Cloning And Expression Patterns Analysis Of MYOZ2 And MYOZ3 Genes In Tianfu Goat Muscle Tissues And Organs

The quality of meat is one of the most important economic traits in domestic animals, and is determined at least in part by muscle fibers and the best quality meat has a high muscle content of mainly slow-twitch muscle fibers, which are under the control of multiple gene products. Calcineurin (CaN), is broadly distributed in various mammalian cells, it is required for the key processes of myocyte differentiation and conversion to the slow (oxidative) muscle phenotype. Many studies have found that MYOZs genes clould influence the expression of calcineurin, which plays an important role in muscle fiber differentiation. In addition, the expression of MYOZ2 and MYOZ3 genes effectively inhibit calcineurin activity, thereby regulating the differentiation of muscle fibers. In order to understand the function of MYOZ2 and MYOZ3 in muscle fibers differentiation, the study was carried out by the used RT-PCR, molecular cloning, RT-qPCR and western blotting on Tianfu goat MYOZ2 and MYOZ3 genes, the results are as follows:1. Sequence of Tianfu goat MYOZ2 gene was shown to be 856 bp in length and to contain an ORF of 795 bp encoding a protein of 264 residues Tianfu goat MYOZ3 gene sequence was 832 bp long with an ORF of 735 bp encoding a protein of 244 residues. And Tianfu goat MYOZ2 and MYOZ3 gene sequences were deposited in GenBank (GenBank:JX573191 and KC537058, respectively).2. Coding nucleotide and protein sequences alignment showed that MYOZ2 was more highly conserved in large mammals than MYZ03. Phylogenetic tree analysis recealed that the highest homology of Tianfu goat MYOZ2 and MYOZ3 was with that of sheep, and the lowest was with that of African clawed frog.3. Properties and structure of protein analysis found that several similarities between the two protein sequences, including the existence of several potential phosphorylation sites, hydrophily and a conserved calsarcin domain but no signal peptides and transmembrane regions.4. RT-qPCR analyses revealed that mRNA expressions of MYOZ2 and MYOZ3 were detected in cardiac muscle, liver, spleen, lung, kidney, rectus abdominis muscle, rectus abdominis muscle, and longissimus dorsi muscle. Particularly high expression levels of MYOZ2 were seen in rectus abdominisand, cardiac muscle, low expression levels were seen in rectus abdominis muscle, longissimus dorsi muscleand very little expression were detected in liver, spleen, lung and kidney. In addition, high expression levels of MYOZ3 were seen in rectus abdominis muscle, rectus abdominis muscle, lungs and kidney, low expression levels were found in longissimus dorsi muscle and spleen and very little expression were detected in cardiac muscle and liver. Temporal mRNA expression results showed that MYOZ2 and MYOZ3 gene expression varied across four muscle tissues with different ages of the goats.5. Western blotting revealed that different levels of MYOZ2 and MYOZ3 proteins were expressed in the cardiac muscle, liver, spleen, lung, kidney, rectus abdominis muscle, rectus abdominis muscle, and longissimus dorsi muscle on day 300. No MYOZ2 and MYOZ3 was detected in liver, spleen, lung, and kidney. However, both proteins were highly expressed in rectus abdominis muscle and rectus abdominis muscle, while lower levels were detected in different tissues. The lowest expression level of MYOZ2 was in longissimus dorsi muscle, and that of MYOZ3 was found in cardiac muscle; MYOZ2 and MYOZ3 proteins were only expressed in goat muscle, with notable temporal expression differences in specialized muscle tissues from five development age stages.