Molecular cloning of hypoxia induced genes barnacle larvae | 低氧誘導之藤壺幼體基因的分子克隆 | Di yang you dao zhi teng hu you ti ji yin de fen zi kelong

Hypoxia is an environmental stress that will trigger a series of gene expression for anorganism to overcome it. Barnacle (Balanus amphitrite), as a cosmopolitan species,plays an important role in the ecosystem. In the current study, molecular response tohypoxia is investigated in barnacle larvae. Barnacle larvae in the nauplii stage weretreated in hypoxic condition (1 mg Oz/L) for 24 hours and the induction/repression ofgene 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 GenBankdatabase and, therefore, they were novel genes of barnacle. The remaining 5 wereidentified to heat shock protein 70 (hsp-70), cell division factor, p76 protein and aprotein kinase. To get the full length sequence of these genes, RACE (Rapidamplification of cDNA ends) was used, with gene specified primers designed from theRAP amplified products. To amplify unique single products, optimization of theconcentration of template and annealing temperature of primer was performed foreach set of primers. The RACE amplified products were subcloned and sequenced.Except protein kinase, overlapping sequence of 5’ RACE and 3’ RACE products wereobtained The length of hsp-70, cell division factor and p76 gene were 2320 bp, 3055bp and 1744 bp respectively. Deduced amino acid sequences showed highsimilarity to human homolog. Northern hybridization was used to investigate thegene expression of these genes in hypoxic adult barnacle. The results showed thathypoxic treatment of 1.0 mg O2/L for 24 hours in barnacle adults induced a 2.5 foldincrease in HSP70 transcript. Whole-mount in situ hybridization of hsp70 in naupliilarvae showed the increase in the expression of hsp70 gene after hypoxia treatment.These results demonstrated that hsp70 gene expression was induced by hypoxia in theboth adult and larva of barnacle, revealing a common response in gene expression tohypoxia.

Thesis (M.Phil.)--City University of Hong Kong, 2004

Includes bibliographical references (leaves 124-135)