MXPA01011262A - Regulation of viral gene expression - Google Patents

Abstract

The present invention relates to methods to alter the expression of a viral gene in a cell using sense and antisense RNA fragments of the gene. The sense and antisense RNA fragments are capable of pairing and forming a double-stranded RNA molecule, thereby altering the expression of the gene. The present invention also relates to cells, plants or animals, their progeny and seeds derived thereof, obtained using a method of the present invention. Preferably, such cells, plants or animals are resistant or tolerant to viruses.

Description

REGULATION OF THE EXPRESSION OF VIRAL GENES DESCRIPTION OF THE INVENTION The present invention relates to methods for altering the expression of viral genes in cells, plants, or animals, using sense and anti-sense RNA fragments of these genes, and cells, plants, or animals with the expression of altered viral genes, obtained using the methods of the present invention. The invention particularly relates to cells, plants, or animals resistant or virus tolerant. One area of deep interest is resistance or tolerance to viruses. Viruses affect most living organisms. In crops, large proportions of the crop can be lost due to virus infections. Farm animals are also frequently infected with viruses, and sometimes must be sacrificed to prevent the spread of the disease that leads to dramatic economic consequences. Pets are also affected by viruses, and finally, viruses infect humans, causing a great deal of suffering. Aungue have developed treatments against viruses, very often they are extremely expensive or of limited efficiency. It is desirable to modify the cells, plants, or REF: 133226 animals, in such a way that the expression of a particular viral gene is altered, to create cells, plants, or animals resistant or tolerant to that virus. Current methods for altering the expression of a gene are usually supported by sense or anti-sense suppression techniques. Unfortunately, these methods are often variable and unpredictable in their ability to alter gene expression, and in many cases complete disruption of the activity of the particular gene is not achieved. Accordingly, there is a long-felt but unsatisfied need for novel methods and compositions that enable the expression of a viral gene to be altered in an effective and predictable manner to obtain cells, plants, or animals with resistance or tolerance to the same. virus. The present invention relates to methods for altering the expression of a viral gene in cells, plants, or animals, using sense and antisense RNA fragments of the gene. It is important that these sense and anti-sense RNA fragments are capable of forming a double-stranded RNA molecule. Particularly, the present invention relates to methods and compositions for conferring resistance or tolerance to viruses to a cell, plant, or animal. Preferably, the invention relates to methods for conferring a plant, resistance or tolerance to viruses. The invention also preferably relates to plant cells obtained using these methods, to plants derived from those cells, to the progeny of these plants, and to seeds derived from such plants. In these plant cells or in these plants, the alteration of the gene expression of a particular viral gene is more effective, selective, and more predictable than the alteration of the genetic expression of a particular viral gene obtained using the current methods known in the art. matter.

Accordingly, the invention provides: A method comprising introducing into a cell, a plurality of sub-sequences, for example RNA fragments or DNA sequences, characterized in that at least two of the sub-sequences have sense and anti-sequence sequences. sense of viral RNAs, and are capable of forming a double-stranded RNA molecule. Preferably, the method comprises introducing into a cell an RNA consisting of a plurality of sub-sequences, characterized in that at least two of the sub-sequences have the sequences of the viral RNAs. Preferably, the RNA contains at least one stop codon located upstream of the 3'-terminal sub-sequence. In another preferred embodiment, the method comprises introducing into a cell, a nucleotide sequence or a DNA molecule that encodes the expression of said RNA.

Accordingly, the invention provides: A method comprising introducing into a cell, an RNA fragment in the sense of a target viral gene, and an anti-sense RNA fragment of said target gene, wherein this sense RNA fragment and this antisense RNA fragment they are able to form a double-stranded RNA molecule, where the expression of the target gene in the cell is altered. In a preferred embodiment, the target gene comprises a viral genome or a portion thereof, and the cell is preferably resistant or virus tolerant. In a preferred embodiment, the virus is selected from the group consisting of tospovirus, potyvirus, potexvirus, tobamovirus, lutein, cucumovirus, bromovirus, closteorvirus, tombusvirus, and furovirus. In another preferred embodiment, the RNA fragments comprise nucleotide sequences derived from a viral coat protein gene, a viral nucleocapsid protein gene, a viral replicase gene, a motion protein gene, or portions of the same. In a further preferred embodiment, a cell is a plant cell, such as a monocot or dicot cell. In another preferred embodiment, the RNA fragments are comprised of two different RNA molecules. In another preferred embodiment, the RNA fragments are mixed before being introduced into the cell. In another preferred embodiment, the RNA fragments are mixed before being introduced into the cell, under conditions that allow them to form a double-stranded RNA molecule. In another preferred embodiment, the RNA fragments are introduced into the cell in sequence. In yet another preferred embodiment, the RNA fragments are comprised in an RNA molecule. In such a case, the RNA molecule is preferably able to fold, such that the RNA fragments comprised therein form a double stranded RNA molecule.

The invention further provides: A method comprising introducing into a cell, a first DNA sequence capable of expressing in that cell, an RNA fragment in the sense of a target viral gene, and a DNA sequence capable of expressing in that cell, an anti-sense RNA fragment of that target gene, wherein the sense RNA fragment and the anti-sense RNA fragment are capable of forming a double-stranded RNA molecule, wherein the expression of the target viral gene is altered in that cell. In a preferred embodiment, the target gene comprises a viral genome or a portion thereof, and the cell is preferably resistant or virus tolerant. In a preferred embodiment, the virus is selected from the group consisting of tospovi-rus, potyvirus, potexvirus, tobamovirus, luteovirus, cucumovi-rus, bromovirus, closteorvirus, tombusvirus, and furovirus. In another preferred embodiment, the DNA sequences comprise a nucleotide sequence derived from a viral coat protein gene, a viral nucleocapsid protein gene, a viral replicase gene, a motion protein gene, or portions thereof. In a further preferred embodiment, a cell is a plant cell, such as a monocot or dicot cell. In a preferred embodiment, the DNA sequences are stably integrated into the genome of the plant cell. In a preferred embodiment, the DNA molecule further comprises a promoter operably linked to the first or second DNA sequences. In another preferred embodiment, the first DNA sequence and the second DNA sequence are comprised in two different DNA molecules, ie, separated. In an alternative way, the first DNA sequence and the second DNA sequence are comprised in a DNA molecule. In this case, the first DNA sequence and the second DNA sequence are preferably comprised in the same DNA strand of the DNA molecule, meaning that the sense RNA fragment and the antisense RNA fragment are comprised in one molecule of RNA. Preferably, the RNA molecule is capable of folding, in such a way that the RNA fragments comprised therein form a double-stranded region. Examples of these RNA molecules comprise the inverted repeat sequence of SEQ ID NO: 13, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 25, or SEQ ID NO: 28. In another preferred embodiment , the RNA fragment in sense and the anti-sense RNA fragment are comprised in, or are expressed as, two RNA molecules. In this case, the first DNA sequence and the second DNA sequence are preferably operably linked to a bidirectional promoter, or alternatively, the first DNA sequence is operably linked to a first promoter, and the second sequence of DNA is operably linked to a second promoter, wherein the first promoter and the second promoter are the same different promoter or promoters. In another preferred embodiment, the first DNA sequence and the second DNA sequence are comprised in complementary strands of said DNA molecule.

In still another preferred embodiment, the first DNA sequence is the DNA strand complementary to the second DNA sequence in said DNA molecule. In this case, the DNA molecule further comprises a first promoter operably linked to the first or second DNA sequences. In a preferred embodiment, the DNA molecule further comprises a first site-specific recombination site between the first promoter and the first or second DNA sequence, and a second site-specific recombination site at the 3 'end of the first sequence of DNA, wherein the first and second site-specific recombination sites are capable of inverting the first or second DNA sequences between the first and second site-specific recombination sites in the presence of a site-specific recombinase. In a further preferred embodiment, and as a result of the inversion, the first promoter is capable of expressing the second (or the first, depending on which DNA sequence was originally linked to the promoter) DNA sequence. The plant cell preferably further comprises a site-specific recombinase capable of recognizing these site-specific recombination sites. In yet another preferred embodiment, the DNA molecule further comprises a first promoter operably linked to the first DNA sequence, and a second promoter operably linked to the second DNA sequence, wherein the first promoter and the second promoter comprise the same promoter, or comprise different promoters. In another preferred embodiment, the promoter in the DNA molecule comprises a promoter native to the cell. In a further preferred embodiment, the promoter is a heterologous promoter, for example a tissue-specific promoter, a developmental regulatory promoter, a constitutive promoter, or an inducible promoter. Optionally, the promoter is a divergent or bidirectional promoter capable of initiating the transcription of DNA sequences on each side of the promoter. In still another preferred embodiment, the sequence of DNA further comprises a linker between the DNA sequences encoding the sense and anti-sense RNA fragments.

The linker comprises, for example, an expression cassette comprising a functional gene, for example a selectable marker gene, or regulatory sequences, for example introns processing signals.

The invention also further provides: A cell comprising the sense and anti-sense RNA fragments of the present invention, wherein the expression of the target viral gene in the cell is altered by these RNA fragments. the cell is resistant or tolerant to viruses In a preferred embodiment, the cell is a plant cell, and the invention further provides a plant and its progeny derived from the plant cell, and seeds derived from the plant.

The invention also provides: DNA constructs comprising the DNA sequences of the present invention. In a preferred embodiment, this DNA construct comprises a first DNA sequence capable of expressing in a cell, an RNA fragment in the sense of a viral genome or a portion thereof, and a second DNA sequence capable of expressing in this cell , an anti-sense RNA fragment of the viral genome or a portion thereof, wherein the sense RNA fragment and the anti-sense RNA fragment are capable of forming a double-stranded RNA molecule. In another preferred embodiment, the expression of the viral genome or a portion thereof in this cell is altered. In another preferred embodiment, the cell is a plant cell. In another preferred embodiment, the virus is selected from the group consisting of tosovirus, potyvirus, potexvirus, tobamovirus, luteovirus, cucu- movirus, bromovirus, closteorvirus, tombusvirus, and furovirus. In another preferred modality, the DNA sequences comprise a nucleotide sequence derived from a viral coat protein gene, a viral nucleocapsid protein gene, a viral replicase gene, a movement protein gene, or portions thereof. In yet another preferred embodiment, the DNA construct further comprises a promoter operably linked to the first or second DNA sequences. In still another preferred embodiment, the DNA construct comprises a first promoter operably linked to the first DNA sequence, and a second promoter operably linked to the second DNA sequence. In yet another preferred embodiment, the DNA construct further comprises a bidirectional promoter operably linked to the first DNA sequence and the second DNA sequence.

The invention further provides: A DNA construct comprising: (a) a first DNA sequence capable of expressing in a cell, an RNA fragment in the sense of a target gene, and a second DNA sequence capable of expressing in the cell , an anti-sense RNA fragment of the target gene, wherein the sense RNA fragment and the anti-sense RNA fragment are capable of forming a double-stranded RNA molecule, wherein the first DNA sequence is the complementary strand of the second DNA sequence in the construction of the DNA, (b) a promoter operably linked to the first or second DNA sequences, (c) a first site-specific recombination site between the promoter and the first or second DNA sequences, and (d) a second site-specific recombination site at the 3 'end of the first or second sequence of DNA, wherein the first and second site-specific recombination sites are capable of inverting the first or second DNA sequence between the first and second site-specific recombination sites, in the presence of a site-specific re-combinase. In a preferred embodiment, the expression of the target gene in the cell is altered.

The invention further provides: A DNA construct comprising: (a) a first DNA sequence capable of expressing in a cell, an RNA fragment in the sense of a target gene, and a second DNA sequence capable of expressing in the cell , an anti-sense RNA fragment of the target gene, wherein the sense RNA fragment and the anti-sense RNA fragment are capable of forming a double-stranded RNA molecule, wherein the first DNA sequence is the complementary to the second DNA sequence in the construction of the DNA, (b) a first promoter operably linked to the first DNA sequence, (c) a second promoter operably linked to the second DNA sequence. In a preferred embodiment, the expression of the target gene in the cell is altered.

A "double stranded RNA (dsRNA)" molecule comprises an RNA fragment in the sense of a target gene, and an anti-sense RNA fragment of the same target gene, wherein both comprise complementary nucleotide sequences for one another, thus allowing the sense and anti-sense RNA fragments to pair and form a double-stranded DNA molecule. "Complementary" refers to two nu-cleotide sequences comprising antiparallel nucleotide sequences capable of pair- ing with one another after the formation of hydrogen bonds between the complementary base residues in antiparallel nucleotide sequences. "Antiparallel" refers herein to two sequences of paired nucleotides through hydrogen bonds between complementary base residues, running phosphodiester bonds in the 5'-3 'direction in a nucleotide sequence, and in the 3 '-5' in the other nucleotide sequence. A "target gene" is any viral gene of known function, or is a gene whose function is unknown, but whose total or partial nucleotide sequence is known. A target gene is a gene native to the cell, or a heterologous gene that had previously been introduced into the cell, preferably by genetic transformation or viral infection of the cell. Preferably, the target gene is a gene in a plant cell. A "native" gene refers to a gene that is present in the genome of the non-transformed cell. An "essential" gene is a gene that encodes a protein, such as, for example, a biosynthetic enzyme, a receptor, a signal transduction protein, a structural gene product, or a transport protein that is essential for growth or the survival of the cell. "Altering" the expression of an objective gene in a cell means that the level of expression of the target gene in a cell after applying the method of the present invention, it is different from its expression in the cell without applying the method. Altering the preferred gene expression means that the expression of the target gene in the cell is reduced, preferably much is reduced, more preferably the expression of the gene can not be detected, resulting in a mutant phenotype eliminated in the cells or plants or animals derived from it. "Isolated", in the context of the present invention, is an isolated nucleic acid molecule that, by the hand of man, exists apart from its native environment, and therefore, is not a product of nature. An isolated nucleic acid molecule can exist in a purified form, or it can exist in a non-native environment, such as, for example, a transgenic host cell. "Plasmid expression", as used herein, means a DNA sequence capable of directing the expression of a particular nucleotide sequence in an appropriate host cell, comprising a promoter operably linked to the nucleotide sequence of interest, which it is operatively linked with termination signals. It also normally comprises sequences required for the proper translation of the nucleotide sequence. The coding region usually codes for a protein of interest, but it can also code for a functional RNA of interest, for example, anti-sense RNA, or an untranslated RNA, in the direction in sense or anti-sense. The expression cassette comprising the nucleotide sequence of interest can be chimeric, meaning that at least one of its components is heterologous with respect to at least one of its components. The expression cassette may also be one that occurs naturally, but has been obtained in a recombinant form useful for heterologous expression. Normally, however, the expression cassette is heterologous with respect to the host, i.e., the particular DNA sequence of the expression cassette does not occur naturally in the host cell, and must have been introduced into the host cell or into a host cell. ancestor of the host cell, through a transforming event. Expression of the nucleotide sequence in the expression cassette can be under the control of a constitutive promoter, or of an inducible promoter that initiates transcription only when it is exposed to the host cell to some particular external stimulus. In the case of a multicellular organism, such as a plant, the promoter may also be specific for a particular tissue and organ or stage of development. "Heterologist", as used herein, means "of different natural origin", or represents a non-natural state. For example, if a host cell is transformed with a nucleic acid sequence derived from another organism, particularly from another species, that nucleic acid sequence is heterologous with respect to that host cell, and also with respect to the descendants of the host cell. that carry that nucleic acid sequence. In a similar manner, heterologous refers to a nucleotide sequence derived from, and inserted into, the same type of natural original cell, but which is present in an unnatural state, eg, a different number of copies, or under the control of different regulatory elements. In its broadest sense, the term "substantially similar", when used herein with respect to a nucleotide sequence, means a nucleotide sequence corresponding to a reference nucleotide sequence, wherein the corresponding sequence encodes a polypeptide. -tido having substantially the same structure and function as the polypeptide encoded by the reference nucleotide sequence, for example, wherein only changes occur in amino acids that do not affect the function of the polypeptide. Desirably, the nucleotide sequence is substantially similar, encodes the polypeptide encoded by the reference nucleotide sequence. The percent identity between the substantially similar nucleotide sequence and the reference nucleotide sequence (number of complementary bases in the complementary sequence divided by the total number of bases in the complementary sequence) is desirably at least 80 percent , more desirably 85 percent, preferably at least 90 percent, more preferably at least 95 percent, and still more preferably at least 99 percent. "Regulatory elements" refer to sequences involved to confer the expression of a nucleotide sequence. The regulatory elements comprise a promoter operably linked to the nucleotide sequence of interest and the termination signals. They also normally encompass sequences required for an appropriate translation of the nucleotide sequence. A "plant" refers to any plant or part of a plant at any stage of development. It also includes cuttings, cell or tissue cultures, and seeds. As used in conjunction with the present invention, the term "plant tissue" includes, but is not limited to, whole plants, plant cells, plant organs, plant seeds, protoplasts, callus, cell cultures, and any groups of plant cells organized into structural and / or functional units. "Resistance or tolerance to viruses" means in the present that a resistant or tolerant cell, plant, or animal is not susceptible, or has a reduced susceptibility, to one or more viruses, compared to a cell, plant, or animal. badly sensitive Resistance or tolerance, for example, means that the usual symptoms of a virus infection are absent or reduced, or that the accumulation or replication of the virus in the cell is prevented or reduced, or that the virus is prevented or reduced. movement of the virus, for example from cell to cell. By "altering the expression of the viral genome or a portion thereof", it is commonly understood that the accumulation, replication, or movement of the virus or a portion or component thereof, for example an RNA, DNA, or protein of the virus, is affected. virus, in the cell. The present invention relates to methods for regulating, i.e., altering the expression of a viral gene in cells, plants, or animals. The methods commonly available to regulate the expression of a gene in cells, plants, or animals, lack predictability, and show variability, depending on which gene is to be regulated. The present method alleviates these problems, and provides for a reproducible and efficient regulation of a gene in cells, plants, or animals. The gene whose expression is regulated is a viral genome or a portion thereof. In a preferred embodiment, a cell is a eukaryotic cell, more preferably a plant cell, such as a monocot or dicot cell, or an animal cell, for example from a mammal, for example from a human, a bovine, a sheep, a pig, a cat, or a dog, or a bird. The present invention utilizes a sense RNA fragment and an anti-sense RNA fragment of a target viral gene, to alter the expression of the gene in a cell. In a first embodiment, the invention provides a method comprising introducing into a cell, a fragment of RNA in the sense of a target gene, and an anti-sense RNA fragment of that target gene, wherein the RNA fragment in sense and the anti-sense RNA fragment are capable of forming a double-stranded RNA molecule, wherein the expression of the target gene in the cell is altered. The RNA fragments are introduced into the cells by different transformation methods, such as particle bombardment, or PEG-mediated transformation, or electroporation. In another preferred embodiment, other techniques are used, such as microinjection of RNA fragments. In a preferred embodiment, the RNA fragments are comprised in two different RNA molecules. In this case, the RNA fragments are mixed before being introduced into the cell, for example under conditions that allow them to form a double-stranded RNA molecule. In another preferred embodiment, the RNA fragments are introduced into the cell in sequence. Preferably, the time interval between the introduction of each of the RNA molecules is short, preferably less than 1 hour. In yet another embodiment, the RNA fragments are comprised in an RNA molecule. By using a single RNA molecule, the two complementary RNA fragments are in close proximity, such that pairing is favored. In such a case, the RNA molecule is preferably capable of folding, such that the RNA fragments comprised therein form a double-stranded region. In this case, the complementary parts of the RNA fragments recognize one another, pair one with the other, and form the double-stranded RNA molecule. In a preferred embodiment, the RNA fragments are incubated under conditions that allow them to form a double stranded RNA molecule before entering the cell. In still another embodiment, the RNA molecule comprises a linker between the sense RNA fragment and the anti-sense RNA fragment. The linker preferably comprises an RNA sequence encoded by an expression cassette comprising a functional gene, for example a selectable marker gene. In another embodiment, the linker comprises an RNA sequence encoded by regulatory sequences, which, for example, comprise introns processing signals. In a further embodiment, the present invention provides a method for altering the expression of a viral genome, which comprises introducing into a cell, a first DNA sequence capable of expressing in this cell, an RNA fragment in the sense of the genome. viral, and a second DNA sequence capable of expressing in this cell, an anti-sense RNA fragment of this viral genome, wherein the sense RNA fragment and the anti-sense RNA fragment are capable of forming a molecule of Double-stranded RNA In a preferred embodiment, the first DNA sequence and the second DNA sequence are stably integrated into the genome of the cell. In another preferred embodiment, the DNA sequences are comprised in two different DNA molecules. In another preferred embodiment, the DNA sequences are comprised in a DNA molecule. In such a case, the DNA molecule preferably encodes a single RNA molecule comprising the sense and anti-sense RNA fragments. By using a single RNA molecule, the two complementary RNA fragments are in close proximity, such that pairing is favored. The DNA molecule encodes two separate RNA molecules, for example an RNA molecule comprising a sense RNA fragment and an RNA molecule comprising an anti-sense RNA fragment. The single RNA molecule or the two different RNA molecules are preferably capable of folding, such that the RNA fragments comprised therein form a double-stranded region, wherein the complementary portions of the RNA fragments are they recognize one another, they pair with one another, and they form the double-stranded RNA molecule. In one embodiment, the single DNA molecule, or each of the two different DNA molecules, comprises a promoter operably linked to the DNA sequences. In a preferred embodiment, the promoter in the DNA sequence comprises a native plant promoter, or the natural promoter of the viral gene to be inactivated, in order to ensure that the double-stranded RNA is expressed in them. tissues and at the same time in the development that the target viral gene. In another modality, the promoter is a heterologous promoter, for example a tissue-specific promoter, a developmentally regulated promoter, a constitutive promoter, or an inducible promoter. In yet another embodiment, the DNA sequence comprises a linker between the DNA sequences encoding these two complementary RNA fragments. The linker preferably comprises an expression cassette comprising a functional gene, for example a selectable marker gene. In another embodiment, the linker comprises regulatory sequences, which, for example, comprise introns processing signals. The DNA molecules of the present invention are transformed into cells using methods well known in the art or described below. The present invention also provides a DNA construct comprising DNA sequences of the present invention, a recombinant vector comprising these DNA constructs, and a composition comprising DNA sequences of the present invention. In the present invention, the complementary region between the sense and anti-sense RNA fragments is desirably at least 15 nucleotides long, more desirably at least 50 nucleotides long, and preferably at least 500 pairs long. long bases. Preferably, the complementary region is less than 5 kb, and more preferably less than 2 kb. In a particular embodiment, the complementary region between sense and antisense RNA fragments comprises the coding region of the target gene. In another preferred embodiment, the complementary region comprises untranslated regions (UTR) of the target gene, for example 5 'UTR or 3' UTR. In yet another preferred embodiment, a DNA sequence encoding a sense or anti-sense RNA fragment of the present invention is derived from a cDNA molecule, or comprises regulatory elements of the target viral gene whose expression is will alter, such as promoter or termination signals. In another preferred embodiment, the complementary region between the sense and anti-sense RNA fragments is identical to the corresponding sequence of the gene whose expression is to be altered. In another preferred embodiment, the complementary region between the sense and anti-sense RNA fragments is substantially similar to the corresponding sequence of the gene whose expression is to be altered, and is still capable of altering the expression of the gene. In this case, the complementary region is desirably at least 50 percent identical to the corresponding sequence of the gene whose expression is to be altered, most desirably at least 70 percent identical, preferably at least 90 percent identical. , more preferably at least 95 percent identical. In this way, the use of a single molecule of double-stranded RNA allows altering the expression of a single gene or a plurality of genes, the single gene comprising sequences identical to double-stranded RNA, or being substantially similar to RNA double chain. In another preferred embodiment, the complementary region between sense and antisense RNA fragments does not contain any mismatch between sense and antisense RNA fragments. In another preferred embodiment, the complementary region between the sense and antisense RNA fragments comprises at least a mismatch between the sense and antisense RNA fragments, and the two RNA fragments are still able to pair and form a double-stranded RNA molecule, thereby altering the expression of the gene. Desirably, there is less than 50 percent mismatch between sense and antisense RNA fragments in the complementary region, more desirably less than 30 percent mismatch, preferably less than 20 percent mismatch, more preferably less of 10 percent of bad coupling, and still most preferably less than 5 percent of bad coupling.

