Studies On Critical Genes Of Melanin Signaling Pathway In Agrotis Ipsilon

Agrotis ipsilon is a destructive agricultural pest in China.At present,the control of Agrotis ipsilon is mainly based on chemical pesticides.However,with the high dose and high frequency of the same kind of pesticides,the resistance of Agrotis ipsilon to chemical pesticides is enhanced and the safety of pesticides is becoming more and more serious.The demand of developing specific and environment-friendly pest management strategy becomes urgent.Tyrosine hydroxylase(TH)catalyzes the conversion of tyrosine to dopa(DOPA),the first step in the insect melanin synthesis pathway,and DOPA is catalyzed by dopa decarboxylase(DDC)to form dopamine.Dopamine is a precursor of synthetic melanin and an important neurotransmitter that regulates the synthesis of melanin and various metabolic activities.In this study,the full-length TH cDNA sequence was cloned from Agrotis ipsilon,and the protein sequence of TH and DDC were analyzed by bioinformatics.qRT-PCR detection was used to analyze the TH and DDC mRNA relatively quantitative expression levels of different developmental stages and different tissues of Agrotis ipsilon.In order to study the gene functions of TH and DDC gene,We used CRISPR/Cas9 genome editing technology to knockout AiTH and AiDDC,then genotyping study showed that there were some typical deleted mutations in the targeted site of AiTH and AiDDC gene locus.We observed there were some phenotypic differences between mutants and wild type,meawhile,we can speculate the regulation of the growth and development of AiTH and AiDDC.The results shows below:1.According to constructing the phylogenetic tree of the TH gene and DDC gene,we can find that TH genes in A.ipsilon,M.separata and S.exigua have a closer relationship,DDC genes in A.ipsilon,M.separata and M.brassicae have a closer relationship,they all in clustered into a branch of other lepidopteran insects.2.The analysis of expression profiles showed that:(1)tissue specific expression results showed that TH and DDC genes in the head and epidermis were higher than other tissues,which was consistent with the place where melanin was produced and functioned.(2)TH gene was expressed in almost every period of development,DDC gene changes significantly before and after molting,which is related to the regulation of ecdysone and juvenile hormone.3.The Cas9 mRNA and sgRNA mixture of the specific gene were injected into the embryo.The genomic detection mutation revealed that the TH and DDC gene had different types of mutations at the base of the target in the mutant.4.Compared with the wild type,the specific phenotype of the TH mutant is as follows: Agrotis ipsilon embryo shrunk inside the egg shell and were difficult to hatch in Cas9/sgRNA-injected group.The body size of the mutant larvae individuals was obviously smaller than the control group individuals under the microscope.Furthermore,the hatched larvae were dehydrated and died within the first day.After knocking out the DDC gene in Agrotis ipsilon,the synthesis of melanin in the mutant individuals was significantly weakened.There was almost no melanin deposition in the hair follicles and heads of the larvae,and the immunity in the later life was low and died in the larval stage.Some of larvae appeared mosaic head traits.The mosaic mutant can survive to the adult stage,and the wings,tentacles and foot color of the mutant adult are faint yellow,the body with reduced melanin phenotypes.5.Real-time quantitative PCR(qRT-PCR)assay was used to analyze the differential expression of TH gene and other key genes in the CRISPR/Cas9-mediated AiTH mutant in the melanin signaling pathway.The analysis showed that AiTH transcripts were significantly down-regulated in mutants.The PO gene was significantly up-regulated,and aaNAT,tan,and ebony were significantly down-regulated.This indicates that AiTH plays an important role in the growth and development of Agrotis ipsilon,and the mutation of TH gene affects the regulation of the melanin pathway.This study mainly used the CRISPR/Cas9 genome editing technology to study the gene and the functional verification of the melanin pathway in Agrotis ipsilon,which indicated that TH and DDC genes play an important role in the melanin synthesis and growth of Agrotis ipsilon.According to the solid and convinced phenotypes,we think DDC gene can be used as a Marker gene for studying the genetic transformation of Agrotis ipsilon.The lethality of TH gene provides a potential target gene for insect population genetic regulation and control of pests,which provides a new idea for effectively preventing and controlling the outbreak and harm of Agrotis ipsilon population.