沙田柚黄龙病病原16S rDNA片段的克隆与序列分析  

Cloning and sequencing of Shatian pomelo Huanglongbing pathogen 16S rDNA

作　　者：冯震[1] 单振菊[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,广东广州510642

出　　处：《广西农业生物科学》2006年第2期107-110,124,共5页Journal of Guangxi Agricultural and Biological Science

基　　金：广东省自然科学基金项目(032262);广东省科技攻关项目(2004B20901010)

摘　　要：采集田间表现斑驳症状的沙田柚叶脉,用CTAB法提取总DNA。根据柑橘黄龙病病原16S rDNA的核苷酸序列设计引物P1/P2,进行PCR扩增,获得1条大小为1 167 bp的片段。酶切分析显示,该片段可被切成大小分别约为640 bp和520 bp的2个片段。扩增产物经纯化,与pM D 18-T V ector连接,转化大肠杆菌(E scherich ia coli)DH 5α,筛选克隆重组子。对PCR产物进行测序及序列分析,结果表明,与柑橘黄龙病病原亚洲种16S rDNA的同源性为99%,与非洲种的同源性为97%,与美洲种的同源性为96%。认为,沙田柚的斑驳症状是由黄龙病病原引致的,称之为沙田柚黄龙病。该沙田柚黄龙病病原属于柑橘黄龙病病原亚洲种(L iberobacter as iaticus)中的一个成员。系统进化树分析显示,沙田柚黄龙病病原与中国柑橘黄龙病病原亲缘关系最近,推测是直接来自中国柑橘黄龙病病原。Total DNA was extracted from mottled leaves＇ vein of Shatian pomelo in field for PCR amplification. A pair of primers P1/P2 in reaction was designed according to 16S rDNA of citrus Huanglongbing pathogen Liberibacter. A 1 167 bp band was amplified with the primers and template. Digestion of PCR product with Xba I produced a 640 bp band and a 520 bp band. Cloning and sequencing of PCR product indicated that cloned DNA shared 99%, 97% and 96% homogeneity of sequence with those of L. asiaticus, L. africanus and L. americanus respectively. The results indicated that pathogen of Shatian pomelo Huanglongbing belonged to L. asiaticus. Phylogenetic tree showed Shatian pomelo Huanglongbing pathogen was very relative to pathogen from China. It speculated that Huanglongbing pathogen in Shatian pomelo maybe came from citrus Huanglongbing pathogen in China.

关 键 词：沙田柚 黄龙病 16S RDNA 克隆 序列分析 

分 类 号：S436.66[农业科学—农业昆虫与害虫防治]