大麦黄矮病毒群体的分子变异
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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。由于中国小麦种植区域的生态地理环境有很大差异,可能造成BYDVs含有较为丰富的变异类型。本试验以我国不同地区的大麦黄矮病株标样为材料,对其进行分子鉴定,明确不同地区分离物的病毒种类(株系)、分子变异和进化关系。
2004年和2005年共采集BYDVs标样279个,提取总RNA经RT-PCR扩增CP基因以及核酸点杂交检测我国近两年的大麦黄矮病发生情况,标样的70%左右为阳性结果,其中,BYDV-GAV占据了阳性样品的大部分比例,BYDV-PAV、BYDV-GPV分别在特定区域优势地发生,如2005年BYDV-PAV主要集中在陕西渭南和杨陵地区,占当地阳性样品的29.41%,比2004年的7%有明显的增多。BYDV-GPV,作为中国特有的大麦黄矮病毒株系,在田间的检测样品中比较少,多发生在山西运城,2005年陕西渭南、陕西凤翔、甘肃甘谷各检测到一例。
RT-PCR产物经克隆测序,进行多序列比对分析不同病毒(株系)的分子变异情况。测序的26个BYDV-GAV核苷酸序列长度为600bp,序列一致程度高,变异程度非常小。根据序列构建的系统进化树具有许多平行分支,表明BYDV-GAV序列之间没有明显的聚类。BYDV-PAV核苷酸序列的分子变异则与BYDV-GAV结果不同,600个碱基中有179个位点发生变异,尤其表现在3'末端。系统进化树形成四个组(A-D)。A、B、C三组同属于PAV-CN分离物,虽从同一进化分支而来,但其各自序列相互间差异程度较大,进而分别聚类成组。BYDV-GPV CP基因核苷酸序列长度为606bp,测得序列间同源性为98.7%-99.8%,变异很小。
在测得的BYDV-PAV序列中,来自甘肃省甘谷的05GG2标样比较特殊,它与PAV-CN核苷酸差异较大,而与国外PAV分离物相似程度较高,根据已公布的BYDV-PAV分离物全基因组序列设计保守引物,扩增出05GG2除末端的近全长基因组序列,由5675个核苷酸组成,与PAV-CN序列同源性仅为78.5%,与PAV-aus全基因组序列同源性高达93.6%。初步分析了05GG2的开放阅读框架及非编码区,并将其与世界各地PAV分离物进行核苷酸、氨基酸比对,相似性分析表明它与国外分离物具有较高的同源性,而与国内PAV-CN差异较大。系统进化树显示,05GG2与PAV-aus等成簇聚类明显,形成独立分支。
Barley yellow dwarf viruses (BYDVs) are members of the plant virus family Luteoviridae. They were phloem-limited and obligately transmitted in the circulative/persistent manner by several species of cereal aphids and can cause significant economic losses worldwide because of damage to barley, wheat, and oats. In China, 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 Shaanxi and Gansu provinces, the last serious epidemic happened in Shaanxi, Shanxi, Gansu, and Hebei provinces as well as Ningxia and the Inner Mongolian autonomous regions in 1998.Because of the different geogaraphy sources, the variability of BYDVs may exist The main objective of the experiments was to identify the molecular variability, strain type and phylogenetic relationship of different BYDVs CP genes using the materials of Chinese wheat isolates which were infected by virus.Two hundred and seventy-nine wheat samples showing barley yellow diseases were collected from wheat field representative of eighteen regions in China.Reverse transcription polymerase chain reaction (RT-PCR) and nucleic acid spot hybridization (NASH) were made using the RNA of samples to detect the CP gene. The results showed that two hundred and one samples had positive reaction which indicate special fragements and positive spots and BYDV-GAV hold the more large proportion and BYDV-PAV BYDV-GPV showed predominance in specific regions.BYDV-PAV occur in Weinan and Yangling regions mainly and the proportion has risen from 7% in 2004 to 29.41% in 2005 .GPV, existed as particular strain in China, had less infection and appears in Yuncheng,Shanxi Province mostly. We presume the proportion of GAV may drop and that of PAV would have ascending tendency according to the quantity of positive GAV and PAV samples.The product of RT-PCR were cloned and sequenced, the molecular variability of GAV,PAV and GPVwere analysised through the mulriple sequences alignments.Comparison of 26 nucletide sequences of BYDV-GAV revealed high conservation of the CP gene. Phylogenetic tree constructed based on the sequences of GAV CP genes from China with several parallel