云南小驳骨丛枝植原体的分子鉴定及相关基因序列分析

Molecular Identification and Related Gene Sequence Analysis of Phytoplasma of Gendarussa vulgaris Witches’-broom in Yunnan Province

【目的】确定采自元谋地区自然表现丛枝病的小驳骨(Gendarussa vulgaris Nees)植株是否感染植原体。【方法】通过利用植原体16S组和亚组通用或半通用特异性引物分别对植原体16S rRNA、rp和secY基因序列进行PCR扩增、克隆及测序分析;此外还对secY蛋白的蛋白特性及其结构进行了分析和预测。【结果】本研究获得了基因片段长度分别为1 248 bp的16S rRNA基因(nested PCR)、1 171 bp的rp基因和1 425 bp的secY基因。基于rp和secY基因核苷酸序列的同源性比对及构建的进化树推断的植原体遗传分化关系几乎与16S rRNA基因的推断相一致,均与植原体16S rⅡ-A亚组各株系的遗传进化关系最为接近,但rp和secY基因序列比16S rRNA基因序列能够呈现出更大的遗传变异程度;此外,对secY蛋白进行了生物信息学分析和初步探讨,发现它具有10个明显且分布相对均匀的跨膜螺旋区域,无信号肽。【结论】小驳骨丛枝病(Gendarussa vulgaris witches’-broom phytoplasma,GvWB-YNym)是由植原体侵染而发生,该植原体株系被划分到16S rⅡ-A亚组,相关的候选种为Candidatus Phytoplasma aurantifolia;secY蛋白生物信息参数的分析表明:secY蛋白在感病小驳骨植株中以疏水性稳定跨膜蛋白的形式存在,含10个明显的疏水跨膜区域,该蛋白不存在信号肽。

[Purpose] To determine whether the pathogen of Gendarusssa vulgaris Nees collected from the Yuanmou area infection with witches’-broom disease was phytoplasma. [Method] The 16S rRNA, rp and secY gene were amplification, cloning and sequencing analysis by using universal or semi-universal specific primers of phytoplasmas 16S groups and subgroups. In addition, we also analyzed and predicted the protein characteristics and structure of secY protein. [Results] In this study, we obtained the 16S rRNA gene(by nested PCR) with gene fragments of 1 248 bp, the rp gene of 1 171 bp, and the secY gene of 1 425 bp. The rp and secY gene-based phylogenetic trees and nucleotide sequences homology alignment were congruent with that inferred from the 16S rRNA gene.They were closely related to the genetic evolutionary relationship of 16S rⅡ-A subgroup phytoplasma strains. But the rp gene and secY gene sequences could exhibit greater variables of genetic variation than the 16S rRNA gene sequence. In addition, bioinformatics analysis and preliminary discussion on secY protein were carried out. We found that it had 10 obvious and relatively uniformly distributed transmembrane spiral regions without signal peptide. [Conclusion] Gendarussa vulgaris witches’-broom phytoplasma disease occurs due to infection by phytoplasma pathogens. This phytoplasma strain was divided into 16S rⅡ-A subgroup, and its related candidate species was Candidatus phytoplasma aurantifolia. By analyzing the biological information of the secY protein, it was found that exists in the form of a hydrophobic stable transmembrane protein in the susceptible Gendarussa vulgaris plant. It contains 10 obvious hydrophobic transmembrane regions, and it has no signal peptide.