Distinctive genes determine different intramuscular fat and muscle fiber ratios of the longissimus dorsi muscles in Jinhua and landrace pigs

Abstract

Meat quality is determined by properties such as carcass color, tenderness and drip loss. These properties are closely associated with meat composition, which includes the types of muscle fiber and content of intramuscular fat (IMF). Muscle fibers are the main contributors to meat mass, while IMF not only contributes to the sensory properties but also to the plethora of physical, chemical and technological properties of meat. However, little is known about the molecular mechanisms that determine meat composition in different pig breeds. In this report we show that Jinhua pigs, a Chinese breed, contains much higher levels of IMF than do Landrace pigs, a Danish breed. We analyzed global gene expression profiles in the longissimus dorsi muscles in Jinhua and Landrace breeds at the ages of 30, 90 and 150 days. Cross-comparison analysis revealed that genes that regulate fatty acid biosynthesis (e.g., fatty acid synthase and stearoyl-CoA desaturase) are expressed at higher levels in Jinhua pigs whereas those that regulate myogenesis (e.g., myogenic factor 6 and forkhead box O1) are expressed at higher levels in Landrace pigs. Among those genes which are highly expressed in Jinhua pigs at 90 days (d90), we identified a novel gene porcine FLJ36031 (pFLJ), which functions as a positive regulator of fat deposition in cultured intramuscular adipocytes. In summary, our data showed that the up-regulation of fatty acid biosynthesis regulatory genes such as pFLJ and myogenesis inhibitory genes such as myostatin in the longissimus dorsi muscles of Jinhua pigs could explain why this local breed produces meat with high levels of IMF.

Figure 1. The Landrace breed grows faster than does the Jinhua breed.

(A) Photographs showing three Jinhua pigs and one Landrace pig at d150. (B) Comparison of the body weight of Jinhua and Landrace pigs at the age of d30, d60, d90, d120 and d150, respectively. Landrace pigs gained weight much faster than Jinhua pigs. Pigs were slaughtered at around the age of d30, d90 and d150 (nine individuals per stage) and d60 and d120 (three individuals per stage) for each breed. Data are presented as means ± standard error. *P<0.05, **P<0.01.

Figure 2. The Jinhua breed has a higher IMF content than the Landrace breed.

(A) Oil Red O staining of longissimus dorsi muscles in Jinhua and Landrace pigs, respectively. Oil Red O stained IMF displayed a red color. (B) Comparison of IMF contents in longissimus dorsi muscles in Jinhua and Landrace pigs at the age of d30, d60, d90, d120 and d150, respectively. Pigs were slaughtered at around the age of d30, d90 and d150 (nine individuals per stage) and d60 and d120 (three individuals per stage) for each breed. Data are presented as means ± standard error. **P<0.01. Scale bars, 100 µm.

Figure 3. Validation of microarray data by qPCR.

Validation by qPCR of 16 genes up-regulated in longissimus dorsi muscles of Jinhua pigs at d90 by qPCR. The qPCR values are shown as expression fold changes after normalization against the control 18s rRNA. Data are presented as means ± standard error. Gene ID was as shown. The full names of gene IDs representing AY589691.1, CO993113, BF712908, CN153105, BF078710, NM_213785.1, NM_213938.1, NM_214392.1, BI399912, U83916.1, NM_214294.1, NM_214236.1 are adiponectin, heat shock 105kDa/110kDa protein 1, lipoprotein lipase, carbonic anhydrase II, leukemia inhibitory factor receptor, tissue factor, 3-oxoacid CoA transferase 1, lysozyme, pyruvate dehydrogenase kinase, connective tissue growth factor, tropomodulin 3 and myoglobin. BX924812, CF365450, BQ600160, CF176622 are novel genes.

Figure 4. pFLJ encodes a novel protein and is highly expressed in longissimus dorsi muscle in Jinhua pigs at d90.

(A) Alignment of amino acid sequences of FLJ homologues from pig (XP_003130310), chimpanzee (XP_001162764), human, rhesus monkey and mouse using the CLUSTAL X programme. (B and C) qPCR analysis of pFLJ expression in different organs/tissues (B) or in longissimus dorsi muscles in Jinhua at different stages as shown (C). The qPCR values are shown as expression fold changes after normalization against the control 18s rRNA. Data are presented as means ± standard error. Gene ID was as shown. ^ab means every two columns with different letters are significantly different (P<0.05).

Figure 5. SR141716 down-regulates pFLJ expression and inhibits fat deposition in cultured intramuscular adipocytes.

(A and B) qPCR analysis of pFLJ expression (A) and measurement of total triglyceride (B) in cultured adipocytes 24 and 48 hours after SR141716 treatment. The qPCR values are shown as expression fold changes after normalization against the control 18s rRNA. Data are presented as means ± standard error. Gene ID was as shown. Cells were stained with Oil-Red O to determine lipid accumulation (total triglyceride). *: P<0.05, **: P<0.01.

Figure 6. pFLJ functions as a positive regulator of fat deposition in intramuscular adipocytes.

(A) Cell images to verify transfection efficiency. Cells were transfected with pSilencer TM 4.1-CMV neo plasmids carrying the sequences fs1, fs2 and fs3. Transfection efficiency was assessed by expression of the reporter gene EGFP (green color) harbored by the plasmid. (B) qPCR analysis of pFLJ expression in cultured adipocytes 24 hours after siRNA treatment. fs1, fs2 and fs3: pFLJ specific siRNAs; ns: negative control siRNA. (C) qPCR analysis of FAS, ACC, ATGL and HSL in cultured adipocytes treated with fs1 siRNA. The qPCR values are shown as expression fold changes after normalization against the control 18s rRNA. Data are presented as means ± standard error. *: P<0.05, **: P<0.01 (C and D) Measurement of total triglyceride (as before) in the cultured adipocytes or free glycerol (the free glycerol release was normalized to total cellular protein and expressed relative to the control group) in the culture medium 36 hours after treatment with fs1 siRNA. ^ab means every two columns with different letters are significantly different (P<0.05).