On-line version ISSN 2221-4062
S. Afr. j. anim. sci. vol.39 n.4 Pretoria Jan. 2009
P.X. NiuI; S.H. ZhaoI; B. FanI,II,#
IKey Laboratory of Agricultural Animal Genetics, Breeding & Reproduction of Ministry of Education, Key Laboratory of Swine Genetics & Breeding of Ministry of Agriculture, Huazhong Agricultural University, Wuhan 430070, P R China
II Department of Animal Science and Centre for Integrated Animal Genomics, Iowa State University, Ames, 50011, USA
The PR domain containing the 16 (PRDM16) gene, also known as the MDS1/EVI1-like gene (MEL1), may act as a bidirectional switch between brown fat and skeletal muscle in mice. The molecular characteristics and possible biological function of porcine PRDM16 gene have been less reported. In this study, the mRNA expression profile, linkage mapping and association analyses of the PRDM16 gene were carried out in the pig. The PRDM16 mRNA was expressed widely in various tissues including fat and the longissimus dorsi muscle. One SNP c.-3284+171C>T in intron 14 was identified and that made the PRDM16 gene being assigned between SWR1130 and SW122 on SSC6. The different genotypes of c.-3284+171C>T were significantly associated with backfat and loin muscle area in the ISU Berkshire × Yorkshire pig resource family, but such associations were not verified in another pig population. The discovery of additional mutations and association studies are warranted before the PRDM16 gene can be recommended for marker assisted selection in the pig.
Keywords: PRDM16, backfat, loin muscle area, linkage mapping, association analysis
The PR domain containing the 16 (PRDM16) gene, comprising of a PRD1-BF1-RIZ1 homologous (PR) domain and several zinc fingers, has been shown to turn on the expression of brown fat-selective genes and turn off the expression of white fat-enriched genes, and therefore acts as an critical regulator during adipose development (Seale et al., 2007; Kajimura et al., 2008). Furthermore, knockdown of the PRDM16 gene from brown fat precursors led to muscle differentiation in mice (Seale et al., 2008). These findings imply that the PRDM16 gene may play a role in the conversion and growth of fat and skeletal muscle tissues. The pig is one of the important livestock species for meat supplies, and it can also easily deposit fat during the growth period. This makes it interesting to explore if the PRDM16 gene is involved in the development of fat and skeletal muscle tissues in pigs. In this study, mRNA expression patterns of the PRDM16 gene were analyzed and further SNP discovery and association analyses were implemented to identify the potential genetic marker associated with fatness and muscle mass in the pig.
The tissues used for mRNA expression analysis were collected from three Yorkshire piglets, and included the heart, liver, spleen, lung, kidney, brain, longissimus dorsi muscle, interior fat, stomach, small intestine and testis. Total RNA was isolated following the instruction of TRIzol reagent kit (Tiangen Biotech, Beijing, China) and mRNA expression patterns were determined by the RT-PCR technique. The PCR mixture consisting of 2 µg total RNA and 5 µL oligo (dT) was incubated at 70 ºC for 5 min to eliminate the RNA secondary structure. The reactions were chilled on ice for 2 min and the remaining reagents including 5 µL dNTPs (10 mM each dNTP), 10 µL 5 × buffer, 2.5 µL RNAase inhibitor with 300 U M-MLV reverse transcriptase (Promega, Madison, WI, USA) were added to a total volume of 50 µL. Reverse transcription reaction was proceeded for 1 h at 42 ºC. Finally, the reverse transcriptase was inactivated by 5 min incubation at 90 ºC. 2 µL single-stranded cDNA was amplified with a pair of primers (F1:5'-CTGGCTGCACGTCTGTTACC-3' /R1: 5'-CCTGAAGGTCACGCCTAGAG-3'). The PCR programme was as follows, 4 min at 94 ºC; 28 cycles of 30 s at 94 ºC, 30 s at 60 ºC, 20 s at 72 ºC, and a final extension of 5 min at 72 ºC. 5 µL PCR products were used to detect the expression profile in agarose gel, and the amplification of the RPL32 gene (F2: 5'-CGGAAGTTTCTGGTACACAATGTAA-3'/R3:5'-TGGAAGAGACGTTGTGAGCAA-3') was performed as a positive control. The mRNA expression patterns of the porcine PRDM16 gene in various tissues are shown in Figure 1. The PRDM16 gene was highly expressed in the brain, lung, testis, interior fat and kidney, and it was moderately expressed in the heart, liver, spleen, longissimus dorsi muscle, stomach and small intestine. In humans it was found that the PRDM16 gene is widely expressed in a variety of normal tissues, especially in the heart, lung, kidney and pancreas (Lahortigav et al., 2004). This is in agreement with the results presented in pigs. In addition, the expression patterns implied that the PRDM16 gene has additional functions besides its roles on brown fat tissues. For instance, the up-regulation of PRDM16 could initiate a leukemogenic cascade (Shing et al., 2007; Modlich et al., 2008), and the copy number of PRDM16 molecules of patients with osteosarcoma was higher than that of the normal (Man et al., 2004).
