本文選題：煙草花葉病毒 + 分子鑒定�。� 參考：《貴州師范大學(xué)》2017年碩士論文

【摘要】：辣椒系茄科辣椒屬一年或有限多年生草本植物,其營養(yǎng)價值極高,堪稱“蔬菜之冠”。作為辣椒產(chǎn)銷大省,貴州辣椒常年栽培面積500萬畝左右,約占中國的20%,產(chǎn)值150億左右,其經(jīng)濟對辣椒產(chǎn)業(yè)的依賴程度有上升之勢。然而,近年來隨著辣椒種質(zhì)的交流,病毒隨之?dāng)U散,并產(chǎn)生越來越嚴(yán)重的危害,造成極大的經(jīng)濟損失。因此,對辣椒病毒病的早期診斷并采取一定的防范措施對減少經(jīng)濟損失具有重要意義。煙草花葉病毒(Tobacco Mosaic Virus,TMV)隸屬于煙草花葉屬(Tobamovirus),為(+)ssRNA病毒,主要通過汁液傳播,病健葉輕微摩擦造成微傷口,病毒即可侵入,對辣椒的危害日益擴大。對植物病毒分離物進行分子鑒定并研究開發(fā)出快速靈敏的檢測方法對病毒病的早期預(yù)防具有重要的理論及實踐意義。本研究對采自貴州的辣椒病樣進行了ELISA檢測,對TMV貴州辣椒分離物(TMV-GZ)進行了分子鑒定,建立了同步檢測TMV、PMMoV和CMV三種辣椒重要病毒的多重RT-PCR方法和檢測TMV的核酸斑點雜交方法,同時,建立并優(yōu)化了TMV外殼蛋白(CP)基因的原核表達體系,以期為TMV抗血清的制備及免疫學(xué)檢測方法的建立奠定基礎(chǔ)。本研究獲得的主要結(jié)論如下:1.成功克隆了TMV-GZ分離物全長CP基因序列,該基因長為480 bp,編碼18 kDa的CP蛋白。TMV-GZ分離物CP基因序列與同屬異種病毒的同源性僅為32%-65%,序列差異明顯;相反,它與11個TMV病毒分離物的同源性在98%-99%之間;TMV及其它病毒分離物聚類形成3個明顯分支,TMV-GZ與其他11個TMV分離物親緣關(guān)系較近,而與同屬其他3種病毒之間的進化距離較遠,證實該分離物確為TMV。2.根據(jù)煙草花葉病毒(TMV)、辣椒輕斑駁病毒(PMMoV)和黃瓜花葉病毒(CMV)的外殼蛋白(CP)編碼序列設(shè)計了3對PCR檢測引物,通過條件優(yōu)化建立了同步檢測以上3種辣椒病毒的多重RT-PCR方法。該多重RT-PCR體系不對ORSV、PVX和PVY等同屬或不同屬的其它病毒發(fā)生反應(yīng),特異性好;可檢測到104×稀釋的病毒cDNA模板,具有較高的靈敏度,可用于PMMoV、TMV和CMV 3種辣椒病毒的同步檢測。3.基于TMV的CP序列,采用隨機引物法制備了地高辛標(biāo)記的TMV雙鏈cDNA雜交探針(DIG-TMV CP),并初步建立了快速檢測TMV的核酸斑點雜交(NASH)方法,該法具有較好的特異性和靈敏度(可檢測到625×稀釋的病毒RNA)。4.構(gòu)建了TMV CP基因的原核表達載體pET32a_TMV CP。該CP基因以融合蛋白的形式獲得了可溶性高效表達。優(yōu)化后的原核表達條件為:0.8 mM IPTG誘導(dǎo)、25℃、200 rpm和9h。以重組的融合CP蛋白免疫小白鼠制備了TMV抗血清。
[Abstract]:Capsicum is an annual or limited perennial herbaceous plant of the family Solanaceae, and its nutritional value is very high. As a big province of capsicum production and marketing, Guizhou pepper cultivation area is about 5 million mu, accounting for about 20% of China, the output value is about 15 billion, and its economic dependence on pepper industry is increasing. However, with the exchange of pepper germplasm in recent years, the virus has spread and caused more and more serious harm, resulting in great economic losses. Therefore, the early diagnosis and preventive measures of capsicum virus disease are of great significance to reduce economic losses. Tobacco Mosaic virus (TMV) belongs to the genus Tobamovirus. The molecular identification of plant virus isolates and the development of rapid and sensitive detection methods have important theoretical and practical significance for the early prevention of virus disease. In this study, ELISA detection was carried out on pepper samples collected from Guizhou, and TMV isolate TMV-GZ was identified by molecular analysis. A multiplex RT-PCR method for simultaneous detection of three important pepper viruses, TMV and CMV, and a nucleic acid dot blot method for TMV detection were established. At the same time, the prokaryotic expression system of TMV capsid protein (TMV) gene was established and optimized in order to lay a foundation for the preparation of TMV antiserum and the establishment of immunological detection method. The main conclusions of this study are as follows: 1. The full-length CP gene sequence of TMV-GZ isolate was cloned successfully. The length of CP gene was 480bp.The CP gene sequence encoding 18 kDa. TMV-GZ isolate had only 32% -65g homology with the homologous virus. The phylogenetic relationship between TMV-GZ and 11 other TMV isolates was close to that of the other 11 TMV isolates, and the phylogenetic distance between TMV and other three other viruses was far from that of the other three viruses, and the homology of them was between 98% and 99%, forming 3 distinct branches of TMV-GZ and the other 11 TMV isolates, and the phylogenetic distance between TMV-GZ and the other 11 TMV isolates was relatively long. It is confirmed that the isolate is TMV.2. Three pairs of PCR detection primers were designed according to the coding sequence of tobacco mosaic virus (TMV), capsicum light mottle virus (PMMoV) and cucumber mosaic virus (CMV). A multiplex RT-PCR method for simultaneous detection of the above three capsicum viruses was established by optimizing the conditions. The multiplex RT-PCR system does not react with other viruses belonging to the same or different genera, such as ORSVV PVX and PVY, and has good specificity, and can detect 104 脳 diluted virus cDNA template, which has high sensitivity and can be used for simultaneous detection of three kinds of capsicum viruses. 3. Based on the CP sequence of TMV, digoxigenin-labeled TMV double-stranded cDNA hybridization probe DIG-TMV-CPH was prepared by random primer method, and a method for rapid detection of TMV by dot blot hybridization was established. The method has good specificity and sensitivity (625 脳 diluted virus RNA.4. The prokaryotic expression vector of TMV CP gene was constructed. The CP gene was expressed in the form of fusion protein. The optimized prokaryotic expression conditions were as follows: 0. 8 mm IPTG induced at 25 鈩，

本文編號：1961362