Expression of calpastatin gene in Kermani sheep using real-time PCR

Document Type : Original Research Articles (Regular Papers)


1 Department of Animal Science, Shahid Bahonar University of Kerman, Kerman, Iran

2 Department of Animal Science, College of Agriculture, Shahid Bahonar University of Kerman. Iran.

3 National University of Water Management and Nature Resources Use

4 Sumy National Agrarian University, Sumy, Ukraine

5 National University of Life and Environmental Sciences of Ukraine, Ukraine.

6 Bila Tserkva National Agrarian University, Ukraine.



The aim of this study was to investigate the calpastatin gene expression in different tissues of Kermani sheep using the real-time PCR. Tissue samples from the brain, humeral muscle, femoral muscle, liver, adipose tissue, rumen and testis were taken from 30 Kermani sheep. Total RNA was extracted using RNXTM plus solution. To determine the quantity (concentration) and quality of the extracted RNA, two methods of RNA; electrophoresis on 1% agarose gel and a Nano drop device were used. A Thermoscientific kit (Iran) was used for cDNA synthesis. After performing normal PCR reactions and obtaining the desired binding conditions and temperature for the genes, real-time PCR was performed to study the relative gene expression. The Beta-actin gene was used as a housekeeping gene. The Pfaffl method was used to analyze the data. The quality of the extracted RNAs was good. The presence of two 18S and 28S bands in the rRNA indicated that the RNA was healthy and the absence of an additional band was an indication of its purity. For the calpastatin gene, the 189bp fragment, and for Beta-actin, the 206bp fragment was observed in all tissues. The real-time PCR findings showed that calpastatin gene was expressed in all tissues (brain, humeral muscle, liver, adipose, femoral tissue, rumen and testis) with the highest level of expression in the humeral and femoral muscles and the lowest level in adipose tissues. This study lays a foundation for further calpastatin research in sheep. It is suggested that this study be conducted on a greater number of animals, and different breeds, sexes, ages and physiological stages to reach a more comprehensive conclusion.


