嗜人按蚊和中华按蚊的ITS_2区段序列分析和比较

SEQUENCE ANALYSIS OF RIBOSOMAL DNA ITS_2 REGION FOR ANOPHELES ANTHRO-POPHAGVS AND ANOPHELES SINEBSIS

目的 分析和比较不同地区中华按蚊和嗜人按蚊的ITS2区段的基因特征。方法 采用特异性ITS2引物对嗜人按蚊和中华按蚊江苏实验株以及从湖北省和越南现场捕获的嗜人按蚊和中华按蚊进行PCR扩增、克隆并对ITS2区段序列进行分析。结果 嗜人按蚊实验株的ITS2区段序列有452bp,与嗜人按蚊现场株的ITS2区段序列相同,中华按蚊实验株的ITS2区段序列有472bp,与中华按蚊现场株的ITS2区段序列也相同;但嗜人按蚊和中华按蚊的ITS2区段基因序列存在明显差异并存在不同的限制性内切酶位点。结论 可依据中华按蚊和嗜人按蚊的ITS2基因序列内限制性内切酶切位点不同的基因特征,采用PCR-RFLP技术建立中华按蚊和嗜人按蚊基因鉴别技术。

Objective To analyze the genetic characteristics of ribosomal DNA ITS2 region for Anopheles anthropophagus and Anopheles sinensis. Methods The ribosomal DNA ITS2 region for An. anthropophagus and An. sinensis both from laboratory lines and field were amplified by using polymerase chain reaction (PCR). The amplified products were purified and cloned into pPCR-Script Amp Cloning vector, then infected with XL10-Gold Kan ultracompetnt cell. The positive clones were selected for sequencing by using the Dyeterminator sequencing method. The sequencing data were analyzed by using the Omega Sequencing analysis program. Results The An. anthropophagus and An. sinensis both from the laboratory lines and field showed the same sequences. However, there was a significant difference between the sequence of ITS2 region from An. anthropophagus and that from An. sinensis. The sequences from An. anthropophagus and An. sinensis were 452 bp and 472 bp respectively. The restriction mapping showed that there was different restriction digest site between the ribosomal DNA ITS2 region sequence from An. anthropophagus and that from An. sinensis. Conclusion According to the different restriction digest site between the ribosomal DNA ITS2 region sequences from An. anthropophagus and An. sinensis, the PCR-RFLP technique can be established for identification between An. anthropophagus and An. sinensis.