MXPA01001796A - Plant expression vectors - Google Patents

Novel combinations of 5', 3'and intron genetic elements are provided for enhanced expression in transgenic plants. The elements are associated with a fructose 1,6-bisphosphatase gene, a chlorophyll a/b binding protein gene, a ubiquitin gene, a nopaline synthase gene, and/or a heat shock gene. Recombinant DNA molecules containing the non-translated 5'and/or 3'non-translated elements of the invention are further provided, as are plant cells, tissues and plants containing those DNA molecules.

VECTORS OF EXPRESSION IN PLANTS FIELD OF THE INVENTION The present invention relates generally to plant genetic engineering. More particularly, it relates to improved gene expression systems for transgenic plants that use different combinations of genetic elements in a plant expression cassette. The present invention also relates to recombinant DNA molecules that contain the genetic elements, and to microorganisms, plant cells and plants transformed with the DNA molecules.

BACKGROUND OF THE INVENTION Recent advances in genetic engineering have provided the necessary tools to transform plants to contain external genes. By building a desired recombinant plant gene and introducing it into plant cells it is now possible to generate transgenic plants that have unique characteristics of agronomic importance. Consistent and reliable genetic elements to be used and to construct recombinant plant genes are of great value in plant genetic engineering. Many such elements can improve the gene expression levels of a particular gene of interest. By doing so, these elements provide several advantages. First, by providing improved expression levels, optimal combinations of genetic elements can result in a more pronounced phenotype. This is due to the relationship observed in many cases between the level of transgene expression in a transgenic plant and the degree to which a desired plant characteristic is altered. Second, untranslated genetic elements that are capable of improving expression can mize some of the steps that limit the speed of production of transgenic plants. The higher the levels of expression that can be obtained, the lower will be the numbers of plants that will need to be produced and evaluated in order to recover those that produce quantities of a target protein or enough RNA molecule to result in the agronomically phenotype wanted. Finally, the identification of a variety of alternative genetic elements provides the additional advantage of reducing vector element redundancies. In this way, as multiple independent transgenes are genetically engineered into lines of transgenic plants, the use of alternate expression vector elements in different transgenes will help to mize the gene-dependent homology inhibition of expression.

BRIEF DESCRIPTION OF THE INVENTION The invention described herein provides novel combinations of genetic elements to be used to construct recombinant DNA molecules that are expressed in plants. A recombinant DNA molecule containing the combinations of genetic elements of the present invention exhibits improved expression levels, as desired for the transformation and regeneration of transgenic plants. The improved expression levels that are obtained using the elements of this invention provide numerous advantages, such as a reduction in the intensive labor selection procedure that is required for the production of transgenic plants and improved phenotypes of the plants produced in this manner. Additionally, the elements are useful alternatives for increasing the gene stacking capabilities in transgenic plants by mizing the repetition of sequences that has been associated with transgene expression instability. Therefore, according to one aspect of the present invention, there is provided a recombinant DNA molecule comprising, chained operably in the 5 'to 3' direction: (a) a promoter sequence; (b) a 5 'untranslated sequence isolated from a nucleotide sequence associated with a gene selected from the group consisting of a wheat fructose-1, 6-bisphosphatase gene, a wheat chlorophyll a / b binding protein gene , a wheat heat shock gene, a wheat peroxidase gene, a rice beta-tubulin gene, a rice amylase gene; c) an isolated intervening sequence from a nucleotide sequence associated with a gene selected from the group consisting of a Rice actin ntron, an intron of rice sucrosasynthase, an intron of phenylalanine amonia lyase of rice, and an intron of rice amylase; d) a DNA coding sequence; and e) a 3 'terminator region isolated from a nucleotide sequence associated with a gene selected from the group consisting of a wheat heat shock protein gene, a wheat ubiquitin gene, a fructose-1 gene, 6-wheat bisphosphatase, a rice glutelin gene, a rice lactate dehydrogenase gene, and a rice beta-tubulin gene. In another aspect of the invention there is provided a method for improving gene expression in plants and increasing the diversity of genetic element comprising: a) transforming plant cells with a recombinant DNA molecule comprising, chained operably in the direction of 'a 3': i) a promoter sequence; I) a 5 'untranslated sequence isolated from a nucleotide sequence associated with a gene selected from the group consisting of a wheat fructose-1, 6-bisphosphatase gene, a chlorophyll a / b binding protein gene of wheat, a wheat heat shock protein gene, a wheat peroxidase gene, a rice beta-tubulin gene and a rice amylase gene. iii) an intervening sequence isolated from a nucleotide sequence associated with a gene selected from the group consisting of an intron of rice actin, an intron of rice sucrosasynthase, an intron of phenylalanine ammonia rice, and an intron of rice amylase. ii) a DNA coding sequence; and v) a 3 'untranslated DNA sequence selected from the group consisting of a wheat heat shock protein gene, a wheat ubiquitin gene, a wheat fructose-1, 6-bisphosphatase gene, a gene of rice glutelin, a rice lactate dehydrogenase gene, and a rice beta-tubulin gene. b) selecting plant cells that have been transformed, and c) regenerating said plant cells to provide a differentiated plant. Plant cells containing the DNA molecules of the invention and the tissues, seeds, and differentiated plants produced therefrom are additionally provided by the invention. Other objects, aspects, and advantages of the present invention will be apparent to those skilled in the art in view of the following descriptions, examples, and claims.

