百合上李属坏死环斑病毒和百合无症病毒的研究
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摘要
百合(Lilium L.)是集观赏、食用、药用于一身的重要经济作物,现已成为国际上重要切花观赏花卉之一。近年来,随着我国观赏百合种植面积的迅速增加,从国外引进种球的数量和批次迅速增长,百合病毒病开始在各百合种植区发生流行。本文以发生在百合上的两种重要的病毒,百合无症病毒(Lily symptomless virus,LSV)和李属坏死环斑病毒(Prunus necrotic ringspot virus,PNRSV)为研究对象,利用分子生物学技术,对上述两种检疫性病毒基因组的分子特征进行了比较研究,分析不同地区LSV的来源、分布及相关性,建立了多重PCR、RNA点杂交和RNA玻片杂交等适用于进境口岸百合种球病毒检疫工作的快速检测方法。研究获得的主要结果和结论如下:
1、揭示了PNRSV外壳蛋白基因序列与其病害症状类型间的相关性,并利用该分子特征建立起的多重RT-PCR检测方法,用于PNRSV轻型和重型两种不同的株系的初诊和快速检测。
根据GenBank中已报道的PNRSV外壳蛋白的基因序列,分析不同致病型株系间核酸水平上的差异,设计了两对特异性引物,并通过扩增条件的优化,建立了可区分PNRSV轻型和重型株系的多重PCR检测方法。该方法对已知的待测样品进行检测,在同一反应体系中扩增出了450bp和170bp两种大小的条带,可区分PNRSV轻型和重型两种不同的株系。揭示了PNRSV外壳蛋白基因序列与其病害症状类型间的具有相关性并可用于不同致病型株系的快速诊断和检测。
2、在分子水平上鉴定了百合上发生的PNRSV,证实PNRSV可通过百合种球带毒传播。
对待测荷兰进口的百合种球进行随机抽样,在温室内种植并观察,长出叶片后,用RNA Extraction Kit (Sangon, China)从幼嫩的叶组织中提取核酸。用根据PNRSV外壳蛋白基因序列设计的特异性引物进行RT-PCR扩增,电泳PCR产物。从待测的两个样品(P-299和P-503)中获得了450bp的目标片段。低熔点胶回收PCR产物,经纯化后克隆至pGEM-TG1载体,测序后进行序列比对分析。将所获得的上述两个分离物的核酸序列与GenBank中已报道的27个PNRSV分离物的CP基因序列进行比对,结果表明,P-299与P-503 CP基因序列相似性为99.8%。P-299和P-503 CP基因序列与27个已报道的分离物CP基因序列的相似性为84.5%～99.1%。据此可知扩增片段为PNRSV的部分CP基因序列,在分子水平上鉴定了百合上发生的PNRSV。将上述两个分离株的CP基因序列登陆GenBank进行了注册,P-299的登陆号为DQ992416;P-503的登陆号为DQ992417。
用插入了P-299分离株的部份CP基因互补序列的质粒P-299B做模板进行PCR,回收PCR产物,制备32P标记探针,对来自浙江丽水县田间的野生百合样品进行RNA斑点杂交检测,在24个百合样品中有5个杂交结果为阳性,证实了PNRSV在自然条件下可通过百合种球带毒传播,说明了百合是PNRSV的一个新寄主。
3、本研究采用RT-PCR法从来自国内不同地区进口百合次生代样品上获得了7个含有LSV基因组3′端部分序列的分离株。经过测序,应用DNAStar软件比较了其与已报道的12个LSV分离株的CP基因和16kD基因的同源性。序列分析结果表明:7个分离株蛋白外壳部分基因序列和16kD蛋白基因序列的相似性分别为87.4%~100%和97.9%~100%;在氨基酸水平上的相似性分别为95.9%~100%和95.7%~100%。系统发育树的比较显示,本研究获得的7个LSV分离株间在系统中有较近的进化关系,但这些分离株间没有明显的地域相关性,也没有发现与块根频繁运输相关的新亚群。
4、建立了RNA玻片杂交检测LSV的新方法。使用该方法对国内不同地区的进口和野生百合上LSV的发生情况进行了检测,结果表明LSV是存在中国境内百合上最为普通的病毒之一。
Lilium is an important economical crop which could be used as ornamental plant, food or medicine. Lilium has already become one of main international ornamental flowers in recent years.As the increasing acreage of Lily, the quantaty and batch of imported lily blubs have been increased tremendously.This trend leads to a prevalence of virus diseases in lily planting areas. This study focuses on Prunus necrotic ringspot virus (PNRSV) and Lily symptomless virus (LSV) occurred on lily. Molecular Characters of these tow viruses (PNRSV and LSV) were compared for analying the relations between origin and distribution. Based on this, several rapid identification methods included RT-PCR, multiple-PCR and RNA silde hybridization were developed for lily virus detection at port quarantine.The main result and conclusion were as follows.
