Identification of Functional and In silico Positional Differentially Expressed Genes in the Livers of High-and Low-marbled Hanwoo Steers

This study identified hepatic differentially expressed genes (DEGs) affecting the marbling of muscle. Most dietary nutrients bypass the liver and produce plasma lipoproteins. These plasma lipoproteins transport free fatty acids to the target tissue, adipose tissue and muscle. We examined hepatic genes differentially expressed in a differential-display reverse transcription-polymerase chain reaction (ddRT-PCR) analysis comparing highand low-marbled Hanwoo steers. Using 60 arbitrary primers, we found 13 candidate genes that were upregulated and five candidate genes that were downregulated in the livers of high-marbled Hanwoo steers compared to low-marbled individuals. A BLAST search for the 18 DEGs revealed that 14 were well characterized, while four were not annotated. We examined four DEGs: ATP synthase F0, complement component CD, insulin-like growth factor binding protein-3 (IGFBP3) and phosphatidylethanolamine binding protein (PEBP). Of these, only two genes (complement component CD and IGFBP3) were differentially expressed at p<0.05 between the livers of highand low-marbled individuals. The mean mRNA levels of the PEBP and ATP synthase F0 genes did not differ significantly between the livers of highand low-marbled individuals. Moreover, these DEGs showed very high inter-individual variation in expression. These informative DEGs were assigned to the bovine chromosome in a BLAST search of MS marker subsets and the bovine genome sequence. Genes related to energy metabolism (ATP synthase F0, ketohexokinase, electron-transfer flavoprotein-ubiquinone oxidoreductase and NADH hydrogenase) were assigned to BTA 1, 11, 17, and 22, respectively. Syntaxin, IGFBP3, decorin, the bax inhibitor gene and the PEBP gene were assigned to BTA 3, 4, 5, 5, and 17, respectively. In this study, the in silico physical maps provided information on the specific location of candidate genes associated with economic traits in cattle. (


INTRODUCTION
Marbling (intramuscular fat) plays an important role in determining the juiciness and tenderness of beef and is one of the main factors used to grade beef quality in the United States (United States Department of Agriculture (USDA), 1989), Japan (Japanese Meat Grading Association (JMGA), 1988), and Korea.Therefore, marbling is a very important and valuable trait in the cattle industry.Muscle is the major target tissue for intramuscular fat development, and several reports have reported gene expression profiling using cDNA microarrays and differential-display reverse transcription-polymerase chain reaction (ddRT-PCR) methods to identify the genes regulating marbling within muscle tissue (Child et al., 2002;Wang et al., 2005).However, no study has examined hepatic gene expression profiles related to these traits.Most dietary long-chain fatty acids (LCFA) and nutrients bypass the liver and produce plasma lipoproteins, such as TG-rich very low-density lipoprotein (VLDL) and chylomicrons (Bauchart, 1993;Grum et al., 2002).These plasma lipoproteins transport lipids from the secreting organ (liver and intestine) to the target tissue (adipose tissue, muscle, and mammary glands).Therefore, it may be important to understand the metabolic mechanism of dietary fatty acids in the liver.Ponsuklili et al. (2005) found that single nucleotide polymorphisms (SNPs) of hepatic differentially expressed genes (DEGs) were associated with economically important traits in the pig, such as obesity and muscularity.Recent advances in high-throughput gene expression technology, such as cDNA microarrays and ddRT-PCR, have facilitated research to identify DEGs related to economic traits, such as marbling.Particularly, the entire bovine genome sequence has been published in GenBank (http://hgdownload.cse.ucsc.edu/goldenPath/bosTau2/chromosomes/), and it is possible to map the physical location of informative DEGs on the genome sequence.In this study, we identified 18 DEGs in the livers of high-and low-marbled Hanwoo cattle (Korean native cattle) using the ddRT-PCR method and mapped the physical location of these DEGs on the bovine genome sequence using an in silico BLAST search.

