新疆柳树花变叶植原体分子鉴定

Molecular Identification of Phy toplasma A ssociated with Willow Phyllody in Xinjiang

为研究新疆7个地区柳树花变叶病为何种病原物所造成,本研究以分子生物学技术对其进行鉴定。通过CTAB法提取柳树总DNA,对其16S rRNA基因和tuf基因进行PCR扩增,将测序得到的基因片段通过Blast比对、同源性比较,构建系统进化树和16S rRNA虚拟RFLP分析。结果显示,PCR扩增分别获得16S rRNA(1238 bp)和tuf(824 bp)的片段,证明该病害与植原体有关,将其命名为柳树花变叶植原体(WPP);16S rRNA序列分析表明,WPP与中国桃黄化植原体(‘Prunus persica’yellows phytoplasma)、枣疯病('Ziziphus jujuba’witches'-broom phytoplasma)、杏褪绿卷叶植原体(Apricot leaf roll phytoplasma)同源性均为99.68%;16S rRNA虚拟RFLP分析结果表明,WPP与枣疯病株系(GenBank登录号:AB052876)有99.8%的相似性,虚拟RFLP模式与16SrV-B亚组的参考模式相同,相似系数为1.00,进一-步证明WPP为16SrV-B亚组成员。WPP tuf基因与榆树黄化组(EY)同源性达99%以上,系统进化树显示,柳树花变叶植原体与榆树黄化组(16SrV)植原体聚为--枝。柳树花变叶植原体属于16SrV.B.tuf-B亚组。

In order to study the pathogenic agents of willow phyllody disease from 7 regions of Xinjiang, molecular biology techniques were used to identify the pathogens. The total DNA of the willow tree was extracted by the CTAB method, and the 16 S rRNA gene and tuf gene were amplified by PCR,and the sequenced gene fragments were compared by Blast comparison and homology comparison to construct a phylogenetic tree, and analyze th virtual RFLP of 16 S rRNA.The results showed that the fragments of 16 S rRNA(1 238 bp) and tuf(824 bp) were obtained by PCR amplification, which proved that the disease was related to the phytoplasma, and was named as willow flower phytoplasma(WPP). The 16 S rRNA sequence analysis showed that the WPP was 99.68% homologous to ‘Prunus persica’ yellows phytoplasma, ‘Ziziphus jujuba’ witches’-broom phytoplasma and apricot leaf roll phytoplasma homology.The results of the 16 S rRNA virtual RFLP analysis showed that the WPP had 99.8% similarity with the JWB strain(GenBank accession number: AB052876). The virtual RFLP model was the same as the reference model of the 16 SrV-B subgroup, and the similarity coefficient was 1.00. It further proved that WPP was a member of the 16 SrV-B subgroup. The WPP tuf gene was more than 99% homologous to the Elm yellows(EY) group. The phylogenetic tree showed that the WPP and the EY group(16 SrV) phytoplasma cluster together, the willow phyllody phytoplasma belonged to the 16 SrV-B and tuf-B subgroups.