利用小RNA介导的基因沉默实现R基因在不同科植物中的应用

发布时间：2018-04-22 06:04

本文选题：人工miRNA + 植物与病原互作　；参考：《华中农业大学》2017年硕士论文

【摘要】：植物内源sRNA(small RNA,小RNA)在调控基因表达过程中起重要作用,它们可以通过激活或抑制特定基因的表达,从而影响植物的多个生物学过程。其中,sRNA在植物病原菌互作中的调控机制引起人们极大的关注。在植物与病原长期互作过程中,植物进化出两个基础免疫系统:病原相关分子模式(PAMP)激发的免疫反应(PTI)和效应蛋白(effector protein)激发的免疫反应(ETI)。病原的effector具有进化快、多样性强的特点,植物在进化过程中积累了大量R基因以应对不同病原微生物的挑战。由于植物内源的R(Resistence)基因受到严格的控制,所以在转基因植物中过量表达R基因往往会导致植株发育异常。小分子RNA是植物整个生命过程中重要的调控因子,在转录、转录后或翻译水平上通过与靶基因序列互补配对的方式调控基因表达,在病原微生物入侵植物过程中有重要的基因表达调控作用。因此我们想通过构建以R基因为靶基因的人工miRNA来降低R基因的表达量,当病原入侵植物时,病原微生物一方面会全面降低sRNA的表达水平,另一方面,病原体会利用植物的RNA诱导沉默复合体来促进自身对植株的侵染,破坏植物体内的sRNA调控机制,此时R基因转录出的mRNA得以翻译,R基因高丰度表达,以此达到抗病的目的。本研究以拟南芥和烟草作为研究材料,以抗烟草花叶病毒的N基因以及抗丁香假单胞杆菌的RPS4基因为研究对象,试图利用小分子RNA介导的基因沉默,来实现两个物种之间抗病基因的利用。主要研究结果如下:1.构建好ami R6019的表达载体,并成功转入哥伦比亚型拟南芥中,获得不同程度表达amiR6019的转基因拟南芥;在此基础上,成功转入烟草N基因,获得生长正常的转基因拟南芥。此结果提供一种抗病研究的新方法:利用R silencer促进物种间R基因的资源利用。2.设计并构建好四个靶定RPS4的artificial microRNA表达载体,并获得四种转基因烟草;构建了RPS4、RRS1、avrRPS4的表达载体并做瞬时表达验证;摸索了丁香假单胞杆菌番茄植病变种DC3000(Pseudomonas syringae pv.tomato DC3000)侵染栽培烟草SR1的最适浓度,这些结果为之后在烟草中研究RPS4与丁香假单胞杆菌的互作奠定了基础。
[Abstract]:Endogenous sRNA(small RNAs (small RNAs) play an important role in the regulation of gene expression. They can affect many biological processes by activating or inhibiting the expression of specific genes. The regulation mechanism of siRNA in the interaction of plant pathogens has attracted much attention. During the long-term interaction between plant and pathogen, plants evolved two basic immune systems: the immune response induced by PAMP-induced immune response (PTI) and the immune response induced by effector protein (ETI). Pathogen effector has the characteristics of rapid evolution and strong diversity. During the evolution process, plants accumulated a large number of R genes to meet the challenge of different pathogenic microorganisms. Due to the strict control of endogenous R resistance genes in plants, overexpression of R genes in transgenic plants often leads to abnormal plant development. Small molecule RNA is an important regulatory factor throughout plant life. It regulates gene expression through complementary pairing with target gene sequences at transcription, post-transcriptional or translation levels. It plays an important role in the regulation of gene expression during the invasion of plants by pathogenic microorganisms. Therefore, we want to reduce the expression of R gene by constructing artificial miRNA with R gene as target gene. When the pathogen invades the plant, the pathogenic microorganism will reduce the expression level of sRNA on the one hand, and on the other hand, The pathogen uses plant RNA induced silencing complex to promote its own infection on plants and destroy the sRNA regulation mechanism in the plant. The mRNA transcribed by R gene can translate the high abundance expression of R gene in order to achieve the purpose of disease resistance. In this study, Arabidopsis thaliana and tobacco were used as the research materials. The N gene of tobacco mosaic virus and the RPS4 gene of Pseudomonas euxomonas were used to study the gene silencing mediated by small molecule RNA. To realize the use of disease resistance genes between two species. The main results are as follows: 1. The expression vector of ami R6019 was constructed and successfully transferred into Colombian type Arabidopsis thaliana to obtain transgenic Arabidopsis thaliana expressing amiR6019 to varying degrees, and on this basis, transgenic Arabidopsis thaliana with normal growth was successfully transformed into tobacco N gene. This result provides a new method for disease resistance research: using R silencer to promote the resource utilization of R gene between species. 2. Four artificial microRNA expression vectors targeting RPS4 were designed and constructed, and four kinds of transgenic tobacco were obtained. The optimal concentration of SR1 infected by DC3000(Pseudomonas syringae pv.tomato DC3000was studied. These results laid a foundation for the study of the interaction between RPS4 and Pseudomonas clovianum in tobacco.
【学位授予单位】：华中农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S432.2

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