云南蔓草虫豆丛枝植原体16S rDNA和secY基因序列分析  

Analysis of the Sequences of 16S rDNA and secY of Cajanus scarabaeoides Witches'-broom Phytoplasma in Yuanmou, Yunnan Province

作　　者：王柱华 刘俊男 杨子祥[2] 赵宇 蔡红[1] 陈海如[1] WANG Zhuhua;LIU Junnan;YANG Zixiang;ZHAO Yu;CAI Hong;CHEN Hairu(Key Laboratory of Agro-biodiversity and Pest Management of Education Ministry of China,Yunnan Agricultural University,Kunming 650201,China;Institute of Tropical Eco-agricultural Sciences of Yunnan Academy of Agricultural Sciences,Yuanmou 651300,China)

机构地区：[1]云南农业大学,农业生物多样性与病虫害控制教育部重点实验室,云南昆明650201 [2]云南省农业科学院热区生态农业研究所,云南元谋651300

出　　处：《云南农业大学学报（自然科学版）》2018年第5期826-835,共10页Journal of Yunnan Agricultural University:Natural Science

基　　金：云南省高校科技创新团队支持计划(云科教[2014]22号);国家自然科学基金项目(31060239)。

摘　　要：【目的】为确定在云南元谋地区蔓草虫豆上发生的疑似丛枝病的病原种类。【方法】利用植原体16S rDNA基因及secY基因通用引物对蔓草虫豆感病植株总DNA进行直接PCR和巢式PCR扩增、克隆和测序,分别获得1.8 kb和1.7 kb的16S rDNA、secY基因片段。【结果】16S rDNA片段i PhyClassifier在线虚拟RFLP及系统进化分析表明:蔓草虫豆丛枝植原体属于16SrII-A亚组成员(相似系数为1.00),与候选种Candidatus Phytoplasma australasiae相关。基于secY基因同源性比对及构建的系统进化树,表明该株系与来源于云南、台湾的花生丛枝植原体组各株系的亲缘关系最近,确定了该株系属于16SrII-A亚组成员。该分类结果与基于16S rDNA基因的分析结果一致。【结论】本研究首次采用分子生物技术确定了云南元谋蔓草虫豆丛枝病的病原是植原体,明确了其分类地位,该结果可为该病害的早期诊断、检测提供理论依据。[Purpose]To determine the pathogen of Cajanus scarabaeoides witches’-broom disease samples which were collected in Yuanmou of Yunnan Province.[Method]Near-full-length phytoplasmal 16S rDNA gene sequences and secY gene sequences were amplified via direct-PCR and nested-PCR using phytoplasma universal primer pairs from DNAs extracted from C.scarabaeoides symptomatic plants.The amplicons were cloned and sequenced.DNA fragments about 1.8 kb of 16S rDNA and 1.7 kb of secY were obtained.[Result]Classification of 16S rDNA by using computersimulated restriction fragment length polymorphism(RFLP)and phylogenetic analysis in iPhyClassi-fier indicated that the pathogen of C.scarabaeoides witches’-broom is a member of phytoplasma subgroup A,group 16SrII(similarity coefficient 1.00),which related to Candidatus Phytoplasma australasiae.From the homology comparison and the phylogenetic tree based on secY gene sequences showed that genetic relationship of C.scarabaeoides witches’-broom phytoplasma strain(CaSWBYNym)was closely related with PnWB from Yunnan and Taiwan Province and confirmed that CaSWB-YNym belongs to the member of subgroup A,group 16SrII.The classification result based on secY gene analysis coincided with those of results based on 16SrDNA gene.[Conclusion]To our best knowledge,it is the first report of a 16SrII group phytoplasma associated with C.scarabaeoides witches’-broom disease in Yunnan Province.And it is the first study to determine the phytoplasma pathogen of C.scarabaeoides witches’-broom disease in Yunnan Province and identify taxonomic status using molecular biological techniques.These results will provide a theoretical basis for early diagnosis and detection of C.scarabaeoides witches’-broom disease.

关 键 词：蔓草虫豆丛枝病 植原体 16S rDNA secY基因 序列分析 

分 类 号：S435.4[农业科学—农业昆虫与害虫防治]