Resistance or Tolerance to Viruses The present invention results in cells, animals, or plants that are resistant or tolerant to viruses. Viruses controlled using the present invention include, but are not limited to, dsDNA viruses, dsRNA viruses, positive and negative strand ssRNA viruses, ambi-sense RNA viruses, and retroviruses. Preferably, plant viruses are controlled, such as astospoviruses (eg tomato spotted wilt virus abbreviated as TS BV), potyviruses (e.g., turnip mosaic virus abbreviated as TuMV, lettuce mosaic virus abbreviated as LMV, watermelon mosaic virus II abbreviated as MVII, zucchini yellow mosaic virus abbreviated as ZYMV, potato Y virus abbreviated as PVY, and papaya ring stain virus open as PRSV), potexvirus, tobamovirus (for example, peppered light virus abbreviated as PMMV, and tomato mosaic virus abbreviated as ToMV), luteovirus (eg, western yellow beet virus abbreviated as B YV, and light beet yellowing virus abbreviated as BMYV), cucumovirus (eg example, cucumber mosaic virus abbreviated as CMV), geminivirus (eg yellow tomato leaflet virus abbreviated as TYLCV), caulimovirus (eg, mosaic virus). cauliflower code abbreviated as CaMV), bromovirus, closteorvirus, tombusvirus, and fu-rovirus (for example, yellow necrotic remnant vein virus abbreviated as VNYVV). Additional classes of viruses that can be controlled using the present invention are described in Zacomer et al. (1995) Journal of General Virology, 76: 231-247, and in Martelli (1992) Plant Disease, 76: 436-441. The preferred DNA sequences of a viral genome to achieve virus control correspond to regions of the genes encoding viral coat proteins, viral nucleocapsid proteins, viral replicates, movement proteins, and the like. Other nucleotide sequences useful for controlling the expression of the virus genome are described in International Publication Number WO 95/09920. Preferred DNA sequences may include portions of the viral genome untranslated in proteins, eg, 5 'or 3' untranslated regions. Preferably, a method of the present invention leads to resistance or tolerance to a broad spectrum of viruses. For example, a method of the present invention leads to resistance or tolerance to the virus encoded by the viral genome and other viruses in the same class, group, or genus of virus. In an alternative way, a method of the present invention leads to resistance or tolerance to the virus encoded by the viral genome and other isolates of the same virus. Also, a method of the present invention leads to resistance or tolerance to the virus encoded by the viral genome and other viruses in the same group or genus of viruses in different species, preferably in the wild. in related species, preferably species where these viruses exist. Optionally, more than one pair is used, that is, when 'minus two pairs of sense and anti-sense RNA fragments are capable of forming a dsDNA. These pairs are derived, for example, from the same viral genome, but from different portions of the same viral genome. In an alternative way, these pairs are derived from different viral genomes. Accordingly, resistance or tolerance to different classes, groups, or genera of viruses is achieved using the present invention. Plant cells and plants derived therefrom, which are resistant or virus tolerant, are preferably dicotyledonous plants. Methods for conferring resistance or tolerance to furoviruses (see, for example, Rusch and Heidel (1995) Plant Disease 79: 868-875) in sugar beet and sugarcane, are disclosed in the present invention, and are described with greater detail for BNYVV, the causative agent of ri-zomania (brittle roots) in sugar beet, in Example 9. Methods for conferring resistance or tolerance to "yellow virus" (see, for example, CRC Handbook on Disease of Sugar Beet, Volume II, pages 35-52), such as BMYV, and western yellow beet virus (BWYV), which infect sugar beet and oilseed rape, respectively, are described in greater detail in Example 8. The methods of this invention also confer tolerance or resistance to viruses in monocotyledonous plants. For example, using the teachings of the present invention and of US Pat. No. 5,569,828, tolerance or resistance to maize chlorotic dwarf virus is obtained. In a similar manner, using the teachings of the present invention and U.S. Patent No. 5,428,144, tolerance or resistance to maize dwarf mosaic virus is achieved.

Plant Transformation Technology The DNA molecules of the present invention are incorporated into plant or bacterial cells, using conventional recombinant DNA technology. In general, a DNA molecule of the present invention is comprised in a transformation vector. A large number of these vector systems known in the art are used, such as plasmids, bacteriophage viruses, and other modified viruses. The components of the expression system are also modified, for example, to increase the expression of sense and anti-sense RNA fragments. For example, truncated sequences, nucleotide substitutions, or other modifications are employed. Expression systems known in the art are used to transform virtually any cell of a crop plant under suitable conditions. A transgene comprising a DNA molecule of the present invention, preferably transforms stably and integrates into the genome of the host cells. In another preferred embodiment, the transgene comprising a DNA molecule of the present invention is located on a self-replicating vector. Examples of self-replicating vectors are viruses, particularly geminiviruses. The transformed cells are preferably regenerated in whole plants. Transformed plants according to the present invention can be monocot or dicot, and include, but are not limited to, corn, wheat, barley, rye, sweet potato, beans, peas, chicory, lettuce, cabbage, cauliflower, broccoli. coli, turnip, radish, spinach, asparagus, onion, garlic, pepper, celery, chayote, pumpkin, hemp, zucchini, apple, pear, quince, melon, plum, cherry, peach, nectarine, apricot-not, strawberry, grape, raspberry, blackberry, pineapple, avocado, papaya, mango, banana, soybean, tomato, sorghum, sugar cane, sugar beet, sunflower, rape seed, clover, tobacco, carrot, cotton, alfalfa, rice, potato, eggplant , cucumber, Arabidopsis, and woody plants, such as co-niferous and deciduous trees. Once a desired nucleotide sequence has been transformed into a particular plant species, it can be propagated in that species, or it can be moved towards other varieties of the same species, particularly including commercial varieties, using traditional breeding techniques.

A. Requirements for the Construction of Expression Cassettes of Plants The genetic sequences intended to be expressed in transgenic plants are first assembled in expression cassettes behind a suitable promoter that can be expressed in plants. The expression cassettes may also comprise any additional sequences required or selected for the expression of the transgene. These sequences include, for example, but are not restricted to, transcription terminators, foreign sequences to improve expression, such as introns, vital sequences, and sequences intended for the direction of the gene product toward organelles and specific compartments. These expression cassettes can then be easily transferred to the plant transformation vectors described below. The following is a description of different components of typical expression cassettes. 1. Promoters The selection of the promoter used in the expression cassettes determines the spatial and temporal expression pattern of the transgene in the transgenic plant. The selected promoters express transgenes in specific cell types (such as leaf epidermal cells, mesophyll cells, root bark cells), or in specific tissues or organs (roots, leaves, or flowers, for example), and the selection reflects the desired location of accumulation of the genetic product. In an alternative way, the selected promoter drives the expression of the gene under different induction conditions. The promoters vary in their strength, that is, in its ability to promote transcription. Depending on the host cell system used, any of a number of suitable promoters known in the art is used. For example, for constitutive expression, the CaMV 35S promoter, the rice actin promoter, or the ubiquitin promoter are used. For example, for regulatable expression, the PR-1 promoter chemically inducible from tobacco or Arabidopsis is used (see, for example, U.S. Patent No. 5,689,044). A preferred category of promoters is one that is wound inducible. Numerous promoters that are expressed at wound sites have been described. Preferred promoters of this class include those described by Stanford et al., Mol. Gen. Genet. 215: 200-208 (1989), Xu and collaborators, Plant Molec. Biol. 22: 573-588 (1993), Logemann et al., Plant Cell 1: 151-158 (1989), Rohrmeier and Lehle, Plant Molec. Biol. 22: 783-792 (1993), Firek et al., Plant Molec. Biol. 22: 129-142 (1993), and Warner et al., Plant J.: 191-201 (1993). Preferred tissue-specific expression patterns include green-specific, stem-specific, stem-specific, and flower-specific tissue. Suitable promoters for expression in green tissue include many that regulate the genes involved in photosynthesis, and many of these have been cloned from both mono- and dicotyledons. A preferred promoter is the corn PEPC promoter from the phosphoenol carboxylase gene (Hudspeth and Gruia, Plant Molec, Biol. 12: 579-589 (1989)). A preferred promoter for specific expression of the root is that described by de Framond (FEBS 290: 103-106 (1991)).; European Patent Number EP 0,452,269), and an additional preferred root-specific promoter is that from the T-l gene provided by this invention. A preferred promoter of the preferred stem is that described in US Pat. No. 5,625,136, and which promotes expression of the maize trpA gene. Preferred embodiments of the invention are transgenic plants that express nucleotide sequences in a specific form of the root. Additional preferred embodiments are transgenic plants that express the nucleotide sequence in a wound-inducible or inducible manner by pathogen infection. 2. Transcription Terminators There are a variety of transcription terminators available for use in expression cassettes. These are responsible for the termination of transcription beyond the transgene and its correct polyadenylation. Suitable transcription terminators are those that are known to work in plants, and include the CaMV 35S terminator, the tml terminator, the nopaline synthase terminator, and the terminator r £ > cS E9 of peas. These are used in both monocotyledonous and dicotyledonous plants. 3. Sequences for improving or regulating expression It has been found that numerous sequences improve gene expression from within the transcription unit, and these sequences can be used in conjunction with the genes of this invention to increase their expression in transgenic plants. For example, it has been shown that different sequences of introns, such as the introns of the Adhl corn gene, improve expression, particularly in monocotyledonous cells. In addition, it is also known that a number of untranslated leader sequences derived from viruses, improve expression, and these are particularly effective in dicotyledonous cells. 4. Optimization of the Coding Sequence The coding sequence of the selected gene can be genetically engineered by altering the coding sequence for optimal expression in the crop species of interest. Methods for modifying the coding sequences in order to achieve optimal expression in a particular culture species are well known (see, for example, Perlak et al, Proc.Na.I.Acid.Sci.U.A. 88: 3324 (1991 ), and Koziel et al., Bio / technol 11: 194 (1993)). In another preferred embodiment, a DNA molecule of the present invention is directly transformed into the plastid genome. Plastid transformation technology is extensively described in U.S. Patent Nos. 5,451,513; 5,545,817; and 5,545,818, in the TPC Application Number WO 95/16783, and in McBride et al. (1994) Proc. Nati Acad. Sci. USA 91, 7301-7305. The basic technique for chloroplast transformation involves introducing regions of cloned plastid DNA flanking a selectable marker together with the gene of interest, into a suitable target tissue, for example, using biolistic or protoplast transformation (e.g. calcium or PEG-mediated transformation). Flanking regions of 1 to 1.5 kb, referred to as targeting sequences, facilitate homologous recombination with the plastid genome, and therefore, allow replacement or modification of specific regions of the plastome. Initially, point mutations are used in the chloroplast 16S mRNA and rpsl2 genes that confer resistance to spectinomycin and / or streptomycin, as selectable markers for transformation (Svab, Z., Hajdukiewivz, P., and Maliga, P. (1990) Proc. Nati, Acad. Sci. USA 87, 8526-8530, Staub, JM, and Maliga, P. (1992) Plant Cell 4, 39-45). The presence of cloning sites between these markers allowed the creation of a plastid targeting vector for the introduction of foreign DNA molecules (Staub, J.M. and Maliga, P. (1993) EMBO J. 12, 601-606). Substantial increases in the frequency of transformation are obtained by replacing the recessive rRNA or the protein-r antibiotic resistance genes with a dominant selectable marker, coding for the bacterial gene aadA the detoxifying enzyme of spectinomycin, aminoglycoside- 3 '-adenyltransferase (Svab, Z., and Maliga, P. (1993) Proc. Na ti. Acad. Sci. USA 90, 913-917). Previously, this marker had been successfully used for the high-frequency transformation of the plastid genome of the green alga Chlamydomonas reinhardtii (Goldschmidt-Clermont, M. (1991) Nucí Acids Res. 19: 4083-4089). Other selectable markers useful for the transformation of plastids are known in the art, and are encompassed within the scope of the invention. In plastid expression, genes are inserted by homologous recombination in the several thousand copies of the circular plastid genome present in each plant cell. In a preferred embodiment, a DNA of the present invention is inserted into a plastid targeting vector, and transformed into the plastid genome of a desired host plant. Homoplasmic plants are obtained for the plastid genomes that contain the DNA molecule of the present invention, and preferably are capable of high expression of the DNA molecule. Preferably, the sense and anti-sense RNA fragments encoded by the DNA molecule are able to pair and form a double-stranded RNA molecule in the plant plastids to alternate the expression of the plastid genes. In a preferred embodiment, the sense and anti-sense fragments do not comprise any bad coupling in the complementary region. In another preferred embodiment, the sense and anti-sense fragments comprise at least a bad coupling in the complementary region. In this case, the DNA sequences in the DNA molecule encoding the RNA fragments are not able to recombine with each other.

B. Construction of Plant Transformation Vectors Ordinary experts in plant transformation techniques know of numerous transformation vectors available for the transformation of plants, and the genes pertinent to this invention can be used in conjunction with any of these vectors . The selection of the vector depends on the preferred transformation technique and on the target species for the transformation. For certain target species, different antibiotic or herbicide selection markers are preferred. The selection markers routinely used in the transformation include the nptll gene, which confers resistance to kanamycin and related antibiotics (Messing and Vierra, Gene 19: 259: 268 (1982); Bevan et al., Nature 304: 184-187 (1983J) , the bar gene, which confers resistance to the herbicide phosphinothricin (White et al., Nucí Acids Res 18_: 1062 (1990), Spencer et al., Theor. Appl. Genet. 19_: 625-631 (1990)), the hph gene , which confers resistance to the hygromycin antibiotic (Blochinger and Diggelmann, Mol Cell Biol 4_: 2929-2931), and the dhfr gene, which confers resistance to methotrexate (Bourouis et al., EMBO J. 2 (7): 1099-1104 (1983 )), and the EPSPS gene, which confers resistance to glyphosate (Patents of the United States of America Numbers 4, 940, 935 and 5,188, 642). 1. Vectors Suitable for Transformation with Agrobacterivan There are many vectors available for transformation using Agrobacterium. turne f aciens. These typically carry at least one T-DNA limit sequence, and include vectors such as pBIN19 (Bevan, Nucí Acids Res. (1984)) and pXYZ. Typical vectors suitable for transformation with Agrobacterium include the binary vectors pCIB200 and pCIB2001, as well as the binary vector pCIBlO, and their hygromycin selection derivatives. (See, for example, U.S. Patent Number 5,639,949). 2. Vectors Suitable for Transformation without Agrobacterium Transformation without the use of Agrobacterium tumefaciens circumvents the requirement for T-DNA sequences in the selected transformation vector, and consequently, vectors lacking these sequences are used in addition to vectors such as those described above, containing T-DNA sequences. Transformation techniques that do not rely on Agrobacterium include transformation by means of particle bombardment, protoplast recovery (e.g., PEG and electroporation), and microinjection. The choice of vector depends largely on the preferred selection for the species being transformed. Typical vectors suitable for transformation without Agrobacterium include pCIB3064, pSOG19, and pSOG35. (See, for example, U.S. Patent Number 5,639,949).

C. Transformation Techniques Once the DNA sequence of interest is cloned into an expression system, it is transformed into a plant cell. Methods for the transformation and regeneration of plants are well known in the art. For example, Ti-plasmid vectors have been used for the delivery of ex-trafficking DNA, as well as direct recovery of DNA, liposomes, electroporation, microinjection, and microprojectiles. In addition, bacteria of the genus Agrobacterium can be used to transform plant cells. Transformation techniques for dicotyledons are well known in the art, and include techniques based on Agrobacterium, and techniques that do not require Agrobacterium. The techniques without Agrobacterium involve the recovery of the exogenous genetic material directly by the protoplasts or the cells. This is done by means of PEG-mediated recovery or electroporation, mediated delivery by particle bombardment, or microinjection. In each case, the transformed cells are regenerated in whole plants using conventional techniques known in the art. The transformation of most monocotyledonous species has now become a routine. Preferred techniques include direct gene transfer into protoplasts using PEG or electroporation techniques, bombardment of particles in callus tissue, as well as Agrobacterium-mediated transformation.

