Introduction of a novel 18S rDNA gene arrangement along with distinct ITS region in the saline water microalga Dunaliella

Mohammad A Hejazi,Nahid Gharajeh,Abolfazl Barzegari,Mohammad S Hejazi

doi:10.1186/1746-1448-6-4

Mohammad A Hejazi, Nahid Gharajeh + Show 2 more

Open Access

https://doi.org/10.1186/1746-1448-6-4

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Abstract

Comparison of 18S rDNA gene sequences is a very promising method for identification and classification of living organisms. Molecular identification and discrimination of different Dunaliella species were carried out based on the size of 18S rDNA gene and, number and position of introns in the gene. Three types of 18S rDNA structure have already been reported: the gene with a size of ~1770 bp lacking any intron, with a size of ~2170 bp consisting one intron near 5' terminus, and with a size of ~2570 bp harbouring two introns near 5' and 3' termini. Hereby, we report a new 18S rDNA gene arrangement in terms of intron localization and nucleotide sequence in a Dunaliella isolated from Iranian salt lakes (ABRIINW-M1/2). PCR amplification with genus-specific primers resulted in production of a ~2170 bp DNA band, which is similar to that of D. salina 18S rDNA gene containing only one intron near 5' terminus. Whilst, sequence composition of the gene revealed the lack of any intron near 5' terminus in our isolate. Furthermore, another alteration was observed due to the presence of a 440 bp DNA fragment near 3' terminus. Accordingly, 18S rDNA gene of the isolate is clearly different from those of D. salina and any other Dunaliella species reported so far. Moreover, analysis of ITS region sequence showed the diversity of this region compared to the previously reported species. 18S rDNA and ITS sequences of our isolate were submitted with accesion numbers of EU678868 and EU927373 in NCBI database, respectively. The optimum growth rate of this isolate occured at the salinity level of 1 M NaCl. The maximum carotenoid content under stress condition of intense light (400 μmol photon m-2 s-1), high salinity (4 M NaCl) and deficiency of nitrate and phosphate nutritions reached to 240 ng/cell after 15 days.

Highlights

Dunaliella is an unicellular halotolerant microalga with a great potential as transgenic bioreactor [1], and more significantly, food source and pharmaceuticals due to its ability to accumulate large amounts of carotenoids [2]
In order to confirm the genus of the isolate, PCR amplification of 18S rDNA gene was performed using MA1-MA2 primers
Restriction Fragment Length Polymorphism (RFLP) and Sequence analysis of 18S rDNA gene The accuracy of the discrepancy from D. salina was approved by RFLP analysis

Summary

Introduction

Dunaliella is an unicellular halotolerant microalga with a great potential as transgenic bioreactor [1], and more significantly, food source and pharmaceuticals due to its ability to accumulate large amounts of carotenoids [2]. On one hand due to environmental adaptation and lacking of a cell wall, a certain Dunaliella isolate may exhibit different morphological and physiological behaviour in different conditions [3]. Identification and classification of the organisms based on conserved and variable regions of 16S or 18S rDNA is a common procedure in taxonomy studies [6]. 18S rDNA gene has been used for molecular identification of different species of Dunaliella as eukaryotic microorganism [79]. Ribosomal spacer sequences, including ITS regions have been frequently utilized for discrimination of genetic variation in green algae [11,12,13,14,15]. Sequence comparison of the ITS region was utilized to predict genetic relatedness and to study phylogeny and taxonomy of Dunaliella [5,15]

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