branches showed that there was no obvious clustering.BYDV-PAV was another case, 179 substitutions in 600 nt implied high variality, especially 3' terminal sequence.The phylogenetic tree constructed based on the sequences of BYDV-PAV CP genes from China formed four branches (A to D).The average similarity within A ,B,C,D group is 98.6%,97.8%,98.8% and 94.9%,respectively,furthermore,the degree of identity among them was 95.5%(A and B),91.5%(A and C),78.4% (A and D) ,90.5%(B and C),78.0% (B and D) ,79.4% (C and D).. A,B and C group were all belong to PAV-CN isolate,even from the same branch,the variability of sequences of three groups is obvious,which resulted in the diversity clusterings. BYDV-GPV comprised 606bp and the degree of identity among sequences is high.The approximately complete nucleotide sequence of genomic RNA of 05GG2, which was differ from other PAV samples was determined. It comprised 5675 nucleotides and contained six open reading frames and four un-translated regions. The size and organization of 05GG2 genome were similar to those
of BYDV PAV-aus. The nucleotide and deduced amino acid sequences of the six ORFs were aligned and compared with those of other BYDV-PAV isolates. The results showed that there was a high degree of identity between 05GG2 and PAV-aus in all ORFs except ORFS and ORF6, which had only 88.7% and 81.7% identities respectively. The reported genomic nucleotide sequence of PAV-aus was longer than that of 05GG2, but the comparison of the genomic nucleotide sequences for PAV-aus and 05GG2 showed 93.6% sequence identity for the same region of the genome. According to the level of sequence similarities, 05GG2 should be closely related to BYDV-PAV-aus.
2004年和2005年共采集BYDVs标样279个,提取总RNA经RT-PCR扩增CP基因以及核酸点杂交检测我国近两年的大麦黄矮病发生情况,标样的70%左右为阳性结果,其中,BYDV-GAV占据了阳性样品的大部分比例,BYDV-PAV、BYDV-GPV分别在特定区域优势地发生,如2005年BYDV-PAV主要集中在陕西渭南和杨陵地区,占当地阳性样品的29.41%,比2004年的7%有明显的增多。BYDV-GPV,作为中国特有的大麦黄矮病毒株系,在田间的检测样品中比较少,多发生在山西运城,2005年陕西渭南、陕西凤翔、甘肃甘谷各检测到一例。
RT-PCR产物经克隆测序,进行多序列比对分析不同病毒(株系)的分子变异情况。测序的26个BYDV-GAV核苷酸序列长度为600bp,序列一致程度高,变异程度非常小。根据序列构建的系统进化树具有许多平行分支,表明BYDV-GAV序列之间没有明显的聚类。BYDV-PAV核苷酸序列的分子变异则与BYDV-GAV结果不同,600个碱基中有179个位点发生变异,尤其表现在3'末端。系统进化树形成四个组(A-D)。A、B、C三组同属于PAV-CN分离物,虽从同一进化分支而来,但其各自序列相互间差异程度较大,进而分别聚类成组。BYDV-GPV CP基因核苷酸序列长度为606bp,测得序列间同源性为98.7%-99.8%,变异很小。
在测得的BYDV-PAV序列中,来自甘肃省甘谷的05GG2标样比较特殊,它与PAV-CN核苷酸差异较大,而与国外PAV分离物相似程度较高,根据已公布的BYDV-PAV分离物全基因组序列设计保守引物,扩增出05GG2除末端的近全长基因组序列,由5675个核苷酸组成,与PAV-CN序列同源性仅为78.5%,与PAV-aus全基因组序列同源性高达93.6%。初步分析了05GG2的开放阅读框架及非编码区,并将其与世界各地PAV分离物进行核苷酸、氨基酸比对,相似性分析表明它与国外分离物具有较高的同源性,而与国内PAV-CN差异较大。系统进化树显示,05GG2与PAV-aus等成簇聚类明显,形成独立分支。
Barley yellow dwarf viruses (BYDVs) are members of the plant virus family Luteoviridae. They were phloem-limited and obligately transmitted in the circulative/persistent manner by several species of cereal aphids and can cause significant economic losses worldwide because of damage to barley, wheat, and oats. In China, 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 Shaanxi and Gansu provinces, the last serious epidemic happened in Shaanxi, Shanxi, Gansu, and Hebei provinces as well as Ningxia and the Inner Mongolian autonomous regions in 1998.Because of the different geogaraphy sources, the variability of BYDVs may exist The main