Analysis Of Genetic Background Of The Growth Related Genes In Banna Miniature Inbreeding Pig

The Banna miniature pig (BNMP)originated from the Xishuangbanna Prefecture,Yunnan Province, China,was characterized by it′s miniature body size and earliermaturity. BNMP has been obtained by inbreeding method since1987, whichcoefficience reached nearly98.6%for its precious genetic resourse in2005.BNMPhas been used widely as laboratory animal and in the xenotransplantation. BNMP isalso an ideal experimental animal as model of dwarf animal.In the present study, Growth hormone (GH), Growth hormone receptor (GHR),Pituitary specific transcription factor-1(PIT-1), Somatostatin (SS) and Ghrelinassumed as candidate genes with purpose to fine the molecular evidences related tothe growth retardation of BNMP. And the main results are as follows.1. The serum GH of3-month-old BNMP were slightly lower than that of otherbreeds, but the difference isn't significant (P﹥0.05). The serum IGF-1of BNMPwere significant lower than that of other breeds (P﹤0.05).2. The complete sequence and cDNA sequence of BNMP GH gene were clonedand submitted to GenBank (Accesssion No.JF276445, No.JF276447). Thereheterozygous transitions(C509T,T597C and T1202C) in exon2and exon4werefound in BNMP GH, which results in anmio acid changes (A9V,R22Q and S104P);The substitutions of A9V and R22Q lie in the sequence encoding signal peptide thataffect intracellular transport and positioning of GH mature protein passably, but thesubstitution of S104P maybe affect the structure and function of mature protein. DNApolymorphism analysis showed that there were24haplotypes in60samples, DNApolymorphism of GH in BNMP was meager (Pi=0.00192±0.00102); Thepolymorphism characteristic of pGH gene was different among swine breeds.3. The cDNA sequence of BNMP GHR gene were cloned and submitted toGenBank (Accesssion No.JF276446). Four anmio acid substitutions were detected inexon10of GHR in BNMP, among these mutations, the mutation of G1225T which causedSer instead of Ala. The different in property between Ser and Ala was significant, somay affected GHR's structure and function. Eighteen genotypes were detected inexon10of GHR in all pig breeds by PCR-SSCP polymorphism analysis. The distributions of the genotypes in amplification with primer P2among porcine breeds weredifferent significantly.Some genotypes only belonged to BNMP.4. By cloning and sequencing,the complete sequences of SS and the majoritysequences of PIT-1and Ghrelin gene were obtained, only one mutation (A110G) inSS was detected which caused Lys instead of Arg, more mutations which lies inintrons not affect the structure and function of mature protein, these results showedthat no significant mutation occurrence in SS, PIT-1and Ghrelin.5. The expressions of GH, GHR and IGF-1genes in tissues were further testedwith Real-Time PCR. Real time PCR analysis indicated that the expression levels ofGH,GHR in tested tissues wasn't significant (P>0.05), and the lowest expressionlevel of IGF-1was found in the muscles of BNMP (P<0.05); Thus we concluded thatmutations detected in GH or GHR interfere signal transduction pathways ofGH-GHR-IGF, ultimately affect the normal growth of the pig.6. The eukaryotic expression vector of BNMP and "Junmu" White pig's GH andGHR gene were constructed, and transfected into PK-15cells. The real time PCR wasapplied to investigate the expression profile of IGF-1gene in cells transfected withconstructed eukaryotic expression vector. The results showed that IGF-1mRNA levelsin the cells transfected with eukaryotic expression vector of BNMP's GHR gene wasthe lowest in all cells transfected with our constructed eukaryotic expression vector(P<0.05). It's suggested that those amino acid substitutions of BNMP's GHR couldinterfere signal transduction pathways of GH-GHR-IGF.The defect of GHR in BNMP inbred was discovered and identified through ourresearch, and the molecular mechanism of growth retardation in BNMP inbred mayberesult from the defect of GHR.