细梢小卷蛾微卫星富集文库的构建与分析

Construction and Analysis of Enriched Microsatellite Library of Rhyacionia leptotubula

目的:构建细梢小卷蛾Rhyacionia leptotubula微卫星富集文库。方法:提取细梢小卷蛾基因组DNA,经限制性内切酶RsaⅠ酶切,用(CT)10和(GT)10生物素探针与其杂交,利用磁珠富集含有微卫星的DNA序列,并对其进行PCR扩增,将扩增产物连接到pMD18-T载体后转入感受态大肠杆菌DH5α中,得到微卫星富集文库。结果:对100个克隆进行随机测序,获得98个微卫星序列。其中具有5次及以上碱基重复次数的微卫星克隆占26%,最高碱基重复次数为33次,非完美型占12%,说明构建的细梢小卷蛾微卫星富集文库是一个高质量的文库。结论:该文库的建立为后续筛选具高多态性的微卫星标记引物研究细梢小卷蛾的种群遗传结构、迁移扩散规律等奠定了基础。

Objective：To construct an enriched microsatellite library of Rhyacionia leptotubula.Method： Genomic DNA of R.leptotubula was extracted and digested with RsaⅠ.It was hybridized with biotin labeled probe（CT）10 and（GT）10.The hybridized products were enriched with magnetic beads.They were amplified by PCR,and the PCR products were cloned into pMD18-T vector and transferred into Escherichia coli DH5α.Finally,an enriched microsatellite library was constructed.Result： 100 clones were randomly sequenced,and 98 microsatellites were obtained.Among them,the microsatellite clones with repeat motif repeated 5 times or more were accounted for 26%,some of the repeat motifs were with highest repeated times of 33,and 12% of them were imperfect microsatellite,indicating that the constructed library was with high quality.Conclusion： This study was beneficial for isolating polymorphic microsatellites to investigate the population genetic structure,migration,and spreading of R.leptotubula.