MXPA00009202A - Plant promoter sequences and methods of use thereof - Google Patents

Abstract

The invention relates to nucleic acid sequences isolated from sugarcane and to methods of using them. In particular, the invention relates to nucleotide sequences which are derived from sugarcane polyubiquitin genes and which are capable of directing constitutive expression of a nucleic acid sequence of interest that is operably linked to the sugarcane polyubiquitin nucleotide sequences. The sugarcane polyubiquitin nucleotide sequences are useful in regulating expression of a nucleic acid sequence of interest in monocotyledonous and dicotyledonous plants.

Description

PROMOTING SEQUENCES OF PLANTS AND METHODS OF USING THEMSELVES • This work was done with the support of the government by the 5 United States Department of Agriculture. The Government of the United States has certain rights in this invention.

FIELD OF THE INVENTION The invention relates to nucleic acid sequences isolated from sugar cane, and to methods for using them. In particular, the invention relates to nucleotide sequences which are derived from sugar cane polyubiquitin genes and which are capable of directing the constitutive expression of a sequence of The nucleic acid of interest that is operably linked to the nucleotide sequences of sugar cane polyubiquitin. The sugarcane polyubiquitin nucleotide sequences are useful for regulating the expression of a nucleic acid sequence of interest in monocotyledonous and dicotyledonous plants. BACKGROUND OF THE INVENTION Much scientific effort has been directed to engineered plants to produce proteins agronomically important. The recombinant genes for Si Producing proteins in plants requires a promoter sequence which is capable of directing the expression of proteins in plant cells. Promoter sequences that direct high levels of • protein expression in plant cells are particularly desirable, since very few numbers of transgenic plants need to be produced and sorted to recover plants that produce agronomically significant amounts of the target protein. In addition, high levels of protein expression help the generation of plants that exhibit phenotypic properties commercially important, such as pest resistance and • diseases, resistance to environmental stress (for example, high water load, drought, heat, cold, light intensity, day length, chemical products, etc.), improved qualities (for example, high fruit production, life Extended storage, form and uniform color of fruit, higher sugar content, higher content of vitamins C and A, lower acidity, etc.). Some promoter sequences that are capable of directing • expression of transgenes (eg, selectable marker genes) in plants are known in the art and are derive from a variety of sources such as bacteria, plant DNA viruses, and plants. Promoters of bacterial origin include the octopine synthase promoter, the nopaline synthase promoter and other promoters derived from native Ti plasmids. Promoters of viral origin include promoters of 35S and 19S RNA of the promoter mosaic virus. Plant promoters include the small subunit promoter, ribulose-1, 3-diphosphate carboxylase, the phaseolin promoter, and the corn polyubiquitin promoter. • Although some promoter sequences that function in plant cells are available, the expression of more than one gene (eg, a selectable marker gene and an agronomically important gene), which is operatively linked to the same promoter sequence is likely to be hindered by silence, dependent on the homology, transgenes in plants. In this way, what is needed are • Additional promoter sequences, which are capable of directing transgene expression. In particular, what is needed are promoter sequences that sanitize the expression of the transgene in both monocotyledonous and dicotyledonous plant cells. 15 COMPENDIUM OF THE INVENTION The present invention provides nucleic acid sequences that have promoter activity. The acid sequences The nucleic acids provided herein direct the expression of operably linked nucleotide sequences in cells, tissues and organs of monocotyledonous and dicotyledonous plants. In one embodiment, the invention provides a substantially purified nucleic acid sequence comprising a sequence of nucleotide selected from the group consisting of SEQ ID NO: 1, * complement of SEC I D NO: 1, homologs of SEC I D NO: 1, complement homologs of SEC I D N O. 1; S EC I D NO: 3, the complement of SEQ ID NO: 3, and complement counterparts of SEC f ID NO: 3. In a preferred embodiment, the nucleotide sequence is characterized as having promoter activity. In a highly preferred embodiment, the promoter activity is constitutive. In another embodiment, the nucleotide sequence has a double-stranded structure. In yet another embodiment, the nucleotide sequence is single chain structure. In another alternative embodiment, the f 10 n -nucleic acid sequence is contained in a plant cell. In a preferred embodiment, the plant cell is derived from a monocotyledonous plant. In a highly preferred embodiment, the monocotyledonous plant is selected from the group consisting of sugarcane, corn, sorghum, pineapple, rice, barley, oats, wheat, rye, sweet potato, onion, banana, coconut, dates, and hops. In an alternative highly preferred embodiment, the plant cell is derived from a monocot plant. In a preferred embodiment, the dicotyledonous plant is selected from the group consisting of tobacco, tomato, soybean and papaya. The invention also provides a substantially purified nucleic acid sequence comprising a portion of a nucleotide sequence selected from the group consisting of SEQ ID NO: 1, and its complement. In a preferred embodiment, the portion is characterized as having promoter activity. In a In a highly preferred embodiment, the promoter activity is constitutive. In a preferred alternative embodiment, the portion is of double chain structure. In another alternative preferred embodiment, the portion is of individual chain structure. In another preferred modality • Alternatively, the portion comprises the nucleotide sequence selected from the group consisting of nucleotides 1 to 242 of SEQ ID NO: 7, 245 to 787 of SEQ ID NO 7, 788 to 1020 of SEQ ID NO: 7, from 1021 to 1084 of SEQ ID NO: 7, from 1085 to 1168 of SEQ ID NO: 7, from 1169 to 1 173 of SEQ ID NO: 7, from 1174 to 1648 of SEQ ID NO: 7, from 1649 to 1805 of SEQ ID NO: 1, from 1 to 378 of SEQ ID NO: 1, of 379 to 444 of SEQ ID NO: 1, and 445 to 1810 of SEQ ID NO: 1. In • an additional preferred alternative embodiment, the nucleic acid sequence is contained in a plant cell. In a highly preferred embodiment, the plant cell is derived from a monocotyledonous plant. In another highly preferred embodiment, the plant The monocot is selected from the group consisting of sugar cane, corn, sorghum, pineapple, rice, barley, oats, wheat, rye, sweet potato, onion, banana, coconut, date, and hops. In another highly preferred embodiment, the plant cell is derived from a dicotyledonous plant. In a still very preferred mode, the plant The dicotyledone is selected from the group consisting of tobacco, tomato, jackfruit and papaya. The invention further provides a substantially purified nucleic acid sequence comprising a portion of a nucleotide sequence selected from the group consists of SEC I D NO: 3 and its complement. In a preferred embodiment, the portion is characterized as having a promoter activity. In a highly preferred embodiment, the promoter activity is constitutive. In an alternative preferred embodiment, the portion is • double chain structure. In another alternative preferred embodiment, the portion is of individual chain structure. In another alternative preferred embodiment, the portion comprises the nucleotide sequence selected from the group consisting of nucleotides 1 to 3600 of SEQ ID NO: 10, 3602 to 3612 of SEQ ID NO: 10, 3614 to 3691 of SEQ ID NO: 3, from 1 to 2248 of SEQ ID NO: 3, from 2249 to 2313 of SEQ ID NO 3, 2314 to 3688 of SEQ ID NO: 3, and 1671 to 2248 of SEQ ID NO: 3. In another alternative preferred embodiment, the nucleic acid sequence is contained in a plant cell. In a highly preferred embodiment, the plant cell is derived from a monocotyledonous plant. In a very modality Preferred, the monocotyledonous plant is selected from the group consisting of sugar cane, corn, sorghum, pineapple, rice, barley, oats, wheat, rye, sweet potato, onion, banana, coconut, dates and hops. In an alternative highly preferred embodiment, the plant cell is derived from a dicotyledonous plant. In a modality still Most preferred, the dicot plant is selected from the group consisting of tobacco, tomato, soybean and papaya. The invention further provides a substantially purified nucleic acid sequence comprising the EcoRI / Xbal fragment isolated from the plasmid pubi4-GUS contained in Escherichia coli deposited as NRRLB-301 15, the fragment complement, fragment homologs, and fragment complement homologs. In a preferred embodiment, the nucleotide sequence is SEC I D NO: 7. In a very modality • preferred, the nucleotide sequence is characterized by having a promoter activity. In another highly preferred embodiment, the promoter activity is constitutive. In an even more preferred alternative mode, the nucleotide sequence is of double stranded structure. In another highly preferred alternative embodiment, the nucleotide sequences are of individual chain structure. In other most preferred alternative mode, the nucleic acid sequence • is contained in a plant cell. In another preferred embodiment, the plant cell is derived from a monocotyledonous plant. In a highly preferred embodiment, the monocotyledonous plant is selected from the group consisting of sugarcane, corn, sorghum, pineapple, rice, barley, oats, wheat, rye, sweet potato, onion, banana, coconut, dates and hops. In an alternative preferred embodiment, the plant cell is derived from a dicotyledonous plant. In another very preferred embodiment, • the dicotyledonous plant is selected from the group consisting of tobacco, tomato, soybean and papaya. Also provided herein is a substantially purified nucleic acid sequence comprising the fragment of H ind lll / Xbal isolated from the plasmid pubi9-G US contained in Escherichia coli cells deposited as N RRLB-301 16, the complement of the fragment, fragment homologs and homologs of the fragment supplement. In a preferred embodiment, the nucleotide sequence is SEC I D NO: 10. In an alternative preferred embodiment, the nucleotide sequence is characterized as having a promoter activity. In another highly preferred embodiment, • promoter activity is constitutive. In a preferred alternative embodiment, the nucleotide sequence is double stranded. In another alternative preferred embodiment, the nucleotide sequence is of individual chain structure. In another alternative preferred embodiment, the nucleic acid sequence is contained in a plant cell. In a highly preferred embodiment, the plant cell 10 is derived from a monocot plant. In a still highly preferred embodiment, the monocotyledonous plant is selected from the group consisting of sugarcane, corn, sorghum, pineapple, rice, barley, oats, wheat, rye, sweet potato, onion, banana, coconut, dates and hops. In another highly preferred embodiment, the cell of plant is derived from a dicotyledonous plant. In another preferred embodiment, the dicot plant is selected from the group consisting of tobacco, tomato, soybean and papaya. The invention further provides a substantially purified nucleic acid sequence comprising a portion of the EcoRI / Xbal fragment isolated from the pubi4-GUS plasmid contained in Escherichia coli cells deposited as NRRLB-301 15 and the fragment complement. In a preferred embodiment, the portion is characterized as having a promoter activity. In a highly preferred embodiment, the promoter activity is constitutive. In a In the preferred embodiment, the portion is of double chain structure. In another preferred alternative modality, the portion is of individual chain structure. In another alternative preferred embodiment, the nucleic acid sequence is contained in a plant cell. In a highly preferred embodiment, the plant cell is derived from a monocotyledonous plant. In another highly preferred embodiment, the monocotyledonous plant is selected from the group consisting of sugarcane, corn, sorghum, pineapple, rice, barley, wheat oats, rye, sweet potato, onion, banana, coconut, dates and hops. In an alternative preferred embodiment, the plant cell is derived from a dicotyledonous plant. In another highly preferred embodiment, the dicot plant is selected from the group consisting of tobacco, tomato, soybean and papaya. The present invention also provides a substantially purified nucleic acid sequence which comprises a portion of the Hindlll / Xbal fragment isolated from the pubi9-GUS plasmid contained in Escherichia coli cells deposited as N RRLB-301 16, and the fragment complement. In a modality Preferred Wp, the portion is characterized as having promoter activity. In a highly preferred embodiment, the promoter activity is constitutive. In another preferred embodiment, the portion is of double chain structure. In another preferred embodiment, the portion is of individual chain structure. In another preferred embodiment, the nucleic acid sequence is contained in a plant cell. In a highly preferred embodiment, the plant cell is derived from a monocotyledonous plant In a still highly preferred embodiment, the monocotyledonous plant is selected from the group consisting of sugarcane, corn, sorghum, pineapple, rice, barley, oats, wheat, rye, sweet potato, onion, banana, coconut, date and hops. In a • Alternately preferred mode, the plant cell is derived from a dicotyledonous plant. In a highly preferred embodiment, the dicot plant is selected from the group consisting of tobacco, tomato, soybean and papaya. The invention further provides a recombinant expression vector comprising a nucleotide sequence selected from the group consisting of SEQ ID NO: 1, the • complement of SEQ ID NO: 1, homologs of SEQ ID NO: 1, homologs of the complement of SEQ ID NO: 1; SEQ ID NO: 3, the complement of SEQ ID NO: 3, homologs of SEQ ID NO: 3, and homologs of the complement of SEQ ID NO: 3. In a modality Preferred, the recombinant expression vector is selected from the group consisting of pubi4-GUS, pubi9-GUS, 4PI-GUS and 9PI-GUS. The invention further provides a recombinant expression vector comprising a portion of a sequence of • nucleotides selected from the group consisting of SEQ ID NO: 1 and its complement. A recombinant expression vector comprising a portion of a nucleotide sequence selected from the group consisting of SEQ ID NO: 3 and its complement is also provided herein. In the present invention there is also provided a cell of * - ^ ~ * ~ * s «- **"> transgenic plant comprising a nucleic acid sequence comprising a nucleotide sequence selected from the group consisting of SEQ ID NO: 1, the complement of SEQ ID NO: 1 , • homologs of SEQ ID NO: 1, complement homologs of SEC I D 5 NO: 1; SEQ ID NO: 3, the complement of SEQ ID NO: 3, homologs of SEQ ID NO: 3, and complement homologs of SEQ ID NO: 3, wherein the nucleotide sequence is operably linked to a nucleic acid sequence of interest. In a preferred embodiment, the transgenic plant cell expresses the sequence of nucleic acid of interest. In a very preferred embodiment, the • expression is constitutive. In an alternative embodiment, the transgenic plant cell is derived from a monocotyledonous plant. In a highly preferred embodiment, the monocotyledonous plant is selected from the group consisting of sugar cane, corn, sorghum, pineapple, rice, barley, oats, wheat, rye, sweet potato, onion, banana, coconut, dates and hops. In another alternative embodiment, the transgenic plant cell is derived from a dicotyledonous plant. In • a highly preferred embodiment, the dicotyledonous plant is selected from the group consisting of tobacco, tomato, soybean and papaya. In other Alternatively, the nucleic acid sequence of interest is a sense sequence. In a highly preferred embodiment, the sense sequence encodes a protein selected from the group consisting of β-glucuronidase, luciferase, β-galactosidase, 1-aminocyclopropane-1-carboxylic acid deaminase, sucrose phosphate, 5-enolpyruvyl-3-phosphoshikimate synthase, -tth I I I I acetolactate synthase, RNase, wheat germ agglutinin, sweetener protein, and toxin proteins of Bacillus thuringiensis crystal. In an additional alternative mode, the nucleic acid sequence of interest is an antisense sequence. In a highly preferred embodiment, the antisense sequence is selected from the group consisting of an antisense sequence for ACC synthase, for sequences capable of ethylene induction, and for polyphenol oxidase. The invention further provides a transgenic plant cell comprising a nucleic acid sequence comprising a portion of a nucleotide sequence selected from the group consisting of SEQ ID NO: 1 and its complement. A plant cell is also provided herein Transgenic comprising a nucleic acid sequence comprising a portion of a nucleotide sequence selected from the group consisting of SEQ ID NO: 3 and its complement f. In addition, the present invention provides a method for expressing a nucleic acid sequence of interest in a plant cell, comprising: a) providing: i) a plant cell; ii) a nucleic acid sequence of interest; and iii) a nucleotide sequence selected from the group consisting of SEQ ID NO: 1, the complement of SEQ ID NO: 1, homologues of SEC I D NO: 1, complement homologs of SEQ ID NO1; SEQ ID NO: 3, the -. t? * ír í í í í í complemento complemento complemento complemento complemento complemento complemento complemento complemento complemento complemento complemento complemento complemento hom complemento hom hom hom hom SEQ ID NO: 3, SEQ ID NO: 3 homologs, and SEQ ID NO: 3 complement homologs; b) operably linking the nucleic acid sequence of interest to the sequence of • nucleotides to produce a transgene; and c) introducing the transgene into the plant cell to produce a cell of transgenic plants under conditions such that the nucleic acid sequence of interest is expressed in the transgenic plant cell. In a preferred embodiment, the method further comprises: d) identifying the transgenic plant cell. In another preferred embodiment, the method further comprises: d) regenerating the tissue of the transgenic plant from the transgenic plant cell. In an alternative preferred embodiment, the method further comprises: d) regenerating a transgenic plant from the transgenic plant cell. In another preferred embodiment, the plant cell is derived from a monocot plant. In another highly preferred embodiment, the monocotyledonous plant is selected from the group consisting of sugar cane, corn, sorghum, pineapple, rice, barley, oats, wheat, rye, sweet potato, onion, banana, coconut, dates and hops. In another highly preferred alternative embodiment, the plant cell is derived from a dicotyledonous plant. In a still highly preferred embodiment, the dicotyledonous plant is selected from the group consisting of tobacco, tomato, soybean and papaya. The invention further provides a method for expressing a nucleic acid sequence of interest in a plant cell, which comprises: a) providing: i) a plant cell; ii) a nucleic acid sequence of interest; and iii) a portion of a nucleotide sequence selected from the group consisting of SEQ ID NO: 1 and its complement; b) operatively link the acid sequence • nucleic of interest to the portion of a nucleotide sequence for producing a transgene; and c) introducing the transgene into the plant cell to produce a transgenic plant cell under conditions such that the nucleic acid sequence of interest is expressed in the transgenic plant cell. In a preferred embodiment, the portion comprises the nucleotide sequence selected from group consisting of nucleotides 1 to 242 of SEC I D NO: 7, of • 245 to 787 of SEQ ID NO: 7, 788 to 1020 of SEQ ID NO: 7, 1021 to 1084 of SEQ ID NO: 7, 1085 to 1 168 of SEQ ID NO: 7, from 1 169 to 1 173 of SEQ ID NO: 7, from 1 174 to 1648 of SEQ ID NO: 7, from 1649 to 1 805 of SEQ ID NO: 1, from 1 to 378 of SEQ ID NO: 1, from 379 to 444 of SEC I D NO: 1, and 445 to 1810 of SEQ ID NO: 1. The present invention also provides a method for expressing a nucleic acid sequence of interest in a cell • plant, which comprises: a) providing: i) a plant cell; ii) a nucleic acid sequence of interest; and iii) a portion of a The nucleotide sequence selected from the group consisting of SEQ ID NO: 3 and its complement; b) operably linking the nucleic acid sequence of interest to the portion of a nucleotide sequence to produce a transgene; and c) introducing the transgene into the plant cell to produce a low transgenic plant cell conditions so that the nucleic acid sequence of interest is expressed in the transgenic plant cell. In a preferred embodiment, the portion comprises the nucleotide sequence selected from the group consisting of nucleotides from 1 to 3600 • of SEQ ID NO.10, 3602 to 3612 of SEQ ID NO: 10, 3614 to 3691 of SEQ ID NO: 3, 1 to 2248 of SEQ ID NO: 3, 2249 to 2313 of SEQ ID NO: 3, from 2314 to 3688 of SEQ ID NO: 3, and from 1671 to 2248 of SEQ ID NO: 3.

BRIEF DESCRIPTION OF THE DRAWINGS 10 • Figure 1 shows an RNA gel stain of hybridized sugar cane polyubiquitin mRNA combinations in specific gene probes for Scubi 221, Scubi 51 1, Scubi 241, Scubi 561, and Scubi 5121 . Figure 2 shows a "Northern Reverse" stain of sugar cane polyubiquitin mRNA combinations. Figure 3 shows the nucleotide sequence of the ubi4 gene of sugar cane polyubiquitin. Figure 3A shows the nucleotide sequence (SEQ ID NO: 1) including the start codon of translation and its upstream sequences. Figure 3B shows the nucleotide sequence (SEQ ID NO: 2) including the translation stop codon and its downstream sequences. The bold letters indicate the putative TATA box, the ATG translation initiation codon, the TAA translation stop codon, and the signal of putative poly-A addition; the underscore indicates the UTRs 5 'and 3' ___ _ ^ _? _ _ ^ _ ____ t-_. putative, based on the homology of cDNAs scubi241 and 51 1; lower case letters indicate that the base is as it is represented, but with some indeterminacy (see also Tables 1 and 2 below).

• Figure 4 is a diagrammatic representation of the initially determined organization of the polyubiquitin genes ubi4 and ubi9. The white boxes listed indicate repeats of polyubiquitin coding, the white boxes not listed are 5 'and 3' untranslated regions, the black boxes are introns, and the lines are flanking DNA, E: EcoRI; N: 10 Nrul; S: Sa / l; H: Hindl ll; M: repeating transposon element • inverted putative miniature. Figure 5 shows the nucleotide sequence (SEQ ID NO: 5) (nucleotides 1 to 5512 of the nucleotide sequence 5551 deposited as GenBank with accession number AF093504) (A) and the sequence of translated amino acid (SEQ ID NO: 6) (B) of the ubiquitous polyubiquitin sugar cane gene. Figure 6 is a diagrammatic representation of the j organization of polyubiquitin genes ubi4 (accession number of GenBank AF093504) and ub19 (GenBank accession number) AF093505). The white boxes listed indicate repetitions of polyubiquitin coding, the white boxes not listed are 5 'and 3' untranslated regions, the black boxes are introns, and the lines are flanking DNA. E: EcoRI; N: Nrul; S: Sa / l; H: Hindlll; M: repeating transposon element inverted putative miniature. k_a-_M_ _M __ * a _ ^ _ ^ _ ^ -_ ^ __ ^^ a Figure 7 shows the nucleotide sequence of the ubi9 gene of polyubiquitin from sugar cane. Figure 7A shows the nucleotide sequence (SEQ ID NO: 3) including the codon of • start of translation and its upstream sequences. Figure 7B 5 shows the nucleotide sequence (SEC I D NO.4) including the translation stop codon and its downstream sequences. The bold letters indicate the putative TATA box, the ATG translation start codon, the TAA translation stop codon, and the putative poly-A addition signal; the underlined indicates the UTRs 5 'and 10 3'; lower case letters indicate that the base is probably as represented (see also Tables 4 and 5 below). Figure 8 shows the nucleotide sequence (SEQ ID NO: 8) (accession number GenBank AF093505) (A) and the translated amino acid sequence (SEQ ID NO: 9) (B) of the ubiquitous 15 polyubiquitin gene. sugar cane. Figure 9 is a graph showing the GUS activity after the transient expression of GUS under the control of the ubi4 promoter of sugarcane (ubi4), the ubi9 promoter of sugarcane (ubi9), and the ubil promoter of corn (mzubi I) in suspension cells of sugarcane (A, B) and tobacco leaves (C, D). The controls did not receive any DNA. The letters inside the parentheses indicate less important difference levels, that is, values of the average GUS activity with the different letters being significantly different at a confidence level of 25 P < 0.05. ii_t__t_I _-__.

