Preliminary Study On The Functions Of CRABP2, TOB1 And TIM4 Genes Affecting Economic Traits

Vitamin A is an essential micronutrient for growth and development and maintenance of life activities, it has a certained role not only in cell differentiation, but also in many other physiological processes, including sperm production, immune response, vision, taste, hearing, appetite and growth, and so on. It include retinol, retinal, retinoic acid. Retinoic acid regulate many physiological and biochemical processes including cell differentiation and proliferation by regulating the transcription of many target genes, but it must combine with some special protein, these special binding proteins include CRABPs family.TOB1 (transducer of ERBB2,1) is a member of the TOB/BTG gene family that have potential to regulate cell growth. The study on the porcine TOB1 gene will contribute to phenotypic differences in muscle between Chinese and foreign breeds of pigs and should be considered a candidate gene for meat production traits.Our laboratory identified that CRABPs and TOB1 had differential expression in embryonic skeletal muscle by LongSAGE library, which suggested that they perhaps played a regulatory role in muscle development. The experiment is carried out to better understand the role of CRABPs and TOB1 in regulating muscle development.In this study, we analyzed the expression of CRABPs and TOB1 during C2C12 differentiation time by RT-PCR and real time PCR, which suggested that the expression level were increased during the myogenic differentiation of C2C12 cells. Subsequently, we constructed CRABP2 and TOB1 expression vector and transfected it into C2C12 and PK15 cells, found that C2C12 cells showed morphological differentiation trend and the TOB1 fusion protein was distributed throughout the nucleus and cytoplasm of the PK15 cells using fluorescence microscopy.Meantime, we analyzed the cell cycle by flow cytometry cycle analysis and found that over-expression group did not change significantly in G1 phase, but the G2/M phase cells decreased, S phase cells increased compared with the control group and negative control group, which in line with the characteristics of the early differentiation of C2C12 cells, so we speculated that CRABP2 perhaps played an important role in the muscle fiber differentiation and development, so as to lay the foundation for further study the role of the gene family in pig muscle development and provide the basis for future molecular marker assisted breeding. The mouse CRABP2 gene promoter was used to do the preliminary research on the transcriptional regulation of gene in the study. We obtained the promoter regions of the mouse CRABP2 gene by PCR, then inserted it into the pGL3-basic vector and found that the reporter gene activity was significantly increased after transfected into C2C12 cells, which demonstrated the validity of the promoter fragment. We predicted possible regulatory elements of CRABP2 promoter using TESS and other promoter analysis software. In order to know its transcriptional regulation, a series of different constructs from 5’-flanking regions were analyzed, thus identified the minimal functional core promoter region and speculated that this potential binding site for MyoD and Sp1 in this region would be important for the expression of the mouse CRABP2 gene. Over-expression and site-directed mutagenesis confirmed the regulation of the mouse CRABP2 gene was mediated also by MyoD and Sp1 transcription factors. Gel retardation electrophoresis experiments also further confirmed the binding activity between MyoD and Sp1 binding sites in CRABP2 promoter and MyoD and Sp1 transcription factor. TIM4 is a member of the TIM(T cell immunoglobulin domain and mucin domain) gene family, which play an important role in immune response, it is also responsible to many auto-immune diseases, such as asthma, rheumatoid arthritis, multiple sclerosis and mouse non-obese diabetes mellitus, and so on. Based on its important roles in many aspects, we performed the characterization and function analysis of the porcine TIM4 gene in this study so as to provide information for its bilolgical function and pig genetics and breeding research.The human and mouse TIM4 gene have been studied, but until now, the study on the porcine TIM4 gene is not reported. In this study, we obtained the partial mRNA sequence, 5’promoter sequence and introns sequences of porcine TIM4 gene, and found that it has high similarity with those of the human TIM4 gene. The porcine TIM4 gene included 9 exons and 8 introns with a 1086 bp ORF encoding 361 amino acids with a molecular mass of 39.9 kDa and an isoelectric point (pI) of 6.75. Real-time PCR analysis revealed that the porcine TIM4 gene had a high expression level in liver, lymph and lung and relatively low expression in other tissues, which confirmed its important role of TIM4 gene in immune system. We also constructed a pEGFP-TIM4 expression vector to observe the subcellular localization of the porcine TIM4 protein in PK15 cells. The result showed that perhaps the fusion protein was localized in the cytoplasm of PK15 cells. Blast analysis revealed that it had 80% similarity between the human and porcine TIM4 promoter region, We also found that the porcine TIM4 promoter region contained TATA box, GATA3, Oct1 and CdxA sites as well as the human TIM4 gene using bioinformatics analysis. A SNP (C/T) at 1553 bp of intron 3 (1 is the first nucleotide base in intron 3) was identified. The Genotyping work was performed for association analysis by PCR products directly sequencing. The association study showed that the SNP (C/T) of the porcine TIM4 gene was associated with several immune traits. This polymorphism site had significant associations with Neutrophile granulocyte percentage (NEI%) (0 day) (p= 0.0009), Intermediate cell percentage (MON%) (0 day) (p=0.0021), Hematocrit (HCT) (17 day) (p=0.0063).