BRIEF DESCRIPTION OF THE DRAWINGS The following drawings are part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention can be better understood by reference to one or more of those drawings in combination with the detailed description of specific embodiments presented herein. Figure 1 illustrates plasmid pMON 19469 Figure 2 illustrates plasmid pMON 26052 Figure 3 illustrates plasmid pMON 26055 Figure 4 illustrates plasmid pMON 26054 Figure 5 illustrates plasmid pMON 19433 Figure 6 illustrates plasmid pMON 32502 Figure J illustrates plasmid pMON 32506 Figure 8 illustrates plasmid pMON 32509 Figure 9 illustrates plasmid pMON 32510 Figure 10 illustrates plasmid pMON 32513 Fig. 1 illustrates plasmid pMON 19437 Fig. 2 illustrates plasmid pMON 32515 figural 3 illustrates plasmid pMON 32516 Figure 14 shows plasmid pMON 32517 Figure 15 shows plasmid pMON 33216 Figure 16 shows plasmid pMON 33210 Figure 17 shows plasmid pMON 33220 Figure 18 shows plasmid pMON 33219 Figure 19 shows plasmid pMON 47901 Figure 20 plasmid pMON 47906 FIGURE 21 FIGURE 21 plasmid pMON 47907 FIGURE 22 illustrates plasmid pMON 47915 FIGURE 23 illustrates plasmid pMON 47916 FIGURE 24 illustrates plasmid pMON 47917 FIGURE 25 illustrates e! plasmid pMON 47919 FIG. 26 illustrates plasmid pMON 32648 FIG. 27 shows plasmid pMON 18364 FIG. 28 illustrates plasmid pMON 19568 DETAILED DESCRIPTION OF THE INVENTION The present invention provides genetic elements for improved expression of recombinant plant genes comprising novel combinations of introns, and 5 'and 3' untranslated genetic elements described herein. The ADNB sequences and methods of the invention allow the production of transgenic plants having increased levels of a desired RNA or protein molecule of interest, thereby facilitating the introduction of agronomically desirable traits in plants through genetic manipulation. "Recombinant plant gene" or "recombinant DNA molecule" as used in the context of this invention, refers to a combination of genetic elements that are operably linked so as to be capable of expressing RNA in a plant cell and / or protein molecule. DNA molecules can be constructed using standard techniques well known to those skilled in the art. In general, a recombinant plant gene comprises, operably linked from the 5 'end to the 3' end: (1) a promoter region that causes the production of a DNA molecule; (2) a 5 'untranslated end sequence; (3) a DNA coding sequence that encodes a Desired RNA and / or protein and (4) a 3 'untranslated region. The region of a gene referred to as the "promoter" is responsible for regulating the transcription of DNA into RNA. The promoters comprise the DNA sequence, which is normally upstream (5 ') to a coding sequence, which regulates the expression of the downstream coding sequence by controlling the production of the messenger RNA (mRNA) by providing the recognition site. for RNA polymerase and / or other factors necessary to initiate transcription at the correct site. The promoter used in a recombinant plant gene of the invention is selected to provide sufficient transcriptional activity to achieve the desired expression levels of the gene or genes of interest. Numerous plant promoters are known in the art and can be obtained from a variety of sources such as plant or plant viruses and can include, but are not limited to, 35S cauliflower mosaic virus (CaMV) promoter (Odell et al., 1985), the Figwort mosaic virus (FMV) 35S (Sanger et al., 1990), the sugarcane bacillary virus promoter (Bouhida et al., 1993), the speckled yellow cornelin virus promoter. (Medberry and Olszewski 1993), the light-inducible promoter from the small subunit of ribulose-1, 5-bis-phosphate carboxylase (ssRUBISCO) (Coruzzi et al., 1984), the cytosolic rice promoter triocephosphate isomerase ( TPI) (Xu et al, 1994), the adenine phosphoribosyltransferase (APRT) promoter from Arabidopsis (Moffatt et al., 1994), the rice actin 1 gene promoter (Zhong et al., 1996) and the manoplna synthase promoters and octopine synthase (Ni et al., 1996). All of those promoters have been used to create several types of recombinant DNA constructs that are expressible in plants. The comparative analysis of constitutive promoters by the expression of reporter genes such as the uidA (ß-glucuronidase) gene from E.coli has been carried out with many of these and other promoters (Li et al., 1997; Wen et al., 1993). ). Other useful promoters include but are not limited to those that are expressed in a tissue specific manner, tissue enhanced, or in a regulated manner of development. Examples of these types of promoters are also known in the art. In addition to the promoter sequence that regulates the expression of operably linked DNA sequences, other genetic elements may play a role in improving gene expression. These elements include, but are not limited to, untranslated regions and intervention sequences (ntrons) that are associated with the genes to which they are linked in a operable. By "associated with" as used herein means that the genetic element is typically associated with a gene during the processing of the gene such as during transcription or translation procedure. The 5 'untranslated regions of an mRNA can play an important role in the initiation of translation and therefore in the regulation of gene expression. A 5 'untranslated leader sequence is characterized as that portion of the mRNA molecule that more typically extends from the 5' CAP site to the translation start codon of AUG protein. For most eukaryotic mRNAs, translation begins with the binding of the CAP binding protein to the mRNA end. This is then followed by the binding of several other translation factors, as well as the pre-ribosome complex or the 43S ribosome. This complex travels to the mRNA molecule while selecting an AUG start codon in an appropriate sequence context. Once it has been found and with the addition of the 60S ribosomal subunit, the complete 80S start complex initiates protein translation (Pain, 1986: Moldave, 1985; Kozak, 1986). A second class of mRNAs has been identified that possesses different translation start characteristics. Translation from these mRNAs starts in an CAP-independent manner and is believed to start with the ribosome binding to internal portions of the 5'-untranslated leader sequence (Sonenberg, 1990; Carrington and Freed, 1990; Jackson et al., 1990). The efficiency of translation initiation can be influenced by characteristics of the 5 'untranslated leader sequence, therefore, the identification and optimization of the 5' leader sequences can provide improved levels of gene expression in transgenic plants. For example, some studies have investigated the use of plant viruses from 5 'untranslated leader sequences for their effects on plant gene expression (Gallie et al., 1987; Jobling and Gehrke, 1987; Skuzeski et al., 1990). Increases in gene expression have been reported using the leader sequence of Tobacco Mosaic Virus Omega (TMV). When compared to other leading viral sequences, such as the leader Alfalfa Mosaic Virus RNA 4 (AMV), improvements were observed in the double triple in gene expression levels using the TMV Omega leader sequence (Gallie et al., 1987); Skuzeski et al, 1990). The 5 'untranslated leader sequences associated with heat shock protein genes have also been shown to significantly improve gene expression in plants (see for example, U.S. Patent 5,362,865). Most of the 5 'untranslated sequences are very rich in A-U and are predicted to lack significant secondary structures. One of the initial steps at the beginning of translation is the relaxation or unwinding of the secondary structure of mRNA (Sonenberg, 1990). The leader sequences of messenger RNA with insignificant secondary mRNA structure may not require this additional step of unfolding and may therefore be more accessible to the translation initiation components. Introductory sequences that can form stable secondary structures can reduce the level of gene expression (Kozak, 1998, Pelletier and Sonenberg, 1985). The ability of a 5 'untranslated leader sequence to interact with translation components could play a key role in affecting subsequent gene expression levels. The 5 'untranslated regions used in this investigation are capable of increasing the level of expression of a transcribable sequence to which they are operably linked. The 5 'untranslated region may be associated with a gene from a source that is either original or that is heterologous with respect to the other untranslated and / or translated elements present in the recombinant gene. The 5 'untranslated sequences provided by this invention are isolated nucleic acid sequences associated with plant genes, preferably monocotyledons including but not limited to wheat and rice. Particularly preferred are the 5 'untranslated regions associated with monocot genes that encode heat shock proteins, fructose-1, 6-bisphosphatases, chlorophyll a / b binding proteins, peroxidases, tubulins and amylases. Those preferred 5 'untranslated regions are illustrated herein by the 5' untranslated wheat heat shock sequence (Ta hsp 5 'leader) of SEQ ID NO: 53, the 5' untranslated sequence associated with the wheat fructose-1, 6- bisphosphatase (leader Ta fbp 5 '), comprising SEQ ID NO: 54, the 5' untranslated region associated with the wheat chlorophyll a / b binding protein gene (Ta leader) cab 5 '), comprising SEQ ID NO: 52, the 5' untranslated region associated with the wheat peroxidase gene (Ta per 5 'leader) comprising SEQ ID NO: 55, the 5' untranslated region associated with the rice amylase gene (r amy 5 'leader) comprising SEQ ID NO: 57, and the 5' untranslated region associated with the rice btub gene (r btub 5 'leader) comprising the SEQ ID NO: 56 The intervening sequences referred to herein as introns are also capable of increasing gene expression. Introns can improve the efficiency of mRNA processing. A number of introns have been reported to increase gene expression, particularly in monocots. In one report, the presence of intron catalasa 1 (Tanaka 1990) isolated from castor seeds resulted in an increase in gene expression in rice but not in tobacco when Gus is used as a marker gene. Further improvements have still been achieved, especially in monocotyledonous plants, by gene constructs having introns in the 5 'untranslated leader placed between the promoter and the structural coding sequence. For example, Callis et al., (1987) reported that the presence of hydrogenase hydrogen introns (Adh-I) or Bronze-Itronics resulted in higher levels of expression. Mascarenkas et al, (1990) reported a 12-fold improvement in CAT expression by using intron Adh. Other introns suitable for use in the DNA molecules of the invention include, but are not limited to, intron of sucrose synthase (Vasil et al 1989), the intron omega TMV (Gallie et al., 1989), the corn intron hsp 70 as shown in SEQ ID NO: 47 (U.S. Patent No. 5,593,874 and U.S. Patent No. 5,859,347 incorporated herein by reference in its entirety), and the rice actin intron (McEIroy et al., 1990 ). A number of factors can influence the degree of improvement of gene expression by an intron including but not limited to the promoter (Jefferson et al., 1987), flanking exon sequences and placement or location of the intron in relation to the gene (Mascerenhas et al. , 1990). The intervention sequences provided by the present invention are associated with a plant gene, preferably genes of monocotyledonous plants including but not limited to wheat and rice. Particularly preferred are intervention sequences associated with monocot genes that encode heat shock proteins, actins, amylases, lyases and synthases. Those preferred intervention sequences are illustrated herein by intervening sequences from a maize heat shock protein comprising SEQ ID NO: 47, the intervention sequence from a rice actin gene comprising SEQ ID NO: 50, the intervention sequence from a rice amylase gene comprising SEQ ID NO: 49, the intervention sequence from a rice ammonia phenylalanine gene comprising SEQ ID NO: 48 and the intervention sequence from a rice sucrose synthase gene comprising SEQ ID NO: 51. Untranslated sequences located at the 3 'end of a gene can also influence expression levels. A 3 'untranslated region comprises a region of the mRNA that generally begins with the translation stop codon and extends at least beyond the polyadenylation site. Ingelbrecht et al (Plant Cell 1: 671-80, 1989) evaluated the importance of these elements and discovered large differences in expression in stable plants depending on the source of the 3 'untranslated region. Using 3 'untranslated regions associated with octopine synthase, 2S seed protein from Arabidopsis, small subunit of rbcS from Arabidopsis, carrot extensin, and chalcone synthase from Antirrinio, a 60-fold difference was observed between the construct that best expresses (containing the untranslated region rbcS 3 ') and the lowest expression construct (which contains the chalcone synthase 3' region). The 3 'untranslated region of the nopaline synthase gene of T-DNA in Agrobacterium tumefaciens (3' nos) comprising SEQ ID NO: 46 has also been used as a terminator region for gene expression in plants. Although it is clear that 3 'untranslated regions can significantly affect the expression of recombinant plant genes, their precise role, and how to better identify and optimize them for maximum expression is an area that is still not well understood. The DNA coding sequences of a recombinant DNA molecule of the invention can encode any transcribable nucleic acid sequence including but not limited to those encoding modified proteins, foreign and / or modified of interest. The selection of this sequence will depend on the objectives for a given application. Typically, the structural DNA sequence encodes a protein molecule capable of modifying one or more characteristics of the plant. Suitable structural genes may include, but are not limited to, genes for controlling insects and other pests, genes for controlling microbial and fungal diseases, genes for herbicide tolerance, and genes for improvements in plant quality, such as production increases, tolerances to the environment, and nutritional improvement. Genes can be isolated from any source including but not limited to plants and bacteria. Alternatively, the DNA coding sequence can effect those phenotypes by encoding a nontranslatable RNA molecule that causes inhibition by target expression of an endogenous gene, for example, through antisense or co-suppression mediating mechanisms (cf. for example, Schuch, 1991; Bird, 1991; Jorgensen, 1990). The RNA could also be a catalytic (i.e., robotic) RNA molecule genetically engineered to cut a desired endogenous mRNA product (see for example, Gibson, 1997). The 3 'untranslated region that is used in a DNA molecule described herein generally causes polyadenylation of the 3' end of the transcribed mRNA sequence and the termination of transcription. The 3 'untranslated region can be associated with a gene from a source that is either original or that is heterologous with respect to the other untranslated and / or translated elements present in the DNA molecule. The 3 'untranslated sequences provided by the present invention are associated with a plant gene, preferably genes from monocotyledonous plants including but not limited to wheat and rice. Particularly preferred are the 3 'untranslated sequences isolated from a nucleotide sequence associated with coding monocot genes, a wheat fructose-1,6-biphosphatase (Ta fbp 3') comprising SEQ ID NO: 60 , a wheat heat shock protein (Ta hsp 3 ') comprising SEQ ID NO: 58, wheat ubiquitin (Ta ubiq 3') comprising SEQ ID NO: 59, a rice glutelin protein (r glut 3 ') comprising SEQ ID NO: 61, a rice lactate dehydrogenase (r lacd 3') comprising SEQ ID NO: 62 and a rice beta-tubulin (r btub 3 ') comprising SEQ ID NO: 63 The 5 'and / or 3' untranslated sequences and intervening sequences of this invention can be isolated by one or more of the numerous methods known to those skilled in the art, or alternatively can be generated synthetically. In one embodiment, the source plant material is plant RNA isolated from plant tissue. In another embodiment, the source material is a synthetic DNA sequence. The standard sequences for the genetic elements include RNA transcripts, cDNA sequences, or genomic DNA. In another embodiment, the PCR primers are synthesized to generate the genetic elements of the present invention. PCR primers can be synthesized to correspond to the terminus of 5 'untranslated or 3' untranslated regions of the target plant transcripts. For example, PCR reactions on the first strand of cDNA products generated by reverse transcription of RNA and PCR fragment containing the desired portion or the entire genetic element can be cloned into an expression vector for testing. Methods for isolation of genes and associated genetic elements are known to those skilled in the art and would include, for example, the PCR methods described herein. A variety of amplification methods are known in the art and are described in, for example, US Patents. Nos. 4, 683,195 and 4,683,202 and Innis et al, 1990. Those skilled in the art are familiar with standard resource materials describing specific conditions and procedures for the construction, manipulation and isolation of macromolecules (eg, DNA molecules, plasmids, etc.), generation of recombinant organisms and selection and isolation of genes (see, for example, Sambrook et al., 1989; Mailga et al., 1995; Birren et al., 1996). Plant molecular methods have also been described in, for example, Pouweis et al., 1985, supp. 1987; Weissbach and Weissbach, 1989; and Gelvin et al., 1990). In addition, one skilled in the art will recognize that the 5 'and / or 3' untranslated regions or intervening sequences of the invention can be modified, such as by base addition, elimination, substitution. , etc. while still providing the benefits described herein. Said modifications are considered to be within the scope of this invention. One type of modification, for example, could involve changes in the leader's nucleotide sequence that leads to a change in secondary structure. The appropriate secondary structure of the 5 'leader sequence may be required for optimal expression. As such, the leader's specific nucleotide sequence may be important as far as the secondary structure is concerned. Therefore, the leader sequence can indeed tolerate modifications in the nucleotide sequence that do not result in changes in secondary structure. Similarly, the introns and the 3 'untranslated sequences of the present invention can be modified accordingly to improve the expression of the gene in a particular system. Sequences surrounding the AUG of a 5 'untranslated region can also affect translation efficiency. For example, a consensus sequence has been identified in plants that can provide an optimal AUG context (Joshi et al., 1987; Koziel et al., 1996). In this way, this region of the 5 'untranslated sequences of the invention can also be modified in this way to further optimize transgenic expression levels. In addition, modifications can be made to other genetic components including but not limited to the 3 'untranslated region or intervening sequences of the recombinant DNA molecule of the invention so that new combinations of elements in the expression vector are further optimized . In addition to those elements discussed above, a recombinant DNA molecule of the invention may also include other regulatory elements such as chloroplast targeting / sequestering sequences, enhancer elements, etc. (for a summary on optimized transgene expression see Koziel et al., 1996). For example, improvements in expression have been obtained using enhancer sequences inserted at the 5 'end of the promoter. A recombinant DNA molecule of the invention may also include a selectable marker. These markers are commonly used to select transformed plants or plant cells that contain the exogenous genetic material of interest, ie, the transgene. Examples of such include, but are not limited to, a neomycin (neo) phosphotransferase gene (Potrykus et al., 1985), which confers resistance to kanamycin. Cells expressing the neomycin phosphotransferase gene can be selected using a suitable antibiotic such as kanamycin or G418. Other selectable markers commonly used include the bar gene that confers resistance to bialaphos; an EPSP synthase mutant gene (Hinchee et al., 1988) that confers aglyphosate resistance; a nitrilase gene that confers resistance to bromoxynil (Stalker et al., 1988); a mutant acetolactate synthase (ALS) gene that confers resistance to imidazolinone or sulfonylurea (European Patent Application 154,204, 1985); and a DHFR gene resistant to methotrexate (Thillet et al., 1988). A recombinant DNA molecule of the invention may also include a selectable marker as a further means by which the expression of the gene can be evaluated. Common selectable markers include a β-glucuronidase or uidA (GUS) gene that encodes an enzyme for which several chromogenic substrates are known (Jefferson, 1987); Jefferson et al., 1987); a luciferase gene (Ow et al., 1986), an R-locus gene that encodes a product that regulates the production of anthocyanin pigments (red color) in plant tissues (Dellaporta 1988); a β-lactamase gene (Sutcliffe et al., 1978) which encodes an enzyme for which several chromogenic substrates are known (eg, PADAC, a chromogenic cephalosporin); a xylE gene (Zukowsky et al., 1983) that encodes a catechol dioxygenase that can convert chromogenic catechols; an α-amylase gene (Ikatu et al., 1990); a tyrosinase gene (Katz et al., 1983) that encodes an enzyme capable of oxidizing tyrosine to DOPA and dopaquinone which in turn condenses to melanin; an α-galactosidase gene that encodes an enzyme whose substrate is chromogenic α-galactose; etc. The terms "selectable" and "evaluable" are also designed to encompass genes encoding "writable" markers whose secretion can be detected as a means of identification or selection for transformed cells. The examples include markers encoding a secretable antigen that can be identified by antibody interaction, or even secretable enzymes that can be detected catalytically. Secretable proteins fall into a number of classes, including small, diffusible, detectable proteins, for example, by ELISA, small active enzymes detectable in extracellular solution (eg, α-amylase, β-lactamase, phosphinothricintransferase), or proteins that are inserted or trapped in the cell wall (such as proteins) which include a leader sequence such as that found in the expression unit of extensin or tobacco PR-S). Other possible selectable / assessable / writable marker genes will be apparent to those skilled in the art. It is understood that the particular nucleotide sequences of the 5 'and 3' untranslated elements that are described herein are representative in the sense that equivalent sequences or portions thereof can be obtained and / or generated after this description. By equivalent means that said gene or portion thereof could function in a manner substantially the same as the element or portion thereof described in the present invention., and would provide a particular benefit or feature to a plant in substantially the same manner. A wide variety of cloning methods and tools are commercially available and have been described extensively (see for example Sambrook et al., 1989; Birren et al., 1996). Such methods are well known and can be readily used by those skilled in the art to construct the DNA molecules of this invention. Any type of vector can be used in the present invention, including but not limited to E. coli plasmid expression vector. More preferably, the combinations of genetic elements of the present invention are operatively linked in a plant transformation vector. In constructing a recombinant DNA molecule of the invention, the various components or fragments thereof are typically inserted using methods known to those skilled in the art into a convenient cloning vector that is capable of duplication in a bacterial host such as E. coli There are numerous vectors that have been described in the literature. After each subcloning, the vector can be isolated and subjected to further manipulation, such as restriction digestion, insertion of new fragments, ligation, elimination, resection, insertion, in vitro mutagenesis, addition of polylinker fragments, and the like, for the purpose of to provide a vector that will fulfill a particular need. Once the construct is complete, the construct can be transferred to an appropriate vector for further manipulation according to the manner of transformation of the plant cell. A number of plant transformation vectors have been described, and the particular vector can be modified depending on the transformation method. In one embodiment, a suitable plant transformation vector can be used for plant transformation mediated by Agrobacterium. A suitable transformation vector can be used for particle bombardment. A typical plant expression vector for Agrobacterium-mediated plant transformation, for example, can include a number of genetic components including but not limited to a promoter, one or more genes of interest, and a terminator sequence. By "genetic component" as used herein is meant any nucleic acid sequence or genetic element which may also be a component or part of a vector. The plant expression vector may also contain the functions for mobilization from E. coli to Agrobacterium and for vector replication in those hosts (ie, E. coli and origin of replication of broad host scales). In addition to one or more selectable marker genes for selection of vector cells containing the vector and for selecting plant cells containing the introduced DNA can be components of plant expression vector. The vector may also typically contain one or more T-DNA boundaries that function to transfer the DNA to the plant cell. A number of vectors suitable for stable transformation of plant cells or for the establishment of transgenic plants has been described in, for example Pouweis et al., Cloning Vectors: a Laboratory Manual, 1985, supp. 1987; Weissbach and Weissbach, Methods for Plant Molecular Biology, Academic Press, 1989; Gelvin et al., Plant Molecular Biology Manual, Kluwer Academic Publishers, 1990; and R.R.D. Croy Plant Molecular Biology LabFax, BIOS Scientific Publishers, 1993. The optimal plant transformation vector can be designed for the particular DNA delivery method and target culture of interest. DNA molecules comprising bacterial or viral cells containing the untranslated sequences of the present invention are also encompassed by the present invention. The introduction of said vectors into a host can be achieved using methods known to those skilled in the art. A DNA molecule of the present invention can be inserted into the genome of a plant by any suitable method. Suitable plant transformation methods include transformation mediated by Agrobacterium, the use of liposomes, electroporation, chemicals that increase the consumption of free DNA, supply of free DNA through bombardment of microprojectiles, transformation using viruses or pollen, etc. The methods to transform in a specific way dicotyledons use mainly Agrobacterium tumefaciens. For example, reported transgenic plants include but are not limited to cotton (patent of E.U.A. No. 5,004,863; patent of E.U.A. No. 5,159,135; patent of E.U.A. ,518,908, WO 97/43430) and soybean (U.S. Patent No. 5,569,834; E.U.A. No. 5,416.01 1). Similarly, a number of transformation and regeneration methods are available for monocots including but not limited to corn (Songstad et al., 1995; Klein et al., 1988); rice (Toriyama et al. 1986) and wheat (Cheng et al 1997, and U.S. Patent No. 5,631, 152 incorporated herein by reference in its entirety). It is evident to those skilled in the art that a number of transformation and regeneration methodologies can be used and modified for production of stable transgenic plants from any number of crops of target interest and methods of plant transformation and regeneration are well known for the expert (for example, see Hinchee et al. (1994), and Ritchie &Hodges (1993) for abstracts). Analyzes for gene expression based on the transient expression of cloned nucleic acid constructs have been developed by introducing the nucleic acid molecules into plant cells by polyethylene glycol treatment, electroporation, or particle bombardment. (Marcotte, et al., (1998), Marcotte, et al., (1989), McCarty, et al., (1991), Hattori, et al., (1992), Goff, et al., (1990). Transient expression systems can be used to rapidly assess gene expression levels and functionally dissect gene constructs (see in general, Mailga et al., (1995).) The present invention also provides plant cells, the genome of which contains one or more recombinant DNA molecules containing a 5 'and / or 3' untranslated sequence described herein The differentiated plants comprising said cells will have the characteristics or benefits provided by the expression of the coding sequence. of DNA which is operably linked to said sequences, said plants may be monocot or dicotyledonous, and may include but are not limited to plants belonging to the selected families of alfalfa, apple, Arabidopsis, barley, breasts, broccoli, squash, citrus, corn, cotton, flax, garlic, lettuce, oats, oilseed turnip, onion, cinnamon, flax, an ornament plant, pea, peanut, pepper, potato, rice, rye, sorghum, strawberry, soy, seed sunflower, cane, sugar beet, tomato, tobacco, wheat, poplar, pine, spruce, eucalyptus, lentil, grape, banana, tea and lawns. Particularly preferred plants include alfalfa, barley, corn, cotton, cinnamon, potato, rice, rye, soybean, sunflower seed, sugar beet and wheat. Even more preferred plants include monocots such as corn, wheat, and rice. The invention will be more readily understood by reference to the following examples which are provided by way of illustration, and are not intended to limit the present invention. The following examples are included to demonstrate examples of certain preferred embodiments of the invention. It should be appreciated by those skilled in the art that the techniques described in the examples that follow represent methods that the inventors have discovered to work well in the practice of the invention, and therefore can be considered to constitute examples of preferred modes for their practice. However, those skilled in the art should appreciate, in light of the present disclosure, that many changes can be made in the specific embodiments described and still obtain an equal or similar result without departing from the spirit and scope of the invention. . In addition to the procedures specifically referred to in this, practitioners are familiar with standard resource materials describing conditions and procedures for the construction, manipulation and isolation of macromolecules (eg, DNA molecules, plasmids, etc.), for the generation of recombinant organisms and for the selection and isolation of clones (see for example, Sambrook et al., (1989); Mailga et al., (1995); Birren et al., (1996).

