PCR-RFLP技术用于鉴别赫坎按蚊复合体近缘种按蚊的研究

IDENTIFICATION OF DIFFERENT ANOPHELINE MOSQUITOES WITHIN ANOPHELES HYRCANUS COMPLEX BY PCR-RFLP

目的 区别赫坎按蚊种团内近缘种。方法 应用聚合酶链式反应连接的限制性片段长度多态性方法 (PCR- RFL P)技术 ,对辽宁省现场捕获的按蚊用特异性 ITS2 引物进行 PCR扩增 ,限制性内切酶 Rsa 和 Hinf 消化 ,琼脂糖凝胶电泳分析。结果 中华按蚊的 PCR扩增产物能被限制性内切酶 Rsa 酶切成 35 0 bp和 2 0 0 bp两条酶切 DNA条带 ;嗜人按蚊核糖体 DNA (r DNA)的 PCR扩增产物能被限制性内切酶 Hinf 酶切成 4 10 bp的酶切 DNA条带 ;雷氏按蚊的 ITS2 基因 PCR扩增产物能分别被限制性内切酶 Rsa 和 Hinf 酶切 ,分别显示 35 0 bp和 4 0 0 bp的酶切 DNA条带 ;八代按蚊的 PCR扩增产物没有显示明显的限制性内切酶 Rsa 或 Hinf 酶切条带。结论 依据r DNA的 ITS2 区段基因特征建立的 PCR- RFL P技术可用于鉴别赫坎按蚊种团的中华按蚊、嗜人按蚊、雷氏按蚊和八代按蚊

Objective To identify the dif fe rent anopheline mosquitoes within Anopheles hyrcanus[WT5”B Z] complex.Methods Field collected anopheline mosquitoes from Liaoning Province were amplified by using the specific ribosoma l DNA ITS 2, digested with restriction enzyme Rsa I and Hinf I and analyzed on an agarose gel. Results PCR products of Anophe les sinensis were only digested with restriction enzyme Rsa I and shown two banes of 350 bp and 200 bp;PCR prod ucts of Anopheles anthropophagus were only diges ted with restriction enzyme Hinf I and shown a 4 10 bp banes; PCR products of Anopheles lesteri w ere digested with both restriction enzyme Rsa I and Hinf I and s hown a 350 bp band and a 400 bp band respectively. PCR products of Anopheles yatsushiroensiswere not digested with restriction enzyme Rsa I or Hinf I.[W T5”HZ] Conclus ion The established PCR-RFLP technique based on the genetic ch a racteristics of ribosomal DNA ITS 2 region sequence can be used for genetic ide ntification of the four anopheline mosquitoes, Anopheles sinensi s, Anopheles anthropophagus, Anopheles lesteri and Anopheles yatsushiroensis within of Anopheles hyrcanuscomplex. [