| White adipose tissue was believed to be just an energy-storage organ, but it is nowrecognized to be a highly active endocrine organ in energy homoeostasis. Visceral adiposetissue-derived serine protease inhibitor （vaspin） is an interesting novel adipocytokine withinsulin-sensitizing effects. Some studies have suggested that vaspin could play an importantrole in the development of obesity and metabolic disorders. So it is an important candidatefactors for the breeding. However, the impact of vaspin gene variants on the growth traits andobesity has not been determined yet. Herein, genetic variants within bovine vaspin gene werestudied by PCR-RFLP and ACRS PCR-RFLP detection. Association analysis and mRNAexpression patten were conducted and the eukaryotic expression vectors（pAdTrack/CMV-vaspin） were constructed.1) Genetic variation of bovine vaspin geneIn the present study, we firstly screened the entire coding region within bovine vaspingene and identified two novel mutations at exon2and exon4in1235individuals of fiveChinese cattle breeds, and could be genotyped by MspI PCR-RFLP and HhaI ACRSPCR-RFLP respectively. The MspI locus （NW001494061: g.1124477G>A）, locating atposition nt501of exon2, generates synonymous mutation at167amino acid position （F167F）,and the HhaI locus （NW001494061: g.1118561T>C）, locating atposition nt144of exon4,leads to a substitution of aspartic acid to glycine at350position （D350G）.2) The association analysis between genetic variation and growth trait in bovine vaspingeneIn the genetic diversity parameters analysis of vaspin gene, we calculated the gene（genotypes） frequencies of the two mutations in five Chinese indigenous breeds. At theg.1124477G>A locus, the QC and NY cattle populations were in accordance with Hardy-Weinberg equilibrium by χ2test, while in g.1118561T>C locus it were NY and CSR cattlepopulations which were in the balance of Hardy-Weinberg. Genotypic frequencies and allelic frequencies at these two loci were found to be similarly in NY, QC, JX and LX breeds,but the CRS population showed low mutational allelic frequency. This situation may be dueto that the NY, QC, JX and LX breeds were all mainly located in the central region of China,but the CRS breed located in the Northern part of China. Relationships between twomutations and growth traits （body weight, body height, body length, and chestgirth） wereanalyzed in NY cattle aged6,12,18and24months. Interestingly, both two mutations weresignificantly associated with growth traits at24months, but no significantly associated at6,12and18months. In the MspI locus （NW001494061: g.1124477G>A）, the individuals withgenotype GG had greater body weight and chest girth than those with genotype AA at24month-old （P<0.05）. Similarly, in the HhaI locus （NW001494061: g.1118561T>C）, theindividuals with genotype TT had greater body weight, body length, chest girth and averagedaily gain than those with genotype CC at24month-old （P<0.05）.3) Expression pattern analysis of bovine vaspin gene.Real-time PCR was performed to assay vaspin gene mRNA expression in new born andadult cattle. The results showed that vaspin was ubiquitously expressed in most tissues,including heart, liver, lung, kidney, small intestine, stomach, fat and skeletal muscle. At newborn stage, vaspin gene was strongly expressed in heart, small intestine, skeletal muscle andfat. Then, at adult stage, vaspin was highly expressed in heart, liver, lung and skeletal muscle,which showed little difference between two developmental stages. Meanwhile, almost novaspin mRNA expression was detected in either in new born or adult cattle spleen.4) Construction of bovine vaspin gene eukaryotic expression vector.Using total cDNA from liver tissue of Qinchuan cattle as PCR template, coding sequenceof bovine vaspin gene was cloned. The pAdTrack/CMV-vaspin recombinant plasmid wasconstructed and identified by sequencing. Then the recombinant plasmid was transfected into293A cell line. Cultured48h after transfection, qRT-PCR was used to detect to expressionefficiency. Results showed the expression level of treated group was significantly higher thenthe control group, which meant pAdTrack/CMV-vaspin vector was successfully constructed. |