EXAMPLES EXAMPLE 1 Construction of expression vectors containing combinations of genetic elements for enhanced transgene expression The construction of expression vectors containing cassettes of genetic elements comprising wheat or rice elements was carried out by annealing synthetic oligonucleotides or by PCR isolation from wheat leaf mRNA or rice genomic DNA and site ligation of restriction upstream and downstream, respectively, of the GUS reporter gene. The plasmids used for cloning and construction of the various expression cassettes are listed in Table 1. All the constructs tested had the e35S promoter which is the promoter for 35S RNA from CaMV which contains a duplication of the region -90 to 300. Other elements contained in the plasmids include the following: origins of replication (ori-M13 and ori-V), marker genes such as GUS or LUC which are the coding sequences for beta-glucuronidase and luciferase, respectively, and coding sequences for selection of antibiotics (bacterial selection AMP) and KAN (which confers resistance to aminoglycoside antibiotics of neomycin and kanamycin). Plasmid pMON32648 (Fig. 26) contains an antifungal protein from large canker (Tfe AFP) as described in patent application 60/097150.

Additional typical transformation vectors would include but are not limited to pMON 18364 (Figure 27) which is a double border Agrobacterium transformation vector and pMON19568 (Figure 28) which is a plasmid that is linearized before a transformation method by bombardment of particles. The PCR conditions used were as recommended by the manufacturer (see, for example, Strategene, La Jolla, CA, PE Biosystems, Foster City, CA). The plasmid DNA was isolated and purified using commercially available equipment (see for example Qiagen Valencia, CA). Synthetic DNA was purchased from Midland Certified Reagent Co., Midland, TX).

