本文選題：煙草叢頂病毒 + RNA依賴的RNA聚合酶��； 參考：《山東農(nóng)業(yè)大學(xué)》2017年碩士論文

【摘要】：煙草叢頂病毒(Tobacco bushy top virus,TBTV)為番茄叢矮病毒科(Tombusviridae)幽影病毒屬(Umbravirus)成員,與黃癥病毒科(Luteoviridae)的煙草扭脈病毒(Tobacco vein distorting virus,TVDV)復(fù)合侵染引起煙草叢頂病。TBTV基因組是一條(+)ssRNA,由4 152個(gè)核苷酸組成,編碼4個(gè)開(kāi)放閱讀框。ORF1編碼35 kDa的蛋白,ORF1的C端與ORF2的N端有8個(gè)密碼子的重疊,ORF1蛋白通過(guò)-1位的移碼翻譯機(jī)制表達(dá)產(chǎn)生RdRp,為ORF1/ORF2融合蛋白形式。在對(duì)云南多地采集的煙草叢頂病毒進(jìn)化分析后發(fā)現(xiàn),煙草叢頂病毒RdRp編碼區(qū)序列一致率相對(duì)較低,推測(cè)RdRp的活性變化可能導(dǎo)致病毒致病力的變化。本研究旨在建立TBTV RdRp介導(dǎo)的體外復(fù)制體系,并初步定位參與復(fù)制調(diào)控的RNA元件。首先通過(guò)重疊PCR擴(kuò)增得到煙草叢頂病毒中國(guó)分離物RdRp的編碼序列。構(gòu)建以pMAL-C2X為基礎(chǔ)載體的原核表達(dá)載體pMAL-TB-RdRp。0.5 mM IPTG誘導(dǎo)可特異性表達(dá)分子量約為120 kDa的MBP-RdRp融合蛋白。不同溫度條件下誘導(dǎo)MBP-RdRp,結(jié)果顯示,相比26℃和37℃,18℃下誘導(dǎo)表達(dá)的MBP-RdRp融合蛋白的可溶性比例較高,約17%;經(jīng)親和層析純化的MBP-RdRp可特異性識(shí)別TBTV正鏈和負(fù)鏈的3'末端序列,催化體外復(fù)制;并且對(duì)正負(fù)鏈的3'末端的體外復(fù)制效率存在差別,識(shí)別負(fù)鏈3'末端的體外復(fù)制效率明顯高于正鏈3'末端。說(shuō)明TBTV RdRp介導(dǎo)的特異性體外復(fù)制的建立。同時(shí),結(jié)合RNA結(jié)構(gòu)體外分析構(gòu)建了3'UTR區(qū)的部分突變體,根據(jù)體外復(fù)制效率的變化初步定位了參與復(fù)制調(diào)控的RNA元件。同時(shí),構(gòu)建了不同分離物TB-JC,TB-MDI,TB-MDII的RdRp的原核表達(dá)載體,并初步驗(yàn)證了純化的不同分離物RdRp的體外復(fù)制活性。為將來(lái)進(jìn)行不同TBTV分離物RdRp的活性比較并定位關(guān)鍵氨基酸突變奠定了基礎(chǔ)。
[Abstract]:Tobacco bushy top virus (TBTV), a member of Tombusviridaevirus, a member of the genus Umbravirus, is infected with tobacco vein distorting virusTV DVV of Luteoviridae.TBTV genome is a single (ssRNAs, composed of 4 152 nucleotides). ORF1, which encodes four open reading frames. ORF1 encodes 35 kDa, is expressed in the form of ORF1/ORF2 fusion protein, with 8 codon overlapped ORF1 proteins at the N end of ORF2 and expressed by the 1-bit frameshift translation mechanism. Based on the evolutionary analysis of tobacco top virus collected in Yunnan province, it was found that the consistent rate of RdRp coding region of tobacco top virus was relatively low. It was speculated that the change of RdRp activity might lead to the change of virulence of the virus. The aim of this study was to establish an in vitro replication system mediated by TBTV RdRp and to locate the RNA elements involved in replication regulation. Firstly, the coding sequence of the Chinese isolate RdRp of tobacco cluster top virus was obtained by overlapping PCR amplification. A prokaryotic expression vector pMAL-TB-RdRp.0.5 mm IPTG based on pMAL-C2X was constructed to induce the specific expression of MBP-RdRp fusion protein with molecular weight of about 120 kDa. The results of induction of MBP-RdRpat at different temperatures showed that the soluble ratio of the MBP-RdRp fusion protein induced at 26 鈩，

本文編號(hào)：1776037