Synthetic nucleic acid molecule for imparting multiple traits

#The present invention relates to DNA constructs containing a modified nucleic acid molecule capable of imparting multiple desired traits to a plant, as well as host cells, plant cells, transgenic plants, and transgenic plant seeds containing such DNA constructs. The present invention also relates to a method of preparing a modified nucleic acid molecule capable of imparting multiple traits to a plant, a method of determining whether multiple desired traits can be imparted to plants by a single modified DNA molecule, and a method for imparting traits to plants.

#Control of plant virus diseases took a major step forward when it was shown that the tobacco mosaic virus (“TMV”) coat protein (“CP”) gene that was expressed in transgenic tobacco conferred resistance to TMV (Powell-Abel et al., “Delay of Disease Development in Transgenic Plants that Express the Tobacco Mosaic Virus Coat Protein Gene,” Science 232:738-43 (1986)). The concept of pathogen-derived resistance (“PDR”), which states that pathogen genes that are expressed in transgenic plants will confer resistance to infection by the homologous or related pathogens (Sanford et al., “The Concept of Parasite-Derived Resistance—Deriving Resistance Genes from the Parasite's Own Genome,” J. Theor. Biol., 113 :395-405 (1985)) was introduced at about the same time. Since then, numerous reports have confirmed that PDR is a useful strategy for developing transgenic plants that are resistant to many different viruses (Lomonossoff, G. P., “Pathogen-Derived Resistance to Plant Viruses,” Ann. Rev. Phytopathol., 33:323-43 (1995)).

#Remarkable progress has been made in developing virus resistant transgenic plants despite a poor understanding of the mechanisms involved in the various forms of pathogen-derived resistance (Lomonossoff, G. P., “Pathogen-Derived Resistance to Plant Viruses,” Ann. Rev. Phytopathol. 33:323-43 (1995)). Although most reports deal with the use of coat protein genes to confer resistance, a growing number of reports have shown that viral replicase (Golemboski et al., “Plants Transformed with a Tobacco Mosaic Virus Nonstructural Gene Sequence are Resistant to the Virus,” Proc. Natl. Acad. Sci. USA 87:6311-15 (1990)), movement protein (Beck et al., “Disruption of Virus Movement Confers Broad-Spectrum Resistance Against Systemic Infection by Plant Viruses with a Triple Gene Block,” Proc. Natl. Acad. Sci. USA, 91:10310-14 (1994)), NIa proteases of potyviruses (Maiti et al., “Plants that Express a Potyvirus Proteinase Gene are Resistant to Virus Infection,” Proc. Natl. Acad. Sci. USA, 90:6110-14 (1993)), and other viral genes are effective. This led to the conclusion that any part of a plant viral genome may give rise to PDR. Furthermore, the viral genes can be effective in the translatable and nontranslatable sense forms, and less frequently, antisense forms (Baulcombe, “Mechanisms of Pathogen-Derived Resistance to Viruses in Transgenic Plants,” Plant Cell 8:1833-44 (1996); Dougherty et al., “Transgenes and Gene Suppression: Telling us Something New?,” Current Opinion in Cell Biology 7:399-05 (1995); Lomonossoff, G. P., “Pathogen-Derived Resistance to Plant Viruses,” Ann. Rev. Phytopathol. 33:323-43 (1995)).

#FIG. 1 shows the scheme of the 216 bp TSWV fragment and the corresponding primers to be named to be used for its synthesis. The diagonal lines indicate the place in the oligonucleotides (“oligos”) where six random nucleotides and six nucleotides corresponding to restriction sites are located: XhoI for the 5′ end (left) and BamHI for the 3′ end (right). The XX in the middle of the oligo name refers to the percentage of change for every single construct according to Tables 3 and 4. The fragment is shown in the sense orientation.

#FIG. 2 is a schematic representation of the cloning/expression cassette vector pEPJ86GFP. The expression vector is located in a pUC backbone between the HindIII and KpnI sites in the multiple cloning site (MCS) adjacent to the lacZ gene. In the same order, from left to right, the expression cassette contains a double 35S CaMV enhancer (the first two ‘hatched’ regions), the 35S Cauliflower Mosaic Virus (CaMV) promoter (striped regions with thin vertical lines), the Alfalfa Mosaic Virus leader sequence (region with thick vertical lines), the green fluorescent protein (GFP) gene (region of horizontal lines), and the CaMV 35S terminator sequence (bubbled region). After sequence confirmation the HindIII/KpnI fragments were subcloned into the plant transformation vector pGA482G.

#FIG. 3 shows a gel electrophoresis of one of the PCR assembly and amplification products obtained in this work (e.g., 5% changes in this case). Row 1: a molecular ladder 100 bp step; Row 2: assembled products after 55 cycles of all mixed different oligonucleotides, as described in examples; Row 3: a unique, single amplification product from the assembled reaction, prior to digestion.

#The present invention relates to a DNA construct containing a modified nucleic acid molecule with a nucleotide sequence which is at least 80%, but less than 100%, homologous to two or more desired trait DNA molecules. Each of the desired trait DNA molecules relative to the modified nucleic acid molecule have a nucleotide sequence similarity value and each of these similarity values differs by no more than 3 percentage points. The modified nucleic acid molecule imparts the desired trait to plants transformed with the DNA construct.

#In one aspect of the present invention, the construct also includes a silencer DNA molecule operatively coupled to the modified nucleic acid molecule. In this embodiment, the modified nucleic acid molecule and silencer DNA molecule collectively impart the desired trait to plants transformed with the DNA construct.

#In another aspect of the present invention, the DNA construct can be a fusion gene (chimera) which includes a plurality of modified DNA molecules at least some of which have a nucleotide sequence that is at least 80%, but less than 100%, homologous to the two or more desired trait DNA molecules. The plurality of trait molecules may be directed to one or more desired traits, and collectively impart the plurality of traits to plants transformed with the DNA construct. At least some of the desired trait DNA molecules, relative to their respective modified nucleic acid molecule, have a nucleotide sequence similarity value and each of these similarity values differs by no more than 3 percentage points.