中国野生毛葡萄芪合成酶基因VqSTS21和VqSTS36功能分析

发布时间：2018-06-04 12:39

本文选题：中国野生葡萄 + 芪合成酶基因　；参考：《西北农林科技大学》2017年硕士论文

【摘要】：芪合成酶(Stilbene synthase,STS)基因是白藜芦醇及其糖苷衍生物生物合成途径中的关键基因。有研究表明,STS基因在植物抗病方面发挥重要作用。但是其抗病的调控机制仍不清楚。此外,有关STS基因的抗渗透胁迫的功能的研究尚未报道。本研究将中国野生毛葡萄VqSTS21和VqSTS36基因转化模式植物拟南芥(哥伦比亚野生型)及番茄(Micro-Tom)过表达,旨在探讨STS基因在抵御生物和非生物胁迫中起到的作用及其抗病机理。取得以下主要研究结果:1.中国野生毛葡萄‘商-24’叶片接种白粉菌后,通过半定量对31个VqSTS基因进行表达分析,发现VqSTS21基因响应白粉菌的诱导并且表达上调。将该基因转入拟南芥后,通过高效液相色谱法检测,发现转基因植株产生的主要芪类化合物是反式白藜芦醇苷(trans-piceid),含量为216-531μg g-1FW。对异源表达的VqSTS21转基因拟南芥接种白粉菌,PstDC3000和叶片离体接种灰霉菌,结果显示白粉病和Pst DC3000侵染后转基因拟南芥活性氧积累和死细胞数量较少,植株发病较轻。但对灰霉菌的侵染,转基因叶片上的病斑较大,植株发病较重。对异源表达的VqSTS21转基因拟南芥种子、幼苗和成年苗进行盐和干旱胁迫处理,结果表明通过保护细胞膜的完整性和增加植株的根系生长量来提高转基因植株抗逆性。实时定量PCR结果显示,转基因拟南芥面对不同的外界刺激时,VqSTS21会通过参与不同信号途径来提高植株的抗性。病原菌的侵染主要激发转基因拟南芥SA信号转导,并诱导细胞活性氧爆发,抑制了活体寄生菌白粉菌和Pst DC3000等病原菌的入侵,减少病症;但对于腐生菌灰霉菌却增加了转基因植株的感病性。外界渗透胁迫诱导VqSTS21参与ABA介导的生物合成途和SOS信号途径径来增强转基因拟南芥对盐和干旱胁迫的抗性。2.中国野生毛葡萄VqSTS36基因cDNA全长1179pb,编码387个氨基酸,通过SMART网站预测,该基因蛋白序列含有与非生物胁迫和生物胁迫相关的结构域。利用tmrpres2d软件分析其跨膜结构域,发现从N端向C端方向的367到384位置处有1个跨膜片段,表示该基因可能参与细胞膜的信号接收或转导。异源表达的VqSTS36转基因番茄和拟南芥接种灰霉菌,发现转基因植株叶片菌斑直径较野生型大,发病较重,转基因番茄和拟南芥对灰霉菌感病。转基因拟南芥叶片接种白粉菌后,发病较轻,接种第三天后叶片中活性氧的积累和死细胞较多。实时定量PCR结果显示,灰霉菌、白粉菌的入侵主要诱导转基因番茄和拟南芥中SA信号途径的响应,SA生物合成途径的响应提高转基因拟南芥对白粉菌的抗性,同时也提高对灰霉菌感病性。对拟南芥种子及幼苗进行盐和干旱胁迫处理,发现转基因拟南芥种子发芽率和幼苗根长均高于野生型,实时定量PCR结果显示,转基因拟南芥幼苗中ABA信号途径重要基因上调,表明VqSTS36可能通过参与ABA信号通路来提高植株抗逆性。
[Abstract]:Stilbene synthase (STS) gene is a key gene in the synthesis of resveratrol and its glycoside derivatives. Studies have shown that STS gene plays an important role in plant disease resistance. However, the regulatory mechanism of its disease resistance is still unclear. In addition, studies on the osmotic resistance of STS genes have not been reported. The purpose of this study was to investigate the role of STS gene in resistance to biotic and abiotic stresses and its mechanism of disease resistance by overexpression of Arabidopsis thaliana (wild type in Colombia) and Micro-Tomato Tomato (Micro-Tomato), the transformation model of VqSTS21 and VqSTS36 genes in Chinese wild grape. Get the following main research results: 1. After inoculated with powdery mildew, 31 VqSTS genes were analyzed by semi-quantitative analysis. It was found that VqSTS21 gene responded to the induction of powdery mildew and up-regulated the expression of VqSTS21 gene. After the gene was transferred into Arabidopsis thaliana, it was found by HPLC that the main compound produced by transgenic plants was trans-resveratrol trans-piceidido, the content of which was 216-531 渭 g / g ~ (-1) FW. Transgenic Arabidopsis thaliana with VqSTS21 heterologous expression was inoculated with powdery mildew PstDC3000 and leaves in vitro. The results showed that the reactive oxygen species accumulation and the number of dead cells in transgenic Arabidopsis thaliana infected by powdery mildew and Pst DC3000 were less, and the disease of plants was lighter. However, the disease spot of transgenic leaves was larger and the disease of plants was more serious in the infection of Aspergillus cinerea. Exogenous