Heat shock protein 70 gene expression and stress response of red-spotted (Epinephelus akaara) and hybrid (E. akaara female × E. lanceolatus male) groupers to heat and cold shock exposure.

The importance of the temperature tolerance of fish is increasing due to climate change caused by global warming. This study examined the expression of the heat shock protein 70 (HSP70) gene, and plasma cortisol and glucose levels, as a stress response in red-spotted and hybrid groupers during exposure to heat and cold shock. Temperature in the tank where fishes acclimated at 20℃ was gradually increased or decreased, respectively, to examine the survival rate of fish. The result showed a higher survival rate of the hybrid than that of the red-spotted grouper upon exposure to a higher temperature. To further analyze the factors associated with temperature-associated stress, fishes were collected from different temperatures which changed from 20 to 30℃ or 10℃, respectively, and then back to 20℃. The expression levels of the gene encoding heat shock protein 70 (HSP70) were analyzed by qPCR using cDNA prepared from RNA extracted from the brain. A higher level of HSP70 transcript was detected in the hybrid during heat shock exposure. Analysis of cortisol and glucose from the blood of fish collected during the acclimation periods clearly indicated that the level of cortisol was increased upon temperature shift although a slight difference in the degrees of changes timing was slightly different between red-spotted grouper and hybrid. The results showed a correlation between the level of HSP70 and survival rate upon exposure to higher temperature shock. This study provides basic information regarding whether HSP70 expression increases the survival rate of fish subjected to rapid temperature changes.

Multiple calmodulin genes of the Pacific abalone, Haliotis discus hannai (Mollusca: Vetigastropoda: Haliotidae).

In this study, we identified four canonical calmodulin genes in the Pacific abalone, Haliotis discus hannai. Their full-length cDNAs were variable in the 5' and 3' untranslated regions, but highly similar (91-97%) in the coding region. Each of the genes encoded 149 amino acids, with 93-97% similarity among themselves and 94-98% similarity with human CAM I. There were 54 substitutions distributed unevenly throughout the coding regions, found mostly in the third codon position. Gene structure analysis revealed that each of the calmodulin genes comprised five exons and four introns. The intron positions and phases were identical and there were no introns in the fourth exon. The corresponding introns differed in their sequences and sizes. Expression profiles of nine tissues from abalone revealed that the calmodulin genes were transcribed in common in gill and mantle tissue, but differentially in the other tissues. A phylogenetic analysis based on the amino acid sequences revealed that calmodulin C was the most common isoform in Gastropoda and calmodulin was the most diverged isoform. An in silico analysis of the calmodulin genes identified paralogous genes in other Haliotis species, indicating that gene duplication might have occurred in the last common ancestor of Haliotis. Abbreviations: ORF: open reading frame; RACE: random amplification of cDNA end; TSA: transcriptome shotgun assembly; UTR: untranslated region.

Molecular Cloning and Co-Expression of Phytoene Synthase Gene from Kocuria gwangalliensis in Escherichia coli.

A phytoene synthase gene, crtB, was isolated from Kocuria gwangalliensis. The crtB with 1,092 bp full-length has a coding sequence of 948 bp and encodes a 316-amino-acids protein. The deduced amino acid sequence showed a 70.9% identity with a putative phytoene synthase from K. rhizophila. An expression plasmid, pCcrtB, containing the crtB gene was constructed, and E. coli cells containing this plasmid produced the recombinant protein of approximately 34 kDa , corresponding to the molecular mass of phytoene synthase. Biosynthesis of lycopene was confirmed when the plasmid pCcrtB was co-transformed into E. coli containing pRScrtEI carrying the crtE and crtI genes encoding lycopene biosynthetic pathway enzymes. The results obtained from this study will provide a base of knowledge about the phytoene synthase of K. gwangalliensis and can be applied to the production of carotenoids in a non-carotenoidproducing host.

Overexpression and characterization of lycopene cyclase (CrtY) from marine bacterium Paracoccus haeundaensis.

Lycopene cyclase converts lycopene to beta-carotene by catalyzing the formation of two beta-rings at each end of the linear carotene structure. This reaction takes place as a two-step reaction in which both sides of of the lycopene molecule are cyclized into beta-carotene rings via the monocyclic gamma-carotene as an intermediate. The crtY gene coding for lycopene cyclase from Paracoccus haeundaensis consists of 1,158 base pairs encoding 386 amino acids residues. An expression plasmid containing the crtY gene (pET44a-CrtY) was constructed and expressed in Escherichia coli, and produced a recombinant protein of approximately 43 kDa, corresponding to the molecular mass of lycopene cyclase. The expressed protein was purified to homogeneity by His-tag affinity chromatography and showed enzymatic activity corresponding to lycopene cyclase. We also determined the lycopene substrate specificity and NADPH cofactor requirements of the purified protein. The Km values for lycopene and NADPH were 3.5 microM and 2 mM, respectively. The results obtained from this study will provide a wider base of knowledge on the enzyme characterization of lycopene cyclase at the molecular level.