VaWRKY55在山葡萄低温和干旱胁迫响应中的功能研究

发布时间：2018-04-12 17:02

本文选题：山葡萄 + WRKY55转录因子　；参考：《中国科学院武汉植物园》2017年硕士论文

【摘要】：非生物胁迫(如低温和干旱)能够通过改变外部环境导致农作物大量减产并限制作物的生长和地理分布。在中国,葡萄是一种重要的果树作物,然而,生产上常用的栽培品种极易遭受低温和干旱胁迫的影响。本实验室前期研究表明WRKY转录因子可能参与了葡萄对低温和干旱胁迫的响应,但是其作用机理仍不清楚。本研究以高抗寒旱的山葡萄为材料,克隆了一个冷胁迫响应的WRKY转录因子基因VaWRKY55,进行VaWRKY55在低温和干旱胁迫条件下的表达模式分析,并通过在拟南芥中过表达VaWRKY55对其功能进行了研究,揭示了调控抗旱性的可能机理。同时通过转录组测序研究VaWRKY55可能参与的抗旱信号通路。取得的主要结果如下:1.依据'黑比诺'葡萄参考基因组序列设计引物克隆了Va WRKY55的编码区序列。序列比对分析表明,VaWRKY55与参考基因组中VvWRKY55的蛋白序列相似度高达92%。利用定量RT-PCR分析VaWRKY55在山葡萄低温和干旱处理下的表达变化,发现VaWRKY55能快速响应低温和干旱,表明其可能在山葡萄低温和干旱胁迫响应过程中起作用。2.构建了以35S为启动子的VaWRKY55过表达载体并转化了拟南芥,获得了 3个T4代转基因株系。表型分析结果表明,在低温条件下,只有一个过表达VaWRKY55的拟南芥植株成活率略显著高于野生型;而所有3个转基因植株在干旱条件下的成活率均显著高于野生型,表明VaWRKY55确实能够参与到植株对干旱胁迫的响应并提高植株的抗旱性。3.生理生化和结构特异性分析表明,和野生型相比,转基因拟南芥在干旱胁迫条件下与可溶性糖和脯氨酸含量升高、过氧化氢和电解质渗透率降低;且转基因植物拥有更低的气孔密度。因此,VaWRKY55可能通过改变转基因拟南芥的叶表面结构和生理反应来增强植株的抗旱性。4.利用转录组测序分析了过表达VaWRKY55调控抗旱性的作用机制。和野生型相比,在干旱处理后,茉莉酸合成以及信号通路相关基因如AtAOC1,AtAOC2,AtAOS,AtLOX2,At3G05727和AtPDF1.2在过表达植株中明显上调表达。此结果表明VaWRKY55可能通过茉莉酸信号通路来提高转基因拟南芥的抗旱性。
[Abstract]:Abiotic stresses (such as low temperature and drought) can reduce crop yield and limit crop growth and geographical distribution by changing the external environment.Grape is an important fruit crop in China. However, common cultivated varieties are susceptible to low temperature and drought stress.Previous studies in our laboratory showed that WRKY transcription factors may be involved in the response of grape to low temperature and drought stress, but its mechanism is still unclear.In this study, a cold stress responsive WRKY transcription factor gene VaWRKY55 was cloned from high cold and drought resistant grapevine, and the expression pattern of VaWRKY55 was analyzed under low temperature and drought stress.The function of VaWRKY55 in Arabidopsis thaliana was studied, and the possible mechanism of drought resistance regulation was revealed.At the same time, transcriptome sequencing was used to study the drought resistance signaling pathway in which VaWRKY55 might be involved.The main results are as follows: 1.According to the reference genome sequence of Pinot Noir grape, a primer was designed to clone the coding region of Va WRKY55.Sequence alignment analysis showed that the similarity between VaWRKY55 and VvWRKY55 protein sequence in reference genome was as high as 92%.Quantitative RT-PCR was used to analyze the expression changes of VaWRKY55 under cold and drought treatment. It was found that VaWRKY55 could quickly respond to low temperature and drought, indicating that VaWRKY55 might play an important role in the response to cold and drought stress.VaWRKY55 overexpression vector with 35s as promoter was constructed and transformed into Arabidopsis thaliana. Three T4 generation transgenic lines were obtained.Phenotypic analysis showed that the survival rate of Arabidopsis thaliana with one overexpression of VaWRKY55 was slightly higher than that of wild type at low temperature, and the survival rate of all three transgenic plants was significantly higher than that of wild type under drought.The results showed that VaWRKY55 could participate in the response of plants to drought stress and improve the drought resistance of plants.Physiological, biochemical and structure-specific analysis showed that compared with wild type, transgenic Arabidopsis thaliana increased the content of soluble sugar and proline and decreased the permeability of hydrogen peroxide and electrolyte under drought stress.And transgenic plants have lower stomatal density.Therefore, VaWRKY55 may enhance the drought resistance of transgenic Arabidopsis thaliana by changing the leaf surface structure and physiological response of transgenic Arabidopsis thaliana.The mechanism of overexpression of VaWRKY55 on drought resistance was analyzed by transcriptome sequencing.Compared with wild type, the genes related to jasmonic acid synthesis and signaling pathway, such as AtAOC1, AtAOC2AAOC2AAOC2AAOS2AAOX2A3G05727 and AtPDF1.2, were upregulated in overexpression plants after drought treatment.These results suggest that VaWRKY55 may enhance drought resistance of transgenic Arabidopsis thaliana through jasmonic acid signaling pathway.
【学位授予单位】：中国科学院武汉植物园
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：Q945.78

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