| The abnormal development of Drosophila melanogaster is mainly regulated by juvenile hormone and 20-hydroxyecdysone.As the primary response gene of juvenile hormone,Kr-h1 gene plays an important role in the signal transduction of juvenile hormone.As a non-coding small miRNA about 22 nt long,miRNAs inhibit the translation or induce degradation of m RNA by binding to specific sites of the target gene m RNA,thus participating in the regulation of growth,development,apoptosis and other important physiological activities in human,animal and plant.Therefore,in this study,Drosophila melanogaster was used as the experimental object to verify the miRNA which targeting the Kr-h1 gene in completely abnormal insects and the effect of miRNA on the development of Drosophila melanogaster,which provided the theoretical basis and guidance for the biological control of mosquitoes and flies.In this study,the miRNA of Kr-h1 gene in Drosophila melanogaster was predicted by several bioinformatics prediction websites,and finally locked miR-927 as the target miRNA that might target the Kr-h1 gene in Drosophila melanogaster.Then the double luciferase reporting system was used to identify the recognition sites between miR-927 and Kr-h1 gene.The results showed that the F/R value of miR-927 mimics and Kr-h1 3’UTR co-transfection group were significantly lower than that of the control group(P<0.01).However,the F/R value of single site mutation of Kr-h1 3’UTR was significantly lower than that of wild-type vector(P<0.05),compared with the wild type,the simultaneous mutation of the two loci showed no significant difference(P>0.05).The results showed that there was a specific binding site of miR-927 mature sequence on the 3’UTR of Kr-h1 gene,there was a target relationship between miR-927 and Kr-h1 gene,and Kr-h1 gene could be specifically bound by miR-927.In order to explore the regulatory relationship between miR-927 and Kr-h1 gene at the transcriptional level,q PCR was used to detect the changes of Kr-h1 gene after overexpression of miR-927 in Drosophila melanogaster Kc cells,the results showed that the expression level was significantly lower than that in the NC group(P<0.05).Combined with the relative expression levels of mir-927 and Kr-h1 at different developmental stages in vivo,it was proved that miR-927 and Kr-h1 showed a mutual inhibitory relationship at certain developmental stages.In this study,the relationship between miR-927 and JH was verified at cell and worm level.Methoprene was used to treat the Kc cells and the white prepupa of Drosophila melanogaster at different time to detect the difference of miR-927 expression.The results showed that JH could inhibit the expression of miR-927 at both the cell level and the worm level(P<0.05).Then the JH was deleted and the expression of miR-927 was detected.The results showed that the expression of miR-927 in the JH deletion group was significantly higher than that in the wild type(P<0.05).The results showed that JH could inhibit miR-927 to a certain extent.In order to better verify the regulation of JH and miR-927 on the growth and development of Drosophila melanogaster,Methoprene was used to treat the abdomen of white prepupa Drosophila melanogaster and observe the phenotype of the back bristles.The results showed that Methoprene treatment could lead to short bristles,plaque and back bristles couldn’t fuse properly,and the flies could not hatch,while the overexpression of miR-927 in drosophila could not prepupate normally.Generally,the nymphs no longer develop,and the body develops slowly,the individual becomes smaller,a few fruit flies became "small pupae",but failed to hatch into adults.Conclusion:(1)Kr-h1 gene is targeted by dme-miR-927.(2)miR-927 can regulate the transcription level of Kr-h1 gene and inhibit the expression of Kr-h1 gene m RNA in Drosophila melanogaster Kc cells and worm body.(3)JH can inhibit the expression of miR-927 at both cell level and worm level.(4)JH and miR-927 play an important role in regulating the growth and development of Drosophila melanogaster. |
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