mRNA and protein expression levels of four candidate genes for ear size in Erhualian and Large White pigs

Abstract

Porcine ear size is an important characteristic for distinguishing among pig breeds. In a previous genome-wide association study of porcine ear size, LEM domain-containing 3 (LEMD3), methionine sulfoxide reductase B3 (MSRB3), high mobility group AT-hook 2 (HMGA2), and Wnt inhibitory factor 1 (WIF1) were implicated as important candidate genes for ear size. This study investigated the expression levels of four candidate genes for ear size in Erhualian and Large White pigs. Ten Erhualian pigs with large ears and eight Large White pigs with small ears at 60 days of age were examined. The mRNA expression levels of the four candidate genes were quantified by real-time polymerase chain reaction. WIF1 mRNA expression was significantly higher in Large White than in Erhualian pigs (P < 0.05), whereas the expression levels of the other three genes were not significantly different between the two breeds. The protein expression levels of the four genes were analyzed using western blot. WIF1 protein expression was significantly higher in Large White than in Erhualian pigs (P < 0.01), whereas MSRB3 protein expression was significantly higher in Erhualian than in Large White pigs (P < 0.05). There were no significant differences between the two breeds in residual protein expression. These results suggest that WIF1 is the main causal gene for ear size in pigs.

Introduction

Porcine ear shape (size and erectness) is regarded as an important characteristic for distinguishing among pig breeds (Rothschild and Ruvinsky, 1998). Many studies have focused on the molecular mechanisms that determine ear size and erectness in pigs. Quantitative trait locus (QTL) mapping studies have discovered the QTLs for porcine ear erectness in Sus scrofa chromosomes (SSC) 1, 5, 6, 7, 9, and 12 (Guo et al., 2004; Wei et al., 2007; Ma et al., 2009). Moreover, QTLs for porcine ear size have been mapped to SSC 1, 4, 5, 6, 7, 8, 9, 11, 12, 16, and X (Wei et al., 2007; Ma et al., 2009). On SSC7, G32E in the PPARD gene has been identified as the causal mutation for porcine ear size (Ren et al., 2011; Duan et al., 2013); however, the causal gene for porcine ear size on SSC5 has not been identified. Therefore, it is important to study the candidate genes on SSC5 for porcine ear size to understand the molecular mechanisms of ear diversity in pigs.

In our previous genome-wide association study, LEM domain-containing 3 (LEMD3) and Wnt inhibitory factor 1 (WIF1) were identified as important candidate genes for porcine ear size (Zhang et al., 2014). Methionine sulfoxide reductase B3 (MSRB3) is adjacent to the most significant single nucleotide polymorphism that is associated with porcine ear size (Zhang et al., 2014), and studies in dogs have found that MSRB3 may be related to ear morphology (Boyko et al., 2010; Vaysse et al., 2011). Furthermore, porcine ear size QTL fine-mapping on SSC5 suggests that HMGA2 is involved in ear size (Wei et al., 2007; Li et al., 2012). Hence, previous studies have identified the possible causal genes for porcine ear size on SSC5 as the four candidate genes WIF1, HMGA2, LEMD3, and MSRB3; however, little is known about the mRNA and protein expression levels of HMGA2, LEMD3, MSRB3, and WIF1 in pig ear tissues. Erhualian pigs have big, floppy ears (Zhang et al., 1986), and the breed is a good model with which to study ear morphology. In this study, we evaluated the mRNA and protein expression levels of the above four candidate genes in Erhualian and Large White pigs.

Material and Methods

Sampling and data collection

Eight Large White and 10 Erhualian pigs at 60 days of age were obtained from the Institute of Animal Science, Chinese Academy of Agricultural Sciences, and the Chuying Agro-Pastoral Group Co. Ltd., respectively. After slaughter, we removed the entire external left ear, traced the shape of each ear on plotting paper to calculate its area, and collected samples for mRNA and protein extraction. All of the animals used in the study were treated in accordance with the guidelines for the use of experimental animals established by the Council of China, and the animal experiments were approved by the Science Research Department of the Institute of Animal Science, Chinese Academy of Agricultural Sciences (Beijing, China).

