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| 淡色库蚊细胞色素P450抗性相关基因克隆与初步鉴定 | |
| 引用本文： | 朱昌亮,李建民,田海生,马磊,李秀兰,吴观陵. 淡色库蚊细胞色素P450抗性相关基因克隆与初步鉴定[J]. 中国寄生虫学与寄生虫病杂志, 2000, 18(5): 263-268 |
| 作者姓名： | 朱昌亮 李建民 田海生 马磊 李秀兰 吴观陵 |
| 作者单位： | 南京医科大学病原生物学与免疫学系,南京,210029 |
| 基金项目： | 国家自然科学基金和江苏省自然科学基金资助项目(39970666和BK97050) |
| 摘 要： | [目的 ]探讨淡色库蚊细胞色素P45 0与溴氰菊酯抗药性的关系。 [方法 ]采用一对昆虫细胞色素P45 0简并引物 ,以反转录 聚合酶链反应从淡色库蚊扩增特异片段 ,T/A直接克隆法筛选阳性克隆 ,对新序列进行cDNA芯片和逆Northern分析。 [结果 ]从淡色库蚊对溴氰菊酯敏感品系和抗性品系获得 112个阳性克隆 ,其中 2 4个阳性克隆测序后显示为细胞色素P45 0新序列 ,由GenBank登录上网 ;经国际细胞色素P45 0命名委员会鉴定分别属CYP4家族CYP4C、CYP4D、CYP4H和CYP4J等 4个亚家族 ;2 4个CYP4cDNA片段中 ,来自敏感品系的 2个片段(NYDS3和NYDS5 )和来自抗性品系的 4个片段 (NYDR6、NYDR9、NYDR15和NYDR17)在两品系间存在差别 ,cDNA芯片信号亮度值均是抗性品系大于敏感品系 ,倍数在 3 .1～ 9.7范围内 ;NYDR17仅与抗性探针杂交 ;逆Northern再鉴定 ,获得了与cDNA芯片一致的结果。 [结论 ]淡色库蚊CYP4与溴氰菊酯抗性相关 ;在淡色库蚊溴氰菊酯抗性机理中 ,可能存在P45 0基因点突变而导致的特异表达。 |
| 关 键 词： | 淡色库蚊 杀虫剂抗性 细胞色素P450 基因克隆 cDNA芯片 逆Northern |
| 文章编号： | 1000-7423(2000)-05-0263-06 |
| 修稿时间： | 2000-03-14 |
CLONING AND IDENTIFICATION OF CYTOCHROME P450 RESISTANCE RELATED GENES IN THE MOSQUITO, CULEX PIPIENS PALLENS |
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| ZHU Chang-liang,LI Jian-min,TIAN Hai-sheng,MA Lei,LI Xiu-lan,WU Guan-ling. CLONING AND IDENTIFICATION OF CYTOCHROME P450 RESISTANCE RELATED GENES IN THE MOSQUITO, CULEX PIPIENS PALLENS[J]. Chinese Journal of Parasitology and Parasitic Diseases, 2000, 18(5): 263-268 | |
| Authors: | ZHU Chang-liang LI Jian-min TIAN Hai-sheng MA Lei LI Xiu-lan WU Guan-ling |
| Affiliation: | Department of Vector Biology and Immunology, Nanjing Medical University, Nanjing 210029. |
| Abstract: | OBJECTIVE: To inquire into the relationship between cytochrome P450 and deltamethrin resistance. METHODS: 24 new cDNA sequences encoding cytochrome P450 were amplified respectively from deltamethrin-susceptible and -resistant strains of Culex pipiens pallens with a pair of degenerate primers according to the conservative amino acid sequences of CYP4 in insects by RT-PCR and the Direct Cloning Method, and then were identified by cDNA chip and reverse Northern. RESULTS: 112 positive clones were obtained, of which 24 were shown to be new sequences encoded for cytochrome P450. They have been lodged in GenBank and were appraised by the Nomenclature Committee of Cytochrome P450, belonging to the subfamily CYP4C, CYP4D, CYP4H and CYP4J in CYP4 family. The hybrid signal values of 6 P450 sequences (NYDS3, NYDS5, NYDR6, NYDR9, NYDR15 and NYDR17) were 3.1-9.7 times higher in the resistance probe than in the susceptible probe, and NYDR17 only reacted with the resistance probe. The result of reverse Northern in NYDR15 was similar to that of cDNA chip. CONCLUSION: CYP4 is related to deltamethrin resistance and the specific expression caused by point mutation of cytochrome P450 gene may exist in deltamethrin-resistant Cx. pipiens pallens. |
| Keywords: | Cx.pipiens pallens insecticide resistance cytochrome P450 gene cloning cDNA chip reverse Northern |
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