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2019 Volume 41 Issue 5
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Gen-tu WU, Xia XU, Si-min CHEN, et al. Molecular Detection and Cloning and Sequence Analysis of the Complete Genome of Tobacco mild green mosaic virus on Pepper Plants in Chongqing[J]. Journal of Southwest University Natural Science Edition, 2019, 41(5): 1-7. doi: 10.13718/j.cnki.xdzk.2019.05.001
Citation: Gen-tu WU, Xia XU, Si-min CHEN, et al. Molecular Detection and Cloning and Sequence Analysis of the Complete Genome of Tobacco mild green mosaic virus on Pepper Plants in Chongqing[J]. Journal of Southwest University Natural Science Edition, 2019, 41(5): 1-7. doi: 10.13718/j.cnki.xdzk.2019.05.001
shu

Molecular Detection and Cloning and Sequence Analysis of the Complete Genome of Tobacco mild green mosaic virus on Pepper Plants in Chongqing

  • 1.

    School of Plant Protection, Southwest University/Chongqing Key Laboratory of Plant Disease Biology, Chongqing 400716, China

  • 2.

    Litong District Agricultural Technology Extension Service Center of Wuzhong, Wuzhong Ningxia 751100, China

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  • Corresponding author: Ling QING
  • Received Date: 07/03/2018
    Available Online: 20/05/2019

  • MSC: S436.418.1+2

  • Pepper (Capsicum annuum L.) is one of the most important cash crops in Chongqing, Tobacco mild green mosaic virus (TMGMV) causes a virus disease in pepper, which brings serious damage to pepper production. To understand the distribution of TMGMV and its genomic characters in Chongqing, leaf samples with mottle, mosaic and distort symptoms were collected from Beibei, Shizhu, Tongnan and Jiulongpo Districts of Chongqing and determined by RT-PCR. The complete genomic sequence of TMGMV was obtained by assembling four fragments amplified from the positive samples by RT-PCR which overlapped the whole TMGMV genome. The results of RT-PCR showed that of the 29 samples 9 were positive, with a detection rate of 31.03%. The detection rate of TMGMV in Shizhu County was the highest, 60.00%. The complete genome sequence analysis revealed that TMGMV of Chongqing isolates consisted of 6, 356 nucleotides with the typical characteristics of the genome of the genus Tobamovirus. Phylogenetic analysis based on TMGMV genome showed that TMGMV-TN29 had a close phylogenetic relationship with isolate Xiamen (JX534224), and they shared a sequence similarity of 99.75%.
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Molecular Detection and Cloning and Sequence Analysis of the Complete Genome of Tobacco mild green mosaic virus on Pepper Plants in Chongqing

    Corresponding author: Ling QING
  • Received Date: 07/03/2018
  • Available Online: 20/05/2019

Abstract: Pepper (Capsicum annuum L.) is one of the most important cash crops in Chongqing, Tobacco mild green mosaic virus (TMGMV) causes a virus disease in pepper, which brings serious damage to pepper production. To understand the distribution of TMGMV and its genomic characters in Chongqing, leaf samples with mottle, mosaic and distort symptoms were collected from Beibei, Shizhu, Tongnan and Jiulongpo Districts of Chongqing and determined by RT-PCR. The complete genomic sequence of TMGMV was obtained by assembling four fragments amplified from the positive samples by RT-PCR which overlapped the whole TMGMV genome. The results of RT-PCR showed that of the 29 samples 9 were positive, with a detection rate of 31.03%. The detection rate of TMGMV in Shizhu County was the highest, 60.00%. The complete genome sequence analysis revealed that TMGMV of Chongqing isolates consisted of 6, 356 nucleotides with the typical characteristics of the genome of the genus Tobamovirus. Phylogenetic analysis based on TMGMV genome showed that TMGMV-TN29 had a close phylogenetic relationship with isolate Xiamen (JX534224), and they shared a sequence similarity of 99.75%.