TABLE 1 CONSTRUCTION OF VECTORS Copstructo (Leader 57lntron / Marker / Terminator 3 ') ** Cloning Sites / genetic element) pMON19469 * none / hsp70 l / GUS / nos 3' (Fig.1) Bglll, Ncol (hsp 70 I) pMON26043 none / GUS / 3 'Bglll, Ncol, Xbal pMON26052 * Ta hsp L hsp 70 l / GUS / nos 3' (Fig. 2) BglII, Ncol (hsp 70 I) pMON25454 vector of rice actintron Stu I / Neo I ( ract 1 1) pMON26044 Ta cab U ractl I / GUS / nos 3 'Ncol / Stu (ract I l). EcoRI / Sma (nos 3') pMON26055 * Ta hsp U ractl I / GUS / nos 3 '(Fig.3) Stu I / Ncol (ractl I) pMON26045 Ta fbp L / GUS / nos 3' Hindlll, Xba, Bglll. Ncol pMON25456 none / ract II / GUS / nos 3 'Hindlll / Bclí (e35S / ract II) pMON26064 ract l 1 / Ta fbp L / GUS / nos 3' ** HindIII, Xba, Bell pMON26054 * Ta cab L / ractl 1 / GUS / nos 3 'Sutl / Ncol (ractl I), see Fig.4 EcoRI / Smal (nos 3') pMON26038 Ta cab L / GUS / nos 3 'Xbal, BglII, Ncol, Pstl, (Pst / Bgllll, nos 3 ') pMON19433 * none / hsp70 I / GUS / nos 3' EcoRI / Bam Hl (nos 3 ') BglIl / EcoRI. see Fig. 5 pMON18375 none / hsp70 I / GUS / Ta hspl 7 3 'EcoRI / Smal (Ta hspl 7 3') pMON32502 * Ta cab U ractl l / GUS / Ta hspl 7 3 'see Fig. 6 pMON32506 * Ta hsp L / ractl I / GUS / Ta hspl 7 3 'see Fig. 7 pMON18377 none / hsp 70 I / GUS / Ta ubiq 3' EcoRI / Sma I (Ta ubiq 3 ') pMON32509 * Ta fbp L / ractl I / GUS / Tab 3 'see Fig.8 pMON32510 * Ta hsp L / ractl I / GUS / Ta ubiq 3' see Fig. 9 pMON 18379 none / hsp70 I / GUS / Ta fbp 3 'pMON32513 * Ta fbp L / ractl I / GUS / Ta fbp 3 'see Fig. 10 pMON19437 * none / hsp 70 I / LUX / nos 3' Ncol / EcoRI (LUX) see Fig. 11 pMON32515 * Ta cab L / ractl I / LUX / Ta hsp 3 'Ncol / EcoRI (LUX) see Fig. 12 pMON32516 * Ta fbp L / ractl I / LUX / Ta ubiq 3 'Ncol / EcoRI (LUX) see Fig. 13 pMON32517 * Ta fbp L / ractl I / LUX / / Ta fbp 3' Ncol / EcoRI (LUX) see Fig. 14 pMON32518 * Ta fbp L / ractl I / LUX / r lac d 3 'see Fig. 15 pMON33210 * none / hsp 70 I / GUS / nos Pmll, Bglll, Xbal (r btubL) see Fig 16 pMON33220 * r btub L / hsp 70 I / GUS / nos 3 'see Fig. 17 pMON26046 Ta per L / none / GUS / nos Bgllll / Ncol (Ta per L) Pstl / Bglll pMON33211 none / r amyl I / GUS / nos 3 'Bglll / Xbal (r amy I) pMON33226 none / r pal I / GUS / nos 3' Bglll / Xba (r pal I) pMON33228 none / r ssl I / GUS / nos 3 'Bglll / Xba (r ssl I) pMON33225 Ta cab L / ractl I / GUS / r glut 3 'EcoRI / Sph (r glut 3') pMON33200 none / hsp 70 I / GUS / nos 3 'Bglll / Pmll PMON33219 * r amy L / hsp 70 I / GUS / see us Fig. 18 PMON47901 * Ta cab U hsp 70 I / GUS / r glut 3 'see Fig. 19 pMON47906 * Ta hsp L / ractl I / GUS / r lac d 3' see Fig.20 pMON47907 * Ta hsp U ractl I / GUS / r glut 3 'see Fig. 21 p MON47915 * Ta per L / ractl I / GUS / r lac d 3' see Fig. 22 PMON47916 * Ta per L / ractl I / GUS / r glut 3 'see Fig 23 PMON47917 * Ta per L / ractl I / GUS / r btub 3 'see Fig. 24 pMON47919 * see Fig. 25 pMON47909 Ta hsp L / ractl I / GUS / r lac d 3' Bglll / Ncol pMON33216 Ta cab U ractl I / GUS / r btub 3 'Stul / Ncol pMON47910 Ta hsp L / ractl I / GUS / r glut 3 'Ncol / BglII (hsp 70 I) pMON47918 Ta per L / hsp 70 I / GUS / r lac d 3' Ncol / BglII (hsp 70 I) pMON47920 Ta per L / hsp 70 I / GUS / r btub BglII / Ncol (hsp 70 I) * Figure ** Different orientation: lntron / leader / marker / 3 ' The intervention sequence from the heat shock protein of corn (intron hsp70) as described in the patents of E.U.A. Nos. 5,593,874 and 5,859,347 incorporated herein by reference) is shown in SEQ ID NO: 47. The synthetic base leader is shown in SEQ ID NO: 45. The 5 'untranslated leader sequence of the wheat mRNA for putative low molecular weight heat shock protein (5' Ta hsp17 L) (Genoteca accession number X13431.gb_pl) (SEQ ID NO: 53) was created by tempering of SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8. The 5 'end of the resulting fragment has a BamHI cohesive end followed by an Xbal restriction site, and the 3' end has a BglII site followed by a Ncol cohesive end for subcloning purposes. This fragment was ligated with the 5,676 kb BglII fragment and the Ncol fragment of pMON19469 (FIG. 1) to create pMON26043. Plasmid pMON 26052 (FIG. 2) was created by subcloning the 884 bp BglII fragment and the Ncol fragment from pMON 19469 (FIG. 1) into the BglII and Ncol sites of pMON26043. pMON26043 contained the HP70 intron and the 5 'leader sequence of wheat (leader Ta hsp). pMON26055 (figure 3) was created by subcloning the 0.449 kb fragment Stul and Ncol containing the intron of rice actin (intron ract 1) (SEQ ID NO: 50) (McEIroy, et al., 1991) of pMON25454 in the BglII and Ncol sites of pMON26043 using adapters to the Stul site that create a Complementary BglII end (SEQ ID NO: 3 and SEQ ID NO: 4).

