【作者】 李建萍；

【导师】 潘洪玉；

【作者基本信息】 吉林大学 ， 作物遗传育种， 2012， 硕士

【摘要】 植物在生长的过程中，常常受到各种不利环境的影响，严重危害植物生长发育及其产量和品质，甚至会导致植物死亡。植物对各种逆境胁迫的响应往往是多途径、多基因协同作用，从不同层次、不同水平调控对逆境的响应。其中，转录水平上的调控越来越受到人们的关注，如今对转录因子的研究已成为植物基因功能研究的一个重点。WRKY基因家族是目前研究比较多、重要的逆境相关转录因子之一。WRKY转录因子不仅参与植物的生长发育、代谢调控，还参与植物对生物胁迫和非生物胁迫的调控。本研究通过生物学信息学分析，在玉米自交系B73cDNA文库中分离鉴定出ZmWRKY25基因，并对ZmWRKY25基因在抗逆方面的功能进行了较系统的研究，研究结果如下：1本研究以玉米自交系B73cDNA文库为模板，根据GenBank中刊登的基因序列设计特异性引物，登录号为NM₀01158417，用PCR方法克隆得到一个WRKY基因。对该基因序列应用生物信息学软件分析表明，该基因编码区全长936bp，编码311个氨基酸。预测编码蛋白的相对分子质量为31.88kD,等电点为9.89，含有跨膜区。通过构建系统进化树进行同源性分析，显示ZmWRKY25与水稻中的OsWRKY25遗传距离较近。通过在线分析，预测该基因编码的蛋白含有信号肽及核输出信号，亚细胞定位于细胞核。2成功构建了植物表达载体pBI121∷ZmWRKY25，并通过农杆菌EHA105介导的方法转化到模式植物拟南芥中，通过50mg/L硫酸卡那霉素的筛选及PCR鉴定，获得了T3代转基因拟南芥植株。3PEG6000模拟的干旱逆境可以快速地诱导玉米中ZmWRKY25基因的表达。在拟南芥中过表达ZmWRKY25基因，可能提高了其对干旱逆境的耐受性。4外源的ABA可以快速地诱导玉米中ZmWRKY25基因的表达。ZmWRKY25基因可能参与ABA介导的信号途径，并且ZmWRKY25可能是ABA信号途径中的正调控因子。综上所述，本研究对WRKY转录因子家族的成员之一ZmWRKY25进行了初步的研究，为研究WRKY转录因子参与的植物响应胁迫调控的分子机制、植物逆境调控机理提供了一定的理论依据。但是，对其调控的下游基因的表达并未做深入探索，以及参与的信号途径并未从分子水平进行研究，因此，本实验还可以从这几个方面进行完善，为植物的抗逆分子机制研究及提高植物的抗逆性奠定基础。更多还原

【Abstract】 Plant was always exposed to a variety of adverse envirment in life of which seriousharmful to plant growth, development, yield and quality, and even lead todeath.Thus,plant generated series mechanisms to resist these damage and theseregulations to various stress stimuli are always not a single pathway but alwaysmulti-pathways,coordinated with multiple genes from different levels response tostresses. The regulations in transcription level are more and more important, and now theWRKY transcription factor has become one of the key point in the gene function research.WRKY transcription factor involved not only in plant growth and development,metabolic control, and it is also involved in the regulation to the process of plants tobiotic stress and abiotic stress.The following progresses were gain in this study:This study successfully cloned a WRKY gene by PCR method,take the maize inbredline B73cDNA library as template, use the primers designed according to the sequencepublished at GenBank, Accession No. is NM₀01158417.The nucleotide sequence totallength is936bp, encoding311amino acids of the coding region, suggesting the relativemolecular mass is31.88kD, isoelectric point is9.89, containing the transmembraneregion of the protein according to bioinformatics softwares.ZmWRKY25protein is closerwith OsWRKY25from rice in the genetic distance of homology analysied with thephylogenetic tree constructed. This protein encoding signal peptide and nuclear exportsignal with online bioinformatics, and subcellular localization prediction nucleus. Thestudy successfully obtained some T3generation of transgenic Arabidopsis plants with themethod of agrobacterium tumefaciens-mediated transformation use the strainEHA105,contianing the vector pBI21∷WRKY25constructed, it has throught screeningunder50mg L-1kanamycin and PCR detected. The expression of ZmWRKY25gene can be rapidly induced by drought stresssimulated by PEG6000in the corn.Overexpression ZmWRKY25gene in Arabidopsis mayincrease its tolerance to drought tolerance.The ZmWRKY25gene can be rapidly induced by added exogenous ABA in the corn.ZmWRKY25may be relyed on and play positive roles in ABA-mediated signalingpathways.In summary, ZmWRKY25gene was studied in this paper,which is one of the WRKYtranscription factors.However, its downstream genes regulated by ZmWRKY25did not dofurther exploration, as well as its roles in the signaling pathways have not studied at themolecular level,so,this experiment can be perfect in these several aspects for the study ofregulation mechanism of WRKY transcription factors involved in plant stress andimproved the resistance of corn.更多还原