The invention will be further described with reference to the following detailed examples. These examples are provided for purposes of illustration only, and are not intended to be limiting, unless otherwise specified.

EXAMPLES The conventional molecular cloning and recombinant DNA techniques used herein are well known in the art, and are described by Sambrook et al., Molecular Cloning, editors, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989 ) and by TJ Silhavy, M.L. Berman, and L.W. Enquist, Experiments with Gene Fusions, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1984), and by Ausubel, F.M. and collaborators, Current Protocols in Molecular Biology, published by Greene Publishing Assoc. and Wiley-Interscience (1987).

Example 1: Regulation of the Expression of a Luciferase Gene Construction of a chimeric DNA molecule encoding a luciferase RNA duplex A 738 base pair "fire" oriented fragment of the firefly luciferase gene from the pLuc + plasmid (Promega) is amplified from the plasmid DNA pPH108, using oligonucleotide primers ds__Lucl (5 '-CGC GGA TCC TGG AAG ACG CCA AAA ACA-3', SEQ ID NO: 1; BamHI restriction site underlined), and ds_Luc2 (5 '-CGG AAG CTT AGG CTC GCC TAA TCG CAG TAT CCG GAA TG-3 ', SEQ ID N0: 2, HindIII restriction site underlined). Thermostable DNA polymerase turboPfu (Stratagene) is used in 50 microliter reactions according to the manufacturers protocol, with five cycles of 95 ° C / 1 minute, 55 ° C / 1.5 minutes, 72 ° C / 2 minutes , followed by 25 cycles of 95 ° C / 1 minute, 72 ° C / 3.5 minutes. In a similar manner, an "antisense" oriented fragment of 737 base pairs of the luciferase luciferase gene is amplified from the pLuc + plasmid by polymerase chain reaction, from the plasmid pPH108 DNA, using the primers of oligonucleotide ds_Luc3 (5 '-CGG TCT AGA GGA AGA CGC CA AAA CAT A-3', SEQ ID NO: 3, restriction site Xbal underlined), and ds__Luc2 (5 '-CGG AAG CTT AGG CTC GCC TAA TCG CAG TAT CCG GAA TG-3 ', SEQ ID NO: 2, Hinldlll restriction site underlined). The resulting DNA fragments are purified by electrophoresis through a 1 percent tris-acetate gene made from low melting point agarose (FMC), followed by extraction with phenol-chloroform from the gel beads. separate ones that contain the products of the polymerase chain reaction. The sense product DNA (ds_Lucl / 2) is digested with BamHI and HindIII, and the anti-sense product DNA (ds_Luc3 / 2) is digested with Xbal and HindIII according to conventional methods (restriction enzymes). were obtained in New England Biolabs). The resulting sticky end DNA fragments are gel purified as described above. A DNA fragment containing the 1 'promoter is obtained (Velten et al. (1984) EMBO J. 3: 2723-2730), digesting the plasmid CSA104 with EcoRI and Hin-cll, and purifying a DNA fragment of 564 pairs of bases.

This fragment is re-digested with BamHI, and the 484 bp base EcoRI-BamHI sub-fragment containing the 1 'promoter is isolated and the gel is purified. In order to construct the plasmid pPH169, the DNA of the cloning vector pLit-mus29 (New England Biolabs) is digested with EcoRI and Xbal, and the isolated fragment is ligated in a four-way reaction using T4 DNA ligase (New England Biolabs) with the EcoRI-BamHI fragment of the lr promoter, and the luciferase gene ds_Luc fragments in sense. { BamHI-HindIII) and anti-sense (H indi I I -Xbal). In order to construct the binary vector pPH170 for the transformation of the plant mediated by Agrobacterium with the construction of the ds_Lucl / 2/3 RNA duplex, the DNA of the binary plasmid pSGCHCl, which carries a kanamycin resistance gene for selection bacterial, and a gene for resistance to hygromycin for the selection of the transgenic plant, is digested with EcoRI and Xbal. The resulting isolated fragment of 11.6 kb from pSGCHCl se. ligated into a four-way reaction using T4 DNA ligase (New England Biolabs) with the EcoRI-BamHI fragment of the plus 1 'promoter, and the luciferase gene fragments ds_Luc in sense (BamHI-HindlII) and anti-sense (HindIII -Xbal).

Transformation of Agrobacterium and Vacuum Infiltration of Arabidopsis Plants Plasmid pPH170 is introduced into Agrobacterium tumefaciens GV3101 by electroporation, and transformed colonies are selected and amplified. Four to five-week-old plants of Arabidopsis thalanga mutant lines expressing luciferase either constitutively 5 (UBQ3 promoter (Norris et al. (1993) PMB 21: 895-906) / UBQ3 + CaMV 35S 5 'UTR / luc +; pPH108), or alternatively in an inducible manner (PR-1 promoter from Arabidopsis / l c +; pPHl35, line 6E) are infiltrated under vacuum with Agrobacterium clones carrying the binary T-DNA vector pPH170. The transformed plants are co-sorted on hygromycin and kanamycin, and are grown under controlled phytotron conditions for the determination of luciferase activity. In addition, luciferase activity in the background pPH135-6E is evaluated 48 hours after induction with BTH (treatment with BTH is essentially as described in Lawton et al., Plant, J. 10: 71-82). The luciferase activity is quantified using a luminescence-based assay in tissue extracts following the addition of the luciferin substrate. Luciferase activity is also monitored inside the plant using a video imaging system cooled with CCD (Hamamatsu).

Example 2: Regulation of the Expression of the GL1 gene of Arab ± dopisis The GL1 gene encodes a transcription factor type m and b that is required for the start of the formation of the normal trichome (leaf hair) (Oppenheimer et al. (1991) Cell 67: 483-493). Removal of GL1 expression early in development results in plants lacking trichomes. The elimination phenotype is easy to identify in young seedlings, and it is not lethal. Three vectors are constructed for constitutive expression, and three vectors for expression regulated by Gal4Cl. The three different vectors to test each promoter are the expression in sense (+), the expression anti-sense (-), and the expression of RNA duplex (+/-) of a fragment of the gene GL1. Vectors (+) and (-) are controls to compare their effect on GLI expression. In each case, a 5 'fragment is used from base # 739 to # 1781 of the GLI sequence (GenBank, Access M79448) for the construction of the vector.

Regulated Expression by Gal4Cl The GL1 gene fragment is cloned into the construction of the inducible vector by crossing pJG304-l as Ncol-SacI fragments. Plasmid pJG304 is derived from pBSSK +. Plasmid pBS SK + (Stratagene, La Jolla) is linearized with Sacl, treated with mung bean nuclease to remove the Sacl site, and ligated again with T4 ligase to make pJG201. Cassette of consensus binding site 10XGAL4 minimum 35S promoter of CaMV / GUS gene / CaMV terminator is removed from pAT71 with Kpnl, and cloned into the Kpnl site of pJG201 to make pJG304. Plasmid pJG304 is partially digested with restriction endonuclease Asp718, to isolate a full-length linear fragment. This fragment is ligated with a molar excess of the 22-base oligonucleotide JG-L (5 '-GTA CCT CGA G TC TAG ACT CGA G-3', SEQ ID NO: 4). Restriction analysis is used to identify a clone with this linker inserted 5 'to the DNA binding site of GAL4, and this plasmid is designated pJG304DXhoI. The Ncol and Sacl sites are added to the ends of the (+) and (-) fragments, synthesizing the polymerase chain reaction primers with the appropriate restriction sites in the 5 'terms. The GL1 (+/-) fragment is produced by first producing two fragments: a (+) fragment with the Ncol site at the 5 'terminus, and a HindIII site at the 3 r term, and a (-) fragment with a HíndIII site in the 5 'term, and a Sacl site in the 3' term. The duplex unit is produced by ligating the resulting fragments in the EcoRI site. The expression unit contains the GAL4 DNA binding domain, followed by a minimum TATA sequence, and the GLL gene fragment oriented (+), (-), or (+/-).

Constitutive Expression The most 1 'promoter of mannopine synthase is used from Agrobacterium (ref), a relatively strong and constitutive one in dicotyledonous plants. As above, the GL (+), (-), and (+/-) fragments are ligated behind the 5 'promoter in pBluescript. The three different expression cassettes are ligated into pCIB200 as EcoRI / SalI fragments (Uknes et al. (1993) Plant Cell 5: 159-169).

Example 3: Regulation of Cystathionine Beta-lyase G-Expression The Cystathionine Beta-lyase (CBL) Gene encodes a step in the path of methionine biosynthesis. The effect of the regulation of its expression in plants is tested using sense and anti-sense constructions, and double-stranded RNA constructions.

Anti-sense Construction: The binary BASTA vector pJG261 containing a fragment from vector pJG304DXhoI is used with an insertion of part of the CBL gene in an anti-sense orientation (nucleotides # 13-1159, Genbank, access # L40511).

Sense Construction: Same as antisense construction, except that the CBL fragment is in the opposite orientation. This construct contains the ATG start codon, and most of the CBL open reading frame, and serves as a control to regulate the expression of the CBL gene.

Construction of double-stranded RNA: A fragment of the CBL gene (# 13-1159) is inserted in the sense orientation in the SalI site of vector pJG304-l downstream of the anti-sense version of the CBL gene. A linker of approximately 10 base pairs is present between the two CBL copies.

Example 4: Requirements for the Construction of Plant Expression Cassettes The genetic sequences intended to be expressed in transgenic plants are first assembled, in expression cassettes behind a suitable promoter, and upstream of a suitable transcription terminator. All the requirement for plant expression cassette constructions is applied to the DNA molecules of the present invention, and is performed using techniques well known in the art.

Selection of the Promoter The selection of the promoter used in the expression cassettes determines the spatial and temporal expression pattern of the DNA molecule in the transgenic plant. The selected promoters express the DNA molecule in specific cell types (such as leaf epidermal cells, mesophyll cells, root bark cells), or in specific tissues or organs (roots, leaves, or flowers, for example), and this selection reflects the desired location of biosynthesis of an RNA fragment encoded by the DNA molecule. Alternatively, the selected promoter can drive the expression of the DNA molecule under a light-induced promoter or other temporarily regulated promoter. An additional alternative is that the selected promoter is chemically regulated. This provides the possibility of inducing the expression of the DNA molecule only when desired and caused by treatment with a chemical inducer.

Transcription Terminators There are a variety of transcription terminators available for use in expression cassettes. These are responsible for the termination of the transcription, and preferably, of the correct polyadenylation. Suitable transcription terminators, and those known to work in plants, include the CaMV 35S terminator, the tml terminator, the nopaline synthase terminator, the pea rbcS E9 terminator. These can be used both in monocroy and dicotyledons.

Sequences for Improving or Regulating Expression Numerous sequences have been found to improve gene expression from within the transcription unit, and these sequences can be used in conjunction with the DNA molecule of this invention to increase their expression in transgenic plants. . It has been shown that different sequences of introns improve expression, particularly in monocotyledonous cells. For example, it has been found that the introns of the Adhl corn gene significantly improve the expression of the wild-type gene under its known promoter when introduced into corn cells. Intron 1 is found to be particularly effective and improves expression in fusion constructs with the chloramphenicol acetyltransferase gene (Callis et al., Genes Develep 1: 1183-1200 (1987)). In the same experimental system, the intron of the Bronzel corn gene had a similar effect to improve expression (Callis et al., Supra). Intron sequences have been routinely incorporated into plant transformation vectors, usually within the nontranslated leader. It is also known that a number of untranslated leader sequences derived from viruses improve expression, and these are particularly effective in dicotyledonous cells. Specifically, it has been demonstrated that the leading sequences of Tobacco Mosaic Virus TMV, the "O-sequence"), Corn Chlorotic Mottled Virus (MCMV), and Alfalfa Mosaic Virus (AMV), are effective to improve expression (for example, Gallie et al., Nucí Acids Res. L5: 8693-8711 (1987); Skuzeski et al., Plant Molec. Biol. 15; 65-79 (1990)).

Example 5: Examples of Construction of Expression Cassettes The present invention encompasses the expression of a DNA molecule of the present invention, under the regulation of any promoter that can be expressed in plants, regardless of the origin of the promoter. Accordingly, the DNA molecule is inserted into any of the expression cassettes, using techniques well known in the art. These expression cassettes can then be easily transferred to plant transformation vectors described below. In addition, the invention also encompasses the use of any plant-expressible promoter in conjunction with any other sequences required or selected for the expression of the DNA molecule. These sequences include, but are not restricted to, transcription terminators, foreign sequences to improve expression (such as introns [eg, intron 1 of Adh], viral sequences [eg, TMV-O]).

Constitutive Expression: CaMV 35S Promoter The construction of plasmid pCGN1761 is described in Published Patent Application Number EP 0,392,225. pCGN1761 contains the "double" 35S promoter, and the tml transcription terminator, with a unique EcoRI site in the promoter and terminator, and has a pUC-like base structure. A derivative of pCGN1761 having a modified polylinker is constructed which includes the Notl and Xhol sites in addition to the existing EcoRI site. This derivative is designated pCGN1761ENX. pCGN1761ENX is useful for the cloning of cDNA sequences or genetic sequences (including the microbial open reading frame sequences) into its polylinker for purposes of its expression under the control of the 35S promoter in transgenic plants. All the cassette of promoter 35S-genetic sequence-terminator Aunt I of this construction can be separated by HindIII, Sphl, Sali, and Xbal, 5 'for the promoter, and the Xbal, BamHI, and BglII sites, 3' for the terminator, to be transferred to transformation vectors. In addition, the double 35S promoter fragment can be removed by 5 'separation with HindIII, SphI, SalI, XbaI, or PstI, and 3' separation with any of the polylinker restriction sites (Eco-RI, NotI, or XhoI) to be replaced with another promoter. In accordance with the foregoing, a DNA molecule of the present invention is inserted into pCGN1761ENX for constitutive expression under the control of the 35S promoter of CaMV.

Expression under a Chemically Regulatory Promoter This section describes the replacement of the double 35S promoter in pCGN1761ENX with any promoter of choice; by way of example, the PR-chemically regulated promoter is described. The promoter of choice is preferably separated from its source by restriction enzymes, but in an alternative manner, it can be amplified with polymerase chain reaction using primers carrying appropriate terminal restriction sites. If the amplification is undertaken with polymerase chain reaction, then the promoter must be re-sequenced to verify amplification errors after cloning of the amplified promoter in the target vector. The chemically-regulable tobacco PR-la promoter is dissociated from plasmid pCIB1004 (see European Patent Number EP 0,332,104), and is transferred to plasmid pCGN1761ENX. pCIB1004 dissociates with Ncol, and the 3 'overhang resulting from the linearized fragment is made blunt by its treatment with T4 DNA polymerase. The fragment is then dissociated with Hinldlll, and the resulting PR-la promoter containing fragments is gel purified, and cloned into pCGN1761ENX, from which the double 35S promoter has been re-moved. This is done by dissociation with XhoI, and blunting with T4 polymerase, followed by dissociation with HindIII and isolation of the fragment containing the terminator of the larger vector, where the promoter fragment pCIB1004 is cloned. This generates a derivative of pCGN1761ENX with the PR-la promoter and the tml terminator, and a polylinker that intervenes, with unique EcoRI and Notl sites. A DNA molecule of the present invention is inserted into this vector, and the fusion product (i.e., pro-motor-gene-terminator) is subsequently transferred to any selected transformation vector, including those described in This application, thereby providing the chemically inducible expression of the DNA molecule.

Constitutive Expression: The Actin Promoter It is known that several actin isoforms are expressed in most cell types, and consequently, the actin promoter is a good choice for a constitutive promoter. In particular, the promoter of the Actl rice gene has been cloned and characterized (McElroy et al., Plant Cell 2: 163-171 (1990)). It is found that a 1.3 kb fragment of the promoter contains all the regulatory elements required for expression in rice protoplasts. In addition, numerous expression vectors based on the Actl promoter have been specifically engineered for use in monocots (McElroy et al., Mol.Gen. Genet, 231: 150-160 (1991)). These incorporate Actl intron 1, the 5 'flanking sequence of Adhl, and Adhl intron 1 (of the maize alcohol dehydrogenase gene), and the sequence from the 35S promoter of CaMV. The vectors showing the highest expression are 35S fu- sions and the Actl intron, or the 5 'flanking sequence of Actl and the Actl intron. The promoter expression cassettes described by McElroy et al. (Mol. Gen. Genet, 231: 150-160 (1991)) are easily modified for the expression of a DNA molecule of the present invention, and are particularly suitable for used in monocotyledonous hosts lines For example, fragments containing the promoter are removed from the McElroy constructs, and used to replace the double 35S promoter in pCGN176lENX, which is then available for the insertion of specific genetic sequences. The fusion genes thus constructed are transferred to appropriate transformation vectors. In a separate report, it has also been found that the Actl rice promoter with its first intron directs high expression in cultured barley cells (Chibbar et al., Plant Cell Rep. 12: 506-509 (1993)). A DNA molecule of the present invention is inserted downstream of this promoter, and the fusion products (i.e., promoter-gene-terminator) are subsequently transferred to any selected transformation vector, including those described in this application.

Constitutive Expression: The Ubiquitin Promoter Ubiquitin is another genetic product that is known to accumulate in many cell types, and its promoter has been cloned from several species for use in transgenic plants (eg, sunflower-Binet et al. , Plant Science 7_9.:87-94 (1991), corn-Christensen et al., Plant Molec, Biol. 12: 619-632 (1989)). The corn ubiquitin promoter has been developed in transgenic monocotyledonous systems, and its sequence and vectors constructed for monocot transformation are disclosed in Patent Publication Number EP 0,342,926. In addition, Taylor et al. (Plant Cell Rep. 12: 491-495 (1993)) describe a vector (pAHC25) comprising the maize ubiquitin promoter and the first intron, and its high activity in cell suspensions of numerous monocots, when it is introduced by means of microprojectile bombardment. The ubiquitin promoter is clearly suitable for the expression of a DNA molecule of the present invention in transgenic plants, especially monocotyledons. Suitable vectors are derivatives of pAHC25 or any of the transformation vectors described in this application, modified by the introduction of the ubiquitin promoter and / or the appropriate introns sequences. Accordingly, a DNA molecule of the present invention is inserted into any of these vectors, and the fusion products (i.e., promoter-gene-terminator) are used for the transformation of plants, resulting in constitutive expression of the DNA molecule.

Root Specific Expression A preferred expression pattern for a molecule of DNA of the present invention, is the expression of the root. The expression of the nucleotide sequence only in the root tissue, has the advantage of altering the expression of a target gene only in the roots, without a concomitant alteration of its expression in the leaf and flower tissue and in the seeds. A suitable root promoter is that described by de Framond (FEBS 290: 103-106 (1991)), and also in published patent application number EP 0,452,269. This promoter is transferred to a suitable vector, such as pCGNl761ENX, and the DNA molecule is inserted into this vector. Subsequently, the entire promoter-gene-terminator cassette is transferred to a transformation vector of interest.

Promoters Induced by Wound Numerous of these promoters have been described (eg, Xu et al., Plant Molec., Biol. 22: 573-588 (1993), Logemann et al., Plant Cell 1: 151-158 (1989), Rohrmeier. and Lehle, Plant Molec, Biol. 22: 783-792 (1993), Firek et al., Plant Molec, Biol. 2_2: 129-142 (1993), Warner et al., Plant J. 3_: 191-201 (1993). ), and all are suitable for use with the present invention. Logemann et al. (Supra) describe the 5 'upstream sequences of the wunl gene of dicotyledonous potato. Xu et al. (Supra) show that a wound-inducible promoter of dicotyledonous potato (pin2) is active in monocotyledonous rice. In addition, Rohrmeier and Lehle (supra) describe the cloning of corn Wipl cDNA that is induced by wound, and whcan be used to isolate the known promoter using conventional techniques. In a similar manner, Firek et al. (Supra) and Warner et al. (Supra) have described a wound-induced gene from the monocot Asparagus officcinalis, whis expressed at sites of local wound and invasion of pathogens. Using cloning techniques well known in the art, these promoters can be transferred to suitable vectors, can be fused to a DNA molecule of this invention, and can be used to express these genes at sites of infection by the pest. of insects.

