Cloning, Functional Analysis And FISH Of Gadd45g Gene In Half-Smooth Tongue Sole (Cynoglossus Semilaevis)

Half-smooth tongue sole (Cynoglossus semilaevis) is considered as one of theeconomic marine fishes which is widely cultured in China. In this study, we tookhalf-smooth tongue sole which cultured in Haiyang, Shandong province as anexperimental fish, and has a systemic study by variety of molecular biologytechniques, which included cloning of the gene sequence, the analysis of its evolution,the expression regulation of the genes in organization and the development stage ofgonads, and the mapping of genes in chromosome. This study fills up the blank in thestudy of Gadd45g gene in half-smooth tongue sole, and lays a foundation for thestudy on sex determination mechanism of half-smooth tongue sole, has a positiveeffect for gender control in half smooth tongue sole.1The gene clone and functional analysis of Gadd45g gene in half-smooth tongue sole(Cynoglossus semilaevis)According to the result of half smooth tongue sole full genome sequencing, threeGadd45g homologous genes were isolated by the RT-PCR and RACE technologies.The whole cDNA sequence for Gadd45g1and Gadd45g2were1270bp and1181bp,respectively, containing a conserved open reading frame (ORF) of480bp which waspredicted to encode a protein of159amino acids residues. The full-length ofGadd45g3gene was1147bp, encoding155amino acids. Although the conservedRibosomal_L7Ae domain was predicted in these three genes, Gadd45g1andGadd45g2, which have high sequence similarity and closely homology with theGadd45g gene of teleost and mammals, were separated from Gadd45g3gene usingthe sequence analysis by the biological software and authoritative website. Gadd45g3has high similarity to Gadd45g-like style from teleost. As determined by qRT-PCR,the tissue distribution of three Gadd45g mRNAs in both female and male adults wasdetected. Gadd45g1was found in female and superlatively detected in liver, lessexpressed in ovary. Gadd45g2mRNA expression level was extreme high in ovary, butlow in other female and male tissues. Gadd45g3has a high expression in the brain,both male and female. Comparing the expression of three Gadd45g homologs ingonads of female, male and neo-male, we can found the expression level ofGadd45g1and Gadd45g2in neo-male and male were approximate, and significantlylower than female; conversely, the expression of Gadd45g3in testis of mature maleand neo-male was significant higher than that in ovary. During the developmentalstages of gonads, the expression profiles of three Gadd45g genes also were detected.At50d period when the gonad of half-smooth tongue sole remained undifferentiatedand sex differentiation would be about to occurred, the increasing expression ofGadd45g1may exert an important influence on gender differentiation. After95d, thehigh expression of Gadd45g1and Gadd45g2imply that these two genes play animportant role in the development of ovary. The expression of Gadd45g3gene, which was highly detected at20d and beginning to reduce at followed period, exert aninfluence for male-determination before the time of gonad differentiation, and is highexpression at the early development stage of ovary and testis.2The chromosomal fluorescence in situ hybridization (FISH) of Gadd45g gene inhalf-smooth tongue sole (Cynoglossus semilaevis)Screening above three Gadd45g genes out from the BAC library, we succeed infinding the single BAC clones which contain these genes respectively. The targetgenes were respectively positioned in the Bam002C14, Hind029A24and Bam027D12clone of BAC library. Then Using the BAC-FISH, probe were prepared successfullyand hybridized with chromosome slide, signals can be seen at picture which mean thatthe position of gene in chromosomes. The FISH result of Gadd45g1show two signalson W chromosomes in female and no signal in male. The FISH result show thatGadd45g1was located on W chromosomes, Gadd45g2was captured on Zchromosome, and Gadd45g3gene was located on autosome.