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黄瓜CsWRKY21对干旱胁迫的应答及其作用
The Roles of Cucumber CsWRKY21 on Plant Drought Stress Responses
【作者】 杨阳；
【导师】 张颖；
【作者基本信息】 沈阳师范大学 ， 生物化学与分子生物学， 2019， 硕士
【摘要】 黄瓜(Cucumis sativus L.)是一种可食用的葫芦科黄瓜属园艺作物,世界上栽培最为重要的园艺作物之一。在生产实践中,干旱对植物影响较为普遍,且大大的影响了作物的产量。与此同时转录因子能够参与植物逆境胁迫的响应并起到了重要的作用。本文以黄瓜及转基因过表达拟南芥作为研究材料,探讨在干旱逆境中黄瓜CsWRKY21的表达模式及功能。通过分析干旱胁迫下黄瓜幼苗的变化,CsWRKY21基因在干旱及ABA影响下的表达变化,转基因过表达CsWRKY21植株在干旱胁迫下的表型及功能分析,探讨该基因参与黄瓜干旱胁迫可能的分子调控机制,为解决园艺作物的生产实践中所存在的问题提供理论依据,所得主要结论如下:1.干旱胁迫对黄瓜幼苗的影响本研究表明:当黄瓜幼苗处于干旱胁迫时植物体内的叶绿素a、叶绿素b、叶绿素总含量都下降。同时在此条件下喷施ABA能够促进上述叶绿素含量上升,进而缓解干旱对植物合成叶绿素的影响。喷施ABA预处理后,可缓解干旱胁迫下黄瓜幼苗中MDA含量下降,可溶性糖、电导率含量上升,抗氧化酶活性提高,证明ABA预处理对黄瓜幼苗抵御干旱有积极作用。此外,我们前期的研究表明,CsWRKY21响应干旱胁迫表达上调,响应外源ABA表达上调。2.黄瓜CsWRKY21的cDNA克隆及生物信息学分析本研究表明:应用RT-PCR克隆得到CsWRKY21的cDNA开发读码框全长序列,将产物连接到pEASY-T1载体中,进行蓝白斑筛选,经菌落PCR鉴定的阳性克隆,扩大培养,提取质粒,双酶切后送上海生工公司测序,得到该基因的cDNA序列。根据CsWRKY21蛋白的亚细胞定位预测,预测CsWRKY21中蛋白有82.6%位于细胞核中,其余13.0%位于细胞质中。该结果表明CsWRKY21是符合转录因子的特性。3.干旱胁迫对转基因植物生理生化指标以及基因表达影响本文研究表明经过干旱胁迫后,野生型(WT)植株大部分枯萎,而过表达转基因(CsWRKY21-OE2、CsWRKY21-OE5)的植株状态保持良好,未受到影响。且干旱胁迫后WT植株含水量明显下降,MDA含量、可溶性糖含量、脯氨酸的含量升高,而转基因过表达植株则没有发生明显的变化。qRT-PCR分析表明,转基因过表达增强了DREB1A,DREB2A,ABF4及ADH等基因的表达,因此可以推测过表达转基因CsWRKY21可能通过ABA依赖途径调控植物抗旱。
【Abstract】 Cucumber is an edible horticultural crop of Cucurbitaceae,one of the most important horticultural crops cultivated in the world.In production practice,drought has a widespread impact on plants,and has a great impact on crop yields.At the same time,transcription factors can participate in plant response to stress and play an important role.This paper takes cucumber and transgenic over expressed Arabidopsis thaliana as research materials,to explore the expression pattern and function of CsWRKY21 in Cucumber under drought stress.By analyzing the changes of Cucumber Seedlings under drought stress,the expression changes of CsWRKY21 gene under drought and ABA,and the phenotype and function analysis of transgenic over expressed plants under drought stress,to explore the possible molecular regulation mechanism of this gene in Cucumber under drought stress,and to provide theoretical basis for solving the problems in horticultural crop production practice.The main conclusions are as follows:1.Effects of Drought Stress on Cucumber SeedlingsThis study shows that:when cucumber seedlings were under drought stress,the contents of chlorophyll a,B and total chlorophyll in plants decreased.At the same time,spraying ABA under these conditions can promote the increase of chlorophyll content,and then alleviate the effect of drought on plant chlorophyll synthesis.Spraying ABA pretreatment could alleviate the decrease of MDA content and increase the contents of soluble sugar,conductivity and antioxidant enzymes in Cucumber Seedlings under drought stress,which proved that ABA pretreatment had a positive effect on Drought Resistance of cucumber seedlings.2.cDNA Cloning and Bioinformatics Analysis of Cucumber CsWRKY21 This study shows that:The full-length reading frame sequence of CsWRKY21 was developed by RT-PCR cloning,the product was linked to pEASY-T1 vector,screened for blue and white spot,positive clones identified by colony PCR,expanded culture,plasmid extraction,double enzyme digestion and sequencing by Shanghai Biotechnology Company to obtain the gene’s DNA sequence.According to subcellular localization of CsWRKY21 protein,measured out that 82.6%of the CsWRKY21 proteins were located in the nucleus and 13.0% in the cytoplasm.The results showed that CsWRKY21 was a transcription factor.3.Effects of Drought Stress on Physiological and Biochemical Indicators and Gene Expression of Transgenic Plants.This study showed that almost all wild type(WT)plants withered after drought stress,overexpression of transgenic plants(CsWRKY-OE2,CsWRKY-OE5)maintained a good state without being affected.The water content of wild type(WT)plants decreased significantly after drought stress,while the content of MDA,soluble sugar and proline increased,while the content of transgenic over expressed plants did not change significantly.qRT-PCR analysis showed that the over expression of DREB1 A,DREB2A,ABF4,ADH and other genes increased,therefore,it can be inferred that over expression of CsWRKY21 may regulate plant drought resistance pathway through ABA-dependent pathway.