Sequence And Tissue Expression Analysis Of Min Pig FoxN1 Gene

Forkhead N1(Forkhead box protein N1, Fox N1) was a member of the winged helix/forkhead transcription factor family, has been occupied a central position in the stablished thymic rudiment genetic network.FoxN1 gene is an important gene related to thymus development and hair growth,its absence could cause severe immune deficiency and thymic hypoplasia. Currently, research on sequences of Min pig FoxN1 gene has not been reported.This study was intended to explore the function of Min pig Fox N1 genes.Min pig FoxN 1 gene was cloned by PCR, it included both Fox N1 genome sequence and entire coding region sequence.The complete coding region of Fox N1 sequence was analyze by bioinformatics methods,the Fox N1 gene sequences of Min pig and Yorkshire were compared.Furthermore,analysed the genetic variation sites.RT-PCR was used to detect expression of the Fox N1 gene in various organizations samples. By 2 months, 4 months, 6 months, 8 months old Min pigs as experimental material,using quantitative Real-PCR detected the expression of Fox N1 at the transcriptional level, using Western-blot detected the expression of Fox N1 at the translational level.The results of this experiment were as follows:(1)The genome sequence of Min pig Fox N1 gene was successfully cloned,a total 14 730 bp.Including 8 exons and 7 introns. Exon position from 1-8 are 1-123,518-976,3 732-3 842,5 512-5642,7 800-7 896,11 390-11 597,11 746-12 237,14 411-14 730.(2)The completely coding sequence of Min pig Fox N1 gene was successfully cloned, it contained 1 941 bp, encoded 646 amino acids,molecular weight was 68.64 k Da, theoretical isoelectric point was 5.81. Fox N1 protein located within the nucleus, signal peptide was not present, not a secreted protein, and there was no transmembrane area. There was a FH domain in269 to 347 amino acid positions. The protein among different species was highly conserved.Utilizing BLAST alignment obtained 42 species Fox N1 gene coding sequence, and builded Fox N1 gene phylogenetic tree by MEGA6.0. Found that Min pig with Bos taurus,Water buffalo,Ovis aries, Chiru and other Artiodactyla animals clustered together, either closely remaining species are also in line with the law of the evolution of species, gathered in a different branch.(3)pEGFP-N1-Fox N1-a/b/c recombinant plasmid was successfully constructed, and it was transiently transfected into pk15 cells. Fox N1 protein was mainly expressed in the nucleus influorescence inverted microscope.(4)Three kinds of transcription splice variants were found in thymus,they were in line with several forms of alternative splicing body.Compared with variant 1,there was deletion in the nucleotide position 124-387 in variant 2,sequence size was 1 677 bp,and GCA insertion in 581 to582 in variant 3,sequence size was 1 944 bp.(5) Compared with Yorkshire, Min pig Fox N1 gene sequences existed four mutations, of which three were synonymous mutations, one was a missense mutation, resulting in the 108 th amino acid changed.(6) Min pig Fox N1 gene expression was detected in 13 organizations, the results show that it was only expressed on the skin、thymus and tongue.(7) Fox N1 gene in different ages of Min pig thymus tissue did Real-Time PCR operation,showed that relative express levels was increased remarkable In 4 month.(8) Fox N1 gene in different ages of Min pig thymus tissue did Western blot operation,showed that relative express levels was increased remarkable in 4 month.