我国葡萄卷叶病病原分子检测技术研究
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摘要
葡萄卷叶病(Grapevine leafroll disease, GLD)是分布最广泛,危害葡萄最为严重的病毒病之一。迄今为止,全世界已报道了11种血清学不相关的葡萄卷叶病毒(Grapevine leafroll-associated virus, GLRaV),分别命名为GLRaV-1, -2, -3, -4, -5, -6, -7, -8, -9, -Pr和-De,均属于长线病毒科,上述病毒单独或复合侵染都能引起葡萄卷叶病的发生。本研究通过田间调查和采样,结合实验室ELISA和RT-PCR检测对我国葡萄卷叶病毒的种类以及葡萄品种的耐病性和抗病性做了调查分析;在研究单一RT-PCR的基础上,建立了能同时检测GLRaV-1,-3,-4,-5等四种葡萄卷叶病毒的多重RT-PCR检测技术;对我国GLRaV-4和GLRaV-5分离物的基因片段的序列进行分析。取得的主要结果摘要如下:
1.为探明我国葡萄卷叶病病原种类,利用ELISA和RT-PCR检测方法对采集于中国农业科学院果树研究所葡萄品种保存圃中,表现典型葡萄卷叶病症状的58份葡萄休眠枝条样品进行了检测,共检测到GLRaV-1,2,3,4,5,7共6种葡萄卷叶病毒,GLRaVs总检出率为81.0%,其中GLRaV-3检出率最高,达到62.1%,GLRaV-1,-2和-7检出率分别是20.7%,17.2%和15.5%;GLRaV-4和-5的检出率最低,分别为3.4%和5.2%,是国内首次报道。2种或3种葡萄卷叶病毒复合侵染现象比较普遍,占所检样品的39.6%。
葡萄品种保存圃症状调查结果表明,欧亚种群中表现葡萄卷叶病症状的品种多,发病率高,发病症状也最为严重;欧美杂种中表现葡萄卷叶病症状的品种发病率和严重度均比欧亚种群低;在保存圃内未见美洲种群的品种表现葡萄卷叶病症状。
2.为提高检测效率,降低检测费用,本研究在单一RT-PCR的研究基础上,对4种葡萄卷叶病毒的多重RT-PCR模板浓度、引物浓度和退火温度等进行优化,建立了同时检测GLRaV-1,-3,-4和-5的多重RT-PCR技术体系。模板浓度,引物浓度、Taq DNA聚合酶浓度、退火温度和循环次数对多重RT-PCR检测结果均有较大影响,而在一定范围内改变dNTP浓度和延伸时间对检测结果影响不大。
3.从“皇家秋天”、“马拉加玫瑰”样品中分别克隆了GLRaV-4的HSP70、CP基因和GLRV-5的CP基因。测序结果表明:
GLRaV-4 HSP70基因片段长442 bp,推测编码146个氨基酸,GenBank登录号为GQ849394。该序列与GenBank上已报道的意大利GLRaV-4(AF039553) HSP70基因核苷酸序列相似性为99%,有2个碱基的差异。
GLRaV-4 CP基因片断长300 bp,推测编码100个氨基酸,GenBank登录号为GQ479041。该序列与GenBank上已报道的智利GLRaV-4(EU746621) CP基因序列相似性为99%,有1个碱基的差异。
GLRV-5 CP基因片断长690 bp,推测编码227个氨基酸,GenBank登录号为GQ246625。该序列与GenBank上已报道的阿根廷GLRaV-5 (EU815935) CP基因序列相似性为95%,有33个碱基的差异。
Grapevine leaf roll disease is one of the most important diseases of grapevines worldwide. At present, eleven serologically distinct viruses belonging to the family Closteroviridae have been found associated with grapevine leafroll disease (GLD). These eleven viruses are named GLRaV-1, -2, -3, -4, -5, -6, -7, -8, -9, -Pr and -De respectively, which can cause grapevine leaf roll disease alone or together.. In this paper, we used ELISA and RT-PCR to identify GLRaVs species, preliminary analyzed the infection condition of GLRaVs in China. And based on the detection of GLRaVs by single RT-PCR, a multiplex RT-PCR was applied to detect 4 grapevine leafroll-associated viruses, GLRaV-1, -3, -4 and -5. The sequences of part genes of GLRaV-4 and GLRaV-5 Chinese isolates were analyzed. The results as follows:
1. In order to identify the GLD Etiological Viruses in China, the dormant grapevine cane samples from 58 individual vines which had shown severe leafroll symptoms were collected from a germplasm collection pot at Research Institute of Pomology, Chinese Academy of Agricultural Sciences. The samples were tested by ELISA and RT-PCR. The results showed that Grapevine leafroll-associated virus (GLRaV) -1, -2, -3, -4, -5 and -7 are present, a total of 81.0% samples were infected and the rate of six GLRaVs was 20.7%, 17.2%, 62.1%, 3.4%, 5.2%, and 15.5% respectively. To our knowledge, this is the first report of GLRaV-4 and -5 in grapevines in China. It is a common phenomenon of mixed infection with two or three GLRaVs. The mixed infection rate was 39.6%.
