We have isolated and characterized the 5' region of the rice actin2 gene (OsAct2), which contains 793 bp of sequence upstream of the OsAct2 transcription initiation site, 58 bp of the first non-coding exon, 1736 bp of the 5' intron and the first 8 bp (non-coding sequence) of the second exon. It was found that the 5' region of OsAct2 is an efficient gene regulatory region for driving the constitutive expression of foreign genes in transgenic rice. In situ histochemical results indicated that OsAct2::GUS (GUS, beta-glucuronidase) gene expression in transgenic rice plants is high in sporophytic and gametophytic tissues. It was demonstrated that a 2.6-kb upstream sequence of the OsAct2 translation initiation codon contains all of the 5' regulatory elements necessary for high-level gus expression in transgenic rice tissues. OsAct2 promoter activity was significantly enhanced by the deletion of a 1590-bp segment from the central region of the first intron. The +96 to +274 region of the intron negatively regulates gus expression in leaves. To identify regulatory elements within the OsAct2 promoter, nested truncations of the promoter region were made and fused to gus. The results showed that the region from -1 to -376 was sufficient for promoter activity. In addition, two OsAct2-based expression vectors for use in monocot transformation were developed to promote the high-level expression of foreign genes.

For thousands of years farm practices have evolved as new innovations have become available. Farmers want more value per unit of land, clean fields, and high yields with less input. Plants with incorporated pest resistance and herbicide resistance help meet these needs through increased yield, reduced chemical use, and reduced soil impacts. Although researchers have developed useful traits for a wide variety of plant species, only a few traits are available commercially; however, global adoption of these traits has and continues to increase rapidly. Availability of future traits will be dependent on input not only from researchers, but from governments, interest groups, processors, distributors and ultimately consumers, in addition to the farmers that drive demand for transgenic seed.

Instability of transgene expression in plants is often associated with complex multicopy patterns of transgene integration at the same locus, as well as position effects due to random integration. Based on maize transposable elements Activator(Ac) and Dissociation(Ds), we developed a method to generate large numbers of transgenic barley (Hordeum vulgare var Golden Promise) plants, each carrying a single transgene copy at different locations. Plants expressing Ac transposase (AcTPase) were crossed with plants containing one or more copies of bar, a selectable herbicide (Basta) resistance gene, located between inverted-repeatDs ends (Ds-bar). F1 plants were self-pollinated and the F2 generation was analyzed to identify plants segregating for transposed Ds-barelements. Of Ds-bar transpositions, 25% were in unlinked sites that segregated from vector sequences, otherDs-bar copies, and the AcTPase gene, resulting in numerous single-copy Ds-bar plants carrying the transgene at different locations. Transgene expression in F2 plants with transposed Ds-bar was 100% stable, whereas only 23% of F2 plants carryingDs-bar at the original site expressed the transgene product stably. In F3 and F4 populations, transgene expression in 81.5% of plants from progeny of F2plants with single-copy, transposed Ds-bar remained completely stable. Analysis of the integration site in single-copy plants showed that transposed Ds-bar inserted into single- or low-copy regions of the genome, whereas silencedDs-bar elements at their original location were inserted into redundant or highly repetitive genomic regions. Methylation of the non-transposed transgene and its promoter, as well as a higher condensation of the chromatin around the original integration site, was associated with plants exhibiting transgene silencing.

Embryogenic cell lines of Gladiolus were bombarded with the bar-uidA fusion gene under the cauliflower mosaic virus (CaMV) 35S promoter (pDM327) or cobombarded with uidA under the CaMV 35S promoter (pBCG) and bar under the CaMV 35S promoter (pDM307). Over 500 cell lines were isolated for either the fusion gene or cobombarded cells following selection on Murashige and Skoog's medium supplemented with 2 mg 1−1 (9 μM) 2,4-dichlorophenoxyacetic acid and 6 mg 1−1 phosphinothricin. The optimum DNA concentration for, isolating stable transformants was one-tenth that for optimal isolation of lines with gus expression, and three times as many cell lines were isolated following cobombardment as compared to bombardment with the bar-uidA fusion gene. Three times as many cell lines (72% of the cell lines) containing the bar-uidA fusion gene expressed gus as compared to cobombarded cell lines (23%) following histological staining. Gus expression ceased after 1 yr in culture for 5% of the cell lines containing the fusion gene and 3% of the cobombarded cell lines. The bifunctionality and utility of the bar-uidA fusion gene were demonstrated, accompanied by enhanced gus expression.

La presente invention se rapporte au promoteur RS324 du mais, ainsi qu'a des compositions comportant cette sequence et a des plantes transformees a l'aide de ces compositions. L'invention se rapporte egalement a des procedes d'expression de transgenes dans des plantes consistant a utiliser ces sequences. Les procedes de cette invention consistent notamment en la creation directe de plantes transgeniques avec le promoteur RS324 par transformation genetique, ainsi qu'au moyen de procedes de selection vegetale. Les sequences de cette invention representent un nouvel outil precieux pour la creation de plantes transgeniques, dotees de preference d'au moins une caracteristique avantageuse ajoutee.

