Sequence And Functional Analysis Of Promoter Osg6B From Rice And Molecular Cloning And Transferring Into Tobacco Of ArgE From E.coli

This study consisted of two parts: sequence and functional analysis of the cloned promoter Osg6B' from rice and molecular cloning and transferring into tobacco ofargE from E.coli.In the part I, the sequence of the cloned promoter Osg6B' was first analyzed. Osg6B' had a whole length of 1688 bp and 98% homology to known sequence of promoter Osg6B. Its transcriptional start point, TATA boxes and the consensus sequences "TGTGG" conserved usually in anther-specific promoters were identical to those of reported Osg6B. However, the cloned promoter had 18 substitution at 18 sites, 22 deletions at 18 sites and 3 insertion at 3 sites between sites 0---1273 bp which was reported to control temporal-spatial specific expression , and 3 substitution at 3 sites , 6 deletions at 6 sites between -1095 bp and -1273 bp, the key functional sequence area, in comparing with known Osg6B. Based on the sequence analysis, the temporal-spatial speciality of Osg6B' was then analyzed with transgenic tobacco plants. A binary plant expression vector with Osg6B' driving report gene GUS was constructed and transferred into tobacco via Agrobacterium tumefaciens mediation. In TO mature transgenic plants tested, GUS staining was observed in different development stages and positions of the anther and some times in calyx, petal, stigma, anther filement and pollens. In 9 TO young plants GUS-tested, 2 plants were negative and the rest were positive: 4 plants stained in root, stem and leaves and 3 plants only in stem and leaves. GUS staining was also found in root, stem and leaves of Kan-resistant Tl seedlings that occurred 3/4 of all Tl generation. These results indicated that the cloned Osg6B' had a constitutive characteristics but not anther specificity, and the transgene was segregated in a Mendelian fashion in the Tl generation.In the part II, a gene, argE, was cloned from E.coli and sequenced. It had 1152 bp in length, and predictably encoded for Af-acetylornithinase (NAO) with total 383 amino acids. Its DNA sequence and predicated amino acid sequence were entirely identical to those of reported argE. This gene, artificially put under the control of CaMV 35S, was introduced into tobacco with the aid of Agrobacterium, and the transgenic plants obtained were identified by GUS staining, PCR and Southern blot analysis. Its function in transgenic plants is being exploited.