葡萄扇叶病和卷叶病的RT-PCR检测技术研究
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摘要
以中国农业科学院郑州果树研究所国家种质资源圃葡萄圃的葡萄品种为试材，对葡萄扇叶病毒(GFLV)和葡萄卷叶病相关病毒Ⅲ(GLRaV-Ⅲ)进行了反转录-聚合酶链式反应(RT-PCR)检测技术研究。采用的技术流程为：通过提取葡萄感病叶片或韧皮部总RNA获得病毒RNA，反转录合成cDNA，经PCR扩增获得病毒cDNA的特征片段，并将其克隆并进行序列分析。本研究对RNA提取方法进行了深入的研究，获得了一种快速简便的提取葡萄组织总RNA的好方法，建立了RT-PCR检测技术规程。现将实验结果归纳如下：
1．提取高质量的RNA是进行RT-PCR检测的基础。酚类化合物、多糖、蛋白质和未知次级代谢产物是影响从葡萄组织提取RNA的几个主要干扰因素。通过对三种提取方法(SDS法、CTAB法和分步离心法)的比较和改进，确立了分步离心法为最佳方法。该法采用磷酸缓冲液，在缓冲液中加入了0.1％2-巯基乙醇，0.15g／100mL BSA，1％的PVP，0.53g Vc／100mL和0.14mol／L NaCl。该法避免了用有机试剂的污染，有效地去除了酚类化合物、多糖、蛋白质和DNA等的污染，在3小时之内可得到高纯度、完整的、适宜进行RT-PCR操作的RNA模板，从而保证了检测的成功。
2．参照已报道的GFLV和GLR_aV-3外壳蛋白基因序列分别设计合成特异引物，采用RT-PCR的方法分别扩增出了722bp和300bp的PCR产物，与预期目的片段一致。
3．为进一步明确PCR扩增的特异性，将PCR产物连接到pGEM-T载体上，转化大肠杆菌E.coli JM109菌株，通过蓝白斑筛选，获得重组质粒，提取质粒DNA，经酶切和PCR对重组质粒进行鉴定，结果证明得到了含有目的DNA片段的重组质粒。对目的片段的序列分析表明GLRaV-Ⅲ的外壳蛋白基因cDNA片段与报道的相应序列具有高度的同源性，其同源率达99.3％。由此证明RT-PCR检测GLRaV-Ⅲ准确可靠，可应用于无毒化栽培的后期检测，对葡萄病毒病的诊断和检疫起重要作用。
The techniques of detecting grapevine fanleaf virus(GFLV) and grapevine leafroll-associated virus type III (GLRaVIII) by RT-PCR were studied. First, extract total RNA from grapevine tisste, then, the cDNA of GFLV and GLRaV III coat protein gene were synthesized with reverse transcriptase using 3' primer, and were amplified by PCR using two primers. At last, the fragments of PCR products were cloned, sequenced and analysised. In this study, a series of methods of extracting total RNA were tried,compared and modified, a simple rapid and useful method was developed and a procedure of detecting grapevine virus was set up.The results are as following:
1. The quality of RNA is the base the process of RT-PCR,we need pure and undegradated RNA. Phenolic compounds, polysacchrides, proteins and unidentified secondary metabolites are the main factors interfering the isolation of RNA and virus detection. Three methods(SDS, CTAB, centrifugation step by step) were compared and modified, and the last method was perfect for RT-PCR. The extracting buffer is phosphate-buffer, consists of 0. 1% ME, 0. 15g BSA/100M1, 1% PVP,and 0.53gVc/100mL, 0. 14 mol/L NaCl. In this way, we can eliminate the inhibeterant substance which I related and obtained high quality RNA within 3 hours.It indicated that the method is simple, convinent and practical.
2. The necessary GFLV and GLRaV Ⅲ nucleotide sequences for PCR primers have been published. The 5' and 3' primers of GFLV and GLRaV III coat protein gene were synthesized according to the published.The reverse transcription step was performed with total RNA extracted from infected grapevine tissue. From the synthesized first cDNA strand, 722 bp and 300 bp nucleotide fragments were amplified by two primers, which are the same as what we had expected.