Cette invention porte sur le promoteur RS81 du mais. Des compositions comprenant la sequence de ce promoteur sont decrites, ainsi que des plantes transformees par ces compositions. L'invention porte en outre sur des procedes d'expression de transgenes dans les plantes comprenant l'utilisation de ces sequences. Les procedes de cette invention comprennent la production directe de plantes transgeniques a l'aide du promoteur RS81 par transformation genetique, ainsi que par des procedes de selection de plantes. Les sequences de cette invention representent un nouvel outil precieux pour la production de plantes transgeniques, et possedent d'autres caracteristiques avantageuses.

Tissue-specific patterns and levels of gene expression were characterized in transgenic Gladiolus plants that contained the phosphinothricin acetyltransferase (bar)-β-glucuronidase (uidA) fusion gene under transcriptional control of the promoter from either the cauliflower mosaic virus 35S (CaMV 35S), duplicated CaMV 35S (2×CaMV 35S), rice actin (Act1), or Arabidopsis ubiquitin (UBQ3) promoters. The bar gene confers resistance to phosphinothricin (PPT)-containing herbicides and allowed selection of transgenic cells. The β-glucoronidase gene encoded by the uidA locus of E. coli functioned as a reporter gene. Maximum levels of β-glucuronidase (GUS) activity in leaves were 173, 112, 50, and 10 nmoles 4-methylumbelliferone h−1 mg−1 protein for transgenic plants with the bar-uidA fusion gene under the control of the CaMV 35S, 2×CaMV 35S, UBQ3, and Act1 promoters, respectively. There was frequently considerable variability in GUS activity between the leaves of a single plant, and levels of uidA expression varied between independently transformed plants for each promoter. Callus derived from transgenic plants showed much less variation in GUS expression than leaves. The mean level of GUS activity was significantly higher (over 3×) for transgenic lines of callus containing the CaMV 35S as compared to the UBQ3 promoter, and this confirmed the higher (2×) level of GUS activity in levels of plants with the CaMV 35S promoter as compared to the UBQ3 promoter. Tissue-specific patterns of uidA expression were determined by histochemical staining. Leaves 5–6 cm long from plants with any of the four promoters tested exhibited uidA expression primarily in the vasculature. Under all four promoters uidA was expressed more frequently in root tips as compared to leaves.

To devise a method for function-based gene isolation and characterization in barley, we created a plasmid containing the maize Activator (Ac) transposase (AcTPase) gene and a negative selection gene, codA, and a plasmid containing Dissociation (Ds) inverted-repeat ends surrounding the selectable herbicide resistance gene, bar. These plasmids were used to stably transform barley (Hordeum vulgare). In vitro assays, utilizing a Ds-interrupted uidA reporter gene, were used to demonstrate high-frequency excisions of Ds when the uidA construct was introduced transiently into stably transformed, AcTPase-expressing plant tissue. Crosses were made between stably transformed plants expressing functional transposase under the transcriptional control of either the putative AcTPase promoter or the promoter and first intron from the maize ubiquitin (Ubi1) gene, and plants containing Ds-Ubi-bar. In F-1 plants from these crosses, low somatic and germinal transposition frequencies were observed; however, in F-2 progeny derived from individual selfed F-1 plants, up to 47% of the plants showed evidence of Ds transposition. Further analyses of F-3 plants showed that approximately 75% of the transposed Ds elements reinserted into linked locations and 25% into unlinked locations. Transposed Ds elements in plants lacking the AcTPase transposase gene could be reactivated by reintroducing the transposase gene through classical genetic crossing, making this system functional for targeted gene tagging and studies of gene function. During the analysis of F-3 plants we observed two mutant phenotypes in which the transposed Ds elements co-segregate with the new phenotype, suggesting the additional utility of such a system for tagging genes.

Efficient negative selection systems are increasingly needed for numerous applications in plant biology. In recent years, various counter-selectable genes have been tested in six dicotyledonous species, whereas there are no data available for the use of negative selection markers in monocotyledonous species. In this study, we compared the applicability and reliability of two different conditional negative selection systems in transgenic barley. The bacterial codA gene encoding cytosine deaminase, which converts the non-toxic 5-fluorocytosine (5-FC) into the toxic 5-fluorouracil (5-FU), was used for in vitro selection of germinating seedlings. Development of codA-expressing seedlings was strongly inhibited by germinating the seeds in the presence of 5-FC. For selecting plants in the greenhouse, a bacterial cytochrome P450 mono-oxygenase gene, the product of which catalyses the dealkylation of a sulfonylurea compound, R7402, into its cytotoxic metabolite, was used. T-1 plants expressing the selectable marker gene showed striking morphological differences from the nontransgenic plants. In experiments with both negative selectable markers, the presence or absence of the transgene, as predicted from the physiological appearance of the plants under selection, was confirmed by PCR analysis. We demonstrate that both marker genes provide tight negative selection; however, the use of the P450 gene is more amenable to large-scale screening under greenhouse or field conditions.

Three selectable marker genes were compared for their efficacy in the production of transgenic wheat plants following microprojectile bombardment of cultured immature embryos. While transformed plants were recovered using the bar (phosphinothricin acetyltransferase) gene in combination with bialaphos, and the aphA (neomycin phosphotransferase) gene in combination with geneticin or paromomycin, no transgenic material was obtained with the hpt (hygromycin phosphotransferase) gene and hygromycin B. Southern analysis revealed single copy as well as multiple copy insertions of the bar and aphA transgenes. Inheritance of these selectable marker genes was demonstrated in the T1 generation progenies.