Isolation And Initial Functional Analysis Of Differentially Expressed Genes In Fat Tissue Between F1 Hybrids And Their Parents From Large White × Meishan Pig Cross Combination

Fat tissue is not only the main location saving up fat, which can store energy, maintain animal heat, protect vital organisms, lighten physical pressure but also an endocrine organ.Due to the difference of origin, genetic background, local environment, feed manner and artificial selection between Chinese indigenous and foreign commercial pig breeds, they have different intrinsic features, and there is a large difference between their fattness traits.When these two breeds are hybridized, F1 hybrids tend to demonstrate hybrid vigor and produce heterosis. Further understanding on gene function and expression regulation has indicated that the difference traits performance and heterosis are in fact the external exhibition of differential genes expression and regulation. Thus, mRNA differential display technique was used to isolate and identify the differentially expressed genes between Large White and Meishan, and their F1 hybrids, Large White×Meishan and Meishan×Large White pigs in the present study, and the gene functions were also primarily analyzed. The results are as follows:1. The gene expression were analyzed between F1 hybrids, White×Meishan and Meishan×Large White, and their parents, Large White and Meishan pigs by mRNA differential display. Eight patterns of gene expression were observed at both four and six months old hybrids and their parents, which included: P1, bands detected in both hybrids and parents; P2, bands occurring in hybrids and one parent; P3, bands occurring in one hybrid and one parent; P4, bands visualized in only hybrids; P5, bands occurring in only one hybrid; P6, bands observed in parents and one hybrid; P7, bands occurring in only parents; and P8, bands occurring in only one parent. We also observed nearly 3000 bands in differentially displayed PAGE gel, and almost 2000 ones can be repreated in duplicate PCR. In addition, the comparison of gene expression patterns between 120 days old and 180 days old hybrids and their parents showed that the gene expression were varied.2. 60 differentially displayed ESTs in PAGE gel were cloned, including 39 ESTs at 120 days old and 21 at 180 days old, between hybrids and their parents. Most ESTs were not homologous to the sequences in GenBank database. The result of validation showed that a majority of ESTs were really differentially expressed, while a few of them weren't by semi-quantitative RT-PCR. Some ESTs were subsequently deposited in GenBank and the accession numbers were CV507051-CV507087.3. We cloned the full-length cDNA of seven genes by rapid amplification of cDNA ends (RACE) in combination with in silico cloning, which included: (1)ACL, which has 4378bp and encodes 1076 amino acids; (2) SMPX, which has 863bp and encodes 86 amino acids; (3)ANGPTL4, which has 1847bp and encodes 412 amino acids; (4) IDH1, which has 2264bp and encodes 402 amino acids; (5) IDH3β, which has three isoforms, IDH3β₁(1247bp), IDH3β₂(1540bp), and IDH3β₃(1445bp), and encode 383, 385 and 385 amino acids, respectively; (6) IDH3γ, which has 1346bp and encodes 392 amino acids; and (7) RPL28, which has 513bp and encodes 137 amino acids. 4. Using DNAStar, CLUSTAL W and some other related software, we analyzed the gene structure, protein structure and conserved motifs of these seven genes. In addition, the corresponding phylogenetic trees were constructed.5. The differential expression of these seven genes between F1 hybrids and their parents were further identification by real-time quantitative PCR. The result showed that the trend of differential gene expression was consonant with the result by semi-quantitative RT-PCR. The mRNA expression level of ACL, IDH3βand RPL28 was higher in F1 hybrids than in both Meishan and Large White pigs, whereas the mRNA of SMPX, ANPTL4, IDH1 and IDH3γwas more abundant in Large White than both two hybrids and Meishan pigs. The tissue distribution of these seven genes in heart, liver, spleen, lung, kidney, stomach, small intestine, uterus, ovary, backfat and longissmus drosi indicated that they were expressed in most tissues and displayed different expression patterns.6. We cloned all introns of four genes (ANGPTL4,IDH1,IDH3βand IDH3γ) and partial introns of other three genes among these seven genes, and analyzed the genomic structure and polymorphism of these 4 genes. The results are as follows: (1) ANGPTL4 gene contains seven exons and six introns. Only two mutations were found. One is G→A transversion in third intron and the other is C→T transversion in sixth intron; (2) IDH1 gene contains ten exons and nine introns. Nine mutations were found in total, including one in second exon (T1112C), five in sixth intron (G13194T, C13228T, C13336T, C13422T and A13477G), three in ten exon (T20531A, G20543A and C inserted mutation at 20534th bp); (3) The genomic structure of IDH3β₁ and IDH3β₂ is organized in twelve exons, while that of IDH3β₃ contains thirteen exons separated by twelve introns. Eight mutations were found in total, including one in first intron (G63A), one in second exon (G187C), two in second intron (G287C and C336T), one in sixth exon (G2732A) and two in eleventh exon (A4277G and 25bp mini-satellite); and (4) IDH3γgene contains ten exons and nine introns. Micro-satellite (GT dinucleotide repeats) was found in second intron.7. Genotyping of a total of six polymorphic locus showed that there are abundant polymorphisms in various pig breeds. Association analysis was performed between polymorphisms and important product traits in Large White×Meishan F2 offspring, and the results showed: (1) For insertion-mutated polymorphism in 24th intron of ACL gene, significant effects were observed on LMP, FMP, CFW, RFT, pH(LD), pH(BF) and WLR; (2) There are significant difference between ACL XhoI-RFLP genotypes and SP, BP, DP, LFW, CFW, pH(LD), pH(BF), pH(SC) and WHC; (3) Statistically significant association were found between ANGPTL4 PCR-SSCP in third intron and LEA, WM, IMF and MM1; (4) For minisatellite polymorphism of IDH3βgene, significant effects were observed on BP, LEA, WM, IMF and pH(SC); (5) For insertion-mutated polymorphism in 5'flanking region of IDH3βgene, significant effects were observed on LFW, LEA, pH(LD), IMF and WM; and (6) For microsatellite polymorphisms of IDH3γgene, significant effects were observed on DP, RNS, AST and WM.8. 936bp 5'flanking sequence ofACL gene and 2447bp 5'flanking sequence of IDH3βgene were obtained by using TAIL-PCR. The former lacks of TATA box and comprise high G+C content (61.75%), whereas the latter lacks both TATA box and CAAT box with a percentage of 55.8 % A+T content.9. To determine the location of the promoter activity of pig ACL and IDH3β, we constructed 8 and 19 recombinants of progressively 5'-deleted DNA fragment linked to the pGL3 reporter, respectively. These recombinants were transiently transfected into PK15 cells. Transcriptional activity of ACL recombinants normalized by Renilla was significant difference with pGL3-Basic expect for construct -27 and -15 (P＜0.01). Construct -919 contains highest activity. 3 times reduction of transcriptional activity from-919 to -679bp indicated that negative regulation factors located probably in this region. The activity started on construct -73 suggested the basal promoter activity was located within the -73 to +77bp region; Transcriptional activity of IDHβrecombinants was not significantly different between the recombinant -58 and pGL3-Basic. The activity started on construct -82, decreasing with the length of the fragment up to -164 in despite of a bit of fluctuation, and kept increasing from construct -164 up to -279. Thus, the basal promoter activity was located within the -82 to +16bp region, whereas the upstream 197bp conferred maximal transcriptional activity. The extension of the 5'flanking sequence up to -2435 diminished the promoter activity. In addition, the IDH3βpromoter with the inserted-mutation possessed higher activity compared with the wild one.