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  • 辣椒(Capsicum annuum L.)属于茄科(Solanaceae)辣椒属(Capsicum)植物，是一类重要的蔬菜作物和经济作物.病毒病是影响辣椒生产的一类重要病害，感病植株表现出叶片畸形、斑驳、花叶和植株矮化等症状，造成辣椒产量下降，品质降低，严重制约辣椒产业发展[1-2].目前，世界上已有60多种植物病毒可侵染辣椒[3]，在我国辣椒上检测到的病毒有33种，归属于10个科12个属[4].其中，已报道有6种烟草花叶病毒属(Tobamovirus)病毒侵染辣椒，分别为烟草花叶病毒(Tobacco mosaic virus，TMV)、番茄花叶病毒(Tomato mosaic virus，ToMV)[1]、辣椒轻斑驳病毒(Pepper mild mottle virus，PMMoV)[5]、烟草轻型绿花叶病毒(Tobacco mild green mosaic virus，TMGMV)[6]、番茄斑驳花叶病毒(Tomato mottle mosaic virus，ToMMV)[7-8]和油菜花叶病毒(Youcai mosaic virus，YoMV)[9].

    烟草轻型绿花叶病毒(Tobacco mild green mosaic virus，TMGMV)，属于烟草花叶病毒属(Tobamovirus)成员，最早在烟草(Nicotiana gluanca)上发现. TMGMV除危害烟草外，还可以侵染辣椒[6]、番茄[10]和南瓜[11]等多种作物及杂草，其在辣椒上发生和危害较为严重. TMGMV侵染辣椒后引起叶片褪绿、黄化及坏死，且在病果上有明显的褪绿条纹等症状.在我国，TMGMV危害辣椒的报道最早于2005年出现在台湾地区；2013年在厦门出现[6]；2015年在江苏出现；2016年在山东大面积发生，严重影响了辣椒的产量[12].这些报道表明该病在我国呈现扩散态势，是当前我国辣椒生产上的潜在危害病毒之一.

    本实验室在前期工作中，从重庆北碚、石柱和潼南等地区采集了表现出花叶、卷曲、斑驳、黄化的辣椒样品，通过高通量测序方法检测到了黄瓜花叶病毒(Cucumber mosaic virus，CMV)、蚕豆萎蔫病毒2号(Broad bean wilt virus 2，BBWV-2)、辣椒潜隐病毒2号(Pepper cryptic virus 2，PCV-2)、辣椒脉斑驳病毒(Chilli veinal mottle virus，ChiVMV)、甜椒脉斑驳病毒(Pepper veinal mottle virus，PVMV)、TMV、PMMoV和TMGMV等8种病毒.虽然在我国已经报道了TMGMV在辣椒上发生危害，但在重庆地区还未见报道.因此，本研究在前期工作的基础上，利用RT-PCR方法对重庆辣椒上的TMGMV进行测定，分析其检出率；通过分段扩增方法获得其基因组全序列，并与国内外报道的TMGMV全序列进行同源性比较，以期明确重庆不同地区TMGMV的发生情况及其与国内外其他分离物间的进化关系.

1.   材料与方法

    1.1.   材料

    1.1.1.   毒源

  • 疑似感染病毒的辣椒样品于2016年6月采自重庆北碚、潼南、石柱和九龙坡4个区县，共29份，保存于-80 ℃冰箱.

    • 1.1.2.   载体和菌株

  • 克隆载体pGEM-T Easy Vector，美国Promega公司；大肠杆菌Escherichia coli Trans5α，北京全式金生物技术有限公司.

    • 1.1.3.   主要试剂

  • Trizol，Invitrogen公司；反转录试剂盒、连接酶，TaKaRa公司；HiFi Taq DNA聚合酶，北京全式金生物技术有限公司；DNA回收试剂盒和质料提取试剂盒，北京天根生化科技公司.

    • 1.1.4.   引物

  • 用于检测TMGMV的引物依据文献[6]报道的序列；根据GenBank中TMGMV的基因组全长序列，比对分析后设计分段扩增TMGMV全基因组引物(表 1).

    • 1.2.   方法

    1.2.1.   植物总RNA提取及cDNA合成

  • 总RNA的提取参考Trizol RNA (TaKaRa)取说明书，最后每份RNA加入30 μL的RNase-free ddH2O溶解RNA，-80 ℃保存，备用.

    以提取样品总RNA为模板，利用反转录试剂盒(PrimeScript RT reagent Kit，TaKaRa)合成cDNA.反应体系为：5×PrimeScript Buffer 2 μL，Oligo dT Primer 0.5 μL，Random 6 mers 0.5 μL，PrimeScript RT Enzyme Mix I 0.5 μL，总RNA 2 μL，加RNase-free ddH2O至10 μL.反应程序为：37 ℃ 30 min，85 ℃ 20 s. cDNA保存于-20 ℃，备用.

