黄瓜绿斑驳花叶病毒的分子鉴定及检测技术研究
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摘要
黄瓜绿斑驳花叶病毒(Cucumber green mottle mosaic virus, CGMMV)是世界上许多国家和地区葫芦科植物上的重要检疫性病毒,严重威胁着西瓜(Citrullus lanatus)、甜瓜(Cucumis melo)、黄瓜(Cucumis sativus Linn.)等葫芦科作物的生产。本研究采用采自广西温室的带有黄瓜绿斑驳花叶病毒的黄瓜叶片进行摩擦接种繁殖,并利用RT-PCR方法进行了检测鉴定。同时,利用RT-PCR方法进行编码区序列扩增,得到了编码区序列(6188bp),并进行同源性分析。另外,应用病毒检测上常用的双抗体夹心酶联免疫吸附测定法(DAS-ELISA)、反转录-聚合酶链反应(RT-PCR)、核酸斑点杂交(NASH)技术,进行了检测灵敏度比较研究。主要研究结果如下:
(1)根据外壳蛋白基因序列保守区域设计了一对引物,经RT-PCR鉴定,证明实验室保存的黄瓜叶片带有黄瓜绿斑驳花叶病毒。经过摩擦接种表明,该病毒分离物在葫芦科植物黄瓜、西瓜、甜瓜、南瓜、福州芋瓠上均产生了系统侵染,在苋色藜上产生局部侵染,不侵染普通烟和心叶烟。这些结果与前人研究结果相同。寄主植物接种后进行RT-PCR检测,结果表明,从接种病毒的葫芦科植物上和苋色藜上均扩增到该病毒的特异性条带,而接种病毒的普通烟、心叶烟和健康对照没有扩增出目的条带,分子生物学鉴定跟生物学鉴定的结果相一致。
(2)编码区全长序列同源性分析表明本实验扩繁的CGMMV-GX分离物与来自各地的CGMMV分离物全长序列的同源性在96%以上,与日本的西瓜株系同源性最高,达97.3%。从不同分离物CP序列同源性比较得出,本实验扩繁的CGMMV-GX与广西的分离物CGMMV-GX-G同源性达100%。CGMMV-GX、CGMMV-GX-G这两个病毒分离物CP序列与CGMMV-France、CGMMV-W-Japan分离物的CP序列,只有3个位点碱基的不同。根据编码区全长序列和CP基因序列构建的进化树来看,不同地区的CGMMV分离物在进化上存在一定的地域相关性。
(3)三种检测方法灵敏度实验结果表明:RT-PCR检测灵敏度高于地高辛标记的核酸斑点杂交技术,以血清学为基础的DAS-ELISA检测灵敏度最低.
(4)评价三种检测方法的优缺点得出,核酸斑点杂交技术在CGMMV检测上具有很好的应用前景。针对目前CGMMV的检疫检测标准没有出台,本文探索能否通过地高辛标记的核酸斑点杂交和鉴别寄主生物学鉴定方法相结合,来建立CGMMV检疫检测标准,旨在为检疫部门鉴定此病毒提供依据和参考。
Cucumber green mottle mosaic virus (CGMMV) is a quarantine virus that is harmful to Cucurbitaceae plants, such as watermelon, muskmelon and cucumber et al..It severely threatens agriculture in many countries and regions over the world. In this study mechanical inoculation test was applied using cucumber leaves infected by CGMMV and reverse transcription polymerase chain reaction (RT-PCR) was applied to detect and identify. The 6188bp coding region of the virus were successfully amplified by RT-PCR,and the sequence homologous was analysed. The detection sensitivity of DAS-ELISA, RT-PCR and NASH (Nucleic acid spot hybridization) were compared.The main results are described as follows:
1. A pair of DNA primers were designed and synthesized based on the nucleotide sequence of the coat protein (CP) gene of CGMMV and comfirmed that the cucumber leaves were CGMMV. Typical systemic symptoms were developed on those hosts of cucumber, watermelon, muskmelon, pumpkin and bottle gourd after inoculated with CGMMV, and local symptoms on Chenopodium amaranitcolor, No symptom on Nicotiana tabacum var. K326 and N. glutinosar.
2. Coding region sequences of the isolate CGMMV-GX had over 96% similarity to the CGMMV isolates from different countries and regions deposited in GenBank. And the 97.3% similarity with CGMMV-W in Japan was the highest.The CP gene sequences were compared with the sequences of the homologous genes of other isolates of CGMMV.The result showed that the highest homology could reach 100% at the nucleic acid level with CGMMV-GX-G (DQ647384). The differences between CGMMV-GX、CGMMV-GX-G with CGMMV-France , CGMMV-W-Japan were lied in 3 base sites of nucleic acid only three differeent nucleic acid bases could be detected between CGMMV-GX, CGMMV-GX-G, CGMMV-France and CGMMV-W-Japan. Phylogenic trees were constructed based on the sequenses of the entire coding regions and CP gene of CGMMV isolates from different regions of the world. Certain relation between different CGMMV isolates and geological distribution was found.
3. The results of comparison of detecting sensitivity showed: RT-PCR was more sensitive than DIG-labeled NASH; DAS-ELISA was lowest sensitive.
4. It was found NASH was suitable to be applied in CGMMV detection after evaluating the three detecting methods.Considering the fact that the quarantine detection standard has not been unveil.This study explored the possibility of using the combination of DIG-Label NASH and differential host test to detect CGMMV.