The untranslated 5 'leader of the wheat mRNA for fructose-1, 6-bisphosphatase (5' Ta fbp L) (Genbank Accession Number) X07780.gb_pl) (SEQ ID NO: 54) was created by tempering SEQ ID NO: 9, SEQ ID NO.10, SEQ ID NO: 11, and SEQ ID NO: 12. The 5 'end of the resulting fragment has a BamHI cohesive end followed by an Xbal restriction site, and the 3' end has a BglII site followed by a Ncol cohesive end for subcloning purposes. This fragment was ligated with the 5,676 kb BglII fragment and the Ncol fragment of pMON 19496 (FIG. 1) to create pMON26045. Plasmid pMON26064 was created by subcloning the 1095 kb Hindlll fragment and the Bell fragment containing the rice actin intron (McEIroy, et al., 1991) and the e35S promoter (Kay et al., 1987) from pMON26045 at the sites Hindlll and Xbal of pMON26045 using adapters to the Bell site that create an end Supplementary Xbal (SEQ ID NO: 13 and SEQ ID NO: 14). pMON26054 (Figure 4) was created by subcloning the 0.449 kb Stul and Ncol fragment containing the rice actin intron (McEIroy et al., 1991) from pMON25454 into the BglII and Ncol sites of pMON20645 using adapters to the Stul site that create a Complementary Bglil end (SEQ ID NO: 3 and SEQ ID NO: 4) The major chlorophyll a / b binding protein of the 5'-untranslated leader (5 'Ta cab L) (Genebank Accession Number) M10144.gb_pl) (SEQ ID NO: 52) was isolated through reverse transcription from wheat leaf RNA followed by 40 cycles of PCR with a denaturing temperature of 94 ° C for 1 minute, a tempering temperature of 50 ° C for 2 minutes, and an extension temperature of 72 ° C for 3 minutes. The primers used SEQ ID NO: 1 and SEQ ID NO: 2 create BamHI and Xbal restriction sites at the 5 'end, and BglII and Ncol restriction sites at the 3' end for subcloning purposes. The 77-base pair fragment containing the 5'-untranslated leader of chlorophyll a / b binding major protein was then digested with BamHI and Ncol and ligated with the fragment of 5.676 kb BglII and Ncol of pMON19469 (FIG. 1) for create pMON26038 Plasmid pMON26044 was created by subcloning the fragment of 0.449 kb Stul and Ncol containing the rice actin ntron (McEIroy, et al., 1991) of pMON25454 at the BglII and Ncol sites of pMON26038 using adapters to the Stul site that create a complementary BglII end (SEQ ID NO: 3 and SEQ ID NO: 4). The 3 'untranslated regions containing the terminator and polyadenylation sequences were isolated and cloned into pMON19433. The plasmid pMON19433 is derived from pUC119 and further contains the improved 35S promoter CAMV, the intron hsp70, the GUS reporter gene, and the untranslated sequence of nopaline synthase (nos) 3 '(SEQ ID NO: 46). This vector contains an EcoRI site and a BamHI site flanking the nos 3 'terminator allowing the removal and replacement of alternating 3' terminator sequences. The cDNA synthesis was performed using an oligo dT primer in the pH regulator of PCR reaction with MgC, 0.2 mM dATP, dCTP, dGTP, and TTP. Five micrograms of total wheat leaf cell RNA were added to the pH regulator of the PCR reaction in a volume of 20 μl. The reaction was initiated by the addition of 4 units of reverse transcriptase (Gibco BRL; Gaithersburg, MD), and incubated at 42 ° C for 2 hours. The reaction was terminated by heat and frozen at -20 ° C. Two microliters of this reaction were added to each pH regulator of PCR reagent containing dNTPs as described above in 100 microliters containing 0.5 units of Taq polymerase according to the manufacturer's specifications (Boerhinger Mannheimm Biochemicals, Indianapolis, IN). The reaction was covered with 50 microliters of mineral oil and cycled in a thermal cycler at 94 ° C for 1 minute, 45 ° C for 2 minutes and 72 ° C for 2 minutes repeatedly for 40 cycles. The 3 'untranslated region of the wheat ubiquitin gene (3' Ta ubiq) (SEQ ID NO: 59) was amplified from the cDNA products with primer sequences SEQ ID NO: 15 and SEQ ID NO: 16. The 3 'untranslated region of the wheat heat shock gene (3' Ta hsp17) (SEQ ID NO: 58) was amplified as described using primer sequences SEQ ID NO: 17 and SEQ ID NO: 18. The 3 'untranslated region of fructose and bisphosphatase (3' Ta fbp) (SEQ ID NO: 60) was amplified as described above using primer sequences SEQ ID NO: 19 and SEQ ID NO: 20. The amplified reaction products were electrophoresed on an agarose gel and analyzed for size. The PCR fragments corresponding to 3 'Ta ubiq (-225 bp), 3' Ta hsp (-240 bp) and 3 'ta fbp (-130 bp) were digested with EcoRi and BamHI, gel purified on a gel 1% agarose and were isolated using the Qiangen PCR preparation according to the manufacturer's specification (Qiagen, Santa Clarita, CA). the digested end PCR fragments were cloned into the 6.5 kb fragment from the base vector pMON 19433 digested with EcoRI BamHI (Figure 5). The resulting transformants with grafts from the 3 'untranslated regions of wheat genes (Ta fbp, Ta hsp, and Ta ubiq) were verified by DNA sequence formation. The expression vector pMON32502 (FIG. 6) was constructed by digestion of pMON26044 and pMON18375 with EcoRI and Smal. The 0.24 kb Ta hsp 3 'fragment from pMON 18375 was ligated with the 6.0 kb fragment of the vector base structure of pMON26044 creating pMON32502. The ligation products were transformed into E. coli DH5 alpha cells by standard procedures, they were plated on LB agar plates containing selective levels of ampicillin (100 μg / ml). The expression vector pMON32506 (FIG. 7) was constructed by digestion of pMON26055 (FIG. 3) and pMON 18375 with EcoRI and Smal. The 0.24 kb Ta hsp 3 'fragment from pMON 18375 was ligated with the 5.9 kb fragment of the vector base structure of pMON26055 (FIG. 3) creating pMON32506. The ligation products were transformed into E.coli DH5 alpha cells by standard procedures. pMON32509 (figure 8) was constructed by digestion of pMON26054 and pMON18377 with EcoRI and Smal. The 0.23 kb Ta ubiq 3 'fragment of pMON 18377 was ligated with the 5.9 kb fragment of base structure of the pMON26054 vector (FIG. 4) creating pMON32509. The ligation products were transformed into E.coli DH5 alpha cells by standard procedures. pMON32510 (figure 9) was constructed by digestion of pMON26055 (figure 3) and pMON18377 with EcoRI and Smal. The 0.23 kb Ta ubiq 3 'fragment from pMON 18377 was ligated with the 5.9 kb fragment of base structure of the pMON26055 vector creating pMON32510 (Figure 9). The ligation products were transformed into E.coli DH5 alpha cells by standard procedures. pMON32513 (figure 10) was constructed by digestion of pMON26054 and pMON 18379 with Ncol and Smal. The 2.0 kb GUS / Ta fbp 3 'sequence fragment from pMON 18379 was ligated with the 4.1 kb fragment of the vector base structure of pMON26054 to create pMON32513. The ligation products were transformed into E.coli DH5 alpha cells by standard procedures. Plasmids pMON32515 (figure 12), pMON32516 (figure 13) and pMON32517 (figure 14) were constructed by digestion of pMON32502, pMON32509, and pMON32513 with Ncol and EcoRI. In each case, the approximate 4.3 kb fragment was then ligated to the 1.8 kb fragment of Ncol luciferase created by a partial digestion with EcoRI of pMON19437 (FIG. 11). The ligation products from these ligations were transformed into E.coli DH5 alpha cells by standard procedures. The 5 'untranslated leader sequence from rice beta-tubulin (5' r btub L) (Genebank Accession Number) L19598.gb_pl) (SEQ ID NO: 56) was created by quination (reaction using kinase T4 polynucleotides to add 5 'phosphates for subsequent ligation steps) and annealing (boiling followed by slow cooling) SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23 and SEQ ID NO: 24. The 5 'end of the resulting fragment had a shaved end Pml I and the 3' end had a BglII cohesive end for subcloning purposes. This fragment was ligated with the 6,507 kb Pmll and BglII fragment of pMON33210 (FIG. 16) to create pMON33220 (FIG. 17). The 5 'untranslated leader sequence from a wheat peroxidase gene (5' Ta per L) (Genebank Accession Number) X56011.gb_pl) (SEQ ID NO: 55) was created by quination and temperate of SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27 and SEQ ID NO: 28. The 5 'end of the resulting fragment had a cogged BglII end followed by an Xbal restriction site, and the 3' end had a BglII site followed by a Ncol cohesive end for subcloning purposes. This fragment was ligated with the fragment of 5.676 kb BglII and Ncol of pMON 19469 (FIG. 1) to create pMON26046. The 5 'untranslated leader sequence from a rice amylase gene (Genebank Accession Number) M24287.gb_pl) (5'r amy L) (SEQ ID NO: 57) was created by tempering SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43 and SEQ ID NO: 44, and phosphorylating and ligating in linearized plasmid of pMON33200 digested with Pml I and Bgl II, to create pMON33219. The first model of the rice amylase gene (r ry amy I) (Genebank Accession Number) X16509.gb_pl) (SEQ ID NO: 49) together with ten base pairs of 5 'and 3 'exon sequence flankers were isolated by PCR from rice genomic DNA (Oryza sativa), using approximately 1 μg of DNA. The amplification was performed using primer sequences SEQ ID NO: 29 and SEQ ID NO: 30. The DNA was denatured for 1 minute at 95 ° C, annealed for 2 minutes at 50 ° C, and extended for 3 minutes at 72 ° C. for a total of 30 cycles. The resulting PCR product was digested with BglII and Xbal and ligated with the 5.687 kb BglII and Xbal fragment of pMON33210 (FIG. 16) to create pMON33211. The first intron of phenylalanine amonia lyase of rice (intron r pal) (Zhu et al, 1995) together with ten base pairs 5 'and 3' flanking exon sequences, (SEQ ID NO: 48) was isolated by rice genomic DNA PCR. Amplification was performed using primer sequences SEQ ID NO. : 31 and SEQ ID NO: 32. The DNA was denatured for 1 minute at 95 ° C, annealed for 2 minutes at 50 ° C, and extended for 3 minutes at 72 ° C for a total of 30 cycles. The resulting PCR product was digested with BglII and Xbal and ligated with the 5.687 kb BglII and Xbal fragment of pMON33210 (FIG. 16) to create pMON33226. The first kernel of the rice sucrose synthase gene (r paltron) (Zhu et al, 1995) together with ten base pairs of 5 'and 3' exon sequence flankers (SEQ ID NO: 51) were isolated by PCR of rice genomic DNA. Amplification was performed using primer sequences SEQ ID NO: 33 and SEQ ID NO: 34. The DNA was denatured for 1 minute at 95 ° C, annealed for 2 minutes at 50 ° C, and extended for 3 minutes at 72 ° C. for a total of 30 cycles. The resulting PCR product was digested with BglII and Xbal and ligated with the 5.687 kb BglII and Xbal fragment of pMON33210 (FIG. 16) to create pMON33228. The 3 'non-translated terminator of rice type II glutelin (3' r glut) (Genebank Accession Number) X05664.gb_pl) (SEQ ID NO: 61) was isolated by PCR from rice genomic DNA. Amplification was performed using primer sequences SEQ ID NO: 35 and SEQ ID NO: 36. The DNA was denatured for 1 minute at 95 ° C, annealed for 2 minutes at 50 ° C, and extended for 3 minutes at 72 ° C. for a total of 30 cycles. The resulting PCR product was digested with Sphl and EcoRI. PMON33225 contains the cloned region Sphl / EcoRI terminator r glut 3 'untranslated. The untranslated 3 'terminator of rice lactate dehydrogenase (3' r lacd) (Genebank Accession Number) D13817.gb_pl) (SEQ ID NO: 62) was isolated by PCR from rice genomic DNA. Amplification was performed using primer sequences SEQ ID NO: 37 and SEQ ID NO: 38. The DNA was denatured for 1 minute at 95 ° C, annealed for 2 minutes at 50 ° C, and extended for 3 minutes at 72 ° C. for a total of 30 cycles. The resulting PCR product was digested with Sphl and EcoRI. PMON33218 (Figure 15) contains the inserted Sphl / EcoRI fragment. The untranslated terminator 3 'of rice beta-tubulin (3' r btub) (Genebank Accession Number) L19598.gb_pl) (SEQ ID NO: 63) was isolated by PCR from rice genomic DNA. Amplification was performed using primer sequences SEQ ID NO: 39 and SEQ ID NO: 40. The DNA was denatured for 1 minute at 95 ° C, annealed for 2 minutes at 50 ° C, and extended for 3 minutes at 72 ° C. for a total of 30 cycles. The resulting PCR product was digested with Sphl and EcoRI. pMON33216 (Figure 13) contains the inserted Sphl / EcoRI fragment. PMON47901 (figure 19) was constructed by ligating the 3,166 kb EcoRI and Pstl fragment from pMON33225, the 0J1 kb Pstl and Bglll fragment from pMON26038, and the 2671 kb BglII fragment and EcoRI from pMON 19433. PMON47906 (figure 20) was constructed ligand the 5 J48 kb Ncol and BglII fragment from pMON47909, the 0.449 kb Stul fragment and Ncol from pMON33216 using adapters to the Stul site that creates a complementary BglII end (SEQ ID NO: 3 and SEQ ID NO: 4). PMON47907 (FIG. 21) was constructed by ligating the 5 J43 kb Ncol and BglII fragment from pMON47910, the 0.449 kb Stul fragment and Ncol from pMON33216 using adapters to the Stul site that creates a complementary BglII end (SEQ ID NO: 3 and SEQ ID NO. : 4). PMON47915 (FIG. 22) was constructed by ligating the 5,758 kb Ncol and BglII fragment from pMON47918, the 0.449 kb Stul fragment and Ncol from pMON33216 using adapters to the Stul site which creates a complementary BglII end (SEQ ID NO: 3 and SEQ ID NO: 4) PMON47916 (figure 23) was constructed by ligating the fragment of 5J53 kb Ncol and BglII from pMON47919 (figure 26) and 0.449 kb Stul and Ncol fragment of pMON33216 using adapters to Stul site which creates a complementary BglII end (SEQ ID NO: 3 and SEQ ID NO: 4). PMON47917 (Figure 24) was constructed by ligating 5,895 kb Ncol and Bglll fragment from pMON47920 and 0.449 kb Stul and Ncol fragment from pMON33216 using adapters to Stul site which creates a complementary BglII end (SEQ ID NO: 3 and SEQ ID NO: 4) PMON47919 (figure 25) was constructed by ligating 3.166 kb EcoRI and Pstl fragment from pMON33225, the 0.726 kb Pstl and BglII fragment from pMON26046 and the 2671 kb BglII fragment and EcoRI from pMON19433 (FIG. 5).

All plasmids were verified by restriction digestion with BglII and tested in transient expression assays in protoplasts derived from wheat Mustang callus or protoplasts derived from corn BMS callus, or were tested as stable integrated constructs in transgenic plants as describe right away.

EXAMPLE 2 Transient transformation and reporter gene expression in Wheat and Maize.

Purified plasmid DNA was prepared by the Qiagen maxi preparation procedure according to the manufacturer's specifications (Qiagen, Valencia, CA). GUS plasmids containing 5 'untranslated leaders or 3' untranslated terminator regions and combinations of 5 'and 3' untranslated regions in vector DNAs and pMON19437 as the internal luciferase control were mixed and electrophoresed. For reverse experiments to examine improvements with additional coding sequences, luciferase was the variable reporter gene. GUS in pMON 19469 was the internal control. The transformations were carried out in duplicate. The elements of the 3 'untranslated region in combination with 5' untranslated leader sequences were also tested in relation to the control base vector pMON19469. The analysis of gene expression in plants has been well documented (Schledzewski et al., 1994, Steinbiss et al., 1991, Stefanov et al., 1991). Protoplast expression assays such as those described herein are often predictive of the expression performance of a recombinant gene in plant cells.

Analysis of gene expression in wheat protoplasts The method used for the isolation and preparation of wheat protoplasts was carried out as described by Zhou et al., 1993. The electrophoresis pH regulator used was as previously described (Li et al., 1995). The culture medium used was MSI WSM (4.4 g Gibco MS / L salts, 1.25 ml Thiamine HCl (0.4 mg / ml), 1 ml 2,4-D (1 mg / ml), 20 g / L sucrose, 0.15 ml Asparagine (15 mg / ml), 0.75 g MgCl2, 109 g 0.6 M mannitol, pH 5.5 Mustang suspensions were used for protoplast isolation approximately four days after subcultivation Briefly, 8 g of wheat cell suspension They were poured into a culture tube and the cells were allowed to settle in. The medium was removed and the remaining cells were resuspended with 40 ml of enzyme solution, transferred to a Petri dish, wrapped in aluminum foil, and incubated at 26 °. C for 2 hours on a spinner at 40 rpm.The suspension was centrifuged at 200g for 8 minutes, washed twice with centrifugation between each wash, resuspended in 10 ml of washing solution and stored on ice.The number of protoplasts was determined and the volume was adjusted to a final concentration of 4x106 protoplasts / ml. They added approximately 0.75 ml of protoplasts to each electrophoresis vessel and added up to about 50 μg of plasmid DNA in 50 μl solution to the protoplasts. The electroforming conditions were 960 μFarrads and 160 volts using a Bio-Rad Gene Pulser pulsator (Bio-Rad Laboratories, Hercules, CA). The samples remained on ice before and during electrophoration. After electrophoration, the samples were left on ice for approximately 10 minutes and then removed and allowed to warm to room temperature for approximately 10 minutes. The electrophoresed cells were then pipetted to MSI WSM medium and incubated in the dark for 18-22 hours at 24 ° C. Cells were harvested by centrifugation at 200-250g for 8 minutes and frozen on dry ice for subsequent analysis for the gene (s) of interest.