Main Subjects

Antonius, A., Ginting, S.P., Elieser, S., Tarigan, A., Solehudin, S., Budisatria, I.G.S., Sari, A.P.Z.N.L., Hariyono, D.N.H., Maharani D., 2020. The association of single nucleotide polymorphism (SNP) g.281G > A of calpastatin gene with meat quality of Boerka Goat. Iranian Journal of Applied Animal Science 10, 303-309.
Barnoy, S., Glaser, T., Kosower, N.S., 1997. Calpain and calpastatin in myoblast differentiation and fusion: ef­fects of inhibitors. Biochimica et Biophysica Acta 1358, 181-188.
Bouhrara, M., Clerjon, S., Damez, J., 2011. Dynamic MRI and thermal simulation to interpret deformation and water transfer in meat during heating. Journal of Agriculture and Food Chemistry 59, 1229-1235.
Bouhrara, M., Lehallier, B., Clerjon, S., 2012. Mapping of muscle deformation during heating: in situ dynamic MRI and nonlinear registration. Magnetic Resonance Imaging 30, 422-430.
Chua, B.T., Guo, K., Li, P., 2000. Direct cleavage by the calcium-activated protease calpain can lead to inactivation of caspases. Journal of Biological Chemistry 275, 5131-5135.
Ebrahimi, Z., Mohammadabadi, M.R., Esmailizadeh, A., Khezri, A., 2015. Association of PIT1 gene and milk protein percentage in Holstein cattle. Journal of Livestock Science and Technologies 3, 41-49.
Enriquez-Valencia, C.E., Pereira, G.L., Malheiros, J.M., de Vasconcelos Silva, J.A.I.I., Albuquerque, L.G., de Oliveira, H.N., Chardulo, L.A.L., Curi, R.A., 2017. Effect of the g.98535683A>G SNP in the calpastatin gene on meat traits of Nellore beef cattle (Bos indicus) and their crosses with Bos taurus. Meat Science 123, 64-66.
Farr, C., 2011. Calpain and calpastatin in a mouse model of acute myeloid leukemia. Dissertation, University of Toronto.
Ghotbaldini, H., Mohammadabadi, M.R., Nezamabadi-pour, H., Babenko, O.I., Bushtruk, M.V., Tkachenko, S.V., 2019. Predicting breeding value of body weight at 6-month age using Artificial Neural Networks in Kermani sheep breed. Acta Scientiarum Animal Science 41, e45282.
Inazawa, J., Nakagawa, H., Misawa, S., Abe, T., Minoshima, S., Fukuyama, R., Maki, M., Murachi, T., Hatanaka, M., Shimizu, N., 1990. Assignment of the human calpastatin gene to chromosome 5 at region q14---q22. Cytogenetics and Cell Genetics 54, 156-158.
Juszczuk-Kubiak, E., Wyszy´nska-Koko, J., Wici´nska, K., Rosochacki, S. 2008. A novel polymorphism in intron 12 of the bovine calpastatin gene. Molecular Biology Reports 35, 29-35.
Kidd, V.J., Lahti, J.M., Teitz, T. 2000. Proteolytic regulation of apoptosis. Cell and Developmental Biology 11, 191-201
Koohmaraie, M., 1996. Biochemical factors regulating the toughening and tenderization process of meat. Meat Science 43, S193-S201.
Leal-Gutiérrez, J.D., Elzo, M.A., Johnson, D.D., 2018. Association of m-calpain and calpastatin polymorphisms with meat tenderness in a Brahman–Angus population. Frontiers in Genetics 9, e56.
Liang, B., Duan, B.Y., Zhou, X.P., 2010. Calpain activation promotes BACE1 expression, amyloid precursor protein processing, and amyloid plaque formation in a transgenic mouse model of Alzheimer disease. Journal of Biological Chemistry 285, 27737-27744.
Masoudzadeh, S.H., Mohammadabadi, M.R., Khezri, A., Kochuk-Yashchenko, O.A., Kucher, D.M., Babenko, O.I., Bushtruk, M.V., Tkachenko, S.V., Stavetska, R.V., Klopenko, N.I., Oleshko, V.P., Tkachenko, M.V., Titarenko, I.V., 2020a. Dlk1 gene expression in different Tissues of lamb. Iranian Journal of Applied Animal Science 10, 669-677.
Masoudzadeh, S.H., Mohammadabadi, M., Khezri, A., Stavetska, R.V., Oleshko, V.P., Babenko, O.I., Yemets, Z., Kalashnik, O.M., 2020b. Effects of diets with different levels of fennel (Foeniculum vulgare) seed powder on DLK1 gene expression in brain, adipose tissue, femur muscle and rumen of Kermani lambs. Small Ruminant Research 193, e106276.
McGinnis, K.M., Gnegy, M.E., Park, Y.H., 1999. Procaspase-3 and poly (ADP) ribose polymerase (PARP) are calpain substrates. Biochemistry and Biophysics Research Communication 263, 94-99.
