Differentially expressed genes identified during salt adaptation in Eucalyptus microcorys : down-regulation of a cDNA sequence coding for α-tubulin

Abstract

Summary Determination of genetic differences and levels of gene expression based on morphological and anatomical characteristics, levels of metabolites, or enzymatic activity is often difficult. However, changes in molecular markers at the nucleic acid level allow polymorphic (genetic and biochemical) variation amongst tissues of individuals and between closely related adaptive clones to be assessed. We describe the use of differential display reverse transcriptase (DDRT)-PCR to screen Eucalyptus microcorys for genes which had been down-regulated as a result of adaptation to elevated levels of NaCl. It was necessary to employ an anchored oligonucleotide-dT primer in combination with a small number of commercially available ten-base oligonucleotide primers before arriving at combinations which clearly distinguished differentially expressed cDNA. The variability of the technique, and in particular the quality of the total RNA extracted, and the need to include betaine in PCR reactions was analysed. We demonstrated that DDRT-PCR was able to detect a number of down-regulated cDNA fragments in E. microcorys during adaptation to salt. An important finding was that one cDNA highly analogous to α-tubulin was strongly repressed during adaptation and NaCl exposure, which was consistent with cell structural changes already observed. Our results show that DDRT-PCR is a versatile and sensitive method, capable of detecting transcriptional changes (i.e. at the mRNA level) in plants.