Abstract

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

Highlights

  • The genus Dunaliella, belonging to Chlorophyceae, has been represented by 27 known species, of which 23 are from salt water

  • This study developed a molecular approach for first monitoring of the diversity of Dunaliella based on PCR amplification, cloning and sequencing of the RuBisCO gene cbbL in metagenomic DNA extracted from the Saltern field, Malahat El-Max, Alexandria, Egypt

  • This work aimed to cover the molecular diversity of almost all Dunaliella species in the collected saline water sample

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Summary

Introduction

The genus Dunaliella, belonging to Chlorophyceae, has been represented by 27 known species, of which 23 are from salt water. Species of Dunaliella occur in freshwater, euryhaline habitats of all continents, oceans including the Dead Sea and even the salt lakes of the Antarctic (Tafreshi and Shariati, 2009). The characterization of economically important algae in solar salterns has become a key focus for many research groups all over the world (Chen and Jiang, 2009). Dunaliella species that are living in these fields, have an economic importance due to its highly production of antioxidants (Oren, 2010). In Egypt, there are several coastal solar salterns, where Dunaliella is living, mainly on and near to the Mediterranean coast. Studies on Dunaliella in Egyptian aquatic habitats have been focused on characterization, environmental stresses and biotechnological applications of isolated species, including Dunaliella bardawil (Adam, 1997), Dunaliella salina

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