Preferred Expression for Sap Patent Application Number WO 93/07278 describes the isolation of the maize trpA gene, whis preferably expressed in the cells of the sap. Using conventional molecular biological techniques, this promoter, or parts thereof, can be transferred to a vector, such as pCGN1761, where it can replace the 35S promoter, and can be used to drive the expression of a DNA molecule of the present invention. in a preferred way by the sap. In fact, the fragments containing the preferred promoter by the sap, or parts thereof, are transferred to any vector, and modified for use in transgenic plants. The preferred expression for the sap of the DNA molecule is achieved by inserting the DNA molecule into this vector.

Specific Expression of Pollen Patent Application Number WO 93/07278 further describes the isolation of the kinase gene of calcium-dependent protein from corn (CDPK), whis expressed in pollen cells. The genetic sequence and the promoter extend up to 1,400 base pairs from the start of transcription. Using conventional molecular biological techniques, this promoter, or parts thereof, is transferred to a vector, such as pCGN1761, where it replaces the 35S promoter, and is used to drive the expression of a DNA molecule of the present invention, from a specific way of pollen. In fact, fragments containing the pollen specific promoter, or parts thereof, can be transferred to any vector, and can be modified for use in transgenic plants.

Specific Leaf Expression A maize gene encoding phosphoenol carboxylase (PEPC) has been described by Hudspeth and Gruvia (Plant Molec Biol 12: 579-589 (1989)). Using conventional molecular biological techniques, the promoter is used for this gene, in order to drive the expression of a DNA molecule of the present invention, in a specific manner of the leaf, in transgenic plants.

Example 6: Construction of Plant Transformation Vectors There are numerous transformation vectors available for transformation of plants, and a DNA molecule of this invention is inserted into any of the expression cassettes described above, so that they are capable of expressing the DNA molecule in the desirable cells, under appropriate conditions. An expression cassette containing the nucleotide sequence is then incorporated into any appropriate transformation vector described below. The selection of the vector to be used will depend on the preferred transformation technique and the target species for the transformation. For certain target species, different antibiotic or herbicide selection markers may be preferred. The selection markers routinely used in the transformation include the nptll gene that confers resistance to kanamycin and related antibiotics (Messing and Vierra, Gene JL9: 259-268 (1982)).; Bevan et al., Nature 304: 184-187 (1983)), the bar gene conferring resistance to the herbicide phosphinothricin (White et al., Nucí Acids Res 18_: 1062 (1990), Spencer et al., Theor Appl Genet 7_9; 625-631 (1990)), the hph gene, which confers resistance to the antibiotic hygromycin (Blochinger and Diggelmann, Mol Cell Biol 4: 2929-2931), and the dhfr gene, which confers resistance to methotrexate (Bourouis et al. EMBO J. 2 (7): 1099-1104 (1983)). Vectors Suitable for Transformation with Agorabacfceriuní There are many vectors available for transformation using Agrobacterium turne faciens. These normally carry at least one T-DNA limit sequence, and include vectors such as pBINl9 (Bevan, Nucí Acids Res. (1984)) and pVictor HINK (SEQ ID N0: 5). The construction of two typical vectors is described below.

Construction of pCIB200 and pCIB2001 The binary vectors pCIB200 and pCIB2001 are used for the construction of recombinant vectors for use with Agrobacterium, and are constructed in the following manner. PTJS75kan is created by digestion with NarI from pTJS75 (Schmid-hauser and Helinski, J Bacteriol 16: 446-455 (1985)), allowing separation of the tetracycline resistance gene, followed by insertion of an Accl fragment from pUC4K carrying an NPTII (Messing and Vierra, Gene 3 ^ 9: 259-268 (1982); Bevan et al., Nature 304: 184-187 (1983); McBride et al., Plant Molecular Biology l_4: 266-276 (1990 )). Xhol linkers are ligated to the EcoRV fragment of pCIB7 which contains the boundaries of left and right T-DNA, a chimeric us / nptll gene selectable in plants, and the pUC polylinker (Rothstein et al., Gene jx3: 153-161 (1987) ), and the fragment digested with XhoI is cloned into pTJS75kan digested with SalI to create pCIB200 (see also European Patent Number EP 0,332,104). pCIB200 contains the following unique polylinker restriction sites: EcoRI, Sstl, Kpnl, BglII, XbaI, and SalI. pCIB2001 is a derivative of pCIB200, which was created by inserting additional restriction sites into the polylinker. The unique restriction sites in the polylinker of pCIB2001 are EcoRI, Sstl, Kpnl, BglII, Xbal, Sal, Mul, BcII, Avrll, Apal, Hpal, and Stul. pCIB2001, in addition to containing these unique restriction sites, also has kanamycin selection in plants and bacterial, left and right T-DNA boundaries for Agrobacterium-mediated transformation, the trfA function derived from RK2 for mobilization between E. coli and other hosts, and the OriT and OriV functions also from RK2. The polylinker pCIB2001 is suitable for the cloning of plant expression cassettes containing their own regulatory signals. Any of the plant expression cassettes described above, and comprising a DNA molecule of the present invention, is inserted into pCIB2001, preferably using the polylinker.

Construction of pCIBlO and Hygromycin Selection Derivatives of The same The binary vector pCIBlO contains a gene that codes for kanamycin resistance for selection in plants, the right and left border sequences of T-DNA, and incorporates sequences from the broad plasmid. host range pRK252, which allows it to replicate in both E. coli and Agrobacterium. Its construction is described by Rothstein et al (Gene 5_3: 153-161 (1987)). Different pCIBlO derivatives have been constructed that incorporate the gene for hygromycin B phospho transferase described by Gritz et al. (Gene 25 ^: 179-188 (1983)). These derivatives make it possible to select cells from transgenic plants only on hygromycin (pCIB743), or on hygromycin and kanamycin (pCIB715, pCIB717). These vectors are used to transform an expression cassette comprising a DNA molecule of the present invention.

Vectors Suitable for Transformation without Agrobacterium Transformation without the use of Agrobacterium tumefa-ciens circumvents the requirement for T-DNA sequences in the selected transformation vector, and consequently, vectors lacking these sequences can be used, in addition to vectors such as those described above, containing T-DNA sequences. Transformation techniques that do not rely on Agrobacterium include transformation by means of particle bombardment, protoplast recovery (e.g. PEG and electroporation), microinjection or pollen transformation (U.S. Patent Number 5,629,183) . The choice of vector depends largely on the preferred selection for the species being transformed. The construction of some typical vectors is described below.

Construction of pCIB3064 pCIB3064 is a vector derived from pUC suitable for directing the techniques of genetic transfer in combination with selection by the herbicide BASTA (or phosphinothricin). Plasmid pCIB246 comprises the CaMV 35S promoter in fusion operable with the E. coli GUS gene and the CaMV 35S transcription terminator, and is described in PCT Published Application No. WO 93/07278. The 35S promoter of this vector con-has two ATG 5 'sequences from the start site. These sites are mutated using conventional polymerase chain reaction techniques, in such a way that the ATGs are removed, and the SspI and PvuII restriction sites are generated. The new restriction sites are at 96 and 37 base pairs from the single Salí site, and at 101 and 42 base pairs from the actual start site. The resulting derivative of pCIB246 is designated pCIB3025. The GUS gene is then separated from pCIB3025 by digestion with Sail and Sacl, the terms blunted and religated to generate the plasmid pCIB3060. Plasmid pJIT82 is obtained from John Innes Center, Norwich, and the 400 base pair Smal fragment containing the bar gene from Streptomyces viridochromogenes is separated and inserted into the Hpal site of pCIB3060 (Thompson et al., EMBO J j >: 2519-2523 (1987)). This generates pCIB3064 comprising the bar gene under the control of the 35S promoter of CaMV and the terminator for the selection of the herbicide, a gene for resistance to ampicillin (for selection in E. coli), and a polylinker with unique sites Sphl, PstI, HindIII, and BamHI. This vector is suitable for the cloning of expression cassettes in plants containing their own regulatory signals, in order to direct the expression of a DNA molecule of the present invention.

Construction of pSOG19 and pSOG35 pSOG35 is a transformation vector that uses the dihydrofolate reductase of the E. coli gene (DHFR) as a selectable marker that confers resistance to methotrexate. Polymerase chain reaction is used to amplify the 35S promoter (approximately 800 base pairs), the 6-intron 6 of the maize Adhl gene (approximately 550 base pairs), and the 18 base pairs of the sequence untranslated leader GUS from pSOGlO. A 250-base pair fragment encoding the type II dihydrofolate reductase gene from E. coli is also amplified by polymerase chain reaction, and these two fragments of the polymerase chain reaction are assembled with a SacI fragment. -PstI from pBI221 (Clontech), which comprises the base structure of the pUC19 vector, and the nopaline synthase terminator. The assembly of these fragments generates pS0G19, which contains the 35S promoter in fusion with the intron 6 sequence, the GUS leader, the DHFR gene, and the nopaline synthase terminator. The GUS leader replacement in pS0G19 with the leader sequence from Corn Chlorotic Speck Virus (MCMV) generated the vector pS0G35. pS0G19 and pSOG35 carry the pUC gene for amphotilin resistance, and have the HindIII, Sphl, PstI, and EcoRI sites available for the cloning of foreign sequences, in particular a DNA molecule of the present invention.

Example 7: Chloroplast Transformation Transformation Vectors For the expression of a DNA molecule of the present invention in plant plastids, the plastid transformation vector pPHl43 is used (International Publication Number WO 97/32011, example 36). The DNA molecule is inserted into pPH143, thus replacing the PROTOX coding sequence. This vector is then used for the transformation of the plastid and the selection of the transformants for spectinomycin resistance. Alternatively, insert the DNA molecule into pPH143, in such a way that it replaces the aadH gene. In this case, the transformants are selected for resistance to PRO-TOX inhibitors.

Chloroplast Transformation "Seeds of Nicotiana tabacum cv. Xanthi nc 'were germinated, seven per dish, in a circular arrangement of 2.54 centimeters, on a medium of T agar, and were bombed 12 to 14 days after sowing with particles of 1 miera tungsten (MIO, Biorad, Hercules, CA) coated with DNA from plasmids pPHl43 and pPHl45, essentially as described (Svab, Z. and Maliga, P. (1993) PNAS 90, 913-917). bombarded were incubated in a medium T for 2 days, after which the leaves were cut and placed with the abaxial side up in bright light (350-500 micromole-photons / m2 / s) on plates with a RMOP medium ( Svab, Z., Hajdukiewicz, P. and Maliga, P. (1990) PNAS 87, 8526-8530) containing 500 micrograms / milliliter of spectinomycin dihydrochloride (Sigma, St. Louis, MO). under bleached leaves 3 to 8 weeks after the bombardment, they were subcloned into the same or selective medium was allowed to form calluses, and secondary shoots were isolated and subcloned. The complete segregation of the copies of the transformed plastid genome (homoplasmicity) in independent subclones was evaluated by conventional Southern blot techniques (Sambrook et al., (1989) Molecular 7 Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor). Total cellular DNA digested with BamHI / EcoRI (Mettler, IJ (1987) Plant Mol Biol Repoter 5, 346-349) was separated on agarose gels of TRIS-borate 1 percent (TBE), transferred to nylon membranes (Amersham), and probed with randomly primed, 2 P-labeled DNA sequences corresponding to a 0.7 kb BamHl / HindIII DNA fragment from pC8 that contained a portion of the address sequence to plastid rps 7/12. The homoplasmic rings were aseptically rooted in an MS / IBA medium containing spectinomycin (McBride, K. E. et al. (1994) PNAS 91, 7301-7305), and transferred to the greenhouse.

Example 8: Construction of a chimeric gene tag encoding a sense and antisense (duplex) RNA fragment for the coat protein gene from BWYV, driven by the RoIC promoter. A 0.6 kb-oriented fragment of the western yellow beet vi-rus (BWYV), a so-called yellow virus, of the coating protein gene (CP), is amplified from the plasmid pZU046, using the HiNK025bis primers ( 5 '-CAÁ TTA CCA TGG ACÁ CGG TCG TGG-3'; SEQ ID NO: 6; restriction site Ncol underlined), and HÍNK226 (5 '-GCC AAA TGT TTG AAC GCT GCC GCC TAT TTG-3', SEQ ID NO: 7, PstI retention site underlined). Alternatively, the fragment is amplified from plasmid pBW17 (Veidt et al., Nucleic Acids Research 16: 9917-9932, 1988, accession number X13063), using primers HiNK025bis2 (5 '-AAT CGT CCA TGG ATA CGG TCG TGG-3 ', SEQ ID NO: 8, nucleotides 3475 to 3498, underlined Ncol restriction site), and HiNK226bis (5'- CTA GGG CCG GGT TCC TCT GCA GCC TAT TTG-3', SEQ ID NO: 9; nucleotides 4114 to 4085, PstI restriction site underlined). Taq DNA polymerase (Life Technologies) is used in 25 microliter reactions, according to the manufacturer's prescription, applying 30 cycles of 94 ° C / 30 seconds, 55 ° C / 30 seconds, 72 ° C / 90 seconds (+ 2 seconds / cycle). The resulting fragment of the polymerase chain reaction is purified by electrophoresis through a 1 percent Tris-acetate gel made from Seakem GTG agarose (FMC), followed by extraction of the gel slices containing the product. of reaction in the polymerase chain reaction, with the QIAquick Gel Extraction Kit (QIAGEN). The polymerase chain reaction product is subsequently digested with Ncol and PstI (all restriction enzymes were supplied by Life Technologies) according to conventional methods, and again gel purified. The purified fragment is ligated between the RoIC promoter and the Nos terminator, using T4 DNA ligase (Life Technologies). The resulting clone is called pHiNK138.

In a manner similar to that described above, a CP fragment of BWYV oriented 'anti-sense' of 1.4 kb is amplified from the plasmid pZU174A, using the primers HINK251 (5 '-CTC CCA GGT TGA GAC TGC CCT GCA GTG CCC A -3 ', SEQ ID NO: 10, underlined PstI restriction site), and HINK228 (5'-TTA CCA TGC ATA CGG TCG TGG GTA GG-3', SEQ ID NO: 11, Nsil restriction site underlined), or from plasmid pBW17, using the primers HINK251 (nucleotides 4844 to 4814), and HiNK228bis (5 '-CGT TAA TGC ATA CGG TCG TGG GTA GG-3', SEQ ID NO: 12; nucleotides 3478 to 3503, Nsi restriction site l underlined). After purification of the gel, the product of the polymerase chain reaction is digested with Nsi I and PstI. The 4.9 kb pHiNK138 plasmid is then linearized with PstI, and dephosphorylated using Termosen-sible Alkaline Phosphatase (Life Technologies). Both the vector and the polymerase chain reaction fragment are gel purified, followed by bidirectional ligation of the polymerase chain reaction fragment in pHiNK138. The orientation generated by the duplex RNA for the CP gene (SEQ ID NO: 13) is identified by restriction site analysis, yielding the plasmid pHiNK152, where the inverted repeat consists of the 0.6 kb CP gene separated by the 0.7 kb separator sequence derived from the BWYV genome downstream of the CP gene, referred to as ORF6. The separating sequence is in the anti-sense orientation. Finally, this genetic cassette is transferred to the proprietary binary vector pVictorHiNK carrying the phosphomannose isomerase as the selectable marker gene (International Publication Number WO 94/20627), producing pHiNK179.

Example 9.1: Construction of a chimeric gene cassette encoding a sense and antisense (duplex) RNA fragment for the replicase gene from BNYW, driven by the Arabidopsis Ubi3int promoter. Total RNA is extracted from Sugar beet root infected with beet necrotic yellow vein virus (BNYVV), a furovirus, using the mini-basket of RNAeasy plants from QIAGEN. In order to amplify the 3 'end of the BNYVV replicase gene (RNA1), the RNA was reverse transcribed to produce a cDNA, using the Supers-cryptMRII RNAse H-Reverse Transcriptase (RT) (Life Technologies), and the reverse primer HÍNK285 (5 '-TCG TAG AAG AG A ATT CAC CCA AAC TAT CC-3', SEQ ID N0: 14). Primer HINK285 is located between nucleotides 6378 and 6405 of the RNA1 sequence of BNYVV (accession number D00115), and is designated to introduce an EcoRI site. The RT reaction is subsequently used as a template for two polymerase chain reactions: Reaction A using the primer HÍNK283 (5 '-AAG AAT TGC AGG ATC CAC AGG CTC GGT AC-3', SEQ ID NO: 15), localizes between nucleotides 5168 bp and 5178 bp of BNYVV RNA1 designated to introduce a BamHI site, and primer HINK284 (5 '-TTC CAGA CGA ATT CGG TCT CAG A-3', SEQ ID NO: 16) located between nucleotides 5597 and 5620 of BNYVV RNA1 designated to introduce an EcoRI site. Reaction B uses the primer HÍNK283 in combination with the primer HINK285, both described above. The products of the reverse transcriptase polymerase chain reaction thus obtained share the sequence of BNYVV RNA1 between nucleotides 5168-5620, which constitute the future RNA duplex. The future separator sequence corresponds to nucleotides 5621-6405 bp of BNYVV RNA1 present in the product of the reverse transcriptase polymerase chain reaction obtained with the primers HÍNK283 and HÍNK285. Taq DNA polymerase (Life Technologies) is used in reactions of 25 microliters according to the prescription of the providers, applying 30 cycles of 94 ° C / 30 seconds, 55 ° C / 30 seconds, 72 ° C / 90 seconds (+ 2 seconds / cycle). The products resulting from the reverse transcriptase polymerase chain reaction are purified by electrophoresis through a 1 percent tris-acetate gel made from Seakem GTG agarose (FMC), followed by an extraction of the gel slices containing the amplification products with the QIAquick Gel Extraction Kit (QIAGEN). After gel purification, the products of the reverse transcriptase polymerase chain reaction are digested with restriction enzymes BamHI and EcoRI (Life Technologies) according to conventional methods, and purified as described above. Finally, the products of the reverse transcriptase polymerase chain reaction are cloned between the Arabidopsis ubiquitin 3 (Ubi3int) promoter and the nos terminator by means of a three-way ligation reaction, using T4 DNA ligase. (Life Technologies). The two resulting clones are called pHiNKldl (separator in anti-sense orientation, see SEQ ID NO: 17) and pHiNK184 (separator in sense orientation, see SEQ ID NO: 18). In order to construct the binary vectors for the Agrobacterium-mediated transformation of sugar beet, the DNA from the binary vector pVictorHiNK carrying the phosphomannose isomerase gene as a selectable marker (International Publication Number WO 94/20627), as well as the plasmids pHiNKldl and pHiNKl84, are digested with AscI and PacI (New England Biolabs), and the vector and the insert fragments are purified by electrophoresis as described above. The resulting 7.7 kb pVictorHiNK vector fragment is ligated using T4 DNA ligase (Life Technologies) to the genetic cassettes encoding the duplex RNA for the BNYVV re-plicase gene, yielding pHiNK187 (anti-sense separator) and pHiNK188 ( separator in sense), respectively.

