| The study was focused on the metabolism and its regulation mechanism as well as adaptation of of branch amino acids in the skeletal muscle of Nile tilapia(Oreochromis niloticus)when nutrition was changing,compared with the liver,kidney,intestinal tract,brain,spleen and heart.The effects of starvation on the metabolism of branch amino acid in the skeletal muscle of Nile tilapia were analyzed using short-term fasting experiments,real-time quantitative PCR,amino acid content determination,related enzyme activity assay and dual luciferase activity assay.The upstream key regulator of micro-RNA-125(mi R-125a-3p)of Kruppel-like factor 15(KLF15)was screened and confirmed using in vivo injection of LNA-125 i.The mi R-125a-3p-KLF15-BCAA pathway and its regulatory mechanisms of branch amino acids was preliminarily elucidated in the skeletal muscle of Nile tilapia.The main results of the experiment were shown as follows:1.In this experiment,the relative expression levels of eight Kruppel-like factor family members in skeletal muscle,liver,kidney,intestine,brain,spleen and heart of Nile tilapia were detected by real-time fluorescent quantitative PCR.The results showed that KLF family members were distributed in various tissues of Nile tilapia.Comparing with the other seven genes,the expression of KLF15 was relatively high in all the tested tissues,and KLF15 was the dominate expressed KLF subtype in Nile tilapia skeletal muscle.2.After 3 days of starvation,the m RNA levels of the KLF15 gene,Branched chain aminotransferase 2(BCAT2)gene and Alanine transaminase(ALT)gene in the skeletal muscle of Nile titifish were significantly higher than those in the control group(P<0.05).Meanwhile,the content of branched-chain amino acids in skeletal muscle of Nile tilapia was significantly lower in the starvation groups compared with normal group(P<0.05),but ALT enzyme activity was higher in starvation groups(P <0.05).3.The relative expression levels of KLF15 gene,BCAT2 gene and ALT gene in skeletal muscle of Nile tilapia for 7 days were significantly lower than those of starvation for 3 days(P<0.05);however,the content of branched chain amino acids in skeletal muscle significantly increased(P<0.05);ALT enzyme activity decreased significantly(P<0.05).The relative expression levels of KLF15 gene,BCAT2 gene and ALT gene in the muscles of Nile tilapia for 15 days were significantly lower than those of hunger for 7 days(P<0.05);however,the content of branched chain amino acids in muscle and ALT enzyme activity was significantly higher than that of hunger for 7 days(P<0.05),and there was no significant difference from the control group starved for 0 days(P＞0.05).5.Double luciferase activity assay confirmed that mi R-125a-3p can negatively regulate the expression of KLF15 gene in muscles,and in vitro experiments confirmed that KLF15 is the target gene of mi R-125a-3p.6.Compared with the control group,the m RNA expression level of Nile tilapia mi R-125a-3p was decreased by intraperitoneal injection of mi R-125a-3p specific inhibitor(LNA-125i);However,m RNA levels of KLF15 gene,BCAT2 gene and ALT gene were decreased.The relative expression was significantly increased(P<0.05);the content of branched-chain amino acids in skeletal muscle after injection was significantly decreased(P<0.05);ALT enzyme activity was significantly increased(P<0.05).In vivo injection experiments demonstrated that mi R-125a-3p can regulate the amino acid metabolism of skeletal muscle branch of Nile tilapia by targeting KLF15 expression.The above results preliminarily revealed the BCAA metabolic adaptation rule of skeletal muscle of Nile tilapia in the process of short-term starvation,and preliminarily elucidated the regulatory function of mi R-125a-3p-KLF15-BCAA pathway in skeletal muscle branched-chain amino acid catabolism,providing scientific basis and reference for further understanding the metabolism and regulatory mechanism of skeletal muscle branched-chain amino acid. |
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