Isolation and molecular identification of β-carotene producing strains of Dunaliella salina and Dunaliella bardawil from salt soil samples by using species- specific primers and internal transcribed spacer (ITS) primers

Abstract

Dunaliella salina and Dunaliella bardawil are unique species of the genus Dunaliella that produce large amounts of â-carotene when cultivated under appropriate conditions. These include high light intensity, high sodium chloride concentration, nitrate deficiency and extreme temperatures. Under these conditions, only D. salina and D. bardawil can accumulate â-carotene to as much as 10% of the cellular dry weight. Because the morphological characterization is based on the environmental factors, the Dunaliella will change the shape, so identification and differentiation of Dunaliella species by morphology is very difficult. In this research study, we isolated, identified and discriminated the different Dunaliella β-carotene producing strains from salt soil samples, by using 18S rDNA and internal transcribed spacer (ITS) gene sequences. The soil samples were collected from four different provinces of the North Eastern part of Thailand.namely: UdonThani, BuriRam, AmnartCharoen and Chaiyaphum. Among the four isolates, only BuriRam KU01 and UdonThani KU01 were D. salina and D. bardawil, respectively whereas AmnartCharoen KU01 and Chaiyaphum KU01 were not these Dunaliella species. At 4 M NaCl, with deficiency of nitrate (KNO 3 ) and phosphate (KH 2 PO 4 ) in the medium, the D. salina strain BuriRam KU01 produced β-carotene at the level of 56.25 ± 0.97 pg·cell -1 and D. bardawil strain UdonThani KU01 produced β-carotene at the level of 52.91 ± 0.29 pg·cell -1 at the 25th day after inoculation. The 18S rDNA and ITS sequences of D. salina strain BuriRam KU01 and D. bardawil strain UdonThani KU01 were submitted to the National Center for Biotechnology Information (NCBI) database with accession numbers of JN052202, JN052203, JN034031 and JN052204, respectively. By using the species-specific primers and ITS primers the â-carotene producing strains of Dunaliella was identified. Key words : 18S rDNA, β-carotene, carotenoid, Dunaliella bardawil, Dunaliella salina , internal transcribed spacer (ITS), salinity.