Example 9.2: Agrobacterium mediated transformation of sugar beet The methods for Agrobacterium-mediated transformation of plant species are well established and are known to a person skilled in the art, and may vary for the type of explant, the Agrobacterium strain , or the selectable marker and regeneration system used. The protocol described below describes Agrobacterium-mediated transformation of sugar beet using cotyledons as a source for explants, and mannose-6-phosphate isomerase as the selectable marker gene (Joersbo et al., Molecular Breeding 4: 111-117, 1998). After surface sterilization, the sugar beet seeds are germinated on water agar and under soft light at a temperature of about 12 ° C. The fully developed cotyledons are removed from the seedling by a cross section just below the nodal region, and both co-tilates are torn by pulling and cutting gently. The cotyledon explants are inoculated by immersing them in a suspension of Agrobacterium strain EHA101, which carries the appropriate transformation vector diluted in an MS medium with a pH of 5.2, supplemented with 20 grams / liter of sucrose, 0.25 milligrams / liter of BA , 0.05 milligrams / liter of NAA, 500 μM of aceto-syringopa diluted to an optical density (OD600) of 0.1 to 0.3. After 5 minutes of incubation, the explants are removed from the Agrobacterium suspension, immersed in a sterile filter paper to remove excess inoculation suspension, and transferred to co-culture dishes. The co-culture plates consist of Petri dishes containing 1/10 of MS medium, pH of 5.7, 30 grams / liter of sucrose, 200 μM of acetosyringone, solidifies with 4.7 grams / liter of agarose, and covered with a filter paper moistened with 1.5 milliliters of TXD medium (MS salts supplemented with vitamins PGO, 0.005 milligrams / liter of kinetin, 4 milligrams / liter of CPA (p-chlorophenoxyacetic acid), 30 grams / liter of sucrose, pH of 5.7) enzyme of the solidified medium. The explants are co-cultivated for 4 days at 21 ° C and under soft light, and subsequently transferred to a selective regeneration medium consisting of an MS medium with a pH of 5.9 supplemented with 20 grams / liter of sucrose, 1.25 grams / liter of mannose, 0.25 milligrams / liter of BA, 0.05 milligrams / liter of NAA, 500 milligrams / liter of carbenicillin, and solidifies with 9 grams / liter of agar. Every third week, the explants were subcultured in a fresh medium containing gradually increasing concentrations of mannose, up to a maximum of 15 grams / liter. After 12 weeks of selection and regeneration at a temperature of 21 ° C, the regenerated shoots are harvested, rooted, and analyzed for PMl activity essentially according to the coupled enzyme assay described by Fera isco et al. (Feramisco and collaborators, Biochem Biophys, Res. Comm. 55: 636-641, 1973). Positive plants are placed in pots on land, and finally transferred to the greenhouse. Example 9.3: Selection to determine resistance to rhizomania After sowing or potting with soil, the seedlings or TO transformants are first allowed to establish a sufficiently developed root system for a period of about four weeks, before being transferred to the ground infested with Polymyxa betae carrying BNYVV. The infested lands are collected from fields infected with rhizomania in Germany. During the resistance test, the plants are grown in 12 cm pots at a temperature of approximately 21 ° C and a light period of 16 hours. Four weeks after transplanting to the infested soil, the plants are pulled from the soil, and the lower half of the root system is cleaned of any adhering dirt by rinsing with water. The sap of a random sample of 0.5 grams of root tissue is collected by means of a Polláhne press, and added to 10 milliliters of extraction regulator consist of phosphate regulated serum with a pH of 7.2, supplemented with 2 percent PVP and 0.2 percent ovalbumin. The amount of virus present in the samples (the clones are obtained by in vitro propagation) is determined by means of the Triple Antibody Sandwich ELISA (TAS) for BNYVV marketed by Adgen Ltd, Scotland, United Kingdom, essentially following the supplier instructions. A standard curve is included in each dish to calculate the virus content of the root samples from the absorbance values measured. The untransformed susceptible sugar beet plants serve as negative controls, and the plants that carry the C28 gene for the natural resistance to rhizomania as positive controls. Table 1 summarizes the results of the transformation events obtained with the plasmid pHINK188. Table 1: Example 10: Construction of a plant transformation vector for potivirus resistance of zucchini yellow mosaic (ZYMV) and papaya ring spot potivium, in melon: The NOS terminator (Bevan, M., et al., 1983 Nucleic Acids Res. 11 (2), 369-385) is cloned as a HindIII / PstI fragment of 260 base pairs in plasmid pZO1560 digested with HindIII / PstI. pZO1560 is a plasmid derived from pUC, where the multiple cloning site has been replaced by a more versatile one. The new construction obtained after insertion of the NOS terminator is called pZU533. The 1728 base pair Ubi3 / intron promoter fragment (Callis, J., et al. (1995 Genetics 139 (2), 921-939) is isolated by digestion with BamHI, followed by treatment with T4 DNA polymerase and digestion with Kpnl This fragment is cloned in pZU533 digested with Smal The new construct is called pZU615.First the additional DNA fragments to be inserted in pZU615 are amplified by polymerase chain reaction, using Pfx DNA polymerase, and The fragments of the polymerase chain reaction obtained are cloned into pBluescript SK +, to amplify an acine fragment (Intron Cintren leader) of 513 base pairs, two primers are designated based on the sequence of An et al. Plant Jourr.al: 10: 107-121, 1996. The 5 'primer (ZUP1563: 5'- GGGCGGATCCGCTAGCCCGCGGCCGCTCTTTCTTTCCAAGG-3', SEQ ID NO: 19) is quenched directly upstream of the 5 'splice site, and adds a site of restriction BamH I, a Nhel, and a SacII at the 5 'end of the Actin2 Intron fragment to be amplified. The 3 'primer (ZÜP1564: 5' -CCCGCCATGGGTCGACGCCATTTTTTA TGAGCTGC-3 ', SEQ ID NO: 20) is quenched directly downstream of the 3' splice site, and adds a Sali restriction site and an Ncol to the 3 'end of the fragment of Actin2 Intron that will be amplified. The fragment of the polymerase chain reaction amplified with ZUP1563 and 1564 is cloned into the EcoRV site of pBluescript. The new plasmid is called pZUdll. To provide the plasmid pZU615 with Acton Intron, a 499 base pair, a fragment of Act2 Intron BamK / NcoI is isolated from pZUßll, and cloned into pZU615 digested with BamHI and Ncol downstream of the Ubi3 / intron promoter, and current above the NOS terminator. The new construction is designated pZU616. The insertion of this fragment simultaneously provides the cassette with five additional restriction sites for the next three cloning steps. To amplify a PRSV CP fragment of 739 untranslated base pairs (nt) (Shyi-Dong, Y., et al., (1992), Journal of General Virology 73, 2531-2541), two primers are designated based on the sequence of an isolated French countryside from Shyi-Dong Yeh. The 5 'primer (ZUP1565: 5'-CCCGCCATGGGATCCGATGATTTCTACCGAGAATTAAGGG-3', SEQ ID NO: 21) is annealed at 285 base pairs downstream of the PRSV CP start codon, and adds a Ncol and a BamHI restriction site to the extreme 5 'of the fragment PRSV CP not translated. The 3 'primer (ZUP1566: 5' -GGGCGCTAGCCTAATGCTTATATAGTACC-30 ', SEQ ID NO: 22) is annealed 55 base pairs downstream of the PRSV CP stop codon, and adds a Nhel restriction site to the 3' end of the fragment PRSV CP not translated. The fragment of the amplified polymerase chain reaction is cloned into the EcoRV site of pBluescript, and the new plasmid is designated pZU612. To amplify a 735 base pair untranslated ZYMV CP fragment (Gal-On, A., et al., (1990), Gene 87, 273-277), two primers are designated based on the sequence of a French field isolate. from Gal-On, A .. The 5 'primer (ZUP1567: 5' -GGGCGCTAGCCTTGCTGGAGTATAAGCCGG-3 ', SEQ ID NO: 23) is annealed at 253 base pairs downstream of the start codon of ZYMV CP, and includes a site Nhel restriction at the 5 'end of the non-translated fragment ZYMV CP. The 3 'primer (ZUP1568: 5'-GGGCGTCGACCGCGGGCTTTAAAGGTGGGAGGCCC-3', SEQ ID NO: 24) is annealed at 89 base pairs downstream of the stop codon ZYMV CP, and includes the Sacll and Salí restriction sites at the 3 'end of the untranslated ZYMV CP fragment. The fragment amplified in the polymerase chain reaction is cloned into the EcoRV site of pBluescript. The new plasmid is called pZU613. Subsequently, a non-translated ZYMV CP fragment is cloned into pZU616 downstream of the Ubi3 / intron promoter, and upstream of Act2 Intron. For this purpose, a non-translated fragment of ZYMV CP, Nhel / SacII of 719 base pairs is isolated from pZU613, and is cloned into the Nhel and Sacll sites of pZU616. The construction is designated pZU617. Subsequently, a non-translated PRSV CP fragment is cloned into pZU617 downstream of the Ubi3 / intron promoter, and upstream of the untranslated ZYMV CP fragment. For this purpose, a non-translated fragment of PRSV CP, BamHI / Nhel of 720 base pairs is isolated from pZU612, and cloned in the BamHI and Nhel sites of pZU617. This new construction is called pZU618. The last fragment to be cloned to complete the genetic cassette contains a non-translated fragment PRSV CP, and downstream thereof, a non-translated fragment of ZYMV CP in the same orientation. A corresponding fragment is amplified from pZU618 as template, using Pfx polymerase chain reaction, and primers ZUP1565 and ZUP1568. The fragment of the polymerase chain reaction is cloned into the EcoRV site of pBluescript SK +, and the resulting plasmid is designated pZU619. To create an inverted genetic repeat cassette, insert the PRSV CP (nt) / ZYMV CP (nt) fragment into pZU618 in the opposite direction of the PRSV CP (nt) / ZYMV CP (nt) fragments already present in the Plasmid pZU618. For this purpose, a Sall / Ncol PRSV CP (nt) / ZYMV CP (nt) fragment of 1445 base pairs is isolated from pZU619, and cloned into pZU618 digested with Sali and Ncol downstream of Act2 Intron, and current above the NOS terminator. The resulting plasmid is designated pZU622. This cassette is inserted into the binary vector? ZU547, a binary vector derived from the pVictorHink plasmid which contains a selection cassette of SMAS / PMI promoter / NOS terminator. For this purpose, the As14 / PacI DNA fragment of 5414 base pairs containing the inverted repeat genetic cassette is isolated from pZU622, and cloned into the AscI and PacI sites of pZU547 in a row orientation. and upstream of the selection cassette. The final construction is designated pZU623 (SEQ ID NO: 25).

Example 11: Construction of a plant transformation vector for resistance to potato virus Y (PVY) in tomato. Based on the sequence of PVYn (a French PVY field isolate), published in the thesis "Engineering resistance against potato virus Y 'of R.A.A. van der Vlugt (1993), two primers (ZUP1598: 5'-CATGCCATGGATCCAATGGCCACGAATTAAAGCTATCACGTC-3 ', SEQ ID NO: 26, and ZUP1590: 5' -ACGCGTCGACCGCGGATTCAAACGATTATTAATTACGATAAAAG-3 ', SEQ ID NO: 27) are used to amplify, by techniques of conventional polymerase chain reaction, using Platinum Pfx DNA polymerase from Life Technologies, a fragment of 804 base pairs containing the cistronic coat protein sequences, and 99 nucleotides of the untranslated region of the end 3 ' The fragment amplified in the polymerase chain reaction is cloned as a blunt-ended fragment in the EcoRV site of pBluescript SK +, the plasmid being designated pZUA. The amplified PVY specific insert is separated as a BamHI / SacII fragment, and cloned into the BamHISacII sites of pZU616 (see example 10), yielding pZUB. pZUB and pZUA are digested both with Ncol and Sali. The specific fragment of PVY from pZUA is purified from the agarose gel, and ligated into the pZUB digested with Ncol and Salí, yielding pZUC containing the following elements: The UBI3 promoter with the intron followed by the reaction product PVY polymerase chain in the sense orientation as a BamHI / SacII fragment, followed by an Act2 intron, SacII / SalI, followed by the product of the PVY polymerase chain reaction in the anti-sense orientation as a fragment Sall / Ncol, and finally a NOS terminator as an NcoI / HindIII fragment. Finally, pZUC is cloned into the binary vector pZU547 which contains the SMAS / PMI promoter selection cassette / NOS terminator derived from the binary vector pHiNK 085. The final construct is designated pZU634 (SEQ ID NO: 28).

Example 12: Transformation of binary vectors for melon and tomato plant material. Methods for transferring binary vectors to plant material are well established, and are known to a person skilled in the art. The variations in the procedures are due, for example, to differences in the strains of Agrobacterium used, to the different sources of explant material, to the differences between the regeneration systems, as well as to the different crops of the plant species that they are going to transform. The binary plant transformation vectors described in Examples 10 and 11 above are used in transformation experiments according to the following procedures. The binary vectors are transferred to Agrobacterium tumefaciens by electroporation, followed by inoculation and cocultivation of the plant explant material with the transformed Agrobacterium strain, selectively killing the Agrobacterium strain using an appropriate antibiotic, selection of transformed cells by culture on a selective medium containing mañosa, transfer of the tissue to the shoot-inducing medium, transfer of the selected shoots to the root-inducing medium, and transfer of the seedlings to the soil. To confirm the presence of the genetic cassettes described in Examples 10 and 11 above, the total DNA is characterized from the transgenic plants, using well-known Southern blot analysis techniques.

Example 13: Selection of transgenic melon plants to determine resistance to ZYMV and PRSV. The transformed plants are grown in the greenhouse under conventional quarantine conditions, in order to prevent any infection by pathogens. The primary transformants self-pollinate, and the seeds are harvested. 100 plants of the progeny SI of the primary transformants are analyzed to determine the segregation of the inserted gene, and subsequently are infected with ZYMV by mechanical inoculation. The tissue of the host plants infected systemically with ZYMV is milled into five volumes of ice cold inoculation regulator (10 mM phosphate buffer), and rubbed in the presence of carborundum powder on the cotyledons and the first leaf of the seedlings. one week old Inoculated plants are monitored to determine the development of symptoms for three weeks after inoculation. Plants containing ZYMV sequences show reduced susceptibility to infection by ZYMV, compared to untransformed control plants, which show severe systemic symptoms of ZYMV within 7 days after inoculation. ZYMV-tolerant plants self-pollinate, and the seeds are harvested. ZYMV resistant transgenic plants are inoculated mechanically with PRSV according to the procedures described above. Plants already resistant to ZYMV also show a reduced susceptibility to infection by PRSV, compared to non-transformed control plants, which show severe systemic symptoms of PRSV within 7 days after inoculation.

Example 14: Selection of transgenic tomato plants to determine the resistance against PVY. The transformed plants are grown in the greenhouse under conventional quarantine conditions, in order to prevent any infection by pathogens. The primary transformants self-pollinate, and the seeds are harvested. 50 plants of the Y progeny of the primary transformants are analyzed to determine the segregation of the inserted gene, and subsequently they are infected with PVY by mechanical inoculation. The tissue of host plants systemically infected with PVY is milled into five volumes of ice-cold inoculation regulator (10 mM phosphate buffer), and rubbed in the presence of carborundum powder on the first two fully-extended seedling leaves. five weeks of age. The inoculated plants are monitored to determine the development of symptoms for three weeks after inoculation. Plants containing PVY sequences show reduced susceptibility to infection by PVY, compared to non-transformed control plants, which show severe systemic symptoms of PVY within 7 days after inoculation. The embodiments disclosed above are illustrative. This disclosure of the invention will put an expert in the field in possession of many variations of the invention. It is intended that all obvious and foreseeable variations be encompassed by the appended claims. It is noted that in relation to this date, the best method known to the applicant to carry out the aforementioned invention, is that which is clear from the present description of the invention.

