Identification And Characterization Of Genes Relating Intramuscular Fat Deposition In Pigs

Intramuscular fat (IMF) content, correlated positively with meat tenderness, juiciness,and taste in pigs, has been revealed to be a major determinant factor affecting sensory meatquality. Conventional breeding is difficult to be carried out, because the IMF content isdifficult to measure in vivo and in piglet. At present, people conducted a large number ofstudies to explore the mechanism of IMF deposition in view of nutritional and genetic point.However, the nutritional control can only improve the IMF deposition, and the key geneaffecting IMF deposition has not yet been determined. Thus, this study was aimed atscreening the genes related to IMF deposition, to further reveal the molecular mechanism ofthe IMF deposition. Laiwu pig and Large White were used to construct high, low IMFLaiwu pig and Laiwu pig, Large White forward and reverse suppression subtractivehybridization (SSH) libraries to identify differentially expressed target genes, and some ofthese genes were varified by real-time PCR. In addition, studies of cloning and expressionwere done in pig NDUFS4gene. The main results are as follows:1. High IMF Laiwu pig and low IMF Laiwu pig positive and negative cDNA librarieswere constructed by using SSH technology.631and486positive clones were obtainedfrom positive and reverse cDNA libraries, respectively. The PCR results showed that theinserted fragments are mainly distributed in the0.1-1.0kb. Forward library, in which highIMF Laiwu Black was Tester and low IMF Laiwu Black was Driver, was screened byreverse Northen blot.35ESTs was obtained from97positive differancial clones throughsequencing. By analysis of BLAST alignment in NCBI, the differential expression geneswere divided to17known functional genes,3hypothetical genes and15unknownsequences.2. Laiwu pig and Large White positive and negative cDNA libraries were constructedby using SSH technology.1039and856positive clones were obtained from positive andreverse cDNA library, respectively. The PCR results showed that the inserted fragments aremainly distributed in the0.1-2.0kb. Forward library, in which Laiwu pig was Tester andLarge White was Driver, and reverse library, in which Large White was Tester and Laiwupig was Driver, were screened by reverse Northen blot.83and55ESTs were obtained from154and93positive differantial clone through sequencing, respectively. By analysis ofBLAST alignment in NCBI, these differential expression genes in positive library were divided to33known functional genes,15hypothetical genes,25unknown sequences and10ESTs which have no apparent homologe with sequences in NCBI; while in reverselibrary that were divided to21known functional genes,8hypothetical genes,18unknownsequences and8ESTs which have no apparent homologe with sequences in NCBI.3. Online classification analysis was done on the known functional genes andhypothetical genes, and found that these genes were involved in a variety of biologicalprocesses. By real-time PCR, we analysis the mRNA expression of fourteen differentiallyexpressed genes, the results showed that thirteen of the genes are consistent with screeningof library, including eleven up-regulated genes, which are SERF2, ATP6, NDUFS4,SERPINF1, h142, ACSL1, ADFP, ACADM, HNRNPA2B1, PDK4, P311, and twodown-regulated genes, which are AMPD1and PGK1.4. Porcine NDUFS4cDNA was cloned by using RT-PCR and5′RACE. PorcineNDUFS4cDNA shares93.56%,92.99%,87.31%and86.55%identity in nucleotidesequence, and92.57%,90.29%,88.57%and86.29%homology in amino acid sequencewith those of cattle, human, mouse and rat, respectively. Tissue expression profile analysiswas carried out using tissue cDNAs of the Laiwu pig as the templates to perform real-timePCR and the results revealed that the pig NDUFS4gene was highly expressed in LD,spleen and kidney, moderately expressed in liver, backfat, brain and spinal cord, weaklyexpressed in heart, and hardly expressed in lung, stomach and large intestine. Real-timePCR demonstrated that the difference of expression was significant between the two breeds(P<0.05), which was higher in Laiwu pig than in Large White.To sum up, we discussed IMF deposition mechanism of Laiwu Black pigs, constructedhigh, low IMF Laiwu Black and Laiwu Black, Large White forward and reverse SSHlibraries, and obtained some differentially expressed target genes, which may affect IMFdeposition. In addition, we firstly cloned the NDUFS4gene of pigs, and analyzed itsexpression profiles in tissues. Real-time PCR analysis indicated that the level of NDUFS4mRNA expression was higher in high IMF Laiwu Black group than in low IMF LaiwuBlack group, and in Laiwu Black than in Large White. The results laid an importantmolecular basis for further study of the pig muscle fat deposition and the geneticmechanism.