Cloning and characterization of two floral-specific genes of sunflower (Helianthus annuus L.).

Sunflower (Helianthus annuus) being an important oil seed crop was chosen for this study, because genes isolated and characterized from sunflower will shed more light on plant development, and can also be useful in biotechnological applications. The study involved the isolation and characterization of two sunflower floral-specific genes: SF15 (an anther-specific gene) and SF17 (a pollen-specific gene). SF15: Northern blot analysis revealed that the probe hybridized to 1.0 kb mRNA transcripts present in the young unopened disc florets, whose development was delineated into seven stages. SF15 transcripts begin to accumulate in stage 1 and reach higher levels in stages 2, 3 and 4, while no expression was observed in late developmental stages 5 to 7 and the expression is confined only to the anthers of male-fertile disc florets. In situ hybridization experiments revealed that SF15 is expressed in a single layer of epidermal cells of the anther. Screening of the floral cDNA library resulted in the isolation of 4 near full-length cDNA clones (SF15-2, SF15-3, SF15-45 and SF15-49). These cDNA clones have inserts of 785 to 934 bp. Screening of a genomic library resulted in the isolation oF a 10.7 kb genomic clone (SF15G). Primer extension analysis revealed that there are two major and, few minor transcription initiation sites. Southern blot analysis showed that SF15 is present as a member of a multigene family in the sunflower nuclear genome and as a single copy in the nuclear genomes of corn and tomato. The DNA sequence information revealed that the genomic and four cDNA clones represent five distinct cognate genes, with nucleotide similarities ranging from 89.9% to 99.7% in the coding regions. SF17: In the Northern blot analysis the 329 bp probe, hybridized to the 2.0 kb transcripts present exclusively in the mature pollen grains. Screening of phase III floral cDNA library resulted in the isolation of 1915 bp cDNA clone. A 15 kb genomic clone (SF17G) was isolated from the genomic library, with the 1915 bp cDNA insert as a probe. A 5.5 kb EcoRI fragment of the genomic clone was subcloned into pUC19 vector for sequence analysis. The DNA sequence information revealed that the cDNA and genomic clones are identical, except for the presence of two small introns in the genomic clone. Primer extension analysis showed that there are two major and a few minor transcription initiation sites. Southern blot analysis showed that SF17 is represented in one or two copies, in the sunflower nuclear genome. The cDNA encodes a leucine-rich-protein of 540 amino acid residues with a molecular mass of 61.7 kDa. SF17 protein did not contain a transit signal sequence, but the SF17 protein consists of five structural features: a hydrophilic domain, a leucine-rich-region (LRR), an acid domain and two trans-membrane domains. (Abstract shortened by UMI).Dept. of Biological Sciences. Paper copy at Leddy Library: Theses & Major Papers - Basement, West Bldg. / Call Number: Thesis1995 .R42. Source: Dissertation Abstracts International, Volume: 56-11, Section: B, page: 5946. Adviser: D. T. N. Pillay. Thesis (Ph.D.)--University of Windsor (Canada), 1995