Study On The Eukaryotic Expression Of Bemisia Tabaci CYP6CX2/3 Gene And Its Degradation Characteristics Of Thiamethoxam

Bemisia tabaci(Gennadius)is an insect of Hemiptera and Whiteflies.Bemisia tabaci,as one of the world's most destructive absorbing agricultural pests,has caused serious economic losses in many regions of the world.At present,the more effective comprehensive insect control method is chemical control,neonicotinoid insecticides are widely used because of their unique mechanism of action,low toxicity and high efficiency.As a representative of the second-generation neonicotinoid insecticide,thiamethoxam has been widely used in the field,however,field Bemisia tabaci has developed serious resistance to drugs due to its long-term and large-scale application.In this study,a series of molecular methods were used to study the function,expression and metabolism of CYP6CX2 and CYP6CX3,and explore the resistance mechanism of Bemisia tabaci to thiamethoxam.The main findings are as follows:The software proves that CYP6CX2 and CYP6CX3 are members of the Bemisia tabaci CYP6 family and have a conserved domain unique to cytochrome P450.The full-length CYP6CX2(open reading frame)is 1575 bp,encoding 524 amino acids;the full-length CYP6CX3 gene is 1524 bp,encoding 507 amino acids.These two genes are highly similar in comparison with the transcriptome database,the full-length sequences of the two genes have been obtained by cloning.Quantitative PCR analysis showed that compared with the susceptible population,the CYP6CX2 gene m RNA transcription level was 2.75-fold in the resistant population;the CYP6CX3 gene m RNA transcription level was 4.45 and 3.11 times in the two resistant populations.Both genes are significantly overexpressed,which initially proves that two genes are related to thiamethoxam.CYP6CX2 and CYP6CX3 are basically not expressed in the egg stage and nymph stage,but they are all expressed in the adult body,and follow-up research focuses on the adult.Using feeding method for RNA interference,both genes in Bemisia tabaci can be interfered.After intervention,fed with thiamethoxam,CYP6CX2 has no difference in mortality;CYP6CX3 gene has significantly increased mortality compared with control ds GFP.Through RNA interference experiments,CYP6CX3 was again related to thiamethoxam resistance.The feeding method was used to verify the induction of genes by pesticide.The expression of CYP6CX2 and CYP6CX3 showed an increasing trend by Quantitative Real-time PCR.This experiment proved that two genes can be induced to express by thiamethoxam under this condition.CYP6CX2 and CYP6CX3 were expressed in Drosophila S2 cells,and the expression of genes after gene transfection into cells was detected by Quantitative Real-time PCR.It was found that the m RNA transcription levels of CYP6CX2 and CYP6CX3 were significantly increased,proving that both genes could overexpress under this condition.Cells were recovered and processed after the expression,mass spectrometry was performed to detect the metabolism of thiamethoxam by CYP6CX2 and CYP6CX3 proteins.The retention time of the thiamethoxam compound is about 6.92,and CYP6CX2 and CYP6CX3 have small peaks at 8.96 minutes and 8.75 minutes,respectively.It was found that both time points were thiamethoxam demethylated methyltriazinone derivative(TMX-dm-tri),which is considered to be the final degradation product of thiamethoxam.Through a series of molecular technology research methods,it is proved that there is a strong correlation between CYP6CX2,CYP6CX3 and the insecticide thiamethoxam,and it is detected that two genes may be involved in the metabolism of the insecticide through foreign cell expression,and the metabolite is thiamethoxam demethylated methyltriazinone derivative.More evidence is needed to prove the metabolic mechanism of thiamethoxam in Bemisia tabaci.