RNA isolation, cDNA production, and quantitative real-time polymerase chain reaction (PCR)

Total RNA was isolated from the tissue samples using an RNAprep Pure Tissue Kit (Tiangen, China) according to the manufacturer’s instructions. Extracted RNA was eluted in water and quantified spectrophotometrically at an A260/280 ratio using a NanoDrop UV-Vis spectrophotometer (Thermo Scientific). The cDNA was amplified using RNA (1 mg) and a PrimeScript™ RT reagent kit (TaKaRa, Japan), according to the manufacturer’s instructions.

Gene mRNA expression levels were assessed by quantitative real-time PCR (QPCR) using a 7900HT Fast Real-Time PCR System (Applied Biosystems). β-actin was used as an endogenous control to normalize the target gene expression. The primer sequences are listed in Table 1. For the QPCR analysis, 1 µL cDNA was used with 7.5 µL SYBR^® Select Master Mix (Applied Biosystems), 0.5 µ L forward primer, 0.5 µL reverse primer, and 5.5 µL RNase-free H₂O. Each QPCR cycle was conducted as follows: 50°C for 2 min, 95°C for 5 min, 95°C for 10 s, and 60°C for 1 min for 40 cycles. Relative quantification was calculated using the 2−∆∆Ct method (Livak and Schmittgen, 2001).

Table 1: Primers used for quantitative real-time polymerase chain reaction amplifications of WIF1, LEMD3, HMGA2, and MSRB3.

Protein isolation and western blotting

The protein expression levels were assessed by western blotting using a Mini Trans-Blot^® System (BIO-RAD). Ear tissue was ground into powder in liquid nitrogen and lysed with radioimmunoprecipitation assay buffer (Beyotime, China). The mixture was then supplemented with protease inhibitor (Pierce, USA) and centrifuged to obtain the total protein. Protein quantification was achieved by conducting a bicinchoninic acid assay. Five primary rabbit monoclonal or polyclonal antibodies (Abcam, UK) were used for western blotting: Anti-beta Actin antibody (ab8227), Anti-MSRB3 antibody (ab180584), Anti-WIF1 antibody (ab155101), Anti-HMGA2 antibody (ab109329), and Anti-LEMD3 antibody (ab124148). The β-actin protein was used as an endogenous control to normalize the target gene expression. After separation by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis, the protein bands were transferred onto a polyvinylidene difluoride membrane (Cell Signaling Technology Inc., USA). The membrane was blocked in defatted milk and incubated at 4°C overnight. The membrane was then incubated with the primary antibody at a 1/1000 dilution for 1 h. Two secondary antibodies, IRDye 800 goat Anti-rabbit IgG (green) and IRDye 680 goat Anti-rabbit IgG (red) (LI-COR Biosciences, USA) were used for WIF1, LEMD3, HMGA2, and β-actin detection at a 1/1000 dilution for 1 h. The protein bands were visualized using chemiluminescence reagents (Millipore, USA), with excitation wavelengths under 800 nm (green) and 700 nm (red). For detecting MSRB3, a secondary antibody, Fluorescein goat Anti-rabbit IgG antibody (Vector, USA), and a BeyoECL Plus kit (Beyotime), were used. Optical density was analyzed using Quantity One^® 4.62 software (BIO-RAD).

Statistical analysis

To statistically determine any significant differences, the relative mean expression levels and standard deviations were compared by Student t-test using SAS version 8.2 (SAS, 2001). All of the figures were plotted using Microsoft Office.

Results

Erhualian pigs had lower body weights but larger ears than Large White pigs

The mean body weights of the Erhualian and Large White pigs were 9.63 kg and 22.61 kg, respectively (Table 2), and their mean ear sizes were 127 cm² and 88.54 cm², respectively. Although the Erhualian pigs had a 12.98-kg lower mean body weight than the Large White pigs (P < 0.0001), their ears were significantly larger than those of the Large White pigs (P < 0.0001).

Table 2: Phenotypic characteristics of Erhualian and Large White pigs.

WIF1 transcript levels were higher in Large White pigs’ ears than in Erhualian pigs’ears

The expression levels of the four genes were similar (Table 3 and Figure 1). No significant differences were observed for LEMD3, HMGA2, or MSRB3, but WIF1 mRNA expression was significantly higher (two-fold) in Erhualian pigs than in Large White pigs (P < 0.05). We detected WIF1 mRNA expression in the livers and muscles of both the Erhualian and Large White pigs, although this was not significantly different (Figure 2). These results provide favorable support for WIF1 as the causative gene for ear size.

