Isolation and characterization of phase-specific cDNAs from carposporophytes of Gracilariopsis andersonii (Gracilariales, Rhodophyta)

Abstract

The carposporophyte life-history phase is unique to florideophycean algae and its morphological development has been thoroughly investigated. Carposporophytes are tiny, generally embedded in female gametophyte tissues, and difficult to investigate via traditional molecular methods. However, subtractive cloning of differentially expressed genes from small amounts of tissue is now possible thanks to recent methodological advances. We applied a cDNA subtraction method using magnetic beads and PCR to isolate carposporophyte-specific cDNAs from Gracilariopsis andersonii (Gracilariales, Rhodophyta). Messenger RNAs extracted from carposporophyte tissues and vegetative thalli of the gametophyte were captured on oligo-dT magnetic beads, followed by first-strand cDNA synthesis in situ. Subtractive hybridization between the amplified second-strand carposporophyte cDNA in solution and the first-strand gametophyte cDNA attached to magnetic beads was conducted to remove common genes present in both gametophyte and carposporophyte tissues. Of 795 cDNA clones examined, 117 showed up-regulation of genes in carposporophyte tissues. Among 32 clones sequenced, we found sequences homologous to glutathione S-transferase (Chondrus crispus) and ESTs from Gracilaria. Full-length sequences for two such genes were obtained by RACE and their functions are discussed.