Figure 10 shows the nucleotide sequence (SEQ ID NO: 7) of the ubi4 gene portion, which was ligated to the gene encoding β-glucuronidase (GUS) in the pubi4-GUS and 4PI-GUS plasmids. SEC ID • NO: 7 corresponds to nucleotides 1 -1802 of SEQ ID NO: 5. Figure 11 shows the nucleotide sequence (SEQ ID NO: 10) of the portion of the ubi9 gene that was linked to the gene encoding β-glucuronidase (GUS) in the pubi9-GUS and 9PI-GUS plasmids. SEQ ID NO: 10 corresponds to nucleotides 1 -3688 of SEQ ID NO: 8. Figure 12 is a graph showing the activity of GUS 10 after stable expression in cane lines of sugarcane • of GUS under the control of the ubi9 promoter of sugar cane (sc ubi9) (A, D), the ubi4 promoter of sugar cane (sc ubi4) (B, D), and the ubil promoter of corn (C, D) ). Figure 13 is a graph showing the gene activity of GUS report on stable transgenic rice key lines expressed under the control of the ubi9 sugarcane promoter in the presence and absence of the putative nuclear matrix (MAR) binding region. Figure 14 shows the nucleotide sequence (SEC I D 20 NO: 1 1) encoding polyphenol oxidase (accession number GenBank S40548). Figure 15 shows the nucleotide sequence (SEC I D NO: 2) encoding the corn sucrose phosphate synthase enzyme (accession number GenBank m97550). 25 ^ ¡Ggj | ¡yj ^ DEFINITIONS To facilitate the understanding of the invention, below • A number of terms are defined. The term "transgenic" when used with reference to a cell, refers to a cell that contains a transgene, or whose genome has been altered through the introduction of a transgene. The term "transgenic" when used with reference to a tissue or a plant, refers to a tissue or plant, respectively, the Which comprises one or more cells that contain a transgene, or whose genome has been altered through the introduction of a transgene. Transgenic cells, tissues and plants can be produced through several methods, including the introduction of a "transgene" comprising a nucleic acid "usually DNA" to a Target cell or the integration of the transgene into a chromosome of a target cell through the intervention of the human being, such as through the methods described herein. The term "transgene" as used herein, refers to any nucleic acid sequence, which is introduced into the genome of a cell through experimental manipulations. A transgene can be an "endogenous DNA sequence", or a "heterologous DNA sequence" (ie, "foreign DNA"). The term "endogenous DNA sequence" refers to a nucleotide sequence, which is naturally found in the cell in where it is introduced as long as it does not contain any modification _., ..-. i ** ^ *. - * - _., _ «. , ni ,,, (for example, a point mutation, the presence of a selectable marker gene, etc.) in relation to the sequence of natural existence. The term "heterologous DNA sequence" is Refers to a sequence of nucleotides, which is linked to, or is manipulated to be linked to, a nucleic acid sequence to which it is not naturally bound, or to which it is linked at a different site by nature. The heterologous DNA is not endogenous to the cell in which it is introduced, but has been obtained from other cells. The heterologous DNA also includes a sequence of endogenous DNA, which includes some modification. Generally, although not necessarily, the heterologous DNA encodes RNA and proteins that are not normally produced by the cell where it is expressed. Example of heterologous DNA include report genes, transcriptional and translational regulation sequences, proteins selectable markers (eg, proteins that confer drug resistance), etc. The term "foreign gene" refers to any nucleic acid (eg, gene sequence), which is introduced into the genome of a cell through experimental manipulations and can include gene sequences found in that cell provided that the introduced gene contains some modification (eg, a point mutation, the presence of a selectable marker gene, etc.) in relation to the naturally occurring gene. The term "transformation" as used herein, is refers to the introduction of a transgene in a cell. The transformation of a cell can be stable or transient. The term "transient or" transiently transformed "refers to the introduction of one or more transgenes into a cell in • Absence of integration of the transgene in the genome of the host cell. Transient transformation can be detected, for example, through the enzyme-linked immunosorbent assay (ELISA), which detects the presence of a polypeptide encoded by one or more of the transgenes. Alternatively, the transient transformation can be detected by detecting the activity of the f 10 protein (e.g., β-glucuronidase) encoded by the transgene (e.g., the uid A gene) as demonstrated herein [e.g., histochemical assay of GUS enzyme activity by dyeing with X gum that provides a blue precipitate in the presence of the GUS enzyme; and a chemiluminescent assay of activity of the GUS enzyme using the GUS-Light kit (Tropix)]. The term "passenger transformant" refers to a cell that has transiently incorporated one or more transgenes. In • contrast, the term "stable transformation" or "stably transformed" refers to the integration and introduction of one or more transgenes in the genome of a cell. Stable transformation of a cell can be detected through hybridization of Southern staining of the genomic DNA of the cell with nucleic acid sequences, which are capable of binding to one or more of the transgenes. Alternatively, the stable transformation of a The cell can also be detected through the reaction of - -i ff -r "polymerase chain of the genomic DNA of the cell to amplify transgen sequences The term" stable transformant "refers to a cell that has stably integrated one or more transgenes into • the A genomic DN. In this manner, a stable transformant is distinguished from a passenger transformant in that, whereas the genomic DNA of the stable transformant contains no or more transgenes, the genomic DNA of the passenger transformant does not contain a transgene. The term "nucleotide sequence of interest" refers to any nucleotide sequence, the manipulation of which may be considered desirable for any reason (eg, confer improved qualities), by one skilled in the art. Said nucleotide sequences include, but are not limited to, structural gene coding sequences (e.g. report, selection marker genes, oncogenes, drug resistance genes, growth factors), and uncoded regulatory sequences, which do not encode an mRNA or protein product (eg, promoter sequence, polyadenylation sequence, sequence of completion, improving sequence, etc. ). The term "isolated" when used in relation to a nucleic acid, as in an "isolated nucleic acid sequence" refers to a nucleic acid sequence that is identified and separated from at least one contaminating nucleic acid with which is ordinarily associated in its natural source. The n-nucleic acid ---- > ** • - - - isolated is the nucleic acid present in a form or binding that is different from that in which it is found by nature In contrast, the non-isolated nucleic acids are nucleic acids • such as DNA and RNA, which are in the state that 5 exist by nature. For example, a given DNA sequence (eg, a gene) is located on the chromosome of the host cell near surrounding genes; RNA sequences, such as a specific mRNA sequence that encodes a specific protein, are found in the cell as a mixture of numerous other mRNAs, which encode a multitude of ^ * w 'proteins. However, an isolated nucleic acid sequence comprising SEQ ID NO: 1, includes, by way of example, said nucleic acid sequences in cells ordinarily containing SEQ ID NO: 1, wherein the nucleic acid sequence is in a chromosomal or extrachromosomal location different from that of natural cells, and is otherwise flanked by a different nucleic acid sequence than that found by F nature. The isolated nucleic acid sequence may be present in a single chain structure form or double chain structure. When an isolated nucleic acid sequence is to be used to express a protein, the nucleic acid sequence will contain at least a portion of the sense or coding strand structure (ie, the nucleic acid sequence may be of chain structure individual) Alternatively, it may contain both sense and antisense strand structures (ie, the nucleic acid sequence may be of double strand structure). As used in the present, the term "purified" is F refers to molecules, either nucleic or amino acid sequences, that are removed from their natural environment, isolated or separated. An "isolated nucleic acid sequence", therefore, is a purified nucleic acid sequence. "Substantially purified" molecules are at least 60% free, preferably at least 75% free, and most preferably at least 90% free from other F 10 components with which they naturally associate. As used herein, the terms "complementary" or "complementarity" are used in reference to related nucleotide sequences through base pair rules. For example, the sequence 5'-AGT-3 'is complementary to the sequence 5'-ACT-3 '. Complementarity can be "partial" or "total". "Partial" complementarity is where one or more nucleic acid bases do not match according to the rules of base pairs. The F "total" or "complete" complementarity between nucleic acids is where each nucleic acid base matches another base under the rules of base pairs. The degree of complementarity between the nucleic acid strand structures has important effects on the efficiency and resistance of hydridation between nucleic acid strand structures. A "complement" of a nucleic acid sequence as used herein, refers to a nucleotide sequence whose _i -__- tU? _ -_a -___- UKteAl Nucleic acids show total complementarity to the nucleic acids of the nucleic acid sequence. The term "homology" when used in relation to patients • Nucleic, refers to a degree of complementarity. There may be partial homology (ie, partial identity) or complete homology (ie, complete identity). A partially complementary sequence is one that at least partially inhibits a sequence completely complementary to the hybridization of an objective nucleic acid and is referred to to use the term Functional "substantially homologous". The inhibition of hybridization of the sequence completely complementary to the target sequence can be examined using a hybridization assay (Southern or Northern staining, solution hybridization, and the like) under conditions of low stringency. A sequence substantially The homologous or probe (i.e., an oligonucleotide that is capable of hydridizing another oligonucleotide of interest) will compete and inhibit the binding (i.e., hybridization) of a completely homologous sequence to a target under conditions of low stringency. This does not mean that conditions of low severity are such that allows the union or specific; Low severity conditions require that the binding of two sequences to each other be a specific (eg, selective) interaction. The absence of non-specific binding can be proven through the use of a second objective that lacks a partial degree of complementarity (for For example, less than about 30% identity); in the absence of non-specific binding, the probe will not hybridize to the second non-complementary target. When used with reference to an acid sequence • double-stranded structure nucleic such as a cDNA or genomic clone, the term "substantially homologous" refers to any probe that can hybridize to either or both of the strand structures of the double-stranded structure nucleic acid sequence under conditions of low severity as described below. • When used with reference to a nucleic acid sequence of individual chain structure, the term "substantially homologous" refers to any probe that can hybridize to the nucleic acid sequence of individual chain structure under conditions of low stringency as it was described above. The term "hybridization" as used herein, includes "any process by which a chain structure of • nucleic acid binds with a complementary strand structure through base pairs. "[Coombs J. (1994) Dictionary of Biotechnology, Stockton Press, New York, NY]. Hybridization and hybridization resistance (ie, the strength of the association between nucleic acids) is impacted by factors such as the degree of complementarity between the nucleic acids, severity of the conditions involved, the Tm of the hybrid formed, and the ratio of G: C within the nucleic acids.

As used herein, the term "Tm" is used with reference to the "melting temperature". The melting temperature is the temperature at which a population of acid molecules • nucleic double-stranded structure is half dissociated in individual chain structures. The equation for calculating the Tm of nucleic acids is well known in the art. As indicated by standard references, a simple value of the value of Tm can be calculated through the equation: Tm = 81.5 + 0.41 (% G + C), where a nucleic acid is in aqueous solution in 1 NaCl [ see, f 10 for example, Anderson and Young, Quantitative Filter Hybridization, in Nucleic Acid Hybridization (1985)]. Other references include more sophisticated computations, which take structural characteristics as well as sequence into account for the calculation of Tm. The conditions of low severity when used with Reference to nucleic acid hybridization, comprise equivalent conditions for binding or for hybridization at 68 ° C in a solution consisting of 5X SSPE (43.8 g / l NaCl, 6.9 g / l • NaH2PO4? 2O and 1.85 g / l EDTA, pH adjusted to 7.4 and NaOH), 1% SDS, 5X Denhardt's reagent [50X of Denhardt contains the following per 500 ml: 5 g of Ficoll (type 400, Pharmacia), 5 g of BSA (Fraction V, Sigma)] and 100 μg / ml of denatured salmon sperm DNA followed by washing in a solution comprising 0.2X SSPE, and 0.1% SDS at room temperature when a DNA probe of about 1000 to about 1000 nucleotides in length, is employed.

High severity conditions when used with reference to nucleic acid hybridization comprise equivalent conditions for binding or hydridation at 68 ° C in a solution consisting of 5X SSPE, 1% SDS, 5X Denhardt's reagent and 100 μg / ml of denatured salmon sperm DNA, followed by washing in a solution comprising 0.1 X SSPE, and 0.1% SDS at 68 ° C when a probe of about 100 to about 1000 nucleotides in length is employed. The term "equivalent" when referring to a hybridization condition as it refers to a hybridization condition of interest, means that the hybridization condition and the hybridization condition of interest result in the hybridization of nucleic acid sequences, which have the same percentage scale of homology. For example, if a hybridization condition of interest results in the hybridization of a first nucleic acid sequence with other nucleic acid sequences having from about 50% to 70% homology to the first nucleic acid sequence, then another hybridization condition it is said to be equivalent to the hybridization condition of interest if this or another hybridization condition also results in the hybridization of the first nucleic acid sequence with the other nucleic acid sequences having 50% to 70% homology with the first nucleic acid sequence.

When used with reference to nucleic acid hybridization, the technique knows very well that they can be used __-_ b__a --_ a_ numerous equivalent conditions to understand severe conditions both low and high; factors such as the length and nature (DNA, RNA, base composition) of the probe and • the nature of the target (DNA, RNA, base composition, present in non-immobilized solution, etc.) and the concentration of salts and other components (eg, the presence or absence of formamide, dextran sulfate, polyethylene glycol) consider and the hybridization solution can be varied to generate hybridization conditions either low or high severity f 10 different from, but equivalent to the conditions listed above. Those skilled in the art know that while higher severities may be preferred to reduce or eliminate non-specific binding between the nucleotide sequences SEQ ID Nos: 1 and 10 and the other nucleic acid sequences, lower severities may be preferred. to detect a larger number of nucleic acid sequences having different homologies to the nucleotide sequence of SEQ ID Nos: 1 and 10. The term "promoter", "promoter element" or "promoter sequence" as used herein , refers to a DNA sequence that when ligated to a nucleotide sequence of interest is capable of controlling the transcription of the nucleotide sequence of interest to mRNA. A promoter is typically (though not necessarily, located 5 '(ie, upstream) of a nucleotide sequence of interest whose mRNA transcription controls it, and provides a site for specific binding via RNA polymerase and others. transcription factors for the initiation of transcription. ^^ Promoters may be specific in the tissue or specific in cells. The term "tissue-specific" as applied to a promoter, refers to a promoter that is capable of directing the selective expression of a nucleotide sequence of interest to a specific type of tissue (e.g., petals) in relative absence of the expression of the same sequence of nucleotide of interest in a different type of tissue (for example, • estate). The tissue-specific character of a promoter can be evaluated, for example, by operatively linking a reporter gene to the promoter sequence to generate a report construct, by entering the report construction into the genome of a plant.

Thus, the report construction is integrated into each tissue of the resulting transgenic plant, and detect the expression of the report gene (e.g., detecting mRNA, protein, or the activity of a f protein encoded by the report gene) in different tissues of the transgenic plant. The detection of a higher level of expression of The reporter gene in one or more tissues in relation to the level of expression of the reporter gene in other tissues, shows that the promoter is specific for tissues where higher levels of expression are detected. The term "specific cell type" as applied to a promoter, refers to a promoter that is direct the selective expression of a nucleotide sequence of interest in a specific cell type in the relative absence of expression of the same nucleotide sequence of interest in a different type of cell within the same tissue. The term "cell type "Specific" when applied to a promoter also means a promoter capable of promoting the selective expression of a nucleotide sequence of interest in a region within a single tissue.The cell-type specificity of a promoter can be analyzed using methods well known in the art, for example, immunohistochemical staining In summary, the tissue sections are embedded in paraffin, and the paraffin sections are reacted with a primary antibody, which is specific for the encoded polypeptide product. by the nucleotide sequence of interest whose expression is controlled by the promoter A labeled secondary antibody (eg, peroxidase) The conjugate, which is specific for the primary antibody, is allowed to attach to the excised tissue and the specific binding is detected through a microscope (e.g., avidin / biotin). The promoters can be constitutive or regulable. The term "constitutive" when referring to a promoter, means that the promoter is capable of directing the transcription of an operably linked nucleic acid sequence in the absence of a stimulus (e.g., heat shock, chemicals, light, etc.). Typically, constitutive promoters are capable of directing the expression of a transgene substantially in any cell and any tissue. In contrast, an "adjustable" promoter is - - ^ b- U- _ ^ _ d_- one that is capable of directing a level of transcription of an operably linked nucleic acid sequence in the presence of a stimulus (e.g., heat shock, chemicals, • light), which is different from the level of transcription of the nucleic acid sequence 5 operably linked in the absence of the stimulus. The terms "infect" and "infection" with a bacterium refer to the co-incubation of an objective biological sample (e.g., cell, tissue, etc.) with the bacterium under conditions such that the nucleic acid sequences contained therein. of the bacteria are introduced into one or more cells of the sample • biological objective. The term "Agrobacterium" refers to a phytopathogenic, rod-shaped, Gram-negative bacterium that is born in the earth, which causes the formation of bile. The term "Agrobacterium" includes, but is not limited to, strains of Agrobacterium tumefaciens, which typically cause bile in infected plants), and Agrobacterium rhizogens, (which causes the disease of hairy roots in infected host plants). The infection of a plant cell with Agrobacterium usually gives As a result, the production of opinas (for example, nopalina, agropina, octopina, etc.) through the infected cell. In this way, the Agrobacterium strains that cause the production of nopaline (for example, strain LBA4301, C58, A208) are referred to as "nopalin-like" agrobacteria; Agrobacterium strains, Those which cause the production of octopine (for example, strain LBA4404, Ach5, B6) are referred to as an Agrobacteria; of "octopi na type"; and Agrobacterium strains, which cause the production of agropin (ie strain EHA105, EHA101.A281) • they are called Agrobacteria of "agropina type". 5 The terms "bombard", "bombardment", and "biolistic bombardment" refer to the process for accelerating the particles towards an objective biological sample (e.g., cell, tissue, etc.) to effect the healing of the cell membrane. a cell in the target biological sample and / or the entry of the particles to the objective biological sample. The methods for the biolistic bombardment • are well known in the art (eg, US patent 5, 584, 807, the contents of which are incorporated herein by reference), and are commercially available (eg, the microprojectile accelerator operated with helium gas) PDS-15 1000 / He) (BioRad). The term "micro-healing" when referring to plant tissue refers to the introduction of macroscopic wounds into that tissue. The formation of microherides can be achieved, for example, through particle bombardment as is described herein. The term "plant" as used herein, refers to a plurality of plant cells, which are greatly differentiated to a structure that is present at any stage of plant development. These structures include, but not limit to, a fruit, a button, stem, leaf, petal of flowers, etc. He The term "plant tissue" includes differentiated and undifferentiated tissues of plants, including, but not limited to, roots, buttons, leaves, pollen, seeds, tumor tissue and various types of plants. • cells in culture (eg, individual cells, protoplasts, embryos, calluses, protocormo bodies, etc.). The plant tissue may be in plant, organ culture, tissue culture or cell culture.

DESCRIPTION OF THE INVENTION 10 • The present invention provides nucleic acid sequences having promoter activity. The nucleic acid sequences provided herein direct constitutive expression of nucleotide sequences operably linked in cells, tissues and organs of monocotyledonous and dicotyledonous plants. The promoter sequences provided herein were discovered by classifying highly expressed j sugar polyubiquitin genes. The sequences of the invention are capable of directing the expression of nucleotide sequences operably linked in plant cells at a level that is comparable to or greater than those levels expressed under the control of prior art corn polyubiquitin promoter sequences. The present invention also provides methods for the constitutive expression of a nucleic acid sequence of interest in plants. Nucleic acid sequences and methods of the -_-____ * -____-- U_t _-_ - _ á _ ^ _ a_ invention allow the generation of transgenic plants that exhibit agronomically desirable characteristics. The invention is further described under (A) sequences • promoters of sugar cane polyubiquitin, (B) the use of probes to identify and isolate homologs of promoter sequences from sugarcane, (C) the use of primers to amplify the nucleotide sequences, and (D) generation of transgenic plants.

A. Sugar Cane Polyubiquitin Promoter Sequences • The implication of ubiquitin has been shown in protein potency, heat shock response and many other important cellular processes (Hershko et al., Annual Review of Biochemistry 61 -808 (1992)]. essential biological, the structure of the protein is very highly conserved in all eukaryotes [Callis et al., Genetics 139, 921 -39 (1995); Callis et al., Oxford Surv Plant Mol. Cell Biol. 6, 1 -f 30 (1989); Sun et al., Plant J 1 1, 1017-27 (1997)]. Many genes that encode ubiquitin contain several numbers of repeats in tandem of the entire protein coding region and, therefore, are called polyubiquitin genes. The primary translation product is a polyprotein, which is processed to form post-translational ubiquitin monomers. The ubiquitin protein is abundant throughout the body of the plants, and it has been shown that several polyubiquitin genes are expressed in most or all cell types under most or all environmental conditions [Kawalleck et al., Plant Mol Biol 21, 673-84 (1993)]. However, numerous genes of • polyubiquitin are expressed in a specific manner in tissue 5 [Callis et al. Proc Nati Acad Sci U. S.A. 91, 6074-7 (1994); Pelase et al., Mol Gen Genet 254, 258-66 (1998)], or in response to environmental signals such as thermal stress [Christensen et al., Plant Molecular Biology 12, 619-632 (1989); Liu et al., Biochem Cell Biol 73, 19-30 (1995)], or both [Almoguera et al., Plant 10 Physiology 107, 765-773 (1995); Binet et al., Plant Mol. Biol 17, • 395-407 (1991): Burke et al., Mol Gen Genet 213, 435-43 (1988); Garbarino et al., Plant Mol Biol 20, 235-44 (1992); Genschik et al., Gene 148, 195-202 (1994); Sun and others (1997) supra; Takimoto et al., Plant Mol Biol 26, 1007-12 (1994)]. Several polyubiquitin promoters have been isolated and have been used to direct the expression of the transgene [Christensen et al., Plant Mol Biol 18, 675-89 (1992); Garbarino et al., Plant Physiology 109, 1371-1378 • (1995)], including some that have been widely used for the constitutive expression of genes in transformation of plants [Christensen et al., Transgenic Research 5, 213-218 (1996); Gallo-Megher et al., Plant Cell Repoter 12, 666-670 (1993); Garbarino et al. (1995) supra; Taylor et al., Plant Cell Reports 12, 491-495 (1993); Quail et al., Patents of E.U.A. Nos. 5,614,399 and 5,510,474]. 25 All Saccharum species are polyploid and most They are at least octoploid (2N = 40-128 or more). The conventional sugarcane crops are all hybrids of the Saccharum species derived from the transmission of chromosome 2N + N • [Sreenivasan and others: Cytogenics, ln: Heinz DJ (ed) Sugarcane 5 Improvement Through Breeding, p. 21 1 -253. Elsevier, Amsterdam (1998)]. This suggests that sugarcane hybrids can have at least twelve alleles for each of the multiple genes that make up the polyubiquitin gene family. The nucleic acid sequences of the invention were discovered during a search by the inventors of promoters that are suitable for the expression of high level constitutive transgene in monocotyledonous and dicotyledonous plants. The inventors isolated five clones of polyubiquitin cDNA (scubi221, 241, 51 1, 561 and 5121) from the stem tissue of the cane. sugar [Albert et al., Plant Physiology 109, 337 (1995)]. Based on the comparison of their 3 'untranslated sequences, the inventors then grouped these 5 genes into four "subfamilies". The research of the inventors of the expression of the two members of these four subfamilies and the isolated genomic clones, led to the isolation of clones containing promoters for two members of the most highly expressed subfamily. The invention provides the nucleic acid sequence of two members of the sugar cane polyubiquitin family, the ubi4 gene and the ubi9 gene. With reference to the ubi4 gene, the acid sequence Initially determined nucleic acid (SEQ ID NO. 1) of the ubi4 gene including the translation start codon (ATG) and the upstream sequence of the translation initiation codon are shown in FIG. 3A, and the nucleic acid sequence (SEQ. ID NO: 2) of the ubi4 gene including the translation stop codon and sequences downstream of the translation stop codon is shown in Figure 3B. The subsequently determined nucleic acid sequence (SEQ ID NO: 5) of the entire ubi4 gene is shown in Figure 5A with the subsequently determined nucleic acid sequence (SEQ ID NO: 7) located upstream of the translation start codon of the ubi4 gene shown in Figure 10. The nucleotide sequence of Figure 5A represents nucleotides 1 to 5512 of the 5551 nucleotide sequence deposited with accession number of GenBank AF093504. Fragments of the ubi4 gene sequence, which were identical in the nucleic acid sequences initially determined and subsequently determined, were as follows (the nucleotide numbers refer to the nucleotide number in SEQ ID NO: 5): Fragment A: 1-242; B fragment: 245-787: fragment C: 788-1020; Fragment D: 1021-1084; Fragment E: 1085-1 168; Fragment F: 1 169-1 173; Fragment G: 1 174-1648; and fragment H: 1649-1805. The nucleotide sequence upstream of the transcription initiation codon of the ub4 gene (ie, nucleotides 1 -1810 of SEQ ID NO: 1, and nucleotides 1 -1802 of SEQ ID NO: 1. NOs: 5 and 7) contained three regions: (a) one upstream of the 5 'UTR sequence (ie, nucleotides 1 -378 of SEQ ID NO.1, and nucleotides 1 -377 of SEQ ID NOs: 5 and 7), (b) the 5 'UTR sequence (ie, nucleotides 379-444 of SEQ ID NO: 1, and nucleotides 378-442 of SEQ ID NOs: 5 and 7); and (c) an intron sequence (is • say, nucleotides 445-1810 of SEQ ID NO: 1, and nucleotides 5 443-1802 of SEC I D NOs: 5 and 7). With respect to the ubi9 gene, the initially determined nucleic acid sequence (SEQ ID NO: 3) of the ubi9 gene including the translation start codon and its upstream sequences is shown in Figure 7A, and the nucleic acid sequence Initially determined (SEQ ID NO: 4) of the ub19 gene including the. The translation stop codon and its downstream sequences are shown in Figure 7B. The subsequently determined nucleic acid sequence (SEQ ID NO: 8) of the entire ubi9 gene is shown in Figure 8A, with the nucleic acid sequence (SEC I D NO: 10) of the ubl9 gene upstream of the translation initiation codon shown in Figure 1 1. It is noted that while the 5'-end of the ubi9 gene was obtained through cleavage with Hindlll, which recognizes the 5'-AAGCTT-3 'sequence, the repeated sequencing of the 5' end of the ubi9 gene was shown instead of the sequence 5? AGTTT-3 '(Figures 8 and 11). In this way, the inventors' point of view is that the sequence of the full-length ubi9 gene, (a) is as shown in Figure 8A (SEQ ID NO: 8) (ie, the total number of nucleotides being of 5174, with the ten nucleotides at the 5 'end being 5'- 25 AAGTTTTGnT-3'), (b) is as shown in Figure 8A (SEQ ID NO: 8) with the exception that it has a total number of nucleotides of 5174, with the ten nucleotides at the 5 'end being 5'- AAGCTTTGnT-3', assuming the substitution of "T" at position 4 • with a "C"), or (c) is as shown in Figure 8A (SEQ ID NO: 8) with the exception that it has a total number of nucleotides of 5175, with all ten nucleotides at the 5 end 'being 5'AAGTTTTGn-3', assuming the insertion of a "C" at position 4. Therefore, any reference here to SEQ ID NO.8 is intended to represent each of the three sequences of nucleotides described in the preceding sentence. • Similarly, it is the point of the inventors that the sequence upstream of the translation start codon of the gene ubi9 (a) is as shown in Figure 11 (SEQ ID NO: 10) (ie, the number total nucleotide being 3688, with all ten nucleotides at the 5 'end being 5'AAGTTTTGnT-3'), (b) is as shown in Figure 1 1 (SEQ ID NO: 10) with the exception that it has a total nucleotide number of 3688, with the ten nucleotides at the 5'final end being 5'-AAGTTTGnT-3 ', assuming the substitution of the "T" at position 4 with a "C", or (c) is as shown in Figure 1 1 (SEQ ID NO: 10) with the exception that it has a total nucleotide number of 3689, with the ten nucleotides at the 5 'end being 5'-AAGCTTTTGn-3', assuming the insertion of a "C" "in position 4. Therefore, any reference herein to S EC ID NO: 10 is intended to represent any and all of the three nucleotide sequences described in the preceding sentence. Fragments of the ubi9 gene sequence, which were identical in the initially determined and subsequently determined nucleic acid sequences were as follows (the nucleotide number refers to the nucleotide number in SEQ ID NO: 8): Fragment A: 1 -3600; Fragment B: 3602-3612; and fragment C: 3614-3691. The nucleotide sequence upstream of the translation initiation codon of ubi9 (ie, nucleotides 1 -3691 of SEQ ID NO: 3, and nucleotides 1 -3691 of SEQ ID NO: 8) contained three regions: (a) one upstream of the 5 'UTR sequence (ie, nucleotides 1 -2248 of SEQ ID NO: 3, and nucleotides 1-242 of SEQ ID Nos: 8 and 10), (b) a 5' UTR sequence (i.e. , nucleotides 2249-2313 of SEQ ID NO: 3 and nucleotides 2249-2313 of SEQ ID NOs: 8 and 10), and (c) an intron sequence (ie, nucleotides 2314-3688 of SEQ ID NO: 3, and nucleotides 2314-3688 of SEQ ID Nos: 8 and 10). A BLAST search of the GenBank database showed 100% homology only for a region of 20-22 bp of nucleotides 1-3688 of SEQ ID NOs: 8 and 10 (i.e., the region of the ubi9 gene that contained the upstream sequence of 5 'UTR, and the intron sequence), 100% homology only to a 20 bp region of nucleotides 1 -2248 of SEQ ID NOs: 8 and 10 (ie, the upstream sequence of 5 'UTR), and 87% homology between a 135 bp fragment of the upstream sequence of 5' UTR of SEQ ID NOs: 8 and 10, and a 1 18 bp fragment of the Saccharum species glucose transport mRNA sp. , 3 'end (access number GenBank L21752). The data presented here demonstrate that plasmids containing the uidA gene encoding β-glucuronidase (GUS) under the control of SEQ ID NO: 7 (which is equivalent to SEQ ID NO: 1 5 initially determined) of the ubi4 gene or under the control of SEQ ID NO: 10 (which is equivalent to SEQ ID NO: 3 initially determined) of the ubi9 gene successfully directs the transient expression of GUS in cultured suspension cells of monocotyledonous sugar cane (Example 3), monocotyledon sorghum callus (Example 5), monocotyledonous pineapple leaves, bodies, roots and fruits of the protocormo type (Example 6), and in dicotyledonous tobacco leaves (Example 4), as well as the stable expression in callus of monocot sugar cane (Example 7), monocotyledonous rice callus (Example 8), and dicotyledonous tobacco leaves (Example 9). The present invention is not limited to SEQ ID NO: 1, but specifically contemplates its portions. As used herein, the term "portion" when referring to a nucleic acid sequence, refers to a fragment of that sequence. The fragment may vary in size from ten (10) nucleotide residues contiguous to the entire nucleic acid sequence minus a nucleic acid residue. In this manner, a nucleic acid sequence comprising "at least a portion of" a nucleotide sequence comprises ten (10) contiguous nucleotide residues of the nucleotide sequence a __a__b__t_-buÁM ..... ... * .. * * _ * - .. the entire nucleotide sequence. In a preferred embodiment, portions contemplated within the scope of the invention include, but are not limited to, portions • greater than 20 nucleotide bases, most preferably greater than 100 bases of nucleotides, the upstream sequence of the 5 'UTR sequence (ie, the nucleotide sequence of position 1 to 377 of SEQ ID NO: 7), the 5 'UTR sequence (ie, the nucleotide sequence of position 378 to 442 of SEQ ID NO: 7), and the intron sequence (i.e., the nucleotide sequence of the position 443 to 1802 of SEQ ID NO: 7). In a preferred alternative embodiment, portions within the scope of the invention include portions greater than 20 nucleotide bases, most preferably greater than 100 nucleotide bases, those sequences that are upstream of the translation initiation codon and which are identical in the initially determined and subsequently determined sequence of the ubi4 gene, and are illustrated by the nucleotide sequence from position 1 to 242, from position 245 to 787, from position 788 to 1020, from position 1021 to 1084 , from position 1085 to 1 168, from position 1 169 to 1 173, from position ,174 to 1648, and from position 1649 to 1802 of SEQ ID NO: 7. In another alternative preferred embodiment, the portion contains the 177 bp sequence, which is upstream of 5 'UTR and which is highly homogeneous (> 90% identity) in both the ubi4 and ubi9 gene sequences, i.e. , the nucleotide sequences from position 1 to 377 of SEC I D NO: 7.

