Progress 10/01/07 to 09/30/12
Outputs
OUTPUTS: Porcine reproductive and respiratory syndrome (PRRS), caused by a small RNA virus (PRRSV) has been devastating and will continue to devastate the U.S. pig industry. This disease is characterized by severe reproductive failure in sows and respiratory disease and high mortality in piglets, causing $644 million in losses annually. At Washington State University, we collaborated with Dr. Bang Liu's lab at Huazhong Agricultural University, China to focus on characterization of porcine SARM1 and its role in regulating TLRs (Toll-like receptors) signaling during highly pathogenic porcine reproductive and respiratory syndrome virus infection in vivo. Both a bioinformatics approach and 5'RACE (Rapid Amplification of cDNA Ends) procedure were used to clone the gene for its full length cDNA sequence. The INRA-UMN porcine Radiation Hybrid (IMpRH) panel was used to map porcine SARM1 gene to pig chromosome. Heart, liver, spleen, lung, kidney, skeletal muscle, fat, stomach, small intestine, lymph node, and brain were collected to examine SARM1 expression. For the expression of porcine SARM1 protein, a full length coding sequence (CDS) of the pig gene was inserted into pcDNA3.1(+) expression vector (Invitrogen). The sequences for the wild-type and four truncated porcine SARM1 were cloned into pEGFP-N1 expression vector (Clontech) and used to observe the subcellular location of porcine SARM1 protein in PK-15 cells. Upstream promoter fragments of porcine SARM1 were inserted into pGL3-Basic Vector (Promega) to test promoter activity. We also collaborated with Dr. Ping Jiang's lab at Nanjing Agricultural University, China to investigate proteome profiles of pulmonary alveolar macrophages (PAMs) infected with PRRSV. Highly pathogenic PRRSV strain BB0907 was used to infect PAMs collected from four, 3-week-old specific-pathogen-free (SPF) piglets that were free of PRRSV, porcine circovirus type 2, pseudorabies virus, classical swine fever virus, swine influenza virus, porcine parvovirus, and Mycoplasma hyopneumoniae infections. Cells were collected at 12, 24, 36 and 48 hours post infection and their proteome was profiled using the two-dimensional liquid chromatography-tandem mass spectrometry coupled with isobaric tags for relative and absolute quantification (iTRAQ) labeling method. PARTICIPANTS: Michal JJ, Zhou X, Jiang T, Du X, Zhou P, Liu B, Lu Q, Bai J, Zhang L, Liu J, He Q, Jiang P. TARGET AUDIENCES: Pork producers, pig-related youth leaders and high school FFA leaders across the state, livestock haulers, processors, retailers, veterinarians, livestock judges and fair management, other swine scientists nationwide (for research and outreach dissemination). PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
By sequencing the PCR and RACE products, we were able to assemble a full-length cDNA of 4370 bp for the porcine SARM1 gene, including a 5'UTR of 395 bp, a coding region of 2,175 bp and a 3'UTR of 1700 bp, respectively. Using the radiation hybrid panel, we mapped the porcine SARM1 gene to chromosome 12. Semi-quantitative RT-PCR revealed that porcine SARM1 was highly expressed in the spleen and the brain, followed by lung, kidney, liver and other tissues. The pig gene was weakly up-regulated when challenged with poly(I:C) in PK-15 cells. Compared with pGL3-Basic plasmid alone as control, the luciferase activity induced by the full promoter sequence (-2193/+341) was up-regulated about 15-20-fold. The promoter (-788/+341) containing CpG-island and core promoter showed higher luciferase activity. We found that porcine SARM1 is located in mitochondria through ARM domain. The luciferase reporter assay revealed that porcine SARM1 negatively regulated NF-κB activity induced by LPS, poly(I:C) stimulation, or HP-PRRSV infection. Gene-interaction network analysis for porcine SARM1 in PAMs showed that down-regulation of SARM1 gene in infected Tongcheng pig may modulate TRIF-depend TLRs signaling and regulate the expression of disease-resistant genes and inflammatory genes. Our findings provide evidence that porcine SARM1 may play an important role in immune regulation with PRRSV infection. As for proteome profiling, the iTRAQ-coupled 2D LC- MS/MS analysis detected a total of 869 proteins in both PRRSV infected and mock infected PAMs at 12, 24, 36 and 48 hours post infection. Among them, 160 proteins displayed significantly altered expression levels at different time points. These differentially expressed proteins were functionally involved in cellular components, metabolic functions, and biological processes. Cellular components-based enrichment analysis identified the differentially expressed proteins that were well-distributed in cell components. Enrichment analysis using biological processes showed that generation of precursor metabolites and energy was affected by virus infection. Metabolic function-based enrichment analysis demonstrated that nucleotide binding, cytoskeletal protein binding, and hydrolyase activity were commonly affected by virus infection. We also observed that some proteins that were significantly altered by PRRSV infection interacted with each other, such as (Txnrd1-Prdx5-Txn1-Prdx6-Cat-Sod2, Rpsa-Rps21-Rps3-Arbp-Gnb2l1-Uba52 and Vim-Anxa2-Anxa1-Lgals1-S100a10-Lgals3). Our work is the first to use iTRAQ proteomic analysis to examine PAM responses to PRRSV infection. These data are helpful for elucidating the molecular mechanisms associated with the interaction between PRRSV and the natural target cells.
