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Cloning and polymorphisms of the 3′ untranslated region of malic enzyme gene in Chinese red cattle
Meat Science 89 (2011) 72–75
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Meat Science j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / m e a t s c i
Cloning and polymorphisms of the 3′ untranslated region of malic enzyme gene in Chinese red cattle G.L. Zhou a, G.L. Zhang b, Y. Cao b, H.G. Jin b,⁎ a b
College of Life Science, Liaocheng University, Liaocheng, 252059, People's Republic of China Branch of Animal Husbandry, Jilin Academy of Agricultural Sciences, Gongzhuling, 136100, People's Republic of China
a r t i c l e
i n f o
Article history: Received 20 December 2010 Received in revised form 26 March 2011 Accepted 29 March 2011 Keywords: Malic enzyme gene Alternative transcripts Polymorphism Chinese red cattle Meat quality and carcass traits
a b s t r a c t The objective of this study was to identify alternative transcripts and single nucleotide polymorphisms (SNPs) in the 3′-untranslated region (3′ UTR) of bovine malic enzyme (ME1) gene and to evaluate the extent to which polymorphisms were associated with meat quality and carcass traits in Chinese red cattle. Two transcripts, long transcript and short transcript that differ in the length of the 3′ UTR were cloned. A single nucleotide polymorphism was detected in 3′ UTR and a restriction site for endonuclease ME1-Dra I was also found. The result revealed that the ME1-Dra I genotypes had a significant effect on cooking loss, pH measured 24 h post-mortem (pH24h) and eye muscle area (P b 0.05). In conclusion, the SNPs may be used as DNA markers to select for meat quality and carcass traits in Chinese red cattle. © 2011 Elsevier Ltd. All rights reserved.
1. Introduction Malic enzyme (ME1) is a lipogenic enzyme. For de novo biosynthesis of fatty acids, considerable amounts of NADPH are required (Guay, Madiraju, Aumais, Joly, & Prentki, 2007). To date, two mitochondrial (NAD+- and NADP+-dependent) and one cytosolic (NADP+-dependent) malic enzyme isoforms have been identified in mammals (Chang & Tong, 2003). Cytosolic ME1 forms part of the tricarboxylate shuttle, which releases acetyl-CoA from the mitochondria to the cytosol. NADPH and acetyl-CoA produced in such a way can be used in fatty acid biosynthesis and many other metabolic processes. Different from human and rodents, in which the principal carbon source for fatty acids biosynthesis is glucose, the acetic acid produced by microorganisms in the rumen acts as the principal carbon source in ruminant species (Bergen & Mersmann, 2005). Therefore, the glucose-pyruvate-acetyl CoA pathway is of little significance in ruminants and the contribution of malic enzyme in NADPH production of ruminants is relatively small in contrast to that of human and rodents (Vernon, 1980). The ME1 gene is known to be expressed in several tissues in rat (Dozin, Magnuson, & Nikodem, 1985), chicken (Ma et al., 1990) and pig (Nunes et al., 1996), and the expression is regulated by both hormones and nutrition levels. In particular, triiodothyronine (Rao, Cronrath, & Rao, 1984) and insulin (Thompson & Drake, 1982)
⁎ Corresponding author. Tel.: + 86 434 6282019; fax: + 86 434 6282020. E-mail address: [email protected] (H.G. Jin). 0309-1740/$ – see front matter © 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.meatsci.2011.03.023
stimulate ME1 in liver and adipose tissue, while glucagon blocks this effect (Siddiqui, Goldflam, & Goodridge, 1981). A high-carbohydrate, low-fat diet after a period of starvation results in increased ME1 expression in liver and adipose tissue (Careche, Obato, Ros, Moreno, & Garcia–Ruiz, 1985). The ME1 locus has been mapped on porcine chromosome1 (SSC1), and two transcript forms have been described (Nunes et al., 1996). Several quantitative trait loci (QTL) affecting growth, backfat and other production traits have been positioned on SSC1. Most of these studies report a single QTL (Milan et al., 2002), but the existence of more than one QTL cannot be ruled out (Quintanilla, Milan, & Bidanel, 2002). Moreover, comparison of glucose-6-phosphate dehydrogenase, acetyl-CoA-carboxylase and ME1 lipogenic enzyme activity in the intramuscular tissue of Meishan and growing Large White pigs revealed that ME activity is much higher in Meishan pigs, being one of the major factors influencing intramuscular fat deposition (Mourot & Kouba, 1999). Vidal et al. (2005) amplified fragments of 1457 and 1459 bp corresponding to the complete coding region and the 3′ UTR, respectively, of the pig ME1 gene. The sequences of these two fragments in pigs from three breeds contained five SNPs in the 3′ UTR and three haplotypes in two generations of a selected Landrace population. Significant associations between ME1 genotype and backfat thickness at 171 days and muscle pH were found in a Landrace population. Therefore, the ME1 gene may be considered as a candidate gene associated with carcass and meat quality traits. The main objective of this study was to investigate the alternative transcripts and polymorphisms in the 3′ UTR of the bovine ME1 gene and to analyze the relationship between polymorphisms and meat quality or carcass traits of Chinese red cattle.
