MOLECULAR ANALYSIS OF FLOWERING GENES OF PHARBITIS NIL

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The goal of this research is to understand the molecular mechanism of floral induction in plants. How photoperiod, a crucial controlling factor, regulates floral induction in a short-day plant Pharbitis nil is the subject of the investigation. Several cDNA libraries will be constructed and screened for the isolation of clones that represent cotyledon and apical meristem mRNAs whose abundance is affected by an inductive photoperiod. The temporal and spacial expression of the mRNAs represented by the cDNA clones will be determined by dot blot, northern blot, and in situ hybridization procedures. The 5' and the 3' ends of the mRNAs will be determined by primer extension and S1 nuclease protection assays. The nucleotide sequence of the cDNA clones will be determined for the analysis of the encoded polypeptides. The genomic clones corresponding to the cDNA clones will be isolated by screening a genomic library using cDNA clones as probe. A physical map of the genomic clone will be constructed by digestion with restriction endonucleases. Southern blot hybridization analysis will be used to identify the transcribed and nontranscribed regions of a gene. The DNA sequences of the 5' nontranscribed region and other region(s) of interest will also be determined. These studies will identify the promoter- regulatory region sequences, and define the intron-exon boundaries, if any. Gene transfer procedures will be used to study the function and regulation of expression of the gene(s) of interest. For functional studies the effect of aberrant expression will be determined. The coding sequences will be cloned in either sense (to overproduce the protein product) or antisense (to produce complementary RNA) orientation downstream from a strong plant promoter in a Agrobacterium Ti plasmid derived binary vector. The chimeric gene will be reintroduced into plants by Agrobacterium-mediated gene transfer procedures. Phenotypic changes occurring in transformant plants resultant of constitutive (over) production of either the encoded gene product or the antisense RNA will be determined. For regulatory studies gene fusions containing the promoter-regulatory region of he gene(s) of interest and a reporter gene (e.g. E. Coli beta-glucuronidase, GUS) will be constructed. Following transfer to plant cell GUS activity will be measured in transformant plants to delineate the regulatory sequences. Sequential deletions will be constructed and analyzed for fine mapping of regulatory sequences. We will also identify, isolate and purify the trans-acting regulatory factors by established procedures. Southern blot hybridization will be used to determine if sequences homologous to the P. nil cDNA clones are present in the long day plant Arabidopsis thaliana and other plant species. If yes, we will isolate and analyze the expression and function of the A. thaliana genes by similar procedures. Induction of flowering is a critical process in angiosperm lifecycle. Understanding how photoperiod control this process will greatly broaden our knowledge on developmental processes in plants.