Publications
- Lu Q., J.Bai, L.Zhang, J.Liu, Z.Jiang, J.J.Michal, Q.He, P.Jiang. 2012. Two-dimensional liquid chromatography-tandem mass spectrometry coupled with isobaric tags for relative and absolute quantification (iTRAQ) labeling approach revealed first proteome profiles of pulmonary alveolar macrophages infected with porcine reproductive and respiratory syndrome virus. Journal of Proteome Research. 11(5):2890-2903.
- Zhou X., T.Jiang, X.Du, P.Zhou, Z.Jiang, J.J.Michal, B.Liu. 2012. Molecular characterization of porcine SARM1 and its role in regulating TLRs signaling during highly pathogenic porcine reproductive and respiratory syndrome virus infection in vivo. Developmental and Comparative Immunology. http://dx.doi.org/10.1016/j.dci.2012.02.001
- Kunej T., D.Jevsinek Skok , M.Zorc, A.Ogrinc, J.J.Michal, Z.Jiang. 2012. Obesity Gene Atlas in Mammals. Journal of Genomics. 1:45-55.
- Pan Z., J.Zhang, J.Zhang, B.Zhou, J.Chen, Z.Jiang, H.L.Liu. 2012. Expression profiles of the insulin-like growth factor system components in liver tissue during embryonic and postnatal growth of Erhualian and Yorkshire reciprocal cross F 1 pigs. Asian-Australasian Journal of Animal Sciences. 25(7):903-912.
- Li R., Q.Sun, Y.Jia, R.Cong, Y.Ni, X.Yang, Z.Jiang, R.Q.Zhao. 2012. Coordinated miRNA/mRNA expression profiles for understanding breed-specific metabolic characters of liver between Erhualian and large white pigs. PLoS One. 7(6):e38716.
- Liu J., J.Bai, Q.Lu, L.Zhang, Z.Jiang, J.J.Michal, Q.He, P.Jiang. 2012. Two-dimensional liquid chromatography-tandem mass spectrometry coupled with isobaric tags for relative and absolute quantification (iTRAQ) labeling approach revealed first proteome profiles of pulmonary alveolar macrophages infected with porcine circovirus type 2. Journal of Proteomics. 79C:72-86.