It is contemplated that the present invention is not limited to SEC I D NO: 3, but specifically includes its portions. In a preferred embodiment, the contemplated portions that are within • of the scope of the invention include, but are not limited to, portions greater than 20 nucleotide bases, most preferably greater than 100 nucleotide bases, the upstream sequence of the 5 'UTR sequence (i.e. position 1 to 2248 of SEQ ID NO: 10), the 5 'UTR sequence (i.e., the nucleotide sequence of position 2249 through 2313 of SEQ.

NO: 10), and the intron sequence (i.e., the nucleotide sequence of position 2314 to 3688 of SEQ ID NO: 10). In an alternative preferred embodiment, portions within the scope of the invention include portions greater than 20 nucleotide bases, most preferably greater than 100 bases of nucleotides, those sequences that are upstream of the translation initiation codon and that are identical in the initially determined and subsequently determined sequence of the ub19 gene, and are illustrated by the nucleotide sequence of position 1 to 3600, from position 3602 to 3612, and from position 3614 to 3688 of SEQ ID NO: 10. In another preferred alternative embodiment, the portion contains the sequence that is upstream of the transcription initiation codon and which is highly homologous (> 90% identity) in both the ubi4 gene sequences and ubi9, ie the nucleotide sequence of position 1671 to 2248 of SEQ ID NO: 10. This sequence includes the MITE (from position 1706 to 1906), which is not homologous to the ubiquitous polyubiquitin ubi4 promoter sequences. The sequences of the present invention are not limited to • SEQ ID NOs: 1 and 10 and their portions, but also include 5 homologs of SEQ ID NOs: 1 and 10, as well as portions of these homologs. A nucleotide sequence, which is a "homologue" of SEQ ID NOs: 1 and 10, is defined herein as a sequence of nucleotides that exhibit greater than 61% identity (but not 100% identity) at the sequence SEQ ID NOs: 1 and 10, respectively. • The present invention also contemplates the functioning or functional homologs of SEQ ID NOs: 1 and 10. A "functional homolog" of SEQ ID NOs: 1 and 10 is defined as a nucleotide sequence having less than 100% homology with SEC ID NOs: 1 and 10, respectively, and which has a promoter activity that has some or all of the characteristics (for example, the constitutive promoter activity) of the SEC promoter activity • ID NOs: 1 and 10, respectively. The homologues of SEQ ID NOs: 1 and 10 and their portions, include, but are not limited to, sequences of nucleotides having deletions, insertions or substitutions of different nucleotides or nucleotide analogs as compared to SEQ ID NOs: 1 and 10, respectively. Such homologs can be produced using methods well known in the art. The invention also contemplates at least a portion of SEC I D NOs: 1 and 10, and their homologues having promoter activity. The term "promoter activity" when made with reference to a nucleic acid sequence refers to the ability of the • nucleic acid sequence to initiate the transcription of a nucleotide sequence operably linked to mRNA. The terms "operably linked", "operable combination" and "operable order", as used herein, refer to nucleic acid sequence binding in a manner such that a nucleic acid molecule is capable of directing the transcription of the The nucleic acid sequence of interest and / or the synthesis of a polypeptide sequence of interest. The promoter activity can be determined using methods known in the art. For example, a candidate nucleotide sequence whose determined promoter base activity is bound in frame to a nucleic acid sequence of interest (eg, a reporter gene sequence, a selectable marker gene sequence) to generate a report vector, enter the report vector into the plant tissue using methods described here, and detect the expression of the report gene (for example, detecting the presence of the encoded mRNA or encoded protein, or the activity of a protein encoded by the report gene). The report gene can express visible markers. Reporting gene systems that express visible markers include β-glucuronidase and its substrate (X-Glue), luciferase and its substrate (luciferin), and β-galactosidase and its substrate (X-Gal), which are widely used not only to identify transformants, but also to quantify the amount of transient or stable protein expression attributable to a system of • specific vector [Rhodes CA et al. (1995) Methods Mol. Biol. 5 55: 121-131]. In a preferred embodiment, the report gene is a GUS gene. The selectable marker gene can confer antibiotic resistance or herbicide. Examples of report genes include, but are not limited to, dhfr, which confers resistance to methotrexate [Wigler M. et al., (1980) Proc. Nati Acad. Sci. 77: 3567-70]; npt, the f 10 which confers resistance to neomycin of aminoglycosides and G-418 [Colbere-Garapin F et al., (1981) J. Mol. Biol. 150: 1-14] and iso or pat, which confer resistance to chlorsulforon and acetyl transferase of phosphinothricin, respectively. The detection of the presence of encoded mRNA or protein, or the activity of a protein encoded by the reporter gene or the selectable marker gene indicates that the candidate nucleotide sequence has promoter activity.

• The sequences within a promoter that affect the promoter activity, can be determined using constructions of Such elimination as those described by Sherri et al., For the determination of HSP70 intron alterations, which impact the transcription of genes operably linked thereto (U.S. Patent No. 5,593,874, incorporated herein by reference). In summary, several expression plasmids are constructed to contain a reporter gene under the regulatory control of different candidate nucleotide sequences, which are obtained either through restriction enzyme removal of internal sequences in SEC I D Nos: 1 and 10, truncation of restriction enzyme sequences • at the 5 'and / or 3' end of SEQ ID Nos: 1 and 10, or through the introduction of individual nucleic acid base changes through PCR to SEQ ID Nos: 1 and 10. The expression of the Report gene through the elimination constructions is detected. The detection of the expression of the report gene in a given elimination construct indicates that the candidate nucleotide sequence in that elimination construct has promoter activity. At the 3 'end of the nucleic acid sequence of interest, other DNA sequences may also be included, eg, a 3' untranslated region containing a polyadenylation site and transcription termination sites. The present invention is not limited to sense molecules of SEQ ID Nos: 1 and 10 but also contemplates within its scope antisense molecules comprising a nucleic acid sequence complementary to at least a portion (eg, a portion greater than 100 nucleotide bases in Length, and most preferably greater than 200 nucleotide bases in length) of the nucleotide sequence SEQ ID Nos: 1 and 1 0. These antisense molecules find use in, for example, the reduction or prevention of the expression of a gene whose expression is controlled through SEQ ID Nos: 1 and 10. _iwt _ «_ a_bH_i_ _ ^ _ i_ The nucleotide sequence of SEC I D Nos: 1 and 10, its portions, its homologues and its antisense sequences, can be synthesized through synthetic chemistry techniques, which • are commercially available and are well known in the art 5 [see Caruthers MH et al., (1980) Nuc. Acids Res. Symp. Ser. 215-223; Horn T, and others, (1980) Nuc. Acids Symp. Ser. 225-232]. In addition, fragments of SEQ ID Nos: 1 and 10 can be made through the treatment of SEQ ID Nos: 1 and 10 with restriction enzymes followed by purification of the fragments through gel electrophoresis. Alternatively, they can also be produced • sequences using the polymerase chain reaction (PCR) as described by Mullis [Patents of E. U.A. Nos. 4,683, 195, 4,683,202 and 4,965, 188, all of which are incorporated herein by reference]. SEC I D Nos: 1 and 10, their portions, their counterparts and Their antisense sequences can be ligated together or to heterologous nucleic acid sequences using methods well known in the art. F. The synthesized sequence nucleotide sequence can be confirmed using commercially available equipment, as well as using methods well known in the art using enzymes such as the Klenow fragment of DNA polymerase I, Sequenase®, Taq DNA polymerase, or thermostable T7 polymerase. Capillary electrophoresis can also be used to analyze the size and confirm the nucleotide sequence of the products of a-ík - ?? __ Í_l_fe_ * Éí_? nucleic acid synthesis, restriction enzyme digestion or amplification by PCR. It is readily appreciated by those skilled in the art that the sequences of the present invention can be used in a variety of ways. For example, these sequences are useful for directing the expression of polypeptide sequences in vitro and in vivo. In plants, this is useful to determine the role of the polypeptide in the development of diseases, as well as in the production of transgenic plants with desirable agronomic characteristics, as described later. In addition, portions of the • sequences of the invention as probes for the detection and isolation of complementary DNA sequences, and for the amplification of nucleotide sequences, as described below. 15 B. Use of Probes to Identify and Isolate Homologs of the Promoter Sequences of Sugarcane. • The invention provided herein is not limited to SEC I D NO: 1 and 10, their counterparts, and their portions, having promoter activity, but also includes sequences that have no promoter activity (ie, non-functional homologs and non-functional portions of homologs). This may be desirable, for example, when a portion of SEC I D NOs: 1 and 10 is used as a probe to detect the presence of SEC I D NOs: 1 and 10, respectively, or your portions in a sample.

As used herein, the term "probe" refers to an oligonucleotide, either naturally occurring as in a purified restriction digestion or synthetically produced, in • recombinant form or through PCR amplification, which is able to hybridize to a nucleotide sequence of interest. A probe can be single chain structure or double chain structure. It is contemplated that any probe used in the present invention will be labeled with any "report molecule", so that it may be detectable in Any detection system including, but not limited to, enzyme (e.g. ELISA, as well as histochemical analysis based on enzymes), fluorescent, radioactive, calorimetric, gravimetric, magnetic and luminescent systems. It is not intended that the present invention be limited to any detection system or private brand. The probes provided herein are useful in the detection, identification and isolation of, for example, sequences such as those listed in SEC I D NOs: 1 and 10, as well as their homologs. The preferred probes are of sufficient length (for example, from approximately 9 nucleotides approximately 20 nucleotides or more, in length), so that high stringency hybridization can be employed. In one embodiment, probes of 20 to 50 nucleotide bases in length are employed.

C. Use of Initiators to Amplify Nucleotide Sequences. The invention provided herein is not limited to SEC I D NO: 1 and • 10, its homologues and its portions, having promoter activity, 5 but also includes sequences that do not have promoter activity. This may be desirable, for example, when a portion of the nucleic acid sequence established as SEC I D NOs: 1 and 10 is used as an initiator for the amplification of nucleic acid sequence through, for example, reactions of polymerase chain (PCR) or polymerase chain reactions- • reverse transcription (RT-PCR). The term "amplification" is defined as the production of additional copies of a nucleic acid sequence and is generally performed using polymerase chain reaction technologies well known in the art (Dieffenbach CW and GS Dveksler (1995) PCR Primer, a Laboratory Manual, Cold Spring Harbor Press, Plainview NY). As used herein the term "polymerase chain reaction" ("PCR") refers to the • method of K. B. Mullis described in the Patents of E. U.A. Nos. 4,683, 195, 4,683,202 and 4,965, 188, all of which are incorporated here by Reference, which describes a method for increasing the concentration of a segment of an objective sequence in a mixture of genomic DNA without cloning or purification. This process to amplify the target sequence consists of introducing a large excess of two oligonucleotide primers into the DNA mixture containing the desired target sequence, followed by a H ^^ precise sequence of thermal cyclization in the presence of DNA polymerase. The two initiators are complementary to their structures ^ chain of the sequence of target of structure of double chain. To effect the amplification, the mixture is denatured and the initiators are then heated and cooled rapidly to their complementary sequences within the target molecule. After heating and rapid cooling, the primers are extended with a polymerase in order to form a new pair of complementary strand structure. The 10 steps of denaturation, heating and cooling of the polymerase primer and extension can be repeated many times (ie, denaturation, heating and cooling and extension constitute a "cycle", there can be numerous "cycles") to obtain a high concentration of an amplified segment of the desired target sequence. The length of the amplified segment of the desired sequence is determined by the relative positions of the initiators with respect to each other, and, therefore, this • Length is a controllable parameter. By virtue of the repetition aspect of the process the method is termed as the "reaction polymerase chain "(hereinafter" PCR "). Since the desired amplified segments of the target sequence become the predominant sequences (in terms of concentration) in the mixture, these will be" amplified by PCR " With PCR, it is possible to amplify an individual copy of a Target specific sequence in the genomic DNA at a level detectable through several different methodologies (eg, hybridization with a labeled probe; incorporation of biotinylated primers followed by detection of avidin-enzyme conjugate; ^ * w and / or the incorporation of deoxyribonucleotide triphosphates labeled with 32P such as dCTP or dATP, in the amplified segment). In addition to genomic DNA, any nucleotide sequence can be amplified with the appropriate set of starter molecules. In particular, the amplified segments created by the same PCR process are, by themselves, efficient templates for subsequent amplifications by PCR. Amplified target sequences can be used to obtain DNA segments (eg, genes) for the construction of vectors, target transgenes, etc. As used herein, the term "initiator" refers to an oligonucleotide, either naturally occurring as in a digestion of purified or synthetically produced restriction, which is capable of acting as a synthesis start point when placed under conditions wherein the synthesis of an initiator extension product, which is complementary to an acid chain structure nucleic acid is induced (that is, in the presence of nucleotides and an induction agent such as DNA polymerase and at a suitable temperature and pH). The initiator is preferably of single chain structure for maximum efficiency in the amplification, but alternatively it can be of double chain structure. If it is of double chain structure, the initiator First, it is treated to separate its chain structures before being used to prepare extension products. Preferably, the initiator is an oligodeoxyribonucleotide. The initiator must be sufficiently long (eg, a length of about '9 nucleotides to about 20 nucleotides or more) to initiate the synthesis of extension products in the presence of the induction agent. Suitable lengths of the primers can be empirically determined and depend on factors such as temperature, source of the initiator and the use of the method. In one embodiment, the present invention employs probes of 20 to 50 bases of nucleotide in length. The primers contemplated by the present invention are useful for, for example, identifying sequences that are homologous to the ubi4 and ubi9 gene sequence of sugarcane in plants and other organisms. 15 D. Generation of Transgenic Plants. The present invention provides methods for constitutively expressing a nucleotide sequence of interest in a cell, tissue, organ, and / or organism. In one modality, the methods provided herein direct the constitutive expression of a nucleotide sequence of interest in cells of monocotyledonous and dicotyledonous plants. In one embodiment, this is accomplished by introducing into a plant cell a vector containing a sequence of nucleotides of interest operably linked to sequences provided herein that have promoter activity. The transformed plant cell is allowed to develop into a transgenic plant wherein the nucleotide sequence of interest is preferably, but not necessarily, expressed in substantially each • tissue. These steps are further described below for 5 specific modes. 1. Vector expression for plants. In one embodiment, the methods of the present invention involve the transformation of the monocotyledonous tissue (cultured ^ F 10 cells of sugar cane suspension, sugar cane callus, rice callus, corn embryos, pineapple leaves, bodies, roots and fruits of the "protocormo" type, and sorghum callus) and dicotyledonous tissue (tobacco leaves, tomato plants and embryonic meristems cut from soybeans) with expression vectors where the ß-glucuronidase (GUS) gene is under the transcriptional control of the promoter sequence of the ubi4 sugarcane illustrative gene (SEC I D NO: 1) or the promoter sequence of the ubi9 sugarcane gene • sugar (SEQ ID NO: 3). As used herein, the terms "vector" and "vehicle" are used interchangeably with reference to nucleic acid molecules that transfer DNA segments from one cell to another. The term "expression vector" as used herein, refers to a recombinant DNA molecule that contains a desired coding sequence and appropriate nucleic acid sequences necessary for the expression of the operably linked coding sequence in a host organism particular. The methods of the invention are not limited to the vectors • of expression described here. Any expression vector that is capable of introducing a nucleic acid sequence of interest into a plant cell is contemplated within the scope of this invention. Typically, the expression vectors comprise the nucleic acid sequence of interest, as well as accompanying sequences that allow transcription of this sequence, and that allow cloning of the vector in a bacterial host or phage. The vector preferably, but not necessarily, contains an origin of replication, which is functional on a wide range of prokaryotic hosts. A selectable marker is generally, but not necessarily, included to allow selection of cells carrying the desired vector. In a preferred embodiment, the promoter sequence is SEC I D NO: 1, which is derived from the ubi4 sugar cane gene. In an alternative preferred embodiment, the promoter sequence is SEC I D NO: 3, which is derived from the ubi9 sugarcane gene. Nevertheless, The invention is limited to the promoter sequences used herein. Any sequence that is a portion, homologue, or homologue of a portion of SEQ ID NOs: 1 and 10 and which has promoter activity is contemplated within the scope of the invention.

In addition to a promoter sequence, the expression vector preferably contains a transcription termination sequence downstream of the nucleic acid sequence of • interest to provide efficient completion. Exemplary 5-termination sequences include the nopaline synthase termination sequence (NOS), and different fragments of the small sugar subunit (scrbcs) carboxylase / ribulose-1-5-biphosphate oxygenase (rubisco) gene. The termination sequences of the expression vectors are not critical for the invention. The termination sequence can be obtained from the same gene as the promoter sequence or can be obtained from different genes. If the mRNA encoded by the nucleic acid sequence of interest is to be efficiently translated, the sequences of Polyadenylation can also be commonly added to the expression vector. Examples of polyadenylation sequences include, but are not limited to, the octopine synthase signal • Agrobacterium, or nopaline synthase signal. When it is preferred that the nucleic acid sequence of interest is not translated into a For example, in which the nucleic acid sequence of interest encodes an antisense RNA, polyadenylation signals are not necessary. The vectors for the transformation of plant cells are not limited to the type or nature of the genes expressed here described. Any nucleic acid sequence of interest can be used to create cells from transgenic plants, tissues, organs and plants. The nucleic acid sequences of interest include sequences that encode a protein of interest. The terms "protein of interest" and "polypeptide of interest" refer to any protein or polypeptide, respectively, the manipulation of which may be considered desirable for any reason, by one skilled in the art. For example, it may be desirable to express a nucleic acid sequence encoding a polypeptide sequence having, for example, enzyme activity. An example of said enzyme is the deaminase enzyme of 1-aminocyclopropan-1-carboxylic acid (ACC), which metabolizes ACC in the tissue of plants, thus reducing the level of ethylene, which is responsible for the wilting of fruits (Patent of E. U.A. No. 5,512,466, the contents of which are incorporated herein by reference). Another enzyme that can be desirably expressed in a plant is the sucrose phosphate synthase enzyme, which f increases the level of sucrose in the fruit. The nucleic acid sequence of the gene encoding this enzyme is known (for example, in corn, Worrell et al. (1991) Plant Cell 3: 1 121 -1 130) (Figure 15) (SEQ ID NO: 12) and has assigned you Genbank access number m97550. In yet another example, more than one enzyme suitable for use in this invention is EPSP synthase (5-25 enolpyruvyl-3-phosphoshikimate, EC: 25.1.19), which is an enzyme involved in the path of shikimic acid of the plants. The path of shikimic acid provides a precursor for the synthesis of essential aromatic amino acids for plants.

• Specifically, the EPSP synthase catalyzes the conversion of 5-phosphovate pyruvate and 3-phosphoshikimic acid to 5-enolpyruvyl-3-phosphoshikimate acid. A herbicide containing N-phosphonomethylglycine inhibits the EPSP synthase enzyme and thus inhibits the shikimic acid path of the plant. The term "glyphosate" is usually used to refer to the herbicide N-phosphonomethylglycine in its acid or anionic forms. Novel EPSP synthase enzymes have been discovered that exhibit an increased tolerance to glyphosate-containing herbicides. In particular, an EPSP synthase enzyme having an individual substitution of glycine to alanine in the highly conserved region having the sequence: -L-15 GNAGTA-, located between positions 80 and 120 in the amino acid sequence of EPSP-type synthase wild, mature, has been shown to exhibit increased tolerance to glyphosate, and is described in the U.S. Patent. No. 4, 971, 908, the teachings of which are incorporated herein by reference. The methods for Transforming plants to exhibit tolerance to glyphosate are discussed in the U.S. Patent. No. 4, 940, 835, incorporated herein by reference. A glyphosate tolerant EPSP synthase plant gene encodes a polypeptide containing a chloroplast transport peptide (CTP), which enables the EPSP synthase polypeptide (or a active portion thereof) to be transported to a chloroplast _ * ta_i_l_tt? _i _ ^ _ b_. inside the plant cell. The EPSP synthase gene is transcribed to mRNA in the nucleus and the mRNA is translated into a precursor polypeptide (CTP / mature EPSP synthase) in the cytoplasm. The precursor polypeptide f is transported to the chloroplast. Additional examples of enzymes suitable for use in this invention are acetolactate synthase, RNase that imparts male sterility [Mariani et al. (1990) Nature 347: 737-741], and wheat germ agglutinin. Still other examples of the desirable nucleic acid sequence are those that encode the sweet protein. The nucleic acid sequence for the gene encoding the sweet protein is known in the art (see, for example, U.S. Patent Application No. 08/670, 186, the contents of which are incorporated herein). by reference). The transformation of plants with the sweet protein is useful for, for example, providing a base level of sweetness in the fruit, thereby reducing the effects of differences in fruit maturity by providing a more uniform sweetness in different parts of the fruit. Other examples of suitable proteins to be used in this invention are glass toxin proteins Bacillus thuringiensis (B .t.), Which when expressed in plants protect plants from insect infestation, since the insect, after eating the plant containing the protein of the Bt toxin , whether he dies or stops feeding. The proteins of the toxin B.t. the which are toxic to both lepidopteran and coleopteran insects, > _._ «_ can be used. Examples of particularly suitable DNA sequences encoding the B.t. toxin protein. are described in Patent Application EP 385,962, entitled "Synthetic Plant Genes and Method for Preparation", (Genes of 5 Synthetic Plants and Preparation Methods), published on September 5, 1990. Alternatively, it may be desirable to express a sequence of nucleic acid encoding an antisense RNA that hybridizes with a DNA sequence of a genomic plant. For example, it may be advantageous to express antisense RNA which is specific for genomic plant DNA sequences that encode an enzyme whose activity is intended to be reduced. Examples of DNA sequences whose reduced expression may be desirable are known in the art and include, but are not limited to, the sequences of ethylene which can be induced in fruits (Patent of U.A. 5, 545,815, all the contents of which are incorporated herein by reference). The expression of antisense RNA, which is homologous With these ethylene sequences capable of induction, it is useful to delay the wilting of fruits and to increase the firmness of the fruit. Another DNA sequence whose expression may be desirably reduced includes the ACC synthase gene, which encodes the ACC synthase enzyme which is the first step and the speed limitation step in ethylene biosynthesis. The nucleic acid sequences for this gene have been described from a number of plant sources (eg, Picton et al. (1993) The Plant J. 3: 469-481; U.S. Patent Nos. 5, 365,015 and 5,723,766, the contents of which are incorporated herein by reference). The expression of the antisense RNA which hybridizes the sequences • ACC synthase genomes in plants may be desirable to delay fruit wilt. Another sequence whose expression can be advantageously reduced is the genomic sequence encoding the polyphenol oxidase enzyme. This enzyme is involved in the staling reaction that occurs during freeze damage. The nucleic acid sequences encoding this enzyme have been • previously described in the art (eg, Shahar et al. (1992) Plant Cell 4: 135-147), as shown in Figure 14 (SEC I D NO: 1 1) (Accession number GenBank s40548). It has been reported that the use of antisense polyphenol antisense sequences inhibit the expression of the polyphenol oxidase (PPO) gene and that inhibits the binding [Bachem et al. (1994) Bio / Technology 12: 1 101-1 105].

• A person skilled in the art knows that the segment of antisense DNA that will be introduced into the plant can include the region of full-length coding of the target gene or a portion thereof. Complete homology between the nucleotide sequences of the antisense RNA and the target genomic DNA is not required. Rather, antisense DNA sequences that encode antisense RNA sequences that are partially homologies to a target genomic DNA sequence are contemplated within the scope of the invention as long as the antisense RNA sequences are capable of repressing the expression of the target genomic DNA sequence. The invention is not limited to vectors expressing a single nucleic acid sequence of interest. Vectors that contain a plurality of (i.e., two or more) nucleic acid sequences under the transcriptional control of the same promoter sequence are expressly contemplated within the scope of the invention. Such vectors may be desirable, for example, when the expression products of the plurality of nucleic acid sequences contained within the vector provide protection against different pathogens, and wherein simultaneous protection against these different pathogens is considered advantageous. Also included within the scope of this invention are 15 vectors containing the same nucleic acid sequences or different sequences under the transcriptional control of different promoter sequences derived from SEC I D NO: 10, and other sequences. Such vectors may be desirable to, for example, control the different expression levels of different nucleic acid sequences of interest in plant tissues. 2. Transformation of Plant Cells. Once the expression vector is prepared, transgenic plants and plant cells are obtained by introducing the vectors expression in plants and plant cells using methods known in the art. The present invention is suitable for any member of the family of monocotyledonous (monocot) plants including, but not limited to, corn, rice, barley, • oats, wheat, sorghum, rye, sugar cane, pineapple, sweet potatoes, onions, 5 bananas, coconut, dates and hops. The present invention is also suitable for any member of the family of dicotyledonous plants (dicot) which include, but are not limited to, tobacco, tomato, soybean and papaya. In one embodiment, the expression vectors are introduced in plant cells through gene transfer mediated by particles. Particle-mediated gene transfer methods are known in the art, are commercially available, and include, but are not limited to, the gas-driven gene delivery system described in the US Pat.

E. U.A. 5, 584, 807 of McCabe, the contents of which are incorporated herein by reference. This method involves coating the nucleic acid sequence of interest on heavy metal particles and accelerating the coated particles under the pressure of compressed gas to be delivered to the target tissue. Other methods of particle bombardment are also available for the introduction of heterologous nucleic acid sequences into plant cells. In general, these methods involve depositing the nucleic acid sequence of interest on the surface of small dense particles of a material such as gold, plati no or tungsten. The coated particles themselves are then placed as a coating either on a rigid surface, such as a metal plate, or on a carrier sheet made of a brittle material such as mylar. The coated sheet is then accelerated towards the objective biological tissue. The use of the flat sheet generates a uniform extension of the accelerated particles, which maximizes the number of cells receiving particles under uniform conditions, resulting in the introduction of the nucleic acid sample into the target tissue. Alternatively, an expression vector can be inserted into • the genome of plant cells by infecting the cells with a bacterium, including, but not limited to, the Agrobacterium strain previously transformed with the nucleic acid sequence of interest. Since most dicotyledonous plants are natural hosts of Agrobacterium, almost all dicotyledonous plants can be transformed through Agrobacterium in vitro. Although monocotyledonous plants, and in particular, • cereals and fats, are not natural hosts for Agrobacterium, the work to transform them using Agrobacterium has also been realized (Hooykas-Van Slogteren et al., (1984) Nature 3: 763- 764. The genera of plants that can be transformed by Agrobacterium include Chrysanthemum, any species of carnation, Gerbera, Euphorbia, Pelaronium, Ipomea, Pasiflora, Ciclamino , Malus, Prunas, Rosa, Rubo, Pópulos, Santalum, Lilium, Daffodils, Ananas, Arachis, Phaseolus and Pisum.

