大麦黄矮病毒三种株系的特异性检测及PAV株系的群体遗传变异
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摘要
大麦黄矮病毒(Barley yellow dwarf viruses,BYDVs)是引起我国麦类病毒病的主要病毒之一,可造成巨大经济损失。本研究以采集自我国不同小麦种植区的大麦黄矮病株样品为材料,进行病毒三种株系的特异性检测鉴定、蚜传分离,明确不同地区的病毒株系分布、优势种类和动态变化,以及PAV株系的群体遗传变异及进化关系。
2007年根据田间症状,在我国四个农业生态区(西北地区、北方地区、中部地区和西南地区)共采集BYDVs样品281份,设计了特异性检测三种病毒株系(BYDV-GAV、BYDV-PAV、BYDV-GPV)的地高辛探针,探针对应病毒株系基因组CP基因与RTP基因,核酸点杂交检测结果表明,GAV、GPV和PAV三种探针能够检测到带毒植物组织的下限分别为25 ug,31.25 ug,62.5 ug,总共有190份样品(占67.6%)鉴定为阳性,其中检测到118份GAV(占41.99%)、54份PAV(占19.22%)、18份GPV(占6.41%),有少数样品还检测到了几种病毒株系混合侵染。GAV占据了田间侵染的大部分比例,是我国目前大麦黄矮病毒的主流株系,在焦作、大同、银川和乌鲁木齐地区普遍发生。PAV和GPV在特定区域呈优势发生,PAV主要分布在洛阳、郑州、天水等地区,GPV在田间侵染较少,在陕西韩城、山西运城零星发生。总体来看,我国麦区黄矮病发病率比较严重,不同地区麦蚜传播同一株系有差异,麦蚜带毒与传毒效率高低直接影响病害流行。
设计了扩增GAV株系、PAV株系CP基因的特异性引物,RT-PCR扩增后克隆测序,将各地样品的CP基因进行序列比对,分析了不同病毒株系的分子变异。测序的11个GAV CP基因均为600 bp,序列保守,系统进化树没有明显聚类。测序的10个PAV CP基因有600 bp和603 bp两种类型,变异程度明显,共有179个位点发生变异,尤其表现在3'末端,系统进化树形成三个组(A、B、C)。A、B两组来自同一进化分支,同属于PAV-CN分离物,CP基因均为600 bp,但序列间差异程度较大,各自聚类成组;样品05GG2和06KM25的CP基因核苷酸为603 bp,聚类成C组,与国外PAV分离物PAV-AUS的序列相似性达93.6%以上。
在大麦黄矮病毒的不同株系中,PAV株系的中国分离物比较特殊。根据GenBank已报道的PAV全基因组序列设计保守引物,经点杂交、RT-PCR鉴定和蚜传生物学分离,克隆测定了有代表性的30个PAV分离物,病毒基因组核苷酸全长为5652-5709 nt(GenBank登录号EU332307-EU332336),核苷酸序列相似性75.1%-99.7%,所编码的RdRp相似性82.2%-99.8%,CP相似性72.3%-99.6%,MP相似性77.6%-99.7%,RTP相似性77.4%-99.7%。中国与世界各地的PAV分离物用分子生物学软件DNAMAN和MEGA构建系统发育树表明,中国PAV至少存在四种群体类型,以PAV-CN为主,是我国PAV的优势类群;另外存在以样品05GG2、06KM14、06KM25、06GY1、06GY5为代表的特殊分离物,它们与PAV-CN核苷酸差异较大(相似性仅为78.5%),ORF和UTR均与国外PAV相似性高(93.6%以上),聚类成簇明显,这表明来自甘肃甘谷、云南昆明和贵州贵阳地区的PAV群体的遗传进化关系复杂特殊,这可能与当地地理环境、气候条件、植物寄主或蚜虫种群有关。地理位置及聚类分析结果表明,我国BYDV-PAV群体遗传多样性丰富,经生物信息学推测,病毒种群处于稳定进化状态,PAV可能起源于西北地区的甘肃、陕西一带,其传播扩散路线可能为:自20世纪60年代在中国甘肃、陕西一带出现后,逐渐向我国北部、东部、中部和南部传播扩散。
In China,Barley Yellow Dwarf Virus(BYDVs) caused mainly yellow dwarf diseases of wheat throughout the northern and northwestern provinces,crop losses of 20%-30%had been observed for many years in many provinces.The main objective of this study was specific detection BYDVs of speices type,incidence rates,species distribution and molecular variability,phylogenetic,genetic variation and evolution relationship for BYDV-PAV.
