本氏烟草RNAi途径基因的amiRNA载体构建，瞬时表达及干扰效果分析
发布时间：2018-08-20 07:38
【摘要】：植物体内有一系列基因抵抗病原侵染,以保证植物的正常生长。RNA干扰(RNAinterference,RNAi)是植物针对病毒侵染的一个重要的应答反应。人工微RNA(artificial microRNA,amiRNA)是一种高效沉默基因表达的第二代RNAi技术,是利用天然miRNA的生成和作用原理设计的,以调控生物体内某一个或多个基因的表达的一项新的基因沉默技术。病毒诱导的基因沉默(VIGS)也是沉默植物基因的有力手段。使用amiRNA的VIGS(MIR VIGS)是一种新开发的植物反向遗传学研究技术,其兼具amiRNA和VIGS技术的优势。本氏烟草(Nicotiana benthamiana)对超过500种不同的病毒敏感,广泛应用于植物与病原微生物相互作用以及高等植物基因功能的研究中,并且可以作为VIGS的良好宿主。植物的RNA沉默途径中的几乎所有的基因都已在拟南芥中发现和功能验证。但是在本氏烟草中RNA沉默途径相关基因的研究鲜有报道。本研究对本氏烟草中RNA沉默途径相关的11条基因(AGO1a、AGO1b、AG02、DCL2、DCL3、DCL4、DRB4、HEN1、RDR1、RDR6、SGS3)进行干扰研究。载体构建成功后通过瞬时表达实验分析,得到了如下结论:1)利用MIR VIGS技术在植物病毒载体pCVA中构建了32个干扰载体,沉默了其中9条基因,沉默效率均在50%以上;2)利用amiRNA技术在植物双元表达载体pBI121(Kpn Ⅰ)中构建了 8个干扰载体,沉默了其中7条基因,沉默效率均在45%以上,其沉默效率整体上不如病毒载体pCVA的沉默效率高;3)农杆菌介导法侵染本氏烟草,病毒载体pCVA和病毒共侵染存在强烈的竞争关系,而植物双元载体pBI121和病毒在侵染液浓度较低(如OD600=0.1)时共侵染则互不影响;4)利用植物双元载体pBI121瞬时表达,在侵染液浓度为OD600=0.1的条件下,NbAG02基因的下调使本氏烟草抗烟草花叶病毒(pJL24)的能力显著减弱,而NbAGO1a、AGO1b、NbRDR1、NbDCL2基因的下调与烟草花叶病毒则没有关系。
[Abstract]:RNA interference (RNAi) is an important plant response to viral infection. Artificial microRNA (amiRNA) is a second-generation RNAi technology that silences gene expression efficiently and utilizes natural microRNA production and production. Virus-induced gene silencing (VIGS) is also a powerful tool for silencing plant genes. VIGS using amiRNA (MIR VIGS) is a newly developed technique for plant reverse genetics, which combines amiRNA and VIGS techniques. Advantages. Nicotiana benthamiana is susceptible to more than 500 different viruses. It is widely used in the study of plant-pathogenic microorganism interactions and gene functions in higher plants. It can also serve as a good host for VIGS. Almost all genes in plant RNA silencing pathways have been found and functioned in Arabidopsis. Verification. However, few studies have been reported on RNA silencing pathway related genes in Ben's tobacco. In this study, 11 genes (AGO1a, AGO1b, AG02, DCL2, DCL3, DCL4, DRB4, HEN1, RDR1, RDR6, SGS3) related to RNA silencing pathway in Ben's tobacco were interfered with. After successful construction of the vector, the following conclusions were obtained: 1) 32 interfering vectors were constructed in plant virus vector pCVA by MIR VIGS technology, 9 of them were silenced, and the silencing efficiency was above 50%. 2) Eight interfering vectors were constructed in plant binary expression vector pBI121 (Kpn I) by amiRNA technology. Seven of them were silenced, and the silencing efficiency was above 45%. For example, the silencing efficiency of virus vector pCVA is high; 3) Agrobacterium tumefaciens-mediated infection of tobacco, virus vector pCVA and virus co-infection have a strong competitive relationship, while the co-infection of plant binary vector pBI121 and virus at low concentration (e.g. OD600 = 0.1) does not affect each other; 4) the use of plant binary vector pBI121 transient expression in the infection solution; Under the condition of OD600=0.1, the down-regulation of NbAG02 gene significantly weakened the resistance of tobacco to tobacco mosaic virus (pJL24), but the down-regulation of NbAGO1a, AGO1b, NbRDR1, NbDCL2 gene had no relationship with tobacco mosaic virus.
【学位授予单位】：内蒙古大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：Q943.2
本文编号：2192900
[Abstract]:RNA interference (RNAi) is an important plant response to viral infection. Artificial microRNA (amiRNA) is a second-generation RNAi technology that silences gene expression efficiently and utilizes natural microRNA production and production. Virus-induced gene silencing (VIGS) is also a powerful tool for silencing plant genes. VIGS using amiRNA (MIR VIGS) is a newly developed technique for plant reverse genetics, which combines amiRNA and VIGS techniques. Advantages. Nicotiana benthamiana is susceptible to more than 500 different viruses. It is widely used in the study of plant-pathogenic microorganism interactions and gene functions in higher plants. It can also serve as a good host for VIGS. Almost all genes in plant RNA silencing pathways have been found and functioned in Arabidopsis. Verification. However, few studies have been reported on RNA silencing pathway related genes in Ben's tobacco. In this study, 11 genes (AGO1a, AGO1b, AG02, DCL2, DCL3, DCL4, DRB4, HEN1, RDR1, RDR6, SGS3) related to RNA silencing pathway in Ben's tobacco were interfered with. After successful construction of the vector, the following conclusions were obtained: 1) 32 interfering vectors were constructed in plant virus vector pCVA by MIR VIGS technology, 9 of them were silenced, and the silencing efficiency was above 50%. 2) Eight interfering vectors were constructed in plant binary expression vector pBI121 (Kpn I) by amiRNA technology. Seven of them were silenced, and the silencing efficiency was above 45%. For example, the silencing efficiency of virus vector pCVA is high; 3) Agrobacterium tumefaciens-mediated infection of tobacco, virus vector pCVA and virus co-infection have a strong competitive relationship, while the co-infection of plant binary vector pBI121 and virus at low concentration (e.g. OD600 = 0.1) does not affect each other; 4) the use of plant binary vector pBI121 transient expression in the infection solution; Under the condition of OD600=0.1, the down-regulation of NbAG02 gene significantly weakened the resistance of tobacco to tobacco mosaic virus (pJL24), but the down-regulation of NbAGO1a, AGO1b, NbRDR1, NbDCL2 gene had no relationship with tobacco mosaic virus.
【学位授予单位】：内蒙古大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：Q943.2
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