The Molecular Basic Research Of Dwarfism In Wuzhishan Inbreeding Pig

Wuzhishan Pig(WZSP) is a kind of miniature pig and mainly distributed previously in the mountain area of Hainan province in China. The mature body-weight of WZSP is only 30-35kg, which is 15-30 percent of normal weight of other pig and is one of the rare and endangered breeds in China. At present, WZSP F₁₈ has been obtained by inbreeding method(Inbreeding coefficient, 0.979). WZSP has been used widely in bio-medical field because its anatomy, physiology and nutrition metabolism is similar to that of human being. WZSP is also an ideal experimental animal model for human diseases. WZSP has been chosen as the preferred animal for xenotransplantation and as national center of miniature pig for laboratorial use. To understand the molecular basic research of WZSP's dwarfism, Homozygotic F₁₆ WZSP was selected for the genetic variation study of the genes relative to GH-IGF-I axis using molecular biology and cytobiology methods. And the main results are as follows:1. The serum GH and IGF-I of three-month-old Large White pig and WZSP were detected by Elisa method. The results showed that: serum IGF-I levels of Large White pig were significantly higher than that of WZSP(P<0.05). Serum GH levels of WZSP were a little lower than that of Large White pig, but the difference isn't significant(P>0.05).2. The full length cDNA sequence of WZSP GH gene(GenBank accession number: DQ415517) was obtained by RT-PCR. The CDS of WZSP GH gene encodes 216 amino acids residues, which molecular weight is 24.4Kda and isoelectric point is 7.175. The predicted protein sequence of alignment of GH CDS for WZSP and pig(GenBank accession number: X53325) by biology software showed that two substitutions were discovered in GH coding region, including C→T mutation at position 26, which causing Val instead of Ala; G→A mutation at position 65, which causing Gln instead of Arg. The two substitutions lie in the sequence encoding signal peptide that could be cut off during the mature process, which not affect the structure and function of mature protein.3. The full length 1917bp cDNA sequence of WZSP GHR gene(GenBank accession number: DQ422962) was obtained by RT-PCR. The CDS of WZSP GHR gene encodes 638 amino acids residues, which molecular weight is 71.1Kda and isoelectric point is 4.509. The predicted protein sequence of alignment of GHR CDS for WZSP and pig(GenBank accession number: NM214254) by biology software showed that five substitutions were discovered in GHR cytoplasmic domain, which maybe influence the GHR downstream transduction. However, the predicted protein sequence of alignment of GHR CDS for WZSP and Large White pig showed two substitutions lie in both extracellular and cytoplasmic domain, especially the substitution at 165 position lies in FN3 conserved domain of GHR, which may affect GHR's structure and function.4. The full length cDNA sequence of WZSP IGF-I gene was obtained by RT-PCR. A synonymous mutation was found in the CDS of WZSP's IGF-1, but didn't cause the substitution of amino acid. The CDS encodes 130 amino acids precursor protein, which molecular weight is 14.4Kda and isoelectric point is 9.144.5. The prokaryotic expression vector of WZSP's GH and GHR genes were constructed, and transferred into E.coli. BL21(DE3) expression strain. Induced 3 hours by 1 mmol/L IPTG, special peptide of WZSP's GH was detected via SDS-PAGE, and which its content was 20 percent of total bacterial protein scanned by Bandscan software(version 4.30). As a result of codon preference, no special signal peptide of WZSP's GHR was found via conventional methods including SDS-PAGE.6. The eukaryotic expression vector of WZSP's GH gene, Large White pig and WZSP's GHR genes were constructed, and transfected into PK-15 cells via Lipofectamine^TM 2000. semi-quantitative RT-PCR was applied to investigate the expression profile of JAK2 gene in four cells transfected with eukaryotic expression vector, and house-keeping geneβ-actin were used as control. The results showed that JAK2 mRNA levels in the cells transfected with LWPGHR-pEGFP-C1 vector was higher than the other three cells transfected with pEGFP-C1, WZSPGH-pEGFP-C1, WZSPGHR-pEGFP-C1 vectors(P<0.05). JAK2 mRNA levels in the cells transfected with WZSPGHR-pEGFP-C1 vector was the lowest, suggested that those amino acid substitutions of WZSP's GHR could influence the expression levels of downstream JAK2 gene and reduce the physiological effect of GH.The defect of GHR of WZSP inbred was discovered and identified through our research, and the molecular mechanism of dwarfism in WZSP inbred maybe result from the defect of GHR.