VqSTS21 transgenic Arabidopsis thaliana seeds, seedlings and adult seedlings were treated with salt and drought stress. The results showed that the resistance of transgenic plants could be improved by protecting the integrity of cell membrane and increasing the growth of root system. The results of real-time quantitative PCR showed that transgenic Arabidopsis thaliana VqSTS21 could increase the resistance of transgenic Arabidopsis thaliana by participating in different signaling pathways when it was exposed to different external stimuli. The infection of pathogenic bacteria mainly stimulated SA signal transduction of transgenic Arabidopsis thaliana and induced cell reactive oxygen species outbreak, which inhibited the invasion of pathogenic bacteria such as powdery mildew and Pst DC3000, and reduced the disease. However, the susceptibility of transgenic plants was increased by Aspergillus graminearum. Exogenous osmotic stress induces VqSTS21 to participate in ABA mediated biosynthesis pathway and SOS signaling pathway to enhance the resistance of transgenic Arabidopsis thaliana to salt and drought stress. The VqSTS36 gene cDNA of wild grape in China is 1179pb in length and encodes 387 amino acids. The protein sequence of this gene is predicted by SMART website to contain the domain related to abiotic stress and biological stress. Tmrpres2d software was used to analyze the transmembrane domain. It was found that there was a transmembrane fragment between 367 and 384 from the N-terminal to the C-terminal, indicating that the gene might be involved in the signal reception or transduction of the cell membrane. The heterologous VqSTS36 transgenic tomato and Arabidopsis thaliana were inoculated with Ash fungus. It was found that the leaf plaque diameter of transgenic plants was larger than that of wild type and the disease was more serious. Transgenic tomato and Arabidopsis thaliana were susceptible to gray mold. Transgenic Arabidopsis thaliana leaves inoculated with powdery mildew, the disease was lighter, the third day after inoculation, the accumulation of reactive oxygen species and the number of dead cells were more. The results of real-time quantitative PCR showed that the resistance of transgenic Arabidopsis thaliana to powdery mildew was enhanced by the response of SA signal pathway in transgenic tomato and Arabidopsis thaliana. At the same time, it also improved susceptibility to Aspergillus. Under salt and drought stress treatment of Arabidopsis thaliana seeds and seedlings, it was found that the germination rate and root length of transgenic Arabidopsis thaliana seeds were higher than that of wild type. The results of real-time quantitative PCR showed that the important genes of ABA signaling pathway were up-regulated in transgenic Arabidopsis seedlings. It is suggested that VqSTS36 may increase plant stress resistance by participating in ABA signaling pathway.
【学位授予单位】：西北农林科技大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S663.1

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