G.L. Zhou et al. / Meat Science 89 (2011) 72–75
2. Materials and methods 2.1. Animals, genomic DNA and total RNA isolation Chinese red cattle are a Chinese indigenous breed, crossbred by British shorthorn (male) and Mongolia cattle (female). They are mainly distributed in Jilin, Inner Mongolia, and Hebei of China. A total of 224 steers were genotyped, randomly selected from three sires in the Department of Animal Husbandry, Jilin Academy of Agricultural Sciences, China from 2007 to 2009. Their ages at slaughter ranged from approximately 24 to 26 months. All phenotypic data of meat quality and carcass traits were determined using the legal grading standard parameters endorsed by professional meat quality graders from the Animal Products Grading Service in China. Blood samples were collected from the aforementioned cattle, and genomic DNA was isolated using standard methods and stored at −20 and/or at 4 °C. Liver samples from Chinese red cattle were obtained within 20 min of dissection and were immediately submerged in liquid nitrogen before storage at –70 °C. A total of 100 mg of each sample tissue was homogenized in Trizol Reagent (Invitrogen) using a high-speed mechanical homogenizer. RNA integrity was monitored by denaturing 1% agarose gel electrophoresis. Concentrations and purities of RNA were measured by spectrophotometry (Amersham Pharmacia Biotech, Buckinghamshire, UK). All experimental procedures involving animals were performed according to authorization granted by the Chinese Ministry of Agriculture. 2.2. The 3′ rapid amplification of cDNA ends (RACE) and RT-PCR of alternatively spliced transcripts Primer pairs ME1F/ME1R (5′-TgT TgC CAC CCT CCT TCA TCA gTC-3′ and 5′-CCA CTC ggA Agg gTA ACg ggA T-3′) and ME2F/ME2R (5′-TgC TgC gAT Tgg Tgg TgC gTT CT-3′ and 5′-CCC gCT Tg CCT TgA CTC Tgg AgA Tg-3′) were designed based on highly conserved regions of the human (Accession no. NM_002395), murine (Accession no. J02652), rat (Accession no. NM_012600), swine (Accession no. X93016), and sheep (Accession no. EU646206) ME1 gene cDNA sequences. The primer pair ME1F/ME1R was used for the amplification of fragment 1, ME2F/ME2R was used for the amplification of fragment 2 (Fig. 1). Primers 3GSP1 (5′-AgC TAT TgT AAC TAC CAC TgA gAA ACC C-3′) and 3GSP2 (5′- ggC TgC CTg Tgg ggT CAA CgT CTT C-3′) were designed based on the sequence assembled of fragment 1 and fragment 2. Synthesis of the liver cDNA was performed using SMARTTM RACE cDNA amplification kit (Clontech) according to the manufacturer's instructions. Two middle cDNA fragments of ME1 gene were obtained by RT-PCR using primers (ME1F/ME1R and ME2F/ME2R) and Advantage® 2 PCR Kit (Clontech) according to the manufacturer's manual. The PCR product was separated, ligated into pGEM-T Easy Vector, and sequenced. The 3′-RACE was performed using SMART™ RACE cDNA amplification kit and Advantage® 2 PCR Kit according to the manufacturer's manual. For 3′-RACE, PCR was performed with a gene-specific primer 3GSP1 and the other gene-specific primer 3GSP2 to amplify different 3′ alternative transcript sequences of fragment 3 and 4, respectively (Fig. 1). All the PCR products were analyzed on 1% agarose gels stained with ethidium bromide. The PCR products were
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then purified using NucleoTrap® gel extraction kit (Clontech), and cloned into the pGEM-T Easy Vector (Promega, Madison, WI, USA). Bidirectional DNA sequences of clone inserts were determined using Big Dye Terminator chemistry on an ABI Prism Cycle sequencing 3730 (Applied Biosystems) in Shanghai Sangon Biological Engineering Technology and Services Co., Ltd of China. All fragments obtained were assembled into a contig by SeqMan program of DNAStar 5.0 software. 2.3. The 3′ UTR SNP identification of bovine ME1 gene For detecting the polymorphisms of 3′ UTR of the bovine ME1 gene, forward (5′-ATT TgT CCg CTC TCA gAT gTA T-3′) and reverse (5′-gAT ggg TTT CTC AgT ggT AgT T-3′) primers were designed according to genbank no. FJ515746 using the oligo 6.0 program. PCR was performed in a 25 μL reaction mixture, consisting of 10 pmol of each primer, 200 μM dNTP and 10 × reaction buffer containing 2.0 mM MgCl2, 1.5 unit of Taq DNA polymerase, and 50 ng template. PCR conditions were 95 °C for 4 min, followed by 35 cycles of 95 °C for 35 s, 54 °C for 35 s, and 72 °C for 35 s. After 33 cycles, reactions were finished by an extension of 7 min at 72 °C, and then 4 °C to terminate the reaction. A 370 bp fragment of the ME1 gene was amplified. The PCR–SSCP method was used for screening mutations within the amplified region. PCR products were half diluted in denaturing loading dye (95% formamide, 0.025% bromophenol blue, and 0.025% xylene cyanol), denatured at 95 °C for 10 min, and then placed on ice for 10 min. The samples were then loaded on 10% nondenaturing polyacrylamide gel with 10% formamide, and run in 1 × TBE buffer at 180 V for 14–16 h at a constant temperature of 4 °C. The gel was stained with 0.1% silver nitrate. PCR fragments harboring various electrophoresis profiles were sequenced and analyzed for nucleotide changes. The sequences obtained were compared with those previously reported in the database using a BLAST search at the NCBI server (http://www.ncbi.nlm.nih.gov/BLAST). A polymorphism was found in 3′ UTR. It can be genotyped using restriction enzyme Dra I. Amplified DNA was digested by Dra I enzyme at 37 °C for 4 h. The reaction mixture comprises 5 μL PCR product, 2 μL buffer, 0.5 μL (5U) Dra I, and 12.5 μL ddH2O. The digestion products were subjected to horizontal electrophoresis (90 V, 30 min) on a 2% agarose gel. 2.4. Statistical analyses Genotype and allele frequencies were calculated for polymorphism locus using the CERVUS 3.0 program (Marshall, Slate, Kruuk, & Pemberton, 1998). The phenotypic data include cooking loss, shear force, drip loss, pH24h, net meat weight, carcass weight, carcass yield ratio, and eye muscle area, determined by standard methods. The traits and ME1 genotypes were statistically analyzed by least squares analysis of variance using the General Linear Model procedure (PROC GLM) of SAS (SAS, 1999). Duncan's multiple range tests from PROC GLM was used to separate means. P b 0.05 was considered significant. The following model was used: Yijkl = μ + Gi + Sj + YSk + eijkl
Fig. 1. Position of the 4 fragment constructs the two ME1 transcripts. Long transcript consists of fragments 1 (1204 bp), 2 (1432 bp), and 3 (682 bp) and short transcript consists of fragments 1 (1204 bp), 2 (partly, 815 bp), and 4 (123 bp). The orientation of the primers used for the amplification of each fragment is indicated by arrows. UP: Universal Primer (from SMART™ RACE cDNA Amplification Kit). TAG: stop code.
