| The body color of insects plays a crucial role in the insects,such as camouflage,warning,attracting the opposite sex,etc.The body color of insects is mostly a reflection of the pigment in the epidermis.Locusta migratoria is an important agricultural pest.There have been many outbreaks of locust plagues in history,causing great harm and loss to ecology and agriculture.There are two forms of locusts in their growth and development,gregarious(black)and solitarious(green and gray).The change of body color is closely related to the behavior of locusts gathering and scattering,and the change of behavior is closely related to the occurrence of locust plagues.Cuticle protein is an important component of insect body wall and plays an important role in insect growth and development,morphology and body color formation.Therefore,the study of the biological function of cuticle protein in the body color formation of locusts is of great significance to the mechanism of insect body color formation and early warning of locust plagues.This paper takes L.migratoria as the research object and uses the cuticle transcriptome database of locusts(20-Hydroxyecdysone treated,20E)to screen out a significantly up-regulated cuticle protein gene LmNCP4.9 and carry out its molecular and expression characteristics analysis,using RNA interference(RNAi)and CRISPR-Cas9 gene editing technology combined with transmission electron microscopy and metabolomics and other series of research methods to explore the biological function of LmNCP4.9 in the body color formation of locusts,the main results are as follows:(1)Molecular characteristics and expression analysis of LmNCP4.9Through the bioinformatics analysis of the differentially expressed gene LmNCP4.9 selected from the transcriptome,it was found that the gene has 2exons;the domain analysis of the protein translated from the gene revealed that it has 3 transmembrane domains;RT-q PCR analysis of the cuticle at different developmental stages of locusts showed that LmNCP4.9 was highly expressed in the middle of each age,and it was highly expressed in the third and fourth instars.The expression analysis of different tissues showed that LmNCP4.9 was on the pronotum.The expression is highest,followed by wings and cuticle.Tissue localization analysis using immunohist-ochemistry technology found that the protein was localized in the cytoplasm,and further precise localization analysis by immunogold labeling revealed that it was localized in pigment granule and mitochondria nearly,and it was speculated that it might play a role in transport.(2)Study on the function of LmNCP4.9 in the body color formation of locustsIn order to explore the role of LmNCP4.9 in the body color formation of locusts,RNAi technology was first used to silence the gene expression in the fourth instar nymph stage.The results showed that compared with the control group,the locusts could molt their skin to the fifth instar nymph after silencing LmNCP4.9,but the body color of the nymph showed two phenotypes of green and gray,the proportions were 30% and 33%,respectively;using frozen sections The technology observed the prontum,abdomen,and wings of the green locust and found that the color of the pronotum,abdomen,and wing epidermis of the green individual locust became lighter than that of the control locust(black).This gene is highly expressed in the third and fourth instars.In order to further analyze the function of this gene in the body color formation of locusts at different instars,the LmNCP4.9 gene was silenced on the third instar nymphs.For locusts,the proportions of part of their body colors turned green and gray were 35% and30% respectively;the same method was used to silence the fifth instar nymphs,and the body color of the locusts that molted into adμLts did not change significantly.It is speculated that LmNCP4.9 was in the third instar of L.migratoria.And the fourth instar period(the key period of locust type transformation)plays an important role in the formation of body color.Using CRISPR/Cas9 gene editing technology to further verify the function of the gene,it was found that the body color of the F0 and F1 generations of the L.migratoria that have been knocked out of the gene changed to green and 13%,respectively,and the gene editing The degree of individual color change is more obvious than RNAi.The frozen section technology is also used to slice and observe the pronotum,abdomen and wing tissues of the gene-edited green locusts.The results show that the gene-edited green locust is compared with the normal black individual,The color of the corresponding tissue part is lighter,indicating that LmNCP4.9 has an important function in the body color formation of locusts.(3)The molecular mechanism of LmNCP4.9 involved in the body color formation of locustsScanning electron microscopy found that the pronotum of which interfered LmNCP4.9 contained more white uric acid particles than the control.In order to explore the molecular mechanism of LmNCP4.9 involved in the body color formation of locusts,metabolomics was used to analyze the cuticle of five-instar green locusts that silenced LmNCP4.9 and black individual locusts in the control group.A total of 22 different metabolites related to changes in insect body color were screened,mainly including uric acid,xanthine acid,phenylalanyl phenylalanine,deoxyuridine,dopa,melanin,carotene and other substances,and found to interfere the LmNCP4.9.The content of uric acid increased significantly,and the content of xanthine,phenylalanyl phenylalanine,deoxyguanosine,carotene,and melanin decreased significantly;the KEGG database was used to annotate and analyze the different metabolites,and the differences were found Metabolites are mainly concentrated in the ommochrome metabolism pathway,phenylalanine metabolism pathway and purine metabolism pathway,while the substance content of tyrosine metabolism pathway has not changed significantly.The tyrosine metabolism pathway related genes of the locust that interfered LmNCP4.9 were detected by RT-q PCR,and it was found that the expression of yellow,tan,and PH genes that played a role in blackening was significantly down-regulated,and the expression of carotene-binding protein was also significantly down-regulated.The combination of the above results and the positioning results indicate that LmNCP4.9 participates in the transport of pigment particles in the body color formation of locusts.(4)Juvenile hormone regulates the expression of LmNCP4.9 and participates in the body color formation of L.migratoriaAccording to the results of the transcriptome database,the fifth-instar locust nymphs were treated with different concentrations of 20 E,and the results showed that 20 E caused an increase the expression of LmNCP4.9;the fifth-instar locust was treated with juvenile hormone analog(JHA),and the results showed that JHA had an effect on the expression of LmNCP4.9 which has a significant inhibitory effect.Juvenile hormone was used to induce the third and fourth instar locusts,and it was found that some locusts turned green.It is speculated that JHA and 20 E may participate in the formation of locust body color by regulating the expression of LmNCP4.9.This paper explores the effect of cuticle protein gene LmNCP4.9 on the body color formation of locusts,and clarifies its mechanism of body color formation,which enriches the research content of insect body color and has important theoretical significance. |
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