嗜人按蚊溴氰菊酯抗性分子机理研究Ⅱ嗜人按蚊CYP4基因cDNA片段的克隆与序列分析

Studies on molecular mechanism of Anop heles anthropo phagus to deltamethrin Ⅱ. Clon ing and sequence an alysis of CYP4 gene cDNA fragments from Anopheles anthropophagus

目的 获得嗜人按蚊CYP4基因家族成员的c DNA片段。方法 采用简并引物对嗜人按蚊溴氰菊酯敏感和抗性品系总RNA进行RT- PCR扩增,得到的特异性片段采用T/ A克隆法克隆并进行序列测定和分析。结果 敏感品系和抗性品系嗜人按蚊均能扩增得到4 5 0 bp和5 30 bp左右的2个片段。经克隆测序后,得到6个基因序列,其中4个来自敏感品系,2个来自抗性品系。其中1个来自敏感品系的序列和1个来自抗性品系的序列完全相同。所得的5个不同基因序列已被Gen Bank收录(登录号:AY2 0 814 1～AY2 0 814 5 ) ,并递交国际细胞色素P4 5 0命名委员会,经鉴定确认均为细胞色素P4 5 0超家族中的CYP4家族成员,其中4个为新基因,另1个为等位基因,分别属于CYP4家族的CYP4 C、CYP4 H和CYP4 J3个亚家族。结论 得到的5个c DNA新序列为CYP4家族新成员的基因片段。

Objective To obtain cDNA fragme nt of CYP4 gene from Anopheles anthropophagus. Methods The degenerate RT-PCR technique was u s ed to amplify the CYP4 mRNA from susceptible and deltamethrin-resistant strains of An.anthropophagus. The specific cDNA fragmen ts were sequenced and analyzed. Results Two spe cific cDNA fragments (450 bp and 530 bp) of CYP4 gene were amplified from the su sceptible and deltamethrin-resistant strains of An.anthropopha g us, and then were cloned and sequenced. Total 6 sequences were o btained, of which 4 were from susceptible strain and 2 from resistant strain. On e sequence from susceptible strain was the same as another sequence from resista nt strain. Compared with P450 sequence databases from GenBank, all of the 6 obta ined sequences were new P450 sequences. After lodging in GenBank (Accession numb e rs: AY208141-AY208145), it was confirmed that all of them were new CYP4 genes i n cluding one allele, and were classified to the subfamily CYP4C, CYP4H and CYP4J in CYP4 family respectively by Nomenclature Committee of Cytochrome P450. Conclusion The cloned 5 sequences are gene fragments from members of CYP4 family.