2. In order to improve detection efficiency and decrease cost, based on the detection of GLRaVs by single RT-PCR, a multiplex RT-PCR was applied to detect 4 grapevine leafroll-associated viruses, GLRaV-1, -3, -4 and -5. In this paper, we studied the mainly factors affecting RT-PCR reaction. The results showed that template concentration, primer concentration, Taq DNA polymerase concentration, annealing temperature and reaction cycles make more influences on the result in the system than that of extension time and dNTP concentration.
3. The HSP70 and CP gene of GLRaV-4 were cloned from“Autumn Royal”. The CP gene of GLRaV-5 were cloned from“Malaga Rose”.
Sequence analysis showed that the HSP70 gene consists of 442 nucleotides and encodes a polypeptide consisting of 146 amino acids. The registered number in GenBank is GQ849394. The sequence showed 99% identical nucleotides with the corresponding region of one GLRaV-4 isolate from Italy (GenBank Accession No. AF039553). There are two distinct nucleotides.
The CP gene consists of 300 nucleotides and encodes a polypeptide consisting of 100 amino acids. The registered number in GenBank is GQ479041. A comparison of the CP gene sequences with the corresponding nucleotide sequences of another GLRaV-4 isolate from Chile (GenBank Accession No. EU746621) showed 99℅ identity . There is one distinct nucleotide.
The CP gene consists of 690 nucleotides and encodes a polypeptide consisting of 227 amino acids. The registered number in GenBank is GQ246625. A comparison of the CP gene sequences with the corresponding nucleotide sequences of another GLRaV-5 isolate from Argentina (GenBank Accession No. EU815935) showed 95℅ identity . There are 33 distinct nucleotides.
1.为探明我国葡萄卷叶病病原种类,利用ELISA和RT-PCR检测方法对采集于中国农业科学院果树研究所葡萄品种保存圃中,表现典型葡萄卷叶病症状的58份葡萄休眠枝条样品进行了检测,共检测到GLRaV-1,2,3,4,5,7共6种葡萄卷叶病毒,GLRaVs总检出率为81.0%,其中GLRaV-3检出率最高,达到62.1%,GLRaV-1,-2和-7检出率分别是20.7%,17.2%和15.5%;GLRaV-4和-5的检出率最低,分别为3.4%和5.2%,是国内首次报道。2种或3种葡萄卷叶病毒复合侵染现象比较普遍,占所检样品的39.6%。
葡萄品种保存圃症状调查结果表明,欧亚种群中表现葡萄卷叶病症状的品种多,发病率高,发病症状也最为严重;欧美杂种中表现葡萄卷叶病症状的品种发病率和严重度均比欧亚种群低;在保存圃内未见美洲种群的品种表现葡萄卷叶病症状。
2.为提高检测效率,降低检测费用,本研究在单一RT-PCR的研究基础上,对4种葡萄卷叶病毒的多重RT-PCR模板浓度、引物浓度和退火温度等进行优化,建立了同时检测GLRaV-1,-3,-4和-5的多重RT-PCR技术体系。模板浓度,引物浓度、Taq DNA聚合酶浓度、退火温度和循环次数对多重RT-PCR检测结果均有较大影响,而在一定范围内改变dNTP浓度和延伸时间对检测结果影响不大。
3.从“皇家秋天”、“马拉加玫瑰”样品中分别克隆了GLRaV-4的HSP70、CP基因和GLRV-5的CP基因。测序结果表明:
GLRaV-4 HSP70基因片段长442 bp,推测编码146个氨基酸,GenBank登录号为GQ849394。该序列与GenBank上已报道的意大利GLRaV-4(AF039553) HSP70基因核苷酸序列相似性为99%,有2个碱基的差异。
GLRaV-4 CP基因片断长300 bp,推测编码100个氨基酸,GenBank登录号为GQ479041。该序列与GenBank上已报道的智利GLRaV-4(EU746621) CP基因序列相似性为99%,有1个碱基的差异。
GLRV-5 CP基因片断长690 bp,推测编码227个氨基酸,GenBank登录号为GQ246625。该序列与GenBank上已报道的阿根廷GLRaV-5 (EU815935) CP基因序列相似性为95%,有33个碱基的差异。
