油菜花叶病毒（ORMV）126kDa复制酶基因在拟南芥中的可诱导表达

发布时间：2017-12-31 12:02

  本文关键词：油菜花叶病毒（ORMV）126kDa复制酶基因在拟南芥中的可诱导表达　出处：《聊城大学》2017年硕士论文　论文类型：学位论文

  更多相关文章： 油菜花叶病毒(ORMV) 126kDa复制酶蛋白 激活应答诱导表达体系 大肠杆菌 感受态细胞

【摘要】：植物病毒危害农作物的形势十分严重,是导致植物病害的主要原因之一。植物病毒对农作物的危害不仅严重威胁农业生产结构,对农业经济也造成了严重损失。因此,如何防治植物病毒病害早已是科学家们所需要研究和解决的重要课题。随着研究发现,植物本身具有能够抵抗植物病毒的防御机制,即RNA干扰机制。RNA干扰是由双链RNA分子介导的、序列特异的转录后基因沉默现象。植物体内的小RNA在植物对抗病毒的RNA干扰机制中起着核心作用,它来自于非蛋白编码RNA的前体。小RNA通过碱基互补配对原则与靶mRNA结合起负调控的作用,利用sRNA抗植物病毒是近年来所研究的一种有效手段。但是病毒为了有效侵染植物,就必须抑制植物体内RNAi机制。因此,植物病毒同时也能够编码蛋白在RNAi的不同步骤起到抑制作用,这类蛋白被称为基因沉默抑制因子。油菜花叶病毒ORMV作为危害油菜等作物生产的病毒之一,属于烟草花叶病毒属。ORMV基因结构中的复制酶基因表达的126kDa蛋白是一种基因沉默抑制因子。126kDa复制酶蛋白在体外(in vitro)可结合21nt左右的小RNA,油菜花叶病毒的侵染不仅能引起21nt小RNA的积累,还对该类小RNA的底物mRNA的表达产生了影响。为了进一步验证复制酶蛋白在体内(in vivo)对21nt小RNA的结合富集作用,同时为了更好地了解复制酶蛋白对植物内源转录本的影响,以及其如何通过影响小RNA进而影响小RNA底物mRNA的表达,我们尝试在植物中过量表达该蛋白。但是前期的实验没有获得持续过量地表达该蛋白的植物,这可能是因为过量表达该蛋白对植物有害。本研究利用植物的可诱导表达技术,在拟南芥中有条件地表达126kDa复制酶蛋白,进而研究ORMV 126kDa复制酶蛋白对植物内源小RNA代谢与功能的影响。这些内源小RNA与植物对病毒侵染的应答有着紧密联系,所以研究这些富集的小RNA类别及作用机制也有助于抗病育种的进一步发展。Silwet-L77是一种非离子型的表面活性剂,常用于植物的转化。本研究发现,SilwetL77的加入也可以显著地提高大肠杆菌的转化效率。同时,我们比较了不同培养温度、不同培养浓度(OD600值)及不同冷冻保护剂对感受态细胞转化效率的影响。我们发现,28℃培养E.coli至OD600值为0.55-0.6之间时制备感受态细胞,利用9%的DMSO做为冷冻保护剂冷冻保存感受态细胞,转化时加入15~20ppm的Silwet-L77,可以获得最高的转化效率。总之,进一步优化了大肠杆菌感受态细胞的制备方法以及转化方法。
[Abstract]:The situation of plant virus harming crops is very serious, which is one of the main causes of plant disease. The harm of plant virus to crops is not only a serious threat to agricultural production structure. Agricultural economy has also caused serious losses. Therefore, how to prevent and cure plant virus diseases has long been an important subject that scientists need to study and solve. Plants have a defense mechanism against plant viruses, I. e., RNA interference mechanism. RNAi is mediated by double-stranded RNA molecules. Sequence-specific post-transcriptional gene silencing. Small RNA in plants play a central role in the mechanism of RNA interference in plants against viruses. It is derived from the precursor of nonprotein encoded RNA. Small RNA play a negative regulatory role by combining the target mRNA with the base complementary pairing principle. It is an effective method to use sRNA to resist plant viruses in recent years, but in order to infect plants effectively, it is necessary to inhibit the mechanism of RNAi in plants. Plant viruses can also encode proteins that inhibit RNAi at different stages. These proteins are known as gene silencing inhibitors. Cauliflower leaf virus (ORMV) is one of the viruses that harms the production of rape and other crops. The 126kDa protein expressed by the replicase gene belonging to the structure of the ORMV gene is a gene silencing inhibitor. 126kDa replicase protein in vitro (. In vitrocan bind to about 21 NT small RNA. The infection of cauliflower leaf virus can not only cause the accumulation of 21nt small RNA. In order to further verify the binding enrichment of replicase protein in vivo to 21nt small RNA. In order to better understand the effects of replicase proteins on endogenous transcripts of plants and how they affect the expression of mRNA substrates of small RNA by affecting small RNA. We tried to over-express the protein in plants, but previous experiments did not produce plants that consistently overexpressed the protein. This may be because overexpression of this protein is harmful to plants. In this study, 126kDa replicase protein was expressed conditionally in Arabidopsis thaliana using plant inducible expression technique. Furthermore, the effects of ORMV 126 kDa replicase protein on the metabolism and function of endogenous small RNA were studied. These small RNA were closely related to the response of plants to virus infection. Therefore, the study of these enriched small RNA types and the mechanism of their action is also helpful to the further development of disease resistance breeding. Silwet-L77 is a non-ionic surfactant. It was found that the addition of Silwet L77 could significantly improve the transformation efficiency of Escherichia coli. At the same time, we compared the different culture temperatures. The effects of different culture concentration (OD600) and different freezing protectants on the transformation efficiency of receptive cells were found. When E. coli was cultured at 28 鈩，

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