Function Analysis Of Wing-specific Cuticle Protein Gene LmACP7 In Locusta Migratoria

Wing is an important subsidiary structure of insects,which is a useful tool for their feeding,courtship,dodging,migration and so on.Locusta migratoria is a kind of agricultural pests in the world as a hemimetabolous insect.Their large-scale outbreak could induce great disaster for their powerful forewing and hindwing.So the study about their wings growth,development and metamorphosis can provide basic information for the prevention of locust plague.In this paper,the total amount of cuticle protein genes was identified from L.migratoria transcriptome.LmACP7 is a wing cuticular protein gene for our target gene.we studied its biological functions by RNAi and TEM.The results were as follows:1)According to the L.migratoria transcriptome,We got 81 cuticle protein genes contained 5 cuticular protein famlies,including 51 CPR genes,2 Tweedle genes with a Tweedle motif,9 CPF/CPFL genes with the diagnostic 44 aa consensus or AAP(A/V)motif,9 CPAPs genes,and ten genes annotated cuticle proteins that not belong to any family as described above.2)Sequenes analysis of LmACP7 showed that the gene consisted of three exons and two introns,which encodes a RR-2 motif belonged to RR-2 subfamliy.Phylogenetic analysis showed LmACP7 is an apparent ortholog to cuticle protein 7 of Ostrinia nubilalis,with 79.6% sequence identity.RT-qPCR results showed that LmACP7 was specifically expressed in the wing pads of 5th instar nymphs.The pattern of different stages showed LmACP7 has a high expression level before molting of each instar from 3rd instar nymph to 5th instar nymph,and low expression level in adult,suggested that it is a pre-ecdysial protein gene,and may be participated in the formation of exocuticle.The immunohistochemistry result indicated that the protein was present in the epidermis cells of wing pads in 5th instar nymph,and was transported to newly formed cuticle.Immunogold labeling for TEM showed LmACP7 protein was detected throughout the exocuticle but not in the enocuticle and epicuticle.3)RNA interference was used to investigate the function of LmACP7.Injection of dsLmACP7 into 4th nymph had influenced 5th nymph wing pads,HE staning showed the epidermal cells layout was disorder.Injection of ds LmACP7 into 5th nymph had influenced adult wings cuticle.TEM observation showed epidermal cells layout was disorder,endocuticle was broken.Then we detected whether apoptosis occurs in the formation of wing cuticle during the nymph-adult morph.All results indicated that deficient of LmACP7 promotes apoptosis through Cytochrome C-caspase-9-mediated pathway.L.migratoria,a kind of hemimetabolous insect,has strong wings with great flight ability.The structural characteristics and development of the wings are different from holometabolous insects such Bombyx mori,Drosophila melanogaster and Tribolium castaneum.In general,We explored the expression pattern of wing-specific cuticule protein gene LmACP7 with the growth and development of L.migratoria,and the function of LmACP7 in the synthesis of wing cuticle.Our research can provide basic information for the control of L.migratoria.