281 wheat samples expressing BYDV-like symptoms were collected from four agro-ecological areas in China during 2007,including Northwestern area,Northern area, Central area,and South-western area.Three digoxigenin-labeled cDNA probes complementary to the coat protein and read-through protein(RTP) gene sequences of barley yellow dwarf virus(one each for three species,namely BYDV-GAV,GPV,and PAV) were synthesized for developing a specific and sensitive dot-blot hybridization detection assay for total RNA extracts from field-infected wheat plants.The sensitivity limit for BYDV-GAV, GPV,and PAV probes corresponded to 25 ug,31.25 ug,and 62.5 ug tissue/spot,respectively. Nucleic acid spot hybridization showed that 190 samples had positive reaction,from which GAV(41.99%) was the most prevalent species in all samples by contrast with PAV(19.22%) and GPV(6.41%),GAV occured mostly in Jiaozuo,Datong,Yinchuan and Wunumuqi regions, PAV occured mostly in Luoyang,Zhengzhou,and Tianshui regions,however,GPV,existed as particular strain in China,had less infection and predominated mostly in specific regions, such as Yuncheng and Hancheng city.It seemed that the incidence rates of GAV may reduce and that of PAV would has been ascending tendency according to the quantity of positive GAV and PAV samples compared with reported data during 2004-2006 from our laboratory. The high sensitivity and reliability of the molecular hybridization assay described introduce an important altemative to serological methods for detecting BYDV.
Nucleic acid spot hybridization and reverse transcription polymerase chain reaction (RT-PCR) were made to amplify the CP gene of BYDV-GAV and BYDV-PAV using the RNA extract of samples.The product of RT-PCR were cloned and sequenced,the molecular variability of GAV and PAV were analysed through the multiple sequence alignments. Comparison of 11 nucletide sequences of GAV revealed high conservation of the CP gene, phylogenetic tree constructed based on the sequences of GAV CP genes showed that there was no obvious cluster,ttowever,PAV was another case,179 substitutions in 600 nt showed high variality,especially in 3'terminal sequence,the phylogenetic tree constructed based on the sequences of PAV CP genes formed three branches(A,B and C),A and B group were both belong to PAV-CN isolate,C group was comprised of isolates 05GG2 and 06KM25,even from the same branch,the variability of sequences of three groups is obvious,which resulted in the diversity clusters.Isolates 05GG2 and 06KM25 were different from other PAV samples, the CP gene comprised 603 nucleotides,and were very similar to those of BYDV PAV-aus, the nucleotide and deduced amino acid sequences showed that there was a high degree of identity between 05GG2/06KM25 and PAV-aus(93.6%identity),according to the level of sequence similarities,05GG2 and 06KM25 would be closely related to BYDV-PAV-aus.
The complete genomic sequences of a selection of 30 BYDV-PAV isolates were cloned and determined,which were comprised of 5652-5709 nucleotides(GenBank accession number: EU332307-EU332336),sharing 75.1%-99.7%identities at the nucleotide level. Phylogeography of PAV was reconstructed from the genomic RNA sequences of these isolates togethered with other PAV isolates reported previously.The variability among BYDV-PAV isolates in China was great,phylogenetic analysis detected four major groups, from which a new cluster of distinct variant was discovered,it is proposed to belong to a new species in China,sharing high sequence identity of individual ORF and UTR with isolates from USA,it suggested the high genetic diversity level and speciality of genetic structure for PAV in China.In contrast,the diversity within PAV isolates from USA was low(97% sequence identity),also when compared to isolates from Sweden,Germany,Australia and Japan.Altogether,it is supported the view that the origin centre of diversification for BYDV-PAV may be Gansu and Shaanxi province and that the virus spread subsequently from Northwest to North,East,Centre and South across China,using bioinformatics methods.