|
Progress 01/01/11 to 12/31/11
Outputs
OUTPUTS: The porcine reproductive and respiratory syndrome (PRRS), caused by a single-stranded RNA virus (PRRSV), is the most economically important disease in pigs worldwide. Studies have shown that PRRSV is able to mutate, thus causing challenges in effective vaccine development. Furthermore, once infected, the virus can evade pig's immune system, delaying protective antibody production for several weeks. Evidence has shown that genetic variation exists in host response to the PRRSV infection, identification of desirable genetic markers or their interactions responsible for genetic variation appears to be a viable option for the pig industry to employ in order to combat the disease. In particular, the PRRS research community has suggested that more studies that focus on the possible pathways of PRRS resistance are needed so that powerful control methods can be found to ease the disease burden and thus increase animal welfare and production profitability. At Washington State University (WSU), we used the public database to compile a list of genes that are differentially expressed in pulmonary alveolar macrophages at different stages post infection with PRRSV and then selected a total of 22 candidate genes involved in three pathways for association analysis. We used comparative annotation procedures to retrieve both cDNA and genomic DNA sequences for 22 genes in pigs. The Splign program was used to determine their exon-intron boundaries and Primer3 was used to design primers for mutation detection in 8 randomly selected animals. Genotyping of single-nucleotide polymorphisms (SNPs)/mutations was carried out on PRRS Host Genetics Consortium trials 1 - 3 pigs (616 animals) using the Sequenom iPLEX assay. The HAPLOVIEW program was used to determine the linkage disequilibrium (LD) of mutations, leading to selection of tag mutations for association analysis. Polynomial curve fitting was used to estimate the area under curve (AUC) values for viral loads at different days post-infection, while a cubic spline interplant program was used to determine the maximum level of virus and the number of days it took the virus to replicate to the maximum level. The association analysis was conducted using the mixed model procedure of SAS v9.2 (SAS institute Inc, Cary, NC). PARTICIPANTS: Michal JJ, Zhang LF, Zhang M, Lunney JK, Rowland, RR. TARGET AUDIENCES: Pork producers, pig-related youth leaders and high school FFA leaders across the state, livestock haulers, processors, retailers, veterinarians, livestock judges and fair management, other swine scientists nationwide (for research and outreach dissemination). PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Among the 22 genes investigated, 132 mutations were discovered in 19 genes and 82 of these mutations were genotyped on 616 samples provided by the PRRS host genetics consortium. Statistical analysis revealed four genes that are significantly associated with virus load at different days post infection. In addition, three of these genes significantly affected pig growth rate. More interestingly, one of these genes is located on SSC4 where a major QTL for both virus load and growth was detected using the GWAS (genome wide association study) approach. Secretion of interferons (IFNs) from virus-infected cells is a hallmark of antiviral immunity. Studies have confirmed that IFNs have antiviral activity against PRRSV. Among these four genes, three encode for IFN-induced proteins. Therefore, our study provides initial evidence that IFN-induced genes also have antiviral activities against PRRSV. The candidate gene list needs to be further expanded, because we have not identified any markers that are associated with viral levels at days 35 and 42 post infection. Our current study also indicates that different sets of genes might be responsible for genetic resistance to PRRSV at different time points in the course of infection. Therefore, our current research would provide two major end-products to the pig industry: a marker panel for genetic selection and potential molecular targets for new drug design, thus helping the industry to efficiently control PRRS disease.
Publications
- Dodson, M.V., G.J.Hausman, L.L.Guan, m.Du, and Z.Jiang. 2011. Potential impact of mature adipocyte dedifferentiation in terms of cell numbers. International Journal of Stem Cells 4:76-78.
- Dodson, M.V., Z.Jiang, m.Du, and G.J.Hausman. 2011. Adipogenesis: It is not just lipid that comprises adipose tissue. Journal of Genomics. 1(1):1-4.
|
Progress 01/01/10 to 12/31/10
Outputs
