Monitoring of uncultured Dunaliella sp. in an Egyptian solar saltern field based on RuBisCO-encoding gene cbbL

Culture-independent molecular approach was used to explore and evaluate the diversity of Dunaliella species living at the salt field Malahat El-Max Alexandria, Egypt. Bulk genomic DNA was extracted directly from the collected salt water samples. Specific PCR primers and methodology were designed to amplify the gene cbbL , which encodes the large subunit of the enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO, EC 4.1.1.39) of only Dunaliella species, from the extracted microbial metagenome. The 700 bp-PCR amplicons were cloned and cbbL clone library was constructed and analyzed by sequencing. Rarefaction curve was saturated at sequence analyses of 23 clones, obtaining 19 phylotypes of Dunaliella cbbL , representing the total composition of Dunaliella in the collected sample. All recorded phylotypes had the known deduced amino acid cbbL motive sequence and catalytic sites. Fingerprint sequence, characterizing Dunaliella cbbL , was recorded. The cbbL phylotypes were grouped into two distinct phylogenetic clusters. The cluster 1, consisting of 18 current  cbbL phylotypes was rooted with a cluster containing cbbLs of Dunaliella salina, Dunaliella bioculata, Dunaliella primolecta and Dunaliella tertiolecta . The single phylotype, uncultured Dunaliella ElMax.3, forming cluster 2, showed a unique phylogenetic lineage in the evolution of Dunaliella cbbL . This study introduced the first functional gene markers for exploring Dunaliella species in salt waters without culture. Keywords: Uncultured Dunaliella , RuBisCO cbbL , solar saltern water, diversity. African Journal of Biotechnology Vol. 12(34), pp. 5361-5369