南方5省区柑橘黄龙病病原16S rDNA片段的克隆与序列分析  

Cloning and Sequence Analysis of 16S rDNA of Citrus Huanglongbing Agents Collected from Five Provinces in South China

作　　者：单振菊[1] 冯震[1] 周根[1] 邓晓玲[1] 

机构地区：[1]华南农业大学资源环境学院

出　　处：《华南农业大学学报》2008年第2期25-29,共5页Journal of South China Agricultural University

基　　金：广东省自然科学基金(06025850);广东省科技计划项目(2004B20901010)

摘　　要：采集了广东、广西、贵州、福建、云南5省区的8个柑橘黄龙病样品，利用黄龙病病原的特异引物对其16SrD—NA片段进行PCR扩增，将其扩增产物纯化、回收，并与pMD18-TVector连接，转化大肠杆菌（Esherichia coli）DH5α，筛选含有黄龙病病原16SrDNA片段的阳性克隆，进行序列测定。利用生物信息学软件对所克隆的序列进行同源性分析和聚类分析．结果表明：来自我国南方5省区的柑橘黄龙病病原16SrDNA的序列与亚洲种（L22532）的同源性为96．9％～98．6％，与非洲种（L22533）的同源性为94．5％～97．1％，与美洲种（AY742824）的同源性为92．5％-94．2％．表明我国南方5省区柑橘黄龙病均属于亚洲种，但在亚洲种内部不同地区的黄龙病病原也发生了微小的变异，其中福建厦门和云南个旧的2个菌株变异较大．In the study, eight samples of Citrus Huanglongbing（HLB） trees showing typical mottle symptoms were collected from Provinces of Guangdong, Guangxi, Guizhou, Fujian, and Yunnan in China. The HLB agent specific primer sets, OI1/OI2c was used to amplify DNA from the loci of 16S rDNA. Amplicons were collected, purified, ligated to pMD18-T and transformed into Escherichia coli DH5α. The cloned DNAs were sequenced and compared to all published DNA sequences in GenBank through BLAST （http：//www. ncbi. nlm. nih. gov） for phylogenetic analyses. The single nucleotide polymorphisms （SNPs） were identified by using BioEdit sofeware. The cluster analyses were performed by MEGA software. It were concluded that eight of 16S rDNA sequences from these provinces were phylogentically most closely related to those of the published HLB agents. The similarities between the eight sequences to that of Candidatus Liberibacter asiaticus were 98.6% to 98.9%, to that of Ca. L. africanus were 95.4% to 97.4%, and to that of Ca. L. americanus were 92. 5% to 94. 5%. The resuhs indicated that the HLB agents collected from these provinces in southern China were Ca. L. asiaticus.

关 键 词：柑橘黄龙病 16S RDNA 基因克隆 序列分析 

分 类 号：S432.41[农业科学—植物病理学]