Mohammadabadi, M.R., 2019a. Dlk1 gene expression in Raini Cashmere goat using real-time PCR. Agricultural Biotechnology Journal 11, 191-205 (In Farsi).
Mohammadabadi, M.R., 2019b. Expression of calpastatin gene in Raini Cashmere goat using real-time PCR. Agricultural Biotechnology Journal 11, 219-235 (In Farsi).
Mohammadabadi, M.R., 2021. Tissue-specific mRNA expression profile of ESR2 gene in goat. Agricultural Biotechnology Journal 12, 169-184 (In Farsi).
Mohammadabadi, M.R., Asadollahpour Nanaei, H., 2021. Leptin gene expression in Raini Cashmere goat using real-time PCR. Agricultural Biotechnology Journal 13, 197-214 (In Farsi).
Mohammadabadi, M., Bordbar, F., Jensen, J., Du, M., Guo, W., 2021. Key genes regulating skeletal muscle development and growth in farm animals. Animals 11, e835.
Mohammadabadi, M.R., Jafari, A.H.D., Bordbar, F., 2017. Molecular analysis of CIB4 gene and protein in Kermani sheep. Brazilian Journal of Medical and Biological Research 50, e6177.
 Morgan, J.B., Savell, J.W., Hale, D.S., Miller, R.K., Griffin, D.B., Cross, H.R., Shackelford, S.D., 1991. National beef tenderness survey. Journal of Animal Science 69, 3274-3283.
Nakagawa, T., Yuan, J., 2000. Cross-talk between two cysteine protease families. Activation of caspase-12 by calpain in apoptosis. Journal of Cell Biology 150, 887-894.
Palmer, B.R., Roberts, N., Hickford, J.G.H., Staffe, R., 1998. Rapid Communication: PCR-RFLP of MSPI and NcoI in the ovine calpastatin gene. Journal of Animal Science 76, 1499-1500.
Pfaffl, M.W., Horgan, G.W., Dempfle, L., 2002. Relative expression software tool (REST©) for group-wise comparison and statistical analysis of relative expression results in real-time PCR. Nucleic Acids Research 30, e36.
Pour Hamidi, S., Mohammadabadi, M.R., Asadi Foozi, M., Nezamabadi-pour, H., 2017. Prediction of breeding values for the milk production trait in Iranian Holstein cows applying artificial neural networks. Journal of Livestock Science and Technologies 5, 53-61
Roudbari, Z., Coort, S.L., Kutmon, M., Eijssen, L., Melius, J., Sadkowski, T., Evelo, C.T., 2020. Identification of biological pathways contributing to marbling in skeletal muscle to improve beef cattle breeding. Frontiers in Genetics 10, e1370.
Ruiz-vela, A., Serrano, F., Gonzalez, M.A., 2001. Transplanted long-term cultured pre-B1 cells expressing calpastatin are resistant to B cell receptor-induced apoptosis. Journal of Experimental Medicine 194, 247-254.
Savell, I.W., Harris, J.J., Cross, H.R., Hale, D.S., Beasley, L., 1991. National beef market basket survey. Journal of Animal Science 69, 2883-2893.
Tang, R., Yu, B., Zhang, K., 2010. Effects of nutritional level on pork quality and gene expression of micro-calpain and calpastatin in muscle of finishing pigs. Meat Science 85, 768-771.
Tohidi nezhad, F., Mohammadabadi, M.R., Esmailizadeh, A.K., Najmi Noori, A., 2015. Comparison of different levels of Rheb gene expression in different tissues of Raini Cashmere goat. Agricultural Biotechnology Journal 6, 35-50 (In Farsi).
Van Ba, H., Reddy, B.V., Hwang, I., 2015. Role of calpastatin in the regulation of mRNA expression of calpain, caspase, and heat shock protein systems in bovine muscle satellite cells. In Vitro Cell and Developmental Biology 51, 447-454.
Wang, D., Zhou, L., Zhou, H., 2015. Effects of nutritional level of concentrate-based diets on meat quality and expression levels of genes related to meat quality in Hainan Black Goats. Animal Science Journal 86,166-173.
Wulf, D.M., Tatum, J.D., Green, R.D., Morgan, J.B., Golden, B.L., Smith, G.C., 1996. Genetic influences on beef longissimus palatability in Charolais and Limousine steers and heifers. Journal of Animal Science 74, 2394-2405. 
Yang, G., Zhou, H., Hu, J., Luo, Y., Hickford, J.G.H., 2012. Variation in the yak calpastatin gene. International Journal of Genetics 4, 72-73.
Zheng, Y.C., Lin, Y.Q., Yue, Y., 2011. Expression profiles of myostatin and calpastatin genes and analysis of shear force and intramuscular fat content of yak Longissimus muscle. Czech Journal of Animal Science 56, 544-550.