Isolation of cowpea genes conferring drought tolerance: Construction of a cDNA drought expression library

I Gazendam,D Oelofse

doi:10.4314/wsa.v33i3.180601

Abstract

The main objective of this study was to identify and isolate the genes conferring drought tolerance in cowpea. A cDNA library enriched for cowpea genes expressed specifically during responses to drought was constructed. A procedure called suppression subtractive hybridisation (SSH) was successfully employed to obtain these differentially expressed transcripts. The library consists of 4 160 individual clones. Preliminary sequencing results identified two clones to be known stress-related plant genes (GST (glutathione-S-transferase) and PR-1 (pathogenesis-related protein-1)). Micro-array analysis will be performed on the library’s clones to verify their differential expression during drought conditions, and to identify additional clones for further investigation. Promising genes can eventually be used to genetically engineer crops for drought tolerance to improve food security in water-stressed areas.Keywords: cowpea, drought tolerance, suppression subtractive hybridisation (SSH)

Highlights

South Africa is classified as a water-scarce country (Department of Water Affairs and Forestry (DWAF), 2004)
This paper addresses the construction of a customised cDNA library that contains cowpea genes expressed during responses to drought
Contaminating genomic DNA was removed with the Turbo DNA-free kit (Ambion) and the RNA cleaned up with the Plant RNeasy kit (Qiagen). mRNA was isolated from 50 μg pools of stressed IT96D-602 RNA and control Tvu7778 RNA using an Oligotex mRNA purification kit (Qiagen). cDNA was synthesised from mRNA using the Roche cDNA synthesis kit

Summary

Introduction

South Africa is classified as a water-scarce country (Department of Water Affairs and Forestry (DWAF), 2004). The utilisation of marginal soils by plants tolerant to drought stress provides the greatest potential for agricultural expansion. Breeding for tolerance must include different strategies, including gene transfer. In order to understand the gene networks that underlie plant stress, it is necessary to identify and characterise the genes that respond to abiotic and biotic stress. Most lines are drought-tolerant and can be grown under the harshest growing conditions. It is, an important crop for subsistence and small-holder farmers (Quass, 1995)

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