Cloning, Expression And Function Prediction Of13Candidate Genes On Genetic Characteristics Of Germplasm In Banna Mini-pig Inbred Line (BMI)

Banna Mini-pig Inbred Line (BMI) is the first successful cultivated large mammal inbred line in the world by highly consecutive inbreeding and strictly scientific breeding methods using unique resources of aboriginal pig breed in China. BMI is optimal large pure experimental animal models for research and application in the field of bio-medicine, xenotransplantation. Over the years, the BMI had been committed the genetic breeding work and formed stable morphological characteristics, but it was relatively little to explore its genetic features of germplasm from the molecular level, especially the genetic characteristics of genes related to its growth, metabolism, immune have not been systematically researched.To reflect its genetic characteristics of germplasm and further insight into the genetic bankground at the molecular level, structure and expression level of13BMI candidate functional genes which are related with its growth and development, metabolism, immune regulation, transplant rejection, pathophysiology, breeding for disease resistance, and sex identification of germplasm genetic characteristics were studied deeply using molecular biology techniques to start with the RNA or DNA. These genes include growth hormone (GH), aquaporin3(AQP3), vascular endothelial growth factor (VEGF), tumor necrosis factor alpha (TNF-alpha), alpha interferon (IFN-alpha), interleukin-6(IL-6), Y chromosome sex-determining gene (SRY), Fas apoptosis inhibitory molecule (FAIM1), polyglutamine binding protein1(PQBP1), synaptotagmin protein6(STXBP6), ribosylation enzymes (RBKS), fat acetyl-CoA reductase (FAR1), centromere protein of P (CENPP). First we isolated and got the complete coding sequence of these genes, second carryed out comparative genomics and bioinformatics analysis, then targeted multi-tissues sample fluorescence quantitative (qPCR) expression analysis according to different genetic characteristics of each gene and in the last predicted and analysed a variety of physical and chemical properties of proteins. In addition, the prokaryotic expression analysis of GH and SRY gene were carried out and GH protein expression was veryfied by Western Blot.Results showed most genes were relatively conservative function genes and had very high amino acid identity among different species, such as the STXBP6identity was100%between BMI and cattle. But we also found some important SNPs, such as the c.26T>C SNP of the BMI GH gene coding region which were different from other12domestic and foreign pig breeds. In addition, we found only by removing signal peptide of GH can it be induced to express the protein. AQP3was100%conservative in amino acids level between different breeds, but had a mutation site (c.204C＞A SNP) in the coding region. VEGF exist a p.A102T amino acid mutation and had two SNPs (c.15G>C and c.438G>A) in the coding region. BMI IFN-a gene sequence was different from other breeds and had many SNPs in nucleic acid and protein levels. We found the BMI SRY owned distinctive haplotype, and screened a pair of specific primers of SRY gene which can be used as the early embryonic sex identification of BMI and found0.5mM IPTG can induce massive SRY protein to produce in prokaryotic expression system. Except GH and SRY genes, multi-tissues of the housekeeping gene18S correction qPCR analysis showed other11gene can express in spleen and lung, but differentially expressed in other tissues of BMI for strong, weak, and no express. This showed that differential express phenomenon of same gene in different tissues and different genes in the same tissue. These proteins' physical and chemical properties and structure such as molecular weight, isoelectric point, secondary structure, tertiary structure, transmembrane structure and signal peptide reflect the special function and effect of each BMI gene.13genes have been submitted to the GenBank database and accession numbers respectively were JQ177096(GH), HQ888860(of AQP3in), JF831364(VEGF), JF831365(TNF-alpha), JQ839262(IFN-alpha), JQ839263(IL-6), GU991615(SRY), Nm₀01245976(FAIM1), Nm₀01246668(PQBP1), Nm₀01246666(STXBP6), JF944892(RBKS), Nm₀01252425(FAR1), JF944894(CENPP). Especially, FAIM1, PQBP1, STXBP6and FAR1genes have been identified as the standard sequence (format********Nm₎, which were mapped to pig chromosome13,X,7and2respectively. These new gene sequences'annotation will be a basis for deeply study the regulation of expression of these genes and functional identification.13genes was the first study in BMI and filled a gap of the study of the related genes.13gene sequences have been submitted to the GenBank database and the coding sequences of GH, AQP3, IFN-a, SRY genes with some SNP sites were different from other breeds of the GenBank sequences, may be unique haplotypes for the BMI. We have developed a pair of specific primers which can be used as a molecular marker for BMI embryonic sex identification. This study established a series of biotechnology methods such as silico cloning BMI new genes and quickly verifying by using relative species gene sequences and splicing pig EST sequences, testing BMI gene expression level in different tissues by fluorescence quantitative PCR (qPCR), studying BMI functional genes and protein by prokaryotic expression system and Western Blot. FAIM1, PQBP1, STXBP6, RBKS, FAR1and CENPP were the first isolated novel pig genes, which related to immune, disease resistance, metabolism and other life activities. This study not only provided a basis of data, methods and ideas for Banna mini-pig inbred line further breeding and studying its unique genetic characteristics at the molecular level, but also played an important role in its exploitation, intellectual property protection and using as experimental animal.