Characterization Of Two Myostatin Genes In Pufferfish Takifugu Bimaculatus:Sequence,Genomic Structure,Expression,and Function

Myostatin(MSTN)is also called growth differentiation factor-8(GDF-8),and it is a member of TGF-β superfamily.As a negative regulator of muscle growth and differentiation,MSTN restrains the proliferation and differentiation of myoblasts by inhibiting the transcriptional activity of the positive regulatory factor,mitogenic determination gene(MyoD).In contrast to the most muscle-specific expression of the single mammalian mstn gene,teleost fish possess at least two mstn genes to be expressed in both muscular and non-muscular tissues.It is generally believed that the two or more copies of the mstn gene in teleost fish are the result of polyploidization.To understand the role of two mstn genes of Takifugu bimaculatus,we identified and characterized mstnl and mstn2 gene in T.bimaculatus(Tbmstnl and Tbmstn2).We also analyzed their expression patterns in detail by quantitative real-time PCR(qRT-PCR)and Whole-Mount Fluorescence in situ Hybridization(WFISH).Meanwhile,different length fragments containing Tbmstn1 5’-regulatory region were constructed,and the expression regulation mechanisms of Tbmstnl at the transcriptional was analyzed by using transient transfection and dual-luciferase reporter gene assays.On the other hand,the CRISPR/Cas9 system was used to attempt the gene editing of Tbmstn1.The main results were as follows:1)The open reading frame(ORF)of Tbmstn1 is 1131 bp,encoding a polypeptide of 376 amino acids.The highly conserved TGF-β domains of the TGF-β superfamily was found at 282-376 residues of TbMSTN1.The ORF of Tbmstn2 is 1080 bp,encoding a polypeptide of 359 amino acids.The highly conserved TGF-β domains of the TGF-β superfamily was found at 265-359 residues of TbMSTN2.Genomic structure comparison,Homology analysis,and phylogenetic analysis all results showed that both MSTN1 and MSTN2 in teleost shared the high identity with other species,respectively,but the length of the encoded amino acid was different.2)The results of qRT-PCR showed that Tbmstn1 was expressed in the eye,kidney,spleen,skeletal muscle,gill,and brain,and the expression level in the skeletal muscle was extremely significantly higher than in other examined tissues.Tbmstn2 was expressed in the skin,skeletal muscle,gill,and brain,and had the highest expression in the skeletal muscle,followed by in the brain.Meanwhile,in different stages of embryonic development,the expression of Tbmstn1 started from the gastrula stage.Its expression in the eye-pigment formation stage and hatching stage was significantly higher than that in other stages.The Tbmstn2 was expressed in all examined embryonic stages with different levels,and the highest expression was detected in the eye-pigment formation stage.3)The result of WFISH showed that Tbmstn1 was significantly expressed in the somite and head at the eye-pigment formation stage and in the somite at the hatching stage.Tbmstn2 was significantly expressed in the somite and head at eye pigment formation stage,and also in the tail of embryos somite at hatching stage,but the expression was not as strong as that in eye pigment formation stage.4)1537 bp 5’ flanking region of Tbmstn was obtained.Bioinformatics analysis showed that the typical TATA boxes and CAAT boxes,eight putative E-box.GRE,Mefl,Mef2c,POU1F1a(Pit1a),CREB,and other putative binding sites were found in this area.Multiple alignment results show that CAAT box and the TATA box are conserved in the upstream of teleost mstn1 5’flanking.5)Five luciferase plasmids with different length deletion fragments were successfully constructed(pGL-mstnl-rl-5).All constructs directed luciferase activity,with the highest activity obtained by the pGL-mstnl-r5 fragment.These experiments showed that all five genomic fragments are functional Tbmstn1 promoters,and differences in promoter activity might be due to presence of enhancers and/or repressor sequences,which regulating Tbmstn1 promoter activity.6)An active sgRNA was successfully edited in zebrafish as a positive control for in vitro detection,and an active sgRNA was successfully selected from several sgRNAs of the Tbmstn1 gene predicted by the software.