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G.L. Zhou et al. / Meat Science 89 (2011) 72–75
where Yijkl is the observation of the trait; μ is the population mean; Gi is the effect of ith genotype (i = 1, 2, and 3); Sj is the effect of jth sire (j = 1, 2, and 3); YSk is the effect of kth seasons of slaughter (l = 1, 2, 3, and 4) and eijkl is the random residue.
Table 1 Least squares means and standard error for meat quality and carcass traits in Chinese red cattle according to ME1-Dra I genotypes. Trait tested
ME1-Dra I genotypes TT (n = 152)
3. Results 3.1. Alternatively spliced transcripts of bovine ME1 gene In order to clone the alternatively spliced transcripts of the bovine ME1 cDNA sequence, the strategy shown in Fig. 1 was adopted. Two transcripts, long transcript and short transcript, differing in the length of the 3′ UTR were cloned. The long transcript consists of fragments 1, 2, and 3 and short transcript consists of fragments 1, 2 (partly) and 4. The above fragments showed 100% identity in their overlapping regions. As already mentioned, joining of the overlapping fragments results in two transcripts that differ in the length of the 3′ UTR. The length of the 3′ UTR is 1448 bp in the long transcript and 335 bp in short transcript. One potential polyadenylation signal AATAAA is located at 2111–2116 bp, that is, 24 bp upstream of the poly (A) attachment sites of the short transcript (FJ515745). The polyadenylation signal for the long transcript is the nonclassical ATTAAA hexanucleotide located 19 bp upstream of the poly (A) attachment site located at position 3229–3234 bp (FJ515746). The sequence of the transcripts was submitted to genbank under the accession numbers FJ515745 and FJ515746. 3.2. Polymorphisms of the 3′ UTR in ME1 gene A single nucleotide polymorphism was revealed by sequencing the 3′ UTR of ME1 gene. A thymine (T)-to-cytosine (C) transition (NW_001495544: g.1536016 T N C) was shown in the SNP. This mutant creates a Dra I restriction site in PCR fragments of the ME1 gene. The DNA samples from 224 Chinese red cattle were genotyped using PCR– RFLP. The following DNA restriction fragments were obtained for the ME1-Dra I polymorphism: 370 bp (no digestion) for the CC genotype, 370, 336, and 34 bp (not shown) for the TC and 336 and 34 bp (not shown) for the TT genotype (Fig. 2). The ME1-Dra I TT genotype had the highest frequency in the herds studied (0.678), followed by the AC genotype (0.228). The least frequent genotype was CC (0.094). 3.3. Association analysis The Dra I PCR–RFLP of ME1 gene was genotyped in the Chinese red cattle population, in order to investigate the possible association between carriers of different genotypes and the trait values. The evaluation of meat quality and carcass traits in Chinese red cattle based on ME1-Dra I genotypes is shown in Table 1. In the meat quality traits, the ME1-Dra I genotypes revealed a significant effect on cooking loss and pH24h (P b 0.05). The Chinese red cattle with the CC genotype have a significantly lower cooking loss than those with the TT and TC genotypes and those with TC genotype have a significantly higher pH24h
Fig. 2. A 2.0% agarose gel displaying a Dra I restriction digest on an amplified portion of the bovine ME1 gene 3′ UTR. Lane PCR: amplification control; Lane CC, TC and TT: different genotypes; Lane M: pBR322 DNA marker.
Cooking loss (%) Shear force (kg) Drip loss (%) pH24h Net meat weight (kg) Carcass weight (kg) Carcass yield ratio (%) Eye muscle area (cm2)
TC (n = 51) a
40.53 ± 2.61 3.80 ± 0.34 2.31 ± 0.76 5.53 ± 0.10b 219.61 ± 3.97 279.66 ± 5.04 0.79 ± 0.01 87.61 ± 0.62b
CC (n = 21) a
40.30 ± 2.22 3.71 ± 0.41 2.33 ± 0.57 5.58 ± 0.10a 221.50 ± 5.55 280.00 ± 7.05 0.78 ± 0.02 88.15 ± 1.11b
39.65 ± 2.13b 3.85 ± 0.23 2.34 ± 0.42 5.49 ± 0.11b 224.52 ± 2.20 284.43 ± 2.79 0.79 ± 0.03 91.42 ± 1.56a
Values with different superscripts within the same line are significantly different at P b 0.05 (a and b).
