secY和nusA基因在泡桐丛枝植原体亚组分类中的应用

Application of secY gene and nusA gene to classification of paulownia witches' broom associated phytoplasma

【目的】利用分子生物学方法分析泡桐丛枝植原体转运蛋白secY基因及转录延伸因子nusA基因,从亚组水平上确定陕西泡桐丛枝植原体的分类地位。【方法】采集陕西杨凌、彬县、旬邑、永寿、周至和宝鸡等6个泡桐栽培区的泡桐丛枝样品,提取病样总核酸;设计引物对secY-F/R和nusA-F/R,对泡桐丛枝植原体secY、nusA基因进行PCR扩增及核苷酸序列测定与系统发育分析。【结果】获得了泡桐丛枝病植原体secY基因序列的全长,大小为1 358bp,编码414个氨基酸;获得了部分nusA基因序列,大小为946bp,编码315个氨基酸。序列同源性分析结果表明,陕西各县区泡桐丛枝植原体的secY、nusA基因序列均一致。一致性分析和系统发育分析表明,陕西泡桐丛枝植原体与台湾泡桐丛枝植原体的亲缘关系最近,secY、nusA基因序列的同源性分别为99.9%和99.6%。【结论】首次报道了泡桐丛枝植原体转运蛋白secY基因及转录延伸因子nusA基因的序列,并以其作为分类标准,将陕西泡桐丛枝植原体归属到16SrⅠ-D亚组。

【Objective】 The research was conducted to divide the paulownia witches＇broom phytoplasma-Shaanxi（PaWB-Shaanxi） into subgroups based on the phytoplasma secY gene and nusA gene.【Method】 The paulownia withces＇broom samples were collected in Yangling,Binxian,Xunyi,Yongshou,Zhouzhi and Baoji,Shaanxi Province.Total nucleic acid of each sample was extracted and used as template to amplify the secY gene and nusA gene by PCR with designed primer pairs secY-F/R and nusA-F/R.The PCR products were sequenced and analyzed.【Result】 Complete secY gene of PaWB-Shaanxi was 1 358 bp,and contained protein of 414 amino acids while the partial nusA was 946 bp with 315 amino acids.The identity analyses and phylogenetic analyses revealed that PaWB-Shaanxi was most closely related to PaWB-Taiwan,and the similarity of secY gene and nusA gene between them was 99.9% and 99.6%,respectively.【Conclusion】 This was the first study of secY gene and nusA gene of paulownia witches＇broom phytoplasma in China.Based on the two genes PaWB-Shaanxi was further classified into subgroup 16SrⅠ-D.