OUTPUTS: To address Objective #1, we characterized the cellular and molecular features of redifferentiation between intramuscular- and visceral-adipocyte derived progeny cells. In the study, mature adipocytes from pig-derived visceral and intramuscular adipose depots were isolated, purified, and allowed to undergo dedifferentiation and redifferentiation in vitro. Wells from progeny cell cultures originating from both adipose depots were collected on days 1, 2, 4, 6, 8, 10, 12, 14 and 16 after initiation of culture. At every time point, 6 wells of cells were harvested for analysis, of which 3 were used to examine lipid content and another 3 were for extracting RNA. Total RNA was extracted from cells with QIAGEN RNeasy Mini Kit (QIAGEN Inc, Valencia, CA) and digested with DNase I (Fermentas Inc., Glen Burnie, MD). Expression levels were analyzed for six lipogenic genes. β-actin was used as an internal standard marker. To address Objective #2, we focused on identification of pathways involved in genetic resistance to PRRSV infection. Research has shown that the major histocompatibility complex (MHC)-antigen production complex is involved in antigen processing and recognition when self and pathogen-derived proteins are degraded and loaded onto MHC class I molecules (or SLA class I in pigs). In particular, studies have found that interaction between disulfide isomerase family A (PDIA3, also known as ERp57 and GRP58) and TAP binding protein (tapasin) (TAPBP) is a key event leading to the generation of such a MHC-antigen production complex. Microarray analysis on genome-wide transcriptional response to the PRRSV infection identified GRP58 (PDIA3) as one of only two genes differentially expressed in primary alveolar macrophages by a minimum of 1.5 fold at 1h post infection with PRRSV. Therefore, the MHC-antigen production complex was selected as our target pathway for the work. In addition, nitric oxide-associated pathway was also selected as the genes in this pathway play a role in plant disease resistance. PARTICIPANTS: Jie Chen, Michael V. Dodson, and Jennifer J. Michal, Washington State University. TARGET AUDIENCES: Pork producers, pig-related youth leaders and high School FFA leaders across the state, livestock haulers, processors, retailers, veterinarians, livestock judges and fair management, other swine scientists nationwide (for research and outreach dissemination). PROJECT MODIFICATIONS: None
Impacts
As for the cellular and molecular features of redifferentiation between intramuscular- and visceral-adipocyte derived progeny cells, we observed that they possess a similar capacity to accumulate lipid at days 1, 2, 4, 6, and 8. However, at days 10, 12, 14, and 16, the latter progeny cells accumulated lipid much faster--the content almost doubled at day 16 (P < 0.05). The faster rate of lipid accumulation in the intramuscular adipose-derived progeny cells was further supported by higher expressions of CCAAT/enhancer binding protein-α (CEBP-α) and peroxisome proliferator-activated receptor-γ (PPAR-γ) at all these nine time points, and diacylglycerol O-acyltransferase homolog 1 (DGAT1), fatty acid binding protein 4 (FABP4) and fatty acid synthase (FASN) at some time points (P < 0.05). These preliminary data suggest that adipose depot differences exist with respect to ability of purified cells of the adipose lineage to redifferentiate and form viable lipid-assimilating cells in vitro. Therefore, our present study might provide a foundation to develop tools for biomedical and agricultural applications, as well as to determine the regulation of depot-specific cells of the adipose lineage. A total of 15 genes were selected as candidates for development of genetic markers associated with genetic resistance to PRRSV infection. Forty-one pairs of primers were designed to target 2 - 5 regions per gene. PCR reactions were carried out on pig samples and direct sequencing revealed mutations (SNPs and insertion/deletions) in 14 of these 15 candidate genes, ranging from 1 to 25 mutations per gene. Forty two mutations were formatted in a Sequenom genotyping assay and genotyped on the PHGC samples. Association analysis between these mutations and PRRSV infection phenotypes is underway. Overall, our focus has been on taking advantages of sequence annotation, SNP genotyping, transcriptome analysis and genome mapping information in identifying QTL and QTN for economically important traits in swine. All markers developed in these studies can be immediately used in marker-assisted selection to optimize production, quality, nutritional value and resistance to diseases in the species.
Publications
- Dodson, M.V., L.L.Guan, M.E.Fernyhough, P.S.Mir, L.R.Bucci, D.C.McFarland, J.Novakofski, J.Reecy, K.Ajuwon, D.Thompson, G.J.Hausman, M.Benson, W.G.Bergen, and Z.Jiang. 2010. Perspectives on the formation of an interdisciplinary research team. Biochemical and Biophysical Research Communications. 391:1155-1157.
- Dodson, M.V., J.L.Vierck, G.J.Hausman, L.L.Guan, M.E.Fernyhough, S.Poulos, P.S.Mir, and Z.Jiang. 2010. Examination of adipose depot-specific PPAR moieties. Biochemical and Biophysical Research Communications. 394:241-242.
- Dodson, M.V., G.J.Hausman, L.L.Guan, m.Du, T.P.Rasmussen, S.Poulos, P.S.Mir, W.G.Bergen, M.E.Fernyhough, D.C.McFarland, R.Rhoads, B.Soret, J.Reecy, S.Velleman, and Z.Jiang. 2010. Skeletal muscle stem cells from animals I. Basic cell biology. International Journal of Biological Sciences. 6(5):465-474.
- Dodson, M.V., G.J.Hausman, L.L.Guan, m.Du, T.P.Rasmussen, S.Poulos, P.S.Mir, W.G.Bergen, M.E.Fernyhough, D.C.McFarland, R.Rhoads, B.Soret, J.Reecy, S.Velleman, and Z.Jiang. 2010. Lipid metabolism, adipocyte depot physiology and utilization of meat animals as experimental models for metabolic research. International Journal of Biological Sciences. 6(7):682-690.
