侵染西番莲的夜来香花叶病毒的分子鉴定及特异性检测

doi:10.3864/j.issn.0578-1752.2017.24.006

摘要/Abstract

摘要： 【目的】鉴定福建果园西番莲上的夜来香花叶病毒（Telosma mosaic virus，TeMV），并建立用于该病毒特异性检测的分子快速检测方法，为该病毒的防治提供参考依据。【方法】采用血清学检测、电镜观察、通用简并引物RT-PCR、特异性引物RT-PCR对福建西番莲样品进行病毒检测，并对阳性样品的PCR产物进行克隆、测序；根据已报道的夜来香花叶病毒基因序列和本研究的序列测定结果，设计一对用于扩增TeMV外壳蛋白（coat protein，CP）基因全长的特异性引物，通过反应条件优化，建立该病毒的特异性RT-PCR检测方法；序列测定结果利用BLAST程序和DNAMAN软件进行比对，同时对获得的CP基因序列采用MrBayes软件的贝叶斯法（Bayesian inference，BI）构建系统发育树并进行系统发育分析。【结果】血清学检测发现，一株表现有花叶、皱缩症状的西番莲样品与马铃薯Y病毒属（Potyvirus）通用抗体反应呈阳性；电镜观察结果表明，该株疑似带毒样品中含有大小约750 nm×12 nm的弯曲线状病毒粒子；Potyvirus通用简并引物RT-PCR从该样品中扩增到一条与预期大小相符的目的片段，克隆、测序获得长度为680 bp的序列，该序列与已报道的TeMV核苷酸序列一致性最高（98.2%）。特异性引物扩增获得的CP基因序列全长为816 bp（命名为BXGFJ-13分离物），与已报道的TeMV核苷酸序列、氨基酸序列一致性分别为86.2%—98.4%和88.2%—97.8%。系统发育分析结果表明，13个TeMV分离物共形成3个类群，相同地区或寄主来源的分离物优先相聚成簇，表现出很强的地理和寄主特异性。本研究获得的BXGFJ-13分离物与中国广西分离物（KJ789129）先以较高的后验概率聚为一个分支，再与泰国2个分离物（AM409188、AM409187）聚为第2类群（Group II），表明其在系统发育关系上与中国广西分离物的亲缘关系最近。利用特异性引物TeMV-CPf/TeMV-CPr建立的RT-PCR方法，具有良好的特异性，仅能从感染TeMV的西番莲样品上扩增出目的片段，而从黄瓜花叶病毒（Cucumber mosaic virus，CMV）、甜菜花叶病毒（Beet mosaic virus，BtMV）、大豆花叶病毒（Soybean mosaic virus，SMV）、虎眼万年青花叶病毒（Ornithogalum mosaic virus，OrMV）、洋葱黄矮病毒（Onion yellow dwarf virus，OYDV）、东亚西番莲病毒（East Asian Passiflora virus，EAPV）等其他病毒样品及阴性对照上均未扩增出目的片段。灵敏度测定结果显示，该方法能从稀释10²倍的RNA上扩增出目的片段。【结论】根据国际病毒分类委员会（ICTV）关于Potyvirus病毒不同成员的分类标准，同时结合血清学检测、电镜观察结果，证实福建果园表现花叶、皱缩症状的西番莲上携带有TeMV；建立的特异性RT-PCR方法能够用于TeMV的快速检测。

关键词: 西番莲, 夜来香花叶病毒, 分子鉴定, 检测

Abstract: 【Objective】 The objective of this study is to identify Telosma mosaic virus (TeMV) on passion fruit in Fujian orchard, develop a molecular method for specific and rapid identification of TeMV, and to provide a reference for prevention and control of the virus.【Method】The Fujian passion fruit samples were detected by using the serology method, electron microscopic observation, universal degenerate primers RT-PCR and specific primers RT-PCR. The PCR products of the positive sample were cloned and sequenced. A set of specific primes amplified the total length of coat protein (CP) gene was designed according to the sequences of reported TeMV and sequence determination of this study, and then specific primers RT-PCR detection method was established after optimizing reaction conditions. The sequence determination results were analyzed with BLAST program and DNAMAN software, and the phylogenetic tree was constructed based on the CP gene sequences obtained in this study by using Bayesian inference (BI) method implemented in MrBayes. 【Result】The serology results showed that one passion fruit sample exhibiting mosaic and crinkle symptom reacted with Potyvirus antiserum. The positive sample was found to have about 750 nm×12 nm linear virions by electron microscopic observation. The expected fragment was amplified from the positive sample by universal degenerate primers RT-PCR, and then cloned and sequenced. Sequence analysis showed that the obtained sequence from the positive sample was identical with the expected size (680 bp), and shared the highest nucleotide sequence identity (98.2%) with the reported TeMV gene sequence. The full-length sequence of CP gene obtained by specific primers RT-PCR was 816 bp (named BXGFJ-13 isolate), and the sequence of nucleotide and amino acid of BXGFJ-13 was 86.2%-98.4% and 88.2%-97.8% identity, respectively, with the reported TeMV isolates. The results of phylogenetic analysis showed that the 13 TeMV isolates could be divided into 3 groups, and the same area or host derived isolates preferentially clustered together, suggesting these isolates had a strong geographical and host specificity. BXGFJ-13 isolate obtained in this paper and Guangxi isolate of China (KJ789129) clustered into a branch with high posterior probability, and then clustered together with two Thailand isolates (AM409188, AM409187) into the 2nd group (Group II), showing that BXGFJ-13 and Guangxi isolate had the closest phylogenetic relationship. The specific primers RT-PCR showed good specificity, which only amplify the expected fragment from TeMV-infected passion fruit sample, and no expected fragment was obtained from Cucumber mosaic virus (CMV), Beet mosaic virus (BtMV), Soybean mosaic virus (SMV), Ornithogalum mosaic virus (OrMV), Onion yellow dwarf virus (OYDV), East Asian Passiflora virus (EAPV) and healthy control. The sensitivity results showed that the target fragment could be amplified from diluted 10² fold RNA.【Conclusion】According to the species demarcation criteria for the Potyvirus given by International Committee on Taxonomy (ICTV) and the results of serological detection and electron microscopic observation, TeMV on the passion fruit sample exhibiting mosaic and crinkle symptom in Fujian orchard was confirmed. The establishedassay of specific RT-PCR could be used for rapid detection of TeMV.

Key words: passion fruit, Telosma mosaic virus (TeMV), molecular identification, detection

谢丽雪，张小艳，郑姗，张立杰，李韬. 侵染西番莲的夜来香花叶病毒的分子鉴定及特异性检测[J]. 中国农业科学, 2017, 50(24): 4725-4734.

XIE LiXue, ZHANG XiaoYan, ZHENG Shan, ZHANG LiJie, LI Tao. Molecular identification and specific detection of Telosma mosaic virus infecting passion fruit [J]. Scientia Agricultura Sinica, 2017, 50(24): 4725-4734.

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