For transformation with Agrobacterium, Agrobacterium cells disassembled with recombinant Ti plasmids of Agrobacterium tumefaciens or plasmids I r of Agrobacterium rhizogenes (such as those described in FIG.

Patent of E. U.A. No. 4,940, 838, the contents of which are incorporated herein by reference), which are constructed to contain the nucleic acid sequence of interest using methods well known in the art [J. Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, f 10 NY]. The nucleic acid sequence of interest is then stably integrated into the plant genome through infection with the transformed Agrobacterium strain. For example, heterologous nucleic acid sequences introduced into plant tissues have been introduced using the Transfer of natural DNA from bacteria Agrobacterium tumefaciens and Agrobacterium rhizogenes (for review see Klee et al. (1987) Ann. Rev. Plant Phys. 38: 467-486). Wß The construction of recombinant Ti and I r plasmids in general follows the methods typically used with vectors most common bacterial, such as pBR322. Additional use can be made of accessory genetic elements and sometimes it is found with native plasmids and some times constructed of foreign sequences. These may include, but are not limited to, structural genes for antibiotic resistance such as selection.

There are two systems of vector systems of recombinant Ti and Ir plasmids now in use. The first system is called the "cointegrated" system in this system the promiscuous vector that • contains the gene of interest is inserted by genetic recombination in a non-oncogenic Ti plasmid that contains both the cis action and trans action elements required for the transformation of plants as, for example, in the promiscuous vector pMLJ 1 and the plasmid Non-oncogenic Ti pGV3850. The second system is the so-called "binary" system, where two f 10 plasmids are used; the gene of interest is inserted into a promiscuous vector that contains the cis-acting elements required for the transformation of plants. The other necessary functions are provided in trans by the non-oncogenic Ti plasmid as illustrated by the promiscuous vector pBI N 19 and the non-oncogenic Ti plasmid PAL4404. Some of these vectors are commercially available. There are three common methods to transform plant cells with Agrobacterium: the first method is through co-cultivation of Agrobacterium with cultured isolated protoplasts.

This method requires a culture system that allows the cultivation of protoplasts and regeneration of plants from cultured protoplasts. The second method is through transformation of cells or tissues with Agrobacterium. This method requires, (a) that the cells or tissues of plants can be transformed through Agrobacterium and (b) that the transformed cells or tissues can be induced to regenerate whole plants. The third method is through transformation of seeds, apices or meristems with Agrobacterium. This method requires micropropagation. • A person skilled in the art knows that the transformation efficiency through Agrobacterium can be improved using a number of methods known in the art. For example, the inclusion of a natural wound response molecule such as acetosyringone (AS) to Agrobacterium culture has been shown to improve transformation efficiency with Agrobacterium tumefaciens [Shahla et al. (1987) Plant Molec. Biol. • 8: 29-298]. Alternatively, the transformation efficiency can be improved by injuring the target tissue that will be transformed. Injury to the tissue of plants can be achieved, for example, through puncture, maceration, bombardment with microprojectiles, etc. [see, for example, Bidney et al. (1992) Plant Molec. Biol. 18: 301-313]. It may be desirable to activate the nucleic acid sequence of interest to a particular site on the plant genome. The • Site-directed integration of the nucleic acid sequence of interest in the genome of plant cells can be achieved, by For example, through homologous recombination using sequences derived from Agrobacterium. In general, plant cells are incubated with an Agrobacterium strain, which contains a target vector in which sequences that are homologous to a DNA sequence within the target site are flanked by DNA (T-DNA) transfer sequences of Agrobacterium, as previously described (Offringa et al., (1996), U.S. Patent 5, 501, 967, the contents of which are incorporated herein by reference). One skilled in the art knows that homologous recombination can be achieved by using target vectors that contain sequences that are homologous to any part of the target plant gene, whether they belong to the gene regulatory elements, or to the coding regions of the gene. Homologous recombination can be achieved in any region of a plant gene as long as the nucleic acid sequence of regions flanking the site to be activated is known. When homologous recombination is desired, the target vector used may be of the replacement or insertion type (Offringa et al. (1996), supra). Replacement type vectors generally contain two regions that are homologous with the target genomic sequence and which flank a heterologous nucleic acid sequence, eg, a selectable marker gene sequence. The replacement type vectors result in the insertion of the selectable marker gene, which in this way divides the target gene. Insertion-type vectors contain an individual region of homology with the target gene and result in the insertion of the entire target vector into the activated gene. Other methods are also available for the introduction of expression vectors into plant tissue, for example, electroinjection (Nan et al. (1995) in "Biotechnology in Agriculture and Forestry." Ed. YPS Bajaj. Springer-Verlag Berlin Heidelberg, Vol. 34: 145-155; Griesbach (1992) HortScience 27: 620); fusion with liposomes, lysosomes, cells, minicells or other fusible lipid surface bodies (Fraley et al. (1982) Proc Nati Acad Sci USA 79: 1859-1863); polyethylene glycol (Krens et al. (1982) Nature 296: 72-74); chemicals that increase the consumption of free DNA; transformation using viruses, and the like. 3. Selection of Transgenic Plant Cells. Plants, plant cells and tissues transformed with a heterologous nucleic acid sequence of interest are readily detected using methods known in the art including, but not limited to, restriction mapping of genomic DNA, analysis by PCR, DNA hybridization. -ADN, DNA-RNA hybridization, DNA sequence analysis, and the like. In addition, the selection of transformed cell plants can be achieved using a selection marker gene. It is preferred, although not necessary, that a selection marker gene be used to select cells from transformed plants. A selection marker gene can confer positive or negative selection. A positive selection marker gene can be used in constructions for random integration and site-directed integration. Positive selection marker genes include antibiotic resistance genes, and herbicide resistance genes, and the like. In one embodiment, the positive selection marker gene is the N PTII gene, which confers resistance to geneticin (G418) or kanamycin. In another modality, the selection marker gene • Positive is the HPT gene, which confers resistance to hygromycin. The selection of the positive selection marker gene is not critical to the invention, since it encodes a functional polypeptide product. Positive selection genes known in the art include, but are not limited to, the ALS (chlorsulfuron resistance) gene, and the DH FR (methotrexate resistance) gene. 10 A negative selection marker gene may also be • included in the constructions. The use of one or more genes of negative selection markers in combination with a positive selection marker gene is preferred in constructs used for homologous recombination. The marker genes of Negative selection is usually placed outside the regions involved in the case of homologous recombination. The negative selection marker gene serves to provide a ^ P disadvantage (preferably lethality) to cells that have integrated these genes into their genome in a form of expression. The cells in where the activation vectors for the homologous recommendation are randomly integrated into the genome will be damaged or annihilated due to the presence of the negative selection marker gene. When a positive selection marker gene is included in the construct, only those cells that have the gene positive selection marker integrated into your genome will survive.

The choice of the negative selection marker gene is not critical to the invention as long as it encodes a functional polypeptide in the cell of transformed plants. The selection gene • Negative, for example, can be chosen from the aux-2 gene of the 5 Ti plasmid of Agrobacterium, the tk gene of SV40, cytochrome P450 of Streptomyces griseolus, the Adh gene of maize or Arabidopsis, etc. any gene that encodes an enzyme capable of converting a substance, which is otherwise innocuous to plant cells in a substance that is dangerous to plant cells, can be used. 4. Regeneration of Transgenic Plants. The present invention provides transgenic plants. The transgenic plants of the invention are not limited to plants in Wherein each cell expresses the nucleic acid sequence of interest under the control of the sequences provided herein. Included within the scope of this invention is any plant that contains at least one cell that expresses the nucleic acid sequence of interest (eg, chimeric plants). It preferred, Although it is not necessary, the transgenic plant expresses the nucleic acid sequence of interest in more than one cell, and most preferably in one or more tissues. Once the transgenic plant tissue, which contains an expression vector, obtained acid, can regenerate plants transgenic from this tissue of this transgenic plant using methods known in the art. The term "regeneration" as used herein, means the growth of an entire plant from a plant cell, a group of plant cells, a plant part or a 5 plant part (e.g., from of a protoplast, callus, body of type protocormo, or part of tissue). The species of the following examples of plant genera can be regenerated from transformed protoplasts. Mayueta, Lotus, Medicago, Onobrichis, Clover, Trigonela, Vigna, f 10 Citrus, Flax, Geranium, Manihot, Daucus, Arabidopsis, Brassica, Rafanus, Sinapis, Atropa, Capsicum, Hiosciamus, Lycopersic, Nicotiana, Solano, Petunia, Digitalis, Marjoram, Cihorio, Liantus, Latuca, Bromus, Asparagus, Antirrinum, Heterocalis, Nemesia, Pelargonium, Panicum, Pennisetum, Ranunculus, Senecio, Salpiglossis, Cucumis, Browaalia, Glycine, Lolium, Zea, Triticum, Sorghum, and Datura. For the regeneration of transgenic plants from transgenic protoplasts, first a suspension of transformed protoplasts or a Petri dish containing transformed explants. The callus tissue is transformed and the shoots can be induced through the callus and subsequently formed into roots. Alternatively, somatic embryo formation in callus tissue can be induced. These somatic embryos germinate as natural embryos to form plants. The media of culture will generally contain several amino acids and plant hormones, such as auxin and cytokinins. It is also advantageous to add glutamic acid and proline to the medium, especially for species such as corn and alfalfa. An efficient regeneration • It will depend on the medium, the genotype and the history of the crop. These five three variables can be empirically controlled to result in a regeneration that can be reproduced. Plants can also be regenerated from cultured cells or tissues. Dicotyledonous plants that have been shown to be capable of regeneration from individual cells transformed to obtain whole transgenic plants include, • for example, apple (Malus pumila), blackberry (Rubus), hybrid blackberry / raspberry (Rubus), red raspberry (Rubus), carrot (Daucus carota), cauliflower (Brassica olerácea), celery (Apium graveolens), cucumber ( Cucumis sativus), eggplant (Solanum melongena), lettuce (Lactuca sativa), potato (Solanum tuberosum), turnip (Brassica napus), wild soybean (Glycine canescens), strawberry (Fragaria x ananassa), tomato (Lycopersicon esculentum), walnut (Juglans regia), melon ( Cucumis meló), grape (Vitis vinifera), and mango (Magnifera indica). The monocotyledonous plants that have shown To be able to regenerate from individual cells to obtain whole transgenic plants include, for example, rice (Oryza sativa), barley (Sécale cereale), and corn. In addition, the regeneration of whole plants from cells (not necessarily transformed) has also been observed in: apricot (Prunus armeniaca), asparagus (Asparagus officinalis), banana (Musa hybrid), bean (Phaseolus vulgaris), cherry (Prunus hybrid), grape (Vitis vinifera), mango (Magnifera indica), melon (Cucumis meló), okra (Abelmoschus esculentus), onion (Allium • hybrid), orange (Citrus sinensis), papaya (Carrica papaya), peach 5 (Prunus persica), plum (Prunus domestica), pear (Pyrus communis), pineapple (Ananas comosus), watermelon (Citrullus vulgaris), and wheat ( Triticum aestivum). The regenerated plants are transformed to standard soil conditions and grown in a conventional manner. After, the expression vector is stably incorporated in plants • Transgenic regenerated, can be transferred to other plants through vegetative propagation or through sexual crossing. For example, in vegetatively propagated crops, mature transgenic plants are propagated by taking cuts or through tissue culture techniques to produce multiple identical plants. In crops propagated by seed, the transgenic plants are themselves crossed to produce a plant • homozygous innate, which is capable of passing the transgene to its progeny through Mendelian inheritance. The innate plant produces the seed containing the nucleic acid sequence of interest. These seeds can be developed to produce plants that can produce the selected phenotype. Innate plants can also be used to create new hybrids by crossing the innate plant with another innate plant to produce a hybrid. t m ^ a ítia aMtl? tm.m ^^ mm ^ tii ^^ ______ - * ^ * ^ ** * **** ... .. .- ***. ... - - ^.

Confirmation of the transgenic nature of the cells, tissues and plants can be carried out through PCR analysis, antibiotic or herbicide resistance, enzymatic analysis and / or staining • Southern to verify the transformation. The progeny of the regenerated plants can be obtained and analyzed to verify if the transgenes can be heritable. The inheritance capacity of the transgene is also a confirmation of the stable transformation of the transgene in the plant.

EXPERIMENTAL PART The following examples serve to illustrate certain preferred embodiments and aspects of the present invention and are not construed as limiting the scope thereof.

EXAMPLE 1 Isolation and characterization of the ubi4 and ubi9 genes of Polyubiquitin from Sugarcane. • A. Plant Materials. The plant materials used for the cDNA collection 20 and for the expression studies were H65-7052 sugarcane hybrid plants grown in the greenhouse of the Hawaii Agriculture Research Center, Aiea, Hawaii. The genomic collection for isolating the genomic clones was made from the hybrid of sugar cane H 32-8560 [Albert et al., Plant Mol. Biol. 20, 663-71-25 (1992)]. The trainings were enumerated consecutively towards _Uk_a_l < below the stem, with the number 1 defined, that the internode hugging the fully expanded younger leaf, as previously described [Moore P. H. : Anatomy and Morphology. In: Heinz DJ (ed) • Sugarcane Improvement Through Breeding, pp. 85-142. Elsevier, 5 Amsterdam (1987)]. Comparisons of 3 'untranslated (UTR) sequences from five polyubiquitin cDNA clones revealed important differences, except for scubi241 and 51 1 clones, which were identical in both 5' and 3 'UTR sequences. Despite these 10 identical regions these clones do not represent transcripts from the same gene, since scubi241 contained four copies of the polyubiquitin coding repeat, while scubi51 contained five of these repeats (data not shown).

B. Plasmid DNA gel stains. The clones of the polyubiquitin cDNA plasmid were diluted with appropriate restriction enzymes and divided in size by gel electrophoresis. The DNA was transferred to Hybond-N + membranes (Amersham) through capillary transfer. The identical DNA gel stains from the five clones were hybridized to T 'UTR probes at high severity. Hybridization at high severity and severe washes at 65 ° C was performed through the method of [Church et al., Proc. Nati Acad. SCI. USA 81, 1991-1995 (1984)]. Probe templates were prepared through PCR amplification of 3 'UTR of each cDNA clone. The probes were labeled with 32P through the method of [Feinberg et al., Anal. Biochem. 132. 6-13 (1983)]. The stains were exposed to a Kodak X-Omat RP XRP-5 film for 5 to 10 minutes at room temperature. 5 C. RNA extraction. Total RNA was extracted from 10 g of each tissue sample through the method of [Bugos et al., Biotechniques 19, 734-737 (1995)]. The concentration of RNA was determined through a spectrophotometer. Poly-A RNA + RNA was isolated from total RNA • using the PoIyATract System (Promega).

D. Analysis of RNA gel staining. 15 μg of each denaturing RNA sample was separated gel electrophoresis as described by [Fourney et al., Focus 10, 5-7 (1988)]. Hybridization and brain washes at 65 ° C were performed by the simplified Northern staining method of f [Virca et al., BioTechniques 8, 370-371 (1990)]. The incorporation of 32P in the probes was verified through two methods, counting by scintillation followed by Sephadex G-50 chromatography and TCA precipitation. An equal amount (1.7 x 107 cpm / ml) of the 32 P-labeled gene-specific probe was used for each hybridization. The autoradiography was with a Kodak X-Omat RP XRP-5 film that was pre-illuminated at an OD540 of 0.15 to to maximize the response linearity, exposed to -80 ° C. _-É _ ^ __? W ^ _____ fc? Four identical RNA gel stains, each containing 15μg of total RNA from mature leaves, immature leaves, stem apices, internode 1, internode 2, internode 3, roots and • callus cultures at control temperatures (26 ° C) and thermal shock 5 (37 ° C), were hybridized to each of the four specific T * UTR probes in the gene, and were exposed to the film for 16 hours as is shown in Figure 1. In Figure 1, 15μg of total RNA was hybridized from each tissue indicated for equal activities of each probe specifies in the gene. The gel stained with ethidium bromide indicates an equal load of RNA from each tissue. M. L., mature daughters; I. L., immature leaves; S.A.; sprout apex; 11, entrenodo 1; 12, entrenode 2; 13, entrenodo 3; R, roots; C, callo 26 ° C; C-HS, callus 37 ° C. The autoradiogram for the scubi221 probe was exposed for 72 hours, all others for 16 hours. With this exposure time of 16 hours, transcript homologues for scubi221 were not detected except in callus tissue under thermal shock at 37 ° C, where an individual band was scarcely detectable. After a 72-hour exposure, the The individual band in the heat shock callus was clear, but no signal was evident for any other tissue. The scubi241 / 51 1 probe detected transcripts of two size classes (in the 16-hour autoradiogram exposure shown in Figure 1, the two bands overlap, however at shorter exposures, the two bands can be resolved), with both transcripts representing approximately equal amounts in all tested fabrics. Transcripts for this subfamily were present at higher levels of all polyubiquitin genes • tested; thermal shock at 37 ° C did not induce a significant change in the accumulation of transcription. The scubi561 probe also detected two kinds of mRNA size; however, the levels of these transcripts were considerably lower than those detected with the 241/51 1 probe. Hybridization of transcription combinations to probe 561 was elevated in an important manner by thermal shock at 37 ° C. The scub5121 probe hybridized to a single size class that was moderately abundant, but significantly smaller than the scub.241 / 51 combination 1. The 5121 levels were very similar in all tissues, except callus. The callus at the control temperature (26 ° C) contained less scubi5121 mRNA than the other tissues; thermal shock at 37 ° C elevated the levels of transcription in the callus to levels approximately equal to those in other tissues at 26 ° C.

• This pattern of reduced transcription levels in the control temperature callus tissue could be seen to a certain degree for all probes, except for scubi561. To confirm the results of the Northern analysis, an "inverse Northern" stain containing the AFN UTR of the five polyubiquitin cDNA clones was hybridized to a first strand cDNA probe, labeled with 32P made from approximately 0.5 μg of mRNA extracted from immature leaves. HE - - - ^ * •• * < 50 ng of each target DNA 3 'UTR were loaded for staining, ensuring that the target DNA could be present in excess and that the hybridization signals should reflect relative abundance of the • different mRNAs in the template mRNA population. The results of this experiment are shown in Figure 2. In Figure 2, 50 ng of 3 'UTR DNA of the specific gene of each cDNA clone was hybridized to a first cDNA probe of total chain structure from immature leaves. The results of this experiment confirmed the Northern results, with the most abundant transcripts scubi241 / 51 1, less abundant scubi221 and scubi 561 and 5121 at intermediate levels (Figure 2). Since these two independent methods for estimating the relative abundance of mRNA for the different subfamilies of polyubiquitin both indicated that the subfamily scubi241 / 51 1 was the higher, and since the difference between scubi241 / 51 1 and the other subfamilies was too large in both measures, this classification of expression levels is accurate. | Although the expression of these polyubiquitin genes from sugar cane was very little affected by the cell type, several of they responded dramatically to an environmental stimulus: tension for heat. Combinations of mRNAs homologous to scubl221, 561 and 5121 in sugarcane callus tissue subjected to thermal shock at 37 ° C, all developed substantially, while the homologous combination for scub 241/1 1 did not show any substantial increase. In summary, scubi241 and scubi51 1 A_k_H_i_. they absolutely fixed the stereotype of "constitutive" genes, with uniformly high levels of mRNA accumulation in all tissues tested and little induction of expression through extension • thermal. 5 Approximately 1 x 106 pfu of a genomic collection of sugar cane in the vector? EMBL4 were classified with a polyubiquitin coding sequence probe. Fifty positive polyubiquitin plaques were again classified and purified with the scubi241 / 51 1 specific probe. Five positive plaques were isolated. scub¡241 / 51 1 and two of these,? Ubi4 and? Ubi ?, were subcloned into plasmid vectors for further analysis and sequencing. The sequence of the pubi4 subclone and the organization were initially determined as shown in Figures 3A, 3B and 4. Tables 1 -2 provide the meaning of symbols of different sequences to the bases G, A, T and C, which indicate ambiguities where two or more bases are equally possible in the sequences shown in Figures 3A and 3B, • 25 __U_i____ _ £ _ > _- Table 1 Ambiguity codes for the sequences of the u b i4 gene of sugar cane polyubiquitine downstream of the translation initiation codon.

Table 2 Ambiguity codes for the sugar cane polyubiquitin ubi4 gene downstream of the translation stop codon.

In the subsequent sequencing of the pubi4 subclone, the nucleotide sequence (SEQ ID NO: 5) and the translated amino acid sequence / SEQ ID NO: 6) were determined as shown in Figure 5. The letter "X" in the Figure 5B refers to any one amino acid. The organization of the ub4 gene was determined as shown in Figure 6. SEQ ID NO: 5 was cloned as two fragments in the plasmid vector pBluescript I I KS + (Stratagene) to generate the pub14a and pubi4b plasmids. The pubi4a and pubi4b plasmids were introduced in the DH5alpha of Escherichia coli host and the cells of • Transformed Escherichia coli were deposited in the Agricultural Research Service Culture Collection (NRRL) under the terms of the Budapest treaty on March 8, 1999 as N RRLB-301 12 (containing pubi4a) and N RRLB-301 14 (containing pubi4b). N RRLB-15 301 12 contains the 2227 bp of the EcoRI / Sall fragment of SEC I D NO: 5, that is, including 1802 bp SEC I D NO: 7, plus the first repetition of coding and part of the second repetition. N RRLB-f. 301 14 contains the 3329 bp Sall / EcoRI fragment, which includes the remainder of the coding region, 3 'UTR and other sequences downstream. An Xbal restriction site was added at the 3 'end of the ubi4 promoter shown in SEQ ID NO: 7 through PCR amplification with an adaptation primer Xbal. The ubiquitous polyubiquitin ubi4 promoter was ligated upstream of a GUS coding sequence and a NOS 3 'terminator in the _A_á__B_U > _ii_ »? pUC 19 vector plasmid to form pubi4-GUS. This plant expression plasmid was transformed into E. coli DH5a host cells and deposited with N RRL under the terms of the Budapest treaty on March 15, 1999 as NRRLB-301 15. Table 3 shows the codes of ambiguity and the bases they represent in the ubi4 sequences shown in Figures 5 and 10.

Table 3 Ambiguity codes for the ubi4 and ubi9 genes of sugar cane polyubiquitin The pubi4 subclone (Figures 3, 4, 5 and 6) contained four copies of the polyubiquitin coding repeat, 238 bp of 3 'UTR, which was approximately 95% identical to the corresponding region of the scub241 / 51 cDNAs 1, a possible 215 bp 3 'poly-A addition signal from the TAA stop codon, and approximately 2.6 kb from another downstream sequence. Immediately 5 'of the codon of appreciation an intron of 1360 bp was found within SEQ ID NO: 5 (intron of 1382 bp within SEQ ID NO: 1), which was preceded by 65 bp in SEQ ID NO: 5 ( 67 bp in SEQ ID NO: 1) that were 98% identical to the 5 'UTR sequence of scubi241 / 51 1. The transcription start site has not yet been determined, and it is not known if the scubi241 and scubi51 1 cDNA clones contain all the 5 'UTR; however, since the cDNA codons both scub1241 and scub151 started in the same nucleotide, this site can serve as a putative transcription start site for description purposes. The pubi4 subclone contained an additional 377 bp upstream of the transcription initiation codon, with a consensus of TATA at -30 bp relative to the start of the cDNAs. Approximately 320 bp upstream of the start codon of transcription were two 10 bp sequences that showed homology to the consensus sequence of heat stress promoter element (HSE) (aGAAnnTTCt) [Scharf et al .: Heat stress Promoters and transcription factors. In: Nover L (ed) Plant Promoters and Transcription Factors, pp. 125-162. Springer-Verlag, Berlin (1994)]. The second of these 10 bp sequences, however, lacked the G residue in position two that was found to be invariant in HSEs [Scharf et al. (1994) supra]. The subclone sequence pubi? and the organization were • initially determined as shown in Figures 7A, 7B and 4. Tables 4-5 provide the meaning of sequence symbols other than bases G, A, T, and C, which indicate ambiguities where two or more bases are equally possible in the sequences shown in Figures 7A and 7B.

Table 4 • Ambiguity codes for the ubiquitous sugar cane polyubiquitin gene downstream of the translation codon.

Table 5 15 Ambiguity codes for the ubi9 polyubiquitin gene from sugar cane downstream of the translation stop codon.

