基于rDNA ITS序列的苹果异胫小卷蛾的分子鉴定

Molecular identification of Thaumatotibia leucotreta (Lepidoptera,Tortricidae) based on ITS sequences

本文首次测定了苹果异胫小卷蛾(Thaumatotibia leucotreta)、坚果异胫小卷蛾(Thaumatotibia batrachopa)、苹果蠹蛾(Cydia pomonella)、梨小食心虫(Grapholita molesta)等8种卷蛾的r DNA ITS的序列,以探索苹果异胫小卷蛾的分子鉴定方法。8种卷蛾的ITS1和ITS2区序列变异较大,其中ITS1区所分析307个位点中可变位点达到212个,ITS2区则在分析的203个位点中可变位点达到151个。根据8种卷蛾ITS区序列差异,设计了针对苹果异胫小卷蛾的特异性引物,应用特异性引物对样品进行PCR扩增,扩增产物经凝胶电泳分析,结果表明只有苹果异胫小卷蛾的样品有目的 DNA扩增条带,其余卷蛾无扩增条带。灵敏度试验结果显示,最低检测限量可达0.01 ng/μL。因此,采用本文设计的ITS区特异性引物可以对苹果异胫小卷蛾进行快速分子鉴定。

The complete sequences of r DNA ITS were determined for eight leaf rollers including the false codling moth Thaumatotibia leucotreta,the macadamia nut borer Thaumatotibia batrachopa,codling moth Cydia pomonella and oriental fruit moth Grapholita molesta etc,in order to explore molecular identification of Thaumatotibia leucotreta. The sequences of r DNA ITS1 and ITS2 extracted from 8 leaf roolers differed significantly. There are 212 variable sites in the 307 analysis sites for the ITS1 region and151 variable sites in the 203 analysis sites for the ITS2 region. Species-specific primer of Thaumatotibia leucotreta was designed based on r DNA ITS sequence variation among the eight leaf rollers. The results of PCR amplification showed that only the sample of Thaumatotibia leucotreta has amplification band.The sensitivity test results showed that the limit of detection can reach to 0.01 ng/μL. Therefore,the molecular identification of Thaumatotibia leucotreta using the species-specific primer base on the r DNA ITS region is feasible.