Molecular genetic analysis of the maize terminal ear1 gene and in silico analysis of related genes : a thesis presented in partial fulfilment of the requirements for the degree of Doctor in Philosophy in Plant Biology at Massey University, Palmerston North, New Zealand
Mutants of the maize terminal ear1 (te1) gene have shortened internodes, abnormal phyllotaxy, leaf pattern defects and partial feminisation of tassels. The te1 gene encodes an RNA recognition motif (RRM) protein, and is expressed in the vegetative shoot apex in semicircular rings that laterally oppose the positions of leaf primordia (Veit 1998). This project aimed to further characterise the molecular biology and function of the te1 gene. Molecular genetic studies aimed to further characterise the genes structure and expression. Genomic clones were sequenced revealing the intron exon structure. 5' RACE was used to predict a 5' transcription start site. Competitive RT-PCR showed that te1 transcripts were highest in vegetative shoot meristems and embryos, lower in ears, roots and tassels, and undetectable in leaves. Two te1 mutant alleles were cloned and the junctions sequenced, a further five alleles were characterised incompletely. The TE1 peptide belongs to a subclass of RRM proteins which includes the Schizosaccharomyces pombe protein MEI2. More than 30 putative plant Mei2-like genes were identified in Genbank, no examples have been found in metazoans. Seven Mei2-like genes were predicted from the completed Arabidopsis genome. Exon structure and amino acid sequence supported three groupings of Mei2-like genes. Structural predictions of Mei2-like proteins indicate that the third RRM contained some novel structural features not present in canonical RRM proteins. Attempts to study the function of the TE1 protein in vitro were limited by the inability of both E. coli and Pichia pastoris expression systems to express the full length protein, probably due to codon bias. Antibodies produced to a C-terminal portion of the protein did not specifically detect the TE1 protein in plant extracts without incurring non-specific activity. The te1 cDNA was ectopically expressed in Arabidopsis from a copper-inducible promoter both with and without the SV40 nuclear localisation signal (NLS). Although both te1 and NLS:te1 transgenes were detected in transformants no phenotypes consistently correlated with transgene expression.