The Effects Of Expression Pattern And Manipulation Of Porcine Myostatin Gene On Muscle Mass In Pigs

Lean meat content is one of the major determinant factors on pork qualitative and is generally associated with the quantity and growth of fibrocyte. Many investigates indicated that myostatin (MSTN) gene is a candidate gene which affect the growth and development of muscle. To determine the expression pattern of MSTN gene in different breed pigs and the effects of nutrient and active immunization against myostatin on the carcass lean percentage and MSTN gene expression,the research included the following contents:1. Effect of Breed on meat quality and MSTN gene expression in pigs: The MSTN gene expression in longissimus of Hampshire and Changsa (LandrancexSaba) at different body weight (20, 50 and 100 Kg) was investigated. The real-time quantitative PCR withβ-actin mRNA as internal standard was applied to determine the levels of myostatin mRNA. At the same time, the pork quality and carcass quality were considerated at 100kg body weight. The results showed the expression of myostatin had an increasing tendency in both breeds of pigs with the increasing of body weight (P>0.05) .The longissimus myostatin gene expression of Changsa pig was greatly significantly or significantly higher than that of the Hampshire at the same body weight. The expression of myostatin had greatly significant positive correlation with backfat thickness (P<0.01) and greatly significant negative correlation with lean percentage (P<0.01) .The results demonstrated that the longissimus myostatin gene increased expression with the growth and its expression was significantly affected by breeds of pig. The results of the correlativity analysis among myostatin, carcass quality and meat quality, indicated that the expression of myostatin had different size correlations coefficients with carcass quality and meat quality.2. Effect of nutritive level on meat quality and MSTN gene expression in pigs:24 DLY (Duroc×Landrance×Yorkshire) with BW 64.24±2.66kg were allotted into 2 groups. One group was fed high nutritive level(DE=13.81MJ/kg, CP=14.19%),and the other was fed low nutritive level(DE=12.55MJ/kg, CP=11.08%).Six pigs were slaughtered in each group at BW lOOkg.The result indicated that the AFI and F/G respectively was decreased 5.07% (p<0.05) and 8.75% (p<0.01) ,and ADG was increased 4.14% (p>0.05) for pig fed high nutrient level compared to low nutritive level. The carcass lean percentage and loin muscle area respectively were increased 4.46% and 4.28%, drop loss and intramuscular fat were increased 16.8% and 37.3%. But the expression of MSTN gene was not obviously influenced by nutrition level. It is concluded that nutrition level could influence carcass quality, but not influence the expression of MSTN gene.3. Effect of Breed and nutrition level on the Expression of Myostatin in pigs: The real-time quantitative PCR withβ-actin mRNA as internal standard was applied to determine the levels of myostatin mRNA. The experimental design was a 2×2 factorial arrangement, including 2 breed (Rongchang pigs and Changrong pigs) and 2 nutrition levels(Feeding standard for Rongchang pigs and China Feeding standard for lean-type pigs). 120 pigs (60 Rongchang pigs and 60 Changrong pigs)were allotted into 4 groups (6 repeats per group and 5 pigs per repeat). Six pigs were killed by electrical stunning followed by exsanguination at BW of 20kg,35kg,50kg,80kg and 110kg respectively. The result indicated that the expression of MSTN had an increasing tendency in both breeds of pigs with the increasing of body weight. And the longissimus MSTN gene expression of Rongchang pigs was greatly significantly or significantly higher than that of the Changrong pigs at the same body weight. The expression of MSTN gene was not obviously influenced by nutrition level, neither by the interaction of breed and nutition levels. The results suggested that the longissimus MSTN gene expression was increased with the growth and its expression was significantly affected by breeds of pig; and the expression of MSTN gene was not obviously influenced by nutrition level.4. Effects of active immunization against myostatin on Muscle Mass in pigs: Twenty-seven pigs were allotted into three groups (9 pigs per group). Three and six pigs were killed by electrical stunning followed by exsanguination at BW of 60 and 100kg respectively. The treated groups were immunized with 1mg and 4mg myostatin and the control group was injected with the same volume of physiological saline. The results indicated that the titer of myostatin antibody was increased in treated groups compared to the control group at D42(p<0.01) and D84 (p< 0.01) respectively. No significant difference was observed in the titer of MSTN antibody between two treated groups .The carcass lean percentage was significantly increased in treated groups compared to the control group at 100 kg BW(p<0.01).The activity of creatine kinase in muscle of pig treated with 1mg and 4mg myostatin obviously decreased compared to the control group at 100kg BW. At 60 kg BW and 100 kg BW the immunization of myostatin obviously decreased the gene expression levels of myostatin in muscle and greatly significant increased the diameter and area of muscle fiber. It was concluded that optimal active immunization against myostatin could increase the content of myostatin antibody and the diameter and area of muscle fiber, suppress the activity of creatine kinase and the expression of myostatin gene, and in result improve the carcass lean percentage for pigs.5. Construction of recombinant yeast wxpression vector of MSTN: Specific primers of MSTN were devised according to the genome sequence of MSTN, and the enzyme-cutting sites were EcoR I and Not I respectively. The target RT-PCR product were cloned into pMD18-T vector, then the MSTN was cutted from the T vector and ligated to the yeast expression vector treated in the same way. The ligated products were transformed in competent cell of Escherichia coli. Restriction enzyme cutting and PCR methords were applied to select positive clone and then the MSTN in recombinant expression vector were sequenced. The bioanalysis software online was applied to analyze the MSTN protein and the recombinant yeast expression plasmid pPIC9K-MSTN. The results demonstrated that the recombinant yeast expression vector was constructed successfully.The research prepared biomaterials for the expression of physiological activity MSTN and the study of MSTN.In summary, we concluded that:(1) MSTN gene expression varied with pig breeds and growing periods. The expression of MSTN had an increasing tendency in all breeds of pigs with the increasing of body weight; the MSTN gene expression for pigs of lean-meat type was obviously lower than that of Chinese local breed pigs or that of cross-breed pigs including Chinese local breed pigs; the MSTN gene expression for cross-breed pigs including Chinese local breed pigs was obviously lower than that of Chinese local breed pigs; and The expression of MSTN gene had greatly significant negative correlation with carcass lean percentage.(2) The expression of MSTN gene was not obviously influenced by nutrition level.(3) Active immunization against MSTN could increase the content of myostatin antibody, the diameter and area of muscle fiber, suppress the activity of CK and the expression of myostatin gene, and in result improve the carcass lean percentage for pigs.