Preliminary Studies On The Function Of Elav-Gal4＞mir-219 Transgenic Drosophila

Objective: In Drosophila,dfmr1 is highly homologous and functionally similar to the human gene Fragile X mental retardation1(FMR1),which is expressed at different levels in different growth stages of Drosophila and regulated by micro RNAs(mi RNAs).The previous research of found that mir-219 can regulate the expression of dfmr1 in Drosophila,but the effects on the viability,phenotype and behavior of Drosophila are not clear.Therefore,this paper conducts a preliminary study on the function of Elav-Gal4＞mir-219 transgenic Drosophila to elucidate the changes in viability,behavior and mir-219-mRNA regulatory pathways in Elav-Gal4＞mir-219 transgenic Drosophila,which provides a theoretical basis to further reveal its pathogenesis in Fragile X syndrome(FXS).Methods: 1.pUAST-attB-mir-219 recombinant vector was constructed by recombinant technology and UAS-mir-219 Drosophila was constructed by embryo microinjection technique.Subsequently,Arm-Gal4 and Elav-Gal4 were used to drive UAS-mir-219 to obtain Arm-Gal4＞mir-219 and Elav-Gal4＞mir-219 transgenic Drosophila,respectively.2.Larval crawling assay,climbing assay,aggression assay and mating preference test were used to study changes in the behavior of Elav-Gal4＞mir-219 transgenic Drosophila.3.The growth and development of Elav-Gal4＞mir-219 transgenic Drosophila at different growth stages and the changes in Drosophila phenotype were studied by lifespan,pupation rate,hatching rate,gender ratio and hetero-wing ratio.4.The mir-219-mRNA axis was predicted and verified in vivo and in vitro by bioinformatics,q RT-PCR and Western Blot,respectively.Result: Part 1: Construction and identification of transgenic Drosophila models: 1.The mir-219 precursor DNA sequence with a size of about 815 bp was obtained by PCR amplification and cloned into the p UAST-att B vector,and the sequencing and Blast analysis showed that the p UAST-att B-mir-219 recombinant vector was successfully constructed.2.PCR amplification of the mir-219 precursor DNA and part vector sequence,the preliminary results showed that the UAS-mir-219 transgenic Drosophila was successfully constructed after the sequencing and Blast analysis.3.The expression levels of mir-219 in Arm-Gal4＞mir-219 and Elav-Gal4＞mir-219 Drosophila were detected by q RT-PCR.Compared with wild-type Drosophila,the expression levels of mir-219 in transgenic Drosophila were increased(P<0.01,P<0.001),further indicating that the UAS-mir-219 transgenic Drosophila was successfully constructed.Part 2: The effect of mir-219 on Drosophila behavior: 1.The crawling ability of the third instar larvae showed that Elav-Gal4＞mir-219 third instar larvae exhibited a frequent turning,and the crawling distance and speed were reduced(P<0.001).2.The adult climbing assay showed that Elav-Gal4＞mir-219 Drosophila had decreased climbing ability on the 15 th and 22 nd days(P<0.05),but there was no significant change on the 1st and8 th days(P＞0.05).3.The aggressive behavior of Drosophila showed that the time to first attack on 7th and 15 th days was shortened and the frequency of attack was increased,indicating an increase in aggressive behavior(P<0.05).4.Mating preference test showed that Elav-Gal4＞mir-219 Drosophila had disordered mating orientation and decreased mating frequency on days 7 and 15(P<0.05).Part 3: The phenotypic changes of Elav-Gal4＞mir-219 transgenic Drosophila: 1.Drosophila pupation rate assay showed that ElavGal4＞mir-219 Drosophila had abnormal development from the third instar larvae to pupa stage,and the pupation rate was significantly reduced(P<0.001).2.The hatchability test of Drosophila showed that the hatching rate of Elav-Gal4＞mir-219 Drosophila was significantly decreased(P<0.001).3.Survival assays indicated that the lifespan test of both female and male Drosophila was reduced in Elav-Gal4＞mir-219(P<0.001).4.The ratio of male and female Drosophila indicated that the number of Elav-Gal4＞mir-219 female Drosophila was significantly decreased(P<0.001).5.Determination of the proportion of abnormal wings in Drosophila showed that Elav-Gal4＞mir-219 female Drosophila had abnormal wings,and the proportion increased significantly(P<0.001).6.Western Blot assay indicated that d FMRP protein levels of Elav-Gal4＞mir-219 were reduced in both third instar larvae and adult stages(P<0.001).Part 4: Prediction and validation of mir-219-mRNA regulatory pathway: 1.Target Scan,RNAhybrid and Pic Tar multiple databases to predict dme-mir-219 target genes,combined with Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)enrichment analysis.Four candidate target genes were initially screened,namely Cip4,para,slo and fra.2.The expression level of candidate target genes in ElavGal4＞mir-219 Drosophila was detected by q RT-PCR.The results showed that the mRNA expression of para and slo increased(P<0.001,P<0.01),the Cip4 mRNA expression decreased(P<0.01),and fra mRNA expression did not change significantly(P＞0.05).when the expression of dfmr1 decreased,the expression of para mRNA increased(P<0.01),the mRNA expression of slo and Cip4 decreased(P<0.01),but the expression of fra mRNA had no significant change(P＞0.05).3.q RT-PCR detected the expression level of hsa-mi R-219 in SK-N-SH cells after transfection with hsa-mi R-219 mimic.The expression level of hsa-mi R-219 was upregulated in the transfected cells compared with the control group(P<0.001).Western Blot analysis indicated that in SK-N-SH cells,after the overexpression of hsa-mi R-219,the protein expression of the SCN1A(SCN1A is a human homolog gene of para)was upregulated(P<0.05),while the protein expression of FMRP had no significant change(P＞0.05).4.Western Blot experiments showed that in SK-N-SH cells after FMR1 small interfering RNA,the protein level of SCN1 A did not change significantly(P＞0.05).Conclusions: 1.Elav-Gal4＞mir-219 transgenic Drosophila was successfully constructed.2.high expression of dme-mir-219 in Drosophila affected its growth as well as phenotype,and behavioral abnormalities were induced.3.dme-mir-219 in Drosophila regulated the expression of para,slo and Cip4 mRNA.4.hsa-mi R-219 regulates the expression of SCN1 A in SK-N-SH cells.