objective of the experiments was to identify the molecular variability, strain type and phylogenetic relationship of different BYDVs CP genes using the materials of Chinese wheat isolates which were infected by virus.Two hundred and seventy-nine wheat samples showing barley yellow diseases were collected from wheat field representative of eighteen regions in China.Reverse transcription polymerase chain reaction (RT-PCR) and nucleic acid spot hybridization (NASH) were made using the RNA of samples to detect the CP gene. The results showed that two hundred and one samples had positive reaction which indicate special fragements and positive spots and BYDV-GAV hold the more large proportion and BYDV-PAV BYDV-GPV showed predominance in specific regions.BYDV-PAV occur in Weinan and Yangling regions mainly and the proportion has risen from 7% in 2004 to 29.41% in 2005 .GPV, existed as particular strain in China, had less infection and appears in Yuncheng,Shanxi Province mostly. We presume the proportion of GAV may drop and that of PAV would have ascending tendency according to the quantity of positive GAV and PAV samples.The product of RT-PCR were cloned and sequenced, the molecular variability of GAV,PAV and GPVwere analysised through the mulriple sequences alignments.Comparison of 26 nucletide sequences of BYDV-GAV revealed high conservation of the CP gene. Phylogenetic tree constructed based on the sequences of GAV CP genes from China with several parallel branches showed that there was no obvious clustering.BYDV-PAV was another case, 179 substitutions in 600 nt implied high variality, especially 3' terminal sequence.The phylogenetic tree constructed based on the sequences of BYDV-PAV CP genes from China formed four branches (A to D).The average similarity within A ,B,C,D group is 98.6%,97.8%,98.8% and 94.9%,respectively,furthermore,the degree of identity among them was 95.5%(A and B),91.5%(A and C),78.4% (A and D) ,90.5%(B and C),78.0% (B and D) ,79.4% (C and D).. A,B and C group were all belong to PAV-CN isolate,even from the same branch,the variability of sequences of three groups is obvious,which resulted in the diversity clusterings. BYDV-GPV comprised 606bp and the degree of identity among sequences is high.The approximately complete nucleotide sequence of genomic RNA of 05GG2, which was differ from other PAV samples was determined. It comprised 5675 nucleotides and contained six open reading frames and four un-translated regions. The size and organization of 05GG2 genome were similar to those
of BYDV PAV-aus. The nucleotide and deduced amino acid sequences of the six ORFs were aligned and compared with those of other BYDV-PAV isolates. The results showed that there was a high degree of identity between 05GG2 and PAV-aus in all ORFs except ORFS and ORF6, which had only 88.7% and 81.7% identities respectively. The reported genomic nucleotide sequence of PAV-aus was longer than that of 05GG2, but the comparison of the genomic nucleotide sequences for PAV-aus and 05GG2 showed 93.6% sequence identity for the same region of the genome. According to the level of sequence similarities, 05GG2 should be closely related to BYDV-PAV-aus.
引文
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