LIST OF SEQUENCES < 110 > Novartis AG < 120 > Regulation of Viral Gene Expression < 130 > S-30959A < 140 > < 141 > < 150 > US 09/309038 < 151 > 1999-05-10 < 160 > 28 < 170 > Patentln Ver. 2.1 < 210 > 1 < 211 > 27 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: ds Lucí < 400 > 1 cgcggatcct ggaagacgcc aaaaaca 27 < 210 > 2 < 211 > 38 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: ds_Luc2 < 400 > 2 cggaagctta ggctcgccta atcgcagtat ccggaatg 38 < 210 > 3 < 211 > 28 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: ds Luc3 < 400 > 3 cggtctagag gaagacgcca aaaacata 28 < 210 > 4 < 211 > 22 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: JG-L < 400 > 4 gtacctcgag tctagactcg ag 22 < 210 > 5 < 211 > 4732 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: pVictorHINK < 400 > 5 gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc 60 caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag 120 agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac 180 tagaaggaca gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt 240 tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa 300 gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg 360 gtctgacgct cagtggaacg aaaactcacg ttaagggatt gattatcaaa ttggtcatga 420 aaggatcttc acctagatcc ttttgatccg gaattaattc ctgtggttgg catgcacata 480 aacggataaa caaatggacg cccttttaaa ccttttcacg ttctaataaa tatccgatta 540 cgctcttttc tcttaggttt acccgccaat atatcctgtc aaacactgat agtttaaact 600 gaaggcggga aacgacaatc tgatcatgag cggagaatta agggagtcac gttatgaccc 660 cgcgggacaa ccgccgatga gccgttttac gtttggaact gacagaaccg caacgctgca 720 gcagcggcca ggaattggcc tttaaatcaa ttgggcgcgc cgaattcgag ctcggtaccc 780 ggggatcctc tagagtcgac catggtgatc actgcaggca tgcaagcttc gtacgttaat 840 taattcgaat ccggagc ggc cgcacgcgtg ggcccgttta aacctcgaga gatctgctag 900 ccctgcagga aatttaccgg tgcccgggcg gccagcatgg ccgtatccgc aatgtgttat 960 taagttgtct aagcgtcaat ttgtttacac cacaatatat cctgccacca gccagccaac 1020 agctccccga ccggcagctc ggcacaaaat caccactcga tacaggcagc ccatcagaat 1080 taattctcat gtttgacagc ttatcatcga ctgcacggtg caccaatgct tctggcgtca 1140 ggcagccatc ggaagctgtg gtatggctgt gcaggtcgta aatcactgca taattcgtgt 1200 ogctcaaggc gcactcccgt tctggataat gttttttgcg ccgacatcat aacggttctg 1260 gcaaatattc tgaaatgagc tgttgacaat taatcatcgg ctcgtataat gtgtggaatt 1320 gtgagcggat aacaatttca cacaggaaac agaccatgag ggaagcggtg atcgccgaag 1380 tatcgactca actatcagag gtagttggcg tcatcgagcg ccatctcgaa ccgacgttgc 1440 tggccgtaca tttgtacggc tccgcagtgg atggcggcct gaagccacac agtgatattg 1500 atttgctggt tacggtgacc gtaaggcttg atgaaacaac gcggcgagct ttgatcaacg 1560 accttttgga aacttcggct tcccctggag agagcgagat tctccgcgct gtagaagtca 1620 ccattgttgt gcacgacgac atcattccgt ggcgttatcc agctaagcgc gaactgcaat 1680 ttggagaatg gcagcgcaat gacattct tg caggtatctt cgagccagcc acgatcgaca 1740 ttgatctggc tatcttgctg acaaaagcaa gagaacatag cgttgccttg gtaggtccag 1800 cggcggagga actctttgat ccggttcctg aacaggatct atttgaggcg ctaaatgaaa 1860 ccttaacgct atggaactcg ccgcccgact gggctggcga tgagcgaaat gtagtgctta 1920 cgttgtcccg catttggtac agcgcagtaa ccggcaaaat cgcgccgaag gatgtcgctg 1980 ccgactgggc aatggagcgc ctgccggccc agtatcagcc cgtcatactt gaagctaggc 2040 aggcttatct tggacaagaa gatcgcttgg cctcgcgcgc agatcagttg gaagaatttg 2100 ttcactacgt gaaaggcgag atcaccaagg tagtcggcaa ataaagctct agtggatccc 2160 cgaggaatcg gcgtgacggt cgcaaaccat ccggcccggt acaaatcggc gcggcgctgg 2220 gtgatgacct ggtggagaag ttgaaggccg cgcaggccgc ccagcggcaa cgcatcgagg 2280 cagaagcacg ccccggtgaa tcgtggcaag cggccgctga tcgaatccgc aaagaatccc 2340 ggcaaccgcc ggcagccggt gcgccgtcga ttaggaagcc gcccaagggc gacgagcaac 2400 cagatttttt cgttccgatg ctctatgacg tgggcacccg cgatagtcgc agcatcatgg 2460 acgtggccgt tttccgtctg tcgaagcgtg accgacgagc tggcgaggtg atccgctacg 2520 cgggcacgta agcttccaga gaggtttcag cag ggccggc cggcatggcc agtgtgtggg 2580 attacgacct ggtactgatg gcggtttccc atctaaccga atccatgaac cgataccggg 2640 aagggaaggg agacaagccc ggccgcgtgt tccgtccaca cgttgcggac gtactcaagt 2700 tctgccggcg agccgatggc ggaaagcaga aagacgacct ggtagaaacc tgcattcggt 2760 taaacaccac gcacgttgcc atgcagcgta cgaagaaggc caagaacggc cgcctggtga 2820 cggtatccga gggtgaagcc ttgattagcc gctacaagat cgtaaagagc gaa'accgggc 2880 ggccggagta catcgagatc gagctagctg attggatgta acagaaggca ccgcgagatc 2940 agaacccgga cgtgctgacg gttcaccccg attacttttt gatcgatccc ggcatcggcc 3000 ccgcctggca gttttctcta cgccgcgccg caggcaaggc tggttgttca agaagccaga 3060 agacgatcta cgaacgcagt ggcagcgccg gagagttcaa gaagttctgt ttcaccgtgc 3120 gcaagctgat cgggtcaaat gacctgccgg agtacgattt gaaggaggag gcggggcagg 3180 ctggcccgat cctagtcatg cgctaccgca gggcgaagca acctgatcga tccgccggtt 3240 cctaatgtac ggagcagatg ctagggcaaa ttgccctagc aggggaaaaa ggtcgaaaag 3300 gtctctttcc tgtggatagc acgtacattg ggaacccaaa gccgtacatt gggaaccgga 3360 tgggaaccca acccgtacat aagccgtaca ttgggaac cg gtcacacatg taagtgactg 3420 atataaaaga gaaaaaaggc gatttttccg cctaaaactc tttaaaactt attaaaactc 3480 ttaaaacccg cctggcctgt gcataactgt ctggccagcg gagctgcaaa cacagccgaa 3540 aagcgcctac ccttcggtcg ctgcgctccc tacgccccgc cgcttcgcgt cggcctatcg 3600 cggccgctgg ccgctcaaaa atggctggcc tacggccagg caatctacca gggcgcggac 3660 aagccgcgcc gtcgccactc gaccgccggc gctgaggtct gcctcgtgaa gaaggtgttg 3720 ccaggcctga ctgactcata atcgccccat catccagcca gaaagtgagg gagccacggt 3780 tgatgagagc tttgttgtag gtggaccagt tggtgatttt gaacttttgc tttgccacgg 3840 gttgtcggga aacggtctgc tctgatcctt agatgcgtga caactcagca aaagttcgat 3900 ttattcaaca aagccgccgt cccgtcaagt cagcgtaatg ctctgccagt gttacaacca 3960 attaaccaat tctgattaga aaaactcatc gagcatcaaa tgaaactgca atttattcat 4020 atcaggatta tcaataccat atttttgaaa aagccgtttc tgtaatgaag gagaaaactc 4080 accgaggcag ttccatagga tggcaagatc ctggtatcgg tctgcgattc cgactcgtcc 4140 caacctatta aacatcaata atttcccctc gtcaaaaata aggttatcaa gtgagaaatc 4200 accatgagtg acgactgaat ccggtgagaa tggcaaaagc tc tgcattaa tgaatcggcc 4260 aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact 4320 cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac 4380 ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa 4440 aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg 4500 caaaaatcga acgagcatcá cgctcaagtc agaggtggcg aaacccgaca ggactataaa 4560 gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc 4620 ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct caatgctcac 4680 gctgtaggta tc cagttcg gtgtaggtcg ttcgctccaa gctgggctgt gt 4732 < 210 > 6 < 211 > 24 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: HiNK025bis < 400 > 6 caattaccat ggacacggtc gtgg 24 < 210 > 7 < 211 > 30 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: HÍNK226 < 400 > 7 gccaaatgtt tgaacgctgc agcctatttg 30 < 210 > 8 < 211 > 24 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: HiNK025bis2 < 400 > 8 aatcgtccat ggatacggtc gtgg 24 < 210 > 9 < 211 > 30 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: HiNK226bis < 400 > 9 ctagggccgg gttcctctgc agcctatttg 30 < 210 > 10 < 211 > 31 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: HÍNK251 < 400 > 10 ctcccaggtt gagactgccc tgcagtgccc a 31 < 210 > 11 < 211 > 26 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: HÍNK228 < 400 > 11 ttaccatgca tacggtcgtg ggtagg 26 < 210 > 12 < 211 > 26 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: HiNK228bis < 00 > 12 cgttaatgca tacggtcgtg ggtagg 26 < 210 > 13 < 211 > 3338 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: BWYV duplex RNA gene < 220 > < 221 > promoter < 222 > (1) .. (1131) < 223 > RolC Promoter < 220 > < 221 > Repetition unit < 222 > (1132) .. (1737) < 223 > Inverted repeat fragment BWYV CP < 220 > < 221 > cycle_tallo < 222 > (1738) .. (2470) < 223 > 0.7 Kb separator fragment downstream of the BWYV CP gene < 220 > < 221 > Repetition unit < 222 > (2471) .. (3074) < 223 > Inverted repeat fragment BWYV CP < 220 > < 221 > terminator < 222 > (3076) .. (3338) < 223 > Terminator NOS < 400 > 13 ggatccggcg tcggaaactg gcgccaatca gacacagtct ctggtcggga aagccagagg 60 tagtttggca acaatcacat caagatcgat gcgcaagaca cgggaggcct taaaatctgg 120 atcaagcgaa aatactgcat gcgtgatcgt tcatgggttc atagtactgg gtttgctttt 180 tcttgtcgtg ttgtttggcc ttagcgaaag gatgtcaaaa aaggatgccc ataattggga 240 ggagtggggt aaagcttaaa gttggcccgc tattggattt cgcgaaagcg cattggcaaa 300 gctgcattca cgtgaagatt agatactttt tctattttct ggttaagatg taaagtattg 360 ccacaatcat attaattact aacattgtat atgtaatata gtgcggaaat tatctatgcc 420 aaaatgatgt attaataata gcaataataa tatgtgttaa tctttttcaa tcgggaatac 480 gtttaagcga ttatcgtgtt gaataaatta ttccaaaagg aaatacatgg ttttggagaa 540 cctgctatag ataatgcca aatttacact agtttagtgg gtgcaaaact attatctctg 600 tttctgagtt taataaaaaa taaataagca gggcgaatag cagttagcct aagaaggaat 660 ggtggccatg tacgtgcttt taagagaccc tataataaat tgccagctgt gttgctttgg 720 tgccgacagg cctaacgtgg ggtttagctt gacaaagtag cgcctttccg cagcataaat 780 aaaggtaggc gggtgcgtcc ggaaaaagca cattattaaa aaagctgaga ttccatagac 840 cacaaaccac cattatt gga ggacagaacc tattccctca cgtgggtcgc tgagctttaa 900 acctaataag taaaaacaat taaaagcagg caggtgtccc ttctatattc gcacaacgag 960 gcgacgtgga gcatcgacag ccgcatccat taattaataa atttgtggac ctatacctaa 1020 ctcaaatatt tttattattt gctccaatac gctaagagct ctggattata aatagtttgg 1080 atgcttcgag ttatgggtac aagcaacctg tttcctactt tgttaccatg gacacggtcg 1140 tgggtaggag aattatcaat ggaagaagac gaccacgcag gcaaacacga cgcgctcagc 1200 gccctcagcc agtggttgtg gtccaaacct ctcgggcaac acaacgccga cctagacgac 1260 gacgaagagg taacaaccgg acaggaagaa ctgttcctac cagaggagca ggttcgagcg 1320 agacatttgt tttctcaaaa gacaatctcg cgggaagttc cagcggagca atcacgttcg 1380 ggccgagtct atcagactgc ccggcattct ctaatggaat gctcaaggcc taccatgagt 1440 ataaaatctc aatggtcatt ttggagttcg tctccgaagc ctcttcccaa aattccggtt 1500 ccatcgctta ccacactgta cgagctggac aactcaactc cctttcctca actatcaaca 1560 agttcgggat cacaaagccc gggaaaaggg cgtttacagc gtcttacatc aacggaacgg 1620 cgttgccgag aatggcacga gaccaattca ggatcctcta caaaggcaat ggttcttcat 1680 cgatagctgg ttctttcaga atcaccatta agtgtcaatt ccacaacccc aaataggctg 1740 cagtgcccaa ctctctttgg tctttctgtc tttacggaac cggatgagcc ttgttcatca 1800 agtgctatgg cgatcatccc tatctccatc atccgatctg tccaggctcc gtacgaaacg 1860 gcgtaattat atttagattg cttctggaca gcgggtccaa caacaaagaa ggaggcattg 1920 tcaccagtcg tcttgatatg acaagtcaaa tctccatctc tttccagaca ctgtccttga 1980 ttgaaatgac agccattgag ttccatgtct gggtggccat acttcaaagt attatcggcc 2040 ttgttgttgg ttatctccac attattgtaa atccccacgt tccaaccttc actaagatca 2100 tcgttgtagg cgatgagacc atcttgtttg tccttgttag gatctgtggt gcttgaggtt 2160 ggttgatacc cctccatcga tatttccacg gtccactcac cttgagggac tggcactatt 2220 tggctttcaa atcatcggga agaattctgg gaataccatc gagaatcgag gttcgtccag 2280 ttcatgttct cgtcctctat gtagcgaaac cgttgggacg gcatatcata caaagagatg 2340 gcatca TCCG tagattgagc gtcacgggga cattatacga gacgataaaa ctccagtata 2400 cgatatttct tttggggtgt gggttgtgga gagggagaag gccctgggct agggccgggt 2460 tcctcgtcta cctatttggg gttgtggaat tgacacttaa tggtgattct gaaagaacca 2520 gctatcgatg aagaaccatt gcctttgtag aggatcctga attggtcctc ggcaacgtcg 2580 tgccattccg ttccgttgat gtaagacgct gtaaacgccc ttttcccggg ctttgtgatc 2640 ccgaacttgt tgatagttga ggaaagggag ttgagtttac agtgtgggtc cagctcgtaa 2700 gcgatggaac cggaattttg ggaagaggct tcggagacga actccaaaat gaccattgag 2760 attttatact catggtaggc cttgagcatt atgccgggca ccattagaga gtctgataga 2820 acgtgattgc ctcggcccga tccgctggaa cttcccgcga tgagaaaaca gattgtcttt 2880 aatgtctcgc tcgaacctgc tcctctggta ggaacagttc ttcctgtccg gttgttacct 2940 cttcgtcgtc gtctaggtcg gcgttgtgtt gcccgagagg tttggaccac aaccactggc 3000 tgagggcgct gagcgcgtcg tgtttgcctg cgtggtcgtc ttcttccatt gataattctc 3060 ctacccacga ccgtatgcag atcgttcaaa catttggcaa taaagtttct taagattgaa 3120 tcctgttgcc ggtcttgcga tgattatcat ataatttctg ttgaattacg ttaagcatgt 3180 aataattaac to tgtaatgca tgacgttatt tatgagatgg gtttttatga ttagagtccc 3240 gcaattatac atttaatacg cgatagaaaa caaaatatag cgcgcaacct aggataaatt 3300 atcgcgcgcg gtgtcatcta tgttactaga tctctaga 3338 < 210 > 14 < 211 > 29 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: HÍNK285 < 400 > 14 tcgtagaaga gaattcaccc aaactatcc 29 < 210 > 15 < 211 > 29 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: HÍNK283 < 400 > 15 aagaattgca ggatccacag gctcggtac 29 < 210 > 16 < 211 > 24 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: HÍNK284 < 400 > 16 ttccaacgaa ttcggtctca gaca 24 < 210 > 17 < 211 > 3648 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: pHiNKldl < 220 > < 221 > promoter < 222 > (1) .. (1738) < 223 > Promoter of ubiquitin 3 plus intron < 220 > < 221 > Repetition unit < 222 > (1739) .. (2166) < 223 > RNA1 repeat fragment of BNYW < 220 > < 221 > various_ characteristics < 222 > (2167) .. (2946) < 223 > splitter fragment < 220 > < 221 > Repetition unit < 222 > (2947) .. (3375) < 223 > RNA1 repeat fragment from BNYW < 220 > < 221 > cycle_tallo < 222 > (1739). . (3375) < 223 > Inverted repeat of BNYW RNA1 fragment separated by separator < 220 > < 221 > terminator < 222 > (3376) .. (3648) < 223 > NOS terminator < 400 > 17 ggtaccggat ttggagccaa gtctcataaa cgccattgtg gaagaaagtc ttgagttggt 60 cagagtagta ggtaatgtaa agaacagaga agagagagag tgtgagatac atgaattgtc 120 gggcaacaaa aatcctgaac atcttatttt agcaaagaga aagagttccg agtctgtagc 180 agaagagtga ggagaaattt aagctcttgg acttgtgaat tgttccgcct cttgaatact 240 tcttcaatcc tcatatattc ttcttctatg ttacctgaaa accggcattt aatctcgcgg 300 gtttattccg gttcaacatt ttttttgttt tgagttatta tctgggctta ataacgcagg 360 cctgaaataa attcaaggcc caactgtttt tttttttaag aagttgctgt taaaaaaaaa 420 aaaagggaat taacaacaac aacaaaaaaa gataaagaaa ataataacaa ttactttaat 480 tgtagactaa aaaaacatag attttatcat gaaaaaaaga gaaaagaaat aaaaacttgg 540 aaaacataca atcaaaaaaa gatcttctaa ttattaactt ttcttaaaaa ttaggtcctt 600 attaggttta tttcccaaca attaaaccaa gagttttgga ctaaaaaata aaagattgtt 660 ctcaaatttg gtagataagt ttccttattt atggtagata taattagtca cttttttttc 720 ttt etttat tagagtagat tagaatettt tatgecaagt tttgataaat taaatcaaga 780 tcataatcaa agataaacta catgaaatta aaagaaaaat etcatatata gtattagtat 840 tetatatata tattat GAIT gcttattctt aatgggttgg gttaaccaag acatagtctt 900 atcttttttg aatggaaaga ttattgatta aactttttcc tagaaaagaa aattetteta 960 agaaattatt tgaggaaaag tatatacaaa aagaaaaata gaaaaatgtc agtgaagcag 1020 atgtaatgga tgaectaate caaccaccac cataggatgt ttctacttga gtcggtcttt 1080 cggtggaaaa taaaaácgca tatgacacgt atcatatgat tccttccttt agtttcgtga 1140 taataatcct caactgatat cttccttttt ttgttttggc taaagatatt ttattetcat 1200 taatagaaaa gacggttttg ggcttttggt ttgcgatata aagaagacct tcgtgtggaa 1260 gataataatt catcctttcg tctttttctg actcttcaat ctctcccaaa gcctaaagcg 1320 atctctgcaa atctctcgcg actctctctt tcaaggtata ttttctgatt ctttttgttt 1380 ttgattcgta tctgatctcc aatttttgtt atgtggatta ttgaatcttt tgtataaatt 1440 gcttttgaca atattgttcg tttcgtcaat ccagcttcta aattttgtcc tgattactaa 1500 gatategatt cgtagtgttt acatctgtgt aatttcttgc ttgattgtga aattaggatt 1560 ttcaaggacg atetattcaa tttttgtgtt ttctttgttc gattetetet gttttaggtt 1620 tcttatgttt agatccgttt ctctttggtg ttgttttgat ttctcttacg gcttttgatt 1680 tggtatatgt tcgctgattg gt ttctactt gttctattgt tttatttcag gtggatccac 1740 cttatttcga aggctcggta aaagtgagaa ategacacaa attctccatc gatagtgttg 1800 cacgtatggt tgetcageta tttgtttctg attgtttggt gccaaatgta gctgatactt 1860 tttctgcttc caatttgtgg .cgaattatgg acaaagetat gcatgacatg gtcgcaaaaa 1920 attaccaagg ccaaatggaa gaggagttta cgcgtaatgc taaactatat cgttttcagt 1980 tgaaggatat tgaaaaacct ttgaaggacc cagagactga tttggcaaag gctggtcaag 2040 ggatattggc atggtctaag gaggcacatg ttaagtttat ggttgctttt agagttttaa 2100 gatttgtt attgaagtca atgttgttta ttaaactcta atgtctgaga