Gene Expression Analysis in Maize Protoplasts A transient maize protoplast analysis system was used to evaluate the GUS / LUX expression of the various constructs. The isolation and electrophoresis of corn leaf protoplasts was performed as described by Sheen, 1991 with the following changes: the seeds were sterilized from the surface, germinated in 1 / 2MS medium (2.2 g / L MS salts, 0.25% gelrite), and cultivated 5 days at 26 ° C in photoperiods of 16/8 day / night, 6 days in complete darkness, 26 ° C and 24 hours under the conditions of the first treatment. The second true leaf of each plant was sliced longitudinally and digested for approximately 2 hours in the light at 26 ° C. After digestion, the plates were rotated at 80-100 rpm for 20-30 seconds, and the protoplast / enzyme solution is pipetted through a 190 μm tissue harvester. The protoplasts were counted using a hemacytometer. Bio-Rad Gene (Bio-Rad, Hercules, CA) push-button containers with a 0.4 cm gap and a maximum volume of 0.8 ml were used for electrophores. Ten to 100 μG of plasmid DNA in addition to 5 μg of DNA containing the luciferase gene as an internal control were added to the vessel. The final protoplast densities were from about 3 million per ml to 4.5 million per ml, with the electrophoration values set at a capacitance of 125 μFarad and 200 volts. The protoplasts were incubated on ice after resuspension in pH electrophoresis buffer and remained on ice until 10 minutes after electrophoration. Protoplasts were added to approximately 7 ml of modified MS medium as described in From et al, 1987, with the addition of 0.6M mannitol in petri dishes stratified with the same medium plus 1.5% SeqPlaque agarose (FMC Bioproducts, Rockland, ME ). The protoplasts were harvested by centrifugation 24 hours after electrophoration and used for analysis of subsequent expression of the gene (s) of interest.

GUS activity The GUS activity (ß-glucuronidase) was determined from 25 μl of cell extract according to the methods of Jefferson et al. (1987) using 2 mM MUG (4-methylumbelliferyl-β-D-glucuronide) in the extraction pH regulator described above. Fluorescence was measured using a Hoescht DNA Fluorometer fluorometer (Model TKO 100). A standard curve of methyllumbellilferone (Sigma) was generated using a 1 μM solution.

Luciferase Activity To determine luciferase activity, ten microliters of each crude test protein extract were served on a microtiter plate. Twenty-five microliters of 2X pH buffer (50mM Tricine (pH 7.8), 30mM MgCl2, 10mM ATP, and 0.5mg / mL) were added to each well containing extract. The reactions were initiated by the addition of 25 μl of 10 mM luciferin. The samples were mixed, and then quantified to chemiluminescence of each sample on a Packard TopoCount micro-scintillation plate counter using a countdown delay of 5 minutes and a count time of 0.2 minutes. The results are expressed as a ratio of experimental reporter gene levels to levels of internal control reporter gene. The control plasmid contained a different reporter gene and was used to correct the variability in the transformation and extraction procedures.

TABLE 2 Effect of 5 'untranslated leaders on the expression of GUS in protoplasts of wheat Mustang The effect of 5 'untranslated leaders on GUS expression in wheat Mustang protoplasts was measured using constructs that contained several 5' untranslated leaders. The level of expression for the control plasmid containing the 5 'base-synthetic sequence (SEQ ID NO: 45), the intron hsp 70 (SEQ ID N0.47), and the 3'nos terminator region (SEQ ID NO; 46) was established as 1.0. GUS expression was increased in wheat protoplasts when wheat heat shock protein (Ta hsp), wheat fructose-1, 6-bisphosphatase (Ta fbp), or chlorophyll binding protein a / b was used. of wheat (Ta cab) the 5 'untranslated leader sequences compared to the synthetic base sequences (table 2). The effect was less pronounced in corn BMS protoplasts (Table 3).

TABLE 2 Effect of untranslated 5 'leaders on the expression of GUS in corn BMS protoplasts Table 4 shows GUS results in wheat Mustang protoplasts using constructs containing 3 'untranslated sequences of heat shock protein (3' Ta hsp), wheat fructose-1, 6-bisphosphatase (3 'Ta fbp), or wheat ubiquitin (3 'Ta ubiq) compared to the vector containing the 3' nos region. Each of the 3 'untranslated regions gave improved GUS expression relative to that observed with the non-translated region nos 3'.

TABLE 4 Effect of terminators of 3 'untranslated region on GUS expression in bombarded wheat leaves.

The combined effects of the described 5 'and 3' untranslated sequences were evaluated in wheat Mustang protoplasts. LUX expression was measured in addition to GUS expression to confirm that increased expression levels were not GUS specific. The expression levels of GUS and LUX were increased when using the constructs containing the Ta cab, Ta hsp, or Ta fbp 5 'untranslated terminators and Ta ubiq, Ta fbp, or Ta hsp 3' untranslated terminators (Table 5).

TABLE 5 Effects in combination of 5 'and 3' untranslated terminator sequences on GUS and LUX expression in wheat Mustang protoplasts The combined effects of the 5 'untranslated leader sequences and the 3' untranslated terminator sequences were also measured in corn BMS protoplasts. The expression of GUS increased in general over the base control (table 6). The results observed in corn corroborate those found in wheat, demonstrating that the beneficial effects of the 5 'and 3' untranslated sequences of the invention are not limited to the species from which the untranslated sequences are derived.

TABLE 6 Combination effects of the 5 'and 3 untranslated terminator sequences on GUS expression in corn BMS protoplasts.

EXAMPLE 3 Stable transformation in wheat plants The effects of the 5 'and 3' untranslated sequences on GUS expression were also evaluated in transgenic wheat plants. The process for transformation and regeneration of wheat was as described in U.S. Patent No. 5,631,152, incorporated herein by reference, but modified for selection of G418. In summary, immature embryos were cultured on CM4C medium for 0-4 days (CM4C components: 4.3 g / L of Gibco MS salts, 10 ml / L of vitamins MS (100X), 0.5 ml / L of 2,4-D , 40 g / L maltose, 0.5 g / L glutamine, 0.75 g / L magnesium chloride, 0.1 g / L casein hydrolyzate, 1.95 g / L MES, 2 g / L Phytagel); cultures were transferred to CM4C Raff / Mann medium for approximately 4 days, bombarded and transferred to CM4C containing 25 mg / L G418 for approximately 5 days; cultures were regenerated on MMSO.2C containing G418 (25 mg / L) for approximately 19 days, regenerated on MMSOC containing 25 mg / L G418 for approximately 33 days and rooted on MMSOC containing 25 mg / L G418 for approximately 57 days and were subsequently transferred to land for approximately 75 days. The CM4C medium (G418) contained 2.2 ml / L of 1 mg / ml piclorama, 1 ml / L G418 (25 mg / ml) and 2 ml / L ascorbic acid (50 mg / ml delivery material). The CM4C Raff / Mann 0.25 medium contained the following components: 4.4 g / L MS salts, 10 ml / L of vitamins MS (100X), 0.5 ml / L 2,4-D, 40 g / L maltose, 74.3 g / L raffinose, 22.78 g / L mannitol, 0.5 g / L glutamine, 0.75 g / L magnesium chloride, 1.95 g / L MES, 0.1 g / L casein hydrosylate, 2 g / L Phytagel, 2.2 ml piclorama (1 mg / ml) and 2 ml of ascorbic acid (50 mg / ml). The medium MMS0.2C contained 4.3 g / L MS salts, 1.95 g / L MES, 2 ml / L of MMS vitamins, 0.2 ml / L 2,4-D, 40 g / L maltose and 2 g / L agar (Schweizer Hall). MM20.2C (G418) contained 1 ml of 25 mg / ml G418 and 2 ml of 50 mg / ml ascorbic acid. MMSOC contained 4.3 g / L of MS salts, 1.95 g / L MES, 2.0 ml / L of MMS vitamins and 40 g / L of maltose. MMSOC (G418) had an additional 1 ml of 25 mg / ml G418 and 2 ml of 50 mg / ml ascorbic acid. Transgenic lines were established using the DNA constructs described in Table 7 below and plants were evaluated for GUS activity levels. The relative GUS levels were comparable or larger than the base control vector for many of the constructs containing the non-translated elements of the invention. In particular, constructs containing a 5 'Ta cab or Ta fbp untranslated leader sequence used in combination with a 3' Ta hsp or Ta fbp untranslated terminator sequence, provided the highest expression levels. Another preferred construct contained an untranslated leader sequence Ta fbp 5 'and a terminator region us not translated 3'.

TABLE 7 Effect of non-translated leader 5 'genetic elements and 3' untranslated terminator on stable expression of GUS in transgenic wheat plants All GUS activity values expressed as pmol / min / mg protein. N = number of independent wheat plants analyzed.

The percentage of positive GUS events in transgenic wheat plants was used to determine the effect of 5 'untranslated leader sequences and 3' untranslated terminator on the recovery of stable GUS expression. The constructs that provided high levels of GUS expression in Table 7 also caused a high percentage of positive GUS events (Table 8).

TABLE 8 Effect of genetic elements Leader not translated 5 'and Terminator not translated 3' in the recovery of stable expression of GUS above threshold levels in transgenic wheat plants.

TABLE 9 Effect of 5 'untranslated leaders on the expression of GUS in corn leaf protoplasts TABLE 10 Effect of untranslated leaders 5 'on the expression of GUS in corn leaf protoplasts The effect of several untranslated 5 'leaders on the expression of GUS in corn leaf protoplasts was measured using constructs that contained several untranslated 5 'leaders (tables 9 and 10). GUS expression was increased in corn leaf protoplasts when the 5 'untranslated leader sequences of chlorophyll a / b binding protein (Ta cab), wheat heat shock protein (Ta hsp), peroxidase wheat (Ta per), or rice beta-tubulin (r btub).

TABLE 11 Effect of introns on GUS expression in corn leaf protopiasts The effect of several introns on GUS expression in corn leaf protoplasts was measured using constructs that contained several introns.

GUS expression was increased in maize leaf protoplasts when the first amylase introns (r amyl), the phenylalanine ammonia lyase (r pal), or the first sucrose synthase (ss I) rice were used.

TABLE 11 Effect of terminators of 2 'untranslated region on GUS expression in corn leaf protoplasts The effect of several 3 'untranslated terminators on GUS expression in corn leaf protoplasts was measured using constructs that contained several 3' untranslated terminators. GUS expression was increased in corn leaf protoplasts using 3 'untranslated terminators of rice type II glutelin (r glut), rice lactate dehydrogenase (r lacd), or rice beta-tubulin (r btub) compared to the control construct that contained the non-translated terminator nos 3 '.