- Chen, J., M.V.Dodson, and Z.Jiang. 2010. Cellular and molecular comparison of redifferentiation of intramuscular- and subcutaneous- adipocyte derived progeny cells. International Journal of Biological Sciences 6:80-88.
|
Progress 01/01/09 to 12/31/09
Outputs
OUTPUTS: The WA station focuses mostly on Objective 1, but we have also started to target Objective 2. The major activities at the WA station include molecular characterization of adipocytes, association analysis of functional genes and in silico construction of QTLs/candidate gene maps for pork quality. In molecular characterization of adipocytes, we obtained mature (lipid-filled) pig-derived adipocytes from the perirenal adipose depot after routine slaughter at the Washington State University (WSU) meats laboratory. The isolated mature adipocytes were purified by using serial differential plating methods to prevent any contamination of mature adipocyte cultures by fibroblast-like cells. At the end of the procedure, mature clonal adipocytes were marked using a fine-tip pen on the bottom of the flask, and photomicrographs of cell transitions were then captured for these marked adipocytes during dedifferentiation. The experiment was performed in collaboration with Dr. Dodson, Animal Sciences Department at Washington State University. Identification of candidate genes for pork quality was done in collaboration with Dr. Chen at Nanjing Agricultural University, China; Dr. Liu at Huazhong Agricultural University, China; and Dr. Peter Dovc at University of Ljubljana, Slovenia. Both Dr. Chen's and Dr. Liu's laboratories used the differential-display reverse transcription-polymerase chain reaction (DDRT-PCR) to identify differentially expressed (DE) genes between longissimus dorsi muscles with extremely different intramuscular fat content. Dr. Dovc sent Dr. Tanja Kunej to the WA station to work on the porcine mitochondrial transcription factor A (TFAM) gene. We used the current PigQTLdb (http://www.animalgenome.org/QTLdb/) for the in silico construction of a pork quality QTL/candidate gene map. The database contains a total of 1831 QTLs from 113 publications representing 316 different pig traits. Among them, at least 520 significant or suggestive QTLs were identified for 86 traits related to fat deposition and fatty acid composition in pigs, such as abdominal fat, backfat (average) thickness, backfat at shoulder, backfat at first rib, backfat between 3rd and 4th rib, backfat at tenth rib, backfat at last rib, backfat at last lumbar, backfat weight, fat percentage in carcass, intermuscular fat percentage, marbling, fat androstenedione level and fatty acid composition. The QTL/candidate gene map was constructed based on their genetic map locations. PARTICIPANTS: Michael V. Dodson and Jennifer J. Michal, Washington State University; Jie Chen, Nanjing Agricultural University, China; Ban Liu, Huazhong Agricultural University, China; Peter Dovc and Tanja Kunej, University of Ljubljana, Slovenia. Two visiting professors, Dr. Chen from Nanjing Agricultural University and Dr. Kunej from University of Ljubljana, received training at the WA station. Under the College's support, three undergraduate students - Kari Von Krosigk, Dor Dor Vuong, and Amy Youngren explored the molecular characterization of the seroyl-CoA desaturase (SCD1) gene in efforts to improve the quality of pork. TARGET AUDIENCES: Genome research community, swine industry professionals, pork producers, pig breeders, veterinarians, livestock judges and fair management, other swine scientists nationwide. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
In cell culture, we observed that clonal cultures of pig-derived mature adipocytes are capable of dedifferentiating and forming proliferative-competent progeny cells in vitro. Mature pig adipocytes extrude lipid before proliferation, whereas beef-derived adipocytes divide without expelling lipid. These observations suggest that dedifferentiation of mature adipocytes relies on species-specific mechanisms, or that different culture conditions are required for pig-derived adipocytes to dedifferentiate in a manner similar to beef adipocytes. This in vitro system will aid in our understanding of lipid metabolism, regulation of single cells, processes involved in dedifferentiation of cells, and/or characteristics of putative stem cells residing in adipose tissue. Dr. Chen's lab identified splicing factor serine-arginine rich protein (SFRS18) and Dr. Liu's lab found cardiomyopathy associated 1 (CMYA1) as the DE genes involved in regulation of intramuscular fat deposition. The mRNA levels of SFRS18 gene showed significant and positive correlation with intramuscular fat content in LD muscle (r = 0.54, P < 0.01). One synonymous mutation and three missense mutations were detected in the porcine CMYA1 gene. Association analysis showed that different genotypes of CMYA1 gene were associated with different back fat thicknesses (P < 0.05). We determined both full-length cDNA and genomic DNA sequences of the porcine TFAM gene. Gene expression analysis revealed an alternative 5' splice site, which excludes exon 4 of the pig gene. We assigned this gene to porcine chromosome 14 (SSC14). A G/A substitution was detected in intron 1 of porcine TFAM gene and genotyped on a total of 252 animals. All these data provided basic genomic information needed for further functional characterization of the porcine TFAM gene. As for the QTL/candidate gene map for the quality of pork, we found that these 469 QTLs for fat deposition and composition are not evenly distributed in the porcine genome, ranging from 2 on SSC16 to 68 on SSC7. Second, among these 14 clusters of traits, 8 are related to backfat measurements. Of them, most QTL data refers to average backfat. Third, although both marbling and intramuscular fat content measure fat stored in muscle and they are well correlated, their genetic backgrounds are quite different. So far, at least 30 candidate genes have been identified that affect fat deposition in pigs. These markers can be immediately used to help producers optimize production, quality, nutritional value and resistance to diseases in pigs.