In the subsequent sequencing of the subclone pubi ?, I nucleotide sequence (SEC I D NO: 8) and the translated nucleotide sequence (SEC I D NO: 9) were determined as shown in the • Figure 8. The letter "X" in Figure 8B refers to any 5 amino acids. The ubi9 gene organization was determined as shown in Figure 6. Table 3, supra, shows the ambiguity codes and the bases representing the gene sequences shown in Figures 8 and 1 1. A fragment of HindI II- EcoRI of approximately 7.2 kb, the A which contains SEQ ID NO: 8 plus an additional downstream sequence of approximately 2 kb, was cloned into the plasmid vector pBluescript II KS + (Stratagene) to form the plasmid pubi ?. This plasmid was transformed into E. coli DH5a host cells and deposited in the Agriculture Research Service collection 15 culture collection (N RRL), under the terms of the Budapest Treaty on March 8, 19TT with accession number NRRLB-301 13 Was a Xbal restriction site added at the 3 'end of the ubi promoter? shown in SEC I D NO: 10 as PCR amplification with an adaptation primer Xbal. The ubi promoter? A) Yes The modified 20 was ligated upstream of a GUS coding sequence and an N OS 3 'terminator in the vector plasmid pUC19 to form pubiγ-GUS. This plant expression plasmid was transformed into E. coli DH5a cells and deposited in N R R L under the terms of the Budapest treaty on March 15, 1999 with accession number N RR LB-301 16. _áÉá_i_ Subclone pubi9 (Figures 4, 6, 7 and 8) contained five copies of the polyubiquitin coding repeat, 244 bp of 3 'UTR, which were 98% identical to the corresponding region of scubi241 / 51 1, and 95 % identical to the corresponding region of pubi4. A possible poly-A addition signal was present 221 bp downstream of the TAA stop codon, and there was an additional downstream sequence of approximately 2 kb. As with the ub4 gene, an intron was located immediately 5 'from the initiation codon; this intron was 1374 bp. 5 'of this intron were 65 f 10 bp (in SEQ ID NO: 8) and 67 bp (in SEQ ID NO: 3) which was 97% identical in both 5' UTR of scubi241 / 51 1 cDNA clones and the corresponding region of the ubi4 gene. The subclone contained an additional 2247 bp of upstream sequence, including a TATA consensus sequence at -30 bp relative to the start of the clones cDNA. Upstream of 5 'UTR, a region of 577 bp of ubi? from position 1671 to 2248 of SEC I D NO: 10 was highly homologous (identity of > T0%) to the corresponding region of ubi4 -f (positions 1 to 377 of SEC I D NO: 5). The partial sequence from an additional subclone of? Ubi4 indicated that this high degree of homology continues at least as much as 2 kb upstream of the transcribed region of the genes (unpublished data). Within this highly homologous region, approximately 344 bp upstream of the start codon of transcription, is an apparent insert of approximately 200 bp not present in the ubi4 promoter of cane of sugar. This region of 200 bp was delimited by imperfect inverted repetitions of 17 bp. A region of 202 bp 82% identical to this insert was also found in 3 'UTR of a clone of glucose transport cDNA from sugar cane, SGTI [Bugos et al., Plant • Physiol. 103, 1469-1470 (1993)]. The nature of this possible case 5 of insertion has not been investigated; however, it has characteristics of miniaturized inverted repeat transposon elements (MITEs) [Wessler et al., Curr Opin Genet Dev 5, 814-21 (1995)]. Without limiting the invention to any particular mechanism, this insertion is not believed to have a functional role in f) promoter activity of ubi9 polyubiquitin promoter, since this insert is inserted in 3 'UTR (not the promoter) of the transport gene of glucose, since it is not present in the ubi4 polyubiquitin gene. As the gene ubi4 the gene ubi? also contained two HSE-type sequences of approximately 320 bp upstream of the start codon of transcription; however, both sequences of HSE type lacked the invariant G residue. A comparison of the sequences upstream of the codon -f of translation start of the ubi4 gene sequence of sugarcane with the corn polyubiquitin promoter (access number of GenBank S94464; Quail et al., Patents of E.U.A. 5,614,399 and 5, 510,474) showed only 51% homology when compared to a fragment containing the upstream sequence of 5 'UTR, the 5' UTR sequence and the intron sequence, only 64% homology when compared to a fragment containing the sequence upstream of the start codon of Transcription, only 65% homology when compared to a fragment containing the 5 'UTR sequence and only 58% homology when compared to a fragment containing the intron sequence. A comparison of the upstream sequences of the translation initiation codon of the ubi9 sugarcane gene sequence with the maize polyubiquitin promoter (accession number of GenBank S94464; Quail et al., Patents of E.U.A. Nos. 5,614.39 and 5,510,474) showed only 65% homology when compared to a fragment containing the upstream sequence of 5 'UTR, the 5' UTR sequence and the intron sequence, only 63% homology when compared to a fragment containing the sequence upstream of the start codon of transcription, only 66% homology when compared to a fragment containing the 5 'UTR sequence, and only a 5T% homology when compared to a fragment containing the intron sequence. It has been found that some polyubiquitin promoters capable of heat shock induction contain [HSEs (Binet, and others, 1991, supra; Christensen et al. (1992) supra]. Both ubi4 and ubi9 genes contained two short sequence elements that have some homology to HSEs; however, three of four of these elements lacked a G residue that was found to be invariant at position 2 of [HSE (aGAAmTTCt) (Scharf et al., (1 TT4) supra.] Given the very marginal induction _á_ßs_i_fa __ = _ a _ ^^ T3 (about twice or less) of the ubi4 and ubi ?, genes when compared with other sugar cane polyubiquitin genes, it is doubtful that these HSE-type elements play a role • important role in the regulation of gene expression. Similar observations have been made regarding the tobacco ubiquitin promoter, U bi. U4; although this promoter also contains "... two elements of degenerate thermal shock type ...", the removal of these elements has no important effect on gene expression (Plesse, et al. (1 TT7) supra]. Both ubi4 and ubi? Genes contained a large intron • immediately upstream of the protein coding region preceded by a fragment of approximately 65 bp, which was highly homologous to 5 'UTR of scub / 241/51 1. An intron in this position (that is, immediately upstream of the codon of initiation) has been found in many other plant polyubiquitin genes (Binet et al., 1 TT1, supra, Christensen and others (1 TT2) supra; Garbarino and others (1 TT5) supra; Norris et al., Plant Mol Biol • 21, 8T5-T06 (1993)], and in some cases it has been shown to be important for high levels of expression (Norris et al. (1993) supra, Garbarino et al. (19T5) supra]. Through analogy, it is the inventors' belief that the intron of the ubi4 and ubi? It can also play an important role in the regulation of gene expression. The cDNA clones scubi241 and scubi51 1 contain three and In five copies of the polyubiquitin coding sequence, T4 respectively, the pubic and pubin9 genomic clones contain four and five copies of the polyubiquitin coding sequence, respectively. Without limiting the invention to any • particular mechanism, this may represent that the subfamily 5 scub¡241 / 511 contains at least three different genes, containing three (ie, scubi241 / 511), four (ie, pubi4) and five (ie pubi). 9) ubiquitin repeats. Alternatively, it is possible that the difference in the number of copies of the polyubiquitin coding sequence reflects a difference between the 10 cultures, with the subfamily scub¡241 / 511 in H65-7052 containing • genes with three and five repeats of ubiquitin, while the same subfamily in H32-8560 contains genes with four and five repeats of ubiquitin.

EXAMPLE 2 Construction of Pubi4-GUS, pubi9-GUS, 4PI-GUS and 9PI-GUS Plasmids Comprising Sugar Cane Polyubiquitin Promoters and an Illustrative Structural Gene Report plasmids were made by placing the uid A gene encoding β-glucuronidase (GUS) [Jefferson et al., Proc. Nati Acad. Sci. USA 83, 8447-8451)] and the nopaline synthase (NOS) terminator under the control of the sugar cane polyubiquitin promoter described herein. The promoter sequence in the pubi4- 25 GUS plasmid contained nucleotides 1-1810 of SEQ ID NO: 1 (i.e. ^, ^ »-. ^. nucleotides 1 -802 of SEQ ID NO: 5) and an Xbal site (TCTAGA) added immediately after 1802 bp, through an adapter in a PCR primer. The promoter sequence in the • plasmid, pubi9-GUS, contained nucleotides 1 -3688 of SEQ ID NO: 3 (ie, nucleotides 1-3688 of SEQ ID NO: 8) and the Xbal site (TCTAGA) added immediately after 3688 bp, through an adapter in a PCR initiator. The Expand ™ PCR system (Boehringer Mannheim) was used to amplify part of 5 'UTR and the intron including the 3' cleavage site; this PCR product i? 10 was used to join the upstream sequence of 5 'UTR, the 5' UTR, the intron to the GUS gene using a unique Nrul site in 5 'UTR and an Xbal site added as an adapter to the 3' PCR primer. pHAT contained a ubiquitous maize promoter that directs a phosphotransferase I I gene of neomycin (N PTI I) and the NOS terminator. This was done by removing the luc gene from pAHC18 described in [Christensen et al., Transgenic Research 5, 213-218 (1 T96)] as the BamH ly fragment and replacing it with an 844 bp BamH I fragment containing the NPTII gene. . 35S-GUS contained the uid A gene encoding GUS under control 20 of the 35S RNA promoter sequence of cauliflower mosaic virus (CaMV), (Clontech). Binary plasmids 9PI-GUS, 4PI-GUS and MPI-GUS were made for transformation by Agrobacterium by ligating the promoter-intron-GUS-NOS cassettes from pubi? -GUS, pubi4-GUS, and pAHC27 [Christensen and others (1 T96) above], respectively, as • HMIllMllÉli ^ bA ^^.

T6 HindI I-EcoRI fragments, to the HindIII-EcoRI sites of pCAMBIA 1300 [Roberts et al., Rockefeller Foundation Meeting of the International Program on Rice Biotechnology, Malacca, Malaysia (1 TT7)]. The binary plasmid pHW537 contained a putative 5 'nuclear matrix (MAR) binding region from the? Ubi4 promoter, ubi? and GUS intron, and the putative 3 * and MAR 39 terminator of? ubi4 as the H indl l-EcoRI fragment in the HindIII-EcoRI sites of pCAMBIA1300.

EXAMPLE 3 • 10 Passenger Expression of pubi4-GUS and pubi9-GUS in Cultivated Cells of Suspension of Sugar Cane Sugarcane suspension cell cultures (variety H50-7209) were maintained as described by [Nickell et al., Physiol. Plant 22, 1 17-125 (196T)]. The DNA report plasmids (pubi4-GUS, pubi? -GUS, or pAHC27) were introduced into sugarcane suspension culture cells through particle bombardment as previously described [Klein et al., Nature 327, 70 -73 (1987)] using a PDS1000 particle accelerator Biolistic (BioRad) at 77.33 kg / cm2. The controls were not bombarded with DNA. Passive trials were performed in a randomized complete block design with four treatments (promoters) and six replications. Each replication consisted of the bombardment of five samples. Two days after the bombing, it analyzed the plant material for the expression of G US Each ÜM ^ IlH ^ aÜM-.

T7 sample was divided into two equal parts, one for histochemical analysis and the other for chemiluminescent measurement of the activity of the GUS enzyme using a GUS-Light kit.

• (Tropix) and a MLX plate reader luminometer (DYNEX). The GUS enzyme activity assays were performed according to the manufacturer's protocol, with 60 minutes of incubation in the GUS reaction pH regulator before measuring the chemiluminescence. The GUS activity was expressed as relative light units (RLU) per nanogram of total protein [Bradford. Anal. Biochem 72: 248-254 (1 T76)]. From each experiment, the most valuable values were discarded • high and low for each plasmid. The analysis of the variation in the data of each group of experiments was carried out; those experiments that exhibited a significant effect (P <0.05) of the promoter treatments were also analyzed through a least significant difference test (P <0.05) to identify which promoters produced significantly different results. The results of histochemical staining and assays of GUS activity of chemiluminescence of suspension culture cells of sugarcane, which had been bombarded with the report plasmids, are shown in Figure?. Figure TA shows that the average number of blue foci detected after bombardment with GUS expression plasmids containing the ubiquitous promoter. was mauro that observed for either the ub4 or ubil corn polyubiquitin promoters [Christensen et al., (1 TT6), supra]. Due to the high levels of T8 variability, it should not be determined from the histochemical staining of these passenger expression experiments if the results seen in the sugarcane callus are actually • significantly different. However, using a chemiluminescent assay to measure G US activity again indicated the average level of expression that was higher for the ubiquitous promoter. that for the ubi4 promoters of sugarcane or maize ubil (Figure TB). The statistical analysis indicated that the difference, as measured by this assay was significant at P < _0.05. 10 These data demonstrated that both the ubi4 promoters and the • ubi? in pubi4-GUS and pubi9-GUS, respectively, were sufficient to direct transient expression in suspension cells of monocot sugar cane.

EXAM PLO 4 Expression Passenger of pubi4-GUS and pubi9-GUS in Tobacco Leaves • A Wisconsin 38 tobacco culture was developed in boxes of 20 Magenta in an MSNT medium [salts of Murashige and Skoog IX (GI BCO BRL # 1 1 17-074), minimum organic products IX (GI BCO BRL # 1 1 1 18-023), 30 g / l sucrose, and 0.8% agar] at 26 ° C under a 16-hour light regime. The DNA report plasmids (pubi4-GUS, pubi? -GUS, or pAHC27) were introduced into the leaves of tobacco through bombardment of particles as described? previously [Klein et al., (1 T87) supra] using a Biolistic particle accelerator PDS1000 (BíoRad) at 45.76 kg / cm2. The controls were not bombarded with DNA. Passenger trials were carried out • in a randomized complete block design with four treatments (promoters) and six replications. Each replication consisted of the bombardment of 5 samples. Two days after the bombing, the plant material was analyzed for GUS expression. Each sample was divided into two equal parts, one for histochemical analysis and one for chemiluminescent measurement for the activity of the GUS enzyme as described above • (Example 3). The results of histochemical staining and GUS chemiluminescent activity assays of the tobacco leaves, which were bombarded with the report plasmids, were show in Figure? The results show that the average G US expression was higher for the ub? 4 sugarcane promoter than for the corn polyubiquitin promoter or the ubiquitous promoter. from • sugar cane when either histochemical (Figure TC) or chemiluminescent (Figure 9D) tests were used. The analysis of chemiluminescent data indicates that the difference between the ubi4 promoters of sugarcane and corn polyubiquitin was statistically different from P < 0.05. These data show that both the ubi4 and ubi? in pubi4-GUS and pubi9-GUS, respectively, were sufficient to direct the transient expression in dicotyledonous tobacco leaves EXAMPLE 5 Passenger expression of pubi4-GUS and pubi9-GUS in Callo de Sorgo • Tripe derived from immature sorghum embryos was cultivated and bombarded as previously described with the ubi4-GUS or ubi-GUS reporter plasmid (prepared as described in Example 2) and with several reporter plasmids where the gene uid A that encodes GUS was placed under the control of each of the various promoters including adh of corn 35S, 35S: 35S, rice actin and corn stalk, as described further • ahead.

A. Culture media Maintenance medium N6 [macro elements (concentration final mg / l), 2830 KNO3, 1650 (N H4) 2SO4, 166 CaCl2-2H2O, 185 MgSO -7H2O, 400 KH2PO4; micro elements (concentration mg / l), 37. 3 Na2 EDTA, 27.8 FeSO4-7H2O, 1.6 H3BO3 0.76 Kl, 3.3 MnSO4, 1.5 ZnSO4-7H2O; carbohydrates (final concentration g / l), sucrose 20; hormones (mg / l concentration), 1.0.2, 4-dichlorophenoxyacetic acid; vitamins (final concentration mg / l), 0.5 thiamine-HCl, 0.25 pyridoxine-HCl, 0.25 nicotinic acid, amino acids (final concentration mg / l), 2875 l-proline, 2.0 glycine, 100 casamino acids; and agar (final concentration g / l), and 2.5 Phytagel], was used. -_____ B. Plant material. A highly embryogenic callus tissue derived from sorghum plants was used (Sorghum bicolor L. Moench, cv Bwheatland • 3TT). To establish the callus cultures, caryopses, 10 to 18 d of 5 posterior anthesis, were sterilized on the surface with 70% ethanol for 20 minutes and 20% chlorine bleach for 15 minutes, followed by two changes of distilled water. Immature embryos, 1.0 to 1.5 mm in length, were aseptically removed using 1 1 cm sterilized forceps in a laminar flow dome under a stereo dissecting microscope. The embryos were placed with the escutiform part exposed in the N6 medium modified for the sorghum cell culture and solidified with 2.5 g / l Phytagel.

C. Preparation of microprojectile bombardment Before bombardment, 1 mm gold particles were coated with transforming DNA through the procedure of Daines (1990). A suspension of supply of gold particles was suspended at 60 mg / ml in absolute ethanol. They were transferred 35 microliters of the suspension to a 1.5 ml microcentrifuge tube, centrifuged at 14,000 g for 3 minutes, and the pellet was suspended in 200 μl of sterile distilled water. After a second centrifugation, the pellet was suspended in 25 ml of tris-EDT containing 25 mg of the transformation plasmid DNA. The The following sterile cooled solutions were added in order: 200 ml of water, 250 ml of 2.5 M of CaCl2, and 50 ml of 0.1 M of Spermidine (sterilized 0.2 μm filter). The microcentrifuge tubes were shaken with a Tomy microtube shaker at 4 ° C for 15 minutes and centrifuged at 16,000 g for 5 minutes. The supernatant was removed, the pellet was washed with 200 ml of ethanol and the gold particles coated with DNA were suspended in 36 ml of ethanol.

D. Establishment and bombardment of target tissue Immature embryos were removed from sorghum cariopses and cultured in a N6 maintenance medium for 7 days. If the immature embryos are smaller than 0.5 mm, they can die in the culture, and if they are greater than 1.5 mm, they can germinate early instead of starting the callus tissue. Four hours before the bombardment, approximately 50 embryo-derived calli were placed in a circle (4 cm diameter) in the center of a Petri dish (15 x 100 mm) containing 0.2 M mannitol and 0.2 M sorbitol in the medium maintenance N6 solidified with 2.5 g of Phytagel. The petri dish containing the target callus tissue was placed in the biolistic device and 10 ml of the DNA-gold suspension was pipetted onto the center of a macroprojectile. The distance between the stop plate and the target callus tissue was adjusted to 13 cm. The fabric was bombarded under vacuum with the resistance to rupture of the disk at 77.33 kg / cm2. Callus tissue was sampled one day after bombardment using the GUS histochemical assay. Approximately 20 replicates were made for each promoter. The average number of blue sites per bombarded plate was determined in each report plasmid using a stereo microscope. 5 The ubiquitous nucleotide sequence was sufficient to direct transient expression in monocotyledonous sorghum. In fact, the ubi4 nucleotide sequences resulted in significantly higher levels of GUS expression as compared to the levels of expression directed by each of the other 10 promoters tested (data not shown). This results • demonstrate that the ubi4 promoter in pubi-GUS was sufficient to direct Ig transient expression in monocotyledonous sorghum callus.

EXEM PLO 6 15 Passenger Expression of pubi9-GUS in Pineapple Leaves, Bodies, Roots and Fruits of the Protocormo Type f Pineapple culture leaves F153, protozoan-type bodies (plbs), roots and fruits were bombarded with a reporter plasmid [pAHC27, pubi-GUS or 35S-GUS], described above (Example 2). The target tissue (leaves, plbs, roots and fruits) was placed in plates in the center (2.5 cm in diameter) of Petri dishes in a modified MS medium, supplemented with 0.8% Difco Bacto agar and 3% sucrose. The bombardments were carried out with a 25 microprojectile accelerator operated by Bio-Rad helium gas (PDS-1000 / He, Bio-Rad) with rupture discs of 77.33 kg / cm2. The gold microcarriers (diameter 1.6 μm, Bio-Rad) were coated with AD N using a CaCl2 precipitation method following the instructions of the • maker. 2 μg of every 5 construction of AD N were used for each shot or shot. GU S histochemical staining was performed 48 hours after the bombardment to determine the transient transformation. They were made or 3 replicas for each promoter in each type of tissue bombarded. The number of blue spotlights / plate of each tissue bombed is shown in Table 4. • Table 4 • a Standard error 15 The above data demonstrates the successful transient expression of GUS under the control of the ubi promoter? of sugar cane polyubiquitin in pubic? -G U S in each of the four monocotyledonous pineapple weave.

EXEM PLO 7 Stable Expression of pubi4-GUS and pubi9-GUS in Transgenic Sugar Cane Callus Sugarcane callus cultures were started from stem apices (variety H62-4671) by sterilizing the surface of the plant material with 70% ethanol, cutting 2 mm transverse slices and developing on MS2 plates [1 X Murashige and Skoog salts (GIBCO BRL # 1 1 17-074)], 1 x minimum organics (GI BCO BRL # 1 1 1 18-023), 2 mg, L 2,4-D, 0.7% agar] low, a light regime of 16 hours. After 1 or 2 months, the calluses were transferred to MS 1 plates [IX salts Murashige and Skoog (GI BCO BRL # 1 1 17-074), 1 X minimum organic products (GIBCO BRL # 1 1 1 18-023) , 1 mg / L 2,4-D, 0.7% agar], and subcultured monthly. The sugarcane callus was co-bombarded with reporter plasmid (pubi-G US, pubi9-G US p pAHC27) and the selection plasmid pHA9, which contained the ubil maize promoter that directs a phosphotransferase II gene from neomycin (N PTI I) (prepared as described above in Example 2). The bombing was with a miera of gold particles and 108.? kg / cm2 of rupture discs. After the bombardment, the callus was kept on MS 1 plates without selection for 2 weeks. After their recovery period, the calluses were transferred to plates of MS 1 with 50 mg / L G418 (Agri-bio) for 1 month, then transferred to plates of MS 1 with 100 mg / L G418 for 2-3 months . The calluses that survived this selection were transferred to plates of MS 1 with 60 mg / L G418 for multiplication. The small calluses that • total about 50 were randomly selected 5 of selected lines. These callus were analyzed for GUS activity using the GUS-Light kit (Tropix) according to the manufacturer's protocol, with 30 minutes of incubation in a GUS reaction pH regulator before measuring chemiluminescence. We analyzed from 2 to 10 samples of 50 mg a from each line, with three chemiluminescence assays for • each sample. The results of the GUS chemiluminescent activity assays are shown in Figure 12. In 10 lines of sugarcane callus independent transgenic selected, the GUS expression of the ubi promoter? of sugarcane averaged 535.4 RLU / ng protein / 30 minutes (Figures 12A, 12D). Seven stable transgenic lines selected by expressing • GUS under the control of the ubi4 promoter of sugarcane averaged 20T.3 RLU / ng of protein / 30 minutes (Figure 7B and 7D), and 7 GUS expression lines under the control of the ubil corn promoter averaged 348.2 RLU / ng protein / 30 minutes (Figure 7C and D). These results show that both the ubiquitous and ubiquitous nucleotide sequences. in pubi4-GU S and pubi? -GUS, respectively, were sufficient to direct the stable pressure in a monocotyledonous sugarcane callus.

EXAMPLE 8 • Stable Expression of 9PI-GUS in Transgenic Rice Callus The EHA105 strain of Agrobacterium was used [Hood et al., J. Bacteriol. 168: 1291-1301 (1986)] to transform rice callus. The report plasmids (TPI-GUS, MPI-GUS, or pHW537, which were prepared as described in Example 2 above) were introduced in the EAH105 strain of Agrobacterium through a • standard procedure. The rice callus was induced from rice scutelle tissue (cv Taipei 30T) and transformed through co-cultivation of Agrobacterium as previously described [Hiei et al., (1T94) above]. After 2-3 months in a selection medium containing 100 mg / l of hygromycin B (CalBiochem), small callus of the selected lines were analyzed for the GUS activity at jf through the chemiluminescence method described above (Example 3). 20 PCR using an initiator within T-DNA and one outside the right border of T-DNA was used to utilize the absence of contamination by Agrobacterium in callus lines tested using methods known in the art. Since the transformation of rice was through Agrobacterium, there is possibility that the Agrobacterium was not annihilated after The transformation, and thus the GUS expression seen is from Agrobacterium, not from the transgenic rice cells. To test this possibility, two PCRs were performed: one to test the presence of the GUS gene, the second to test the vector plasmid sequences outside the T-DNA. The Agrobacterium hosts a binary plasmid, a section of which contains the GUS coding gene under the control of promoter sequences of sugarcane. This section of the vector plasmid is referred to as T-DNA, and only this part is ordinarily transferred to the plant genome. A positive PCR ^ p for GUS indicates that the isolated plant DNA can be successfully amplified by PCR and the GUS gene is present (ie, confirming the observation of GUS activity). A negative PCR for the vector plasmid outside the T-DNA confirms that all the plasmid, which is present in Agrobacterium, is no longer present, ie, that the T-DNA (which contains the GUS sequences under the control of sugarcane promoter sequences) is successfully integrated into the plant genome. One, seven, four and two callus lines of transgenic rice stably transformed following the co-cultivation of the rice callus with Agrobacterium, which had been transformed with the report plasmids 4PI-GUS, 9PI-GUS, pHW537, were selected. and MPI-GUS, respectively. The expression of GUS in six 25 transgenic lines transformed with the promoter sequence ubi? ._____-_ k_t _ - _, -___ * _ Miii _ * _ ii. is shown in Figure 1 3. The expression of GUS in six transgenic lines transformed with the ubiquitous promoter sequence. it averaged 681.0 RLU / ng of protein / 30 minutes. • Four stalk lines of transgenic rice 5 were stably transformed following the co-cultivation of the rice callus with Agrobacterium, which had been transformed with the report plasmid pHW537. The expression of GUS in these transgenic lines averaged 567.7 RLU / ng protein / 30 minutes. Since the reporter plasmid pHW537 differs from the TPI-GUS plasmid in that it also contains the nuclear matrix binding regions of • putative 5 'and 3' flanking (MARs), and since both the TPI-GUS and pHW537 report plasmids successfully resulted in the expression of comparable levels of GUS in rice callus, these results demonstrate that 5 'nuclear MARs and 3 'putative no. 15 are necessary for the promoter activity of the pubic sequences. The results also suggest that either the promoter of f | ub4 or ubi? of polyubiquitin (with or without putative MARs) directs GUS expression at levels comparable to the ubiquitous 20-corn promoter in stable transgenic rice callus. The previous results show that the promoter ubi? in 9PI-GUS and pHW537 was sufficient to direct stable expression in monocotyledonous rice callus. -b_b_ta_ai_M- EXAMPLE 9 Stable Expression of? ubi4-GUS and pubi9-GUS in Transgenic Tobacco Sheets Agrobacterium tumefaciens was used to transform tobacco leaves. The report plasmids (9PI-GUS, 4PI-GUS, MPI-GUS, or 35S-GUS, which were prepared as described in Example 2, above), were introduced into the leaf discs through a procedure of transformation mediated by Agrobacterium adapted from Horsch and others Science 227: 122? -1231 • (1T85). The controls were not transformed. In summary, 2 ml of the overnight cultures of Agrobacterium tumefaciens were pelleted by short centrifugation, decanted and suspended in 2 ml of medium.

MSNTS liquid (per liter of medium: one package of MS salt mix [GIBCO BRL # 11117-066], 30 g of sucrose, 1.0 ml of 1000X of vitamins B5, 50 μl, 2 mg / ml of acid a- Naphthalene Acetate [GIBCO BRL • # 21570-015], and 50 μl, 20 mg / l of benzyladenine [GIBCO BRL # 1610-017]). The aseptically developed tobacco leaves were harvested and placed in Petri dishes containing 20 ml of MSNTS with 0.6 ml of resuspended Agrobacterium and cut into approximately 1 cm squares with a sterile scalpel. After 1 to 5 minutes, the pieces of leaves were removed from the liquid, stained moderately on a dry sterile paper, and placed on MSNT plates of 0.8% agar (per liter of medium: one MS salt mix package [GI BCO BRL # 1 1 17-066], 30 g of sucrose , 1.0 ml of 1000X of vitamins B5, 8 g of Bacto-Agar) with 500 μg / ml of Cefotaxime (Phyto Technology Laboratories). After 2 days, the 5 pieces of leaves were transferred to MSNTS plates of 0.8% agar with 500 μg / ml of Cefotaxime and 100 μg / ml of Canamycin (Phyto Technology Laboratories). Outbreaks appeared for 2 to 3 weeks, at that time, the outbreaks were transferred to Magenta boxes containing the MSNT medium without antibiotics. To confirm the resistance to kanamycin and to reduce the likelihood of obtaining • Chimeric plants, a "trigger test" was used: leaves of putative transgenic plants were placed on MSNTS plates of 0.8% agar containing 100 μg / ml kanamycin. The outbreaks that emerged from these "firing tests" were transferred to non-selective root formation media (MSNT) and were developed in Magenta boxes. The expression of GUS was determined as described in • Example 3 above. The results show that both ubiquitous and ubiquitous nucleotide sequences in 4PI-GUS and TPI-GUS, respectively, were sufficient to direct stable expression in dicotyledonous tobacco leaves. In addition, the levels of GUS expression under the control of either the ubiquitous nucleotide sequences as ubi? in 4PI-GUS and TPI-G US were equal to or greater than the levels of GUS expression under the control of the promoter ubi l. On the other hand, the expression of GUS under the control of the CaMV 35 S promoter was greater than the control of either the ubi4 or ubi? Promoters. of sugar cane (data not shown).

