ITS-RFLP分析进行昆虫病原线虫斯氏属和异小杆属的分类鉴定

MOLECULAR IDENTIFICATION OF ENTOMOPATHOGENIC NEMATODE SPECIES (STEINERNEMA AND HETERORHABDITIS)BY RFLP ANALYSIS OF PCR AMPLIFIED rDNA INTERNAL TRANSCRIBED SPACER (ITS)REGION

本研究通过PCR扩增和六种限制性内切酶 (AluⅠ、HinfⅠ、MboⅠ、RsaⅠ、HaeⅢ和PvuⅡ )酶切 ,对国内害虫防治上常用的几种昆虫病原线虫 ,包括斯氏属S.car pocapsae、S.feltiae和S.glaseri以及异小杆属H .bacteriophora、H .zealandica、H .indicus和H .megidis等 8个品系rDNA -ITS进行分析 ,建立起可以区分各线虫种的标准RFLP图谱。该方法快速简便、稳定可靠 ,需要的样品量少 ,可以用于新鲜的、或冻存的样品 ,甚至分析单条的线虫。不仅可进行昆虫病原线虫的快速分类鉴定 ,而且进一步可以应用于线虫田间释放的辅助监测 ,实际田间感染率的测定和线虫毒力的比较。

A molecular technique for identification of 7 species of entomopathogenic nematodes (EPN)currently used in insect biocontrol was provided. The rDNA internal transcribed spacer (ITS)regions of S.feltiae, S.glaseri,H.bacteriophora, H.zealandica, H.indicus, H.megidi and two strains of S.carpocapsae were amplified with universal primers from the single worm or the purified genomic DNA. The RFLP analysis was generated with six restriction endonucleases(Alu Ⅰ,Hinf Ⅰ,Mbo Ⅰ,Hae Ⅲand PvuⅡ)following by 2.0% agarose gel electrophoresis.Four enzymes (AluⅠ,HinfⅠ,RsaⅠ and MboⅠ)yielded patterns that could be used for differentiating all 7 species of EPN examined, while Hae Ⅲand PvuⅡ provided little taxonomic information. Two strains of S.carpocapsae (All and A24) gave the same patterns for the six enzymes examined, so do the axenic and monoxenic H.bacteriophora. This study showed for the first time that the presence of symbiotic bacteria had no effect on the PCR-RFLP pattern of ITS of nematodes. It also demonstrated that ITS was an ideal marker for species identification and PCR-RFLP technique provided an accurate and reliable molecular means for the taxonomy of EPN. With this technique, unambiguous and rapid identification of EPN could be achieved even using single infective juvenile. Therefore, the technique provided a simpler and more rapid means for monitoring field release of EPN and assessing biologic virulence of nematodes than the bioassay test used previously.