Animals, diet and sampling
Ninety Hanwoo male were selected at 1 month old with the similar birth weight (24.8±3.8 kg) and castrated at 3 months old.All steers were fed the same diets in a feedlot house.The concentrates were offered at 1.5% and 1.8% of body weight on growing (-6 months) and early fattening stage (6-12 months), respectively, and in late fattening stage (13-27 months) steers were fed high concentrate grain diet ad libitum.Steers were fed forage ad libitum and had free access to fresh water during the whole period.The animals were humanely killed at 27 months old at the slaughter house of NIAS (National Institute of Animal Science).All Hanwoo tissue samples (liver, longissimus dorsi, rump and subcutaneous adipose tissue) were taken and immediately frozen in liquid nitrogen and stored at -80°C until analysis.Of ninety Hanwoo steers, we have taken each seven liver samples (three individuals for prescreening, and four individuals for validation) in high (marbling grading 6-7) and low (marbling grading 2-3) marbled Hanwoo steers (a marbling score of Beef Marbling Standard was assessed on a 1 to 7 scale, with 7 the highest (Kim et al., 2003)).

Total RNA extraction
The total RNA samples was prepared from liver for the ddRT-PCR prescreening using Trizol reagent (Life Technologies Inc., Grand Island, NY, USA) according to the manufacturer's instructions and quantified by absorbance at 260 nm.

Cloning and sequence analysis
Differentially expressed bands were extracted and cloned into a TOPO TA cloning vector (Invitrogen, Karlsruhe, Germany).To confirm the identities of insert DNA, isolated plasmids were sequenced using a ABI3730XL sequencer (Applied Biosystems, USA).Complete sequences were analyzed by searching for similarities using a BLASTX program (Altschul et al., 1990) at the National Center for Biotechnology Information (NCBI).

Quantitative real-time PCR confirmation for selected genes
In this study, we identified 18 differentially expressed genes in each three samples of marbling high and low individuals.Of these genes, we selected 4 differentially AGG CAG ATG GAT CAG CCA expressed genes to validate ACP ddRT-PCR prescreening.
The selected genes for ACP ddRT-PCR analysis validation were insulin like growth factor 3, phosphatidyl ethanolamine binding protein, ATP synthase F0 and complement component CD.Sequence-specific primers were designed to amplify products ranging from 150 to 300 bp using the program primer3 (http://frodo.wi.mit.edu/cgibin/primer3/primer3_www.cgi;Table 1).First strand cDNA was synthesized from 3 µg total RNA from each four individual animal using the random hexamer primers (Promega Co., Madison, USA) and the superscript reverse transcriptase enzyme (Invitrogen Co., Grand Island, NY, USA).The mRNA expression levels for the selected genes were analysed by quantitative real-time PCR with specific primer (Table 1).18S rRNA gene (GenBank accession No. AY779626) was used as an internal control.Realtime RT-PCR amplification mixtures (20 µl) contained 1 µl cDNA, 2×SYBR Green I Master Mix (10 µl) (Qiagen., GmbH, Germany), and 10 pM forward and reverse primers.The real-time PCR reactions started at 95°C for 15 min for predenaturation, and the condition was set at 95°C for 10 second, 58°C for 20 second and 72°C for 30 second.The PCR cycle performed 40 cycles.The PCR were conducted in ABI 7500 system (Applied biosystems, USA).The relative quantification of target genes expression were evaluated using the ∆C T method.The ∆C T value was determined by nomalizing the 18S ribosomal C T value for each sample from the target C T value.Fold change in the relative gene expression of target were determined by calculating the 2 -∆CT .

In silico mapping of differentially expressed genes
The differentially expressed genes (DEG) and subset of MS marker from 1997 MARC map (Kappes et al., 1997) were compared by BLASTN to the draft assembly of the bovine whole-genome sequence using the NLRI (National Institute of Animal Science, RDA, Korea) interface (http://www.nabc.go.kr).An E-value of e -4 was considered significant for identification of bovine orthologues.Significant matches were returned for 16 of the 18 differentially expressed genes (89%).Distribution of these DEG sequences and nearest MS marker informations on the bovine genome sequence is presented in Table 2.

Statistical analysis
Results of realtime PCR analysis are given as means±SD.Statistical significance was determined using the Student's t-test.The level of significance used in all studies was p<0.05.