Publications
- Wang, X.X., C.Y.Xue, X.N.Wang, H.L.Liu, Y.X.Xu, R.Q.Zhao, Z.Jiang, M.V.Dodson, and J.Chen. 2009. Differential display of expressed genes reveals a novel function of SFRS18 in regulation of intramuscular fat deposition. International Journal of Biological Sciences 5:28-33.
- Hausman, G.J., M.V.Dodson, K.Ajuwon, M.Azain, K.M.Barnes, L.L.Guan, Z.Jiang, S.P.Poulos, R.D.Sainz, S.Smith, M.Spurlock, J.Novakofski, M.E.Fernyhough, and W.G.Bergen. 2009. Board Sponsored Review: The biology and regulation of preadipocytes and adipocytes in meat animals. Journal of Animal Science. 87(4):1218-1246.
- Xu, X.L., X.W.Xu, P.W.Pan, K.Li, Z.Jiang, M.Yu, M.F.Rothschild, and B.Liu. 2009. Porcine skeletal muscle differentially expressed gene CMYA1: isolation, characterization, mapping, expression and association analysis with carcass traits. Animal Genetics. 40:255-261.
- Chen, J., M.Guridi, M.E.Fernyhough, Z.Jiang, L.L.Guan, G.J.Hausman, and M.V.Dodson. 2009. Clonal mature adipocyte production of proliferative-competent daughter cells requires lipid export prior to cell division. International Journal of Stem Cells 2:76-79.
- Chen, J., M.Guridi, M.E.Fernyhough, Z.Jiang, L.L.Guan, G.J.Hausman, and M.V.Dodson. 2009. Initial differences in lipid processing leading to pig- and beef-derived mature adipocyte dedifferentiation. Basic and Applied Myology 19:243-246.
- Kunej, T., X.L.Wu, J.J.Michal, T.M.Berlic, Z.Jiang, and P.Dovc. 2009. The Porcine Mitochondrial Transcription Factor a Gene: Molecular Characterization,Radiation Hybrid Mapping and Genetic Diversity among 12 Pig Breeds. American Journal of Animal and Veterinary Sciences 4(4):129-135.