EXAMPLE 10 5 Stable Expression of pubi-GUS and? Ubi9-GUS in Transgenic Corn Embryos and Regeneration of Transgenic Maize Plant Embryogenic corn cultures were started from immature corn embryos, bombarded simultaneously with a report plasmid (pubi4-GUS, pubi9-GUS, or mzubM-GUS) • and a selection plasmid (pHAT), and stably transformed maize plants regenerated as previously described by Brown et al., U.S. Patent. 5, 5T3,874, incorporated herein by reference. 15 In summary, embryogenic maize cultures were initiated from immature maize embryos of the "Hi-lr" genotype, which was cultivated 18-33 days in a medium of N6 2- 100-25-Ag modified jf to contain 2 mg / l 2,4-dichlorophenoxyacetic acid, 180 mg / casein hydrolyzate, 25 mm L-proline, 10 μm nitrate silver, pH 5.8, solidified with Phytagel ™ (Sigma) at 0.2%. These embryogenic cultures were used as the target tissue for transformation thr bombardment of particle guns. A 1: 1 mixture of the report vector (pubi-4-GUS, pubi-G US or mzubi I. GUS) and the selection plasmid (pHAT) was precipitated on M 10 particles of tungsten by adding 12.5 μl of particles (25 mg / ml in 50% glycerol), 2.5 μl of plasmid DNA (1 μg / μl), 12.5 μl of 1 M calcium chloride, and 5 μl 0.1 M of spermidine, and swirled briefly. The particles were allowed to settle for • 20 minutes, after which 12.5 μl of 5 supernatant were removed and discarded. Each sample of tungsten-DNA was applied sound briefly and 2.5 μl were bombarded in embryogenic cultures using a particle gun PDS-1000 Biolisitics (DuPont). The bombarded tissue is transferred to a non-selective medium, fresh, the day after the bombing. Six days after • bombardment, the material is transferred to selective media containing 50 mg / L G418 (Agri-bio). After 2-3 weeks, the cultures are transferred to fresh media which contains 200 mg / L G418. The cultures were maintained in the media of 200 mg / L G418, were transferred at intervals of 2-3 weeks, until the callus resistant to G418 could be distinguished. The calluses resistant to G418 were recovered from the embryogenic material. The lines • resistant to G418 were pooled and analyzed for GUS expression using a histochemical or chemiluminescent assay as described above (Example 3). The plants were regenerated from callus resistant to G418, which expressed GUS activity in a three step regeneration protocol. All regeneration was performed at 200 mg / L G418. The first two steps were carded in the dark to 28 ° C, and the final step under a photo period of 16: 8 hours, at about 25 ° C. The small green shoots that formed in the regeneration medium 3 in Petri dishes of 100X 25 mm were transferred to the regeneration medium 3 in 200X 25 mm • PyrexTM or PhytatraysTM to allow the further development of 5 glands and root formation. After the formation of a sufficient root system, the plants were carefully removed from the medium, the root system was washed under running water, and the plants were placed in 6.35 cm pots containing the Metromix 350 growth medium. they kept for several days in a high humidity environment, and then the humidity was gradually reduced to harden the plants. The plants were transplanted from the 6.35 cm pots into 15.24 cm pots, and finally into 25.4 cm pots during growth. Corn plants regenerated from embryonic calluses resistant to G418, expressing GUS activity, were tested for GUS expression using histochemical or chemiluminescent assays as described above (Example 3). The expression of GUS (for example, as determined by staining with blue color in the histochemical test or through luminescence in the chemiluminescent assay) by one or more maize plant tissues that were generated from calluses that were bombarded with pubi4-GUS or pubi? -GUS, demonstrates that the ubiquitous and ubiquitous nucleotide sequences are present. in pubi4-GUS and pubi-GUS, respectively, are sufficient to direct stable expression in corn plants regenerated monocots.

EXAMPLE 11 Stable Expression of 4PI-GUS and 9PI-GUS in Transgenic Tomato Plants # 5 A report plasmid (4PI-GUS or TPI-GUS, prepared as described in Example 2) or a control plasmid (ie, a plasmid containing the uid A gene encoding GUS and lacking a promoter sequence) were transformed into Agrobacterium, and the transformed Agrobacterium was used to infect plants of tomato as previously described in Theologis et al., Patent • from E.U.A. No. 5,723,766 incorporated herein by reference. Briefly, a report plasmid or a control plasmid was introduced into Agrobacterium strain LBA4404 as follows: it was grown overnight at 28 ° C Agrobacterium tumefaciens LBA-15 4404 (2 ml), in LB broth and this was used to inoculate 50 ml of the LB broth to obtain the desired culture. The inoculated medium was developed at 28 ° C until OD.6oo is 0.5-1.0. The cells were harvested by centrifugation and the pellet was resuspended in 1 ml of 20 mM ice-cold CaCl2. To 100 μl of the cell suspension, 1 μg of the plasmid was added, and the mixture was incubated on ice for 30 minutes before freezing in liquid nitrogen. The cells were then thawed at 37 ° C for 5 minutes and used to inoculate 1 ml of LB. After 2 hours of growth at 28 ° C with shaking, they were placed in plates 100 μl of the culture in a medium of LB + kanamycin5", was expected - * - ** - - - * ----- colonies appear in 2-3 days at 28 ° C. The cells were again cultured by collecting several colonies and scratching the LB + kanamycin 50 medium; again, 3-4 striped colonies were collected • independent and 5 ml of cultures in the medium of LB + kanamycin50 were developed. Fixed phase cultures were used for the transformation of tomato plants, which were developed as described above. The transformed Agrobacterium cells were frozen using 15% glycerol at -80 ° C for final use. To prepare host tomato plants, tomato seeds were sterilized using a protocol consisting of treatment with 70% ethanol for 2 minutes with mixing; followed by treatment with 10% sodium hypochlorite and 0.1% SDS for 10 minutes with mixing, followed by treatment with 1% sodium hypochlorite, 0.1% SDS for 30 minutes with mixing, and washing with sterile water three times for 2 minutes per wash. For germination of the sterilized seeds, 0.8 g of the sterilized seeds were placed in a seed germination medium and grown for two weeks with low light in a quarter of development. After two weeks, when the seeds germinated, the cotyledons of the crops were cut by cutting the tips of the cotyledon and then cutting the stem. This process was conducted in large Petri dishes containing 5-10 ml of the MSO medium. Feeding plates were prepared from a tobacco cell suspension in a liquid medium at 25 ° C, prepared with stirring at 130-150 rpm. The suspension was transferred to a fresh medium at 1: 10 dilution every 3-5 days. 1 ml of • Rapidly dividing culture on the feed plate, covered with 5 filter paper and placed in low light in a growing room. The feeding plates were supplemented with 10 ml of the feeding medium. The transformed Agrobacterium cultures, which contain the report or control plasmids, were inoculated in 50 ml of LB containing kanamycin with a single colony of the strain. The culture was developed by shaking vigorously at 30 ° C until • saturation (OD> 2.0 to 600 m). The strain was selected to fully develop in less than 24 hours. The culture was then diluted 5 times and divided into 50 ml portions in plastic tubes. 15 The cotyledons from two of the feeding plants were scraped into each tube and rotated moderately during -30 minutes. The cotyledons were then removed from the bacterial AFK culture on a filter paper (abaxial top part) in a tobacco feed plate and incubated for 48 hours. with low light in a growing room. The cotyledons were then transferred upward axially to the callus induction medium. In the callus induction medium, approximately four plates were used per Magenta box, and the explants were swirled. The box was placed in a growing room during 3 weeks, and it was expected that small masses of change would form on the surface of the cotyledons. The explants were transferred to fresh plates containing the callus induction medium every 3 weeks. When the calluses exceeded 2 ml, these were • transferred to plates containing an outbreak induction medium. 5 When the stem structure is evident, the shoots are cut from the calluses and the shoots are transferred to plates containing the root induction medium. After a vigorous root system is formed in the plants, the seedlings are transferred to the soil by taking the seedlings from the plates, removing the agar that is possible and placing the plants in a land with a high content • peat in a small peat pot, which is fixed in a Magenta box with a cover. When the sowing leaves reach the top of the box, the lid loosens to reveal the box slowly over a period of 4-5 days. The plants are then transferred to a light chart and larger pots, and they stay moist. The flowers of these regenerated plants are polinated and tomatoes are grown.

• GUS expression is measured in tissues of regenerated tomato plants transformed with report or control plasmids using a histochemical or luminescent assay as described above (Example 3). The expression of G US by one or more tissues of tomato plants, which were generated from the tissue that was transformed with 4PI-GUS or TPI-GUS, demonstrates that the nucleotide sequences ubi4 and ubi? in 4PI-GUS and TPI-GUS, respectively, are sufficient to direct stable expression in regenerated dicotyledonous tomato plants.

EXAMPLE 12 Stable Expression of Pubi4-GUS and Pubi-GUS in Embryonic Meristems Cut from Transgenic Soy and Regeneration of Transgenic Soy Plant Soy explants from cut meristems were derived, bombarded simultaneously with a report plasmid (pubi4-10 GUS, pubi? -GUS or mzubi1 -GUS) and a selection plasmid (pHAT), • and stably transformed soybean plants were regenerated as previously described by Christou et al., U.S. Patent. A. 5,015, 580, incorporated herein by reference. In summary, the soybean explants of the Williams 82 culture are derived from cut meristems of the embryonic axes of immature seeds. The primary leaves were removed and the explant was placed in a plate on a target plate containing agar from • 1% water. The explants were transformed with a reporter plasmid or a selection plasmid loaded at 1.0-0.001 μg / ml of pearls The particle accelerator was charged at 13-16 kV. The carrier was loaded with 0.05-0.40 mg of charged beads per cm2, with a preferred loading level of 0.2 mg / cm2. The bombed explants were then plated in the dark in a modified basal MS medium, which has a High level (ie, 13.3 μM) of cytokinin benzylaminopurine.

After incubation for 1 to 2 weeks in the dark, the tissues were transferred on the same basal medium at a lower level (1.7 μM) of cytokinin to promote elongation of the • outbreak The shoots were harvested at a height of 0.5 to 1 cm. The success of the transformation protocol is verified by fixing the transformed explants at each stage to analyze the GUS activity as described above (Example 3). Two days after the injection of the DNA particle, it was expected that dozens of active G-US cells were detected in each explants. At 6 to 8 weeks, the plants were analyzed for • GUS activity in the outbreak. Most plants were expected to be chimeric, having blue stripes (ie, GUS expression cells) when analyzed using histochemical analysis. The transformed cut meristem tissue was used to regenerate sexually mature and fully mature chimeric plants, using methods known in the art such as those described in the U.S. patent. A. No. 5, 015, 580 of Christou et al. (Incorporated herein by reference). The expression of G US through one or more tissues of soy plants, which were regenerated from cut embryonic meristems that were transformed with pubi-4-GUS or pubi? -GUS, demonstrates that the ubiquitous and ubiquitous nucleotide sequences? in pubi4-G US and pubi? - G U S, respectively, are sufficient to direct the expression stable in regenerated dicotyledonous soybean plants.

From the foregoing, it is evident that the invention provides promoter sequences that are capable of directing transgene expression in both monocotyledon plant cells and • Dicotyledonous, and that are useful to generate transgenic plants with desirable agronomic characteristics.