    • 1.2.2.   RT-PCR及DNA纯化

  • 以合成的cDNA为模版进行PCR扩增，反应体系为：10×HiFi buffer (Mg2+) 2.5 μL，dNTPs (2.5mM) 2.0 μL，primer F(10 μM) 0.3 μL，primer F(10 μM) 0.3 μL，cDNA 1 μL，HiFi Taq (500 U) 0.2 μL加ddH2O至25 μL. PCR反应程序：94 ℃预变性3 min，94 ℃变性30 s，退火30 s，72 ℃延伸1 000 bp/min，循环35次，72 ℃延伸10 min.取5 μL PCR产物用1%琼脂糖凝胶电泳检测.

    PCR产物纯化参考天根DNA纯化回收试剂盒(Universal DNA Purification Kit，TIANGEN)进行PCR产物回收，最后用30 μL ddH2O溶解DNA，12 000 r/min离心2 min，收集DNA溶液. DNA回收产物保存于-20 ℃，备用.

    • 1.2.3.   T载体连接、转化及检测

  • 参考T载体快速连接试剂盒(pGEM-T Easy Vector System I，Promega)和本实验室体系[13]进行回收的DNA片段连接.反应体系为：2×Ligation Buffer 5 μL，pGEM-T载体1 μL，DNA片段3 μL，T4 DNA Ligase(3 U/μL)1 μL.置室温反应2 h或4 ℃反应8 h.将重组质粒转化至大肠杆菌DH5α感受态细胞，培养后经蓝白斑筛选，挑选白色单克隆菌落至LB液体培养基(含100 μg/mL Amp)中培养4~6 h后PCR检测阳性克隆用于测序.

    • 1.2.4.   序列测定与分析

  • 菌液经过PCR鉴定为阳性克隆后委托华大基因科技有限公司进行序列测定.利用DNAStar[14]软件对所获得的基因序列进行处理，利用BLAST程序进行序列相似性搜索，并用DNAStar MegAlign程序的Clustal W方法与GenBank已登录的病毒核苷酸序列进行多序列比较分析，采用MEGA 5.0[15-16]的邻接法(neighbor joining，NJ)构建进化树，重复次数设置为1 000次.

2.   结果与分析

    2.1.   TMGMV在重庆辣椒上的发生情况

  • 对采集自重庆4个区县的29份辣椒疑似病毒病害样品进行总RNA抽提，反转录合成cDNA，利用TMGMV CP基因特异性引物进行PCR扩增，获得目的大小的PCR产物(图 1).阳性结果分析发现共有9个样品检测到TMGMV，检出率为31.03%，表明TMGMV在重庆北碚、九龙坡、潼南和石柱均有发生(表 2).进一步分析发现，从石柱采集的5份样品中检测到TMGMV的样品有3份，检出率高达60.00%；潼南TMGMV检出率为40.00%；九龙坡TMGMV检出率为30.00%；而北碚9份样品中仅有1份样品检测到TMGMV，检出率最低，为11.11%(表 2).

    • 2.2.   重庆辣椒上TMGMV CP克隆及序列分析

  • 采用RT-PCR方法成功克隆重庆辣椒上TMGMV CP基因，通过连接pGEM-T载体，转化大肠杆菌、菌液PCR检测，DNA测序等过程，获得重庆地区分离物TMGMV CP基因全序列，命名为TMGMV-CQ.对已获得的分离物与GenBank中19个TMGMV分离物的CP基因进行系统进化树分析(图 2).结果表明，这20个TMGMV分离物主要聚为3支.其中，巴拿马辣椒分离物(EU934035)与西班牙等欧洲分离物聚为一支(group Ⅰ)；TMGMV-CQ与2个韩国分离物(AF103782和AF103783)、美国烟草分离物(AF131908)、美国鲸鱼花分离物(EF469769)和2个中国辣椒分离物(KM596785、JX534224)聚为一支(group Ⅱ)；韩国矮牵牛分离物(AM262165)、美国烟草分离物(AF132907)、中国台湾辣椒分离物(DQ821941)和日本烟草分离物(AB078435)聚为一支(group Ⅲ)，系统进化分析结果表明TMGMV分离物间的亲缘关系具有一定的地理相关性. TMGMV-CQ与厦门辣椒分离物(JX534224)聚集在同一个小分支，表明两者的亲缘关系最近.

