Functional Analysis Of Transcription Factors AHR And CncC In The Antennae Of Locusta Migratoria

Locusta migratoria is a typical representative of the world's agricultural pests.It prefers to eat poaceae,such as wheat,corn,and sorghum.Swarm outbreaks of L.migratoria often cause tremendous damage to agricultural production and lead to significant economic losses.The behaviors of L.migratoria,such as feeding,migration and aggregation,are tightly related to its powerful olfactory system.Many proteins present in the sensilla play important roles in insect olfaction mechanisms,such as odorant-binding proteins,odorant receptors,and odorant-degrading enzymes.The genes encoding these proteins can be regulated by the transcription factors AHR(aryl hydrocarbon receptor)and Cnc C(cap ‘n' collar isoform C),especially odorant-degrading enzymes.AHR is transcription factor belonging to the b HLH-PAS family.It has a basic helix-loop-helix(b HLH)domain and a Per-ARNT-Sim(PAS)domain.It can be activated by endogenous and exdogenous substances and regulates the expression of certain genes.Many evidences in the literature have demonstrated that AHR can regulate the expression of many metabolism-related genes in insects,such as P450,GST,UGT,etc.Therefore,insect AHR has an important role in the xenobiotic metabolism.The transcription factor Cnc C was first studied in Drosophila melanogaster.It is homologous to vertebrate nuclear factor Nrf2(erythroid2-related factor 2)protein and nematode SKN-1 protein.Like AHR,after Cnc C is activated by endogenous or exogenous substances,it enters the nucleus and forms a heterodimer with the Maf protein to regulate the expression of downstream genes.Previous studies have shown that Cnc C regulates the expression of insect detoxifying enzyme genes to adapt to the environment.Therefore,we speculated that the transcription factors AHR and Cnc C play important roles in the antennae of L.migratoria.The following two aspects of research have been carried out.The specific research results are asfollows:1.Molecular characteristics of transcription factors AHR and Cnc C in L.migratoriaThe multiple alignments and phylogenetic trees were generated using the amino acid sequences of AHR and Cnc C and AHR and Cnc C from other species.The results showed that the amino acid sequences of AHR and Cnc C were highly homologous to AHR and Cnc C of other insects.Phylogenetic analysis showed that AHR and Cnc C in L.migratoria first clustered with the insects in Blattaria and then with those in Coleoptera.These results indicate that AHR and Cnc C in L.migratoria are highly conserved and have similar functions to other species.The expressions of AHR and Cnc C were detected by RT-q PCR.The results showed that AHR was highly expressed in the antennae of L.migratoria,suggesting that this gene might play an important role in the antennae of L.migratoria.Cnc C is widely expressed in the antennae,foregut,midgut,hindgut,gastric caecum,fat body,malpighian tube and hemolymph of L.migratoria.The wide distribution of Cnc C indicated that the gene has important physiological functions in L.migratoria.AHR and Cnc C were expressed in the whole developmental stages of antennae in L.migratoria.This further indicated these two genes played crucial roles in the antennae of L.migratoria.2.Functional analysis of transcription factors AHR and Cnc C in the antennae of L.migratoriaThe silencing efficiency of antennae AHR and Cnc C were detected after they were knocked down for 24 h and 48 h by RNAi.The results showed that both genes were significantly silenced.The electroantennogram responses to gramineae volatiles were investigated after AHR silencing.The results showed that the EAG response of the female L.migratoria to 1-Heptanol was decreased while those to ?-pinene,decanal,trans-2-Hexen-1-al,nonanal,1-octen-3-ol and linalool have no significant changes after silencing of AHR.The EAG response of the male locusts to 1-octen-3-ol was reduced while those to the other gramineae volatiles tested had no significant changes.Thebehavioral responses to gramineae volatiles were determined after AHR silencing.There were no significant changes in the behavioral responses of female locusts to decanal,trans-2-Hexen-1-al,1-Heptanol,and 1-octen-3-ol.The attraction of 1-octen-3-ol to the male locusts was decreased while those to decanal,trans-2-Hexen-1-al and 1-Heptanol had no significant changes.The EAG responses of the female L.migratoria to nananal and1-Heptanol were decreased while those to ?-pinene,decanal,trans-2-Hexen-1-al,1-octen-3-ol and linalool have no significant changes after silencing of Cnc C.The EAG responses of the male locusts to1-octen-3-ol,trans-2-Hexen-1-al,nananal and 1-Heptanol were reduced while those to the other gramineae volatiles tested had no significant changes.The behavioral responses to gramineae volatiles were determined after Cnc C silencing.The behavioral responses of the female locusts to nananal and1-Heptanol were declined while those to decanal,trans-2-Hexen-1-al and1-octen-3-ol had no significant changes.The attraction of nananal and trans-2-Hexen-1-al to the male locusts were decreased while those to decanal,1-octen-3-ol and 1-Heptanol had no significant changes.The above results show that the transcription factors AHR and Cnc C play vital roles in the olfactory behavior of antennae to gramineae volatiles in L.migratoria.In this paper,the conserved domains of AHR and Cnc C and their phylogenetic relationships with other species were studied by sequence alignments and phylogenetic analysis.RT-q PCR was used to study the expression of these two transcription factors in different tissues and the developmental stages of antennae in L.migratoria.The function of AHR and Cnc C in the antennae of L.migratoria was studied by RNAi combined with EAG and behavioral responses.These results provide an important theoretical basis for the development of olfactory-based green control technology of L.migratoria.