Molecular Cloning And Expression Analysis Of JUNCTOPHILIN Family Of Megalobrama Amblycephala

Junctophilin?JPH?is considered to be the main component of junctional membrane complex,and plays important roles in maintaining the ultrastructure of striated muscle cells,excitation contraction coupling,Ca²⁺signaling,and so on.JPH1 and JPH2 are mainly expressed in skeletal muscle,which is an important source of protein in human food.However,information about Jphs function in fish is still limited.Therefore,it is important to explore the potential molecular mechanism of Jphs in fish.In the present study,the cDNA sequences of the jph1a,jph1b,jph2 and jph3 genes of Megalobrama amblycephala were amplified and characterized.The quantitative expression of jphs in different tissues and different developmental stages were analyzed,and the subcellular localization of Jph1a,Jph1b and Jph2 was identified.In addition,effect of jph1b knockdown on expression of myogenic regulatory factors and cell proliferation was also analyzed.The main results were as follows:1)Molecular cloning and sequence analysis of M.amblycephala jphs gene familyThe jph1a,jph1b,jph2 and jph3 cDNA contained an open reading frame?ORF?of2031 bp,2016 bp,2358 bp and 2361 bp encoding 676,671,785 and 786 amino acids,respectively.These four genes composed of five exons and four introns,which are similar to the JPHs gene structure of other species.They all had eight membrane occupation recognition nexus?MORN?motifs and one transmembrane motif?TM?,and showed higher similarity with homologues of other species.The phylogenetic tree analysis showed that JPHs proteins in vertebrates were generally fell into four distinct clades,and had the closer relationship between JPH1 and JPH2.Furthermore,multiple sequence alignment also found that JPH1 and JPH2 proteins were very conservative.2)Expression of jphs in different development periods and tissues of M.amblycephalaThe real-time quantitative PCR results showed that the jph1a,jph1b,jph2 and jph3genes had tissue-specific expression patterns,which jph1a,jph1b and jph2 mRNAs were mainly expressed in skeletal muscle and heart,and jph3 was mainly expressed in heart,blood and brain.In addition,higher expression levels appeared in blood,brain and spleen for jph1a,in brain and spleen for jph1b,and in blood for jph2.The expression patterns of jph1a,jph1b,jph2 and jph3 were similar during early development.At embryonic stages,their expression was up-regulated during blastocyst stage to gastrula stage,where appeared the first peak,followed by a quick down-regulation at the body segment appearance stage,then gradually increased to the second peak at the heartbeat stage.After hatching,the mRNA levels were always low after hatching until 15 dph,and reached the third peak at 25 dph.3)The subcellular localization of M.amblycephala Jph1a,Jph1b and Jph2The ORF sequences of jph1a,jph1b and jph2 genes were cloned into pEGFP-N1and pEGFP-C1 vector,and transfected into FHM cells,respectively.The cells were incubated with DAPI for staining nuclear,and observed under fluorescence microscope.The subcellular localization showed that the Jph1a,Jph1b and Jph2 proteins were all distributed in the whole cells including the nucleus and cytoplasm.4)Effect of Ca²⁺treatment on jph1b expressionFHM cells were treated with CaCl₂ at 50,100,500,1000??mol/L?concentrations for 12 h.It was found that the expression of jph1b was inhibited at 100?mol/L and 500?mol/L concentrations of Ca²⁺.5)Effects of jph1b inhibition on muscle development-related genes and cell proliferationThe jph1b knockdown significantly increased myod expression,but decreased the expression of myf5,myf6 and myog.In addition,our result also showed that the gene knockdown could promote cell proliferation based on cell counting kit-8?CCK8?assays.