(1)根据外壳蛋白基因序列保守区域设计了一对引物,经RT-PCR鉴定,证明实验室保存的黄瓜叶片带有黄瓜绿斑驳花叶病毒。经过摩擦接种表明,该病毒分离物在葫芦科植物黄瓜、西瓜、甜瓜、南瓜、福州芋瓠上均产生了系统侵染,在苋色藜上产生局部侵染,不侵染普通烟和心叶烟。这些结果与前人研究结果相同。寄主植物接种后进行RT-PCR检测,结果表明,从接种病毒的葫芦科植物上和苋色藜上均扩增到该病毒的特异性条带,而接种病毒的普通烟、心叶烟和健康对照没有扩增出目的条带,分子生物学鉴定跟生物学鉴定的结果相一致。
(2)编码区全长序列同源性分析表明本实验扩繁的CGMMV-GX分离物与来自各地的CGMMV分离物全长序列的同源性在96%以上,与日本的西瓜株系同源性最高,达97.3%。从不同分离物CP序列同源性比较得出,本实验扩繁的CGMMV-GX与广西的分离物CGMMV-GX-G同源性达100%。CGMMV-GX、CGMMV-GX-G这两个病毒分离物CP序列与CGMMV-France、CGMMV-W-Japan分离物的CP序列,只有3个位点碱基的不同。根据编码区全长序列和CP基因序列构建的进化树来看,不同地区的CGMMV分离物在进化上存在一定的地域相关性。
(3)三种检测方法灵敏度实验结果表明:RT-PCR检测灵敏度高于地高辛标记的核酸斑点杂交技术,以血清学为基础的DAS-ELISA检测灵敏度最低.
(4)评价三种检测方法的优缺点得出,核酸斑点杂交技术在CGMMV检测上具有很好的应用前景。针对目前CGMMV的检疫检测标准没有出台,本文探索能否通过地高辛标记的核酸斑点杂交和鉴别寄主生物学鉴定方法相结合,来建立CGMMV检疫检测标准,旨在为检疫部门鉴定此病毒提供依据和参考。
Cucumber green mottle mosaic virus (CGMMV) is a quarantine virus that is harmful to Cucurbitaceae plants, such as watermelon, muskmelon and cucumber et al..It severely threatens agriculture in many countries and regions over the world. In this study mechanical inoculation test was applied using cucumber leaves infected by CGMMV and reverse transcription polymerase chain reaction (RT-PCR) was applied to detect and identify. The 6188bp coding region of the virus were successfully amplified by RT-PCR,and the sequence homologous was analysed. The detection sensitivity of DAS-ELISA, RT-PCR and NASH (Nucleic acid spot hybridization) were compared.The main results are described as follows:
1. A pair of DNA primers were designed and synthesized based on the nucleotide sequence of the coat protein (CP) gene of CGMMV and comfirmed that the cucumber leaves were CGMMV. Typical systemic symptoms were developed on those hosts of cucumber, watermelon, muskmelon, pumpkin and bottle gourd after inoculated with CGMMV, and local symptoms on Chenopodium amaranitcolor, No symptom on Nicotiana tabacum var. K326 and N. glutinosar.
2. Coding region sequences of the isolate CGMMV-GX had over 96% similarity to the CGMMV isolates from different countries and regions deposited in GenBank. And the 97.3% similarity with CGMMV-W in Japan was the highest.The CP gene sequences were compared with the sequences of the homologous genes of other isolates of CGMMV.The result showed that the highest homology could reach 100% at the nucleic acid level with CGMMV-GX-G (DQ647384). The differences between CGMMV-GX、CGMMV-GX-G with CGMMV-France , CGMMV-W-Japan were lied in 3 base sites of nucleic acid only three differeent nucleic acid bases could be detected between CGMMV-GX, CGMMV-GX-G, CGMMV-France and CGMMV-W-Japan. Phylogenic trees were constructed based on the sequenses of the entire coding regions and CP gene of CGMMV isolates from different regions of the world. Certain relation between different CGMMV isolates and geological distribution was found.
3. The results of comparison of detecting sensitivity showed: RT-PCR was more sensitive than DIG-labeled NASH; DAS-ELISA was lowest sensitive.
4. It was found NASH was suitable to be applied in CGMMV detection after evaluating the three detecting methods.Considering the fact that the quarantine detection standard has not been unveil.This study explored the possibility of using the combination of DIG-Label NASH and differential host test to detect CGMMV.
引文
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2.王鸣,文生仓,阎友辉. (1997).主要瓜类作物的抗病毒育种(上) .长江蔬菜, (1):2-5.
3.古勤生,范在丰,李怀方. (2002).葫芦科作物病毒名录.中国西瓜甜瓜, (1) :45-47.
4.古勤生.(2007).防治监控黄瓜绿斑驳花叶病毒,确保瓜类安全生产.中国瓜菜, 2 (1): 47-48.
5.农业部.中华人民共和国农业部第788号公告〔EB〕.〔2006-12-21〕.
6.孙伟,焦奎. (2002).酶联免疫吸附分析法在植物病毒病检测中的应用用.化学研究与应用, 14 (5):511-514.
7.张永江,李明福. (2008).基于外壳蛋白核酸序列的中国黄瓜绿斑驳花叶病毒分离物起源分析.江西农业大学学报, 30 (4):601-604.
8.张永江. (2006).黄瓜绿斑驳花叶病毒研究进展.河南农业科学,8: 9-12.
9.张仲凯,李毅. (2001) .云南植物病毒.北京:科学出版社.
10.陈红运,白静,朱水芳,赵文军,李明福. (2006).黄瓜绿斑驳花叶病毒辽宁分离物外壳蛋白基因与3’非编码区的序列分析.中国病毒学, 21 (5):516-518.
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