Inosine monophosphate (IMP) is a key aroma component present in livestock and poultry muscle and an indicator of meat quality   . The de novo synthesis of IMP in livestock and poultry requires a ten-step reaction, of which adenosuccinate lyase (ADSL) and 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase/IMP cyclohydrolase (ATIC) catalyze the last three steps     . AMP deaminase 1 (AMPD1), which is highly expressed in muscle, catalyzes the hydrolysis of AMP into IMP and ammonia post-slaughter   . Studies on IMP metabolism have mainly focused on the polymorphisms and mRNA levels of AMPD1, ADSL, and ATIC and their association with IMP content in chickens  -  . The study findings revealed that the expression levels of these genes are higher in muscle than in other tissues   and that IMP content is associated with these three genes       . The polymorphisms of ADSL, AMPD1, and ATIC in different swine species and their associations with IMP metabolism have been reported      . In porcine muscle, researchers have studied the factors that affect IMP content, including swine species and storage temperature post-slaughter    . However, no study has focused on the relationship between ADSL, AMPD1, and ATIC and IMP content. The correlation between mRNA levels of ADSL and IMP content has been reported  , but not the correlation between mRNA levels of ATIC and AMPD1 and IMP content.
To determine the molecular mechanism of aroma component formation in native pigs and support the breeding of high-quality pigs, we measured the muscle mRNA levels of ADSL, AMPD1, and ATIC and determined their correlations with muscle IMP content in Dapulian (DPL), Landrace × Dapulian (LDPL), and Duroc × Landrace × Dapulian (DLDPL) hybridized pigs.
2. Materials and Methods
2.1. Animals and Sampling
All experimental procedures with animals were conducted in accordance with the standards specified by Administrative Committee for Laboratory Animals of the Institute of Animal Husbandry & Veterinary Science, Shandong Academy of Agricultural Sciences (IACC20060101, 1 Jan, 2006). DPL, LDPL, and DLDPL hybridized pigs of similar parity (~25.0 kg; n = 8 per species) were bred in Dongsan Dapulian Pig Seed Farm (Jining, Shandong, China). The animals had ad libitum access to water and feed and were sacrificed in Yinbao Food Co. Ltd. (Shandong, China), when the body weight was ~
2.2. Measurement of IMP Content
Muscle IMP content was measured by HPLC   . Briefly,
2.3. Total RNA Isolation and RT
Total RNA was isolated from longissimus dorsi using RNAiso Plus reagents (TaKaRa) and subjected to 0.8% denaturing agarose gel electrophoresis. RNA content was measured using a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific). Residual DNA was removed, and cDNA was synthesized using a PrimeScript RT reagent kit (RR
2.4. Primer Design and Synthesis and Real-Time PCR
Eleven genes (ACTB, GAPDH, B
Quantitative real-time PCR (qPCR) of cDNA was performed in triplicate using LightCycler® 480 SYBR Green I Master (Roche). The reaction mixture (20 µL) contained Blue-SYBR-Green mix (10 μL), 1 µL of each forward and reverse primers (10 μM/μL), cDNA (1 μL), and distilled water (7 μL). The PCR program consisted of a pre-denaturation step at 95˚C for 5 min, followed by 40 cycles at 95˚C for 10 s (denaturation) and 60˚C for 20 s (annealing and extension). The melting curve was performed at 95˚C for 5 s (denaturation), 65˚C for 60 s (annealing), and 97˚C for 10 s (melting). The CT values of the internal genes and target genes were quantified using Roche LightCycler® 480 (Roche).
2.5. Statistical Analysis
For the candidate internal gene, the minimal Ct value was set to 1. The relative value of genes was calculated by 2−ΔCt, where ΔCt = Ct value of other samples −
Table 1. Selected reference genes.
Table 2. Target genes.
minimal Ct value. For stability analysis, data were analyzed using geNorm V3.5 software.
The relative expression of the target genes in DPL, LDPL, and DLDPL hybridized pigs was expressed as 2−ΔCt, where ΔCt = Ct value of the target gene ? Ct value of internal gene. ΔCt > 0 is indicative that the level of the target gene is higher than that of the internal gene, while ΔCt < 0 is indicative that the level of the target gene is lower than that of the internal gene. Statistical analyses were performed with SPSS19.0 statistical software package for Windows (SPSS Inc., Chicago, IL, USA). Results were expressed as mean ± SD. One-way analysis of variance (ANOVA) was used for the comparison of muscle IMP content and expression of AMPD1, ADSL, ATIC between Dapulian and hybridized pig species.