|
Progress 01/01/08 to 12/31/08
Outputs
OUTPUTS: In 2008, we focused our research activities on Objective #1: Understand the dynamic genetic mechanisms that influence production efficiency and quality of pork. In collaboration with Dr. Chen at Nanjing Agricultural University, China, we investigated how expression of sterol regulatory element binding transcription factor 1 (SREBF1) and nuclear receptor coactivators 1 (NCOA1), 2 (NCOA2) and 3 (NCOA3) genes affect intramuscular fat (IMF) deposition in pigs. Two experiments were performed for the SREBF1 gene. In Exp. 1, longissimus muscle (LM) samples were removed from 4 male and 4 female Erhualian (EHL) piglets at 3, 20 and 45 d of age and SREBF1 mRNA expression level and IMF content were measured. In Exp. 2, 100 Sutai finishing pigs, a synthetic line produced by crossing EHL and Duroc pigs were used to establish relationships between the SREBF1 mRNA level and IMF content. For three nuclear receptor coactivator genes, longissimus Dorsi (LD) samples at the last rib were collected from 60 castrated male F1 offspring of Landrace X Large White pigs. A portion of the sample was put immediately into liquid nitrogen and then stored at −80 degrees C for mRNA study, whereas the rest of the sample was used to determine muscle IMF content. Full-length cDNA sequences for these porcine genes were obtained using in silico cloning combined with PCR amplification. The pig whole genome is currently being sequenced by The Wellcome Trust Sanger Institute through funding provided by the Cooperative State Research, Education and Extension Service at the United States Department of Agriculture (CSREES-USDA). In 2008, we started to annotate the genomic sequences of Sus scrofa chromosome 1 (SSC1). We downloaded a total of 1,317 clones derived from the chromosome. Sequences of these clones were used as references for BLAST searches against the Human Genome Maps (Build 36.3), thus identifying their orthologous start and stop sites as well as orientations along each human chromosome. When the human orthologous regions harbor annotated genes, their existence in the pig clones were also verified on a one-to-one gene basis using the BLAST 2 Sequences program at NCBI. The swine genome research at WSU has generated two peer-reviewed publications and a poster presented at the International Society of Animal Genetics Conference, which was held in Amsterdam, the Netherlands. Two undergraduate students were involved in the project to gain hands-on experience. PARTICIPANTS: Natashia M. Robinson, Tyler F. Daniels, John P. McNamara and Jan R. Busboom, Washington State University; Xiuxing Wang, Honglin Liu and Jie Chen, Nanjing Agricultural University, China; and Chong Liu, Meiying Fan, China Agricultural University, China. TARGET AUDIENCES: Genome research community, swine industry professionals, pork producers, pig breeders, pig-related youth leaders and high School FFA leaders, livestock haulers, processors, retailers, veterinarians, livestock judges and fair organizers. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
We observed association of mRNA expression level in SREBF1, NCOA1, NCOA2 and NCOA3 genes with IMF in pigs. In Erhualian pigs, IMF content and expression of SREBF1 mRNA was greater (p<0.05) in females than in males at different stages of age. In Sutai pigs, there was a positive correlation between the SREBF1 mRNA level and IMF content (r = 0.67, p<0.01). In three nuclear receptor coactivator genes, we found that both NCOA1 transcript variant 2 (r=-0.554, p<0.01) and total NCOA1 (r=-0.516, p<0.01) expression levels were negatively correlated with IMF contents, while NCOA2 transcript variant 1 (r=0.605, p<0.01) and NCOA3 (r=0.435, p<0.05) were positively associated with IMF content in Longissimus Dorsi muscle. These results indicate that target candidate gene expression analysis might lay a foundation to identify variations within genes that cause the expression differences. Evidence has shown that variants affecting gene expression (expression QTL, eQTL) play a substantial role in understanding genetic basis of complex phenotypes. In annotation of SSC1, we found that among these 1,317 clones, 1,312 (99.62%) were painted to unique regions in the human genome. Such a comparative sequencing painting also revealed that SSC1 has one synteny block with HSA6 and HSA18, 2 synteny blocks with HSA14, 3 with HSA15 and 5 with HSA9. In addition, SSC1 might have slight syntenies with HSA1, HSA10, HSA11 and HSA20. The orthologous regions on HSA6, HSA9, HSA14, HSA15 and HSA18 have a total of 1,412 genes annotated to date (Build 36.3), including 917 known function genes, 170 genes similar to the other genes, 137 hypothetical genes, 114 ORF genes and 74 pseudogenes. However, one-to-one orthologous hits only confirmed 820 (89%) known function genes, 47 (28%) genes similar to other genes, 66 (48%) hypothetical genes, 82 (72%) ORF genes, and 27 (36%) pseudogenes in pigs. These results indicate that different categories of genes might have different evolutionary mechanisms.
Publications
- Chen, J., Yang, X.J., Xia, D., Chen, J.P., Wegner, J., Jiang, Z., and Zhao, R.Q. 2008. SREBF1 expression and genetic polymorphism significantly affect intramuscular fat deposition in the longissimusmuscle of Erhualian and Sutai pigs. Journal of Animal Science. 86:57-63.