    • 2.3.   重庆辣椒上TMGMV全基因组序列分析

  • 将GenBank中TMGMV的基因组全长序列经过序列比对，根据其保守序列设计分段扩增TMGMV基因组全长的引物(表 1)，采用分段扩增再拼接的方法，分成4个片段进行扩增(图 3a).对采自重庆潼南的辣椒样品(编号TN29)进行TMGMV基因组分段扩增，获得大小约为1.6 kb，1.6 kb，1.8 kb和1.9 kb的4个片段(图 3b)，分别连接pGEM-T载体，转化大肠杆菌、菌液PCR检测，DNA测序等过程.测序结果获得了TMGMV-TN29基因组片段分别为1 588 nt，1 648 nt，1 852 nt和1 915 nt.这4个片段经过DNAStar的Segman程序拼接，最终得到TMGMV-TN29基因组全长序列为6 356 nt，其5’和3’末端分别有71 nt和210 nt的非编码区(Untranslated Regions，UTR)，包括4个开放阅读框(Open Reading Frame，ORF)，分别编码1.83×105/1.26×105的复制相关蛋白，2.8×104的移动蛋白(Movement protein，MP)和1.7×104的外壳蛋白(Coat protein，CP)(图 4).

    将TMGMV-TN29分离物与已报道的其他TMGMV全基因序列进行同源性分析表明，TMGMV各分离物之间的同源性在97.00%~99.7%之间.序列分析结果表明，TMGMV-TN29与厦门辣椒分离物Xiamen(JX534224)基因组核苷酸序列及其编码的氨基酸序列的相似性都为最高(表 3).系统进化树显示TMGMV-CQ分离物与厦门辣椒分离物Xiamen(JX534224)聚为一组，表明这两者的亲缘关系最近(图 5).

3.   结论

  • 虽然在重庆辣椒上检测到了CMV，TMV，TuMV，ToMV，BBWV-2，PCV-2和TSWV等病毒[17-21]，但还未见TMGMV侵染重庆辣椒的报道.近年来，TMGMV在我国各地报道不断增多，为明确重庆辣椒是否受TMGMV侵染，本研究从重庆4个不同辣椒种植区采集了29份疑似病毒病样品，经RT-PCR及克隆测序发现，这4个地区均有TMGMV发生，且发病率介于11.11%~60.00%之间.石柱是重庆重要的辣椒生产地[22]，TMGMV检出率高达60.00%，值得植保工作者注意.本文首次在重庆辣椒上检测到TMGMV，说明TMGMV在重庆辣椒上也存在，可能是其早就侵入重庆而未被发现，或近几年来才扩散到重庆.鉴于TMGMV在我国辣椒上流行的风险，有必要对TMGMV发生情况加以监测.

    TMGMV最早在烟草上发现，属Tobamovirus成员.本研究首次从重庆辣椒上检测并克隆了TMGMV的全基因组序列，共包含6 356 nt，由4个开放阅读框组成，ORF1和ORF2分别编码1.83×105，1.2×105复制酶相关蛋白，ORF3编码运动相关蛋白，ORF4编码外壳蛋白，具有Tobamovirus病毒特征.大多数的Tobamovirus病毒能通过种子传播，近年来随着贸易的发展，我国各地辣椒种植地区对辣椒种子的引进和交换也越来越频繁. 2013年厦门出入境检验检疫局从进境辣椒种子中检测到TMGMV，说明该病毒有较高的传入风险.根据以往的报道结果显示Tobamovirus病毒进化缓慢，基因组通常比较保守[23-24]，而目前对TMGMV亲缘关系的研究集中在CP ORF水平，为更全面地了解TMGMV遗传结构，本研究对重庆地区侵染辣椒的TMGMV测定了其全基因组序列，进一步发现TMGMV重庆辣椒分离物TMGMV-TN29与厦门辣椒分离物Xiamen(JX534224)的基因组核苷酸序列相似性高达99.75%，基于基因组全长的进化树分析也表明，TMGMV-TN29与Xiamen(JX534224)的亲缘关系较近，由此推测它们可能来源于同一个进化祖先.

Figure (5)  Table (3) Reference (24)

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