Isolation, Imprinting Identification And Methylation Analysis Of Five Genes In Porcine Embryo

Imprinted genes are the genes whose expression is dependent on parental origin that is only one allele from the father or mother is expressed and the other allele is not expressed or lowly expressed. In mammals, they play an essential role in the regulation of fetal growth, development, placental function and reproduction traits. They not only control the gene expression related to quality traits, but also affect the phenotypic variance of many quantitative traits and the phenotypic values. Studies of genome-wide scanning for QTL revealed that many QTLs are maternally or paternally imprinted, which significantly affect growth, backfat thickness, carcass composition and reproduction. However, comparing to the extensive research of imprinting in human and mouse, there is still a dearth of knowledge about imprinting in swine. Therefore, the identification of new imprinted genes in swine is very important, not only for completing the imprinting study in livestock but also for the comparative genomic analysis of genomic imprinting among different species.In this study, the RT-PCR-RFLP method were used to detect the imprinting status of five porcine gene at developmental stages of day 30,65 and 100 of gestation. Using PCR-RFLP, we detected SNPs of four genes in different pig breeds and performed associations with carcass and meat quality traits in Large White and Meishan F2 hybrids. Using Real-time PCR and semi-quantitative RT-PCR, we analysed the tissue expression of candidate genes. We obtained promoter sequences of four genes, and analysed the promoter activity of two genes.in addition, we analyzed the methylation status of CpG islands. The main results are as follows:1. We isolated and cloned the porcine DIO3,RTL1,DIRAS3 and COPG2 genes, which all contain the complete open reading frame (ORF), the deduced amino acid sequences have high similarity with human and mouse.2. The reciprocal Yorkshire×Meishan F1 hybrid model and the RT-PCR-RFLP method were used to detect the imprinting status of porcine DIO3, RTL1, NNAT, DIRAS3 and COPG2 genes at the developmental stages of day 30,65 and 100 of gestation. Porcine DIO3, RTL1 and DIRAS3 genes were paternally expressed in day 30 fetuses, day 65 and day 100 fetal tissues, including heart, liver, spleen, lung, kidney, stomach, small intestine, skeletal muscle, brain and placenta; NNAT gene escapes imprinting in the day 65 and day 100 fetal spleen, but paternally expressed in the other tissues; COPG2 gene was not imprinted in all developmental stages.3. We detected the tissue expression profile of DIO3 gene in the two month Yorkshire sow by Real-time PCR, and found that it high expressed in adipose tissue. Semi-quantitative RT-PCR analysis was performed to detect the relative mRNA expression profile of RTL1 gene in 13 different tissuses, which were collected from day 65 fetuses of Yorkshire, the result showed that the RTL1 gene was expressed at a high leveal in the small intestine, biceps femoris and semitendinosus. we used the real-time PCR technique to examine the mRNA expression in two pig breeds (Yorkshire and Meishan) at four stages (fetal 60 days and postnatal 35,60 and 120 days) and three types of muscles (longissimus dorsi muscle, LD; biceps femoris, BF; semitendinosus,ST). Moreover, we found the expression of RTL1 was reduced as the differentiation of C2C12 cells. Five different transcripts were detected in COPG2 gene, named COPG2-1, COPG2-2, COPG2-3, COPG2-4 and COPG2-5 respectively. The expression level of different COPG2 isoforms in different ten tissues of two develepent stages (fetal 65 days and postnatal 60 days) were detected by semi-quantitative RT-PCR.4. According to the sequences of database, PCR primers were designed to amplify the promoter sequences of porcine RTL1, NNAT and DIRAS3 gene. The promoter regions of porcine LCAT gene were obtained by genomics walking. Several binding sites for MyoD,YY1 and NF-kappaB which are involved in muscle development were observed in RTL1 and NNAT promoter regions. In addition, we analyzed the promoter activety by dual luciferase reporter system. Using the bioinformatics software, we predicted the CpG islands in the RTL1 and NNAT promoter regions, and analyzed the methylation of the CpG islands by Bisulfite Sequencing PCR. We speculated that the two CpG islands in NNAT gene promoter region maybe the differentially methylated region.5. Using PCR-RFLP, we detected SNPs of DIO3, RTL1, NNAT and COPG2 genes in different pig breeds and performed associations with carcass and meat quality traits in Large White and Meishan F2 hybrids. The results showed:(1) DIO3, A687C-Acy I-RFLP is significant association with ratio of lean to fat (RLF), fat meat percentage (FMP), lean meat percentage (LMP), shoulder fat thickness (SFT),6-7th rib fat thickness (RFT), buttock fat thickness (BFT), loin eye area (LEA), longissimus dorsi drip loss rate (LD DLR), longissimus dorsi water holding capacity (LD WHC), Water Moisture (m. longissimus Dorsi, LD) (WM), meat color value (LD), meat color value (BF), meat marbling (LD) and meat marbling (BF) (P<0.05), is higher significant association with intramuscular fat (LD) (P<0.01); (2) RTL1,G1209A-Sat I-RFLP is significant association with skin percentage (SP), internal fat rate (IFR),6-7 rib fat thickness (RFT), thorax-waist fat thickness (TFT), Meat pH (m.longissimus Dorsi, LD) and meat color value (BF) (P<0.05), is higher significant association with bone percentage (BP), shoulder fat thickness (SFT), buttock fat thickness (BFT), average backfat thickness (ABT), longissimus dorsi drip loss rate (LD DLR), longissimus dorsi water holding capacity (LD WHC) and meat color value (LD) (P<0.01); RTL1, A3929G-Van91 I-RFLP is significant association with internal fat rate (IFR), shoulder fat thickness (SFT), loin eye height (LEH), loin eye area (LEA), Water Moisture (m.longissimus Dorsi, LD) (WM), meat color value (LD) and intramuscular fat (LD) (P<0.05), is higher significant association with skin percentage (SP), longissimus dorsi drip loss rate (LD DLR), longissimus dorsi water holding capacity (LD WHC) meat color value (BF) (P<0.01); (3) A-107G in the promoter of NNAT gene is significant association with loin eye height (LEH), Loin Eye width (LEW), loin eye area (LEA), Water Moisture (m.longissimus Dorsi, LD) (WM), meat marbling (LD) and meat marbling (BF) (P<0.05); (4) G/A polymorphism in the 3'UTR of COPG2 is significant association with fat percentage (FP), thorax-waist fat thickness (TFT), buttock fat thickness (BFT), average backfat thickness (ABT), Meat pH (m.longissimus Dorsi, LD), Meat pH (m.Biceps Femoris, BF) and Meat pH (m.Semispinalis Capitis, SC) (P<0.05).