發(fā)布時間：2017-12-31 12:02

  本文關(guān)鍵詞：油菜花葉病毒（ORMV）126kDa復(fù)制酶基因在擬南芥中的可誘導(dǎo)表達　出處：《聊城大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 油菜花葉病毒(ORMV) 126kDa復(fù)制酶蛋白 激活應(yīng)答誘導(dǎo)表達體系 大腸桿菌 感受態(tài)細胞

【摘要】：植物病毒危害農(nóng)作物的形勢十分嚴重,是導(dǎo)致植物病害的主要原因之一。植物病毒對農(nóng)作物的危害不僅嚴重威脅農(nóng)業(yè)生產(chǎn)結(jié)構(gòu),對農(nóng)業(yè)經(jīng)濟也造成了嚴重損失。因此,如何防治植物病毒病害早已是科學(xué)家們所需要研究和解決的重要課題。隨著研究發(fā)現(xiàn),植物本身具有能夠抵抗植物病毒的防御機制,即RNA干擾機制。RNA干擾是由雙鏈RNA分子介導(dǎo)的、序列特異的轉(zhuǎn)錄后基因沉默現(xiàn)象。植物體內(nèi)的小RNA在植物對抗病毒的RNA干擾機制中起著核心作用,它來自于非蛋白編碼RNA的前體。小RNA通過堿基互補配對原則與靶mRNA結(jié)合起負調(diào)控的作用,利用sRNA抗植物病毒是近年來所研究的一種有效手段。但是病毒為了有效侵染植物,就必須抑制植物體內(nèi)RNAi機制。因此,植物病毒同時也能夠編碼蛋白在RNAi的不同步驟起到抑制作用,這類蛋白被稱為基因沉默抑制因子。油菜花葉病毒ORMV作為危害油菜等作物生產(chǎn)的病毒之一,屬于煙草花葉病毒屬。ORMV基因結(jié)構(gòu)中的復(fù)制酶基因表達的126kDa蛋白是一種基因沉默抑制因子。126kDa復(fù)制酶蛋白在體外(in vitro)可結(jié)合21nt左右的小RNA,油菜花葉病毒的侵染不僅能引起21nt小RNA的積累,還對該類小RNA的底物mRNA的表達產(chǎn)生了影響。為了進一步驗證復(fù)制酶蛋白在體內(nèi)(in vivo)對21nt小RNA的結(jié)合富集作用,同時為了更好地了解復(fù)制酶蛋白對植物內(nèi)源轉(zhuǎn)錄本的影響,以及其如何通過影響小RNA進而影響小RNA底物mRNA的表達,我們嘗試在植物中過量表達該蛋白。但是前期的實驗沒有獲得持續(xù)過量地表達該蛋白的植物,這可能是因為過量表達該蛋白對植物有害。本研究利用植物的可誘導(dǎo)表達技術(shù),在擬南芥中有條件地表達126kDa復(fù)制酶蛋白,進而研究ORMV 126kDa復(fù)制酶蛋白對植物內(nèi)源小RNA代謝與功能的影響。這些內(nèi)源小RNA與植物對病毒侵染的應(yīng)答有著緊密聯(lián)系,所以研究這些富集的小RNA類別及作用機制也有助于抗病育種的進一步發(fā)展。Silwet-L77是一種非離子型的表面活性劑,常用于植物的轉(zhuǎn)化。本研究發(fā)現(xiàn),SilwetL77的加入也可以顯著地提高大腸桿菌的轉(zhuǎn)化效率。同時,我們比較了不同培養(yǎng)溫度、不同培養(yǎng)濃度(OD600值)及不同冷凍保護劑對感受態(tài)細胞轉(zhuǎn)化效率的影響。我們發(fā)現(xiàn),28℃培養(yǎng)E.coli至OD600值為0.55-0.6之間時制備感受態(tài)細胞,利用9%的DMSO做為冷凍保護劑冷凍保存感受態(tài)細胞,轉(zhuǎn)化時加入15~20ppm的Silwet-L77,可以獲得最高的轉(zhuǎn)化效率�？傊�,進一步優(yōu)化了大腸桿菌感受態(tài)細胞的制備方法以及轉(zhuǎn)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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