Identifying novel salt-tolerant genes from Dunaliella salina using a Haematococcus pluvialis expression system

Abstract

Dunaliella salina (Dunal) Teod, a unicellular eukaryotic green alga, is a highly salt-tolerant organism. To identify novel genes with potential roles in salinity tolerance, a salt stress-induced D. salina cDNA library was screened based on the expression in Haematococcus pluvialis, an alga also from Volvocales but one that is hypersensitive to salt. Five novel salt-tolerant clones were obtained from the library. Among them, Ds-26-16 and Ds-A3-3 contained the same open reading frame (ORF) and encoded a 6.1 kDa protein. Transgenic tobacco overexpressing Ds-26-16 and Ds-A3-3 exhibited increased leaf area, stem height, root length, total chlorophyll, and glucose content, but decreased proline content, peroxidase activity, and ascorbate content, and enhanced transcript level of Na+/H+ antiporter salt overly sensitive 1 gene (NtSOS1) expression, compared to those in the control plants under salt condition, indicating that Ds-26-16 enhanced the salt tolerance of tobacco plants. The transcript of Ds-26-16 in D. salina was upregulated in response to salt stress. The expression of Ds-26-16 in Escherichia coli showed that the ORF contained the functional region and changed the protein(s) expression profile. A mass spectrometry assay suggested that the most abundant and smallest protein that changed is possibly a DNA-binding protein or Cold shock-like protein. Subcellular localization analysis revealed that Ds-26-16 was located in the nuclei of onion epidermal cells or nucleoid of E. coli cells. In addition, the possible use of shoots regenerated from leaf discs to quantify the salt tolerance of the transgene at the initial stage of tobacco transformation was also discussed.