2007年根据田间症状,在我国四个农业生态区(西北地区、北方地区、中部地区和西南地区)共采集BYDVs样品281份,设计了特异性检测三种病毒株系(BYDV-GAV、BYDV-PAV、BYDV-GPV)的地高辛探针,探针对应病毒株系基因组CP基因与RTP基因,核酸点杂交检测结果表明,GAV、GPV和PAV三种探针能够检测到带毒植物组织的下限分别为25 ug,31.25 ug,62.5 ug,总共有190份样品(占67.6%)鉴定为阳性,其中检测到118份GAV(占41.99%)、54份PAV(占19.22%)、18份GPV(占6.41%),有少数样品还检测到了几种病毒株系混合侵染。GAV占据了田间侵染的大部分比例,是我国目前大麦黄矮病毒的主流株系,在焦作、大同、银川和乌鲁木齐地区普遍发生。PAV和GPV在特定区域呈优势发生,PAV主要分布在洛阳、郑州、天水等地区,GPV在田间侵染较少,在陕西韩城、山西运城零星发生。总体来看,我国麦区黄矮病发病率比较严重,不同地区麦蚜传播同一株系有差异,麦蚜带毒与传毒效率高低直接影响病害流行。
设计了扩增GAV株系、PAV株系CP基因的特异性引物,RT-PCR扩增后克隆测序,将各地样品的CP基因进行序列比对,分析了不同病毒株系的分子变异。测序的11个GAV CP基因均为600 bp,序列保守,系统进化树没有明显聚类。测序的10个PAV CP基因有600 bp和603 bp两种类型,变异程度明显,共有179个位点发生变异,尤其表现在3'末端,系统进化树形成三个组(A、B、C)。A、B两组来自同一进化分支,同属于PAV-CN分离物,CP基因均为600 bp,但序列间差异程度较大,各自聚类成组;样品05GG2和06KM25的CP基因核苷酸为603 bp,聚类成C组,与国外PAV分离物PAV-AUS的序列相似性达93.6%以上。
在大麦黄矮病毒的不同株系中,PAV株系的中国分离物比较特殊。根据GenBank已报道的PAV全基因组序列设计保守引物,经点杂交、RT-PCR鉴定和蚜传生物学分离,克隆测定了有代表性的30个PAV分离物,病毒基因组核苷酸全长为5652-5709 nt(GenBank登录号EU332307-EU332336),核苷酸序列相似性75.1%-99.7%,所编码的RdRp相似性82.2%-99.8%,CP相似性72.3%-99.6%,MP相似性77.6%-99.7%,RTP相似性77.4%-99.7%。中国与世界各地的PAV分离物用分子生物学软件DNAMAN和MEGA构建系统发育树表明,中国PAV至少存在四种群体类型,以PAV-CN为主,是我国PAV的优势类群;另外存在以样品05GG2、06KM14、06KM25、06GY1、06GY5为代表的特殊分离物,它们与PAV-CN核苷酸差异较大(相似性仅为78.5%),ORF和UTR均与国外PAV相似性高(93.6%以上),聚类成簇明显,这表明来自甘肃甘谷、云南昆明和贵州贵阳地区的PAV群体的遗传进化关系复杂特殊,这可能与当地地理环境、气候条件、植物寄主或蚜虫种群有关。地理位置及聚类分析结果表明,我国BYDV-PAV群体遗传多样性丰富,经生物信息学推测,病毒种群处于稳定进化状态,PAV可能起源于西北地区的甘肃、陕西一带,其传播扩散路线可能为:自20世纪60年代在中国甘肃、陕西一带出现后,逐渐向我国北部、东部、中部和南部传播扩散。
In China,Barley Yellow Dwarf Virus(BYDVs) caused mainly yellow dwarf diseases of wheat throughout the northern and northwestern provinces,crop losses of 20%-30%had been observed for many years in many provinces.The main objective of this study was specific detection BYDVs of speices type,incidence rates,species distribution and molecular variability,phylogenetic,genetic variation and evolution relationship for BYDV-PAV.
281 wheat samples expressing BYDV-like symptoms were collected from four agro-ecological areas in China during 2007,including Northwestern area,Northern area, Central area,and South-western area.Three digoxigenin-labeled cDNA probes complementary to the coat protein and read-through protein(RTP) gene sequences of barley yellow dwarf virus(one each for three species,namely BYDV-GAV,GPV,and PAV) were synthesized for developing a specific and sensitive dot-blot hybridization detection assay for total RNA extracts from field-infected wheat plants.The sensitivity limit for BYDV-GAV, GPV,and PAV probes corresponded to 25 ug,31.25 ug,and 62.5 ug tissue/spot,respectively. Nucleic acid spot hybridization showed that 190 samples had positive reaction,from which GAV(41.99%) was the most prevalent species in all samples by contrast with PAV(19.22%) and GPV(6.41%),GAV occured mostly in Jiaozuo,Datong,Yinchuan and Wunumuqi regions, PAV occured mostly in Luoyang,Zhengzhou,and Tianshui regions,however,GPV,existed as particular strain in China,had less infection and predominated mostly in specific regions, such as Yuncheng and Hancheng city.It seemed that the incidence rates of GAV may reduce and that of PAV would has been ascending tendency according to the quantity of positive GAV and PAV samples compared with reported data during 2004-2006 from our laboratory. The high sensitivity and reliability of the molecular hybridization assay described introduce an important altemative to serological methods for detecting BYDV.