3.1. Determination of RNA and qPCR Results
We obtained three RNA bands (28S, 18S, and 5S) via agarose gel electrophoresis. The A260/A280 ratio and content of total RNA in muscle was 1.97 ± 0.02 and 268.9 ± 72.6 ng/μL, respectively. For the target and internal genes, the amplification curve had a typical S-shape, the melting curve had a standard peak at 85˚C - 90˚C, and the amplification efficiency of the standard curve ranged from 0.95 to 1.1, which met the requirements of relative quantitative analysis.
3.2. Stability Analysis of Internal Genes
The Ct values of the 11 internal genes in swine muscle (n = 24) were measured using qPCR and analyzed using geNorm software. The stabilities of the internal genes were sorted according to the M values obtained from geNorm software (the smaller the M value, the higher the stability). The average stability degree of the two most stable internal genes was determined after successively removing the most unstable internal gene and analyzing the average of the stability degree of the remaining internal genes (M value). In decreasing order of M value (Figure 1), the internal genes were SDHA (0.770) > B
GeNorm software allows the determination of the number of internal genes by analyzing the matching variation, V value, and the selection of the optimal combination of internal genes other than a single internal gene. The Vn/Vn + 1 ratio was compared with the default V value (0.15). At Vn/Vn + 1 ratio > 0.15, it is important to consider the n + 1 gene, whereas the new internal gene might not be considered. It would be optimal for the correction of system deviation to obtain more reliable results and analyze the subtle changes in gene expression. As shown in Figure 2, V2/V3 = 0.094 < 0.15, which revealed that the third internal gene might not be considered, and the average Ct value of YWHAZ and RPL4 may accurately correct and normalize the expression of the target gene.
Figure 1. Average expression stability values of candidate reference genes.
Figure 2. Determination of the optional number of candidate reference genes for normalization.
3.3. IMP Content and Expression of Genes Involved in IMP Metabolism
YWHAZ and RPL4 were selected as internal genes. The expression levels of ADSL, AMPD1, and ATIC were calculated by 2-ΔCt, where ΔCt = Ct value of the target gene − Ct value of internal gene (Vandesompele, et al., 2002). As shown in Table 3, DPL had the highest IMP content in muscle, followed by LDPL and DLDPL. DPL had 25.00% (P < 0.05) and 15.56% more IMP than DLDPL and LDPL, respectively, and LDLP had 8.17% more IMP than DLDPL (P > 0.05).
The muscle mRNA levels of ADSL and ATIC were higher in DPL and LDPL than in DLDPL, with no significant differences between DPL and LDPL. The mRNA levels of ADSL were higher (P < 0.05) in DPL and LDPL than in DLDPL (24.14% and 12.07%, respectively). The mRNA levels of ATIC were higher (P < 0.05) in DPL and LDPL than in DLDPL (66.67% and 33.33%, respectively). The mRNA levels of AMPD1 were higher in DPL than in LDPL and DLDPL (14.49% and 33.26%, respectively), while the difference between LDPL and DLDPL was not significant. Therefore, muscle IMP content in DPL, LDPL, and DLDPL had a similar trend to the mRNA levels of ADSL, AMPD1, and ATIC.
3.4. Correlation between IMP Content and mRNA Levels of Genes Involved in IMP Metabolism
Table 4 shows that muscle IMP content was positively correlated with the mRNA levels of ADSL, AMPD1, and ATIC with correlation coefficients of 0.327, 0.335, and 0.342, respectively (P < 0.05). The mRNA levels of ADSL were positively correlated with the mRNA levels of AMPD1 and ATIC with correlation coefficients of 0.501 and 0.391, respectively (P < 0.01). The mRNA levels of AMPD1 were not significantly correlated with the mRNA levels of ATIC.
Data correction with the appropriate internal gene is critical to obtain reliable
Table 3. IMP content and expression of ADSL, AMPD1, and ATIC.
Different lowercase superscripts represent significant differences within the same row (P < 0.05). DPL, Dapulian pig; LDPL, Landrace × Dapulian hybridized pigs; DLDPL, Duroc × Landrace × Dapulian hybridized pigs.
Table 4. Correlation between IMP content and expression of ADSL, AMPD1, and ATIC.
*P < 0.05. **P < 0.01.
data in mRNA expression analysis  . The expression of any internal gene cannot be consistent under different experimental conditions      . In this study, YWHAZ and RPL4 were selected as internal genes to correct the targets gene.
