Identification Of Transcript Factor Acj6 In Helicoverpa Armigera And Its Role In The Regulation Of Olfactory Development In Male Moths

Helicoverpa armigera is an agricultural pest worldwide.Olfactory sense plays an important role in feeding,mating and oviposition of H.armigera.The olfactory system of H.armigera includes peripheral sensory system and central nervous system.Odor molecules enter hemolymph through peripheral receptors,bind with odor binding proteins,transport to odor receptors and interact with each other,so as to activate olfactory receptor neurons,generate membrane potential,and realize the process of chemical signals into electrical signals.Olfactory receptor neurons transmit electrical signals into the central nervous system through axons,and insects respond to the integration of odor information.Transcription factors play an important role in the expression of olfactory related proteins in insects and the targeting regulation of nerve axons.Therefore,the study of olfactory related transcription factors is helpful to elucidate the olfactory mechanism of insects.In this study,the head transcriptome data of H.armigera were analyzed,transcription factors related to olfactory regulation were mined,and a POU family transcription factor abnormal Chemosensory Jump 6(acj6)was obtained.The bioinformatics and expression pattern of this transcription factor was analyzed.In addition,CRISPR/Cas9 mediated the knockout of acj6 in H.armigera to explore its biological function in H.armigera.The specific research results are as follows:(1)Screening transcription factors related to olfaction of H.armigera: RNA-seq was used to analyze the transcriptome data of H.armigera’s head,and compared with the transcription factor database.A total of 313 transcription factors from 54 families were obtained from the transcriptome data,and 18 transcription factors that may be involved in olfactory neural development and olfactory related protein expression in cotton bollworm were identified.A transcription factor,acj6,was selected for followup study.(2)Biological analysis of Haacj6: It was found by sequence cloning that Haacj6 contained five exon regions and two alternative spliceosomes.The sequence length of Haacj6 transcript variant A is 1071 bases,encoding 357 amino acids.The sequence length of Haacj6 transcript variant B is 1048 bases,encoding 349 amino acids.Both Haacj6 isoform A and Haacj6 isoform B are hydrophilic unstable proteins belonging to the Homeodomain POU family,which contains three domains,POU IV box,POU specific domain and POU Homeodomain.According to the amino acid homology analysis and phylogenetic tree construction results,the homology of acj6 in lepidoptera is high,up to 95%.The expression patterns of Haacj6 were analyzed by semiquantitative PCR and fluorescence quantitative PCR.The results showed that Haacj6 was expressed in larvae,pupae and adults of H.armigera.In adults,Haacj6 expression was highest in antennae and lower lip,followed by head and thorax.(3)CRISPR-mediated Haacj6 gene knockout: Two sg RNA sites were designed on exon 1 of Haacj6 gene with a distance of 29 bp,and the eggs of Cotton bollworm were injected with sg RNA and Cas9 proteins by microinjection.By comparing the knockout rate of single target and double target,it is found that the knockout rate of double target is 20%-40%higher than that of single target.Five homozygous males were obtained by double-target knockout.Antennae and antennae lobe morphology of these five mutants were observed.The antennae were deformed to different degrees,and the top of the whip was scorched and enlarged.The morphology of olfactory glomerulus in the antennae lobe of the mutant was diffused compared with that of the wild type,and the volume of Dorsomedial glomerulus in the enlarged Macroglomerular complex was significantly reduced compared with that of the wild type.(4)Transcriptome difference analysis of Haacj6 mutant: RNA-Seq technology was used to obtain transcriptome data of Haacj6 mutant head and compare with wildtype data to screen out genes with significant differences after Haacj6 knockout.After CRISPR knockout,a total of 41 genes were upregulated and 105 genes were downregulated.Through annotation in GO database,13 genes related to neural development and 5 genes related to chemical information perception were discovered.These results indicate that Haacj6 plays an indispensable role in the formation of antennae receptors and olfactory glomerulus in antennae lobe,as well as the expression of olfactory related proteins,which lays a foundation for further study on the olfactory regulation mechanism of H.armigera.