Comparative Studies On Expression And DNA Methylation Of The GH/IGF Axis Genes In Diploid And Tetraploid Loach Misgurnus Anguillicaudatus

Cytogenetic studies on Cobitid loach Misgurnus anguillicaudatus(Cantor,1842)have demonstrated that,in addition to the most common bisexual diploids(2n=50),large amount of tetraploid individuals(4n=100)also can be found in the Yangtze River basin,China.With respect to diploids,the tetraploids exhibit larger body size and superior growth performance.However,the molecular mechanisms associated with the superior growth performance of tetraploids are unclear at present.The growth hormone/insulin-like growth axis factor(GH/IGF-1)include growth hormone(GH),growth hormone receptors(GHR)and insulin-like growth factors 1(IGF-1),which were confirmed to play important role in stimulating and modulating fish growth.Studies on polyoloidy organisms suggested that,polyploidization often accompanied with alteration of DNA methylation,which has a close relationship with the gene expression(gene silencing or gene activation).In this study,the diploid and tetraploid M.anguillicaudatus were uesd to compare the GH/IGF-1 axis gene expression difference and the DNA methylation veriation between-ploidy;the obtained results were used to elucidatethe mechanism of DNA methylation regulation on gene expression of the polyploid M.anguillicaudatus.The main results were as the following:1.c DNAs clone and analysis for GH/IGF-1 axis genes1)In both diploids and tetraploids,the length of c DNA for GH gene were 690 bp,containing 633 bp open reading frame(ORF)and 57 bp 5’UTR.The ORF encode 210 amino acids,which including 22 aa signal peptide and 188 aa mature peptides.7 bp variations were found between-ploidy,but no difference was found in the protein sequences.2)The c DNA lengths of GHR gene in both diploids and tetraploids were 1913 bp,containing 1818 bp ORF and 95 bp 5’UTR.The ORF encode 605 aa.5bp variations were found between-ploidy,but no difference was found in the the protein sequences.3)For both diploids and tetraploids,the length of IGF-1 genes were 695 bp,containing 483 bp ORF,165 bp 5’UTR and 47 bp 3’UTR.The ORF encode 160 aa,containing 44 aa signal peptide and 116 aafunction domain(B,C,A,D and E).1bp variation was found between-ploidy,but also,no difference was found in the the protein sequences.2.Expression difference of GH/IGF-1 axis genes between-ploidy1)In both diploids and tetraploids,GH gene expressed specifically in pituitary.No expression can be found in other tissues,such as liver,heart and muscle.Expression level was compared between-ploidy,and the results showed a significantly higher expression in tetraploids than in diploids(P<0.05).2)In both diploids and tetraploids,GHR gene expressed in all tissues tested in the present study,but expression level varied between-tissue.The highest expression was found in liver.Expression level was compared between-ploidy,and the results showed a significantly higher expression in tetraploids than in diploids(P<0.05).3)Similar with GHR,the IGF-1 gene also expressed in all tissues,and the highest expression tissue was liver.Expression level in tetraploids was significantly highly in tetraploids than in diploids(P<0.05).3.Clone and analysis of 5’flanking sequences for GH/IGF-1 axis genes1)5’flanking sequence of GH gene were cloned with TAIL method.1147 bp sequences were obtained for both diploids and tetraploids,containing 1081 bp promoter and 56 bp 5’UTR.TATA-box can be predicted in the promoter sequence,but no CAAT-box,GC-box or E-box can be found.In the promoter sequence,some transcription factor binding site can be predicted,such as a pituitary-specific transcription factor(Pit-1),one or more AP-1,GATA1,GATA5,SRY,STAT4,STAT5 a,YY1,Gfi1 b,and so on.The sequence similarity was 98.5% between-ploidy.Compared with tetraploids,a C/EBP-β transcription factor binding site was missing in diploids due to base deletion or substitution.2)5’flanking sequences of GHR were cloned,and 1528 bp were obtained,which include 1345 bp promoter and 95 bp 5’UTR.GHR promoter of M.anguillicaudatus exhibited typical conserved structure of eukaryote’s promoter.Near the transcription initiation sites,the TATA-box,CCAAT-box,GC-box and E-box can be found.And also,some transcription factor binding site can be predicted,such as SP1,C/EBP,STAT5,C/EBP,HNF-5a,and so on.3)5’flanking sequences of IGF-1 was 1988 bp,including 1778 bp promoter sequence and 162 bp 5’UTR.IGF-1 promoter of M.anguillicaudatus did not exhibited typical conserved structureof eukaryote’s promoter,neither TATA-box,CAAT-box,nor GC-box can be predicted.But one or more transcription factors binding sites can be predicted,such as C/EBP,Myo D and Sp1.The sequence similarity was 97.4% between-ploidy.Compared with diploids,the tetraploids miss partial sequence,which containing a NFIX and C/EBP transcription factor binding sites.4.DNA methylation variation in 5’flanking sequences of GH/IGF-1 axis gene between-ploidyUsing BSP method,the DNA methylation variation in 5’flanking sequences were compared between-ploidy.The results showed that,the DNA methylation of GH,GHR and IGF-1 in diploids were 88.9%,1.0% and 0%,respectively.While in tetraploids,the values were 90.7%,0% and 7.4%,respectively.The results obtained in the present study showed that,the gene expression levels of GH/IGF-1 gene in tertaploids were significantly higher than in diploids,but compared with the DNA methylation,no clear relationship can be found.The results obtained in the present study need to be further analyzed.