TABLE 13 Combination effects of 5 'and 3' untranslated sequences on GUS expression in corn leaf protoplasts TABLE 14 Effects of several leader sequences on GUS expression in corn leaf protoplasts The level of expression enhancement for structural DNAs may vary due to reasons other than the 5 'untranslated leader sequence, the 3' untranslated terminator sequence, or the neutron sequences. These reasons may include transcription processing sites, polyadenylation sites, transcription termination sites, transport signals within the coding region, etc. The same sequence may provide variable expression levels depending on the species in which it is being expressed and the precise composition of the sequence, and may require some degree of routine optimization for better results in different plant species. Certain features and sub-combinations of the present invention can be used without reference to other features and sub-combinations. This is contemplated by and is within the scope of the claims. Because many possible embodiments of the invention can be made without departing from the scope thereof, it is to be understood that all of the subject matter hereof which is set forth or shown in the accompanying drawings is to be construed as illustrative and not as an illustration. limiting sense. All of the compositions and methods described and claimed herein may be made and executed without undue experimentation in light of the present disclosure. Although the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those skilled in the art that variations can be applied to the DNA molecules and in the steps or in the sequence of steps of the methods to be applied. describe in the present without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents that are chemically and physio-logically related can be substituted for the agents described herein while obtaining the same or similar results. All of these similar substitutes and modifications obvious to those skilled in the art are contemplated to be within the spirit, scope and concept of the invention as defined in the appended claims.