than those with the TT and CC genotypes at the ME1-Dra I locus (P b 0.05). In the carcass traits, the ME1-Dra I genotypes revealed a significant effect on eye muscle area (P b 0.05). Chinese red cattle with the CC genotype have a significantly higher eye muscle area than those with the TT and TC genotypes at the ME1-Dra I locus (P b 0.05). 4. Discussion In the study, two different 3′ UTR transcripts were detected by RACE method in the bovine ME1 gene. The short transcript contains the polyadenylylation signal AATAAA at the position 2111/2116 which was found in most eukaryotic mRNAs. The long transcript contains the second polyadenylylation signal ATTAAA at the position 3229/3234 which was found in about 10% of eukaryotic mRNAs (Breathnach & Chambon, 1981). These results indicate that the size differences of the two ME1 mRNA are due to heterogeneity at the 3′ untranslated region and originate from the use of alternative polyadenylylation signals. The existence of two transcripts in mouse (Bagchi et al., 1987), pig (Nunes et al., 1996), and sheep (Stefos, Argyrokastritis, Bizelis, & Rogdakis, 2008) is suggested to be caused by the occurrence of two distinct polyadenylation signals. SNP analysis is a well-established tool for the identification of genes associated with traits of economic interest in livestock populations. The present study is the first report on polymorphisms of the bovine ME1 gene. A novel SNP of 3′ UTR was identified in the Chinese red cattle ME1 gene. The SNP was located at a mutual region of the 3′ UTR in two different transcripts of the bovine ME1 gene (NW_001495544: g.1536016 T N C). The genotype and allele frequencies of the SNP sites in Chinese red cattle were calculated. The frequency of the C allele is 0.208. The frequency of mutation alleles is lower. The fact that the mutant allele is rare suggests that they might be recent mutations that have not spread far. The result of least square analysis confirmed the significant association with meat quality or carcass traits in Chinese red cattle. In meat quality traits, Chinese red cattle with the CC genotype have significantly lower cooking loss than those with the TT and TC genotypes, and those with the TC genotype have significantly higher pH24h than those with the TT and CC genotypes at the ME1-Dra I locus at the same time (P b 0.05). In carcass traits, Chinese red cattle with the CC genotype have significantly higher eye muscle area than those with the TT and TC genotypes at the ME1-Dra I locus (P b 0.05). The bovine ME1 locus maps on the chromosome 9, where a quantitative trait loci affecting fat deposition has been described (Casas, Shackelford, & Keele, 2003). Because no references of fine map for bovine ME1 gene were found, it is unclear whether the ME1 gene maps to the region of the reported QTL. It can be deduced that bovine the ME1 gene is linked with the reported QTL. In conclusion, the present study identified a new SNP of the ME1 gene in Chinese red cattle populations. The results provide evidence that the ME1 gene might have potential effects on carcass and meat
G.L. Zhou et al. / Meat Science 89 (2011) 72–75
quality traits in Chinese red cattle. Further work will be necessary to use the SNP for marker assisted selection in larger populations in the breeding program of cattle. It is also necessary to investigate whether the ME1 gene plays a role in development of these traits and whether it is in linkage disequilibrium with other causative mutations. Acknowledgements The authors thank workers of branch of animal husbandry for the assistance in samples collection. We thank Zhaozhi LI and Jin LI for help in genotyping. This project was supported by the National High Technology Research and Development Project of China (No. 2008AA101010) and Natural Science Fund of Shandong province of P. R. China (Y2007D33). References Bagchi, S., Wise, L. S., Brown, M. L., Bregman, D., Sul, H. S., & Rubin, C. S. (1987). Structure and expression of murine malic enzyme mRNA. Differentiation–dependent accumulation of two forms of malic enzyme mRNA in 3 T3–L1 cells. Journal of Biological Chemistry, 262, 1558–1565. Bergen, W. G., & Mersmann, H. J. (2005). Comparative aspects of lipid metabolism: Impact on contemporary research and use of animal models. Journal of Nutrition, 135, 2499–2502. Breathnach, R., & Chambon, P. (1981). Organization and expression of eucaryotic split genes coding for proteins. Annual Review of Biochemistry, 50, 349–383. Careche, M., Obato, M. F. L., Ros, M., Moreno, F. J., & Garcia–Ruiz, J. P. (1985). Influence of starvation/refeeding transition on lipogenesis and NADPH producing systems in adipose tissue, mammary gland and liver at mid–lactation. Hormone and Metabolic Research, 17, 226–229. Casas, E., Shackelford, S. D., & Keele, J. W. (2003). Detection of quantitative trait loci for growth and carcass composition in cattle. Journal of Animal Science, 81, 2973–2983. Chang, G. G., & Tong, L. (2003). Structure and function of malic enzymes, a new class of oxidative decarboxylases. Biochemistry, 42, 12721–12733. Dozin, B., Magnuson, M. A., & Nikodem, V. M. (1985). Tissue–specific regulation of two functional malic enzyme mRNAs by triiodothyronine. Biochemistry, 24, 5576–5581.