• • S ECUENCE OF S ECUENCES < 110 > Albert, Henrik H. Wei, Hairong • < 120 > PROMOTING SEQUENCES OF PLANTS AND METHODS OF USE OF SAME ZAS < 130 > UH-03648 < 140 > XX / XXX.XXX < 141 > 1999-03-17 < 160 > 12 < 170 > Patentln Ver. 2.0 < 210 > 1 • < 211 > 1813 < 212 > DNA < 213 > Hybrid cultivation of Saccharum H32-8560 < 400 > 1 gaattcatta tgtggtctag gtaggttcta tatataagaa aacttgaaat gttctaaaaa 60 aaaattcaag cccatgcatg attgaagcaa acggtatagc aacggtgtta acctgatcta 120 gtgatctctt gcaatcctta acggccacct accgcaggta gcaaacggcg tccccctcct 180 cgatatctcc gcggcgacct ctggcttttt ccgcggaatt gcgcggtggg gacggattcc 240 acnanaccgc gacgcaaccg cctctcgccg ctgggcccca caccgctcgg tgccgtagcc 300 tcacgggact ctttctccct cctcccccgt tataaattgg cttcatcccc tccttgcctc 360 atccatccaa atcccagtcc ccaatcccat cccttcgtcg gagaaattca tcgaagcgaa 420 gcgaatcctc gcgatcctct caaggtactg cgag ttcg atccccctct cgacccctcg 480 tatgtttgtg tttgtcgtac gttttagtag gtatgctttc cctgtttgtg ttcgtcgtag 540 • cgtttgatta ggtatgcttt ccctgttcgt gttcatcgta gtgtttgatt aggtcgtgtg 600 aggcgatggc "ctgctcgcgt ccttcgatct gtagtcgatt tgcgggtcgt ggtgtagatc 660 gatgáagtta tgcgggctgt tttggtgtga tctgctcgcc tgattctgcg ggttggctcg 720 agtagatatg gatggttgga ccggttggtt cgtttaccgc gctagggttg ggctgggatg 780 atgttgcatn gcgccgttgc gcgtgatccc gcagcaggac ttgcgtttga ttgccagatc 840 tcgttacgat tatgtgattt ggtttggact tattagatct gtagcttctg cttatgttgc 900 shits gcgcc tactgctcca tatgcctgat gataatccat aaatggcagt ggaaatcaac 960 tagttgattg cggagtcatg tatcagctac aggtgtaggg actagctaca ggtgtaggga 1020 attgtttggt ctngcgtcta ccttaactca tgtgcaatta tgcaatttag tttagatgtt 1080 tgttccaant catctaggct gtaaaaggga cactggttag attgctgttt aatcttttta 1140 gtagattata ttatattggt aac tattaa cccntattaa catgccataa cgtggattct- 1200 gctcatgcct gatgataatc atagatcact gtggaattaa ttagttgatt gttgaatcat 1260 cataccacgg gtttcatgta cacaattgct tagttcctta attttactga acaaatgcaa 1320 tccatgtatg atttgcgtgg ttctctaatg tgaaatacta tagctacttg ttagtaag aa 1380 tcaggttcgt atgcttaatg ctgtatgtgc tgcctgatga cttctgctca taatcatata 1440 tcactggaat taattagttg atcgtttaat catatatcaa gtacatacca tggcacaatt 1500 tttagtcact taacccatgc agattgaact ggtccctgca tgttttgcta aattgttcta 1560 ttctgattag accatatatc aggtattttt ttttggtaat ggttctctta ttttaaatgc 1620 tatatagttc tggtacttgt tagaaagatc tggttncata gtttagttgc ctatccttcg 1680 • aattaggatg ctgagcagct gatcctatag ctttgtttca tgtatcaatt cttttgtgtt 1740 caacagtcag tttttgttag attcattgta acttatgttc gcttactctt ctggtcctca 1800 atgcttgcag atg 1813 < 210 > 2 < 211 > 2804 < 212 > DNA < 213 > Hybrid cultivation of Saccharum H32-8560 < 400 > 2 taatcctggg ccatgancag ctgtccttcc aggttcacaa gtctggtgcc ttcttctgtc 60 cctccgatgg agattatctg catgtcgtgg tcgtgtcctg atcgaatcct cgttgaatcc 120 • ctatgttttt cttcaagaaa tgtgagtcct atgtcagtct ggttgcgttt gtgaacattt 180 ctgctgctga gcagcacttt ggctggaact gtgcaatgaa ataaatggaa ccctggtttc 240 tggttatgtg tgtgttagct aatgtttttg aagtggaagc tctaatcttc tatcgcgttg 300 ctactacaat tctgcttgtg ttttgatggt tcttggtttc tgttagttgg ttcagaggaa 360 gttttgcttc cacagactaa gatgcagttg aactttggtt gccctggttt ctagatttca 420 ttgagtgata tttgtgctgg gtaagaaaca accggtgttc acatataatc aggttttgtg 480 ctgctcgagt gatcgtcaaa aaccaccggt gttcacatct aaaaaggttt cgatccccag 540 gtttagatct cccgtttaat tccaaaaaaa aagttctgtg tacttgcatt tagttgggtg 600 gttgatgctg gaaagagtaa ctttcaagag taataatctt tggtgactac tctgtttcaa 660 ctgatcaatc cctaggaaag gtacaccttt gaaattctta acttagggaa gaaccttgca 720 actgataata ctttgtttca gtatacttta tag taaaa aatattcaga tatattagac 780 accggatgtc atccactcat ccttacaaac ctctgtcatg gtcctgcaga aatgtttgcc 840 agctccagtg gcttcctgat aaatctgtgg agtgcctgtt AATC gctgc caatttttgc 900 tgagcactgt atatatgtta g aag act ttgggccacc aattccattt tgacacagca 960 • ctattggtcc tcctgacaca accaattcga gcactgcata atttgaaacg tttttgctcc 1020 cattttgcaa ggctacaaat ttagatcatg tttascatyc tgtgggatac aatatatgga 1080 tatcgaacaa acttggtatg tcagagaaaa aatagtttat tttcaaaact aacattttta 1140 tgaactttaa aagccttcta accttcagca tttgggatca agatgagtgc tcgaacaaga 1200 gtgcactttt tctccaaaat aatctactac agagttcttt tttatatata aaaaaactta 1260 tacttaacag ataaatcaga cctcttctgc tccatatcac cttgacaaat caaagaagca 1320 gcaccagcga agggtattat tattgaggta aatataagat ctcgtttact gaaaaagacc 1380 gcgtgtttac ctaaactacc attttgcttt gatagcagca tacatgtgat agaattgcgg 1440 atcctaccgt gctgactgtg aangtggtaa gggtgagaga ttggtgggcg aggtctgaac 1500 gagcgaaaac agtactgcat ttactgttca caaggaggcg gcttaggttt tggtctccca 1560 gctctctaag ggaagctgag aattatgatt ctcttgctta attatttctt aaccaaagtt 1620 ataaatatat agcctatgag atcctaattt atggaaataa ctaaactatt ttaaggaaat 1680 atataaatag ataatcagcc cactaacggg cntagcgccc actaacaggc ctggtgctga 1740 gcccgacata acatctctcc ccgcctggrg aaacagctcg tcctcgagct gaaat ctggt 1800 agaagcatc tcaaccaaca ccggggtcat gctggaacac tgcatcaggc gctaccgcag 1860 ctggtacgtc gtcgtcgagg aagtcagccg actccaagta gaacagtcgc ttacaactga 1920 tgtccgcgga cgtagggctc atcacaattg taaacaaagc cctygacgac ggcactccaa 1980 cggtgtgaga acagctcttc cgacgaaacg agggagctag agcgggtagt ggcgcgggga 2040 acagccagtg tagcgcctgt agtcaccgag ggatggggcg gtcgggcgcc gcgaggctgc 2100 ggtgccaggt ggaggttcaa cattcttcaa acgcccgtgc caagtacatg gcggactgga 2160 • ggtcgggcgg tgcgcggagc tgaacctgct tacggaggtg gtccggcagc ccacccacgt 2220 acaactccgc cttttggcga gcggagaggt tgtgggcatg gcacaggacg gcgttgtaac 2280 gctccgagta atcctgaacg gaagaaccaa aaggaaggcg ggcaagctcc gccaaccgag 2340 tgcccaaaac aggaggcccg aagcgaagcg agcataattc gcggaagcgc tcccaaggag 2400 gcataccctc gtcttgctcc agggcgtagt accatgtctg ggcaacaccc cgaagatggt 2460 ccatgtgcga aggacgcgag gcggaggcga gcgtntgctg gccgcggaag aactgctcgc 2520 actggttcaa ccaattcagg ggatcggtcg aaccgtcgta cgtagggaac tccagtttgt 2580 agaatttggg ccccgcctgg gcgcctgcga gggcagcagc aagggctggg tcgagccccc 2640 cctgcggctg gccgcccgag ggagagggcg cccgaagaac agcggcccgt ccaccccccc 2700 gaaaagagtg ctggcggggg gtagggagga catcgttgtc gccgccgccg tcgtgtaggc 2760 tgtcggggag ggcgacgtgg ccatcgagta gatccgggga ATTC 2804 • < 210 > 3 < 211 > 3691 < 212 > DNA < 213 > Hybrid cultivation of Saccharum H32-8560 < 400 > 3 aagttttgnt aaaatgaaca aagaattggg gaaactatag ccaaagtggg tggggaatgg 60 tgccaaacaa aacttcgtaa accaacccaa aaagatccgg aaaacaaatg gatacgtgca 120 gggcatgcat gcaatagccc agccataaaa agcggcgagc caatgcccgg gtgtcaaaca 180 aaatggcgcc tgtgccggct ctggctgctt ccggctcagc tttcggaacg atccgccgca 240 gtttggcctc gcatatgatg acgatgatgg tctcctcttc tcgatttgta gctccggcat 300 gggagccacc tcctgtcggc tcacacatag cacgcgcctt agcccgtgct cgctctcccc 360 acctgcgcca tagatgcttc atcagtgtga tcagatggta gcccatcgtg ctcgtacgta 420 tggagtaacg tgataccaca acacgtacac tggtcagaat tgatagtata tgatcctgtc 480 • gacccgatgt gttttagtac cttgcagtgg ccggagagga gtggccgcgc gcatgcggcg 540 caggggttct ccgcgctcgc tgatcgcttc ctcactgtgc gctcgtttag gaacaccacc 600 tcgtggtcgc tcaccatgtg tgactgcatg caacgctacg aatcaggacc cagatggaaa 660 cgaagcgcct ctcgaccacc tctgcctcgg tgatggttgg tgtgcagtgc gtacgcatgc 720 acgctaccaa tatcatacct ggatgccggt gcaatcgaac agcttcaggt tgtcgacgcg 780 gacggcgaag caggacgcgt acttccatat ctttgggttc cattacgtac cgtcaatcga 840 ataaataaag agaagagttt gagatcagct tgttgggagc aggtgaccgc ccgacatgca 900 tgccgattgt cgacggcacg gaaataaaca acacatttgt gagggagcca gggaggcagt 960 ggcggcacag cgtcgcggca cagtogatgc agaagtggtt cttgtcgttc ttgcgctccc 1020 cccgggtgtg cagcgcacgc ctttgaaaaa ctccgatagc aggccacaca gccattgcgg 1080 acggccgcca ggcgccgcgc gctgcatccc cgtttgttcg cacatgcgct aggtggtcct 1140 gcggccgttc cttgcaccgc ggagacgcgg ggtggaccag tgggggaatg gatgaactgc 1200 tggtaggttt ggttggattg gcgagtgcgt agagggggca tgggcaacga tagactcgat 1260 tcaattcaaa gactgaaaat agtggagttc taacaccatt ctgtgcggcg ctaattctcg 13 20 acatggcagg cgtaagcata ataccgacat ggcatgcaac gatgttcgtg aacagtggtg 1380 acacatggat atggtggccg tccaggggat tcgttccatt caattcaaag accgaaaatc 1440 gcggggttcc gtagcatttt gtgcggtgct aattctcgaa catgcgagac gtaagcctaa 1500 taccgagatg atgttcgtga gcatgcaaca acaacagtga cacgtggatg cggtggccgt 1560 ctagggattc gcgttctaag ctggtatatg tgcggtgtta attcttgaca tgcggggcgt 1620 ccaagatgaa aagtgtaata cggtgacacg tggacgcggg ggtcgtcaaa caattcattc 1680 cgtggtctag ggtaggttat atataaaggc cagtcttagt gggggatttt atggccatgt 1740 tattaatgca acccatattt ggaaaacagt gcaggaagag tttcatcttc gtaaaactct 1800 ctctaattcc atgaaactct tatcatctct ctcttcatca atacggtgcc acatcagcct 1860 atttaatgtc catgaaactc tgatgaaatc cactgagacg ggcctcagaa aacttgaaat 1920 aaattcaagt cttctaaaaa ccatgcatga ttgaagcaaa cggtatagca acggtgttaa 1980 cctgatctag tgatctcttg taatccttaa cggccaccta ccacaggtag caaacggcgt 2040 ccccctcctc gatatctccg cggcggcctc tggctttttc cgcggaattg cgcggtgggg 2100 acggattcct cgagaccgcg acacaaccgc ctttcgccgc tgggccccac accgctcggt 2160 g ccgtagcct cacgggactc tttctccctc ctcccccgct ataaattggc ttcatcccct 2220 tccatccaaa ccttgcctca tcccagtccc caatcccagc ccatcgtcgg agaaattcat 2280 agaagcgaag cgaatcctcg cgatcctctc aaggtagtgc gagttttcga ttcccctctc 2340 gacccctcgt atgctttccc tgtttgtgtt tcgtcgtagc gtttgattag gtatgctttc 2400 • cctgtttgtg ttcgtcgtag cgtttgattt ggtatgcttt ccccgttcgt gttcctcgta 2460 gtgtttgatt aggtcgtgtg aggcgatggc ctgctcgcat ccttcgatct gtagtcgatt 2520 tgcgggtcgt ggtgtagatc tgcgggctgt gatgaagtta tttggtgtga tcgtgctcgc 2580 ctgattctgc gggttggctc gagtagatat gatggttgga ccggttggtt tgtttaccgc 2640 gctagggttg ggctgggatg atgttgcatg cgccgttgcg cgtgatcccg cagcaggact 2700 tgegtttgat tgccagatct cgttacgatt atgtgatttg gtttggactt tttagatctg 2760 ttatgtgcca tagcttctgc gatgcgccta ctgctcatat gcctgatgat aatcataaat 2820 ggctgtggaa ctaactagtt gattgcggag tcatgtatca gctacaggtg tagggactag 2880 ctacaggtgt agggacttgc gtctaaattg tttggtcctg tactcatgtt gcaattatgc 2940 aatttagttt agattgtttg ttccactcat ctaggctgta aaagggacac tgcttagatt 3000 gctgtttaat ctttttagta gattatatat tatattggta acttattacc cttattacat 3060 gccatacgtg acttctgctc atgcctgatg ataatcatag atcactgtgg aattaattag 3120 ttgattgttg aatcatgttt catgtacata ccacggcaca attgcttagt tccttaacaa 3180 atgcaaattt tactgatcca tgtatgattt gcgtggttct ctaatgtgaa atact atagc 3240 • tacttgttag taagaatcag gttcgtatgc ttaatgctgt atgtgccttc tgctcatgcc 3300 tgatgataat catatatcac tggaattaat tagttgatcg tttaatcata tatcaagtac 3360 ataccatggc acaat.tttta gtcacttaac ccatgcagat tgaactggtc cctgcatgtt 3420 ttgctaaatt gttctatttc tgattagacc atatatcatg taattttttt tttgggtaat 3480 ggttctccta ttttaaatgc tatatagttc tggtacttgt tagaaaaatc tgcttccata 3540 gtttagttgc ttatccctcg aattatgatg ctgagcagct gatcctatag ctttgtttca 3600 ggtatcaatt ctngtgttca acagtcagtt tttgttagat tcattgtaac ttatggtcgc 3660 ttactcttct ggtcctcaat gcttgcagat g 3691 < 210 > 4 < 211 > 343 < 212 > DNA < 213 > Hybrid cultivation of Saccharum H32-8560 . , .._... »._ .. < 400 > 4 taagtcctgg gccatgagca gctgtccttc cagggttcac aagtagtggt gccttcttnc 60 tgtccctccg atggagatta tctgcatgtc gtggtcgtgt cctgatcgag tcgtcgttga 120 tttttcttca gtccctatgt agaaatgtga gtcctatgtc agtctggttg cgtttgtgaa 180 cattttctgc tgctgcgcag cagtttggtt ggaactgtgc aatgaaataa attgaaccct 240 ggtttctggt tatgtgtgtt agctaatgtt tttgaagtgg aagctntaat cttntatcgc 300 gttgctacta caattctgnt tgtgttttga 343 tgttcttgtt tet < 210 > 5 < 211 > 5512 < 212 > DNA < 213 > Cultivation of Saccharura Hybrid H32-8560 < 400 > 5 gaatteatta tgtggtctag gtaggttcta tatataagaa aacttgaaat gttctaaaaa 60 aaaattcaag cccatgcatg attgaagcaa acggtatagc aacggtgtta acctgatcta 120 gtgatctctt gcaatcetta acggccacct accgcaggta gcaaacggcg tccccctcct 180 cgatatctcc gcggcgaect ctggcttttt ccgcggaatt gcgcggtggg gacggattcc 240 • acaaccgcga cgcaaccgcc tctcgccgct gggccccaca ccgctcggtg ccgtagcctc 300 acgggactct ttctccctcc tcccccgtta taaattggct tcatcccctc cttgcctcat 360 ccatccaaat cccagtcccc aatcccatcc cttcgtcgga gaaattcatc gaagcgaagc 420 gatcctctca gaatcctcgc aggtactgcg agttttcgat ccccctctcg acccctcgta 480 tgtttgtgtt tgtcgtacgt ttgattaggt atgctttccc tgtttgtgtt cgtcgtagcg 540 tttgattagg tatgetttec ctgttcgtgt tcatcgtagt gtttgattag gtcgtgtgag 600 gcgatggcct gctcgcgtcc ttcgatctgt agtcgatttg cgggtcgtgg tgtagatctg 660 cgggctgtga tgaagttatt tggtgtgatc tgctcgcctg attctgcggg ttggctcgag 720 tagatatgga tggttggacc ggttggttcg tttaccgcgc tagggttggg * ctgggatgat 780 gttgcatgcg ccgttgcgcg tgatcccgca gcaggacttg cgtttgattg ccagatctcg 840 ttaegattat gtgatttggt ttggacttat tagatctgta gcttctgctt atgttgccag 900 atgcgcctac tgetecatat gcctgatgat aatccataaa tggcagtgga aatcaactag 960 ttgattgcgg agtcatgtat cagetacagg tgtagggact agctacaggt gtagggactg 1020 cgtctaattg tttggtcctt aactcatgtg caattatgea atttagttta gatgtttgtt 1080 • ccaatcatct aggctgtaaa agggacactg gttagattgc tgtttaatct ttttagtaga 1140 ttggtaactt ttatattata attaacecta ttacatgeca taacgtggat tctgctcatg 1200 cctgatgata atcatagatc actgtggaat taattagttg attgttgaat catgtttcat 1260 gtacatacca cggcacaatt gcttagttcc ttaacaaatg caaattttac tgatccatgt 1320 atgatttgcg tggttctcta atgtgaaata etatagetac ttgttagtaa gaatcaggtt 1380 cgtapgctta atgctgtatg tgccttctgc tcatgcctga tgataatcat atatcactgg 1440 aattaattag ttgatcgttt aatcatatat caagtacata ccatggcaca atttttagtc 1500 acttaaccca tgcagattga actggtccct gcatgttttg ctaaattgtt ctattctgat 1560 tagaccatat atcaggtatt tttttttggt aatggttctc ttattttaaa tgctatatag 1620 ttctggtact tgttagaaag atctggttca tagtttagtt gcctatcctt cgaattagga 1680 tgctgagcag ctgatcctat agctttgttt catgtatcaa ttcttttgtg ttcaacagtc 1740 agtttttgtt agatteattg taacttatgt tcgcttactc ttctggtcct caatgcttgc 1800 agatgcagat cttcgttaag accctcactg gcaagaccat cacccttgag gttgagtctt 1860 cagacamtat tgactnatgtc roaggctaaga tacaggacaa ggaaggcatt cct ccggatc 1920 agcagaggct gatctttgct ggcaagcagc tcgaggatgg gytgactaca ccgtacccta 1980 acatccagaa ggagtccacc stccacctgg tgctcaggct caggggaggc atgcaaatct 2040 tcgtcaagac cctcactggc aagactatca cgcttgaggt cgagtcttct gacacgatcg 2100 acaacgtgaa ggccaagatc caggacaagg agggaatccc cccggaccag cagcgtctca 2160 tcttcgctgg caagcagctc gaggatggcc gcaccctcgc tgactacaac atccagangg 2220 agtcgantnt ccaccttgtg ctcaggttna ggggtggcat gcagattttt gtcaagacct 2280 • tnactggcaa gaccatcacc ttggaggtgg agtcttcgga caccatngac aatgtgaagg 2340 ggacaaggaa pgaagatcca cagaccagca ggaatccccc tttgctggca gcgtcttatt 2400 agcagcttga ggatggccgc accctagcag actacaacat ccagaaggag tccacccttc 2460 acctggtgct ccgcttncgc ggtggtatgc agatcttcgt caagaccctc accggcaaga 2520 ccatcaccct ggaggtggag tcctctgaca ccatogacaa tgtgaaggcg aagatccagg 2580 acaaggaggg catccccccg gaccagcagc gtctcatctt cgccggcaag cagctggagg 2640 atggccgcac cctggcagac tacaacatcc agaaggagtc cactctccac ctggtgctcc 2700 gtctccgtgg tggccagtaa tcctgggcca tgaagctgtc cttccaggtt cacaagtctg 2760 gcgccttctc ctgtccctcc gatggagatt atctgcatgt cgtggtcgtg tcctgatcga 2820 atcctcgttg aatccctatg tttttcttca agaaatgtga gtcctatgtc agtctggttg 2880 cgtttgtgaa catttctgct gctgagcagc actttggctg gaactgtgca atgaaataaa 2940 tggaaccctg gtttctggtt atgtgtgtgt tagctaatgt ttttgaagtg gaagctctaa 3000 • tcttctatcg cgttgctact acaattctgc ttgtgttttg atgttcttgg tttctgttag 3060 ggaagttttg ttggttcaga cttccacaga ctaagatgca gttgaacttt ggttgccctg 3120 gtttctagat ttcatttgtg ctggttgagt gatagtaaga aacaaccggt gttcacatat 3180 aatcaggttt tgtgctgctc gagtgatcgt caaaaaccac cggtgttcac atctaaaaag 3240 gtttcgatcc ccaggtttag atctcccgtt taattccaaa aaaaaagttc tgtgtacttg 3300 catttagttg ggtggttgat gtaactttca gctggaaaga tctttggtga agagtaataa 3360 ctactctgtt tcaactgatc aatccctagg aaaggtacac ctttacttag ggaagaaatt 3420 cttagaacct tgcactttgt ttcaactgat aatagtatac tttattagat aaaaaatatt 3480 agacaccgga cagatatatt tgtcatccac tcatccttac aaacctctgt catggtcctg 3540 cagaaatgtt tgccagctcc agtggcttcc tgataaatct gtggagtgcc tgttaatcgg 3600 ttgctgagca ctgccaattt ctgtatatat gttagtaagt actattgggc caccaattcg 3660 attttgacac agcactattg gtccaccaat tcgattctga cacagcactg cataatttga 3720 aacgtgttgc tccattttgc aaggctacaa atttagatca tgtttagcat tctgtgggat 3780 acaatatatg gatatcgaac aaacttggta tgtcagagaa aaaatagttt attttcaaaa 3840 • ctaacatttt taaagccttc tatgaacttt aaaccttcag catttgggat caagatgagt 3900 gctcgaacaa gagtgcactt tttctccaaa ataatctact acagagttct tttttatata 3960 taaaaaaact tatacttaac agataaatca gactttttct gctccatatc accttgacaa 4020 atcaaagaag cagcaccagc gaagggtatt attattgagg taaatataag atctcgttta 4080 ctgaaaaaga ccgcgtgttt acctaaacta ccattttgct ttgatagcag catacatgtg.4140 atagaattgc ggatcctacc gtgctgactg tgaaggtggt aggggtgaga gattggtggg 4200 acgagcgaga cgaggtctga acagtactgc atttactgtt cacaaggagg cggcttaggt 4260 tttgggtctc ccagctctct aagggaagct gagaattatg attctcttgc ttaattattt 4320 gttataaata cttaaccaaa tatagcctat gagatcctaa tttatggaaa taactaaact 4380 aatatataaa attttaagga tagataatca gcccactaac gggcctagcg cccactaaca 4440 ggcctggtgc tgagcccgac ataacatctc tccccgcctg gagaaacagc tcgtcctcga 4500 ggtagaagca gctgaaatct acaccggggt tcatcaacca catgctggaa cactgcatca 4560 ggcgctaccg cagctggtac gtcgtcgtcg aggaagtcag ccgactccaa gtagaacagt 4620 cgcttacact gatgtccgcg gacgtagggc tcatcacaat tgtaacaaag CCCT tgacga 4680 cggcactcca acagctcctc cggtgtgaga cgacgaaacg agggagctag agcgggtagt 4740 ggcgcgggaa cagccagtgt agcgcctgta gtcaccgagg gatggggcgg tcgggcgccg 4800 cgaggctgcg gtgcaggtgg aggtttcaca ttcctcaaac gcccgtgcca agtacatggc 4860 ggactggagg tcgggcggtg cgcggagctg aacctgctta cggaggtggt ccggcagccc 4920 acccacgtac aactccgcct tttggcgagc ggagaggttg tgggcatggc acaggacggc 4980 gttgtaacgc tccgagtaat cctgaacgga agaaccaaaa ggaaggcggg caagctccgc 5040 caaccgagtg cccaaaacag gaggcccgaa gcgaagcgag cataattcgc ggaagcgctc 5100 ccaaggaggc ataccctcgt cttgctccag ggcgtagtac catgtctggg caacaccccg 5160 aagatggtag gacgcgagcc atgtgcgagc ggaggcgagc gtctgctggc cgcggaagaa 5220 ctgctcgcac tggttcaacc aattcagggg atcggtcgaa ccgtcgtacg tagggaactc 5280 cagtttgtag aatttgggcc ccgcctgggc gcctgcgagg gcagcagcaa gggctgggtc 5340 gagccccccc tgcggctggc cgcccgaggg agagggcgcc cgaagaacag cggcccgtcc 5400 acccccccga aaagagtgct ggcggggggt agggaagaca tcgttgtcgc cgccgccgtc 5460 gtgtaggctg tcggggaagg cgacgtggcc atcgagtaga tccggggaat you 5512 < 210 > 6 < 2ll > 305 < 212 > PRT < 213 > Hybrid cultivation of Saccharum H32-8560 < 400 > 6 Met Gln lie Phe Val Lys Thr Leu Thr Gly Lys Thr He Thr Leu Glu 1 5 10 15 Val Glu Ser As As Xaa He Asp Xaa Val Xaa Ala Lys He Gln Asp 20 25 30 Lys Glu Gly Pro Pro Asp Gln Gln Arg Leu He Phe Wing Gly Lys 35 40 45 Gln Leu Glu Asp Gly Arg Thr Leu Xaa Asp Tyr Aen He Gln Lys Glu 50 55 60 Being Thr Xaa, His fceu Val Leu Arg Leu Arg Gly Gly Met Gln He Phe 65 70 75 80 Val Lys Thr Leu Thr Gly Lys Thr He Thr Leu Glu val Glu Ser Ser 85 90 95 Asp Thr He Asp Asn Val Lys Wing Lys He Gln Asp Lys Glu Gly He 100 105 110 Pro Pro Asp Gln Gln Arg Leu He Phe Wing Gly Lys Gln Leu Glu Asp 115 120 125 Gly Arg Thr Leu Wing Asp Tyr Asn He Gln Xaa Glu Ser Xaa Xaa His 130 135 140 Leu Val Leu Arg Xaa Arg Gly Gly Met Gln He Phe Val Lys Thr Xaa 145 150 155 160 Thr Gly Lys Thr He Thr Leu Glu Val Glu Ser Ser Asp Thr Xaa Asp 165 170 175 Asn Val Lys Xaa Lys He Gln Asp Lys Glu Gly He Pro Pro Asp Gln 180 185 190 Gln Arg Leu He Phe Wing Gly Lys Gln Leu Glu Asp Gly Arg Thr Leu 195 200 205 Wing Asp Tyr Asn He Gln Lys Glu Ser Thr Leu His Leu Val Leu Arg 210 215 220 Xaa Arg Gly Gly Met Gln He Phe Val Lys Thr Leu Thr Gly Lys Thr 225 230 235 240 He Thr Leu Glu Val Glu Ser Ser Asp Thr He Asp Asn Val Lys Wing 245 250 255 Lys He Gln Asp Lys Glu Gly He Pro Pro Asp Gln Gln Arg Leu He 260 265 270 Phe Wing Gly Lys Gln Leu Glu Asp Gly Arg Thr Leu Wing Asp Tyr Asn 275 280 285 He Gln Lys Glu Ser Thr Leu His Leu Val Leu Arg Leu Arg Gly Gly 290 295 300 • Gln 305 < 210 > 7 < 211 > 1802 < 212 > DNA < 213 > Hybrid cultivation of Saccharum H32-8560 < 400 > 7 gaattcatta tgtggtctag gtaggttcta tatataagaa aacttgaaac gttctaaaaa 60 aaaattcaag cccatgcatg attgaagcaa acggtatagc aacggtgtta acctgatcta 120 gtgatctctt gcaatcctta acggccacct accgcaggta gcaaacggcg tccccctcct 180 cgatatctcc gcggcgacct ctggcttttt ccgcggaatt gcgcggtggg gacggattcc 240 acaaccgcga cgcaaccgcc tctcgccgct gggccccaca ccgctcggtg ccgtagcctc 300 acgggactct ttctccctcc tcccccgtta taaattggct tcatcccctc cttgcctcat 360 ccatccaaat cccagtcccc aatcccatcc cttcgtcgga gaaattcatc gaagcgaagc 420 gaatcctcgc gatcctctca aggtactgcg agttttcgat ccccctctcg acccctcgta 480 tgtttgtgtt tgtcgtacgt ttgattaggt atgctttccc tgtttgtgtt cgtcgtagcg 540 tttgattagg tatgctttcc ctgttcgtgt tcatcgtagt gtttgattag gtcgtgtgag 600 • gcgatggcct gctcgcgtcc ttcgatctgt agtcgatttg cgggtcgtgg tgtagatctg 660 cgggctgtga tgaagttatt tggtgtgatc tgctcgcctg attctgcggg ttggctcgag 720 tagatatgga tggttggacc ggttggttcg tttaccgcgc tagggttggg ctgggatgat 780 gttgcatgcg ccgttgcgcg tgatcccgca gcaggacttg cgtttgattg ccagatctcg 840 ttacgattat gtgatttggt ttggacttat tagatctgta gcttctgctt atgttgccag 900 atgcgcctac tgctccatat gcctgatgat aatccataaa tggcagtgga aatcaactag 960 ttgattgcgg agtcatgtat cagctacagg tgtagggact agctacaggt gtagggactg 1020 cgtctaattg tttggtcctt aactcatgtg atttagttta caattatgca gatgtttgtt 1080 ccaatcatct aggctgtaaa agggacactg gttagattgc tgtttaatct ttttagtaga 1140 ttggtaactt ttatattata attaacccta ttacatgcca taacgtggat tctgctcatg 1200 cctgatgata atcatagatc actgtggaat taattagttg attgttgaat catgtttcat 1260 gtacatacca cggcacaatt gcttagttcc ttaacaaatg caaattttac tgatccatgt 1320 atgatttgcg tggttctcta atgtgaaata ctatagctac ttgttagtaa gaatcaggtt 1380 • cgtatgctta atgctgtatg tgccttctgc tcatgcctga tgataatcat atatcactgg 1440 aattaattag ttgatcgttt aatcatatat ccatggcaca caagtacata atttttagtc 1500 tgcagattga acttaaccca actggtccct gcatgttttg ctaaattgtt ctattctgat 1560 tagaccatat atcaggtatt tttttttggt aatggttctc ttattttaaa tgctatatag 1620 ttctggtact tgttagaaag atctggttca tagtttagtt gcctatcctt cgaattagga 1680 tgctgagcag ctgatcctat agctttgttt catgtatcaa ttcttttgtg ttcaacagtc 1740 agtttttgtt agattcattg taacttatgt tcgcttactc tcctggtcct caatgcttgc 1800 AG 1802 < 210 > 8 < 211 > 5174 < 212 > DNA < 213 > Cultivation of Saccharum Hybrid H32-8560 • < 400 > 8 aagttttgnt aaaatgaaca aagaattggg gaaactatag ccaaagtggg tggggaatgg 60 tgccaaacaa aacttcgtaa accaacccaa aaagatccgg aaaacaaatg gatacgtgca 120 gggcatgcat gcaatagccc agccataaaa agcggcgagc caatgcccgg gtgtcaaaca 180 aaatggcgcc tgtgccggct ctggctgctt ccggctcagc tttcggaacg atccgccgca 240 gtttggcctc gcatatgatg acgatgatgg tctcctcttc tcgatttgta gctccggcat 300 gggagccacc tcctgtcggc tcacacatag cacgcgcctt agcccgtgct cgctctcccc 360 acctgcgcca tagatgcttc atcagtgtga tcagatggta gcccatcgtg ctcgtacgta 420 tggagtaacg tgataccaca acacgtacac tggtcagaat tgatagtata tgatcctgtc 480 gacccgatgt gttttagtac cttgcagtgg ccggagagga gtggccgcgc gcatgcggcg 540 caggggttct ccgcgctcgc tgatcgcttc ctcactgtgc gctcgtttag gaacaccacc 600 tcgtggtcgc tcaccatgtg tgaetgcatg caacgctacg aatcaggacc cagatggaaa 660 cgaagcgcct ctcgaccacc tctgcctcgg tgatggttgg tgtgcagtgc gtacgcatgc 720 acgctaccaa tatcatacct ggatgccggt gcaatcgaac agcttcaggt tgtcgacgcg 780 gacggcgaag caggacgcgt acttccatat ctttgggttc cattacgtac cgtcaatcga 840 ataa ataaag agaagagttt gagatcagct tgttgggagc aggtgaccgc ccgacatgca 900 tgccgattgt cgacggcacg gaaataaaca acacatttgt gagggagcca gggaggcagt 960 ggcggcacag cgtcgcggca cagtcgatgc agaagtggtt cttgtcgttc ttgcgctccc 1020 cccgggtgtg cagcgcacgc ctttgaaaaa ctccgatagc aggccacaca gccattgcgg 1080 • ggcgccgcgc acggccgcca gctgcatccc cgtttgttcg cacatgcgct aggtggtcct 1140 gcggccgttc cttgcaccgc ggagacgcgg ggtggaccag tgggggaatg gatgaactgc 1200 tggtaggttt ggttggattg gcgagtgcgt agagggggca tgggcaacga tagactcgat 1260 tcaattcaaa gactgaaaat agtggagttc taacaccatt ctgtgcggcg ctaattctcg 1320 acatggcagg cgtaagcata ataccgacat ggcatgcaac gatgttcgtg aacagtggtg 1380 acacatggat atggtggccg tccaggggat tcgttccatt caattcaaag accgaaaatc 1440 gcggggttcc gtagcatttt gtgcggtgct aattctcgaa catgcgagac gtaagcctaa 1500 taccgagatg atgttcgtga gcatgcaaca acaacagtga cacgtggatg cggtggccgt 1560 ctagggattc gcgttctaag ctggtatatg tgcggtgtta attcttgaca tgcggggcgt 1620 ccaagatgaa aagtgtaata cggtgacacg tggacgcggg ggtcgtcaaa caattcattc 1680 cgtggtctag ggtaggttat atataaaggc cagtcttagt gggggatttt atggccatgt 1740 tattaatgca acccatattt ggaaaacagt gcaggaagag tttcatcttc gtaaaactct 1800 • ctctaattcc atgaaactct tatcatctct ctcttcatca atacggtgcc acatcagcct 1860 atttaatgtc catgaaactc tgatgaaatc cactgagacg ggcctcagaa aacttgaaat 1920 cttctaaaaa aaattcaagt ccatgcatga ttgaagcaaa cggtatagca acggtgttaa 1980 cctgatctag tgatctcttg taatccttaa cggccaccta ccacaggtag caaacggcgt 2040 ccccctcctc gatatctccg cggcggcctc tggctttttc cgcggaattg cgcggtgggg 2100 acggattcct cgagaccgcg acacaaccgc ctttcgccgc tgggccccac accgctcggt 2160 gccgtagcct cacgggactc tttctccctc ctcccccgct ataaattggc ttcatcccct 2220 tccatccaaa ccttgcctca tcccagtccc caatcccagc ccatcgtcgg agaaattcat 2280 agaagcgaag cgaatcctcg cgatcctctc aaggtagtgc gagttttcga ttcccctctc 2340 gacccctcgt atgctttccc tgtttgtgtt tcgtcgtagc gtttgattag 'gtatgctttc 2400 cctgtttgtg ttcgtcgtag cgtttgattt ggtatgcttt ccccgttcgt gttcctcgta 2460 gtgtttgatt aggtcgtgtg aggcgatggc ctgctcgcat ccttcgatct gtagtcgatt 2520 tgcgggtcgt ggtgtagatc tgcgggctgt gatgaagtta tttggtgtga tcgtgctcgc 2580 ctgattctgc gggttggctc gagtagatat gatggttgga ccggttggtt tgtttaccgc 2640 gctagggttg ggctgggatg atgttgcatg cgccgttgcg cgtgatcccg cagcaggact 2700 • tgcgtttgat tgccagatct cgttacgatt atgtgatttg gtttggactt tttagatctg 2760 ttatgtgcca tagcttctgc gatgcgccta ctgctcatat gcctgatgat aatcataaat 2820 ggctgtggaa ctaactagtt gattgcggag tcatgtatca gctacaggtg tagggactag 2880 ctacaggtgt agggacttgc gtctaaattg tttggtcctg tactcatgtt gcaattatgc 2940 aatttagttt agattgtttg ttccactcat ctaggctgta aaagggacac tgcttagatt 3000 gctgtttaat ctttttagta gattatatat tatattggta acttattacc cttattacat 3060 gccatacgtg acttctgctc atgcctgatg ataatcatag atcactgtgg aattaattag 3120 ttgattgttg aatcatgttt catgtacata ccacggcaca attgcttagt tccttaacaa 3180 atgcaaattt tactgatcca tgtatgattt gcgtggttct ctaatgtgaa atactatagc 3240 tacttgttag taagaatcag gttcgtatgc ttaatgctgt atgtgccttc tgctcatgcc tga 3300 gataat catatatcac tggaattaat tagttgatcg tttaatcata tatcaagtac 3360 ataccatggc acaattttta gtcacttaac ccatgcagat tgaactggtc cctgcatgtt 3420 ttgctaaatt gttctatttc tgattagacc atatatcatg taattttttt tttgggtaat 3480 ggttctccta ttttaaatgc tatatagttc tggtacttgt tagaaaaatc TGCT tccata 3540 gtttagttgc ttatccctcg aattatgatg ctgagcagct gatcctatag ctttgtttca 3600 kgtatcaatt cttgtgttca acagtcagtt tttgttagat tcattgtaac ttatggtcgc 3660 ttactcttct ggtcctcaat gcttgcagat gcagattttc gttaagaccc tcactggcaa 3720 gaccatcacc cttgaggttg agtcctcaga cactattgac aatgtcaagg ctaagatcca 3780 ggcattcctc ggacaaggaa cagatcagca gaggctgaty tttgctggca agcagctcga 3840 ggatggccgt accctagctg actacaacat ccagaaggag tccaccctcc acctggtgct 3900 caggcttagg ggaggcatgc agattttcgt caagaccctc actggcaaga ctatcacgct 3960 tcttctgaca tgaggtcgag cgatcgacaa cgtgaaggcc aagatccagg acaaggaggg 4020 aatccccccg gaccagcagc gtytcatttt cgctggcaag cagctcgagg atggccgcac 4080 cctcgctgac tacaacatcc agaaggagtc gactctccac cttgtgctca ggctcagggg 4140 tggcatgcag atcttcgtca agaccctcac tggcaagacc atcaccttgg aggtggagtc 4200 ctcggacacc attgacaatg tgaaggcgaa gatccaggac aaggagggca tccccccgga 4260 ccagcagcgt ctcatyttcg ccggcaagca rcttgaggat ggccgcaccc ttgcgganta 4320 aargagtcca caacatccag cccttcacct ggtgctccgc cttcgtggtg gtatgcaga t 4380 tttcgtcaag accctcaccg gcaagaccat caccctggag gtggagtcct ctgacaccat 4440 tgacaatgtg aaggcgaaga tccaggataa ggagggcatc cccccggacc agcagcgtyt 4500 tatctttgct ggcaagcagc ttgaggatgg ccgcaccctg gcagantaca acatccagaa 4560 ggagtccacc cttcacctgg tgctccgcct tcgcggtggt atgsagatyt tcgtcaagac 4620 aagaccatca cctcaccggc ccctggaggt ggagtcctct gacaccatcg acaatgtgaa 4680 ggcgaagatc caggacaagg agggcatccc cccggaccag cagcgtctca tcttcgccgg 4740 caagcagctg gaggatggcc gcaccctggc agactacaac atccagaagg agtccactct 4800 ccacctggtg ctccgtctcc gtggtggcca gtaagtcctg ggccatgagc agctgtcctt 4860 ccagggttca caagtagtgg tgccttcttn ctgtccctcc gatggagatt atctgcatgt 4920 cgtggtcgtg tcctgatcga gtcgtcgttg agtccctatg ttttttcttc aagaaatgtg 4980 agtcctatgt cagtctggtt gcgtttgtga acattttctg ctgctgcgca gcagtttggt 5040 tggaactgtg caatgaaata aattgaaccc tggtttctgg ttatgtgtgt tagctaatgt 5100 ttttgaagtg gaagctntaa tcttntatcg cgttgctact acaattctgn ttgtgttttg 5160 5174 atgttcttgt TTCT < 210 > 9 < 211 > 381 < 212 > PRT < 213 > Hybrid cultivation of Saccharum H32-8560 < 400 > 9 Met Gln He Phe Val Lys Thr Leu Thr Gly Lys Thr He Thr Leu Glu 1 5 10 15 Val Glu Being Ser Asp Thr He Asp Asn Val Lys Wing Lys He Gln Asp 20 25 30 Lys Glu Gly Pro Pro Asp Gln Gln Arg Leu He Phe Wing Gly Lys 35 40 45 Gln Leu Glu Asp Gly Arg Thr Leu Wing Asp Tyr Asn He Gln Lys Glu 50 55 60 --H-t-M-tUH-t -_a -_ ^! _.

