Cloning And Prokaryotic Expression Of Porcine Sox6 And Its Role In Muscle Fiber Type Transformation

At present, pork is the world's largest consumption of meat products. Especially in China, pork products have long dominated the top of the meat consumption because of traditional food habits. With the fast development of swine industry and living standards, consumer demand for pork quality is getting higher and higher. Meat quality is closely correlated with muscle fiber types. According to the myosin heavy chain (MyHC) isoforms, muscle fibers can be divided into types ?,?a, ?x and ?b, mainly expressing MyHC ?, MyHC ?a, MyHC ?b and MyHC ?x, respectively. Increasing the proportion of MyHC I can efficiently improve pork quality. Sox6 is an important member of the Sox gene family of transcription factors involved in regulating muscle fiber type conversion, which can affect the proportion of MyHC I in muscles. Thus, Sox6 has potential function in pork quality. However, there is no report of porcine Sox6 (pSox6) in the literature. This study aims to clone pSox6, construct pSox6 prokaryotic and eukaryotic expression vector, prepare recombinant pSox6 protein, and explore the effect of pSox6 overexpression and recombinant pSox6 protein in muscle fiber type conversion. Main contents and results are as follows:Experiment 1:Cloning of the full coding region of pSox6 and its tissue expression pattern analysisSox6 belongs to a member of the Sox gene family of transcription factors and is an inhibitor of slow-twith specific genes, which is involved in the regulation of muscle fiber type conversion. Sox6 can affect the content of type I muscle fibers in muscle. However, cloning of pSox6 has not been previously reported. In this experiment,500mg longissimus dorsi (LL) muscle from a 3-day-old DLY (Duroc x Landrace x Yorkshire) piglet was used for total RNA extraction and reverse transcribed into cDNA. According to the known Sox6 coding sequence of mouse, rat and human, a pair of degenerate primers was designed and used to amplificate pSox6. In this study, the pSox6 cDNA was cloned by degenerate PCR. The entire open reading frame (ORF) of pSox6 is 2406 bp. The nucleotide sequence of pSox6 was deposited in GenBank with accession number KF933861. This nucleotide sequence shares 87.7%,86.71%,90.91% and 89.49% homology with the known Sox6 sequences of human, mouse, rat and cattle, respectively. The predicted protein is composed of 801 amino acids. The degree of sequence identity between pSox6 and those of human, mouse, rat and cattle were 89.9%,90.74%,95.26% and 90.53%, respectively. There is 100% sequence identity in the functionally critical domains (the HMG box, the leucine-zipper motif, the glutamine-rich Q-box in the N-terminal region, the Q-box in the C-terminal region) among those proteins, indicating that pSox6 may have the same function as that of the reported Sox6. Next, three 10-week-old female DLY pigs were used to detect the expression level of pSox6 mRNA in the heart, liver, kidney, fat, LL muscle, extensor digitorum longus (EDL) muscle, soleus (SOL) muscle, psoas major muscle, and anterior tibialis muscle. Real-time quantitative PCR showed that pSox6 transcript was more abundantly expressed in the heart and skeletal muscle than in the kidney, fat, liver, spleen and lung. Real-time quantitative PCR also showed that pSox6 mRNA was more abundantly expressed in the LL and EDL muscles than in the SOL muscle.Experiment 2:Prokaryotic expression and purification of pSox6Prokaryotic expression vector pET-30a(+)-pSox6 expressing a fusion protein corresponding to pSox6 carrying the extra 6-histidine C-terminal tag was successfully constructed. The pSox6 fusion protein with a molecular weight of about 90 kDa was expressed in E. coli Rosetta (DE3). In order to obtain optimal conditions for protein expression,0 mmol/L,0.25 mmol/L,0.5 mmol/L,0.75 mmol/L,1.0 mmol/L,2.0mmol/L and 3.0 mmol/L IPTG concentrations and 1 h,3 h,4 h,5 h,6 h,8 h time points were set, respectively. SDS-PAGE results showed that the optimal condition was 1 mM IPTG,5 h and 30?. Solubility analysis results showed that the recombinant protein existed in the target protein existed in the supernatant, which means it was expressed in a soluble form. The protein was purified by Ni affinity chromatography, yielding approximately 5?g/mL. The purified pSox6 protein was then identified by Western blot analysis using the monoclonal anti-His (C-term) antibody and confirmed by matrix-assisted laser desorption/ionization time-of-flight/time-of-flight (MALDI-TOF/TOF) mass spectrometer analysis, suggesting that the purified recombinant protein was the pSox6 protein.Experiment 3:Effect of pSox6 on muscle fiber type transformationOn the basis of successful construction of the eukaryotic expression vector pcDNA3.1(+)-pSox6, preparation of recombinant pSox6 protein and isolation of porcine primary satellite cells, this study investigated the effect of pSox6 on muscle fiber type transformation. Firstly, pcDNA3.1(+)-pSox6 plasmid was injected into tibialis anterior muscle of mice by using the method of intramuscular injection to examine the effect of pSox6 overexpression on MyHC isoform expression in vivo. Real-time quantitative PCR results showed that overexpression of pSox6 led to downregulation of MyHC ? mRNA expression and upregulation of MyHC Ha, MyHC ?x, and MyHC ?b mRNA expressions. Western blot results showed that overexpression of pSox6 downregulated MyHC ? protein expression and upregulated MyHC ?b protein expression. Secondly, C2C12 cells were used as a cell model to to examine the effect of pSox6 overexpression on MyHC isoform expression in vitro. Western blot results showed that overexpression of pSox6 obviously downregulated MyHC ? protein expression and upregulated MyHC ?b protein expression in C2C12 myotubes. Finally, this study investigated that effect of recombinant pSox6 protein on the expression of slow-twitch muscle fiber specific genes in myotubes derived from primary porcine satellite cells. Real-time quantitative PCR results showed that recombinant pSox6 downregulated MyHC ?, Tnntl, Tnncl, and Tnnil mRNA expressions in porcine myotubes. Taken tegother, these results suggest that pSox6 plays an important role in muscle fiber type transformation.In conclusion, this study successfully cloned the pSox6 gene and found that it was most abundant in the heart and skeletal muscles of pigs. This study first prepared recombinant pSox6 protein with biological activity by using E. coli expression system, affinity chromatography purification and the method of dilution renaturation. The present study also confirmed that pSox6 had played an important role in regulating muscle fiber type transformation. This study fills the gap of pig Sox6 gene and provides an important theoretical basis and new strategies in improving pork quality.