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Guay, C., Madiraju, S. R., Aumais, A., Joly, E., & Prentki, M. (2007). A role for ATP–citrate lyase, malic enzyme, and pyruvate/citrate cycling in glucose–induced insulin secretion. Journal of Biological Chemistry, 282, 35657–35665. Ma, X. J., Salati, L. M., Ash, S. E., Mitchell, D. A., Klautky, S. A., Fantozzi, D. A., et al. (1990). Nutritional regulation and tissue–specific expression of the malic enzyme gene in the chicken. Transcriptional control and chromatin structure. Journal of Biological Chemistry, 265, 18435–18441. Marshall, T. C., Slate, J., Kruuk, L. E. B., & Pemberton, J. M. (1998). Statistical confidence for likelihood-based paternity inference in natural populations. Molecular Ecology, 7, 639–655. Milan, D., Bidanel, J. P., Iannuccelli, N., Riquet, J., Amigues, Y., Gruand, J., et al. (2002). Detection of quantitative trait loci for carcass composition traits in pigs. Genetics Selection Evolution, 34, 705–728. Mourot, J., & Kouba, M. (1999). Development of intra– and intermuscular adipose tissue in growing Large White and Meishan pigs. Reproduction and Nutrition Development, 39, 125–132. Nunes, M., Lahbib–Mansais, Y., Geffrotin, C., Yerle, M., Vaiman, M., & Renard, C. (1996). Swine cytosolic malic enzyme: cDNA cloning, sequencing, and localization. Mammalian Genome, 7, 815–821. Quintanilla, R., Milan, D., & Bidanel, J. P. (2002). A further look at quantitative trait loci affecting growth and fatness in a cross between Meishan and Large White pig populations. Genetics Selection Evolution, 34, 193–210. Rao, G. S., Cronrath, C. M., & Rao, M. L. (1984). Induction of malic enzyme in explants of human adipose tissue by L–3, 5, 3¢–triiodothyronine. Hormone and Metabolic Research, 14, 16–20 Suppl. SAS (1999). SAS/STAT software for PC. Release8.01. Cary, NC, USA: SAS Institute Inc. Siddiqui, U. A., Goldflam, T., & Goodridge, A. G. (1981). Nutritional and hormonal regulation of the translatable levels of malic enzyme and albumin mRNAs in avian liver cells in vivo and in culture. Journal of Biological Chemistry, 256, 4544–4550. Stefos, G. C., Argyrokastritis, A., Bizelis, I., & Rogdakis, E. (2008). Molecular cloning and characterization of the sheep malic enzyme cDNA. Gene, 423, 72–78. Thompson, E. W., & Drake, R. L. (1982). Induction of hepatic malic enzyme in response to insulin. Molecular and Cellular Endocrinology, 26, 309–314. Vernon, R. G. (1980). Lipid metabolism in the adipose tissue of ruminant animals. Progress in Lipid Research, 19, 23–106. Vidal, O., Varona, L., Oliver, M. A., Noguera, J. L., Sanchez, A., & Amills, M. (2005). Malic enzyme 1 genotype is associated with backfat thickness and meat quality traits in pigs. Animal Genetics, 37, 28–32.
Contents lists available at ScienceDirect
Meat Science j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / m e a t s c i
Cloning and polymorphisms of the 3′ untranslated region of malic enzyme gene in Chinese red cattle G.L. Zhou a, G.L. Zhang b, Y. Cao b, H.G. Jin b,⁎ a b
College of Life Science, Liaocheng University, Liaocheng, 252059, People's Republic of China Branch of Animal Husbandry, Jilin Academy of Agricultural Sciences, Gongzhuling, 136100, People's Republic of China
a r t i c l e
i n f o
Article history: Received 20 December 2010 Received in revised form 26 March 2011 Accepted 29 March 2011 Keywords: Malic enzyme gene Alternative transcripts Polymorphism Chinese red cattle Meat quality and carcass traits
a b s t r a c t The objective of this study was to identify alternative transcripts and single nucleotide polymorphisms (SNPs) in the 3′-untranslated region (3′ UTR) of bovine malic enzyme (ME1) gene and to evaluate the extent to which polymorphisms were associated with meat quality and carcass traits in Chinese red cattle. Two transcripts, long transcript and short transcript that differ in the length of the 3′ UTR were cloned. A single nucleotide polymorphism was detected in 3′ UTR and a restriction site for endonuclease ME1-Dra I was also found. The result revealed that the ME1-Dra I genotypes had a significant effect on cooking loss, pH measured 24 h post-mortem (pH24h) and eye muscle area (P b 0.05). In conclusion, the SNPs may be used as DNA markers to select for meat quality and carcass traits in Chinese red cattle. © 2011 Elsevier Ltd. All rights reserved.
