Expression Pattern And Functional Study Of Epidermal Construction-related Gene CP12,CP23 And BLOS2 In Antheraea Pernyi

The insect cuticle is a highly ordered layered structure formed by the secretions of insect epidermis cells,which it is mainly composed of cuticular proteins(CP)and chitin.The main factors affecting cuticular structure and mechanical properties are the types and number of CP genes.Among CP genes,the CPR family is the most abundant in all types of insects,and has R&R conservative sequence with the ability to bind to chitin,which is divided into three subfamilies,including RR-1,RR-2 and RR-3,are related to the formation of insect cuticle.On the other hand,the translucent insect integument is related to the accumulation of urate granules in insect epidermal cells,which can reduce the stimulation of insects by photooxidation.The urate granules transport-related BLOS2 gene plays a very important role in this process.Studies have found that BLOS2 mutations can make insect integument translucency,but none translucency integument larval mutant has been found due to urate granules accumulate or transport in A.pernyi.To explore the expression pattern and its role of CP and BLOS2 genes in the construction of the cuticle of Antheraea pernyi,the molecular characteristics and expression patterns of three genes were studied using molecular biology techniques.The RNAi technology was used to explore the function of genes and lay a foundation for the construction of epidermis of A.pernyi.The results are as follows.1.In this study,two CP genes with RR-1 consensus in A.pernyi were cloned and named ApCP12(Genebank accession number: MF318874)and ApCP23(Genebank accession number: MF318875),respectively.Bioinformatics analysis indicated that ApCP12 is 690 bp in length with an open reading frame(ORF)of 336 bp,encoding a protein of 111 amino acid residues with a calculated molecular weight of 12 kD,while ApCP23 is 1 243 bp in length with an ORF of 594 bp,encoding a protein of 197 amino acid residues with a calculated molecular weight of 23 kD.Both proteins contain RR-1 consensus and are classified in different clusters with RR-2 cuticular protein by phylogenetic analysis.Tissue-specific mRNA expression profiling showed that ApCP12 was more widely distributed than ApCP23.In the progress of embryonic development,the expression level of ApCP12 increased gradually.During larval ecdysis stage,the expression level of ApCP12 increased by 3-fold,and that of ApCP23 increased by 13-fold in 3 d after ecdysis than in molting stage.During pupal melanization,the expression level of ApCP12 was higher than that of ApCP23.The expression levels of the two genes had no significant changes during the earlier stage of pupal eclosion,while on last day before eclosion,the expression level of ApCP12 and ApCP23 increased3.5-and 3-fold,respectively,as compared with that on the first day of 20 E injection.After RNAi treatment,the expression level of ApCP12 and ApCP23 in the 3rd instar larvae were down-regulated 5-and 3-fold,respectively.The results suggest that these two CPs of RR-1 consensus participated in the cuticle build of larva,pupa and adult.They have close relationships with whole life cycle of A.pernyi development stages.2.The biogenesis of lysosome-related organelles complex-1 subunit 2(BLOS2)has been cloned in this study.This gene named ApBLOS2(GenBank accession No.MT038419).The full gene sequence has 4312 bp with 4 exon and 3 intron,which containing an open reading frame(ORF)of 438 bp,encoding 145 amino acids with 16.9 kD theoretical molecular weight.Homology analysis revealed that the predicted amino acid sequences of BLOS2 have 84.03% identical with Philosamia cynthia ricini(Saturniidae).The ApBLOS2 expressed in whole embryo development stage,while in newly-hatched silkworm have some decline by using real-time quantitative PCR(qPCR).Tissue expression analysis showed that ApBLOS2 expressed in epidermis,spermaries/ovaries and hemolymph,showing higher expression quantity in A.pernyi larvae.In the progress of changing to fourth instar larvae,ApBLOS2 significantly increased4.13-fold from trimolter to the first day of 4th instar larvae.After RNAi treatment on the 3rd day of 2nd instar larvae,the expression level of ApBLOS2 was down-regulated 2.01-fold in 48 h(inject dsBLOS2)compared with control group(inject dsEGFP).Except ApBLOS2 gene expression declines 31.77% in tetramolter after RNAi treatment,the uric acid content in hemolymph of dsBLOS2 group increased1.51-fold than control.This research investigate gene expression pattern of ApBLOS2 uric acid change after RNAi treatment.The results indicate ApBLOS2 gene has important function in urate granules transport in A pernyi.