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Colleen G < 120 > New Expression Vectors in Plants < 130 > monocot elements < 140 > < 141 > < 150 > 60/097150 < 151 > 1998-08-19 < 160 > 63 < 170 > Patent in Ver. 2.0 < 210 > 1 < 211 > 36 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Artificial sequence description: synthetic < 400 > 1 gatggatcct ctagaaccat cttccacaca ctcaag 36 < 210 > 2 < 211 > 34 < 212 > DNA < 213 > Artificial sequence < 220 > < 223 > Description of Artificial sequence: synthetic < 400 > 2 gatccatggc gcagatctta tggtgtgttg tccc 34 < 210 > 3 < 211 > 13 < 212 > DNA < 213 > Artificial sequence < 220 > < 223 > Description of Artificial sequence: synthetic < 400 > 3 gatccaaggg agg 13 < 210 > 4 < 211 > 9 < 212 > DNA < 213 > Artificial sequence < 220 > < 223 > Description of Artificial sequence: synthetic < 400 > 4 cctcccttg < 210 > 5 < 211 > 48 < 212 > DNA < 213 > Artificial sequence < 220 > < 223 > Description of Artificial sequence: synthetic < 400 > 5 gatcctctag agaattccct tttcctacct acgatccgat accgaatt 48 < 210 > 6 < 211 > 37 < 212 > DNA < 213 > Artificial sequence < 220 > < 223 > Description of Artificial sequence: synthetic < 440 > 6 ttccgagcgc acaagccaaa ccaaagcaag atctgac 37 < 210 > 7 < 211 > 35 < 212 > DNA < 213 > Artificial sequence < 220 > < 223 > Description of Artificial sequence: synthetic < 400 > 7 tcggatcgta ggtaggaaaa gggaattctc tagag 35 < 210 > 8 < 211 > 50 < 212 > DNA < 213 > Artificial sequence < 220 > < 223 > Description of Artificial sequence: synthetic < 400 > 8 catggtcaga tcttgctttg gtttggcttg tgcgctcgga aaattcggta 50 < 210 > 9 < 211 > 49 < 212 > DNA < 213 > Artificial sequence < 220 > < 223 > Description of Artificial sequence: synthetic < 400 > 9 gatcctctag agggccacca ccacggtgcg cgccaagaca aggcagggg 49 < 210 > 10 < 211 > 31 < 212 > DNA < 213 > Artificial sequence < 220 > < 223 > Description of Artificial sequence: synthetic < 400 > 10 agagaaattc gtcaatccgc agcagatctg c 31 < 210 > 11 < 211 > 35 < 212 > DNA < 213 > Artificial sequence < 220 > < 223 > Description of Artificial sequence: synthetic < 400 > 11 gtcttggcgc gcaccgtggt ggtggccctc tagag 35 < 210 > 12 < 211 > 45 < 212 > DNA < 213 > Artificial sequence < 220 > < 223 > Description of Artificial sequence: synthetic < 400 > 12 catggcagat ctgctgcgga ttgacgaatt tctctcccct gcctt 45 < 210 > 13 < 211 > 12 < 212 > DNA < 213 > Artificial sequence < 220 > < 223 > Description of Artificial sequence: synthetic < 400 > 13 gatctacggg gt 12 < 210 > 14 < 211 > 12 < 212 > DNA < 213 > Artificial sequence < 220 > < 223 > Description of Artificial sequence: synthetic < 400 > 14 ctagaccccg ta 12 < 210 > 15 < 211 > 31 < 212 > DNA < 213 > Artificial sequence < 220 > < 223 > Description of Artificial sequence: synthetic < 400 > 15 aggaattcgc tcctggccat ggagctgctt c 31 < 210 > 16 < 211 > 55 < 212 > DNA < 213 > Artificial sequence < 220 > < 223 > Description of Artificial sequence: synthetic < 400 > 16 agggatccaa aaaacacaca cagatctccg ctcacttatt catagttcac caaag 55 < 210 > 17 < 211 > 31 < 212 > DNA < 213 > Artificial sequence < 220 > < 223 > Description of Artificial sequence: synthetic < 400 > 17 aggaattctg catgcgtttg gacgtatgct c 31 < 210 > 18 < 211 > 56 < 212 > DNA < 213 > Artificial sequence < 220 > < 223 > Artificial sequence description: synthetic < 400 > 18 agggatccaa aaaacacaca cagatctaat tccttttttt ttgcactcaa aatcag 56 < 210 > 19 < 211 > 32 < 212 > DNA < 213 > Artificial sequence < 220 > < 223 > Description of Artificial sequence: synthetic < 400 > 19 aggaattcaa caagaacgag ggagggatac ac 32 < 210 > 20 < 211 > 54 < 212 > DNA < 213 > Artificial sequence < 220 > < 223 > Description of Artificial sequence: synthetic < 400 > 20 agggatccaa aaaacacaca cagatctctt gacctcacaa tccaattgga attc 54 < 210 > 21 < 211 > 35 < 212 > DNA < 213 > Artificial sequence < 220 > < 223 > Description of Artificial sequence: synthetic < 400 > 21 gtgtccaccc acccctcgat ctctcgctcg ccgcc 35 < 210 > 22 < 211 > 43 < 212 > DNA < 213 > Artificial sequence < 220 > < 223 > Description of Artificial sequence: synthetic < 400 > 22 gccgatcgga tcgcgtggtt ggatcatcac aactcggcaa aga 43 < 210 > 23 < 211 > 43 < 212 > DNA < 213 > Artificial sequence < 220 > < 223 > Description of Artificial sequence: synthetic < 400 > 23 cgatcggcgg cggcgagcga gagatcgagg ggtgggtgga cac 43 < 210 > 24 < 211 > 38 < 212 > DNA < 213 > Artificial sequence < 220 > < 223 > Description of Artificial sequence: synthetic < 400 > 24 gatctctttg ccgagttgtg atgatccacc acgcgatc 38 < 210 > 25 < 211 > 56 < 212 > DNA < 213 > Artificial sequence < 220 > < 223 > Description of Artificial sequence: synthetic < 400 > 25 gatcctctag aaccaccaca ccactccacc agtaagaagt gcagcaggta gctagt 56 < 210 > 26 < 211 > 39 < 212 > DNA < 213 > Artificial sequence < 220 > < 223 > Description of Artificial sequence: synthetic < 400 > 26 aagccggcgt agctttgctc ttgcagctag agatctaac 39 < 210 > 27 < 211 > 42 < 212 > DNA < 213 > Artificial sequence < 220 > < 223 > Description of Artificial sequence: synthetic < 400 > 27 tgctgcactt cttactggtg gagtggtgtg gtggttctag ag 42 < 210 > 28 < 211 > 53 < 212 > DNA < 213 > Artificial sequence < 220 > < 223 > Description of Artificial sequence: synthetic < 400 > 28 catggttaga tctctagctg caagagcaaa gctacgccgg cttactagct acc 53 < 210 > 29 < 211 > 32 < 212 > DNA < 213 > Artificial sequence < 220 > < 223 > Description of Artificial sequence: synthetic < 400 > 29 tagtagagat ctcctgtttc aggtaagaga te 32 < 210 > 30 < 211 > 32 < 212 > DNA < 213 > Artificial sequence < 220 > < 223 > Description of Artificial sequence: synthetic < 400 > 30 tagtagtcta gaagttgaat ccctgcatca te 32 < 210 > 31 < 211 > 29 < 212 > DNA < 213 > Artificial sequence < 220 > < 223 > Description of Artificial sequence: synthetic < 400 > 31 tagtagagat ctgagctcat caggtgagg 29 < 210 > 32 < 211 > 32 < 212 > DNA < 213 > Artificial sequence < 220 > < 223 > Description of Artificial sequence: synthetic < 400 > 32 tagtagtcta gaccgggatt gaggaatctg ce 32 < 210 > 33 < 211 > 31 < 212 > DNA < 213 > Artificial sequence < 220 > < 223 > Description of Artificial sequence: synthetic < 400 > 33 tagtagagat ctccaccatt gggtatgttg c 31 < 210 > 34 < 211 > 35 < 212 > DNA < 213 > Artificial sequence < 220 > < 223 > Description of Artificial sequence: synthetic < 400 > 34 tagtagtcta gaatttcagg aactgcaaag aaagg 35 < 210 > 35 < 211 > 31 < 212 > DNA < 213 > Artificial sequence < 220 > < 223 > Description of Artificial sequence: synthetic < 400 > 35 tagtaggaat tcgttggcaa tgcggataaa g 31 < 210 > 36 < 211 > 33 < 212 > DNA < 213 > Artificial sequence < 220 > < 223 > Description of Artificial sequence: synthetic < 400 > 36 tagtaggcat gcccataaga taagggaggg ttg 33 < 210 > 37 < 211 > 29 < 212 > DNA < 213 > Artificial sequence < 220 > < 223 > Description of Artificial sequence: synthetic < 400 > 37 tagtaggaat tctaaatctt attattatc 29 < 210 > 38 < 211 > 31 < 212 > DNA < 213 > Artificial sequence < 220 > < 223 > Description of Artificial sequence: synthetic < 400 > 38 tagtaggcat gctcgacaat aagtacttgt c 31 < 210 > 39 < 211 > 31 < 212 > DNA < 213 > Artificial sequence < 220 > < 223 > Description of Artificial sequence: synthetic < 400 > 39 tagtaggaat tcggtggctt ttgcttggtg g 31 < 210 > 40 < 211 > 31 < 212 > DNA < 213 > Artificial sequence < 220 > < 223 > Description of Artificial sequence: synthetic < 40D > 40 tagtaggcat gcaagatcca tatgcctata g 31 < 210 > 41 < 211 > 40 < 212 > DNA < 213 > Artificial sequence < 220 > < 223 > Description of Artificial sequence: synthetic < 400 > 41 gtgatccatc atctacaaga gatcgatcag tagtggttag 40 < 210 > 42 < 211 > 47 < 212 > DNA < 213 > Artificial sequence < 220 > < 223 > Description of Artificial sequence: synthetic < 400 > 42 gttgctgcta accactactg atcgatctct tgtagatgat ggatcac 47 < 210 > 43 < 211 > 41 < 212 > DNA < 213 > Artificial sequence < 220 > < 223 > Description of Artificial sequence: synthetic < 400 > 43 cagcaactca ctatcgaaca cggtttcagc ttacacagat a 41 < 210 > 44 < 211 > 38 < 212 > DNA < 213 > Artificial sequence < 220 > < 223 > Description of Artificial sequence: synthetic < 400 > 44 gatctatctg tgtaagctga aaccgtgttc gatagtga 38 < 210 > 45 < 211 > 29 < 212 > DNA < 213 > Artificial sequence < 220 > < 223 > Description of Artificial sequence: synthetic < 400 > 45 cacgctgaca agctgactct agcagatct 29 < 210 > 46 < 211 > 253 < 212 > DNA < 213 > Agrobacterium tumefaciens < 400 > 46 gatcgttcaa acatttggca ataaagtttc ttaagattga atcctgttgc cggtcttgcg 60 atgattatca tataatttct gttgaattac gttaagcatg taataattaa catgtaatgc 120 atgacgttat ttatgagatg ggtttttatg attagagtcc cgcaattata catttaatac 180 gcgatagaaa acaaaatata gcgcgcaaac taggataaat tatcgcgcgc ggtgtcatct 240 253 atc atgttactag < 210 > 47 < 211 > 804 < 212 > DNA < 213 > Zea mays < 400 > 47 accgtcttcg gtacgcgctc actccgccct ctgcctttgt tactgccacg tttctctgaa 60 tgctctcttg tgtggtgatt gctgagagtg gtttagctgg atctagaatt acactctgaa 120 atcgtgttct gcctgtgctg attacttgcc gtcctttgta gcagcaaaat atagggacat 180 aacgaagata ggtagtacga agcaatacga gaacctacac gaaatgtgta atttggtgct 240 tatttaagca tagcggtatt catgttggtg ttatagggca cttggattca gaagtttgct 300 gttaatttag gcacaggctt catactacat gggtcaatag tatagggatt catattatag 360 gcgatactat aataatttgt tcgtctgcag agcttattat ttgccaaaat tagatattcc 420 tattctgttt ttgtttgtgt gctgttaaat tgttaacgcc tgaaggaata aatataaatg 480 acgaaatttt gatgtttatc tctgctcctt tattgtgacc ataagtcaag atcagatgca 540 cttgttttaa atattgttgt ctgaagaaat aagtactgac agtattttga tgcattgatc 600 tgcttgtttg ttgtaacaaa atttaaaaat aaagagtttc ctttttgttg ctctccttac 660 ctcctgatgg tatctagtat ctaccaactg acactatatt gcttctcttt acatacgtat 720 cttgctcgat gccttctccc tagtgttgac cagtgttact cacatagtct ttgctcattt cattgtaatg cagataccaa GCGG 780 804 < 210 > 48 < 211 > 149 < 212 > DNA < 213 > Oryza sativa < 400 > 48 gatctgagct catcaggtga ggattaggat tccaaataag cgataacgtt tacctggtca 60 ctgcgattag ttcagtttac tgtgaaattc tttggaccct tcttaattat aaatttgctt 120 gttttctcgg cagattcctc aatgccggt 149 < 210 > 49 < 211 > 128 < 212 > DNA < 213 > Oryza sativa < 400 > 49 gatctcctgt ttcaggtaag agatcgccat gagttgggtt tcaggcttca gtgaactgat 60 cgggttttgt actgagccta agagaatgat gcagtgatgc tcttgtgttt gatgatgatg 120 cagggatt 128 < 210 > 50 < 211 > 491 < 212 > DNA < 213 > Oryza sativa < 400 > fifty cctccgccgc cgccggtaac caccccgccc ctctcctctt tctttctccg tttttttttc 60 cgtctcggtc tcgatctttg gccttggtag tttgggtggg cgagaggcgg cttcgtgcgc 120 gcccagatcg gtgcgcggga ggggcgggat ctcgcggctg gggctctcgc cggcgtggat 180 ccggcccgga tctcgcgggg aatggggctc tcggatgtag atctgcgatc cgccgttgtt 240 gggggagatg atggggggtt taaaatttcc gccgtgctaa acaagatcag gaagagggga 300 aaagggcact atggtttata tttttatata tttctgctgc ttcgtcaggc ttagatgtgc 360 tagatctttc tttcttcttt ttgtgggtag aatttgaatc cctcagcatt gttcatcggt 420 agtttttctt ttcatgattt gtgacaaatg cagcctcgtg cggagctttt ttgtaggtag 480 aagtgatcaa 491 <; 210 > 51 < 211 > 1186 < 212 > DNA < 213 > Oryza sativa < 400 > 51 ccaccattgg gtatgttgct tccattgcca aactgttccc ttttacccat aggctgattg 60 atcttggctg tgtgattttt tgcttgggtt tttgagctga ttcagcggcg cttgcagcct 120 cttgatcgtg gtcttggctc gcccatttct tgcgattctt tggtgggtcg tcagctgaat 180 cttgcaggag tttttgctga catgttcttg ggtttactgc tttcggtaaa tctgaaccaa 240 gaggggggtt tctgctgcag tttagtgggt ttactatgag cggattcggg gtttcgagga 300 aaaacctcaa aaaccggcaa atcctcgacc tttagttttg ctgccacgtt gctccgcccc 360 attgcagagt tctttttgcc cccaaa ttt tttttacttg gtgcagtaag aatcgcgcct 420 cagtgatttt ctcgactcgt agtccgttga tactgtgtct tgcttatcac ttgttctgct 480 taatcttttt tgcttcctga ggaatgtctt ggtgcctgtc ggtggatggc gaaccaaaaa 540 'tgaagggttt tttttttttg aactgagaaa aatctttggg tttttggttg gattctttca 600 tggagtcgcg accttccgta ttcttctctt tgatctcccc gcttgcggat tcataatatt 660 cggaacttca tgttggctct gcttaatctg tagccaaatc ttcatatctc cagggatctt 720 tcgctctgtc ctatcggatt taggaattag gatctaactg gtgctaatac taaagggtaa 780 tgccattata tttggaacca attttgcaaa gtttgagata atctcaatga tgccatcggt 840 tacttactaa aacc caacaa atccatttga taaagctggt tcttttatcc ctttgaaaac 900 attgtcagag tatattggtt caggttgatt tattttgaat cagtactcgc actctgcttc 960 gatgctttca gtaaaccata gttgtgtaga tgaaacagct gtttttagtt atgttttgat 1020 cttccaatgc ttttgtgtga tgttattagt gttgatttag catggctttc ctgttcagag 1080 atagtcttgc aatgcttagt gatggctgtt gactaattat tcttgtgcaa gtgagtggtt 1140 ttggtacgtg ttgctaagtg taacctttct ttgcagttcc tgaaat 1186 < 210 > 52 < 211 > 71 < 212 > DNA < 213 > Triticum aestivum < 400 > 52 gatcctctag aaccatcttc cacacactca agccacacta ttggagaaca cacagggaca 60 acacaccata to 71 < 210 > 53 < 211 > 66 < 212 > DNA < 213 > Artificial sequence < 220 > < 223 > Description of Artificial sequence: synthetic < 400 > 53 gaattccctt ttcctaccta cgatcagata ccgaattttc cgagcgcaca agccaaacca 60 aagcaa 66 < 210 > 54 < 211 > 68 < 212 > DNA < 213 > Artificial sequence < 220 > < 223 > Artificial sequence description: synthetic < 400 > 54 tctagagggc caccaccacg gtgcgcgcca agacaaggca ggggagagaa attcgtcaat 60 ccgcagca 68 < 210 > 55 < 211 > 82 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of Artificial sequence: synthetic < 400 > 55 tctagaacca ccacaccact ccaccagtaa gaagtgcagc aggtagctag taagccggcg 60 tagctttgct cttgcagcta ga 82 < 210 > 56 < 211 > 70 < 212 > DNA < 213 > Artificial sequence < 220 > < 223 > Description of Artificial sequence: synthetic < 400 > 56 tccacccacc cctcgatctc tcgctcgccg ccgccgatcg gatcgcgtgg ttggatcatc 60 acaactcggc 70 < 210 > 57 < 211 > 74 < 212 > DNA < 213 > Artificial sequence < 220 > < 223 > Description of Artificial sequence: synthetic < 400 > 57 atccatcatc tacaagagat cgatcagtag tggttagcag caactcacta tcgaacacgg 60 tttcagctta poop 74 < 210 > 58 < 211 > 234 < 212 > DNA < 213 > Triticum aestivum < 400 > 58 aattctgcat gcgtttggac gtatgctcat tcaggttgga gccaatttgg ttgatgtgtg 60 tgcgagttct tgcgagtctg atgagacatc tctgtattgt gtttctttcc ccagtgtttt 120 ctgtacttgt gtaatcggct aatcgccaac agattcggcg atgaataaat gagaaataaa 180 ttgttctgat tttgagtgca aaaaaaaagg aattagatct gtgtgtgttt ttg 234 < 210 > 59 < 211 > 231 < 212 > DNA < 213 > Triticum aestivum < 400 > 59 aattcgctcc tggccatgga gctgcttctg tctctgggtt cacaagtctc ggtgtctccg 60 gtatcctcca atggagtctg gtctgtgtct gtcgttgcct gactgtcttt gtttctgtac 120 tgcagtgtta catactgtga tcgtttgtat cttcaaactt ctgctggtgt ggagcagctt 180 tggtgaacta tgaataagtg agcggagatc tgttgtgtgt tttttggatc c 231 < 210 > 60 < 211 > 131 < 2? 2 > DNA < 213 > Triticum aestivum < 400 > 60 aattcaacaa gaacgaggga gggatacaca ggctgtttct tccaagaaat tattgtaact 60 aatatataat gtagcccttt tcttgtgatg cggaaaatat atttgaagaa ttccaattgg 120 attgtgaggt c 131 < 210 > 61 < 211 > 236 < 212 > DNA < 213 > Oryza sativa < 400 > 61 ctaagttggc aatgcggata aagaataact aaataaataa ataaataaat tgcaagcaat 60 tgcgttgctg ctatgtactg taaaagtttc ttataatatc agttctgaat gctaaggaca 120 tccctcaaga tggtctttct atttttgtgt tcccgttcca atgtactgtt cgtatcctct 180 tggagattca tcaatatgag aaaacagaga atggacaacc ctcccttatc ttatgg 236 < 210 > 62 < 211 > 241 < 212 > DNA < 213 > Oryza sativa < 400 > 62 ttctaaatct tattattatc atcgtcgtcg tcgtctcgtc acggaattaa ttaaagtacc 60 tactccgtac ttagctagct acaataataa ggattcattg atcactacaa gagtgatcga 120 ctcgactgta gtatgtgtgt gcaatataat gtgctgtcta tcaacaacta ctagtattgt 180 catttttttc gaaccaggga actttttaat gataagaaga aaaagacaag tacttattgt 240 c 241 < 210 > 63 < 211 > 381 < 212 > DNA < 213 > Oryza sativa < 400 > 63 attcggtggc ttttgcttgg tggttctagg gcagggtttt gtgtgcttgg tgtttccgtc 60 ttacattatc accgtattac cgcctcgtac gccaccgccg gttcctatgt cttcgctttg 120 ttttttcgtc tgtgctatgg gaaccttttt gggtactgta ttacttgatg ctggtctgcg 180 attgttgata tttcgggatg aattttacct ttccgcgttg gtcctcgtgt gtaatatttg 240 caaattacgg aactaggaag gtagcccgcg cattcgcgtg ggcatgtatc gtaggctgta 300 tcgtaagtaa tttgagataa taggctgatt gtgttaaaat gttgcatttg ttatatagta 360 381 t aactataggc atatggatct

NOVELTY OF THE INVENTION CLAIMS