Being Thr Leu His Leu Val Leu Arg Leu Arg Gly Gly Met Gln He Phe 65 70 75 80 Val Lys Thr Leu Thr Gly Lys Thr He Thr Leu Glu Val Glu Ser Ser 85 90 95 • Asp Thr He Asp Asn Val Lys Wing Lys He Gln Asp Lys Glu Gly He 100 ios no Pro Pro Asp Gln Gln Arg Xaa He Phe Wing Gly Lys Gln Leu Glu Asp 115 120 125 Gly Arg Thr Leu Wing Asp Tyr Asn He Gln Lys Glu Ser Thr Leu His 130 135 140 Leu Val Leu Arg Leu Arg Gly Gly Met Gln He Phe Val Lys Thr Leu 145 150 155 160 • Thr Gly Lys Thr He Thr Leu Glu Val Glu Ser Ser Asp Thr He Asp 165 170 175 Asn Val Lys Ala Lys He Gln Asp Lys Glu Gly He Pro Pro Asp Gln 180 185 190 Gln Arg Leu He Phe Wing Gly Lys Gln Leu Glu Asp Gly Arg Thr Leu 195 200 205 Ala Xaa Tyr Asn He Gln Lys Glu Ser Thr Leu His Leu Val Leu Arg 210 215 220 Leu Arg Gly Gly Met Gln He Phe Val Lys Thr Leu Thr Gly Lys Thr 225 230 235 240 • He Thr Leu Glu Val Glu Ser Ser Asp Thr He Asp Asn Val Lys Wing 245 250 255 Lys He Gln Asp Lys Glu Gly He Pro Pro Asp Gli. Gln Arg Xaa He 260 265 270 Phe Wing Gly Lys Gln Leu Glu Asp Gly Arg Thr Leu Wing Xaa Tyr Asn 275 280 285 He Gln Lys Glu Ser Thr Leu His Leu Val Leu Arg Leu Arg Gly Gly 290 295 300 Met Gln He Phe Val Lys Thr Leu Tbr Gly Lys Thr He Thr Leu Glu 305 310 315 320 Val Glu Ser Ser Asp Thr He Asp Asn Val Lys Wing Lys He Gln Asp 325 330 335 Lys Glu Gly Pro Pro Asp Gln Gln Arg Leu He Phe Wing Gly Lys 340 345 350 Gln Leu Glu Asp Gly Arg Thr Leu Wing Asp Tyr Asn He Gln Lys Glu 355 360 365 Being Thr Leu His Leu Val Leu Arg Leu Arg Gly Gly Gln 370 375 380 < 210 > 10, 211.- 3688 '< 212. > DNA < 213 Hybrid Cultivation of Saccharum H32-8560 < 400 > 10 aagttttgnt aaaatgaaca aagaattggg gaaactatag ccaaagtggg tggggaatgg 60 tgccaaacaa aacttcgtaa accaacccaa aaagatccgg aaaacaaatg gatacgtgca 120 gggcatgcat gcaatagccc agccataaaa agcggcgagc caatgcccgg gtgtcaaaca 180 aaatggcgcc tgtgccggct ctggctgctt ccggctcagc tttcggaacg atccgccgca 240 gtttggcctc gcatatgatg acgatgatgg tctcctcttc tcgatttgta gctccggcat 300 gggagccacc tcctgtcggc tcacacatag cacgcgcctt agcccgtgct cgctctcccc 360 acctgcgcca tagatgcttc atcagtgtga gcccatcgtg tcagatggta 'ctcgtacgta 420 tggagtaacg tgataccaca acacgtacac tggtcagaat tgatagtata tgatcctgtc 480 gacccgatgt gtt tagtac cttgcagtgg ccggagagga gtggccgcgc gcatgcggcg 540 caggggttct ccgcgctcgc tgatcgcttc ctcactgtgc gctcgtttag gaacaccacc 600 tcgtggtcgc tcaccatgtg tgactgcatg caacgctacg aatcaggacc cagatggaaa 660 cgaagcgcct ctcgaccacc tctgcctcgg tgatggttgg tgtgcagtgc gtacgcatgc 720 acgctaccaa tatcatacct ggatgccggt gcaatcgaac agcttcaggt tgtcgacgcg 780 gacggcgaag caggacgcgt acttccatat ctttgggttc cat acgtac cgtcaatcga 840 ataaataaag agaag agttt gagatcagct tgttgggagc aggtgaccgc ccgacatgca 900 tgccgattgt cgacggcacg gaaataaaca acacatttgt gagggagcca gggaggcagt 960 ggcggcacag cgtcgcggca cagtcgatgc agaagtggtt cttgtcgttc ttgcgctccc 1020 cccgggtgtg cagcgcacgc ctttgaaaaa ctccgatagc aggccacaca gccattgcgg 1080 acggccgcca ggcgccgcgc gctgcatccc cgtttgttcg cacatgcgct aggtggtcct 1140 gcggccgttc cttgcaccgc ggagacgcgg ggtggaccag tgggggaatg gatgaactgc 1200 tggtaggttt ggttggattg gcgagtgcgt agagggggca tgggcaacga tagactcgat 1260 tcaattcaaa gactgaaaat agtggagttc taacaccatt ctgtgcggcg ctaattctcg 1320 acatggcagg cgtaagcata ataccgacat ggcatgcaac gatgttcgtg aacagtggtg 1380 acacatggat atggtggccg tccaggggat tcgttccatt caattcaaag accgaaaatc 1440 gcggggttcc gtagcatttt gtgcggtgct aattctcgaa catgcgagac gtaagcctaa 1500 taccgagatg atgttcgtga gcatgcaaca acaacagtga cacgtggatg cggtggccgt 1560 ctagggattc gcgttctaag ctggtatatg tgcggtgtta attcttgaca tgcggggcgt 1620 ccaagatgaa aagtgtaata cggtgacacg tggacgcggg ggtcgtcaaa caattcattc 1680 cgtggtctag ggtaggttat to tataaaggc cagtcttagt gggggatttt atggccatgt 1740 tattaatgca acccatattt ggaaaacagt gcaggaagag tttcatcttc gtaaaactct 1800 ctctaattcc atgaaactct tatcatctct ctcttcatca atacggtgcc acatcagcct 1860 • atttaatgtc catgaaactc tgatgaaatc cactgagacg ggcctcagaa aacttgaaat 1920 aaattcaagt cttctaaaaa ttgaagcaaa ccatgcatga tagca CGGT acggtgttaa 1980 cctgatctag tgatctcttg taatccttaa cggccaccta ccacaggtag caaacggcgt 2040 ccccctcctc gatatctccg cggcggcctc tggctttttc cgcggaattg cgcggtgggg 2100 acggattcct cgagaccgcg acacaaccgc ctttcgccgc tgggccccac accgctcggt 2160 gccgtagcct cacgggactc tttctccctc ctcccccgct ataaattggc ttcatcccct 2220 tccatccaaa ccttgcctca tcccagtccc caatcccagc ccatcgtcgg agaaattcat 2280 agaagcgaag cgaatcctcg cgatcctctc aaggtagtgc gagttttcga ttcccctctc 2340 gacccctcgt atgctttccc tgtttgtgtt tcgtcgtagc gtttgattag gtatgctttc 2400 cctgtttgtg ttcgtcgtag cgtttgattt ggtatgcttt ccccgttcgt gttcctcgta 2460 gtgtttgatt aggtcgtgtg aggcgatggc ctgctcgcat ccttcgatct gtagtcgatt 2520 tgcgggtcgt ggtgtagatc tgcgggctgt gatgaagtta tttggtgtga tcgtgctcgc 2580 • ctgattctgc gggttggctc gagtagatat gatggttgga ccggttggtt tgtttaccgc 2640 gctagggttg ggctgggatg atgttgcatg cgccgttgcg cgtgatcccg cagcaggact 2700 tgcgtttgat tgccagatct cgttacgatt atgtgatttg gtttggactt tttagatctg 2760 ttatgtgcca tagcttctgc gatgcgccta ctgctcatat gcctgatgat aatcataaat 2820 ggctgtggaa ctaactagtt gattgcggag tcatgtatca gctacaggtg tagggactag 2880 ctacaggtgt agggacttgc gtctaaattg tttggtcctg tactcatgtt gcaattatgc 2940 aatttagttt agattgtttg ttccactcat ctaggctgta aaagggacac tgcttagatt 3000 gctgtttaat ctttttagta gattatatat tatattggta acttattacc cttattacat 3060 gccatacgtg acttctgctc atgcctgatg ataatcatag atcactgtgg aattaattag 3120 ttgattgttg aatcatgttt catgtacata ccacggcaca attgcttagt tccttaacaa 3180 atgcaaattt tactgatcca tgtatgattt gcgtggttct ctaatgtgaa atactatagc 3240 tacttgttag taagaatcag gttcgtatgc ttaatgctgt atgtgccttc tgctcatgcc 3300 tgatgataat catatatcac tggaattaat tagttgatcg tttaatcata tatcaagtac 3360 ataccatggc acaattttta gtcacttaac ccatgcagat tgaactggtc cctgc atgtt 3420 ttgctaaatt gttctatttc tgattagacc atatatcatg taattttttt tttgggtaat 3480 • ggttctccta ttttaaatgc tatatagttc tggtacttgt tagaaaaatc tgcttccata 3540 gtttagttgc ttatcGctcg aattatgatg ctgagcagct gatcctatag ctttgtttca 3600 kgtatcaatt cttgtgttca acagtcagtt tttgttagat tcattgtaac ttatggtcgc 3660 ttactcttct ggtcctcaat gcttgcag 3688 < 210 > 1-1 < 211 > 2146 < 212 > DNA < 213 > Lycopersicon esculentupt < 400 > 11 ttatcngcaa agatctacaa cgtgttacac attttgtgct acaatatacc ttcaccattt 60 tgtgtatata taaaggttgc atctcttcaa ctccatcaca acaaaaatca acacaatgtc 120 ttcttcttct tctattacta ctactcttcc tttatgcacc aacaaatccc tctcttcttc 180 cttcaccacc accaactcat ccttgttatc aaaaccctct caacttttcc tccacggaag 240 gcgtaatcaa agtttcaagg tttcatgcaa cgcaaacaac gttgacaaaa accctgacgc 300 tgttgataga cgaaacgttc ttttagggtt aggaggtctt tatggtgcag ctaatcttgc 360 accattagcg actgctgcac ctataccacc tcctgatctc aagtcttgtg gtactgccca 420 tgtaaaagaa ggtgttgatg taa atacag ttgttgccct cctgtacccg atgatatcga 480 • tagtgttccg tactacaagt tcccttctat gactaaactc cgcatccgcc cccctgctca 540 tgcggcggat gaggagtacg tagccaagta tcaattggct acgagtcgaa tgagggaact 600 tgataaagac ccctttgacc ctcttggctt taaacaacaa gctaatattc attgtgctta 660 ttgcaacggt gcttacaaag ttggtggcaa agaattgcaa gttcatttct cgtggctttt 720 ctttcccttt catagatggt acttgtactt ttacgaaaga attttgggat cacttattaa 780 tgatccaact tttgctttac cttactggaa ttgggatcat ccaaaaggca tgcgtatacc 840 tcccatgttt gatcgtgagg gatcatctct ttacgatgag aaacgtaacc aaaatcatcg 900 caatggaact attattgatc ttggtcattt tggtaaggaa gttgacacac ctcagctaca 960 aataatttaa gataatgact ccctaatgta ccgtcaaatg gttactaatg ctccttgccc 1020 ttcccaattc ttcggtgctg cttacctctg ggttctgaac ccaagtccgg gtcagggtac 1080 atccctcata tattgaaaac ctccggttca catctggacc ggtgacaaac ctcgtcaaaa 1140 gacatgggta aaacggtgaa atttctactc agccggttta tttactgcca gatccgattt 1200 • ccatgccaat gtggacagga tgtggaatga attggcggga atggaaatta aaagaaggga 1260 tttaacagat aaagattggt tgaactctga attctttttc tacgatgaaa atcgtaaccc 1320 ttaccgtgtg aaagtccgtg atgttttgga cagtaaaaaa atgggattcg attacgcgcc 1380 aatgcccact ccatggcgta attttaaacc aatcagaaag tcatcatcag gaaaagtgaa 1440 tacagcgtca attgcaccag ttagcaaggt gttcccattg gcgaagctgg accgtgcgat 1500 atcacgcggc ttcgttctct aaggacaaca cagcctcgtc atgagcagga caagagaaaa 1560 acattcaata ggagattctg aaatatcgta tgatgatagg aactatgtaa ggttcgatgt 1620 gtttctgaac gtggacaaga ctgtgaatgc agatgagctt gataaggcgg agtttgcagg 1680 gagttatact agcttgccgc atgttcatgg aagtaatact aatcatgtta ccagtgttac 1740 tttcaagctg gcgataactg aactgttgga ggatattgga ttggaagatg aagatactat 1800 cgcggtgact ttaattccaa aagctggcgg tgaaggtgta tccattgaaa gtgtggagat 1860 gattgttaaa caagcttgag gtctgcatga gttggtggct atggagccaa atttatgttt 1920 aattagtata attatgtgtg gtttgagtta tgttttatgt taaaatgtat cagctcgatc 1980 gatagctgat tgctagttgt gttaatgcta tgtatgaaat aaataaatgg ttgtc ttcca 2040 • ttcagtttat cattttttgt cattctaatt aacggttaac ttttttttct actatttata 2100 cgaagctact atactatgta tatcatttgg aaaattatat attatt 2146 < 210 > 12 < 211 > 3509 < 212 > DNA < 213 > Zea mays < 400 > 12 gaattccggc gtgggcgctg ggctagtgct cccgcagcga gcgatctgag agaacggtag 60 agttccggcc gggcgcgcgg gagaggagga gggtcgggcg gggaggatcc gatggccggg 120 tcaatgggta aacgagtgga cctggaggcg atcctcgaca gccacacctc gtcgcggggt 180 gccggcggcg gcggcggcgg gggggacccc aggtcgccga cgaaggcggc gagcccccgc 240 tgaacttcaa ggcgcgcaca cccctcgcac tacttcgtcg aggaggtggt caagggcgtc 300 gacgagagcg acctccaccg gacgtggatc aaggtcgtcg ccacccgcaa cgcccgcgag 360 ggctcgagaa cgcagcacca catgtgctgg cggatctggc acctcgcgcg caagaagaag 420 cagctggagc tggagggcat ccagagaatc tcggcaagaa ggaaggaaca ggagcaggtg 480 cgtcgtgagg cgacggagga cctggccgag gatctgtcag aaggcgagaa gggagacacc 540 atcggcgagc ttgcgccggt tgagacgacc aagaagaagt tccagaggaa cttctctgac 600 cttaccgtct ggtctgacga caataaggag aagaagcttt acattgtgct catcagcgtg 660 • catggtcttg ttcgtggaga aaacatggaa ctaggtcgtg attctgatac aggtggccag 720 gtgaaatatg tggtcgaact tgcaagagcg atgtcaatga tgcctggagt gtacagggtg 780 gacctcttca ctcgtcaagt gtcatctcct gacgtggact ggagctacgg tgagccaacc 840 gagatgttat gcgccggttc caatgatgga gaggggatgg gtgagagtgg cggagcctac 900 attgtgcgca taccgtgtgg gccgcgggat aaatacctca agaaggaagc gttgtggcct 960 tacctccaag agtttgtcga tggagccctt gcgcatatcc tgaacatgtc caaggctctg 1020 ggagagcagg ttggaaatgg gaggccagta ctgccttacg tgatacatgg gcactatgcc 1080 gatgctggag atgttgctgc tctcctttct ggtgcgctga atgtgccaat ggtgctcact 1140 ttgggaggaa ggccactcac caagctggaa caactgctga agcaagggcg catgtccaag 1200 gaggagatcg attcgacata caagatcatg aggcgtatcg agggtgagga gctggccctg 1260 gatgcgtcag agcttgtaat cacgagcaca ttgatgagca aggcaggaga gtggggattg 1320 tacgatggat ttgatgtcaa gcttgagaaa gtgctgaggg cacgggcgag gcgcggggtt 1380 • agctgccatg gtcgttacat gcctaggatg gtggtgattc ctccgggaat ggatttcagc 1440 aatgttgtag ttcatgaaga cattgatggg gatggtgacg tcaaagatga tatcgttggt 1500 ttggagggtg cctcacccaa gtcaatgccc ccaatttggg ccgaagtgat gcggttcctg 1560 accaaccctc acaagccgat gatcctggcg ttatcaagac cagacccgaa gaagaacatc 1620 actaccctcg tcaaagcgtt tggagagtgt cgtccactca gggaacttgc aaaccttact 1680 ctgatcatgg gtaacagaga tgacatcgac gacatgtctg ctggcaatgc cagtgtcctc 1740 accacagttc tgaagctgat tgacaagtat gatctgtacg gaagcgtggc gttccctaag 1800 catcacaatc aggctgacgt cccggagatc tatcgcctcg cggccaaaat gaagggcgtc 1860 ttcatcaacc ctgctctcgt tgagccgttt ggtctcaccc tgatcgaggc tgcggcacac 1920 ggactcccga tagtcgctac caagaatggt ggtscggtcg acattacaaa tgcattaaac 1980 aacggactgc tcgttgaccc acacgaccag aacgccatcg ctgatgcact gctgaagctt 2040 gtggcagaca agaacctgtg gcaggaatgc cggagaaacg ggctgcgcaa catccacctc 2100 tactcatggc cggagcactg ccgcacttac ctcaccaggg tggccgggtg ccggttaagg 2160 ggctgaagga aacccgaggt cacaccagca gatgccggag ccgatgagga ggagttcctg 22 20 gaggattcca tggacgctca ggacctgtca ctccgtctgt ccatcgacgg tgagaagagc 2280 tcgctgaaca ctaacgatcc actgtggttc gacccccagg atcaagtgca gaagatcatg 2340 aacaacatca agcagfecgtc agcgcttcct ccgtccatgt cctcagtcgc agccgagggc 2400 ccatgaacaa acaggcagca atacccactc ctgcgccggc gccggcgctt gttcgtcata 2460 gctaccagga gctgtggact cgatggccgt gctagcaaga agatgctgca ggtgatccag 2520 gaagttttca gagcagtccg atcggactcc cagatgttca agatctcagg gttcacgctg 2580 tcgactgcca tgccgttgtc cgagacactc cagcttctgc agctcggcaa gatcccagcg 2640 accgaettcg acgccctcat ctgtggcagc ggcagcgagg tgtactatcc tggcacggcg 2700 aactgcatgg acgctgaagg aaagctgcgc ccagatcagg actatctgat gcacatcagc 2760 caccgctggt cccatgacgg cgcgaggcag accatagcga agctcatggg cgctcaggac 2820 ggttcaggcg acgctgtcga gcaggacgtg gcgtccagta atgcacactg tgtcgcgttc 2880 ctcatcaaag acccccaaaa ggtgaaaacg gtcgatgaga tgagggagcg gctgaggatg 2940 cgtggtctcc gctgccacat catgtactgc aggaactcga caaggcttca ggttgtccct 3000 caaggtcaca ctgctagcat ggcactcagg TATC ttccg tgcgctgggg CGTA tctgtg 3060 gggaacatgt atctgatcac cggggaacat ggcgacaccg atctagagga gatgctatcc 3120 gggctacaca agaccgtgat cgtccgtggc gtcaccgaga agggttcgga agcactggtg 3180 aggagcccag gaagctacaa gagggacgat gtcgtcccgt ctgagacccc cttggctgcg 3240 tacacgactg gtgagctgaa ggccgacgag atcatgcggg ctctgaagca agtctccaag 3300 acttccagcg gcatgtgaat ttgatgcttc ttttacattt tgtccttttc ttcactgcta 3360 gttgtgaaca tataaaataa gtaccgcggg tgtgtatata tatattgcag tgacaaataa 3420 • aacaggacac tgctaactat actggtgaat atacgactgt caagattgta tgctaagtac 3480 tccatttctc aatgtatcaa tcggaattc 3509 •

Claims (9)

1 .- A nucleic acid sequence substantially • purified comprising a nucleotide sequence selected from the group consisting of SEC I D NO: 1, the complement of SEC I D NO: 1, homologs of SEQ ID NO: 1, complement homologs of SEC I D NO: 1; SEQ ID NO: 3, the complement of SEQ ID NO: 3, homologs of SEQ ID NO: 3, and complement homologs of SEC I D NO: 3. 10

2.- The nucleic acid sequence substantially • purified according to claim 1, wherein said nucleotide sequence is characterized as having promoter activity.

3. The substantially purified nucleic acid sequence according to claim 2, wherein the promoter activity is constitutive.

4. The purified substantially f nucleic acid sequence comprising a portion of a nucleotide sequence selected from the group consisting of SEQ ID NO: 1, and its complement.

5. The substantially purified nucleic acid sequence according to claim 4, wherein the portion is characterized as having promoter activity. 6 - The substantially purified nucleic acid sequence according to claim 5, wherein the promoter activity is constitutive. The substantially purified nucleic acid sequence according to claim 5, wherein the portion comprises the nucleotide sequence selected from the group consisting of nucleotides 1 to 242 of SEQ ID NO: 7, from 245 to 787 of SEQ ID NO: 7, 788 to 1020 of SEQ ID NO: 7, 1021 to 1084 of SEQ ID NO: 7, 1085 to 1168 of SEQ ID NO: 7, 1169 to 1173 of SEQ ID NO: 7 , from 1174 to 1648 of SEQ ID NO.7, from 1649 to 1805 of SEQ ID NO: 1, from 1 to 378 of SEQ ID NO: 1, 379 to 444 of SEQ ID 10 NO: 1, and 445 to 1810 of SEQ ID NO: 1. • 8.- A substantially purified nucleic acid sequence comprising a portion of a nucleotide sequence selected from the group consisting of SEQ ID NO: 3 and its complement. 9. The substantially purified nucleic acid sequence according to claim 8, wherein the portion is characterized as having promoter activity. 10. The nucleic acid sequence substantially • purified according to claim 9, wherein the activity 20 promoter is constitutive. 11. The substantially purified nucleic acid sequence according to claim 8, wherein the portion comprises the nucleotide sequence selected from the group consisting of nucleotides from 1 to 3600 of SEQ ID NO: 10, from 3602 to 25 3612 of SEQ ID NO: 10, 3614 to 3691 of SEQ ID NO: 3, 1 to 2248 of SEQ ID NO: 3, 2249 to 2313 of SEQ ID NO: 3, 2314 to 3688 of SEQ ID NO : 3, and from 1671 to 2248 of SEQ ID NO: 3. 12.- A transgenic plant cell comprising a • nucleic acid sequence comprising a 5 nucleotide sequence selected from the group consisting of SEQ ID NO: 1, the complement of SEQ ID NO: 1, homologs of SEQ ID NO: 1, complement homologs of SEQ ID NO: 1; SEQ ID NO: 3, the complement of SEQ ID NO: 3, homologues of SEQ ID NO: 3, and complement homologs of SEQ ID NO: 3, wherein said nucleotide sequence is operably linked to a • nucleic acid sequence of interest. 13. A transgenic plant cell comprising a nucleic acid sequence comprising a portion of a nucleotide sequence selected from the group consisting of 15 SEQ ID NO: 1, the complement of SEQ ID NO: 1, SEQ ID NO: 3, and the complement of SEQ ID NO: 3. 14. A method for expressing a nucleic acid sequence of interest in a plant cell, comprising: a) providing: i) a plant cell; ii) a nucleic acid sequence of interest; and iii) a nucleotide sequence selected from the group consisting of SEQ ID NO: 1, the complement of SEQ ID NO: 1, homologs of SEQ ID NO: 1, 25 homologs of the complement of SEQ ID NO: 1; SEQ ID NO: 3, the complement of SEQ ID NO: 3, homologs of SEQ ID NO: 3, and homologs of the complement of SEQ ID NO: 3; • b) operably linking said nucleic acid sequence of interest to the nucleotide sequence to produce a transgene; and c) introducing said transgene into the plant cell to produce a cell of transgenic plants under conditions such that the acid sequence The nucleic acid of interest is expressed in the plant cell • transgenic. 15. The method according to claim 14, further comprising, d) identifying said transgenic plant cell. 1

6. The method according to claim 14, further comprising, d) regenerating the transgenic plant tissue from said transgenic plant cell. 1

7. The method according to claim 14, further comprising, d) regenerating a transgenic plant of said • cell of transgenic plant. 1

8. A method for expressing a nucleic acid sequence of interest in a plant cell, comprising: a) providing: i) a plant cell; ii) a nucleic acid sequence of interest; and iii) a portion of a nucleotide sequence ___ ^ É_É ^ -liÉ selected from the group consisting of SEQ ID NO: 1, and its complement; b) operably linking said nucleic acid sequence • of interest to the portion of a nucleotide sequence 5 to produce a transgene; and c) introducing said transgene into the plant cell to produce a cell of transgenic plants under conditions such that the nucleic acid sequence of interest is expressed in the plant cell. 10 transgenic. 1

9. The method according to claim 18, wherein the portion comprises the nucleotide sequence selected from the group consisting of nucleotides 1 to 242 of SEQ ID NO: 7, 245 to 787 of SEQ ID NO: 7 , from 788 to 1020 of SEQ ID NO: 7, from 1021 to 1584 of SEQ ID NO: 7, from 1085 to 1168 of SEQ ID NO: 7, from 1169 to 1173 of SEQ ID NO: 7, from 1174 to 1648 of SEQ ID NO: 7, 1649 to 1805 of SEQ ID NO: 1, 1 to 378 of SEQ ID NO: 1, 379 to 444 of SEQ ID NO: 1, and 445 to 1810 of SEQ ID NO: 1. 20. A method for expressing a nucleic acid sequence of interest in a plant cell, comprising: a) providing: i) a plant cell; ii) a nucleic acid sequence of interest; and iii) a portion of a nucleotide sequence 25"selected from the group consisting of SEQ ID NO. "• * -» - - • - - - • »NO: 3, and its complement, b) operably linking said nucleic acid sequence of interest to the portion of a nucleotide sequence to produce a transgene, and 5 c) introducing the transgene into the plant cell to produce a cell of transgenic plants under conditions so that the nucleic acid sequence of interest is expressed in the transgenic plant cell 21 - The method according to claim 20, wherein The portion comprises the selected nucleotide sequence of the • group consisting of nucleotides 1 to 3600 of SEQ ID NO: 10, 3602 to 3612 of SEQ ID NO: 10, 3614 to 3691 of SEQ ID NO: 3, 1 to 2248 of SEQ ID NO: 3, from 2249 to 2313 of SEQ ID NO: 3, from 2314 to 3688 of SEQ ID NO: 3, and from 1671 to 2248 of SEQ ID NO: 3. 15 •