Cloning And Functional Analysis Of CncC And Keapl Genes In Helicoverpa Armigera

Helicoverpa armigera(Hübner)is an important agricultural pest in the world,with a variety of host plants.In respond to plant defenses,it develops a series of sophisticated counter-defense strategies or mechanisms(detoxification enzyme system).Moreover,due to the extensive application of chemical insecticides,insecticide resistance has developed rapidly in field populations of H.armigera.Keap1/Cnc C-ARE signaling pathway plays important roles in cell antioxidant and xenobiotic detoxification.Therefore,it is of great significance for us to elucidate mechanism of insecticide detoxification and insights into the evolution of insects to study the genes related to the pathway.In this paper,the effects of plant allelochemicals(flavone and xanthotoxin)on the growth and development of H.armigera was investigated,and the expression of detoxification enzyme genes were also studied after treatment with plant allelochemicals.The Cnc C and Keap1 genes of H.armigera were cloned,and their functions were analyzed.The main results are as follows:1.Effects of plant allelochemicals on the growth and development of H.armigera larvaeAfter treatment with 1 mg/g flavone,the larval body weight was not changed significantly at 48 h,72 h and 96 h after feeding.Compared with the control group,the larval body weight was significantly decreased at 48 h,72 h and 96 h after treatment with 3mg/g flavone.After treatment with different concentrations of xanthotoxin,the larval body weight decreased significantly at all treatment compared with the control group.Plant allelochemical-treated(1 mg/g)larvae exhibited faster weight increase than plant allelochemical-treated(3 mg/g)larvae.The abnormal rate was significantly increased after treatment with flavone and xanthotoxin and were higher with the increase of treatment concentration.These results indicate that flavone and xanthotoxin have a certain inhibitory effect on the growth of H.armigera,and cotton bollworms can tolerate a relatively low concentrations of flavone and xanthotoxin.2.Effects of plant allelochemicals on the expression of detoxification enzymes in the larvae of H.armigeraAfter treatment with flavone,the CYP321A1,CYP6AE14,CYP6B7 and Car E1 genes were significantly up-regulated in the midgut of H.armigera,and the expression of GST1 and Car E1 genes was increased in the fat body,but the difference was not significant.After treatment with xanthotoxin,the CYP6AE12,CYP6AE14 and CYP6B7 genes were significantly up-regulated in the midgut,and the CYP6AE12 and CYP6B7 genes were significantly up-regulated in the fat body.The results showed that these genes may be involved in the detoxification of flavone and xanthotoxin.3.Cloning of Cnc C and Keap1 gene and expression analysisThe full length of Ha Cnc C was 3265 bp,the open reading frame(ORF)was 2196 bp that encodes a protein of 731 amino acid residues with a predicted molecular weight of 82.2k Da and a theoretical isoelectric point of 5.75.The full-length of Ha Keap1-X1 and Ha Keap1-X2 are 3053 bp and 3028 bp,respectively.The ORF is 1935 bp and 1989 bp,encoding 645 and 663 amino acid residues.The molecular weight of Ha Keap1-X1 is 71.6k Da,and isoelectric point is 5.9.Ha Keap1-X2 protein has a molecular weight of 73.5 k Da and an isoelectric point of 5.83.Phylogenetic analysis and multiple sequence alignment showed that both Ha Cnc C and Ha Keap1 genes have high homology with Lepidoptera.The Ha Cnc C gene has the highest expression in the midgut,and a higher expression level in the head and foregut.The Ha Keap1 gene has the highest expression in the midgut and lower expression in other tissues.The expression of Ha Cnc C was significantly higher in the first instar larvae stage,and the expression level decreases with age.The expression level of Ha Keap1 gene was the highest in eggs,and the expression level decreased gradually with age.After treatment with flavone,the transcription level of Ha Cnc C gene was significantly increased in the midgut of H.armigera.After treatment with xanthotoxin,the transcription level of Ha Cnc C gene was significantly increased in the midgut and fat body of H.armigera.After treatment with flavone and xanthotoxin,the transcription level of Ha Keap1 gene was not significantly changed in the midgut of H.armigera,however,the expression level of Ha Keap1 gene was significantly decreasedin fat body.4.Effects of Ha Cnc C and Ha Keap1 RNAi on plant allelochemicals-induced detoxification genes expression in H.armigeraThe Keap1 knockdown increased flavone induction of CYP321A1,CYP6AE14,CYP6B7 and Car E1 genes,however,Cnc C knockdown blocked flavone induction of these four genes.After the knockdown of Cnc C and Keap1,xanthotoxin induction of CYP321A1,CYP6AE12 and CYP6AE14 was similar to results that after treatment with flavone.The results showed that Cnc C/keap1 regulates the expression of flavone-induced CYP321A1,CYP6AE14,CYP6B7 and Car E1 genes in H.armigera.Cnc C/keap1 regulates the expression of xanthotoxin-induced CYP321A1,CYP6AE12 and CYP6AE14 genes in H.armigera.