DEGs in the livers of high-and low-marbled Hanwoo steers
The ddRT-PCR method has a high false positive rate.Recently, a new annealing control primer (ACP) ddRT-PCR method was developed.The ACP ddRT-PCR method is based on the unique tripartite structure of a specific primer, which has a 3'-end region with a target core nucleotide sequence, a 5'-end region with a nontarget universal nucleotide sequence, and a poly (dI) linker bridging the 3'-and 5'-end sequences (Hwang et al., 2003;Kim et al., 2004).These 5'-end nontarget and poly (dI) linker sequences do not interrupt nonspecific hybridization.Therefore, this method results in a highly reproducible PCR reaction.We used ACP ddRT-PCR to isolate DEGs from the livers of high-and low-marbled Hanwoo steers using a combination of 60 arbitrary primers and two anchored oligo (dT) primers from the ACP-based GeneFishing PCR kit (Seegene, Korea).Using the 60 arbitrary primers, we amplified about 500 bands.Of these 500 amplified bands, we found 18 differentially expressed bands, of which 13 were upregulated in high-marbled meat and five were downregulated.The 18 differentially expressed band fragments were extracted from gels and cloned into TOPO-TA cloning vector (invitrogen, Karlsruhe, Germany).These cloned band fragments were sequenced using 3730XL DNA sequencer (Applied Biosystems, USA) (Figure 1 and Table 2).The DNA sequences of these clones were analyzed using the BLAST search interface in the TIGR gene index, which showed that 14 of the differentially expressed clones were well characterized and four clones were not annotated in bovine gene pools.The 14 differentially expressed clones were classified using a GeneOntology search.Of these, NADH hydrogenase, ketohexokinase, ATP synthase F0, and electron-transfer flavoprotein-ubiquinone oxidoreductase are related to the mitochondrial respiratory chain.The major functions of mitochondria are to produce ATP energy and conduct oxidative phosphorylation and fatty acid βoxidation.The mitochondrial metabolic cycle in human muscle and liver is related to disease and aging (Honokio et al., 1995;Nicoletti et al., 2005).Ketohexokinase or hepatic fructokinase catalyzes the first step in the metabolism of dietary fructose to produce energy, converting fructose into fructose-1-phosphate; malfunction of this enzyme causes diabetes mellitus (Hayward et al., 1996).Mitochondrial ATP synthase F0 plays a role in synthesizing ATP, which is the energy source derived from the proton gradient in mitochondria.The ATP synthase F0 subunit transports protons across the mitochondrial inner membrane to F1-ATPase (Fernando et al., 2005).Electron transfer flavoprotein exists in the mitochondrial matrix and has two heterodimer forms, involving 30 and 28 kDa subunits, and one flavin adenine dinucleotide (FAD) and one adenosine 5'-monophosphate (AMP).This enzyme is required for electron transfer from at least nine mitochondrial flavincontaining dehydrogenases to the main respiratory chain.Therefore, these mitochondrial respiratory genes are very important for generating ATP or FADH energy.Currently, in livestock genomics, these mitochondrial genes have not been associated with economically important traits.However, many genes derived from the mitochondrial genome were detected in our previous 23 K Affymetrix chip or ddRT-PCR analysis of the age-and fat-dependent phenotypes of Hanwoo cattle (data not shown).Therefore, it is necessary to study the function and structure of these DEGs derived from mitochondria further, to confirm their contribution to important livestock traits.
In addition, we identified phosphatidylinositol 4-kinase (PI4-K), which is involved in the phosphatidyl inositol cycle, which in turn is tied to cellular regulation by growth factor and hormones.The phylogenetic tree of the phosphoinositide superfamily contains three families: the phosphatidylinositol 4-kinase, phosphoinositol 3-kinase, and phosphatidylinositol related lipid/protein kinase families (Gehrmann et al., 1998).PI4-K (phosphoinositide-4-kinase) participates in mitogenesis, glucose transport, the regulation of hepatic glucose output, and insulin exocytosis in pancreatic beta cells (Gehrmann et al., 1998;Gromada et al., 2005).Recently, Kim et al. (2005) reported that SNPs within the PI3-K (phosphatidylinositol 3-kinase) gene were significantly associated with back and carcass fat in F 2 animals of a cross between Korean native and Landrace pigs.The phosphatidylethanolamine-binding protein (PEBP) is a raf kinase inhibitor protein (RKIP) that plays a role in the proliferation and differentiation of different cell types (Yeung et al., 1999).RKIP suppresses the mitogenactivated protein kinase (MAPK) pathway.Recently, the RKIP gene was reported to play a role in cancer and Alzheimer's disease (Chen et al., 2006;Lee et al., 2006).
The SNARE domain of syntaxin mediates a membrane fusion process, involving vesicular transport along the exocytic and endocytic pathways.Syntaxin 4 is the major plasma membrane SNARE for insulin-stimulated GLUT4 translocation in adipocytes.In addition, Min et al. (1999) found that syntaxin-binding protein 4 controls glucose transport and GLUT4 vesicle translocation.Therefore, the syntaxin gene may be related to fat deposition in cattle.
triplicate for eight individual samples.Of these four selected genes, only two genes (complement component CD and IGFBP3) were differentially expressed at p<0.05 between the livers high-and low-marbled Hanwoo steers.However, these genes are necessary to do further gene expression study using more many Hanwoo steers.Figure 2 shows the mRNA expression of these four genes, which were upregulated in high-marbled individuals.Particularly, the mean hepatic mRNA level of complement component CD in high-marbled individuals was twice as high as in low-marbled individuals, and the insulin-like growth factor binding protein 3 gene was 1.5 times higher than in low individuals.Complement component CD protein is a component of the complement activation pathway, which is part of the innate immune system that is capable of killing microorganisms directly and modulating phagocytosis, inflammation, and the humoral immune response (Wimmers et al., 2003).Generally, the immune system is closely connected to disease.Interestingly, there are several reports that the MHC haplotype influences growth traits in the mouse (Simpson et al., 1982), chicken (Briles, 1954), and pig (Jung et al., 1989).Immune system-related genes, such as MHC and complement genes, may be related to the disease or stress susceptibility of animals.Therefore, we need to identify SNPs of the complement component CD genes and examine their association with meat quality.The insulin-like growth factors, their receptors, and their binding proteins play key roles in regulating cell proliferation and apoptosis.Particularly, insulin-like growth factor (IGF) affects the proliferation and differentiation of muscle cells (Florini et al., 1996).IGFBP3 regulates IGF bioactivity to binding IGF (Jones and Clemmons, 1995).Johnson et al. (1999) reported that the IGFBP3 mRNA level is decreased significantly during differentiation of the myogenic cell line.Lee et al. (2005) reported that IGF-1 and IGFBP-3 have positive correlations with the live weight of cattle.In addition, Kim et al. (2005) reported 22 SNPs within IGFBP3 of Hanwoo cattle.Until now, no study has examined the relationship between IGFBP3 and meat quality.Therefore, we need to study the association between the SNPs of IGFBP3 and meat quality in Hanwoo cattle.In addition, although the mean mRNA levels of the PEBP gene and ATP synthase F0 gene were slightly higher in highmarbled individuals than in low-marbled individuals, the difference in the expression of these two genes was not significant.

