Preliminary Screening Of Three Detoxifying Enzyme Genes And Expression Analysis Of GST Gene In Agrilus Zanthoxylumi

Agrilus zanthoxylumi(Hou)is an important stem borer in Zanthoxylum bungeanum producing area in northern China.Its larvae overwinter in the trunk and gnaw on the phloem mainly.Its occurrence in a large number causes serious decline in the tree vigor,gum flow and rot in the branches,which has seriously affected the economic development of Z.bungeanum industry.At present,the most common control means are chemical pesticides combined with artificial control.However,the replacement and long-term use of pesticides will make pests extremely easy to develop drug resistance,which increases the difficulty of pest control.After the insects eat the plants,the secondary substances in the plants can induce the detoxification enzymes in the insects to carry out metabolic degradation function and escape from the adverse environment.It has been clarified that the detoxification resistance and host adaptability to pesticides are closely related to the proteins related to detoxification metabolism.The three types of detoxification enzymes that play the main roles are cytochrome P450 monooxygenase,glutathione S-transferase,and carboxylesterase.They detoxify in insects in different ways and with many varieties.The clarification of their detoxification metabolism mechanism will provide ideas and basis for the drug resistance monitoring and control of A.zanthoxylumi.In this study,we screened and identified three detoxification enzyme genes with detoxification metabolism function based on the existing transcriptome data of A.zanthoxylumi.The enzyme activities of the three detoxification enzymes in male and female adults of A.zanthoxylumi were determined by systematic bioinformatics analysis,and then the GST gene was cloned and identified based on the transcriptome data,and the expression of GST gene in different gender and different tissue parts of A.zanthoxylumi was determined.The main results achieved are:1.Preliminary screening of three major detoxification enzymes: a total of 13 A.zanthoxylumi GST genes,43 CYP genes and 22 carboxylesterase genes were identified,two families of Delta and Sigma were identified by GST genes,five families of CYP3,CYP4,CYP6,CYP9 and CYP12 were identified by cytochrome P450,four subfamilies of genes were identified by Car Es,12 were α-esterase subfamily,two were acetylcholinesterase subfamily,four were juvenile hormone esterase and four were unclassified families.The results of phylogenetic analysis indicated that the evolutionary relationship of GST gene of A.zanthoxylumi was the closest to that of Agrilus planipennis and Tenebrio molitor.Cytochrome P450 and carboxylesterase-related genes had the highest confidence and the closest evolutionary relationship with A.planipennis.2.Enzyme activity analysis: Enzyme activity analysis: There is a difference between the enzyme activity of male and female worms of A.zanthoxylumi.The ability of catalyzing the binding of 1μmol CDNB and GSH per gram of sample per minute of male worms of Agrilus zanthoxylumi was higher than that of female worms as a whole,and the GST enzyme activity of male worms was better than female.The concentration of cytochrome P450 in the tissues of male A.zanthoxylumi was slightly higher than that of the female,so it was preliminarily speculated that the cytochrome P450 activity of A.zanthoxylumi was higher in the male.The increase rate of female catalytic absorbance value is generally higher,and it was preliminarily speculated that the Car E enzyme activity of the female was stronger than that of the male.3.Cloning and tissue expression analysis of GST genes: Five GST genes with complete open reading frames were finally successfully cloned.Azan GST gene was differentially expressed in different tissues and sexes,and its expression level in each tissue of the male was higher than that of the female as a whole,especially in the head.The expression levels of five genes in the head of the male were higher than those in the head of the female.The expression levels of Azan GST3 and Azan GST5 in the head of the male were about 27 times and 13 times that in the head of the female,respectively.The expression levels in the head,abdomen and wings of the male were relatively high,the distribution value of male abdomen is about 19 times that of female.The part with the highest expression in the female was the wing,followed by the foot and abdomen,and the part with the highest expression in the male was the head,followed by the abdomen and wing.The relevant genes mined from the research results could lay a data basis for the research on the adaptability of A.zanthoxylumi to pesticides,the resistance generation mechanism and the function of host secondary biomass metabolism,and provide new ideas for formulating sustainable green prevention and control strategies of A.zanthoxylumi.