cDNA cloning and expression of vitamin-K Epoxide Reductase (VKOR) gene from orange spotted grouper Epinephelus coioides

Abstract

The cDNA sequence encoding Vitamin-K Epoxide Reductase (VKOR) was constructed from hypothalamus and pituitary cDNA libraries of Orange spotted Grouper Epinephelus coioides. VKOR gene contains 610 nucleotides including the open reading frame (ORE) of length 399 bp. ORE was started with ATG codon at the position of 199 and terminated with a stop codon TAA of 585. ORE sequence had 128 amino acids and the predicted protein molecular weight is approximately 14.08 kDa (GenBank: FJ939281.1). Expression of VKOR gene in different tissues was further examined using one-step RT-PCR. RT PCR result revealed that, VKOR mRNA expression was detected only in liver, kidney, hypothalamus and pituitary. VKOR gene sequence of E. coioides aligned with related sequences by multiple sequence alignment by clustal X and phylogenetic tree was generated by Neighbor Joining method using Genious v 5.4. E. coioides VKOR had the highest identity and homology with the puffer fish, Tetraodon nigroviridis, Takifugu rubripes and Zebra fish Danio rerio. Based on the VKOR expression in the kidney, liver hypothalamus and pituitary of the seven year's old (sex reversal stage) E. coioides, it may be involved in sex reverse.The cDNA sequence encoding Vitamin-K Epoxide Reductase (VKOR) was constructed from hypothalamus and pituitary cDNA libraries of Orange spotted Grouper Epinephelus coioides. VKOR gene contains 610 nucleotides including the open reading frame (ORE) of length 399 bp. ORE was started with ATG codon at the position of 199 and terminated with a stop codon TAA of 585. ORE sequence had 128 amino acids and the predicted protein molecular weight is approximately 14.08 kDa (GenBank: FJ939281.1). Expression of VKOR gene in different tissues was further examined using one-step RT-PCR. RT PCR result revealed that, VKOR mRNA expression was detected only in liver, kidney, hypothalamus and pituitary. VKOR gene sequence of E. coioides aligned with related sequences by multiple sequence alignment by clustal X and phylogenetic tree was generated by Neighbor Joining method using Genious v 5.4. E. coioides VKOR had the highest identity and homology with the puffer fish, Tetraodon nigroviridis, Takifugu rubripes and Zebra fish Danio rerio. Based on the VKOR expression in the kidney, liver hypothalamus and pituitary of the seven year's old (sex reversal stage) E. coioides, it may be involved in sex reverse