中华白蛉微卫星DNA序列的分离和多态位点筛选的初步研究

中国寄生虫学与寄生虫病杂志 ›› 2009, Vol. 27 ›› Issue (6): 11-507.

中华白蛉微卫星DNA序列的分离和多态位点筛选的初步研究

张丽,樊勇,马雅军*

第二军医大学病原生物学教研室，上海 200433

收稿日期:1900-01-01 修回日期:1900-01-01 出版日期:2009-12-30 发布日期:2009-12-30

Isolation of Microsatellite DNA and the Polymorphic Locus Screeningfrom Phlebotomus chinensis （Diptera ∶ Psychodidae）

ZHANG Li,FAN Yong,MA Ya-jun*

Department of Pathogen Biology, The Second Military Medical University, Shanghai 200433, China

Received:1900-01-01 Revised:1900-01-01 Online:2009-12-30 Published:2009-12-30

摘要/Abstract

摘要： 目的分离中华白蛉的微卫星DNA序列，并筛选其中具有多态性的位点。方法应用中华白蛉基因组DNA的酶切片段与生物素标记的寡核苷酸探针（AAT）₁₇、（GA）₂₅、（CCT）₁₇和（TG）₁₈杂交，亲合素富集和超滤离心浓缩目的片段，扩增放大后克隆并测序，构建中华白蛉微卫星DNA库。挑选合适的微卫星位点，建立PCR扩增体系。应用中华白蛉现场标本对不同的微卫星DNA进行扩增，聚丙烯酰胺凝胶电泳筛选具有多态性的位点。结果本研究分离中华白蛉微卫星DNA的方法效率高，重组克隆阳性率为78.6%。构建的微卫星DNA库含有118条序列，GenBank登录号为FJ919812～FJ919932（登录号为FJ919833、FJ919836和FJ919869除外），其中典型微卫星DNA序列72条（占61.0%），非典型序列46条。在构建的中华白蛉微卫星DNA库中选择22个位点进行多态性筛选，电泳结果显示14个为多态位点，双核苷酸重复的位点比三核苷酸和多核苷酸重复位点的多态性高。结论首次构建了含有118条序列的中华白蛉微卫星DNA库，共获得14个新的多态微卫星位点。

关键词: 中华白蛉, 微卫星DNA, 多态位点

Abstract: Objective To isolate the microsatellite DNA of Phlebotomus chinensis and screen the polymorphic loci. Methods Genomic DNA fragments were hybridized with biotinylated oligonucleotide probes（AAT）₁₇,（GA）₂₅, （CCT）₁₇，and（TG）₁₈. The hybridized fragments were captured with Vectrex Avidin D and enriched by centrifugal ultrafiltration with ultra-4-column ultrafiltrate. The target fragments were amplified, cloned and sequenced． The suitable microsatellite loci were chosen in Ph. chinensis’s library to establish PCR amplification assay. The polymorphism screening was conducted by PAGE with Ph. chinensis field specimens. Results The enrichment protocol used in this study was efficient, with a percentage of recombinant clone as 78.6%. There were 118 microsatellite sequences in library, the GenBank accession numbers were from FJ919812 to FJ919932（except GenBank accession numbers FJ919833, FJ919836，and FJ919869）. There were 72 typical microsatellite sequences occupying 61.0% and the rest were 46 nontypical microsatellite sequences in the library. Twenty-two loci were chosen to polymorphism screening and PAGE showed that 14 loci were polymorphic. The loci of dinucleotide repeat were more polymorphic than those of trinucleotide and polynucleotide repeat. Conclusion The microsatellite-containing library of Ph. chinensis has been constructed with 118 sequences, and 14 new polymorphic microsatellite loci are reported.

Key words: Phlebotomus chinensis, Microsatellite DNA, Polymorphic locus

张丽;樊勇;马雅军. 中华白蛉微卫星DNA序列的分离和多态位点筛选的初步研究[J]. 中国寄生虫学与寄生虫病杂志, 2009, 27(6): 11-507.

ZHANGLi;FANYong;MAYa-jun*. Isolation of Microsatellite DNA and the Polymorphic Locus Screeningfrom Phlebotomus chinensis （Diptera ∶ Psychodidae）[J]. Chinese Journal of Parasitology and Parasitic Diseases, 2009, 27(6): 11-507.

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