Characterization of the AOX gene and cyanide-resistant respiration in Pyropia haitanensis (Rhodophyta)

Abstract

In order to characterize the structure and function of the alternative oxidase (AOX) gene in Pyropia haitanensis, full-length complementary DNA (cDNA) of the locus (denoted as PhAOX) was obtained by in silico cloning, rapid amplification of cDNA ends, and reverse transcription PCR. The PhAOX sequence was 3,192 bp including a 5′-untranslated region (UTR) of 1,484 bp, a 3′-UTR of 274 bp, and an open reading frame of 1,434 bp predicted to encode a protein of 477 amino acids with a molecular mass of 50.47 kDa and a putative isoelectric point of 9.75. PhAOX is predicted to be a precursor protein with mitochondrial-targeting peptides. The deduced amino acid sequence of PhAOX was most similar to PyAOX from Porphyra (= Pyropia) yezoensis (77 % identity); however, gene sequences from PhAOX and PyAOX showed obvious divergence. PhAOX had a 315-bp intron but PyAOX had no intron. Expression of mRNA in filamentous thalli and the foliose thallus, which are different life cycle stages, was examined by real-time fluorescent quantitative PCR (qPCR). The highest PhAOX expression was in filamentous thalli and was about 10 times the expression in the foliose thallus. We showed that an alternative respiration pathway also existed in P. haitanensis using a noninvasive microsensing system. The contribution of the alternative pathway to total respiration in filamentous thalli was greater than the contribution in the foliose thallus. This result was consistent with the level of PhAOX gene expression observed in different life cycle stages.