1. Introduction Malic enzyme (ME1) is a lipogenic enzyme. For de novo biosynthesis of fatty acids, considerable amounts of NADPH are required (Guay, Madiraju, Aumais, Joly, & Prentki, 2007). To date, two mitochondrial (NAD+- and NADP+-dependent) and one cytosolic (NADP+-dependent) malic enzyme isoforms have been identified in mammals (Chang & Tong, 2003). Cytosolic ME1 forms part of the tricarboxylate shuttle, which releases acetyl-CoA from the mitochondria to the cytosol. NADPH and acetyl-CoA produced in such a way can be used in fatty acid biosynthesis and many other metabolic processes. Different from human and rodents, in which the principal carbon source for fatty acids biosynthesis is glucose, the acetic acid produced by microorganisms in the rumen acts as the principal carbon source in ruminant species (Bergen & Mersmann, 2005). Therefore, the glucose-pyruvate-acetyl CoA pathway is of little significance in ruminants and the contribution of malic enzyme in NADPH production of ruminants is relatively small in contrast to that of human and rodents (Vernon, 1980). The ME1 gene is known to be expressed in several tissues in rat (Dozin, Magnuson, & Nikodem, 1985), chicken (Ma et al., 1990) and pig (Nunes et al., 1996), and the expression is regulated by both hormones and nutrition levels. In particular, triiodothyronine (Rao, Cronrath, & Rao, 1984) and insulin (Thompson & Drake, 1982)
⁎ Corresponding author. Tel.: + 86 434 6282019; fax: + 86 434 6282020. E-mail address: [email protected] (H.G. Jin). 0309-1740/$ – see front matter © 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.meatsci.2011.03.023
stimulate ME1 in liver and adipose tissue, while glucagon blocks this effect (Siddiqui, Goldflam, & Goodridge, 1981). A high-carbohydrate, low-fat diet after a period of starvation results in increased ME1 expression in liver and adipose tissue (Careche, Obato, Ros, Moreno, & Garcia–Ruiz, 1985). The ME1 locus has been mapped on porcine chromosome1 (SSC1), and two transcript forms have been described (Nunes et al., 1996). Several quantitative trait loci (QTL) affecting growth, backfat and other production traits have been positioned on SSC1. Most of these studies report a single QTL (Milan et al., 2002), but the existence of more than one QTL cannot be ruled out (Quintanilla, Milan, & Bidanel, 2002). Moreover, comparison of glucose-6-phosphate dehydrogenase, acetyl-CoA-carboxylase and ME1 lipogenic enzyme activity in the intramuscular tissue of Meishan and growing Large White pigs revealed that ME activity is much higher in Meishan pigs, being one of the major factors influencing intramuscular fat deposition (Mourot & Kouba, 1999). Vidal et al. (2005) amplified fragments of 1457 and 1459 bp corresponding to the complete coding region and the 3′ UTR, respectively, of the pig ME1 gene. The sequences of these two fragments in pigs from three breeds contained five SNPs in the 3′ UTR and three haplotypes in two generations of a selected Landrace population. Significant associations between ME1 genotype and backfat thickness at 171 days and muscle pH were found in a Landrace population. Therefore, the ME1 gene may be considered as a candidate gene associated with carcass and meat quality traits. The main objective of this study was to investigate the alternative transcripts and polymorphisms in the 3′ UTR of the bovine ME1 gene and to analyze the relationship between polymorphisms and meat quality or carcass traits of Chinese red cattle.
G.L. Zhou et al. / Meat Science 89 (2011) 72–75
2. Materials and methods 2.1. Animals, genomic DNA and total RNA isolation Chinese red cattle are a Chinese indigenous breed, crossbred by British shorthorn (male) and Mongolia cattle (female). They are mainly distributed in Jilin, Inner Mongolia, and Hebei of China. A total of 224 steers were genotyped, randomly selected from three sires in the Department of Animal Husbandry, Jilin Academy of Agricultural Sciences, China from 2007 to 2009. Their ages at slaughter ranged from approximately 24 to 26 months. All phenotypic data of meat quality and carcass traits were determined using the legal grading standard parameters endorsed by professional meat quality graders from the Animal Products Grading Service in China. Blood samples were collected from the aforementioned cattle, and genomic DNA was isolated using standard methods and stored at −20 and/or at 4 °C. Liver samples from Chinese red cattle were obtained within 20 min of dissection and were immediately submerged in liquid nitrogen before storage at –70 °C. A total of 100 mg of each sample tissue was homogenized in Trizol Reagent (Invitrogen) using a high-speed mechanical homogenizer. RNA integrity was monitored by denaturing 1% agarose gel electrophoresis. Concentrations and purities of RNA were measured by spectrophotometry (Amersham Pharmacia Biotech, Buckinghamshire, UK). All experimental procedures involving animals were performed according to authorization granted by the Chinese Ministry of Agriculture. 