Molecular characterization and functional analysis of a pollen-specific gene in Brassica napus.
|Title:||Molecular characterization and functional analysis of a pollen-specific gene in Brassica napus.|
|Authors:||Treacy, Brian K.|
|Abstract:||cDNA and genomic clones a new pollen-specific gene, Bnm1, have been isolated of from Brassica napus L. cv. Topas. The gene contains an open reading frame of 546 bp and a single intron of 362 bp. A comparison of the deduced amino acid sequence with sequences in data banks did not show similarity with known proteins. Northern blot analysis of developing pollen showed that Bnm1 mRNA was first detected in bicellular pollen and accumulated to higher levels in tricellular pollen: Bnm1 mRNA was not detected in leaves, stems, roots, pistils, seeds or pollen-derived embryos. RNA in situ hybridization of whole flower buds confirmed that Bnm1 was pollen-specific and expressed late in development. A promoter fragment of the Bnm1 gene fused to the gusA reporter gene yielded similar patterns of tissue specificity and developmental regulation in transgenic B. napus cv Westar plants; however, the promoter was also active during the early stages of pollen development. The Bnm1 gene, cloned in this study, was derived from the A genome of the allotetraploid species B. napus (AACC). Southern blot analysis indicated that sequences similar to the Bnm1 gene were found in both A and C Brassica genomes. Related sequences were found in all 10 members of the Brassiceae tribe examined, but were not present in all tribes of the Brassicaceae family. Antisera raised against the BNM1 protein detected a single band of predicted size in tricellular and germinating pollen. BNM1 protein was not detected in other developmental stages including; tetrad, unicellular microspores and bicellular pollen. Protein samples extracted from somatic tissues, leaf and stem were not recognized by the anti-BNM1 primary antibody. To investigate the function of BNM1 in pollen, B. napus plants were transformed with a construct containing the Bnm1 promoter, fused to the complete cDNA in reverse orientation. Seven transgenic plants showed lower levels of Bnm1 when compared with wild-type. No obvious morphological lesions in transgenic antisense plants were observed. No differences in viability or in vitro germinations were observed between these downregulated plants and wild-type. In vivo germination assays suggested that, although BNM1 is not essential to germination and pollen tube growth, BNM1 may modulate the growth/guidance of the pollen tube towards the ovule.|
|Collection||Thèses, 1910 - 2010 // Theses, 1910 - 2010|