cgataacaca 2160 ccgaattcac ccaaactatc ctcaacgggt ggcaatgaca taaaaacttc ccgtatcggg 2220 aacatcatca taaattgctc acgcccgatt cgagacacag agataatage atccaaccaa 2280 cgttgaacat catcaacatt gcgacaagat aagctagcgt tacactccaa aaaatcagca 2340 taaacagctg ggtctttggg aagattttta atccaatcac gcaaagattc ctggtattca 2400 caaaaatgct tataetcaeg gaacctgtgt gctgctatct tcgtcaattt acgcgaaaca 2460 aatgtccatt cttggaaaca taaccacaaa agataaagea aagtgatagg aacatttaaa 2520 tccaatttga aatccaagac agtttccttt ttaatcaact ttaacatttg atcgttaatt 2580 ttcaaattag cctgcctttt aaaaccatca tcgcccttca tggccataca aaatggtccg 2640 gtcccacgaa geatageatt taacatagea cccattaaaa tggtattacc aagcaaagtg 2700 ccgggttctc cactagtctt aacataagac atatgtgctc tgacatatct ggactgcata 2760 acatatttct cacgaaatga tccaaaaaga gaaataccaa agtcagaaat gcccaaagca 2820 gcataaatat gtctttctat caattgggtg aaaacccctt gcccagaatc gcaageagea 2880 gcatcgataa ccccgtttat ageactatet ggtactgtat tcatggcggc atttattttt 2940 ccaacaaa tt cggtctcaga cattgtgtta tcgtaaacaa cattagagtt taatgacttc 3000 aataacaaat catttaaaac tctaaaagca accataaact taacatgtgc ctccttagac 3060 catgecaata tcccttgacc agcctttgcc aaatcagtct ctgggtcctt caaaggtttt 3120 tcaatatcct tcaactgaaa acgatatagt ttageattac gcgtaaactc ctc tccatt 3180 tggccttggt aattttttgc gaccatgtca tgcatagctt tgtccataat tcgccacaaa 3240 ttggaagcag aaaaagtatc agetacattt ggcaccaaac aatcagaaac aaatagctga 3300 gcaaccatac gtgcaacact atcttgtgtc gatgatggag aatttctcac ttttcgaaat 3360 aagtaccgag cctgtggatc ccccgaattt ccccgatcgt tcaaacattt ggcaataaag 3420 tttettaaga ttgaatcctg ttgccggtct tgcgatgatt ateatetaat ttctgttgaa 3480 ttacgttaag catgtaataa ttaacatgta atgcatgacg ttatttatga gatgggtttt 3540 tatgattaga gtcccgcaat tatacattta ataegegata gaaaacaaaa tatagcgcgc 3600 aaactaggat aaattatcgc gcgcggtgtc atctatgtta ctagatcc 3648 < 210 > 18 < 211 > 3648 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: pHiNK184 < 220 > < 221 > promoter < 222 > (1) .. (1738) < 223 > ubiquitin 3 promoter plus intron leader < 220 > < 221 > Repetition unit < 222 > (1739) .. (2166) < 223 > RNA1 repeat fragment from BNYW < 220 > < 221 > various_ characteristics < 222 > (2167) .. (2946) < 223 > separator < 220 > < 221 > unit__repetition < 222 > Complement ((2947) .. (3375)) < 223 > RNA1 repeat fragment from BNYW < 220 > < 221 > cycle_tallo < 222 > (1739) .. (3375) < 223 > Inverted repeat of BNYW RNA1 fragment separated by separator < 220 > < 221 > terminator < 222 > (3376) .. (3648) < 223 > NOS terminator < 400 > 18 ggtaccggat ttggagccaa gtctcataaa cgccattgtg gaagaaagtc ttgagttggt 60 cagagtagta ggtaatgtaa agaacagaga agagagagag tgtgagatac atgaattgtc 120 gggcaacaaa aatcctgaac atcttatttt agcaaagaga aagagttccg agtctgtagc 180 agaagagtga ggagaaattt aagctcttgg acttgtgaat tgttccgcct cttgaatact 240 tcttcaatcc tcatatattc ttcttctatg ttacctgaaa accggcattt aatctcgcgg 300 gtttattccg gttcaacatt ttttttgttt tgagttatta tctgggctta ataacgcagg 360 cctgaaataa attcaaggcc caactgtttt tttttttaag aagttgctgt taaaaaaaaa 420 aaaagggaat taacaacaac aacaaaaaaa gataaagaaa ataataacaa ttactttaat 480 tgtagactaa aaaaacatag attttatcat gaaaaaaaga gaaaagaaat aaaaacttgg 540 aaaacataca atcaaaaaaa gatcttctaa ttattaactt ttcttaaaaa ttaggtcctt 600 attaggttta tttcccaaca attaaaccaa gagttttgga aaagattgtt ctaaaaaata 660 ctcaaatttg gtagataagt ttccttattt taattagtca atggtagata cttttttttc 720 ttttctttat tagagtagat tagaatcttt tatgccaagt tttgataaat taaatcaaga 780 tcataatcaa agataaacta catgaaatta aaagaaaaat ctcatatata gtattagtat 840 tctctatata tattatg att gcttattctt aatgggttgg gttaaccaag acatagtctt 900 atcttttttg aatggaaaga ttattgatta aactttttcc tagaaaagaa aattcttcta 960 agaaattatt tgaggaaaag tatatacaaa aagaaaaata gaaaaatgtc agtgaagcag 1020 atgtaatgga tgacctaatc caaccaccac cataggatgt ttctacttga gtcggtcttt 1080 cggtggaaaa taaaaacgca tatgacacgt atcatatgat tccttccttt agtttcgtga 1140 taataatcct caactgatat cttccttttt ttgttttggc taaagatatt ttattctcat 1200 taatagaaaa gacggttttg ggcttttggt ttgcgatata aagaagacct tcgtgtggaa 1260 gataataatt catcctttcg tctttttctg actcttcaat ctctcccaaa gcctaaagcg 1320 atctctgcaa atctctcgcg actctctctt tcaaggtata ttttctgatt ctttttgttt 1380 ttgattcgta tctgatctcc aatttttgtt atgtggatta ttgaatcttt tgtataaatt 1440 gcttttgaca atattgttcg tttcgtcaat ccagcttcta aattttgtcc tgattactaa 1500 gatatcgatt cgtagtgttt acatctgtgt aatttcttgc ttgattgtga aattaggatt 1560 ttcaaggacg atctattcaa tttttgtgtt ttctttgttc gattctctct gttttaggtt 1620 tcttatgttt agatccgttt ctctttggtg ttgttttgat ttctcttacg gcttttgatt 1680 tggtatatgt tcgctgattg gtt tctactt gttctattgt tttatttcag gtggatccac 1740 cttatttcga aggctcggta aaagtgagaa attctccatc atcgacacaa gatagtgttg 1800 tgctcagcta cacgtatggt tttgtttctg attgtttggt gccaaatgta gctgatactt 1860 tttctgcttc caatttgtgg cgaattatgg acaaagctat gcatgacatg gtcgcaaaaa 1920 attaccaagg ccaaatggaa gaggagttta cgcgtaatgc taaactatat cgttttcagt 1980 tgaaggatat tgaaaaacct ttgaaggacc cagagactga tttggcaaag gctggtcaag 2040 ggatattggc atggtctaag gaggcacatg ttaagtttat ggttgctttt agagttttaa 2100 attgaagtca atgatttgtt ttaaactcta atgttgttta cgataacaca atgtctgaga 2160 tggaaaaata ccgaatttgt aatgccgcca tgaatacagt accagatagt gctataaacg 2220 gggttatcga tgctgctgct tgcgattctg ggcaaggggt tttcacccaa ttgatagaaa 2280 gacatattta tgctgctttg ggcatttctg acttcttttt ggattggtat ttctcatttc 2340 gtgagaaata tgttatgcag tccagatatg tcagagcaca tatgtcttat gttaagacta 2400 gtggagaacc cggcactttg cttggtaata ccattttaat gggtgctatg ttaaatgcta 2460 • tgcttcgtgg gaccggacca ttttgtatgg ccatgaaggg cgatgatggt tttaaaaggc 2520 gaaaattaac aggctaattt gatca aatgt taaagttgat taaaaaggaa actgtcttgg 2580 atttcaaatt ggatttaaat gttcctatca ctttttgtgg ttatgcttta tctaatggac 2640 atttgtttcc aagtgtttcg cgtaaattga cgaagatagc agcacacagg ttccgtgagt 2700 ataagcattt ttgtgaatac caggaatctt tgcgtgattg gattaaaaat cttcccaaag 2760 acccágctgt ttatgctgat tttttggagt gtaacgctag cttatcttgt cgcaatgttg 2820 atgatgttca acgttggttg gatgctatta tctctgtgtc tcgaatcggg cgtgagcaat 2880 ttatgatgat gttcccgata cgggaagttt ttatgtcatt gccacccgtt gaggatagtt 2940 tgggtgaatt cggtctcaga cattgtgtta tcgtaaacaa cattagagtt taatgacttc 3000 aataacaaat catttaaaac tctaaaagca accataaact taacatgtgc ctccttagac 3060 catgccaata tcccttgacc agcctttgcc aaatcagtct ctgggtcctt caaaggtttt 3120 tcaatatcct tcaactgaaa acgatatagt ttagcattac gcgtaaactc ctcttccatt 3180 tggccttggt aattttttgc gaccatgtca tgcatagctt tgtccataat tcgccacaaa 3240 ttggaagcag aaaaagtatc agctacattt ggcaccaaac aatcagaaac aaatagctga 3300 gcaaccatac gtgcaacact atcttgtgtc gatgatggag aatttctcac ttttcgaaat 3360 aagtaccgag cctgtggatc ccccgaattt ccccgatcgt tcaaacattt ggcaataaag 3420 tttcttaaga ttgaatcctg ttgccggtct tgcgatgatt atcatctaat ttctg ttgaa 3480 ttacgttaag catgtaataa ttaacatgta atgcatgacg ttatttatga gatgggtttt 3540 tatgattaga gtcccgcaat tatacattta atacgcgata gaaaacaaaa tatagcgcgc 3600 aaactaggat aaattatcgc gcgcggtgtc atetatgtta etagatec 3648 < 210 > 19 < 211 > 41 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: ZUP1563 < 400 > 19 gggcggatcc gctagcccgc ggccgctctt tctttccaag g 41 < 210 > 20 < 211 > 35 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: ZUP1564 < 400 > 20 cccgccatgg gtcgacgcca ttttttatga gctgc 35 < 210 > 21 < 211 > 40 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: ZUP1565 < 400 > 21 cccgccatgg gatccgatga tttctaccga gaattaaggg 40 < 210 > 22 < 211 > 29 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: ZUP1566 < 400 > 22 gggcgctagc ctaatgctta tatagtacc 29 < 210 > 23 < 211 > 30 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: ZUP1567 < 400 > 23 gggcgctagc cttgctggag tataagccgg 30 < 210 > 24 < 211 > 35 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: ZUP1568 < 400 > 24 gggcgtcgac cgcgggcttt aaaggtggga ggccc 35 < 210 > 25 < 211 > 12766 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: pZU623 < 220 > < 221 > promoter < 222 > (55) .. (1781) < 223 > ubiquitin 3 promoter plus intron leader < 220 > < 221 > characteristic__various < 222 > (1790) .. (2430) < 223 > CPS region of PRSV nt < 220 > < 221 > various_ characteristics < 222 > (2511) .. (3111) < 223 > CP region of ZYMV nt < 220 > < 221 > intron < 222 > (3245) .. (3686) < 223 > Intron actin2 < 220 > < 221 > various_ characteristics < 222 > Complement ((3822) .. (4422)) < 220 > < 221 > various_ characteristics < 222 > Complement ((4503) .. (5143) < 223 > CP region of PRSV nt < 220 > < 221 > terminator < 222 > (5160) .. (5429) < 223 > NOS terminator < 220 > < 221 > promoter < 222 > (5498) .. (6669) < 223 > SMAS promoter < 220 > < 221 > gene < 222 > (6691) .. (7866) < 223 > Phosphomannose isomerase A coding sequence < 220 > < 221 > terminator < 222 > (7928) .. (8202) < 223 > nopaline synthase terminator < 220 > < 221 > various_ characteristics < 222 > (8254) .. (8385) < 223 > fragment of the left limit of nopaline < 220 > < 221 > gene < 222 > (8664) .. (9452) < 223 > Spectinomycin coding sequence (aadA) < 220 > < 221 > various_ characteristics < 222 > (9462) .. (11549) < 223 > pVSl ORÍ < 220 > < 221 > various_ characteristics < 222 > (11550) .. (12484) < 223 > pUC19 ORÍ < 220 > < 221 > various_ characteristics < 222 > (12500) .. (12755) < 223 > region of the nopalina right boundary < 220 > < 221 > cycle_tallo < 222 > (1790). . (5143) < 223 > inverted repeat fragment PRSV-ZYMV < 400 > 25 ggccgcagcg gccatttaaa tcaattgggc gcgccgaatt cgagctcggt accccggatt 60 tggagccaag tctcataaac gccattgtgg aagaaagtct tgagttggtg gtaatgtaac 120 gaacagagaa agagtagtaa gagagagagt gtgagataca tgaattgtcg ggcaacaaaa 180 tcttatttta atcctgaaca gcaaagagaa agagttccga gtctgtagca gaagagtgag 240 agctcttgga gagaaattta cttgtgaatt gttccgcctc ttgaatactt cttcaatcct 300 catatattct tcttctatgt tacctgaaaa ccggcattta atctcgcggg tttattccgg 360 ttcaacattt tttttgt tt gagttattat ctgggcttaa taacgcaggc ctgaaataaa 420 ttcaaggccc aactgttttt tttttaagaa gttgctgtta aaaaaaaaaa aagggaatta 480 acaacaacaa caaaaaaaga taaagaaaat aataacaatt actttaattg tagactaaaa 540 aaacatagat tttatcatga aaaaaagaga aaagaaataa aaacttggat caaaaaaaaa 600 aacatacaga tcttctaatt attaactttt cttaaaaatt aggtcctttt tcccaacaat 660 taggtttaga gttttggaat taaaccaaaa agattgttct aaaaaatact caaatttggt 720 agataagttt ccttatttta attagtcaat ggtagatact tttttttctt ttctttatta 780 gaatctttta gagtagatta tgccaagttt tgataaatta aatcaagaag ataaactatc 840 tgaaattaaa ataatcaaca agaaaaatct catatatagt attagtattc tctatatata 900 ttatgattgc ttattcttaa tgggttgggt taaccaagac atagtcttaa tggaaagaat 960 cttttttgaa ctttttcctt attgattaaa ttcttctata gaaaagaaag aaattatttg 1020 tatacaaaaa aggaaaagta gaaaaataga aaaatgtcag tgaagcagat gtaatggatg 1080 accaccacca acctaatcca taggatgttt ctacttgagt cggtctttta aaaacgcacg 1140 gtggaaaata tgacacgtat catatgattc cttcctttag ataatcctca tttcgtgata 1200 actgatatct tccttttttt gttttggcta aagatatttt attctcatta atagaaaaga 1260 cggttttggg cttttggttt gcgatataaa gaagaccttc taataattca gtgtggaaga 1320 tcctttcgtc tttttctgac tcttcaatct ctcccaaagc ctaaagcgat ctctgcaaat 1380 ctctcgcgac tctctctttc aaggtatatt ttctgattct ttttgttttt gattcgtatc 1440 tgatctccaa tttttgttat g tggattatt gaatcttttg tataaattgc ttttgacaat 1500 attgttcgtt tcgtcaatcc agcttctaaa ttttgtcctg attactaaga tatcgattcg 1560 tagtgtttac atctgtgtaa tttcttgctt gattgtgaaa ttaggatttt caaggacgat '1620 ctattcaatt tttgtgtttt ctttgttcga ttctctctgt tttaggtttc ttatgtttag 1680 atccgtttct ctttggtgtt gttttgattt ctcttacggc ttttgatttg gtatatgttc 1740 gctgattggt ttctacttgt tctattgttt tatttcaggt gggggatccg atgatttcta 1800 ccgagaatta agggaaagac tgtccttaat ttaaatcatc ttcttcagta taatccgcaa 1860 caaattgaca tttctaacac tcgtgccact cagtcacaat ttgagaagtg gtatgaggga 1920 gtgaggaatg attatggtct caatgataat gaaatgcaag tgatgctaaa tggcttgatg 1980 gtttggtgta tcgagaatgg tacatctcca gacatatctg gtgtctgggt tatgatggat 2040 ggggaaaccc aagttgatta tccaatcaag cctttaatag agcatgctac tccgtcattt 2100 aggcaaatta tggctcactt tagtaacgcg gcagaagcat acattgcgaa gagaaatgct 2160 actgagagat acatgccgcg gtatggaatc aagagaaatt tgactgacat tagtcttgct 2220 agatacgctt tcgacttcta tgaggtgaat tcgaaaacac ctgatagggc tcgtgaagct 2280 cacatgcaga tgaaagctgc AGCGCT gcga aacactagtc gcagaatgtt tggtatggac 2340 gtaacaagga ggcagtgtta agaaaacacg gagagacaca tgtcaataga cagtggaaga 2400 gacatgcact ctctcctggg tatgcgcaac tgaatactcg cgcttgtgtg tttgtcgagt 2460 ctgactcgac cctgtttcac cttatggtac tatataagca ttaggctagc cttgctggag 2520 tataagccgg atcaaattga gttatacaac acacgagcgt ctcatcagca attcgcctct 2580 tggttcaacc aagttaaaac agaatatgat ctgaatgagc aacagatggg agttgtaatg 2640 aatggtttca tggtttggtg cattgaaaat ggcacgtcac ccgacattaa cggagtatgg 2700 acggtaatga gttatgatgg gcaggttgaa tatcctttga tgaaaatgca aaccaatagt 2760 aagccaacgc tgcgacaaat aatgcatcac ttttcagatg cagcggaggc atatatagag 2820 atgagaaatg cagaggcacc atacatgccg aggtatggtt tgcttcgaaa cttacgggat 2880 aggagtttgg cacgatatgc tttcgacttc tacgaagtca attccaaaac tccggaaaga 2940 ctgttgcgca gcccgcgaag gcagccctta gatgaaagca gcaatgtttc ttcaaggttg 3000 tttggccttg atggaaatgt tgccaccact agcgaagaca ctgaacggca cactgcacgt 3060 gatgttaata ggaacatgca ggtgtgaata caccttgcta caatgcagta aagggtaggt 3120 ggttatcgtt cgcctaccta tcgctgccga cgtaattcta atatttaccg ctttatgtga 3180 tgtctttaga tttctagagt gggcctccca cctttaaagc ccgcggccgc tctttctttc 3240 caaggtaata ggaactttct ggatctactt tatttgctgg atctcgatct tgttttctca 3300 atttccttga gatctggaat tcgtttaatt tggatctgtg aacctccact aaatcttttg 3360 aatcgatcta gttttactag agttgaccga tcagttagct cgattatagc taccagaatt 3420 tggcttgacc ttgatggaga gatccatgtt catgttacct gggaaatgat ttgtatatgt 3480 gaattgaaat ctgaactgtt gaagttagat tgaatctgaa cactgtcaat gttagattga 3540 atctgaacac tgtttaagtt agatgaagtt tgtgtataga ttcttcgaaa ctttaggatt 3600 tgtagtgtcg tacgttgaac agaaagctat ttctgattca atcagggttt atttgactgt 3660 attgaactct ttttgtgtgt ttgcagctca taaaaaatgg cgtcgaccgc gggctttaaa 3720 ccactctaga ggtgggaggc catcacataa aatctaaaga attagaatta agcggtaaat 3780 gaaacgataa cgtcggcagc cctaggtagg cgacctaccc tttactgcat tgtattcaca 3840 cctagcaagg tgtgcatgtt cctattaaca tcacgtgcag tgtgccgttc agtgtcttcg 3900 ctagtggtgg caacatttcc atcaaggcca aacaaccttg aagaaacatt gctaagggct 3960 gctgctttca tctgcgcaac agcttcgcgg gctctt tccg gagttttgga attgacttcg 4020 tagaagtcga aagcatatcg tgccaaactc ctatcccgta agtttcgaag caaaccatac 4080 ctcggcatgt atggtgcctc tgcatttctc atctctatat atgcctccgc tgcatctgaa 4140 aagtgatgca ttatttgtcg cagcgttggc tttgcatttt caactattgg tttcaaagga 4200 tattcaacct gctcattacc gtccatcata acccatactc cgttaatgtc gggtgacgtg 4260 tgcaccaaac ccattttcaa catgaaacca ttcattacaa ctcccatctg ttgctcattc 4320 agatcatatt ctgttttaac ttggttgaac caagaggcga attgctgatg agacgctcgt 4380 gtgttgtata actcaatttg atccggctta tactccagca aggctagcct aatgcttata 4440 aggtgaaaca tagtaccata gggtcgagtc agactcgaca aacacacaag cgcgagtatt 4500 cagttgcgca tacccaggag agagtgcatg tctctattga catcttccac tgtgtgtctc 4560 tccgtgtttt cttccttgtt actaacactg ccgtccatac caaacattct gcgactagtg 4620 tttcgcagcg ctgcagcttt catctgcatg tgagcttcac gagccctatc aggtgttttc 4680 gaattcacct catagaagtc gaaagcgtat ctagcaagac taatgtcagt caaatttctc 4740 ttgattccat accgcggcat gtatctctca gtagcatttc tcttcgcaat gtatgcttct 4800 gccgcgttac taaagtgagc cataatttgc ctaaatgacg gagtag catg ctctattaaa 4860 ggcttgattg gataatcaac ttgggtttcc ceatecatea taacccagac accagatatg 4920 tctggagatg taccattctc gatacaccaa accatcaagc catttagcat caettgeatt 4980 tcattatcat tgagaccata atcattcctc actccctcat accacttctc aaattgtgac 5040 tgagtggcac gagtgttaga aatgtcaatt tgttgcggat tatactgaag aagatgattt 5100 aaattaagga cagtctttcc ettaattetc ggtagaaatc atcggatcca tggtgatcac 5160 tgcagatcgt tcaaacattt ggcaataaag tttettaaga ttgaatcctg ttgccggtct 