The Molecular Mechanism Of The Transcription Factor CncC In The Regulation Of CYP315A1 Mediated Resurgence In Tetranychus Cinnabarinus(Boisduval)

Tetranychus cinnabarinus is an important agricultural pest with a wide range of hosts and serious damage.The control of T.cinnabarinus has been mainly based on the application of chemical pesticides for many years,and the problem of resistance has become increasingly serious.The resurgence and resistance of pest(mites)are common undesirable problems after the use of chemical pesticides.In a long-term study of fenpropathrin-resistant strains of T.cinnabarinus,our research team proved that the enhanced activity of detoxification enzyme cytochrome P450 is an important factor in mediating resistance,and the expression of related cytochrome P450 is regulated by the transcription factor CncC.At the same time,it was also found that this strain had increased fertility,and reproduction-related genes represented by P450(CYP314A1 and CYP315A1)were also overexpressed.Based on this,this study combined with the physiological functions of the transcription factor CncC to conduct a systematic and in-depth study on the expression regulation of a reproduction-related gene TcCYP315A1 of T.cinnabarinus,in order to explain the regulation mechanism of the resurgence T.cinnabarinus.The specific research results are as follows:1.The mRNA expression patterns of TcCncC in different strains of T.cinnabarinus was analyzed by Quantitative Real-time PCR(qPCR).The results showed that:(1)The expression level of TcCncC in YN-Fe R was 2.2 times that of YN-SS.(2)In the growth and development period of T.cinnabarinus,TcCncC has a higher expression level in the egg stage.(3)The expression level of TcCncC under high temperature(34℃)compared with room temperature has a significant difference,which is 1.57 times.2.The leaf disc feeding method was used to conduct RNAi research on transcription CncC,its ligand protein Maf,and CYP315A1.The results showed that when the silencing efficiency of TcCncC was 49%,the expression of TcCYP315A1 was down-regulated by73%,and the expression of TcCYP314A1 was down-regulated by 50%;When the silencing efficiency of TcMaf was 75%,the expression of TcCYP315A1 was downregulated by 59%,and the expression of TcCYP314A1 was down-regulated by 26%.When the silencing efficiency of TcCYP315A1 was 38%,the expression of Tcvg1 was down-regulated by 21%,and the expression of Tcvg2 and Tcvgr was not significantly different from the negative control.Then the leaf disc feeding method and the injection method were used to silence the expression of the TcCYP315A1 gene.The daily egg production of a single adult mite was observed under these two methods.The fecundity of the treatment group was observed under the two interference methods.The curves were lower than those of the control group,indicating that after silencing the expression of the TcCYP315A1 gene,the egg production of T.cinnabarinus decreased and the fertility decreased.3.Combining Tetranychus urticae genome data and T.cinnabarinus transcriptome data to clone and obtain(1)CYP315A1 of T.cinnabarinus.The c DNA of this gene is1590 bp in length,encoding 529 amino acids,and the predicted protein molecular weight is 60.53 KDa,the theoretical isoelectric point is 8.30,named TcCYP315A1;(2)The binding site of transcription factor CncC/Maf was identified on promoter sequences of TcCYP314A1 and TcCYP315A1.Using luciferase reporter gene analysis,the binding of CncC/Maf on the promoters of TcCYP314A1 and TcCYP315A1 genes was studied.The results showed that the expression of TcCYP315A1 was regulated by the transcription factor CncC,while TcCYP314A1 was not regulated.Then,the binding site(CATGATACATAAATT)was further verified.4.In P.citri,the leaf disc feeding method was used to conduct RNAi research on PcCncC.The results showed that when silencing efficiency of PcCncC was 80%,the expression of PcCYP315A1 also decreased by 62.5%.At the same time,the number of eggs laid by a single female was recorded.Compared with the control,the fecundity curve of the treatment group was lower than that of the control.It shows that PcCncC can also affect the expression of PcCYP315A1,which in turn affects its reproductive ability.This regulatory pathway may be widespread in mites.The transcription factor CncC up-regulates the expression of genes related to P450 detoxification and metabolism,and at the same time increases the expression of growth and development-related genes TcCYP315A1,indicating that CncC can regulate both resistance genes and reproductive genes,and is a co-regulator of different P450 genes,When T.cinnabarinus became resistant to certain agents(fenpropathrin),it became resurgence again.