PCR-RFLP鉴别微小按蚊亲缘种A和C

ESTABLISHMENT OF PCR-RFLP METHOD BASED ON 28S-D3 REGION OF RIBOSOIBOSOMAL DNA FOR DIFFERENTIATION OF ANOPHELES MINIMUS A AND C

目的 建立微小按蚊复合体不同亲缘种A和C的分子鉴别方法—PCR产物酶切片段长度多态性分析 (PCR RFLP)。 方法 用单蚊蚊腿消化提取基因组DNA ,PCR特异扩增核糖体 2 8S第 3结构域 (D3 )基因 ,对PCR产物进行纯化、克隆和测序 ;分析微小按蚊A和CD3基因的酶切图谱 ,选择合适的限制性内切酶对两者PCR产物进行特异性切割 ,按照酶切片段长度区分两亲缘种。 结果 微小按蚊A被限制性内切酶MboII切割后在约 3 76bp、2 68bp和 10 8bp处出现 3条条带 ,而微小按蚊C因第 96bp、97bp位点突变 ,不存在限制性内切酶MboII的特异识别位点而未被消化 ,仅在 3 76bp处存在唯一条带。 结论 本实验建立的PCR RFLP分子鉴别方法能有效地将形态难以鉴别的微小按蚊亲缘种A和C(GenBank :AF416782 ,AF415 5 94)区分开来。

Objective To establish a molecular method for differentiation of two sibling species, Anopheles minimus A and An. minimus C, of Minimus Complex: polymerase chain reaction-restriction fragment length polymorphism analysis (PCR-RFLP). Methods Genomic DNA was extracted from the individual mosquito’s legs by the GNT-K method and the rDNA-28S-D3 gene was amplified by specific primers and PCR products were purified, cloned, sequenced and analyzed. MboII restriction enzyme was chosen based on the restriction maps of 28S D3 gene of An. minimus A and C to cut PCR products. Results Two different D3 sequences (GenBank: AF416782, AF415594), blasted as An. minimus A and C, were detected in this study. An. minimus A and C can be visually distinguished according to the electrophoresis pattern: the three fragments in 376 bp, 268 bp and 108 bp were observed in the lanes of An. minimus A while only a band in 376 bp for An. minimus C because of mutation in 96 bp, 97 bp. Conclusion Two sibling species, An. minimus A and C, were found in southern China and they can be distinguished by the PCR-RFLP method established in this study.