| Ghrelin is a peptide hormone secreted by the gastrointestinal tract and initially found that it promots the role of growth hormone secretion,appetite and gastric acid secretion.Unpon the interaction with a variety of adipocyte derived factors, ghrelin also involves in many biological processes including energy metabolism and the pathogenesis of obesity.AdipoQ play a key role in the down-regulation of blood lipid,glucose,insulin resistance and atherosclerosis development.Threrefore, adiponectin or its agonist can be used for the treatment of insulin resistance-related diseases and cardiovascular diseases.In this study,the ghrelin and adipoQ coding sequence were cloned with purpose to fine the molecular evidences related to the growth and development difference among different pig breeds.Two pairs of primers were designed for ghrelin and adiponectin cDNA amplification according the sequences released in the GenBank.Total RNAs purified from Guangxi Bama Mini-pig gastric tissue or adipotissue was used as the reverse transcription templates and the strands of cDNA were synthesized by using MLV reverse transcription enzyme.Each segment was amplified by the polymerase chain reaction(PCR),then ligated into pMD18-T vector after purification.The recombined pMD18-T vector was transformed into the coliform DH5a for sequencing.The results showed that the cloned ghrelin and adiponectin cDNA contained 357bp and 732bp,respectively which included the entire coding sequences.The ghrelin or adiponectin cDNA from Bama mini-pig shared 99.7%,85.3%,81.4%and 82.1%or 99.7%,86.6%,79.1%and 84.5%homologue to the previously reported pig,human being,mice and cow's ghrelin or adiponectin cDNA in Genbank,respectively.Mutations of C→A(T→N)in ghrelin and C→T(P→S)and G→A(V→I)in adiponectin were detected.The PCR-SSCP polymorphisms analysis of ghrelin and adipoQ gene in the five pig breeds showed that:(1)Three genotypes(AA,AB and BB)were detected in ghrelin exon3.In Luchuan-pig and Guangxi Bama mini-pig,only genotype BB and AA was detected respectively,while in Duroc,Landrace and Yorkshire pig,three genotype including AA,AB and BB were detected.The B allele frequency in Duroc,Landrace and Yorkshire pig were 0.633,0.575 and 0.667,respectively.The genetic polymorphism information content(PIC)were 0.3567,0.3693,0.3456,0 and 0 in Duroc,Landrace,Yorkshire,Bama Mini-pig and Luchuan-pig,respectively.The result ofχ~2 test(χ~2=3.9278 in Duroc and 0.065 in Yorkshir,P＞0.05)showed that the allele in the state of Hardy-Weinberg equilibrium was detected in Duroc and Yorkshir but not in the other three pig breeds.(2)No polymorphism was observed in adipoQ coding sequence but AA,GG and AG genotypes were detected in intron 2 of adipoQ gene.In Landrace and Yorkshire breeds,only allele A was detected.The predominant allele G with frequency of 0.925 and 0.861 respectively existed in Guangxi Bama mini-pig and Luchuan-pig was observed.In Duroc pig,only AA and AG genotype were found and the predominant allele with frequency of 0.8 was A.The PIC value were 0.2688,0,0,0.1291 and 0.2108 in Duroc,Landrace,Yorkshire,Bama Mini-pig and Luchuan-pig,respectively.The result ofγ~2 test(γ~2=0.8098 in Duroc and 0.0769 in Luchuan-pig,P＞0.05)showed that the allele in the state of Hardy-Weinberg equilibrium was detected in Duroc and Luchuan-pig but not in the other three pig breeds.
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