| As a high-quality local sheep breed in China,Hu sheep has many advantages,such as multiple births,fast growth,resistance to heat and humidity.The high prolificacy performance of Hu sheep is particularly prominent.It has always been a hotspot for research and application on the characteristics of Hu sheep.FSHR is a major gene for mammalian prolificacy traits by regulating follicular development and ovulation,and its expression level and SNP are closely related to the fecundity traits of Hu sheep.In this study,the non-coding region(5'-UTR and 3'-UTR)of Hu sheep FSHR gene was selected as the main research object.The non-coding region of FSHR gene was obtained by cloning and sequencing,and then its sequence characteristics were analyzed by bioinformatic softwaress.The regulation of transcription factors,mutations and miRNAs on the transcriptional activity of Hu sheep FSHR gene was detected by luciferase reporter gene system,EMSA and overexpression technology.The results provided a theoretical basis for exploring the regulation mechanism of FSHR gene expression and further revealing the mechanism of high prolificacy traits in Hu sheep.The maim results in this study were summarized as follows:(1)Cloning and Analysis of FSHR Gene 5'-UTRA 730bp 5'-flanking sequence of FSHR gene was obtained by cloning and sequencing,in which the 5'-UTR was 161bp in length.The sequence alignment results showed that the 5'-UTR sequence of Hu sheep FSHR gene was fully consistent with that of the Texel sheep(100%),and also indicated a highly evolutionary conservation with other mammalian species,such as cattle and mice.Multiple transcription factor binding sites was found in the 5'-UTR of the FSHR gene,such as IRF-1,Sp-1,E4F,PAX4,GATA2.Among them,the transcription factor PAX4 has not been reported for the regulation of mammalian FSHR gene transcription.(2)Transcription regulation of PAX4 on the transcriptional activity of FSHR gene in Hu sheepTissue expression profiling showed that the PAX4 was expressed in the heart,liver,spleen,lung,kidney,stomach,muscle,large intestine,small intestine,uterus and ovary inHu sheep.Especially,PAX4 was highly expressed in the ovarian tissue.After the construction of Hu sheep PAX4 overexpressing vector pcDNA3.1-PAX4 and transfection with ovarian granulosa cells,Flow cytometry analysis indicated that PAX4 significantly promoted ovarian granulosa cell apoptosis.This demonstrated that PAX4 was a pro-apoptotic factor for ovarian granulosa cells.FSHR gene 5'-UTR luciferase reporter vector pGL3-730 was constructed,which was then co-transfected with pcDNA3.1-PAX4 in ovarian granulosa cells and COS7 cells.The results showed that the expression of pGL3-730 luciferase activity was significantly down-regulated by pcDNA3*1-PAX4,indicating that PAX4 could inhibit the transcriptional activity of FSHR gene in Hu sheep.EMSA detection also confirmed that PAX4 protein could bind to the FSHR gene 5'-UTR region.In summary,Transcription factor PAX4 would target to the 5'-UTR region of the FSHR gene,significantly downregulate the transcriptional activity of FSHR,and finally promote ovarian granulosa cell apoptosis.(3)Characteristics of the FSHR gene 3'-UTR sequenceA 877bp 3'-regulatory region of FSHR gene was obtained by cloning and sequencing.The homology analysis showed that the 3'-UTR nucleotide sequence of mammalian FSHR gene was conserved between different mammalian species,with a 99.12%similarity between Hu and Texel sheep.A tail signal(ATAAA)was found at*290nt?*294 nt,and two additional ARE elements at*105nt?*109 nt and*462nt-*466 nt by further analysis.miRNA binding sites were also predicted in Hu sheep FSHR gene 3'-UTR.7 potential miRNA binding sites were found,including miR-633,miR-126*,miR-2352,miR-539-5p,miR-145-5p and miR-96.(4)Mutation of the 3'-UTR region of the FSHR gene affects its transcriptional activityThree SNP loci were found in the 3'-UTR of Hu sheep FSHR gene by pooled-DNA sequencing,which were named as*88A>G,*158C>A and*159T>G(*158C>A and*159T>G sites completely linkaged,so together named as*158C>A).The pmir-GLO double fluorescent reporter recombinant vector for above two sites was constructed respectively and then transfected with 293T cells.The results showed:At the*88A>G site,the luciferase activity of type A was significantly higher than that of type G.At the*158C>A site,the luciferase activity of type C was significantly higher than that of A type.These results indicated that the mutation of the above 2 sites could affect the transcriptional activity of FSHR gene in Hu sheep.Then,the luciferase gene was detected by qRT-PCR atOh,1h,2h,3h and 4h after actinomycin D(Act D)treatment.The results showed that at the*88A>G site,the half-life of luciferase gene type A(1.15h)was higher than that of type G(0.42h),The C-type(1.15h)was higher than the A-type(0.51h)at the*158C>A site.So,the mutation of the above 2 sites could affect the transcriptional activity of FSHR gene in Hu sheep by affecting the stability of mRNA.Finally,ARE element mutant dual fluorescent reporter recombinant vector for the two mutant sites was constructed,and transfected with 293T cells.When the ARE1 locus was destroyed,there were no significant differences in luciferase activity between different genotype.The results showed that the mutations of the above 2 sites could affect the stability of mRNA by ARE1,which could affect the transcriptional activity of FSHR gene.(5)Targeting regulation of FSHR gene by miRNAThe*158C>A site were found to locate within the binding site of miR-633 at the 3'-UTR of Hu sheep FSHR gene by online software,miR-633 mimics were co-transfected with 293T cells in C,A-type pmir-GLO double fluorescent reporter vector at the*158C>A site respectively.Luorescease activity analysis showed that miR-633 could significantly down-regulate the transcriptional activity of the two vectors,and the difference between the two vectors was not significant.The results indicated that miR-633 could inhibit the FSHR gene in Hu sheep,but not participate in*158C>A mutation regulation for FSHR transcriptional activity. |
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