1. Study revealed correlations between symptom type and the nucleotide sequences of 3b (coat protein) open reading frame.
According to nucleotide sequences of PNRSV coat protein open reading frame (ORF), we designed two paires of“strain-specific”primers and established a multiple-PCR method with optimized RT-PCR reaction conditions for discrimination of rugose from mild strains. Two RT-PCR products with different sizes were simutaneously amplified, which could distinguish rugose type from mild type. It revealed correlations between symptom type and the nucleotide sequences of 3b (coat protein) open reading frame.
2. PNRSV from lily corm was obtained and identified on molecular levels.It shown that Lily is a new carrier of PNRSV.
Lily corms were randomly selected and grown in a greenhouse. Total RNA was extracted from young leaves with RNA Extraction Kit (Sangon, China). Amplification of partial CP gene was performed by RT-PCR with the primer pairs specific for PNRSV. An expected 450 bp fragment was harvested, cloned into pGEM-T-easy vector, sequenced, and compared with 27 PNRSV isolates reported previously. This 450 bp fragment amplified from lily shared 84.5% to 99.1% homology with 27 PNRSV isolates reported previously at the nucleotide level.
3. Comparison and identification of 7 LSV isolates were conducted on molecular levels for analying the relations between origin and distribution.Results shown that isolates are not regional relevant.
Seven LSV isolates from secondary generations of imported Lily in different domestic areas were obtained by RT-PCR and sequenced. These sequences are corresponded to the 3’end region of LSV genome including a partial CP gene and a entire gene encoding a 16 kD protein. Compared with 12 other isolates reported on GenBank, the 7 isolates sequence homologies of partial CP and 16 kD protein genes were 87.4% to 100% and 97.9% to 100%, respectively. At the amino acid level, they shared 95.9% to 100% and 95.7% to 100% sequence similarity, respectively. Comparison of their phylogenetic tree of the CP gene sequences and amino caid sequences show that there are no apparent regional relationships among these isolates.
4.An RNA slide hybridization assay was developed and used for detection of the natural occurrence of LSV in lily corm originated from both imported cultivars and from wild lily spp.Results show that LSV is one of the ordinary virues occurred on Lily in China.
1、揭示了PNRSV外壳蛋白基因序列与其病害症状类型间的相关性,并利用该分子特征建立起的多重RT-PCR检测方法,用于PNRSV轻型和重型两种不同的株系的初诊和快速检测。
根据GenBank中已报道的PNRSV外壳蛋白的基因序列,分析不同致病型株系间核酸水平上的差异,设计了两对特异性引物,并通过扩增条件的优化,建立了可区分PNRSV轻型和重型株系的多重PCR检测方法。该方法对已知的待测样品进行检测,在同一反应体系中扩增出了450bp和170bp两种大小的条带,可区分PNRSV轻型和重型两种不同的株系。揭示了PNRSV外壳蛋白基因序列与其病害症状类型间的具有相关性并可用于不同致病型株系的快速诊断和检测。
2、在分子水平上鉴定了百合上发生的PNRSV,证实PNRSV可通过百合种球带毒传播。
对待测荷兰进口的百合种球进行随机抽样,在温室内种植并观察,长出叶片后,用RNA Extraction Kit (Sangon, China)从幼嫩的叶组织中提取核酸。用根据PNRSV外壳蛋白基因序列设计的特异性引物进行RT-PCR扩增,电泳PCR产物。从待测的两个样品(P-299和P-503)中获得了450bp的目标片段。低熔点胶回收PCR产物,经纯化后克隆至pGEM-TG1载体,测序后进行序列比对分析。将所获得的上述两个分离物的核酸序列与GenBank中已报道的27个PNRSV分离物的CP基因序列进行比对,结果表明,P-299与P-503 CP基因序列相似性为99.8%。P-299和P-503 CP基因序列与27个已报道的分离物CP基因序列的相似性为84.5%～99.1%。据此可知扩增片段为PNRSV的部分CP基因序列,在分子水平上鉴定了百合上发生的PNRSV。将上述两个分离株的CP基因序列登陆GenBank进行了注册,P-299的登陆号为DQ992416;P-503的登陆号为DQ992417。
用插入了P-299分离株的部份CP基因互补序列的质粒P-299B做模板进行PCR,回收PCR产物,制备32P标记探针,对来自浙江丽水县田间的野生百合样品进行RNA斑点杂交检测,在24个百合样品中有5个杂交结果为阳性,证实了PNRSV在自然条件下可通过百合种球带毒传播,说明了百合是PNRSV的一个新寄主。
3、本研究采用RT-PCR法从来自国内不同地区进口百合次生代样品上获得了7个含有LSV基因组3′端部分序列的分离株。经过测序,应用DNAStar软件比较了其与已报道的12个LSV分离株的CP基因和16kD基因的同源性。序列分析结果表明:7个分离株蛋白外壳部分基因序列和16kD蛋白基因序列的相似性分别为87.4%~100%和97.9%~100%;在氨基酸水平上的相似性分别为95.9%~100%和95.7%~100%。系统发育树的比较显示,本研究获得的7个LSV分离株间在系统中有较近的进化关系,但这些分离株间没有明显的地域相关性,也没有发现与块根频繁运输相关的新亚群。
