辣椒烟草花叶病毒的分子鉴定及检测技术研究

发布时间：2018-05-31 21:36

  本文选题：烟草花叶病毒 + 分子鉴定　； 参考：《贵州师范大学》2017年硕士论文

【摘要】：辣椒系茄科辣椒属一年或有限多年生草本植物,其营养价值极高,堪称“蔬菜之冠”。作为辣椒产销大省,贵州辣椒常年栽培面积500万亩左右,约占中国的20%,产值150亿左右,其经济对辣椒产业的依赖程度有上升之势。然而,近年来随着辣椒种质的交流,病毒随之扩散,并产生越来越严重的危害,造成极大的经济损失。因此,对辣椒病毒病的早期诊断并采取一定的防范措施对减少经济损失具有重要意义。烟草花叶病毒(Tobacco Mosaic Virus,TMV)隶属于烟草花叶属(Tobamovirus),为(+)ssRNA病毒,主要通过汁液传播,病健叶轻微摩擦造成微伤口,病毒即可侵入,对辣椒的危害日益扩大。对植物病毒分离物进行分子鉴定并研究开发出快速灵敏的检测方法对病毒病的早期预防具有重要的理论及实践意义。本研究对采自贵州的辣椒病样进行了ELISA检测,对TMV贵州辣椒分离物(TMV-GZ)进行了分子鉴定,建立了同步检测TMV、PMMoV和CMV三种辣椒重要病毒的多重RT-PCR方法和检测TMV的核酸斑点杂交方法,同时,建立并优化了TMV外壳蛋白(CP)基因的原核表达体系,以期为TMV抗血清的制备及免疫学检测方法的建立奠定基础。本研究获得的主要结论如下:1.成功克隆了TMV-GZ分离物全长CP基因序列,该基因长为480 bp,编码18 kDa的CP蛋白。TMV-GZ分离物CP基因序列与同属异种病毒的同源性仅为32%-65%,序列差异明显;相反,它与11个TMV病毒分离物的同源性在98%-99%之间;TMV及其它病毒分离物聚类形成3个明显分支,TMV-GZ与其他11个TMV分离物亲缘关系较近,而与同属其他3种病毒之间的进化距离较远,证实该分离物确为TMV。2.根据烟草花叶病毒(TMV)、辣椒轻斑驳病毒(PMMoV)和黄瓜花叶病毒(CMV)的外壳蛋白(CP)编码序列设计了3对PCR检测引物,通过条件优化建立了同步检测以上3种辣椒病毒的多重RT-PCR方法。该多重RT-PCR体系不对ORSV、PVX和PVY等同属或不同属的其它病毒发生反应,特异性好;可检测到104×稀释的病毒cDNA模板,具有较高的灵敏度,可用于PMMoV、TMV和CMV 3种辣椒病毒的同步检测。3.基于TMV的CP序列,采用随机引物法制备了地高辛标记的TMV双链cDNA杂交探针(DIG-TMV CP),并初步建立了快速检测TMV的核酸斑点杂交(NASH)方法,该法具有较好的特异性和灵敏度(可检测到625×稀释的病毒RNA)。4.构建了TMV CP基因的原核表达载体pET32a_TMV CP。该CP基因以融合蛋白的形式获得了可溶性高效表达。优化后的原核表达条件为:0.8 mM IPTG诱导、25℃、200 rpm和9h。以重组的融合CP蛋白免疫小白鼠制备了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，

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