Nucleic acid spot hybridization and reverse transcription polymerase chain reaction (RT-PCR) were made to amplify the CP gene of BYDV-GAV and BYDV-PAV using the RNA extract of samples.The product of RT-PCR were cloned and sequenced,the molecular variability of GAV and PAV were analysed through the multiple sequence alignments. Comparison of 11 nucletide sequences of GAV revealed high conservation of the CP gene, phylogenetic tree constructed based on the sequences of GAV CP genes showed that there was no obvious cluster,ttowever,PAV was another case,179 substitutions in 600 nt showed high variality,especially in 3'terminal sequence,the phylogenetic tree constructed based on the sequences of PAV CP genes formed three branches(A,B and C),A and B group were both belong to PAV-CN isolate,C group was comprised of isolates 05GG2 and 06KM25,even from the same branch,the variability of sequences of three groups is obvious,which resulted in the diversity clusters.Isolates 05GG2 and 06KM25 were different from other PAV samples, the CP gene comprised 603 nucleotides,and were very similar to those of BYDV PAV-aus, the nucleotide and deduced amino acid sequences showed that there was a high degree of identity between 05GG2/06KM25 and PAV-aus(93.6%identity),according to the level of sequence similarities,05GG2 and 06KM25 would be closely related to BYDV-PAV-aus.
The complete genomic sequences of a selection of 30 BYDV-PAV isolates were cloned and determined,which were comprised of 5652-5709 nucleotides(GenBank accession number: EU332307-EU332336),sharing 75.1%-99.7%identities at the nucleotide level. Phylogeography of PAV was reconstructed from the genomic RNA sequences of these isolates togethered with other PAV isolates reported previously.The variability among BYDV-PAV isolates in China was great,phylogenetic analysis detected four major groups, from which a new cluster of distinct variant was discovered,it is proposed to belong to a new species in China,sharing high sequence identity of individual ORF and UTR with isolates from USA,it suggested the high genetic diversity level and speciality of genetic structure for PAV in China.In contrast,the diversity within PAV isolates from USA was low(97% sequence identity),also when compared to isolates from Sweden,Germany,Australia and Japan.Altogether,it is supported the view that the origin centre of diversification for BYDV-PAV may be Gansu and Shaanxi province and that the virus spread subsequently from Northwest to North,East,Centre and South across China,using bioinformatics methods.
引文
1.范在丰,李怀方.植物病毒的分类与命名现状[J].植物病理学报,2003.32(2):112-115.
2.晋治波.大麦黄矮病毒GAV基因组全序列分析及运动蛋白基因介导的抗病毒小麦的研究[D].北京:中国农业科学院研究生院,2003.
3.晋治波,王锡锋,常胜军等.大麦黄矮病毒GAV基因组全序列测定及其结构分析[J].中国科学(C辑),2003,33(6):505-513.
4.廖寓荣.水稻条纹病毒及其介体灰飞虱的遗传多样性[D].福州:福建农林大学,2004.
5.刘芳.两种病毒的基因组全序列测定以及辣椒轻斑驳病毒的DIG标记点杂交检测[D].长沙:湖南农业大学,2005.
6.刘艳.BYDV-GPV与GAV混合侵染蚜传专化性研究[D].北京:中国农业科学院研究生院,2004.
7.孙博.大麦黄矮病毒群体的分子变异[D].北京:中国农业科学院研究生院,2006.
8.张世贤.我国北方稻区水稻条纹病毒CP、SP基因的分子变异和品种抗病性研究[D].北京:中国农业科学院研究生院,2006.
9.施曼玲.芜菁花叶病毒单克隆抗体制备及病毒基因组变异研究[D].杭州:浙江大学,2005.
10.王锡锋.麦蚜传播大麦黄矮病毒的机制研究[D].北京:中国农业科学院研究生院,1998.
11.魏太云,林含新,谢联辉.植物病毒分子群体遗传学研究进展[J].福建农林大学学报(自然科学版),2003,32(4):27-30.
12.魏太云.水稻条纹病毒的基因组结构及分子群体遗传[D].福州:福建农林大学,2003.
13.杨军.小麦黄色花叶病毒RNA1的自然变异[D].北京:中国农业大学,2003.
14.周广和,张淑香,钱幼亭.小麦黄矮病毒4种株系鉴定与应用[J].中国农业科学,1987,20(4):7-12.
