CDNA Cloning And Tissue Expression Of CTSD、CSTB In Tianfu Goat

After a period of time when animal was slaughtered, muscles turn into meat by a series of reaction. Cathepsin D (CTSD) was one of the important factors affecting the muscles soften, and Cystatin B (CSTB) is an intracellular inhibitor that regulates the activity of cysteine proteases. In this study, we selected the most representative gene of cathepsin and cystatin to research. We cloned CTSD and CSTB gene in Tianfu goat for the first time and analyzed their tissue expression. And then submitted them to NCBI, two Genbank ID were obtained (KJ004415 and KF991243). Real-time fluorescent quantitative RT-PCR technology analysis was used to detect CTSD and CSTB gene in heat, liver, spleen, lung, kidney, biceps femoriss, abdominal muscle and longissimus dorsi of Tianfu goat. And the expression of CTSD and CSTB gene in different time of Tianfu goat was detected, too. Then we used western bloting to detective the expression of protein in different tissues.The results showed that CTSD gene complete open reading frame (ORF) for 1239 bp, encodes 412 amino acids. Protein secondary structure was mainly composed by random crimp and alpha screw. It existed twenty phosphorylation sites. The signal peptide, Al_Propeptide and Asp structural domain was found in CTSD protein which conformed to reports in other mammals. A phylogenetic tree analysis revealed that the Tianfu goat CTSD protein has a evolutional distance and close genetic relationship with CTSD in other mammals. By using real time quantitative technique, we found that CTSD gene express in Tianfu goat eight organization, and lung express quantity highest, then low level of expression were observed in liver and spleen, and lowest level of expression were observed in biceps femoriss, rectus abdominis and longissimus dorsi muscle. In addition, the temporal mRNA expression analysis further showed that gene expression of CTSD was increased in longissimus dorsi muscle from month 6 to month 12, then decreased from month 12 to month 24. Western blotting results detected the CTSD protein in skeletal muscles. No signal was detected in heart, liver, spleen, lung and kidney.The length of the Tianfu goat CSTB cDNA was 425 bp which contained a 306 bp long coding region that encoded a 101 amino sequence. It had three phosphorylation sites and not exist any signal peptide. Protein secondary structure was mainly composed by random crimp and alpha screw. The Cystatin-like domain was forecasted in CTSD protein. The result of phylogenetic tree analysis revealed that the Tianfu goat CSTB protein has a close genetic relationship and evolutional distance with cattle CSTB. Analysis of mRNA expression by RT-qPCR showed that the CSTB mRNA was detected in heart, liver, spleen, lung, kidney, biceps femoris, abdominal muscle and longissimus dorsi. In particular, high expression levels were detected in liver, spleen, lung and kidney, and low level of expression were observed in heart, biceps femoris, abdominal muscle and longissimus dorsi.In addition, the temporal mRNA expression analysis further showed that gene expression of CSTB was increased in longissimus dorsi muscle from month 6 to month 12, then decreased from month 12 to month 24. Western blotting results detected the CSTB protein in heart, liver, spleen, lung and kidney. No signal was detected in skeletal muscles.