IMP, an indicator of meat quality, differs among porcine species. The IMP content of native pigs, including Laiwu and Yanan pigs, is significantly higher than that of lean species or native × hybridized pigs     . In this study, muscle IMP content was the highest in DPL, followed by LDPL and DLDPL, which revealed that muscle IMP content decreased in the DPL bloodline. However, Zhang et al.   reported that muscle IMP content was higher in Danish landrace than in thin Wei pig or Anqing six-white pig, which was not in agreement with other studies     . This result might be attributed to slaughter age, dietary components, and feeding methods, which need further validation by increasing the sample size.
ADSL, AMPD1, and ATIC code for enzymes involved in IMP metabolism       . Studies have reported that there are significant differences in muscle IMP content within the same chicken species due to point mutations in ADSL, AMPD1, or ATIC       . The association between muscle IMP content and point mutations in ATIC or AMPD1 in swine    and the association between muscle IMP content and point mutations in ADSL in duck have been investigated, which can be used as a molecular marker for breeding strategies  . However, limited studies have focused on the relationship between IMP content and IMP-metabolic genes in chicken, duck, and swine or on the relationship between IMP content and AMPD1 in chicken, ADSL in duck, or ADSL in swine     . No study has focused on the relationship between IMP content and ADSL, AMPD1, or ATIC in swine. In this study, the muscle mRNA levels of ADSL and ATIC were higher in DPL and LDPL than in DLDPL, while the mRNA levels of AMPD1 were significantly higher in DPL than in LDPL or DLDPL. Muscle IMP content had a similar trend to that of mRNA levels of ADSL, AMPD1, and ATIC. Muscle IMP content is affected by the expression of ATIC or AMPD1 in swine    and by the expression of ADSL, AMPD1, and ATIC in chicken       , consistent with our study findings. However, Zhang et al.   reported that the expression of ADSL in Danish landrace was lower than that in Wei pig and higher than that in Anqing six-white pig without significant differences. Additionally, the expression of ADSL in Wei pig was higher than that in Anqing six-white pig, which was not consistent with our results. In this study, the muscle mRNA levels of ADSL were the highest in DPL, followed by LDPL and DLDPL. The different feeding methods between DPL and Wei and Anqing six-white pigs might affect the muscle IMP content and mRNA levels of ADSL resulting from. The muscle mRNA levels of AMPD1 in chickens and of ADSL in ducks are positively correlated with IMP content   , while the muscle mRNA levels of ADSL in swine are not highly correlated with IMP content   . Our results showed that muscle IMP content was positively correlated with the mRNA levels of ADSL, AMPD1, and ATIC. The mRNA levels of ADSL were positively correlated with the mRNA levels of AMPD1 and ATIC, while the mRNA levels of AMPD1 were not significantly correlated with the mRNA levels of ATIC. The de novo synthesis of IMP consists of 10 reactions catalyzed by 10 enzymes. Muscle IMP can also be formed from the progressive hydrolysis of ATP post-slaughter, which involves several enzymes, such as ATPase, creatine kinase, adenosine deaminase, and phosphatase. Future studies should analyze the factors that affect muscle IMP content.
The relative expression of ADSL in DPL, LDPL, and DLDPL was positively correlated with IMP content. IMP has medium heritability  , which is important for the breeding of high-quality pigs. In this study, the mRNA levels of ADSL, AMPD1, and ATIC were different in the DPL hybridized pigs, which revealed that some key factors may regulate these genes. MicroRNA (miRNA) and siRNA are key molecules that regulate gene expression. MiRNAs are endogenous non-coding RNA molecules (22 nucleotides in length) with regulatory function in eukaryotes. MiRNAs recognize mRNA by base pairing and degrade the target mRNA or inhibit the translation of the target mRNA  . Specific miRNAs are involved in the regulation of ADSL, AMPD1, and ATIC. If the correlation between the relative expression of other enzymes involved in IMP synthesis and the IMP content in the corresponding tissues is identified, the genes that code for those enzymes may represent suitable molecular markers that assist in the selection of animal species with high IMP content.
Muscle mRNA levels of ADSL, AMPD1, and ATIC and muscle IMP content were the highest in DPL followed by LDPL and DLDPL. Muscle IMP content was positively correlated with the mRNA levels of ADSL, AMPD1, and ATIC.
This work was supported by the Youth Foundation of Shandong Academy of Agricultural Sciences [grant number 2014QNM41], the Key Research and Development Plan of Shandong Province, China [grant number 2015GNC111011], the National Natural Science Foundation of China [grant number 31501928, 31501927], and the Natural Science Foundation of Shandong Province, China [grant number ZR2015CM007].
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