Preliminary Study On The Metabolism Of Imidacloprid By Aphis Glycines CYP6CY7 And CYP6CY48

Soybean aphid,Aphis glycines,is a worldwide agricultural pest,which causes huge economic losses and makes the control of A.glycines an urgent problem to be solved.Imidocloprid is highly effective against A.glycines because of its unique characteristics and low toxicity to humans.However,the resistance of insects to imidacloprid will decrease the control effect.Cytochrome P450 is an important detoxification enzyme in insects,which is related to the resistance of insects to insecticides.In this study,the cDNA sequences of 12 novel cytochrome P450 genes,which may be related to the resistance of A.glycines,were obtained by transcriptome sequencing,the sequences were cloned and analyzed and their molecular characteristics were studied.The expression response of A.glycines to imidacloprid was used to verify whether the genes were related to resistance and to screen out the main resistance genes.Then,the function of the main resistance genes was further studied by RNA interference.This study provides a theoretical basis for the study of resistance of A.glycines to insecticides,and a new way to develop new pesticides.The main findings are as follows:?1?In this study,12 cDNA sequences of cytochrome P450 CYP6 family of A.glycines were obtained.They were named CYP6CY7,CYP6CY8,CYP6CY9,CYP6CY12,CYP6CY14,CYP6CY16,CYP6CY18,CYP6CY20,CYP6CY48,CYP6CZ1,CYP6DB1 and CYP6DD1 by the International P450Nomenclature Committee,which has been registered in Gen Bank,the Genbank accession were MN055997,MN056000,MN055999,MN055998,MN056003,MN055995,MN056002,MG829856,MN055996,MN055993,MN056001 and MN055994,respectively,the lengths were1735,1913,2071,1686,2468,1769,1556,2040,1639,1837,2287 and 2108 bp,including the lengths of 1554,1545,1569,1518,1542,1542,1527,1530,1536,1536,1542 and 1539 bp open reading frames,encoding 517,514,522,505,513,513,509,509,511,511,513 and 512 amino acids,respectively.The predicted isoelectric points of amino acids were 8.40,8.60,8.34,8.68,7.56,8.38,8.76,9.10,8.87,8.86,9.11 and 8.27,the molecular weights were 59.20,59.50,59.44,58.21,58.96,59.27,59.38,59.20,59.48,59.37,59.58 and 58.10 k Da,respectively.The amino acid sequences of 12 A.glycines P450 CYP6 included the conserved domain of insect P450.The evolutionary analysis showed that the amino acid sequences of A.glycines P450 CYP6 were first clustered with those of the P450 amino acid sequences of insects,which also belongs to aphididae in Hemiptera.The results were accorded with the classification character of insect cytochrome P450 as well as morphology.?2?The relative expression levels of 12 genes were significantly up-regulated within 48 h after the LC₅₀ concentration of imidacloprid treated with A.glycines at different times.When imidacloprid induced A.glycines for 3,6,12,and 48 h,the relative expression of 4,1,10 and 8 genes increased significantly,respectively.The relative expression of CYP6CY7 and CYP6CY48 genes were the highest,the relative expression of CYP6CY7 gene at 12 h was 3.55-fold higher than that of uninduced,and the relative expression of CYP6CY48 gene at 48 h was 3.36-fold higher than that of uninduced.The results showed that these 12 CYP6 genes were involved in the detoxification of imidacloprid by A.glycines,and the CYP6CY7 and CYP6CY48 genes were the major regulators.?3?RNA interference successfully inhibited the expression of CYP6CY7 and CYP6CY48 genes in A.glycines,and led to the decrease of P450 enzyme activity.When RNA interfered with CYP6CY7 gene for 3,6 and 12 h,the relative expression of CYP6CY7 gene was significantly decreased by 58.49%,73.80%and 69.30%,and the P450 enzyme activity was significantly decreased by 19.38%and 21.96%for 12 and 24 h.When RNA interfered with CYP6CY48 gene for1,2 and 3 h,the relative expression of CYP6CY7 gene was significantly decreased by 33.59%,25.60%and 25.70%,and the P450 enzyme activity was significantly decreased by 39.71%,32.79%and 16.75%for 6,12 and 24 h.?4?After RNA interference,the insecticidal activity of imidacloprid against A.glycines was significantly increased.The mortality of A.glycines treated with imidacloprid was significantly increased by 14.71%at 24 h after RNA interference of CYP6CY7 gene,and by 13.13%at 12 h after RNA interference of CYP6CY48 gene.This indicated that the genes were involved in the detoxification and metabolism of imidacloprid by A.glycines.