- Wang, X.X., Chen, J., Liu, H.L., Xu, Y.X., Wang, X.N., Xue, C.Y., Yu, D.B. and Jiang, Z. 2008. The pig p160 co-activator family: full length cDNA cloning, expression and effects on intramuscular fat content in Longissimus Dorsi muscle. Domestic Animal Endocrinology. 35:208-216.
|
Progress 01/01/07 to 12/31/07
Outputs
OUTPUTS: Comparative mapping of the porcine genome. Pigs were among the first animals to be domesticated and pork is one of the most widely eaten meats in the world today. The pig has also been an excellent biomedical model for understanding a variety of human health issues such as obesity, diabetes, cancer, female reproductive health, cardiovascular disease, and infectious diseases. Genome sequencing, mapping, expression and functional analyses have significantly advanced our ability to unravel the secrets of the pig. Based on several radiation hybrid (RH) maps developed by the swine genome mapping community, we constructed a comparative genome map of pig with an identification of over 170 conserved segments between the human and pig genomes, which will help to further determine the evolutionary relationship between these two species. In January 2007, I organized and chaired the swine workshop at the Plant and Animal Genome Conference in San Diego and edited a special issue to
celebrate the year of the pig 2007. Sterol regulatory element binding transcription factor 1 expression and genetic polymorphism significantly affect intramuscular fat deposition in the longissimus muscle of Erhualian and Sutai pigs. This is a collaboration work between Washington State University and Nanjing Agricultural University, China. Two experiments were performed to elucidate the role of sterol regulatory element binding transcription factor 1 (SREBF1) in intramuscular fat (IMF) deposition in pigs. In Exp. 1, the longissimus muscle (LM) samples were removed from 4 male and 4 female Erhualian (EHL) piglets at 3, 20 and 45 d of age and SREBF1 mRNA expression level and IMF content were measured. Intramuscular fat content and expression of SREBF1 mRNA was greater (P < 0.05) in females than males at all three stages of age, providing initial evidence that the level of SREBF1 mRNA expression is related to IMF deposition in muscle of suckling pigs. Additionally, in Exp. 2 there was a
significantly positive correlation between the SREBF1 mRNA level and IMF content (r = 0.67, P < 0.01) in 100 Sutai finishing pigs, a synthetic line produced by crossing EHL and Duroc pigs. Single strand conformation polymorphism (SSCP) analysis of the reverse transcription PCR (RT-PCR) products of SREBF1 gene revealed 3 genotypes in Sutai pigs with frequencies of 50% for AA, 36% for AB and 14% for BB, respectively. Both SREBF1 mRNA level and IMF content in muscle were greater (P < 0.05) in AB and BB animals than in AA animals, while no difference in backfat thickness was observed among the 3 genotypes. Sequencing analysis identified two single nucleotide polymorphisms (SNP) at T1006C and C1033T within the open reading frame of SREBF1 gene (NM_214157). Although both are silent mutations, they affected the secondary structure of SREBF1 mRNA. These results suggest that SREBF1 might play an important role in regulation of muscle fat deposition during postnatal growth of pigs.
PARTICIPANTS: Nanjing Agricultural University: J. Chen, X. J. Yang, D. Xia, J. Chen and R. Q. Zhao. Iowa State University: M.F. Rothschild and Z.Hu.
TARGET AUDIENCES: Pig breeders and producers, pork processors and consumers, genome mapping community
Impacts
Comparative maps of the porcine genome will assist in its genome sequence assembly. The SNPs identified in the SREBF1 gene suggest that it could be used as a genetic marker to improve intramuscular fat content in pigs. During the last five decades, selection for lean meat production has been an important objective for improving pork quality. However, this effort might have caused intramuscular fat content to decrease, thus making pork tougher and less flavorful. As the SREBF1 gene is not associated with subcutaneous fat depth, our study provides an important tool for the pig industry to produce lean pork with a reasonable intramuscular fat content, without significantly increasing other fat depots.
Publications
- Jiang, Z., and Rothschild, M.F. 2007. Swine genome science comes of age. International Journal of Biological Sciences 3:129-131.
- Rothschild, M.F., Hu, Z., and Jiang, Z. 2007. Advances in QTL mapping in pigs. International Journal of Biological Sciences 3:192-197.
- Chen, J., Yang, X.J., Xia, D., Chen, J.P., Wegner, J., Jiang, Z., and Zhao, R.Q. 2007. Sterol regulatory element binding transcription factor 1 expression and genetic polymorphism significantly affect intramuscular fat deposition in the longissimus muscle of Erhualian and Sutai pigs. J. Anim. Sci. 86:57-63.
|
|