基于16S rDNA序列比对分析赣南脐橙上柑橘黄龙病病原的分子特征

Molecular characterization of Citrus Huanglongbing Bacteria in Gannan navel orange based on 16S rDNA sequences alignment

运用PCR技术以柑橘黄龙病病原亚洲种、非洲种和美洲种特异性引物对赣南脐橙上7个代表性柑橘黄龙病样品进行扩增检测．结合限制性内切酶Xba I对赣南脐橙上的黄龙病病原16SrDNA基因RFLP分析和测定的16Sr DNA基因序列同GenBank登录的亚洲种、非洲种和美洲种的相应基因序列进行比对分析。结果表明，在赣南脐橙上检测到黄龙病病原亚洲种．未检测到非洲种和美洲种：赣南脐橙上黄龙病病原16Sr DNA基因Xba I酶切具有亚洲种图谱：16SrDNA基冈序列比对分析表明7个赣南脐橙上的黄龙病病原间同源性为99.％～100％，与亚洲种具有98．4％，99．9％的序列同源性，高于与非洲种／美洲种的序列同源性；与亚洲种问遗传距离小于与非洲种／美洲种间的遗传距离：系统聚类分析表明7个赣南脐橙上的黄龙病病原同亚洲种聚类在同一组群，具有较高的同源性和较近的亲缘关系，而与非洲种和美洲种在系统发育树中分属不同的组群，试验结果表明赣南脐橙上感染的黄龙病病原为亚洲种。

Seven Citrus Huanglongbing （HLB） bacteria of Gannan navel orange in Jiangxi Province were detected by PCR with primers specific to Asian, African and American liberibacters. The Xba I restriction endonucleases were used to digest the HLB 16S rDNA fragment of Gannan navel orange, and the sequences of 16S rDNA were sequenced and compared with those corresponding sequences of Asian, African and American liberibacters from GenBank by BioEidt and MEGA software. The results showed that the HLB pathogen of Gannan navel orange could be detected by the specific primers of Candiatus L. asiaticus. RFLP analysis demonstrated that the digested Xba I profiles of 16S rDNA of the HLB isolates from Gannan navel orange had similar profiles with Ca. L. asiaticus. The 16S rDNA sequences of the 7 HLB isolates have identity ranging from 99.8% to 100%, and 98.4% to 99.9% with Asian liberibacters were higher identity than those with African liberibacters or American liberibacters. The same results were found by analyzing the sequences average nucleotide distance of HLB isolates of Gannan navel orange, Asian, African and American liberibacters. Phylogenetic analysis revealed that the 7 HLB isolates of Gannan navel orange were located in different phylogenetic clusters with African and American liberibacters, and classified into one cluster with Asian liberibacters, and shared higher homology and closer relationships. The results indicated that the HLB isolates of Gannan navel orange were Ca L. asiaticus.