云南花生丛枝植原体16S rRNA和核糖体蛋白基因序列分析

 利用植原体16S rRNA基因及核糖体蛋白基因(ribosomal protein, rp)通用引物对发生在云南元谋的花生丛枝病病株DNA进行PCR扩增,并对扩增片段进行序列测定。扩增获得的云南元谋花生丛枝植原体(PnWB-YNym)16S rDNA、16S-23S rDNA和23S DNA片段总长1 806 bp,rp基因扩增片段长1 171 bp。云南株系与来源于台湾和海南的花生丛枝植原体均有较高同源性。比较16S rDNA片段,发现云南株系在5个位点上与来自台湾或海南的株系存在碱基差异,其中有1个位点的差异是云南元谋株系特异的;再分别比较核糖体蛋白rplV-rpsC 2个基因所编码的氨基酸序列,发现云南株系rpsC编码的第194位氨基酸与台湾和海南的株系存在差异。经16S rDNA片段系统进化及iPhyClassifier在线分析,表明PnWB-YNym在分类上属于16SrII-A亚组成员,与候选种‘Candidatus Phytoplasma australasiae’相关;基于rp基因构建的系统进化树表明,PnWB-YNym与16SrII-A亚组各成员聚为同一亚进化支(iii)。

Sequence analysis of 16S rRNA and ribosomal protein gene of peanut witches’-broom phytoplasma disease in Yunnan Province

Using PCR with phytoplasma universal primer pair for 16S rRNA and ribosomal protein gene (rp) was used to detect phytoplasma associated with peanut witches’-broom disease found in Yuanmou County, Yunnan Province. DNA fragments about 1 806 bp of 16S rDNA, 16S-23S rDNA and 23S rDNA and 1 171 bp of rp gene were amplified from the total DNA of diseased peanut respectively. The phytoplasma was designed yuanmou strain of peanut witches’-broom phytoplasma (PnWB-YNym). Homology analysis showed that PnWB-YNym shared high similarity with PnWB collected in Taiwan and Hainan. Comparison of 16S rDNA sequences found that there were five different sites between PnWB-YNym and the strains from Taiwan and Hainan, and among them, the one site was specific for PnWB-YNym. Compared the amino acid sequences encoded by rplV and rpsC in rp genes, it was found that the amino acid located in the 194^th of rpsC gene of PnWB-YNym was different with the strains from Taiwan and Hainan. Phylogenetic tree and online analysis of iPhyClassifier for 16S rDNA indicated that the phytoplasma PnWB-YNym was belonged to the subgroup 16SrII-A and related to ‘Candidatus Phytoplasma australasiae’. The phylogenetic analysis result of ribosomal protein gene, revealed that the PnWB-YNym was clustered in the same rp subclade(iii) with the members of subgroup 16SrII-A.