基于16S rDNA序列的Wolbachia的检测及分型

Detection and type determination of Wolbachia based on 16S rDNA sequences

Wolbachia是广泛分布于节肢动物体内的一类共生细菌。采用16S rDNA特异片段的PCR-RFLP方法对烟粉虱Bemisia tabaci(Gennadius)不同生物型及米蛾Corcyra cephalonica(Stainton)共生菌Wolbachia进行了检测与分型分析。基于wsp基因对烟粉虱共生菌B组Wolbachia以及米蛾共生菌Wolbachia进行了系统树分析,并对相应的Wolbachia16S rDNA特异片段进行了克隆、测序以及序列比对。结果表明:16S rDNA的特异片段经NheⅠ酶切后RFLP图谱可有效检测与鉴别Wolbachia。烟粉虱共生菌Wolbachia的16S rDNA特异片段经VspⅠ酶切后可得到预期RFLP图谱,而米蛾共生菌B组Wolbachia(基于wsp序列分析为B组)则产生不同的RFLP图谱。序列分析表明,Nauru型烟粉虱体内B组Wolbachia的16S rDNA片段序列与已知B组Wolbachia对应序列(DQ278884)同源性为100%;米蛾体内B组Wolbachia16S rDNA特异片段有碱基变异,并存在于VspⅠ识别位点内,这是导致VspⅠ酶切后RFLP图谱不同的原因。结果提示,B组Wolbachia16S rDNA特异片段经VspⅠ酶切的RFLP图谱存在多态性。本研究结果可为今后Wolbachia的检测与分型提供借鉴。

Wolbachia are intracellular bacteria widely iniecting the arthropod species. In tins study, the 16S rDNA specific fragment for Wolbachia was digested with endonucleases Nhe I and Vsp I, respectively, to detect and determine the type of Wolbachia within different biotypes of Bemisia tabaci （Gennadius） and Corcyra cephalonica （Stainton）. The phylogenetic analysis of the B-type Wolbachia within B. tabaci and that within C. cephalonica was carried out based on the wsp gene. The 16S rDNA fragments of the corresponding Wolbachia were cloned, sequenced and analyzed. The results showed that the 16S rDNA fragments digested with Nhe I can be used to detect Wolbachia. The RFLP patterns of 16S rDNA digested with Vsp I of Wolbachia within B. tabaci were consistent with the expected, while within C. cephalonica not. The sequence analysis showed that genetic variation occurred within 16S rDNA fragment for B-group Wolbachia within C. cephalonica, which resulted in a different RFLP pattern from that of B. tabaci biotype Nauru. The results suggest that the RFLP pattern with Vsp I for B group Wolbachia is polymorphic. The procedure developed here may facilitate the future research on the Wolbachia detection and type determination.