15.周广和.世界大麦黄矮病毒研究概况[J].世界农业.1989,7:36-38.
16.Adrian J.Gibbs,Charles H.Calisher,Femando Garcfa-Arenal,Molecular basis of virus evolution.Camberidge university press.2005.
17.Ahlquist P.RNA-dependent RNA polymerase,viruses,and RNA silencing.Science,2002,296:1270-1273.
18.Aranda M.A.,Fraile A,Dopazo J,Malpica JM,1997.Contribution of mutation and RNA recombination to the evolution of a plant pathogenic RNA.Journal of Molecular Evolution,44(1):81-88.
19.Azzam O,Arboleda M,Umadhay KML,2000a.Genetic composition and complexity of virus populations at tungro-endemic and outbreak rice sites.Archives of Virology,145(12):2643-2657.
20.Azzam O,Yambao MLM,Muhsin M,Mcnally KL,Umadhay KML,2000b.Genetic diversity of rice tungro spherical virus in tungro-endemic provinces of the Philippines and Indonesia.Archives of Virology,145(6):1183-1197.
21.Bencharki B,El Yamani M,Zaoui D(2000) Assessment of transmission ability of barley yellow dwarf virus-PAV isolates by different populations of Rhopalosiphum padi and Sitobion avenae.
2.晋治波.大麦黄矮病毒GAV基因组全序列分析及运动蛋白基因介导的抗病毒小麦的研究[D].北京:中国农业科学院研究生院,2003.
3.晋治波,王锡锋,常胜军等.大麦黄矮病毒GAV基因组全序列测定及其结构分析[J].中国科学(C辑),2003,33(6):505-513.
4.廖寓荣.水稻条纹病毒及其介体灰飞虱的遗传多样性[D].福州:福建农林大学,2004.
5.刘芳.两种病毒的基因组全序列测定以及辣椒轻斑驳病毒的DIG标记点杂交检测[D].长沙:湖南农业大学,2005.
6.刘艳.BYDV-GPV与GAV混合侵染蚜传专化性研究[D].北京:中国农业科学院研究生院,2004.
7.孙博.大麦黄矮病毒群体的分子变异[D].北京:中国农业科学院研究生院,2006.
8.张世贤.我国北方稻区水稻条纹病毒CP、SP基因的分子变异和品种抗病性研究[D].北京:中国农业科学院研究生院,2006.
9.施曼玲.芜菁花叶病毒单克隆抗体制备及病毒基因组变异研究[D].杭州:浙江大学,2005.
10.王锡锋.麦蚜传播大麦黄矮病毒的机制研究[D].北京:中国农业科学院研究生院,1998.
11.魏太云,林含新,谢联辉.植物病毒分子群体遗传学研究进展[J].福建农林大学学报(自然科学版),2003,32(4):27-30.
12.魏太云.水稻条纹病毒的基因组结构及分子群体遗传[D].福州:福建农林大学,2003.
13.杨军.小麦黄色花叶病毒RNA1的自然变异[D].北京:中国农业大学,2003.
14.周广和,张淑香,钱幼亭.小麦黄矮病毒4种株系鉴定与应用[J].中国农业科学,1987,20(4):7-12.
15.周广和.世界大麦黄矮病毒研究概况[J].世界农业.1989,7:36-38.
16.Adrian J.Gibbs,Charles H.Calisher,Femando Garcfa-Arenal,Molecular basis of virus evolution.Camberidge university press.2005.
17.Ahlquist P.RNA-dependent RNA polymerase,viruses,and RNA silencing.Science,2002,296:1270-1273.
18.Aranda M.A.,Fraile A,Dopazo J,Malpica JM,1997.Contribution of mutation and RNA recombination to the evolution of a plant pathogenic RNA.Journal of Molecular Evolution,44(1):81-88.
19.Azzam O,Arboleda M,Umadhay KML,2000a.Genetic composition and complexity of virus populations at tungro-endemic and outbreak rice sites.Archives of Virology,145(12):2643-2657.
20.Azzam O,Yambao MLM,Muhsin M,Mcnally KL,Umadhay KML,2000b.Genetic diversity of rice tungro spherical virus in tungro-endemic provinces of the Philippines and Indonesia.Archives of Virology,145(6):1183-1197.
21.Bencharki B,El Yamani M,Zaoui D(2000) Assessment of transmission ability of barley yellow dwarf virus-PAV isolates by different populations of Rhopalosiphum padi and Sitobion avenae.