Grapevine leaf roll disease is one of the most important diseases of grapevines worldwide. At present, eleven serologically distinct viruses belonging to the family Closteroviridae have been found associated with grapevine leafroll disease (GLD). These eleven viruses are named GLRaV-1, -2, -3, -4, -5, -6, -7, -8, -9, -Pr and -De respectively, which can cause grapevine leaf roll disease alone or together.. In this paper, we used ELISA and RT-PCR to identify GLRaVs species, preliminary analyzed the infection condition of GLRaVs in China. And based on the detection of GLRaVs by single RT-PCR, a multiplex RT-PCR was applied to detect 4 grapevine leafroll-associated viruses, GLRaV-1, -3, -4 and -5. The sequences of part genes of GLRaV-4 and GLRaV-5 Chinese isolates were analyzed. The results as follows:
1. In order to identify the GLD Etiological Viruses in China, the dormant grapevine cane samples from 58 individual vines which had shown severe leafroll symptoms were collected from a germplasm collection pot at Research Institute of Pomology, Chinese Academy of Agricultural Sciences. The samples were tested by ELISA and RT-PCR. The results showed that Grapevine leafroll-associated virus (GLRaV) -1, -2, -3, -4, -5 and -7 are present, a total of 81.0% samples were infected and the rate of six GLRaVs was 20.7%, 17.2%, 62.1%, 3.4%, 5.2%, and 15.5% respectively. To our knowledge, this is the first report of GLRaV-4 and -5 in grapevines in China. It is a common phenomenon of mixed infection with two or three GLRaVs. The mixed infection rate was 39.6%.
2. In order to improve detection efficiency and decrease cost, based on the detection of GLRaVs by single RT-PCR, a multiplex RT-PCR was applied to detect 4 grapevine leafroll-associated viruses, GLRaV-1, -3, -4 and -5. In this paper, we studied the mainly factors affecting RT-PCR reaction. The results showed that template concentration, primer concentration, Taq DNA polymerase concentration, annealing temperature and reaction cycles make more influences on the result in the system than that of extension time and dNTP concentration.
3. The HSP70 and CP gene of GLRaV-4 were cloned from“Autumn Royal”. The CP gene of GLRaV-5 were cloned from“Malaga Rose”.
Sequence analysis showed that the HSP70 gene consists of 442 nucleotides and encodes a polypeptide consisting of 146 amino acids. The registered number in GenBank is GQ849394. The sequence showed 99% identical nucleotides with the corresponding region of one GLRaV-4 isolate from Italy (GenBank Accession No. AF039553). There are two distinct nucleotides.
The CP gene consists of 300 nucleotides and encodes a polypeptide consisting of 100 amino acids. The registered number in GenBank is GQ479041. A comparison of the CP gene sequences with the corresponding nucleotide sequences of another GLRaV-4 isolate from Chile (GenBank Accession No. EU746621) showed 99℅ identity . There is one distinct nucleotide.
The CP gene consists of 690 nucleotides and encodes a polypeptide consisting of 227 amino acids. The registered number in GenBank is GQ246625. A comparison of the CP gene sequences with the corresponding nucleotide sequences of another GLRaV-5 isolate from Argentina (GenBank Accession No. EU815935) showed 95℅ identity . There are 33 distinct nucleotides.
引文
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