| Animal sexual maturity is a complex trait that is affected by many factors.At present,the study of molecular regulation mechanism on sexual maturity of poultry remains largely unknown.The chicken follicles are regulated by multiple genes during maturation,including regulation of miRNAs.miRNAs are non-coding RNAs of about 22 nucleotides in length that play an important regulatory role in primordial follicle formation,follicle recruitment,follicle selection,follicular atresia,oocyte exchange,granulocyte function and corpus luteumization.In our previous study,the Hiseq small RNA deep sequencing was performed on sexually mature(162d)and sexually immature(42d)ovary tissues of single Comb White Leghorn chickens.It was found that miR-26a-5p was differentially expressed in the sexually mature and sexually immature ovary tissues.Further study found that miR-26a-5p regulates TNRC6 A expression and facilitates theca cell proliferation in chicken ovarian follicles.And then,this study further studied the regulation of miR-26a-5p expression and its effects on the function of follicle cells,The results were as follows:(1)Previous studies have found that miR-26a-5p expressed in all levels of follicles.We further found that the expression of miR-26a-5p in theca cells,granulosa cells were significantly higher than that of pre-theca cells and pre-granulosa cells(P<0.05).(2)After overexpression of miR-26a-5p in chicken follicle granulosa cells,the results of RT-PCR showed that after overexpression of miR-26a-5p in granulosa cells,the expression of BCL-2 gene was significantly up-regulated(P<0.05),and the expression of Caspase-3 gene was down-regulated(P>0.05).In combination with the result of CCK-8,these results showed that mi R-26a-5p can promote the granulosa cells proliferation in chicken ovarian follicles.(3)The differentially expressed genes were selected based on the transcriptome sequencing after over-expression of miR-26a-5p in theca cells,and they are NLK,IGF1,MARK1,GSK3 B,SMAD1,SEMA6 D,SEMA3A,SEMA3 D,KDR,NRP1,plexin-A4,THSB2,TLR2,STAR,MMP11,MMP13,VEGFA and TNRC6 A.RT-PCR confirmed that IGF1(P<0.05),SEMA6D(P<0.05),SEMA3D(P<0.01),NRP1(P<0.05),TLR2(P<0.001),MMP11(P<0.05),VEGFA(P<0.01),TNRC6A(P<0.01)gene expression at m RNA Levels were down-regulated after over-expression of miR-26a-5p in theca cells.And NLK(P<0.01),SEMA3A(P<0.05),KDR(P<0.01),and VEGFA(P<0.05)gene expression at the mRNA level in granulosa cells were down-regulated,while plexin-A4 was up-regulated.(4)In combination with the predicted results of Targetscan,the NLK gene was selected as a candidate target gene for miR-26a-5p.The results of RT-PCR showed that the expression patterns of miR-26a-5p and NLK gene mRNA showed opposite trends in the single Comb White Leghorn chickens of different development stages.The activity of double luciferase activity analysis showed that the luciferase activity significantly increased after the second NLK 3'UTR target mutation(P<0.05).These result indicating that mi R-26a-5p is binding to the 3'UTR region of the NLK gene and negatively regulated its expression.(5)Ten single nucleotide polymorphism sites in pri-miR-26a-5p were obtained by sequencing the PCR products: rs14130969(C>T),rs735817649(C>T),rs314567798(C>T),rs316124568(T>A),rs14130971(T>C),rs14130972(A>G),rs316365438(C>T),rs14130973(G>A),rs13534877(G>C),rs15056408(G>A).And there were two highest frequency of haplotypes TCTACGCACG and TTTATATACA.The two different haplotypes altered the free energy,and resulted in the change of predicted secondary structure.And the results of RT-PCR showed that the expression of mature mi R-26a-5p in TCTACGCACG was significantly higher than that in TTTATATACA(P<0.05),indicating that the haplotype combination significantly affects the expression of mature miR-26a-5p.The association analysis with the egg production traits in White Recessive Rock population showed that the individuals with the TT genotype at rs316365438(C>T)had earlier ages than the CC genotypes and higher egg production(32 weeks of age). |
PDF Full Text Request