Table 3: Normalized mRNA expression levels of four genes in Erhualian and Large White pigs.

WIF1 and MSRB3 protein levels were significantly different in Erhualian and Large White pigs’ ears

The expression levels of the four proteins in the ear tissues are shown in Figure 3 and Table 4. WIF1 was significantly more expressed in Large White than in Erhualian pigs (P < 0.01), whereas MSRB3 was significantly more expressed in Erhualian than in Large White pigs (P < 0.05). There were no significant differences between the two breeds in residual protein expression. These results suggest that WIF1 and MSRB3 protein expression affect pig ear size more than HMGA2 and LEMD3 protein expression.

Table 4: Western blot analysis of four genes in Erhualian and Large White pigs.

Discussion

In this study, two pig breeds, Erhualian and Large White, were used to examine the mRNA and protein expression levels of four candidate genes, WIF1, LEMD3, HMGA2, and MSRB3. In previous studies, the genomic regions on SSC5 harboring QTLs for ear size havebeen obtained from both White Duroc x Erhualian F2 resource populations and Large White Minzhu intercross populations (Li et al., 2012; Zhang et al., 2014). In the present study, the Erhualian pigs’ ears were about 127 cm² in area, which was 1.5-fold larger than those of Large White pigs. With such phenotypic differences between Erhualian and Large White pigs, the two breeds are good models with which to study differences in candidate gene mRNA and protein expression levels.

Both the mRNA and protein expression levels of WIF1 were significantly higher in Large White than in Erhualian pigs. There were no significant differences in the mRNA or protein expression levels of LEMD3 or HMGA2 between the two breeds. However, the MSRB3 protein expression level was significantly higher in Erhualian than in Large White pigs. A previous study reported that MSRB3 mRNA expression is significantly higher in the ears of Minzhu pigs, which have large ears, than in the ears of Large White pigs, which have small ears (Zhang et al., 2015). In the present study, the limitation of only using two pig breeds to analyze four candidate genes for ear size was evident, and a comparison of these genes’ expression levels among more pig breeds with a greater diversity in ear size is necessary to identify gene functions further. Nevertheless, the Erhualian breed has almost the largest ears among pig breeds and are obviously larger than those of Large White pigs. Therefore, we can conclude that WIF1 is the prime candidate gene for porcine ear size and morphology.

Previous studies have shown that WIF1 is a secreted protein that can directly bind with Wnts and/or Fzds to interfere with functional interactions between agonist and transmembrane receptors, and affect their activity (Hsieh et al., 1999). WIF1 can also inhibit β-catenin expression, induce human and mouse osteoblastic differentiation, and suppress osteosarcoma growth (Kansara et al., 2009). The Wnt/β-catenin pathway can suppress chondrocyte hypertrophy and endochondral ossification in the nascent stage (Tamamura et al., 2005). WIF1 also stimulates osteoblasts and regulates bone formation (Westendorf et al., 2004), and is also expressed in the marginal periarticular perichondrium of the metacarpal (Witte et al., 2009). Loss of WIF1 expression increases sensitivity to osteoblasts (Kansara et al., 2009). Recently, some studies have reported that WIF1 interferes with the Wint-3a-mediated inhibition of chondrogenesis in embryonic chick limb bud cells (Surmann-Schmitt et al., 2009; Stock et al., 2013). Therefore, low WIF1 expression might allow the Wnt/β-catenin pathway to increase cartilage cell proliferation during the process of outer ear growth, resulting in the large ears of Erhualian pigs.

In summary, we detected different mRNA and protein expression levels of WIF1, LEMD3, HMGA2, and MSRB3 in Erhualian pigs with large ears and Large White pigs withsmall ears. There were no significant differences in LEMD3 or HMGA2 mRNA or protein expression levels between the breeds. At the protein level, MSRB3 was significantly more highly expressed in Erhualian than in Large White pigs, whereas WIF1 mRNA and protein expression levels were both significantly higher in Large White than in Erhualian pigs. These results provide useful information for further functional analyses of candidate genes that influence ear size in pigs.

Conflicts of interest

The authors declare no conflict of interest.

Acknowledgments

Research supported by the Agricultural Science and Technology Innovation Program (#ASTIP-IAS02), the National Key Technology R&D Program of China (#2015BAD03B02), the Earmarked Fund for Modern Agro-Industry Technology Research System, and the National Technology Program of China.