2.2. The 3′ rapid amplification of cDNA ends (RACE) and RT-PCR of alternatively spliced transcripts Primer pairs ME1F/ME1R (5′-TgT TgC CAC CCT CCT TCA TCA gTC-3′ and 5′-CCA CTC ggA Agg gTA ACg ggA T-3′) and ME2F/ME2R (5′-TgC TgC gAT Tgg Tgg TgC gTT CT-3′ and 5′-CCC gCT Tg CCT TgA CTC Tgg AgA Tg-3′) were designed based on highly conserved regions of the human (Accession no. NM_002395), murine (Accession no. J02652), rat (Accession no. NM_012600), swine (Accession no. X93016), and sheep (Accession no. EU646206) ME1 gene cDNA sequences. The primer pair ME1F/ME1R was used for the amplification of fragment 1, ME2F/ME2R was used for the amplification of fragment 2 (Fig. 1). Primers 3GSP1 (5′-AgC TAT TgT AAC TAC CAC TgA gAA ACC C-3′) and 3GSP2 (5′- ggC TgC CTg Tgg ggT CAA CgT CTT C-3′) were designed based on the sequence assembled of fragment 1 and fragment 2. Synthesis of the liver cDNA was performed using SMARTTM RACE cDNA amplification kit (Clontech) according to the manufacturer's instructions. Two middle cDNA fragments of ME1 gene were obtained by RT-PCR using primers (ME1F/ME1R and ME2F/ME2R) and Advantage® 2 PCR Kit (Clontech) according to the manufacturer's manual. The PCR product was separated, ligated into pGEM-T Easy Vector, and sequenced. The 3′-RACE was performed using SMART™ RACE cDNA amplification kit and Advantage® 2 PCR Kit according to the manufacturer's manual. For 3′-RACE, PCR was performed with a gene-specific primer 3GSP1 and the other gene-specific primer 3GSP2 to amplify different 3′ alternative transcript sequences of fragment 3 and 4, respectively (Fig. 1). All the PCR products were analyzed on 1% agarose gels stained with ethidium bromide. The PCR products were
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then purified using NucleoTrap® gel extraction kit (Clontech), and cloned into the pGEM-T Easy Vector (Promega, Madison, WI, USA). Bidirectional DNA sequences of clone inserts were determined using Big Dye Terminator chemistry on an ABI Prism Cycle sequencing 3730 (Applied Biosystems) in Shanghai Sangon Biological Engineering Technology and Services Co., Ltd of China. All fragments obtained were assembled into a contig by SeqMan program of DNAStar 5.0 software. 2.3. The 3′ UTR SNP identification of bovine ME1 gene For detecting the polymorphisms of 3′ UTR of the bovine ME1 gene, forward (5′-ATT TgT CCg CTC TCA gAT gTA T-3′) and reverse (5′-gAT ggg TTT CTC AgT ggT AgT T-3′) primers were designed according to genbank no. FJ515746 using the oligo 6.0 program. PCR was performed in a 25 μL reaction mixture, consisting of 10 pmol of each primer, 200 μM dNTP and 10 × reaction buffer containing 2.0 mM MgCl2, 1.5 unit of Taq DNA polymerase, and 50 ng template. PCR conditions were 95 °C for 4 min, followed by 35 cycles of 95 °C for 35 s, 54 °C for 35 s, and 72 °C for 35 s. After 33 cycles, reactions were finished by an extension of 7 min at 72 °C, and then 4 °C to terminate the reaction. A 370 bp fragment of the ME1 gene was amplified. The PCR–SSCP method was used for screening mutations within the amplified region. PCR products were half diluted in denaturing loading dye (95% formamide, 0.025% bromophenol blue, and 0.025% xylene cyanol), denatured at 95 °C for 10 min, and then placed on ice for 10 min. The samples were then loaded on 10% nondenaturing polyacrylamide gel with 10% formamide, and run in 1 × TBE buffer at 180 V for 14–16 h at a constant temperature of 4 °C. The gel was stained with 0.1% silver nitrate. PCR fragments harboring various electrophoresis profiles were sequenced and analyzed for nucleotide changes. The sequences obtained were compared with those previously reported in the database using a BLAST search at the NCBI server (http://www.ncbi.nlm.nih.gov/BLAST). A polymorphism was found in 3′ UTR. It can be genotyped using restriction enzyme Dra I. Amplified DNA was digested by Dra I enzyme at 37 °C for 4 h. The reaction mixture comprises 5 μL PCR product, 2 μL buffer, 0.5 μL (5U) Dra I, and 12.5 μL ddH2O. The digestion products were subjected to horizontal electrophoresis (90 V, 30 min) on a 2% agarose gel. 2.4. Statistical analyses Genotype and allele frequencies were calculated for polymorphism locus using the CERVUS 3.0 program (Marshall, Slate, Kruuk, & Pemberton, 1998). The phenotypic data include cooking loss, shear force, drip loss, pH24h, net meat weight, carcass weight, carcass yield ratio, and eye muscle area, determined by standard methods. The traits and ME1 genotypes were statistically analyzed by least squares analysis of variance using the General Linear Model procedure (PROC GLM) of SAS (SAS, 1999). Duncan's multiple range tests from PROC GLM was used to separate means. P b 0.05 was considered significant. The following model was used: Yijkl = μ + Gi + Sj + YSk + eijkl
Fig. 1. Position of the 4 fragment constructs the two ME1 transcripts. Long transcript consists of fragments 1 (1204 bp), 2 (1432 bp), and 3 (682 bp) and short transcript consists of fragments 1 (1204 bp), 2 (partly, 815 bp), and 4 (123 bp). The orientation of the primers used for the amplification of each fragment is indicated by arrows. UP: Universal Primer (from SMART™ RACE cDNA Amplification Kit). TAG: stop code.
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where Yijkl is the observation of the trait; μ is the population mean; Gi is the effect of ith genotype (i = 1, 2, and 3); Sj is the effect of jth sire (j = 1, 2, and 3); YSk is the effect of kth seasons of slaughter (l = 1, 2, 3, and 4) and eijkl is the random residue.
