| Locusta migratoria is an important agricultural pest which has the ability of long-distance and large-scale migration depending on its powerful function of fore-and hind wings.The dense migratory caused the locust disaster and a serious food crisis,which is one of the most harmful agricultural production in history.The insect wings also play an important role in evading enemies and breeding foraging,making it more suitable for complex and changing living environments.Wing development is regulated by multiple genes and signal pathways.The connection between cells and the interaction between cells and extracellular matrix also play a key role in the growth and development of insect wings.Integrin is a kind of cell adhesion molecules on the surface of the cells which compose of ? and ? subunit,it plays an important role in insect immune response,cytoskeleton formation,and growth and development.However,the mechanism of integrin action is still unclear in the development of insects,especially the gradual metamorphosis of migratory pests.Therefore,the exploring the function of integrin ? subunit currently found in the transcriptome database in the development of locust wing will provide new targets for biological control of migratory pests.In this study,the Integrin gene encoding Lm Integrin?-PS protein in locust was used as the research target to investigate the biological function in the development of Locusta migratoria wings by using RNAi technology,and the conclusions were as follows:1.Sequence analysis and expression analysis of Lm Integrin?-PSThe c DNA sequence of Lm Integrin? was obtained from the locust transcriptome and genomic database,which is consistent with the previously coding sequence(KJ535130.1)of the integrin protein from NCBI that the function has not been identified.Analysis of its genetic structure revealed that it contained 12 exons,encoding a total of 864 amino acids with a signalpeptide(SP),von Willebrand factor(v WF)type A domain,epidermal growth factor(EGF),a tail domain,a shorter transmembrane domain(TM),and a cytoplasmic domain(beta cytoplasm).Multiple sequence alignments revealed that it is conserved among different species,all of which contain metal ion-adhesive regions(MIDAS),cysteine repeats(cys-repeats),a transmembrane domain(TM),and binding site of cytoplasmic tail(Nxx Y/F).Based on the similarity of the encoded amino acid sequence with the homologous protein sequence in other insects,it was named as Lm Integrin?-PS,and the cluster analysis results showed that it was clustered with the Blattaria.The RT-q PCR was used to determine the different tissues and stage expression pattern of Lm Integrin?-PS in the fifth instar nymphs,and the results showed that Lm Integrin?-PS was highly expressed in wing pads and had a peak expression level in the mid-stage of fifth instar nymphs.2.Biological function analysis based on RNA interferenceRNA interference(RNAi)is a simple and efficient gene silencing technology.By taking advantage of the RNAi sensitivity of L.migratoria,locust nymphs were injected with ds RNA on the first day of the fourth and fifth instar,respectively.The results showed that the fourth-instar nymphs injected with ds Lm Integrin?-PS were able to successfully molt to the fifth instar nymphs,but about 65% of them have the abnormal wing morphological.Among them,40% of the nymphs have severe blister wing and 25% of the wing pads could not close normally.Similarly,about 58% of the fifth instar nymphs injected with ds Lm Integrin?-PS could not successfully molt to adult and died with blister in the base of wings.About30% of the nymphs could successfully molt to adult,but their wings appeared serious blister.The results of immunohistochemistry showed that Lm Integrin?-PS was mainly located at the basement membrane at the bottom of the epidermal cells,suggesting that it might involve in the junction between cells and basement membrane.3.Effects of Lm Integrin?-PS on the structure of wing cuticleIn order to further explore the mechanism of Lm Integrin?-PS in the growth and development of locust wings,H&E staining was performed according to the results of RNAi in the fifth instar nymphs.Compared with the control group,it was found that the epidermal cell layer of the locust wing in the treatment group obviously lost the junction with the basement membrane,resulting in the loss of junction between the two layers of epidermal cells in the dorsal and ventral wing,and forming a huge cavity.Subsequently,the changes of wing ultrastructure were observed by scanning electron microscope(SEM)and transmission electron microscope(TEM).The results of SEM analysis showed that,the wing surface of the insects treated with ds Lm Integrin?-PS was severely shrunk,the bristles were reduced and its ends were flattened and bent,compared with the normal structure of the wing surface in the control group.TEM results showed that the cell arrangement of wings was disordered,and the wing structure was obviously damaged after silencing of Lm Integrin?-PS.Meanwhile,RT-q PCR analysis showed that the expression of cell junction-related genes in the ds Lm Integrin?-PS-injection group was significantly reduced compared with that of the control,suggesting that Lm Integrin?-PS may regulate the expression of cell junction-related genes to ensure normal cell junction and cytoskeletal homeostasis to participate in the growth and development of the locust wing.In summary,Lm Integrin?-PS may be involved in the junction between the epidermal cell layer and the basement membrane of the locust wing,and then participate in the development of the wing pads and wings during nymphs to nymphs and nymphs to adults transition.The results will provide a theoretical basis for the research on the mechanism of integrin in insect wing development,and also provide a basis for pest control with migratory pest wing as the target. |
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