Molecular Cloing, Identification And Functional Characterization Of Glutathione S-transferase And Glutathione Peroxidase In The Fresheater Mussel Cristaria Plicata

The freshwater mussel Cristaria plicata, which is of great economical importance and known as "pearl bivalves" in the aquaculture industry of China, has been suffering serious problems due to the outbreak of diseases. Thus, understanding of the immunity of freshwater mussel is crucial for diseases management and development of sustainable mussel culture and pearl production.Glutathione S-transferases (GSTs) are multifunctional phaseⅡdetoxification enzymes that catalyze the attachment of electrophilic substrates to glutathione, play an important role in protecting organisms against the toxicity of reactive oxygen species (ROS). The piGST cDNA was cloned and sequenced by rapid amplification of cDNA ends (RACE) from the freshwater mussel Cristaria plicata. The comparison of the deduced amino acid sequences with GSTs from other species showed that the enzymes belonged to the pi-class and the amino acids defining the binding sites of glutathione (G-site) and for xenobiotic substrates (H-site) were highly conserved. The Cp-piGST cDNA is 816 nucleotides (nt) in length and contained a 615 nt open reading frame (ORF) encoding 205 amino acid residues, and has 19 nt of 5'.untranslated region (UTR) and a 3' UTR of 182 nt including a tailing signal (AATAAA) and a poly (A) tail. The molecular weight of the predicted piGST is 23.4 kD, with the calculated PI being 5.2. Cp-piGST mRNA was expressed highly levels in haemocytes, gill, liver, muscle and mantle of normal bivalves and among the expression levels in the liver were higher than the other tissues. After 0-48 h of challenge, the tissues expression increased, and reached the culminating point for 12 h, then gradually reduced to previous expression for 48 h. Furthermore, the recombinant Cp-piGST with high enzyme activity was induced to be expressed as a soluble form by IPTG at 20℃for 8 h, and then was purified by using the native Ni2+ affinity chromatography. The specific activity of the purified soluble Cp-piGST enzyme into pET30 was 2.396μmoL/min/mg, and which into pET32 was 1.706μmoL/min/mg. The recombinant Cp-piGST had a maximum activity at approximately pH 8.0, and its optimum temperature was 37℃. The recombinant Cp-piGST enzyme activity became lower gradually with the denaturant concentration increasing.Glutathione peroxidase (GPX) is one family of important antioxidant metalloenzymes involved in scavenging the high level of reactive oxygen species (ROS) to protect cells. The Se-GPX cDNA was cloned and sequenced by rapid amplification of cDNA ends (RACE) from the freshwater mussel Cristaria plicala. The comparison of the deduced amino acid sequences with GPXs from other species showed that the enzymes belonged to the Se-GPX and the amino acids which defining the function and biochemical properties of GPX (Gln106, Trp179, Argl23 and Arg195 and of the three loop structures) were conserved in Cristaria plicata. The Cp-GPX cDNA is 919 nucleotides (nt) in length and contained a 696 nt open reading frame (ORF) encoding 232 amino acid residues, and has 68 nt of 5'. untranslated region (UTR) and a 3'UTR of 155 nt including a tailing signal (AATAAA) and a poly (A) tail. The 72nd amino acid corresponds to a selenocysteine encoded by a TGA codon. The molecular weight of the predicted piGST is 26.4 kD, with the calculated PI being 6.4. The Se-GPX transcripts are expressed at high levels in the liver, but at very low levels in the other tissues. The Genome of Se-GPX from Cristaria plicata was 6403 bp, which contained two introns. The first intron was 2130 bp, the second intron was 3354 bp.