| The integument covers the entire body and is the outermost tissue of insects.It has important functions such as maintaining the morphology of the insects,resisting the invasion of foreign substances,and preventing evaporation of water in the body.The insect cuticle consists of the epicuticle,procuticle and epidermals.The integument mainly consists of chitin,cuticle proteins and lipids.The function of cuticle is different in different tissue,and even in different developmental stages,there are significant differences in chemical composition and physical properties of the same sites,which is related to chemical composition.Cuticle proteins are the most important component and is essential for the growth and development of insects.It affects cuticle morphology and mechanical properties of insects and is a potential target for pest control.In this paper,we take the locusta as the research object,using proteomics analysis to identify three endocuticle protein genes LmNCP1、LmNCP2 and LmCP6 as research targets,using RNAi technology to explore its biological function in the cuticle development of L.migratoria,the following results:（1）Proteomics analysis of the cuticle of L.migrotoriaAccording to reports,the endocuticle and exocuticle formation time of L.migrotoria was used to determine by H&E staining.The total protein of the cuticle was detected by SDS-PAGE.The label free method was performed to determine time node.Three cuticle protein genes that were highly expressed after molting were screened out,presumed to be involved in the formation of the endocuticle.（2）The three endocuticle protein genes of L.migratoria were used as research targets for bioinformatics analysis.Unigene 3699 contains 2 exons encoding 101 amino acids,a signal peptide and 3 repeat motifs conserved among homologous proteins of different species.LmNCP1 is closely related to BcNCP1,named as LmNCP1.LmNCP2 contains 3 exons encoding 205amino acids,and a signal peptide and a chitin binding domain（ChtBD4）.LmNCP2 is highly related to the Larval Cuticle Protein 2（CP2）and named as LmNCP2.LmCP6 contains 7 exons encoding 359 amino acids,a signal peptide and a chitin binding domain（ChtBD4）,LmCP6 and insect Cuticle Protein 6 are clustered together and named LmCP6.（3）Analysis of the mRNA expression characteristics of three endocuticle protein genes.According to the obtained cDNA sequence,the corresponding primers were designed,and the full-length cDNA was amplified by RT-PCR to verify the sequence.The RT-qPCR technique was used to analyze the expression of the spatial and temporal.The expression of LmNCP1,LmNCP2 and LmCP6 in different tissues showed high specific expression in integument and wing pads;Expression at different developmental stages showed that LmNCP1 and LmNCP2 were highly（4）Functional studies of three endocuticle protein genes LmNCP1,LmNCP2 and LmCP6.Because the expression of LmNCP1,LmNCP2 and LmCP6 is consistent with the time of formation of the endocuticle,it is speculated that they may participate in the formation of endocuticle.After dsLmNCP1 and dsLmNCP2 were injected on the 4th and 5th instar nymphs by RNAi method,the locustas could still molt without obvious phenotype,but the cuticles was stained by H&E staining,and its endocuticle was thinner compared with the control group that leads to a significant thinning of the cuticle.After silencing the ds LmCP6,the locustas can develop into adults,but it is found that the composition of the adult wings becomes curled and shrunk.L.migratoriais a typical incomplete metamorphosis insect,and it is also one of the world’s agricultural pests.Therefore,the study on the cuticlce protein gene of L.migratoria is helpful to understand its role in the growth and development of L.migratoria,and provides a theoretical basis for the selection of pest control targets. |
