Molecular cloning of hypoxia induced genes barnacle larvae

Abstract

Hypoxia is an environmental stress that will trigger a series of gene expression for an organism to overcome it. Barnacle (Balanus amphitrite), as a cosmopolitan species, plays an important role in the ecosystem. In the current study, molecular response to hypoxia is investigated in barnacle larvae. Barnacle larvae in the nauplii stage were treated in hypoxic condition (1 mg Oz/L) for 24 hours and the induction/repression of gene expressions were studied by RAP (RNA fingerprinting by arbitrary primed PCR). Thirty-nine differential PCR products were obtained from 46 arbitrary chosen primers. After cloning and sequencing, 34 products showed no homolog in the GenBank database and, therefore, they were novel genes of barnacle. The remaining 5 were identified to heat shock protein 70 (hsp-70), cell division factor, p76 protein and a protein kinase. To get the full length sequence of these genes, RACE (Rapid amplification of cDNA ends) was used, with gene specified primers designed from the RAP amplified products. To amplify unique single products, optimization of the concentration of template and annealing temperature of primer was performed for each set of primers. The RACE amplified products were subcloned and sequenced. Except protein kinase, overlapping sequence of 5’ RACE and 3’ RACE products were obtained The length of hsp-70, cell division factor and p76 gene were 2320 bp, 3055 bp and 1744 bp respectively. Deduced amino acid sequences showed high similarity to human homolog. Northern hybridization was used to investigate the gene expression of these genes in hypoxic adult barnacle. The results showed that hypoxic treatment of 1.0 mg O2/L for 24 hours in barnacle adults induced a 2.5 fold increase in HSP70 transcript. Whole-mount in situ hybridization of hsp70 in nauplii larvae showed the increase in the expression of hsp70 gene after hypoxia treatment. These results demonstrated that hsp70 gene expression was induced by hypoxia in the both adult and larva of barnacle, revealing a common response in gene expression to hypoxia.