柑橘黄龙病菌亚洲种16S rDNA和16S-23S rDNA间隔区的PCR-RFLP及序列分析

为明确柑橘黄龙病菌的遗传多样性,PCR扩增了6个不同地区黄龙病菌的16SrDNA和16S-23S rDNA间隔区(intergenic spacer regions,ISR),分别通过4种内切酶对其进行了限制性片段长度多态性分析,发现不同分离物的16S rDNA和16S-23S rDNA间隔区各酶切产物的电泳谱带完全一致,未表现多态性。对16S-23SrDNA ISR进行了克隆和测序,经DNAman和NCBI Blast比对分析发现,6个分离物16S-23SrDNAISR序列之间不存在碱基差异,与数据库中柑橘黄龙病菌亚洲种(Candidatus Liberibacter asiaticus)同源性高达99.0%～100%。系统发育分析显示所有的Ca.L.asiaticus归为一个类群,而后与Ca.L.africanus、Ca.L.americanus和Ca.L.psyllaurous组成韧皮部杆菌属(Candidatus Liberibacter)一个大的类群。结果表明,同一种柑橘黄龙病菌不同分离物16S rDNA和16S-23S rDNAISR无分子变异或变异较小,不存在遗传多样性。

PCR-RFLP and Sequence Analysis of 16S rDNA and 16S-23S rDNA Intergenic Spacer Regions of Candidatus Liberibacter asiaticus

In order to determine the genetic diversity of Citrus huanglongbing pathogen, 16S rDNA and 16S-23S rDNA intergenic spacer regions of Candidatus Liberibacter asiaticus from 6 different regions were amplified, and the PCR products were analyzed by restriction fragment length polymorphism （RFLP） with 4 restriction enzymes. The electrophoresis bands of digested products of 16S rDNA and 16S-23S rDNA intergenic spacer regions （ISR） were identical and showed no polymorphisms. The 16S-23S rDNA ISR were cloned and sequenced, and the sequences were aligned and analyzed. The results suggested that the 16-23S rDNA ISR of 6 isolates had no base difference between each other, and the homology level was 99.0% to 100% with other Ca. L. asiaticus. Phylogenetic analysis showed that all Ca. L. asiaticus isolates were clustered into one group, and then clustered in larger group with Ca. L. africanus, Ca. L. americanus and Ca. L. psyllaurous. The results showed that there was no or slight molecular variation of 16S rDNA and 16S-23S rDNA IRS between different isolate of the same specie of citrus huanglongbing pathogen.