| Asian swamp eel(Monopterus albus)is one of the important freshwater economically fish in China,and 70%of its body is muscle.Skeletal muscle development of Asian swamp eel is not only related to the quality of meat,but also closely related to the quality of life.At present,people don’t know much about the molecular regulation mechanism of muscle development and growth metabolism of Asian swamp eel.Therefore,it is necessary to further explore the genes related to skeletal muscle development and growth metabolism of Asian swamp eel and study its function and regulation mechanism.REDD1 is a conserved stress response gene,which is upregulated by various physiological and pathological stresses such as hypoxia,DNA damage,glucocorticoids,arsenite,Parkinson’s disease,diabetes,etc.,thereby inhibiting mTORC1 signaling pathways come into play.REDD2,as a similar gene of REDD1,also responds to stress such as energy stress and axonal damage,is functionally complementary to REDD1,and participates in the regulation of the mTORC1 signaling pathway.In mammals,REDD1 and REDD2 play an important role in skeletal muscle metabolism through the TSC1/TSC2-mTORC1 signaling pathway.In Asian swamp eel,do redd1 and redd2 regulate skeletal muscle development and growth metabolism through the mTORC1 signaling pathway?What are the specific mechanisms?What are the expression patterns of the two in Asian swamp eel and their regulation in hunger-induced muscle atrophy?It’s not clear.In this study,the REDD genes of Asian swamp eel was cloned by RACE method;further studied its expression in skeletal muscle of different tissues,different body weights,and different starvation times using real-time fluorescence quantitative PCR.The pEGFP-N1 vector of redd1 gene was constructed to study its subcellular localization,and the prokaryotic expression vector of REDD genes was successfully constructed,which laid a foundation for the follow-up study on the function of REDD genes.The results are as follows.1、The reddl and redd2 of Asian swamp eel were cloned,and two novel REDD genes were found and identified:redd1l and redd2l.redd1 and redd2 are orthologous genes of mammals REDD1 and REDD2.The two novel REDD genes are found only in bony fish,and no homologous genes have been found in quadruped vertebrates such as mammals and reptiles.The two new REDD genes are only found in teleost fish,and their homologous genes have not been found in mammals,reptiles and other four-limbed vertebrates.The full-length of redd1 cDNA is 1416 bp,including 156 bp 5’-UTR,684 bp ORF and 576 bp 3’-UTR,encoding 227 amino acids.The full-length of redd2-x1 cDNA is 1247 bp,including 128 bp 5’-UTR,582 bp ORF and 537 bp 3’-UTR,encoding 193 amino acids;In the second intron,113 bp and 187 bp bases were retained,respectively,to generate two different alternative spliceosomes:redd2-x2 and redd2-x3.The full-length of redd1l cDNA is 1140 bp,including 121 bp 5’-UTR,681 bp ORF and 338 bp 3’-UTR,encoding 226 amino acids.The full-length of redd1l cDNA is 1140 bp,including 121 bp 5’-UTR,681 bp ORF and 338 bp 3’-UTR,encoding 226 amino acids.The full-length of redd2l cDNA is 1154 bp,including 184 bp 5’-UTR,624 bp ORF and 346 bp 3’-UTR,encoding 207 amino acids.The four REDD proteins have similar RTP801_C domains at the carboxy terminus,and have three conserved amino acid fragments EPCGL/IRG,PTF,KK/RKKLY.2、In different tissues of healthy Asian swamp eel,4 RED’D genes were expressed.reddl is highest expressed in the spleen and lowest in the hindgut.The expression of redd11 is highest in the liver and lowest in the spleen and hindgut.In the liver,the expression levels of the three alternative spliceosomes of redd2 are the highest,followed by skeletal muscle,body kidney,and head kidney.In the heart,spleen,foregut,and hindgut,the expression of these alternative spliceosomes is relatively low.The expression of redd2-xl is highest in all tissues except skeletal muscle;in skeletal muscle,the expression levels of redd2-xl and redd2-x2 are similar,both being five-fold higher than redd2-x3.In the kidney,the expression of redd2-x2 and redd2-x3 is much lower than that of redd2-x1,while the expression of redd2-x1 in the body kidney is higher than that of the head kidney.The expression of redd21 is highest in the head kidney,followed by the body kidney,spleen,foregut and hindgut,and the lowest in skeletal muscle.3、The REDD genes expression of Asian swamp eel in different body weight ranges was analyzed,revealed that four REDD genes is expressed in all body weights.The expression of redd1 was highest at 5 g,and gradually decreased with the increase of body weight,and the lowest expression was at 80 g.The expression of redd11 was higher at 5 g,then gradually decreased,reached the lowest at 40 g,then increased,and reached the highest level at 80 g.The expression levels of the three transcripts of redd2 showed different trends.The expression of redd2-x1 was higher at 5 g and 10 g,and then decreased gradually,to a minimum at 60 g and 80 g.The expression of redd2-x2 is also high at 5 g,10 g,and reduced to a minimum at 60 g,but rebounded again at 80 g.The expression of redd2-x3 was higher at 5 g and 10 g,and decreased to a minimum at 20 g,and then the expression level began to increase,reaching the maximum at 80 g.4、Fasting has an important effect on the transcriptional level of four REDD genes.The expressions of redd1,redd11,redd2 and redd2l all increased,then decreased,then increased,and then decreased.After 7 days of fasting,the expression of all REDD genes except redd2l was significantly up-regulated,and then returned to normal levels after 15 days of fasting.Although the expression of redd2l did not increase significantly compared to the control at 7 days of fasting,it had a clear increasing trend,and it significantly increased compared to the expression of 3 days of fasting.Next,with the exception of redd2l and redd2-x3,the expression levels of other transcripts increased significantly at 90 d,and then decreased significantly after 210 days of fasting.On day 15,the expression of redd2l was significantly reduced and remained unchanged at normal levels.The expression of redd2-x3 increased significantly at 90 d and decreased significantly at 210 d.5、Three prokaryotic expression vectors and pEGFP-N1 expression vectors for each REDD gene of Asian swamp eel were successfully constructed,and the recombinant protein and subcellular localization of redd1 were investigated.Only the cold shock expression vector pCold-TF-redd1 can express a highly soluble recombinant protein.redd1 protein of Asian swamp eel is expressed in the nucleus and cytoplasm.In summary,there are four REDD genes in Asian swamp eel,redd1 and redd2 are homologous genes of mammalian REDD1 and REDD2.redd1 protein is located in the nucleus and cytoplasm.redd2 has three different alternative spliceosomes because of intron retention.redd1l and redd2l are two novel fish-specific genes.These four genes are expressed in all tissues and play an important role in the early development and metabolism of skeletal muscle,as well as immunity. |
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