In silico physical gene mapping
The differentially expressed genes (DEG) and subset of MS marker from 1997 MARC map (Kappes et al., 1997;Ihara et al., 2004) were compared by BLASTN to the draft assembly of the bovine whole-genome sequence using the NIAS (National Institute of Animal Science, RDA, Korea) interface (http://www.nabc.go.kr).An E-value of e -4 was considered significant for identification of bovine orthologues.Significant matches were returned for all of the 18 differentially expressed genes (100%).Distribution of these DEG sequences and nearest MS marker informations on the bovine genome sequence is presented in Table 3. Differentially expressed genes related to energy metabolism (ATP synthase F0, Ketohexokinase, Electron-transfer flavoprotein-ubiquinone oxidoreductase, and NADH 1 Starting position is the nucleotide number on the bovine whole-genome assembly for the given chromosome.
2 Percent ID is the sequence similarity determined from the aligned sequence match.
3 Nearest MS marker position with the differentially expressed genes.
hydrogenase) were assigned to BTA 1, 11, 17, 22.The syntaxin, insulin-like growth factor binding protein 3, decorin, bax inhibitor gene, and phosphatidylethanolamine binding protein gene were assigned to BTA 3, 4, 5, 5 and 17, respectively.In this study the in silico physical maps provided information on the specific location of candidate genes associated with economic traits in cattle (Table 3).

Figure 1 .
Figure 1.Gel image for the differential banding patterns obtained from Hanwoo high and low marbled liver samples.The ACP DDRT-PCR products are separated on 1.5% agarose gel and stained with ethidium bromide.The red box indicated differential expressed bands selected in high and low marbled Hanwoo liver.

Table 1 .
Primer for quantitative real-time reverse transcription PCR

Table 2 .
Identification of differentially expressed gene between marbling high and low cattle liver

Table 3 .
Summary of in silico physical mapping of bovine differentially expressed genes related intramuscular fat development