乳酸脱氢酶与蚊虫对溴氰菊酯抗性相关性的研究

目的研究乳酸脱氢酶(LDH)与蚊溴氰菊酯抗性之间的关系。方法应用RT-PCR及cDNA末端快速扩增(rapid amplification of cDNA end,RACE)技术扩增白纹伊蚊LDH全长基因;用实时荧光定量PCR检测敏感和抗性蚊C6/36细胞中LDH基因的表达水平;用乳酸脱氢酶活性检测试剂盒检测敏感和抗性蚊细胞中LDH酶活性。结果 LDH基因全长共1831 bp,其中ORF为996 bp,共编码331个氨基酸。该基因与埃及伊蚊、黑腹果蝇、小家鼠和人的乳酸脱氢酶的同源性分别为89%、72%、66%和65%。实时荧光定量PCR结果表明,LDH基因在抗性蚊细胞中高表达,为敏感蚊细胞的1.87倍(P<0.05)。LDH酶活性检测结果显示,LDH在敏感蚊细胞中的酶活性范围为1～95 U/gprot,均值为40 U/gprot;在抗性蚊细胞中的酶活性范围为20～150 U/gprot,均值为75 U/gprot(n=15,P<0.01)。结论抗性蚊细胞中的LDH表达量和酶活性均高于敏感蚊细胞,提示LDH可能与蚊溴氰菊酯抗性相关。

Correlation of LDH with mosquitoes deltamethrin-resistance

Objective To investigate the correlation between LDH and deltamethrin（DM）-resistance in mosquitoes. Methods Cloning the full-length of LDH from mosquito C6/36 cell line and checking the expression level of LDH between DM-susceptible and resistant mosquito cells using quantitative PCR. Then, assaying the enzyme activi- ties of LDH between DM-susceptible and resistant mosquito cells using lactate dehydrogenase assay kit. Results The full-length of LDH（ 1 831 bp） was cloned from Aedes albopictus using RT-PCR and RACE-PCR. An open reading frame （ORF） of 996 bp was found to encode 331 amino acids. The sequence shared 89% , 72% , 66% and 65% ho- mology with LDH of Aedes aegypti ,Drosophila melanogaster,Mus musculus and Homo sapiens. Quantitative PCR dem- onstrated that the expression level of LDH was 1.87 folds （P 〈 0.05） higher in DM-resistant cells than that in-sus- ceptible cells. The enzyme activities of LDH in DM-susceptible cells was 1 -95 U/gprot,the average level was 40 U/ gprot;the enzyme activities of LDH in DM-resistant cells was 20 -150 U/gprot, the average level was 75 U/gprot（ n = 15 ,P 〈 0.01 ）. Conclusion Both the expression and the enzyme activities of LDH in DM-resistant cell are higher than that in DM-susceptible cell,which indicates that LDH might be associated with DM-resistance in mosqui- toes.