3. To check the specificity and efficiency of GFLV and GLRaV Ⅲ cDNA
fragments amplified, the fragments were isolated from agarose gels and ligated to pGEM-T, transfect E.coli JM 109,the -galactosidase and ampicillin-resistant recombinant plasmids were obtained, these clones were further characterized by restriction enzyme digestion and PCR for the presence of the insert PCR products.The recombinant plasmids were then sequenced by the dideoxy chain termination method. The results showed that there is 99.3% between what we obtained by RT-PCR and the published. So, we consider that the technique of RT-PCR is exact and reliable, and it will give us an important preparation for virus diagnosis and quarantine on grapevine in the future.
1．提取高质量的RNA是进行RT-PCR检测的基础。酚类化合物、多糖、蛋白质和未知次级代谢产物是影响从葡萄组织提取RNA的几个主要干扰因素。通过对三种提取方法(SDS法、CTAB法和分步离心法)的比较和改进，确立了分步离心法为最佳方法。该法采用磷酸缓冲液，在缓冲液中加入了0.1％2-巯基乙醇，0.15g／100mL BSA，1％的PVP，0.53g Vc／100mL和0.14mol／L NaCl。该法避免了用有机试剂的污染，有效地去除了酚类化合物、多糖、蛋白质和DNA等的污染，在3小时之内可得到高纯度、完整的、适宜进行RT-PCR操作的RNA模板，从而保证了检测的成功。
2．参照已报道的GFLV和GLR_aV-3外壳蛋白基因序列分别设计合成特异引物，采用RT-PCR的方法分别扩增出了722bp和300bp的PCR产物，与预期目的片段一致。
3．为进一步明确PCR扩增的特异性，将PCR产物连接到pGEM-T载体上，转化大肠杆菌E.coli JM109菌株，通过蓝白斑筛选，获得重组质粒，提取质粒DNA，经酶切和PCR对重组质粒进行鉴定，结果证明得到了含有目的DNA片段的重组质粒。对目的片段的序列分析表明GLRaV-Ⅲ的外壳蛋白基因cDNA片段与报道的相应序列具有高度的同源性，其同源率达99.3％。由此证明RT-PCR检测GLRaV-Ⅲ准确可靠，可应用于无毒化栽培的后期检测，对葡萄病毒病的诊断和检疫起重要作用。
The techniques of detecting grapevine fanleaf virus(GFLV) and grapevine leafroll-associated virus type III (GLRaVIII) by RT-PCR were studied. First, extract total RNA from grapevine tisste, then, the cDNA of GFLV and GLRaV III coat protein gene were synthesized with reverse transcriptase using 3' primer, and were amplified by PCR using two primers. At last, the fragments of PCR products were cloned, sequenced and analysised. In this study, a series of methods of extracting total RNA were tried,compared and modified, a simple rapid and useful method was developed and a procedure of detecting grapevine virus was set up.The results are as following:
1. The quality of RNA is the base the process of RT-PCR,we need pure and undegradated RNA. Phenolic compounds, polysacchrides, proteins and unidentified secondary metabolites are the main factors interfering the isolation of RNA and virus detection. Three methods(SDS, CTAB, centrifugation step by step) were compared and modified, and the last method was perfect for RT-PCR. The extracting buffer is phosphate-buffer, consists of 0. 1% ME, 0. 15g BSA/100M1, 1% PVP,and 0.53gVc/100mL, 0. 14 mol/L NaCl. In this way, we can eliminate the inhibeterant substance which I related and obtained high quality RNA within 3 hours.It indicated that the method is simple, convinent and practical.
2. The necessary GFLV and GLRaV Ⅲ nucleotide sequences for PCR primers have been published. The 5' and 3' primers of GFLV and GLRaV III coat protein gene were synthesized according to the published.The reverse transcription step was performed with total RNA extracted from infected grapevine tissue. From the synthesized first cDNA strand, 722 bp and 300 bp nucleotide fragments were amplified by two primers, which are the same as what we had expected.
3. To check the specificity and efficiency of GFLV and GLRaV Ⅲ cDNA
fragments amplified, the fragments were isolated from agarose gels and ligated to pGEM-T, transfect E.coli JM 109,the -galactosidase and ampicillin-resistant recombinant plasmids were obtained, these clones were further characterized by restriction enzyme digestion and PCR for the presence of the insert PCR products.The recombinant plasmids were then sequenced by the dideoxy chain termination method. The results showed that there is 99.3% between what we obtained by RT-PCR and the published. So, we consider that the technique of RT-PCR is exact and reliable, and it will give us an important preparation for virus diagnosis and quarantine on grapevine in the future.
引文
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