本文選題：山葡萄 + WRKY55轉(zhuǎn)錄因子��； 參考：《中國科學(xué)院武漢植物園》2017年碩士論文

【摘要】：非生物脅迫(如低溫和干旱)能夠通過改變外部環(huán)境導(dǎo)致農(nóng)作物大量減產(chǎn)并限制作物的生長和地理分布。在中國,葡萄是一種重要的果樹作物,然而,生產(chǎn)上常用的栽培品種極易遭受低溫和干旱脅迫的影響。本實驗室前期研究表明WRKY轉(zhuǎn)錄因子可能參與了葡萄對低溫和干旱脅迫的響應(yīng),但是其作用機理仍不清楚。本研究以高抗寒旱的山葡萄為材料,克隆了一個冷脅迫響應(yīng)的WRKY轉(zhuǎn)錄因子基因VaWRKY55,進行VaWRKY55在低溫和干旱脅迫條件下的表達(dá)模式分析,并通過在擬南芥中過表達(dá)VaWRKY55對其功能進行了研究,揭示了調(diào)控抗旱性的可能機理。同時通過轉(zhuǎn)錄組測序研究VaWRKY55可能參與的抗旱信號通路。取得的主要結(jié)果如下:1.依據(jù)'黑比諾'葡萄參考基因組序列設(shè)計引物克隆了Va WRKY55的編碼區(qū)序列。序列比對分析表明,VaWRKY55與參考基因組中VvWRKY55的蛋白序列相似度高達(dá)92%。利用定量RT-PCR分析VaWRKY55在山葡萄低溫和干旱處理下的表達(dá)變化,發(fā)現(xiàn)VaWRKY55能快速響應(yīng)低溫和干旱,表明其可能在山葡萄低溫和干旱脅迫響應(yīng)過程中起作用。2.構(gòu)建了以35S為啟動子的VaWRKY55過表達(dá)載體并轉(zhuǎn)化了擬南芥,獲得了 3個T4代轉(zhuǎn)基因株系。表型分析結(jié)果表明,在低溫條件下,只有一個過表達(dá)VaWRKY55的擬南芥植株成活率略顯著高于野生型;而所有3個轉(zhuǎn)基因植株在干旱條件下的成活率均顯著高于野生型,表明VaWRKY55確實能夠參與到植株對干旱脅迫的響應(yīng)并提高植株的抗旱性。3.生理生化和結(jié)構(gòu)特異性分析表明,和野生型相比,轉(zhuǎn)基因擬南芥在干旱脅迫條件下與可溶性糖和脯氨酸含量升高、過氧化氫和電解質(zhì)滲透率降低;且轉(zhuǎn)基因植物擁有更低的氣孔密度。因此,VaWRKY55可能通過改變轉(zhuǎn)基因擬南芥的葉表面結(jié)構(gòu)和生理反應(yīng)來增強植株的抗旱性。4.利用轉(zhuǎn)錄組測序分析了過表達(dá)VaWRKY55調(diào)控抗旱性的作用機制。和野生型相比,在干旱處理后,茉莉酸合成以及信號通路相關(guān)基因如AtAOC1,AtAOC2,AtAOS,AtLOX2,At3G05727和AtPDF1.2在過表達(dá)植株中明顯上調(diào)表達(dá)。此結(jié)果表明VaWRKY55可能通過茉莉酸信號通路來提高轉(zhuǎn)基因擬南芥的抗旱性。
[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.
【學(xué)位授予單位】：中國科學(xué)院武漢植物園
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：Q945.78

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本文編號：1740633