Identification of drought-responsive genes by cDNA-amplified fragment length polymorphism in maize

The cDNA-AFLP (amplified fragment length polymorphism) technique was performed to detect transcript-derived fragments related to drought tolerance in maize inbred lines X178 and B73 to identify and understand drought-responsive genes at the transcriptional level. By comparing the sequences of fragments differentially expressed between these lines under water-stressed and well-watered conditions, 72 drought-responsive unique expressed sequence tags (ESTs) were identified with 41 primer combinations targeting PstI/MseI restriction sites. Most of the fragments isolated in these experiments were found to be associated with genes responsive to abiotic or biotic stresses. Subsequently, a putative R1-type MYB transcription factor gene corresponding to ZmMYB-R1 on bin 9.03 was obtained by in silico cloning by homology to EST sequence P36M87-366, which matched one of the AFLPs identified in this study. The 1881 bp sequence encodes a protein of 626 amino acids that contains a single conserved MYB-like DNA-binding domain. Expression of green fluorescence protein fusions showed that ZmMYB-R1 was localised in the cell nucleus. Quantitative real-time polymerase chain reaction revealed that ZmMYB-R1 was induced by drought, high salt, heat, cold and exogenous ABA in maize. The expression of ZmMYB-R1 initially reaches its highest levels in leaves, then is subsequently also detected in stems and roots. This work revealed genes potentially responsible for drought tolerance in maize, and a new gene, ZmMYB-R1, was shown to be associated with water stress.