Cloning, Chromosome Mapping, Characterization Of Porcine Visfatin, Omentin And GNA14

Meishan breed is one of the best local porcine breeds of China, and is also an important breed resource in the world. Cloning, characterization and functional analysis of significant genes would benefit accumulating and exploring the gene resource information of important local porcine breeds in China, which would shed light on theories related to the further genetic improvement of excellent porcine breeds.Proteins coding by visfatin, omentin and GNA14 play important roles in the signal transduction pathway of adipose metabolism. Primes based on the homogenous sequences of these genes among different species were designed; cDNAs of specific genes were amplified through RT-PCR technology; the 3' end sequence of the cDNA was got through 3'RACE technology; genomic sequences of specific genes were got through genomic PCR; genes were mapped to specific chromosomes by radio hybridization mapping; gene expression patterns among different tissues were revealed through RT-PCR technology; additionally, eukaryotic and prokaryotic expression vectors were constructed through molecular cloning technologies and a target gene was overexpressed in a cell line through cell culturing and gene transfection. The results are as follows:1. A 2184 bp visfatin cDNA from Meishan pigs containing a full coding region of 1473 bp was obtained, its two related alternate splicing variants: 1958 bp and 1258 bp were also isolated (variant 2 and variant 3); Variant 2 is generated by a deletion of 226 bp from the wild type visfatin (visfatin variant 1), Variant 3 of visfatin is generated by a deletion of 926 bp from the wild type visfatin; visfatin of Meishan pig shares 95.5% homology with human visfatin. RT-PCR detection showed that visfatin variant 1 and variant 3 are expressed in heart, brain, muscle, adipose tissue, stomach, lung, testis, gut, kidney and liver, while visfatin variant 2 was not detected in testis and liver. A search of human, mouse and rat EST databases and RT-PCR detection of total mRNA from mouse indicated that the splice event of visfatin occurs exclusively in pig.2. A 1213 bp omentin cDNA from Meishan pigs containing a full coding region of 975 bp was obtained, omentin of Meishan pig shares 79.4% homology with human omentin. RT-PCR detection showed that omentin of Meishan pigs is expressed in gut and muscle. Porcine omentin includes 8 exons and 7 introns and it locates in 4q15-q16 of porcine chromosome.3. A 1359 bp GNA14 cDNA from Meishan pigs containing a full coding region of 1068 bp was obtained. GNA14 of Meishan pig shares 91.5% homology with human GNA14. RT-PCR detection showed that GNA14 of Meishan pigs is expressed in kidney and lung. GNA14 locates in lq21-27 of porcine chromosome.4. It is effective to overexpress omentin in PK15 (Porcine Kidney-15) cell line through transfection with lipofectamine. The eukaryotic fusion expression vectors for full length or partial visfatin/red fluorescent protein, the eukaryotic non-fusion expression vector for full length visfatin/green fluorescent protein (plRES2-EGFP-visfatin), the prokaryotic fusion expression vectors for full length visfatin (pTYB12-visfatin) and the eukaryotic non-fusion expression vector for full length omentin/ green fluorescent protein was constructed (plRES2-EGFP-omentin) by molecular cloning technologies.