Table 1 Least squares means and standard error for meat quality and carcass traits in Chinese red cattle according to ME1-Dra I genotypes. Trait tested
ME1-Dra I genotypes TT (n = 152)
3. Results 3.1. Alternatively spliced transcripts of bovine ME1 gene In order to clone the alternatively spliced transcripts of the bovine ME1 cDNA sequence, the strategy shown in Fig. 1 was adopted. Two transcripts, long transcript and short transcript, differing in the length of the 3′ UTR were cloned. The long transcript consists of fragments 1, 2, and 3 and short transcript consists of fragments 1, 2 (partly) and 4. The above fragments showed 100% identity in their overlapping regions. As already mentioned, joining of the overlapping fragments results in two transcripts that differ in the length of the 3′ UTR. The length of the 3′ UTR is 1448 bp in the long transcript and 335 bp in short transcript. One potential polyadenylation signal AATAAA is located at 2111–2116 bp, that is, 24 bp upstream of the poly (A) attachment sites of the short transcript (FJ515745). The polyadenylation signal for the long transcript is the nonclassical ATTAAA hexanucleotide located 19 bp upstream of the poly (A) attachment site located at position 3229–3234 bp (FJ515746). The sequence of the transcripts was submitted to genbank under the accession numbers FJ515745 and FJ515746. 3.2. Polymorphisms of the 3′ UTR in ME1 gene A single nucleotide polymorphism was revealed by sequencing the 3′ UTR of ME1 gene. A thymine (T)-to-cytosine (C) transition (NW_001495544: g.1536016 T N C) was shown in the SNP. This mutant creates a Dra I restriction site in PCR fragments of the ME1 gene. The DNA samples from 224 Chinese red cattle were genotyped using PCR– RFLP. The following DNA restriction fragments were obtained for the ME1-Dra I polymorphism: 370 bp (no digestion) for the CC genotype, 370, 336, and 34 bp (not shown) for the TC and 336 and 34 bp (not shown) for the TT genotype (Fig. 2). The ME1-Dra I TT genotype had the highest frequency in the herds studied (0.678), followed by the AC genotype (0.228). The least frequent genotype was CC (0.094). 3.3. Association analysis The Dra I PCR–RFLP of ME1 gene was genotyped in the Chinese red cattle population, in order to investigate the possible association between carriers of different genotypes and the trait values. The evaluation of meat quality and carcass traits in Chinese red cattle based on ME1-Dra I genotypes is shown in Table 1. In the meat quality traits, the ME1-Dra I genotypes revealed a significant effect on cooking loss and pH24h (P b 0.05). The Chinese red cattle with the CC genotype have a significantly lower cooking loss than those with the TT and TC genotypes and those with TC genotype have a significantly higher pH24h
Fig. 2. A 2.0% agarose gel displaying a Dra I restriction digest on an amplified portion of the bovine ME1 gene 3′ UTR. Lane PCR: amplification control; Lane CC, TC and TT: different genotypes; Lane M: pBR322 DNA marker.
Cooking loss (%) Shear force (kg) Drip loss (%) pH24h Net meat weight (kg) Carcass weight (kg) Carcass yield ratio (%) Eye muscle area (cm2)
TC (n = 51) a
40.53 ± 2.61 3.80 ± 0.34 2.31 ± 0.76 5.53 ± 0.10b 219.61 ± 3.97 279.66 ± 5.04 0.79 ± 0.01 87.61 ± 0.62b
CC (n = 21) a
40.30 ± 2.22 3.71 ± 0.41 2.33 ± 0.57 5.58 ± 0.10a 221.50 ± 5.55 280.00 ± 7.05 0.78 ± 0.02 88.15 ± 1.11b
39.65 ± 2.13b 3.85 ± 0.23 2.34 ± 0.42 5.49 ± 0.11b 224.52 ± 2.20 284.43 ± 2.79 0.79 ± 0.03 91.42 ± 1.56a
Values with different superscripts within the same line are significantly different at P b 0.05 (a and b).
than those with the TT and CC genotypes at the ME1-Dra I locus (P b 0.05). In the carcass traits, the ME1-Dra I genotypes revealed a significant effect on eye muscle area (P b 0.05). Chinese red cattle with the CC genotype have a significantly higher eye muscle area than those with the TT and TC genotypes at the ME1-Dra I locus (P b 0.05). 4. Discussion In the study, two different 3′ UTR transcripts were detected by RACE method in the bovine ME1 gene. The short transcript contains the polyadenylylation signal AATAAA at the position 2111/2116 which was found in most eukaryotic mRNAs. The long transcript contains the second polyadenylylation signal ATTAAA at the position 3229/3234 which was found in about 10% of eukaryotic mRNAs (Breathnach & Chambon, 1981). These results indicate that the size differences of the two ME1 mRNA are due to heterogeneity at the 3′ untranslated region and originate from the use of alternative polyadenylylation signals. The existence of two transcripts in mouse (Bagchi et al., 1987), pig (Nunes et al., 1996), and sheep (Stefos, Argyrokastritis, Bizelis, & Rogdakis, 2008) is suggested to be caused by the occurrence of two distinct polyadenylation signals. SNP analysis is a well-established tool for the identification of genes associated with traits of economic interest in livestock populations. The present study is the first report on polymorphisms of the bovine ME1 gene. A novel SNP of 3′ UTR was identified in the Chinese red cattle ME1 gene. The SNP was located at a mutual region of the 3′ UTR in two different transcripts of the bovine ME1 gene (NW_001495544: g.1536016 T N C). The genotype and allele frequencies of the SNP sites in Chinese red cattle were calculated. The frequency of the C allele is 0.208. The frequency of mutation alleles is lower. The fact that the mutant allele is rare suggests that they might be recent mutations that have not spread far. The result of least square analysis confirmed the significant association with meat quality or carcass traits in Chinese red cattle. In meat quality traits, Chinese red cattle with the CC genotype have significantly lower cooking loss than those with the TT and TC genotypes, and those with the TC genotype have significantly higher pH24h than those with the TT and CC genotypes at the ME1-Dra I locus at the same time (P b 0.05). In carcass traits, Chinese red cattle with the CC genotype have significantly higher eye muscle area than those with the TT and TC genotypes at the ME1-Dra I locus (P b 0.05). The bovine ME1 locus maps on the chromosome 9, where a quantitative trait loci affecting fat deposition has been described (Casas, Shackelford, & Keele, 2003). Because no references of fine map for bovine ME1 gene were found, it is unclear whether the ME1 gene maps to the region of the reported QTL. It can be deduced that bovine the ME1 gene is linked with the reported QTL. In conclusion, the present study identified a new SNP of the ME1 gene in Chinese red cattle populations. The results provide evidence that the ME1 gene might have potential effects on carcass and meat
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