stress.After analyzing the transcriptome data of microarray,we found that:The early salt responses of wheat root transcriptome were mainly composed of the up-regulation of regulatory genes and the down-regulation of transporter genes.The transcriptome changes under NaCl and PEG treatment shared common in a large extent,but more genes were affected under PEG treatment in comparison with NaCl treatment,and the regulatory genes（genes involved in transcriptional regulation or hormone biosynthesis and metabolism） also changed greater under PEG treatment.Some probes only responded to NaCl but not to PEG and some transporter genes were more suppressed under NaCl treatment than under PEG treatment.These findings confirmed that osmotic stress was the main stress conferred by salt treatment at the earlier stage.It was surprising that osmotic stress itself caused larger transcriptomal changes than the combination of osmotic and ionic stress.This suggests a salt stress specific way to moderate the osmotic impaction.The differently expressed genes may be involved in early salt signal transduction or ionic stress specific signal transduction.4.Cloning and functional analysis of early salt responsive genes TaDi19A and TaDi19B from SR3Based on the differential screening of SSH cDNA library,TaDi19A and TaDi19B were cloned from SR3,which belonged to Di19（DROUGHT INDUCED 19） gene family.TaDi19A was localized in long arm of the chromosome 3B and its protein products mainly presented in nucleus.It was constitutively expressed in both the roots and leaves of wheat seedlings grown under non-stressed conditions,but was substantially up-regulated by the imposition of stress（salinity,drought and cold）,or the supply of stress-related hormones （ABA and ethylene）.The heterologous over-expression of TaDi19A in Arabidopsis thaliana increased the plants’sensitivity to salinity stress,ABA and mannitol during the germination stage.Root elongation in these transgenic lines showed a reduced tolerance to salinity stress and a reduced sensitivity to ethophon.Flowering was accelerated in the transgenic lines when stressed with H₂O₂.The expression of the ABA signal pathway genes ABI1,RAB18,ERD15 and ABF3,and SOS2（SOS pathway） was altered in transgenic lines.These results suggest that TaDi19A plays a role in the plant’s response to abiotic stress,and some possible mechanisms of its action are proposed.For TaDi19B,we analyzed its sequence character,subcellular localization and expression patterns.The heterologous over-expression Arabidopsis transgenic lines were generated.TaDi19B protein was found to be presented in nucleus and was transcriptionally up-regulated by salt,osmotic and cold stress.Its stress response was greater in SR3 than in JN177.This suggests that it may contribute to the salinity tolerance of SR3 and is involved in abiotic stress response.5.Cloning and preliminary functional analysis of TaERD15A and TaERD15B, two stress responsive genes from SR3Also based on the differential screening of SSH cDNA library,TaERD15A and TaERD15B were isolated from SR3.By homology analysis of their protein sequences,it was found that this type of proteins was less conserved among species.The only known homologous gene in Arabidopsis is ERD15A（EARLY RESPONSE TO DEHYDRATION 15）, a negative regulator of ABA pathway.Gene structure,subcellular localization,expression patterns of the two genes were analyzed and heterologous over-expression Arabidopsis transgenic lines were generated respectively.TaERD15A localized on the short arm of chromosome 1A and the protein presented in nucleus.It was up-regulated by NaCl and PEG treatments and more expressed in JN177 than in SR3.It also responded to cold but not to exogenous ABA.The salinity tolerance of transgenetic Arabidopsis seedlings was reduced.These results indicate that TaERD15A is a negative regulator of salinity tolerance and functions in an ABA independent manner.Its less expression in SR3 under salt stress may results in the significant salt tolerance.The subcellular localization of TaERD15B was similar to TaERD15A.It was also induced by PEG and cold treatment but not by NaCl treatment.Under PEG treatment,its transcripts accumulated more in JN177 than in SR3.These imply that it is involved in osmotic and cold stress response.更多还原