4、建立了RNA玻片杂交检测LSV的新方法。使用该方法对国内不同地区的进口和野生百合上LSV的发生情况进行了检测,结果表明LSV是存在中国境内百合上最为普通的病毒之一。
Lilium is an important economical crop which could be used as ornamental plant, food or medicine. Lilium has already become one of main international ornamental flowers in recent years.As the increasing acreage of Lily, the quantaty and batch of imported lily blubs have been increased tremendously.This trend leads to a prevalence of virus diseases in lily planting areas. This study focuses on Prunus necrotic ringspot virus (PNRSV) and Lily symptomless virus (LSV) occurred on lily. Molecular Characters of these tow viruses (PNRSV and LSV) were compared for analying the relations between origin and distribution. Based on this, several rapid identification methods included RT-PCR, multiple-PCR and RNA silde hybridization were developed for lily virus detection at port quarantine.The main result and conclusion were as follows.
1. Study revealed correlations between symptom type and the nucleotide sequences of 3b (coat protein) open reading frame.
According to nucleotide sequences of PNRSV coat protein open reading frame (ORF), we designed two paires of“strain-specific”primers and established a multiple-PCR method with optimized RT-PCR reaction conditions for discrimination of rugose from mild strains. Two RT-PCR products with different sizes were simutaneously amplified, which could distinguish rugose type from mild type. It revealed correlations between symptom type and the nucleotide sequences of 3b (coat protein) open reading frame.
2. PNRSV from lily corm was obtained and identified on molecular levels.It shown that Lily is a new carrier of PNRSV.
Lily corms were randomly selected and grown in a greenhouse. Total RNA was extracted from young leaves with RNA Extraction Kit (Sangon, China). Amplification of partial CP gene was performed by RT-PCR with the primer pairs specific for PNRSV. An expected 450 bp fragment was harvested, cloned into pGEM-T-easy vector, sequenced, and compared with 27 PNRSV isolates reported previously. This 450 bp fragment amplified from lily shared 84.5% to 99.1% homology with 27 PNRSV isolates reported previously at the nucleotide level.
3. Comparison and identification of 7 LSV isolates were conducted on molecular levels for analying the relations between origin and distribution.Results shown that isolates are not regional relevant.
Seven LSV isolates from secondary generations of imported Lily in different domestic areas were obtained by RT-PCR and sequenced. These sequences are corresponded to the 3’end region of LSV genome including a partial CP gene and a entire gene encoding a 16 kD protein. Compared with 12 other isolates reported on GenBank, the 7 isolates sequence homologies of partial CP and 16 kD protein genes were 87.4% to 100% and 97.9% to 100%, respectively. At the amino acid level, they shared 95.9% to 100% and 95.7% to 100% sequence similarity, respectively. Comparison of their phylogenetic tree of the CP gene sequences and amino caid sequences show that there are no apparent regional relationships among these isolates.
4.An RNA slide hybridization assay was developed and used for detection of the natural occurrence of LSV in lily corm originated from both imported cultivars and from wild lily spp.Results show that LSV is one of the ordinary virues occurred on Lily in China.
引文
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