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Expression Patterns of an Abiotic Stress-inducible Dehydration Responsive Element Binding Protein-2 (dreb2) Gene in Tomato

M. S. Islam
Department of Genetics and Plant Breeding, Sher-e-Bangla Agricultural University, Dhaka-1207, Bangladesh.

M. H. Wang
School of Biotechnology, Kangwon National University, Chuncheon, Kangwon-do, 200-701, South Korea

Abstract/ Executive Summary:

A novel DREB (dehydration-responsive element binding) gene, designated as LeDREB2, was isolated from tomato. Based on multiple sequence alignment and phylogenetic characterization, LeDREB2 gene was classified into an A-2 group member of the DREBs family. We examined the expression pattern of LeDREB2 gene in tomato under different abiotic stresses. Southern blot analysis showed that LeDREB2 is a single copy gene in tomato genome. The organ specific expression profiling indicated that LeDREB2 gene was strongly expressed in young leaves and roots but weak expression in mature leaves and shoots. Northern blot analysis revealed that various kinds of environmental stress such as salt, drought and cold were significantly induced by LeDREB2 gene after application of time courses treatments except little increase in ABA. Our findings showed that the expression of LeDREB2 gene was induced by high salinity, drought and cold but not by the abscisic acid (ABA) treatment. These results indicated that the LeDREB2 gene is a member of the DREB transcription factors, which may play a role in both abiotic and oxidative stress responses.

Keywords: Abiotic stresses, LeDREB2, Expression analysis, Tomato

Location:

Start Date:

End Date:

Program Area: Variety and Species

Commodity: Tomato

Objectives:

The implication of this investigation is that the DREB2 genes in tomato will become another target for engineering biotech crops with enhanced tolerance to abiotic stress.

Methodology:

Plant materials, growth conditions, and stress treatments Seeds of tomato (Lycopersicum esculentum L.) were surface-sterilized for 5 min in 1% (w/v) sodium hypochloride and finally washed with distilled water. Then seeds were cultured in Murashige and Skoog (MS) medium (pH 5.8) including 3% sucrose and 0.8% agar. The germinated plants were transferred to pots and kept in culture room at 25°C for 4 weeks. Drought was induced by removing plants from the pots and placing them on filter paper at 25°C under dim light for 48 h. Tomato leaves were collected after 0 (untreated, control), 3, 6, 12, 24 and 48 h after drought treatment. For cold treatment, the leaves were placed in distilled water and kept in a 4°C cold chamber under dim light and photoperiodic conditions described above upto 48 h. For oxidatives stresses such as salinity, ABA treatments were applied by submerging the whole seedlings continuously in a water solution of 250 mM NaCl (salt), 100 μM ABA (abscisic acid) until 48 h respectively. Treated samples were collected after indicated time periods. Sterile water was used as a control for all treatments. All stress treated plant materials were immediately frozen in liquid nitrogen and stored at −80°C until further use. DNA isolation and Southern blot analysis Genomic DNA was isolated from mature tomato leaves. Genomic DNA samples (10 μg) were completely digested with EcoRI and HindIII. Digested genomic DNA was separated by electrophoresis on a 1% agarose gel, denatured, and blotted onto a nylon membrane (Amersham Pharmacia, Uppsala). Membranes were then hybridized with the full-length of LeDREB2 cDNA probe labeled with [α-32P] dCTP. Hybridization was performed overnight at 65ºC in 5% dextran sulfate, 0.25 M disodium phosphate (pH 7.2), 7% (w/v) SDS, and 1 mM EDTA. After hybridization, the blot was washed twice with 2 × SSC and 0.1% SDS for 10 min each at room temperature and twice with 0.1 × SSC and 0.1% SDS for 5 min each at 65ºC. The blots were then dried and developed on X-ray film incubated at –80ºC for 1 week. RNA isolation and Northern blot analysis Total RNA was isolated from stress treated and control tomato plants using Triozol-reagent according to the manufacturer’s instructions (MRC, USA). To ensure approximately equal loading of RNA, 20 μg of total RNA for salt and drought while 10 μg for cold and ABA were loaded onto 1.2% (w/v) denaturing formaldehyde agarose gels and transferred to Hybond-N+ membranes (Amersham Pharmacia, UK) with 20 × SSC (3 M NaCl, 0.3 M sodium citrate, pH 7.0) as transfer solution. It was cross-linked to the membranes by baking at 2 h in 80°C. RNA blots were pre-hybridized overnight at 65°C in hybridization buffer: 50% formamide, 5 × SSPE, 5 × Denhardt’s, 0.1% SDS. PCR products corresponding to LeDREB2 cDNAs were labeled with [α-32P] dCTP by random priming (Promega, USA). All membranes were hybridized at 65°C with hybridization buffer containing labeled DNA probe. Hybridization was performed 3-4 days at 65oC in 5% dextran sulfate, 0.25 M disodium phosphate (pH 7.2), 7% (w/v) SDS, and 1 mM EDTA. Following hybridization, the filter was washed twice with 2 × SSC and 0.1% SDS for 10 min each at room temperature, and twice with 0.1 × SSC and 0.1% SDS for 5 min each at 65oC. After the washing steps, the blots were exposed to X-ray film (Kodak) at –80°C for a week and developed. For tissue-specific expression of LeDREB2 gene, total RNA was prepared from tomato seedling mature leaves, young leaves, roots, and stems. Northern blot was performed according to the above mentioned procedures.

Source of Information: Bangladesh J. Pl. Breed. Genet., 25(1): 01-09, 2012

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Achievement/Findings:

These findings demonstrate that LeDREB2 gene, a homolog of the transcription factor gene that is induced after application of stress treatments in tomato which is an abiotic stress related gene in tomato. The implication of this investigation is that, this LeDREB2 gene will become another target for engineering biotech crops with enhanced tolerance to abiotic stress. We intend to test whether overexpression of LeDREB2 gene in transgenic tomato plants enhances their resistance to abiotic stresses. In addition, an in vivo analysis of different gene-silencing phenotypes will help to clarify the role of LeDREB2 gene in its responses to abiotic stresses.

Knowledge Management: Journal