The human PRDM16 transcript (ENST00000378389) retrieved from the Ensemble database (http://www.ensembl.org/index.html) was used as an entry to search for a homologous pig sequence (http://www.ncbi.nlm.nih.gov/projects/genome/guide/pig). The pig genome sequence fragments showing above 85% identity with the entry sequence, were obtained. The pairs of primers were designed for PCR amplification from the pig genomic DNA. PCR products were commercially sequenced. SNP discovery was implemented by sequencing the pooled PCR products amplified from six DNA samples and each two were from Yorkshire, Landrace and Tongcheng pigs. Three SNPs, c.-3284+171C>T, c.-3284+207C>T, c.-3284+208C>T, in intron 14 were identified and then deposited into a dbSNP database (dbSNP acc. no. ss136268357, ss136268362 and ss136268366). The SNP c.-3284+171C>T could be distinguished by the restriction enzyme, AciI. A PCR-AciI RFLP technique was developed using the redesigned primers F3/R3 (F3: 5'-AGAGCTAGGGCCACAGAG-3'/R1: 5'-CCAACAGCGAGATGAACC-3'), with allele T revealing a 280bp and allele C revealing a 215bp and a 70bp fragment. The PCR-RFLP procedure for SNP genotying was as follows: PCR mixture (10 µL) included 1×PCR buffer, 0.2 µM each primer, 150 µM each dNTP, 1.5 mM MgCl2, 2U Taq DNA polymerase (Takara Company, Dalian, China) and 12.5 ng genomic DNA; PCR reaction comprised of the initial denature at 95 ºC for 5 min, 30 cycles with 94 ºC for 30 s, 60 ºC for 30 s, 72 ºC for 15 s, followed by a final extension at 72 ºC for 5 min; RFLP reaction mixture (10 µL) consisted of 1 µL 10×buffer, 1U restricted enzyme AciI (NEB, Ipswich, MA, USA) and 3 µL PCR products. Samples were incubated overnight at 37 ºC. SNP genotyping was performed in two different pig populations. Population A (n = 205) included the Yorkshire (Y, n = 26), Landrace (L, n = 26) and Tongcheng (T, n = 49) breeds, and crossbreds L×(Y×T, n = 54) and Y × (L × T, n = 50). The association analysis was implemented using the mixed procedure (SAS, 2006) and this model treated population/population combination, sex, slaughter date and marker genotyping as fixed effects, dam as random effect and body weight as covariate (Tang et al., 2008). The genotyping was also performed in the ISU Berkshire × Yorkshire (B×Y) pig resource family comprising of 515 F2 animals (Malek et al., 2001). The association analyses were implemented using the mixed model procedure, including sex, slaughter date and marker genotypes as fixed effects, dam (litter) as random effect and body weight as covariate. In population A, fat traits including average backfat, backfat at shoulder, 6 - 7th rib, loin, last rib and rump were used for association analysis, as well as muscle mass traits including lean meat ratio and loin eye area. The details about the measurement of these traits were described by Tang et al. (2008). In population B, the analyzed traits included average backfat, backfat at 10th rib, lumber and last rib and loin eye area (Malek et al., 2001).
The SNP c.-3284+171C>T showed high polymorphism in both populations A and B. In population B c.-3284+171C>T was highly significantly (P <0.01) associated with loin eye muscle area and was significantly (P <0.05) associated with lumbar backfat, carcass weight and body length. It was also suggestively significantly (P <0.1) associated with average backfat and last rib backfat (Table 1). The zinc-finger PR domain-containing protein encoded by the PRDM16 gene can induce expression of a number of genes associated with white and brown fat (such as adipokine resistin, PGC-1α and UCP-1), mitochondrial biogenesis and oxygen consumption. Most recently the PRDM16 gene has been shown to interact with either PGC-1α or CtBPs to activate brown genes or to suppress white gene expression, respectively (Seale et al., 2007; Kajimura et al., 2008). PRDM16 binds and co-activates the transcriptional function of PPAR-γ, which is known to be involved in adipose tissue development. In addition, loss of PRDM16 from brown fat cells could cause an increase in myogenic gene expression and bona fide skeletal muscle differentiation (Seale et al., 2008), but the exact mechanisms need to be further addressed. It is possible that in pigs the PRDM16 gene functions in fat and lean meat growth with the same molecular mechanisms. However, there was no significant association between SNP c.-3284+171C>T genotypes and the analyzed traits in population A (data not shown), which suggested that additional SNP mining and association analyses in pig populations with different genetic backgrounds are necessary.
The human PRDM16 gene locates at HSA 1p36.23-p33, where it corresponds to the SSC6 of pigs (https://www-lgc.toulouse.inra.fr/pig/compare/compare.htm). The SNP c.-3284+171C>T was used for linkage mapping in the B×Y population. The mapping procedures were performed with two-point linkage analyses using an improved CRIMAP (Ver 2.5) developed by Evans I and Maddox J (16 Park Square Port Melbourne, 3207 Australia, E-mail: firstname.lastname@example.org). The PRDM16 gene was mapped on SSC6 and the order of makers was SWR1130 (10.6cM)-PRDM16-(3.1cM) SW122. Numbers of QTL related to fat and loin muscle area have been mapped around this region (http://www.animalgenome.org/cgi-bin/QTLdb; Paszek et al., 2001; Yue et al., 2003; Edwards et al., 2008). Combing the association analyses and linkage mapping promote the porcine PRDM16 gene as a candidate gene for fat deposit and muscle production. Further work on causative mutation discovery and function analysis of PRDM16 gene in pigs is required.
This study was financially supported by the National Natural Science Foundation of China (30771169), Wuhan Youth Chenguang Project (200750731282) and Key Project of National Basic Research and Developmental Plan (2006CB102105) of China. The authors thank the members of Max F Rothschild's research group at Iowa State University for their assistance in this research.
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