Characterisation and functional analysis of pollen-specific cDNAs encoding putative transcriptional regulators from Nicotiana tabacum.
thesisposted on 19.11.2015, 09:10 by Justin Paul. Sweetman
The significance of putative transcriptional regulators and their expression patterns in the developing male gametophyte (or pollen grain) have been investigated in Nicotiana tabacum. A reverse genetic approach was used to isolate genes involved with the development of the pollen grain, after initial attempts to isolate transcription factors directly by expression library screening proved unsuccessful. Oligonucleotides corresponding to a part of the MYB and LIM domain sequences from other plant proteins were used as probes to screen a cDNA library prepared from poly A+ RNA isolated from mature tobacco pollen. Two clones from each class isolated were found to have unique nucleotide sequences (myb.Nt1, myb.Nt2, Nt11 and Nt12). Homology searches revealed the presence of MYB and LIM domain repeats in each pair of clones respectively. Detailed Northern blot analysis showed that the myb.Nt1 and Nt11 transcripts were present specifically in mature and germinating pollen. Both transcripts were detectable immediately before the asymmetrical division Pollen Mitosis I, reaching a maximal level in mature pollen. Transgenic plants containing the whole myb.Nt1 and Nt11 cDNA clones in sense and antisense orientations to the pollen-specific lat52 promoter and in sense orientation to the widely and stronghly expressing cauliflower mosaic virus 35S promoter (but whose expression in pollen is relatively weak) were generated. Analysis of transgenic seed from second generation plants, revealed significantly reduced transmission through the pollen grain. Analysis of these plants showed a reduction in endogenous myb/lim transcript abundance. These results suggest that the MYB.NT1 protein is crucial for pollen function. Plants harbouring the 35S CaMV-myb.Nt1 construct exhibited a stably inherited dwarf phenotype, ranging from partial to severe dwarfism in second generation plants. Analysis of these plants showed among other traits a reduction in internodal growth and a change in leaf morphology. These results suggest that MYB.NT1 is involved with plant hormone biosynthesis and/or hormone induced gene expression in the pollen grain.