5220 tgcgatgatt ateatataat ttctgttgaa ttacgttaag catgtaataa ttaacatgta 5280 atgcatgacg ttatttatga gatgggtttt tatgattaga gtcccgcaat tatacattta 5340 gaaaacaaaa ataegegata tatagcgcgc aaactaggat aaattatege gcgcggtgtc 5400 atetatgtta etagatetet agaaagette gtacgttaat taattcgaat ccggagcggc 5460 cgcagggcta gcatcgatgg taccgagctc gagactatac aggccaaatt cgctcttagc 5520 cgtacaatat tactcaccgg tgcgatgccc cccatcgtag gtgaaggtgg aaattaatga 5580 tecatettga gaccacaggc ccacaacagc taccagtttc ctcaagggtc caccaaaaac 5640 gtaagcgctt acgtacatgg tegataagaa aaggcaattt gtagatgtta to catccaacg 5700 tcgctttcag ggatcccgaa ttccaagctt ggaattcggg atectacagg ccaaattcgc 5760 tcttagccgt acaatattac tcaccggtgc gatgcccccc atcgtaggtg aaggtggaaa 5820 ttaatgatcc atcttgagac cacaggccca caacagctac cagtttcctc aagggtccac 5880 caaaaacgta agcgcttacg tacatggtcg ataagaaaag gcaatttgta gatgttaaca 5940 ctttcaggga tccaacgtcg caagcttgga tcccgaattc ctacaggcca attcgggatc 6000 tagccgtaca aattcgctct atattactca ccggtgcgat ccccccatcg taggtgaagg 6060 tggaaattaa tgatecatet tgagaccaca ggcccacaac agetaccagt ttcctcaagg 6120 gtccaccaaa aacgtaagcg cttacgtaca tggtcgataa gaaaaggcaa tttgtagatg 6180 ttaacateca acgtcgcttt cagggatccc gaattccaag cttgggctgc aggtcaatcc 6240 cattgctttt gaageagetc aacattgatc tetttetega gggagatttt tcaaatcagt 6300 gcgcaagacg tgacgtaagt atccgagtca gtttttattt ttctactaat ttggtcgttt 6360 atttcggcgt gtaggacatg gcaaccgggc ctgaatttcg cgggtattct gtttctattc 6420 ttgatccgca caactttttc gccattaacg acttttgaat agataegetg acacgccaag 6480 caaaagtgta cctcgctagt ccaaacaacg ctttacagca agaacggaat gcgcgt GACG 6540 cgccatttcg ctcgcggtga aatggataaa ccttttcaga tectattata tageettget 6600 tcttcccaaa ttaccaatac attacactag catctgaatt tetegataca tcataaccaa 6660 ccaaatcgag atctgcaggg atccccgatc atgcaaaaac teattaaetc agtgcaaaac 6720 tatgcctggg gcagcaaaac ggcgttgact gaactttatg gtatggaaaa tccgtccagc 6780 cagccgatgg ccgagctgtg gatgggcgca catccgaaaa gcagttcacg agtgcagaat 6840 gccgccggag atatcgtttc actgcgtgat gtgattgaga gtgataaatc gactctgctc 6900 ggagaggccg ttgccaaacg ctttggcgaa ctgcctttcc tgttcaaagt attatgcgca 6960 tctccattca gcacagccac ggttcatcca aacaaacaca attctgaaat cggttttgcc 7020 aaagaaaatg ccgcaggtat cccgatggat gccgccgagc gtaactataa agatectaac 7080 cacaagccgg agctggtttt tgcgctgacg cctttccttg cgatgaacgc gtttcgtgaa 7140 ttttccgaga ttgtctccct actccagccg gtcgcaggtg cacatccggc gattgetcae 7200 tttttacaac agcctgatgc cgaacgttta agcgaactgt tcgccagcct gttgaatatg 7260 cagggtgaag aaaaatcccg cgcgctggcg attttaaaat cggccctcga tagecageag 7320 ggtgaaccgt ggcaaacgat tcgtttaatt tctgaatttt acccggaaga cagcggtctg 7 380 ttctccccgc tattgctgaa tgtggtgaaa ttgaaccctg gcgaagcgat gttcctgttc 7440 gctgaaacac cgcacgctta cctgcaaggc gtggcgctgg aagtgatggc aaactccgat 7500 aacgtgctgc gtgcgggtct gacgcctaaa tacattgata ttccggaact ggttgccaat 7560 gtgaaattcg aagccaaacc ggctaaccag ttgttgaccc agccggtgaa acaaggtgca 7620 gaactggact tcccgattcc agtggatgat tttgccttct cgctgcatga ccttagtgat 7680 ccattagcca aaagaaacca gccattttgt gcagagtgcc tctgcgtcga aggcgatgca 7740 aaggttctca acgttgtgga gcagttacag cttaaaccgg gtgaatcagc gtttattgcc 7800 gccaacgaat caccggtgac tgtcaaaggc cacggccgtt tagcgcgtgt ttacaacaag 7860 ctgtaagagc ttactgaaaa aattaacatc tcttgctaag ctgggagctc gtcgacggat 7920 cgaattcctg cagatcgttc aaacatttgg caataaagtt tettaagatt gaatcctgtt 7980 gccggtcttg cgatgattat catataattt ctgttgaatt acgttaagca tgtaataatt 8040 aacatgtaat gcatgacgtt atttatgaga tgggttttta tgattagagt cccgcaatta 8100 acgcgataga tacatttaat aaacaaaata tagcgcgcaa actaggataa attatcgcgc 8160 gcggtgtcat ctatgttact agatetetag aactagtgga tctgctagcc ctgcaggaaa 8220 tttacc ggtg cccgggcggc cagcatggcc gtatccgcaa tgtgttatta agttgtctaa 8280 gtttacacca gcgtcaattt caatatatec tgccaccagc cagccaacag ctccccgacc 8340 cacaaaatca ggcagctcgg ccactcgata caggcagccc atcagaatta attctcatgt 8400 ttgacagctt ateategact gcacggtgca ccaatgcttc tggcgtcagg cagccatcgg 8460 aagctgtggt atggctgtgc aggtcgtaaa teactgeata attcgtgtcg ctcaaggcgc 8520 actcccgttc tggataatgt tttttgcgcc gacatcataa cggttctggc aaatattctg 8580 aaa gagctg ttgacaatta atca cggct cgtataatgt gtggaattgt gagcggataa 8640 caattteaca caggaaacag accatgaggg aagcggtgat cgccgaagta tcgactcaac 8700 tatcagaggt agttggcgtc atcgagcgcc atctcgaacc gacgttgctg gccgtacatt 8760 tgtacggctc cgcagtggat ggcggcctga agccacacag tgatattgat ttgctggtta 8820 cggtgaccgt aaggcttgat gaaacaacgc ggcgagcttt gatcaaegac cttttggaaa 8880 cttcggcttc ccctggagag agegagatte tccgcgctgt agaagtcacc attgttgtgc 8940 acgacgacat cattccgtgg cgttatccag ctaagcgcga actgcaattt ggagaatggc 9000 cattcttgca agcgcaatga ggtatcttcg agccagccac gatcgacatt gatctggcta 9060 tcttgctgac aaaagcaaga gaacatagcg ttgccttggt aggtccagcg gcggaggaac 9120 tctttgatcc ggttcctgaa caggatctat ttgaggcgct aaatgaaacc ttaacgctat 9180 ggaactcgcc gcccgactgg gctggcgatg agcgaaatgt agtgcttacg ttgtcccgca 9240 tttggtacag cgcagtaacc ggcaaaatcg cgccgaagga tgtcgctgcc gactgggcaa 9300 tggagcgcct gccggcccag tatcagcccg tcatacttga agctaggcag gcttatcttg 9360 gacaagaaga tcgcttggcc tcgcgcgcag atcagttgga agaatttgtt cactacgtga 9420 aaggcgagat caccaaggta gtcggcaaat aaagctctag tggatccccg aggaatcggc 9480 gtgacggtcg caaaccatcc ggcccggtac aaatcggcgc ggcgctgggt gatgacctgg 9540 tggagaagtt gaaggccgcg caggccgccc agcggcaacg catcgaggca gaagcacgcc 9600 ccggtgaatc gtggcaagcg gccgctgatc gaatccgcaa agaatcccgg caaccgccgg 9660 cagccggtgc gccgtcgatt aggaagccgc ccaagggcga cgagcaacca gattttttcg 9720 ttccgatgct ctatgacgtg ggcacccgcg atagtcgcag catcatggac gtggccgttt 9780 tccgtctgtc gaagcgtgac cgacgagctg gcgaggtgat ccgctacgag cttccagacg 9840 ggtttcagca ggcacgtaga gggccggccg gcatggccag tgtgtgggat tacgacctgg 9900 tactgatggc ggtttc ccat ctaaccgaat ccatgaaccg ataccgggaa gggaagggag 9960 acaagcccgg ccgcgtgttc cgtccacacg ttgcggacgt actcaagttc tgccggcgag 10020 ccgatggcgg aaagcagaaa gacgacctgg tagaaacctg cattcggtta aacaccacgc 10080 acgttgccat gcagcgtacg aagaaggcca agaacggccg cctggtgacg gtatccgagg 10140 gtgaagcctt gattagccgc tacaagatcg taaagagcga aaccgggcgg ccggagtaca 10200 tcgagatcga gctagctgat tggatgtacc gcgagatcac agaaggcaag aacccggacg 10260 tgctgacggt tcaccccgat tactttttga tcgatcccgg catcggccgt tttctctacc 10320 ccgcgccgca gcctggcacg ggcaaggcag aagccagatg gttgttcaag acgatctacg 10380 cagcgccgga aacgcagtgg gagttcaaga agttctgttt caccgtgcgc aagctgatcg 10440 ggtcaaatga cctgccggag tacgatttga aggaggaggc ggggcaggct ggcccgatcc 10500 tagtcatgcg ctaccgcaac ctgatcgagg gcgaagcatc cgccggttcc taatgtacgg 10560 agcagatgct agggcaaatt gccctagcag gggaaaaagg tcgaaaaggt ctctttcctg 10620 tggatagcac gtacattggg aacccaaagc cgtacattgg gaaccggaac ccgtacattg 10680 ggaacccaaa gccgtacatt gggaaccggt cacacatgta agtgactgat ataaaagaga 10740 aaaaaggcga tttttccgcc taaaactctt taaaacttat taaaactctt aaaacccgcc 10800 tggcctgtgc ataactgtct ggccagcgca gctgcaaaaa cagccgaaga gcgcctaccc 10860 ttcggtcgct gcgctcccta cgccccgccg cttcgcgtcg gcctatcgcg gccgctggcc 10920 ggctggccta gctcaaaaat cggccaggca atctaccagg gcgcggacaa gccgcgccgt 10980 cgccactcga ccgccggcgc tgaggtctgc ctcgtgaaga aggtgttgct gactcatacc 11040 cgccccatca aggcctgaat tccagccaga aagtgaggga gccacggttg atgagagctt 11100 tgttgtaggt ggaccagttg gtgattttga acttttgctt tgccacggaa cggtctgcgt 11160 tgtcgggaag atgcgtga c actcagcaaa tgatccttca attcaacaaa agttcgattt 11220 cgtcaagtca gccgccgtcc gcgtaatgct ctgccagtgt tacaaccaat taaccaattc 11280 aactcatcga tgattagaaa gcatcaaatg aaactgcaat ttattcatat caggattatc 11340 aataccatat ttttgaaaaa gccgtttctg taatgaagga gaaaactcac cgaggcagtt 11400 ccataggatg gcaagatcct ggtatcggtc tgcgattccg catcaataca actcgtccaa 11460 acctattaat ttcccctcgt caaaaataag gttatcaagt gagaaatcac catgagtgac 11520 gactgaatcc ggtgagaatg gcaaaagctc tgcattaatg aatcggccaa cgcgcgggga 11580 gag gcggttt gcgtattggg cgctcttccg cttcctcgct cactgactcg ctgcgctcgg 11640 tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag 11700 taacgcagga aatcagggga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc 11760 gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca 11820 aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt 11880 ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc 11940 tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca atgctcacgc tgtaggtatc 12000 tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc 12060 ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact 12120 tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg 12180 ctacagagtt cttgaagtgg tggcctaact acggctacac tagaaggaca gtatttggta 12240 tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca 12300 aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa 12360 aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg 12420 aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc 12480 ttttgatccg gaattaattc ctgtggttgg catgcacata caaatggacg aacggataaa 12540 ccttttcacg cccttttaaa tatccgatta ttctaataaa cgctcttttc tcttaggttt 12600 acccgccaat atatcctgtc aaacactgat agtttaaact gaaggcggga aacgacaatc 12660 tgatcatgag cggagaatta agggagtcac gttatgaccc ccgccgatga cgcgggacaa gccgttttac 12720 12766 gtttggaact gacagaaccg caacgctgca ggaatt < 210 > 26 < 211 > 42 < 212 > DNA < 213 > Artificial Sequence < 220 > . < 223 > Description of the Artificial Sequence: ZUP1598 < 400 > 26 catgccatgg atccaatggc cacgaattaa agctatcacg te 42 < 210 > 27 < 211 > 44 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: ZUP1590 < 400 > 27 acgcgtcgac cgcggattca aacgattatt aattacgata aaag 44 < 210 > 28 < 211 > 11461 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: pZU634 < 220 > < 221 > promoter < 222 > (55) .. (1786) < 223 > ubiquitin 3 promoter plus intron leader < 220 > < 221 > various_ characteristics < 222 > (1790) .. (2574) < 223 > CP region of PVY nt < 220 > < 221 > intron < 222 > (2595) .. (3036) < 223 > Intron actin2 < 220 > < 221 > various_ characteristics < 222 > Complement ((3057) .. (3847)) < 223 > CP region of PVY nt < 220 > < 221 > terminator < 222 > (3855) .. (4124) < 223 > NOS terminator < 220 > < 221 > promoter < 222 > (4193) .. (5364) < 223 > SMAS promoter < 220 > < 221 > gene < 222 > (5386) .. (6561) < 223 > Phosphomannose isomerase A coding sequence < 220 > < 221 > terminator < 222 > (6623) .. (6897) < 223 > nopaline synthase terminator < 220 > < 221 > various_ characteristics < 222 > (6949) .. (7080) < 223 > fragment of the left limit of nopaline < 220 > < 221 > gene < 222 > (7359) .. (8147) < 223 > Spectinomycin coding sequence (aadA) < 220 > < 221 > various_ characteristics < 222 > (8157) .. (10244) < 223 > pVSl, ORI < 220 > < 221 > various_ characteristics < 222 > (10245) .. (11179) < 223 > pUC19 ORÍ < 220 > < 221 > various_ characteristics < 222 > (11195) .. (11450) < 223 > region of the nopalina right boundary < 220 > < 221 > Stem cycle < 222 > (1790) .. (3847) < 223 > inverted repeat region PVY < 400 > 28 ggccgcagcg gccatttaaa tcaattgggc gcgccgaatt cgagctcggt acccgtaccg 60 gatttggagc caagtctcat aaaegecatt gtggaagaaa gtcttgagtt ggtggtaatg 120 taacagagta gtaagaacag agaagagaga gagtgtgaga tacatgaatt gtcgggcaac 180 aaaaatcctg aacatcttat tttagcaaag agaaagagtt ccgagtctgt ageagaagag 240 tgaggagaaa tttaagctct tggacttgtg aattgttccg cctcttgaat acttcttcaa 300 tcctcatata ttettettet atgttacctg aaaaccggca tttaatctcg cgggtttatt 360 ccggttcaac attttttttg ttttgagtta ttatctgggc ttaataaege aggcctgaaa 420 taaattcaag gcccaactgt tttttttttt aagaagttgc tgttaaaaaa aaaaaaaggg 480 aattaacaac aacaacaaaa aaagataaag aaaataataa caattaettt aattgtagac 540 taaaaaaaca tagattttat catgaaaaaa agagaaaaga aataaaaact tggatcaaaa 600 acagatette aaaaaaacat cttttcttaa taattattaa ctttttccca aaattaggtc 660 acaattaggt ttagagtttt ggaattaaac caaaaagatt gttctaaaaa atactcaaat 720 agtttcctta ttggtagata ttttaattag tcaatggtag atactttttt ttcttttctt 780 gattagaatc tattagagta agttttgata ttttatgcca aattaaatca agaagataaa 840 ctatcataat caacat gaaa ttaaaagaaa aatetcatat atagtattag tattetetat 900 atatattatg attgcttatt cttaatgggt tgggttaacc aagacatagt cttaatggaa 960 agaatctttt ttgaactttt tccttattga ttaaattctt ctatagaaaa gaaagaaatt 1020 atttgaggaa aagtatatac aaaaagaaaa atagaaaaat gtcagtgaag cagatgtaat 1080 atccaaccac ggatgaccta caceatagga tgtttctact tgagtcggtc ttttaaaaac 1140 aaa gcacggtgga atgaca cgtatcatat gattccttcc tttagtttcg tgataataat 1200 cctcaactga tatetteett tttttgtttt ggctaaagat attttattct cattaataga 1260 aaagacggtt ttgggctttt ggtttgcgat ataaagaaga ccttcgtgtg gaagataata 1320 attcatcctt tcgtcttttt ctgactcttc aa ctctccc aaagcctaaa gcgatctctg 1380 caaatctctc gcgactctct ctttcaaggt atattttctg attctttttg tttttgattc 1440 gtatctgatc tccaattttt gttatgtgga ttattgaatc ttttgtataa attgcttttg 1500 acaatattgt tcgtttcgtc aatccagctt ctaaattttg tcctgattac taagatatcg 1560 attcgtagtg tttacatctg tgtaatttct tgcttgattg tgaaattagg attttcaagg 1620 acgatctatt caatttttgt gttttctttg ttegattetc tetgttttag gtttettatg 1680 tttagatccg tttctctttg gtgttgtttt gatttctctt acggcttttg atttggtata 1740 ttggtttcta tgttcgctga cttgttctat tgttttattt caggtggggg atccaatggc 1800 cacgaattaa agetatcaeg tccaaaatga gaatgeccaa gagtaagggt gcaactgtac 1860 taaatttgga acacctactc gagtatgetc cacagcaaat tgaaatctca aatactcgag 1920 caactcaatc acagtttgat acatggtatg aagcagtaca acttgcatac gacataggag 1980 aaactgaaat gccaactgtg atgaatgggc ttatggtttg gtgeattgaa aatggaacct 2040 cgccaaatat caatggagtt tgggttatga tggatggaga tgaacaagtc gaatacccac 2100 tgaaaccaat cgttgagaat gcaaaaccaa aatcatggca cacttaggca catttctcag 2160 agegtatata atgttgcaga gaaatgegea acaaaaagga accatatatg ecaegatatg 2220 gtttagt cg taatctgcgc gatggaagtt tggctcgcta tgcttttgac ttttatgaag 2280 ttacatcacg tacaccagtg agggctagag aggcacacat tcaaatgaag gccgcagctt 2340 tcaatctcga taaaatcagc ctttt cggat tggatggtgg cattagtaca caagaggaaa 2400 acacagagag gcacaccacc gaggatgttt ctccaagtat gcatactcta cttggagtga 2460 agaacatgtg attgtagtgt ctttccggac gatatataga tatttatgtt tgcagtaagt 2520 attttggctt ttcctgtact acttttatcg taattaataa tcgtttgaat ccgcggccgc 2580 tctttctt c caaggtaata ggaactttct ggatctactt tatttgctgg atctcgatct 2640 atttccttga tgttttctca gatctggaat tcgtttaatt tggatctgtg aacctccact 2700 aaatcttttg gttttactag aatcgatcta agttgaccga tcagttagct cgattatagc 2760 taccagaatt tggcttgacc ttgatggaga gatccatgtt catgttacct gggaaatgat 2820 ttgtatatgt gaattgaaat ctgaactgtt gaagttagat tgaatctgaa cactgtcaat 2880 gttagattga atctgaacac tgtttaagtt agatgaagtt tgtgtataga ttcttcgaaa 2940 ctttaggatt tgtagtgtcg tacgttgaac agaaagctat ttctgattca atcagggttt 3000 atttgactgt attgaactct ttttgtgtgt ttgcagctca taaaaaatgg cgtcgaccgc 3060 ggattcaaac gattattaat tacgataaaa gtagtacagg aaaagecaaa ataettactg 3120 tateta caaacataaa ata tcgtccggaa atcacatgtt agacactaca cttcactcca 3180 agtagagtat gcatacttgg agaaacatec tcggtggtgt gcctctctgt gttttcctct 3240 tgtgtactaa tgccaccatc caatcegaaa agtegagatt gagctgattt taaagctgcg 3300 geetteattt gaatgtgtge ctctctagcc ctcactggtg taegtgatgt aaetteataa 3360 aagtcaaaag catagegage caaacttcca tcgcgcagat tacgaactaa accatategt 3420 ggcatatatg gttccttttt gttgcgcatt tetatataeg cttctgcaac atctgagaaa 3480 tgtgccatga tttgcctaag tgttggtttt geattetcaa cgattggttt cagtgggtat 3540 tcgacttgtt catctccatc catcataacc caaactccat tgatatttgg cgaggttcca 3600 ttttcaatgc accaaaccat aagcccattc atcacagttg gcatttcagt ttctcctatg 3660 tcgtatgcaa gttgtactgc ttcataccat gtatcaaact gtgattgagt tgctcgagta 3720 tttgagattt caatttgctg tggagcatac tcgagtaggt gttccaaatt tagtacagtt 3780 gcacccttac tcttgggcat tctcattttg gacgtgatag ctttaattcg tggccattgg 3840 atccatggtg atcactgcag atcgttcaaa catttggcaa taaagtttct taagattgaa 3900 tcctgttgcc ggtcttgcga tgattatcat ataatttctg ttgaattacg ttaagcatgt 3960 atgtaatgca aataattaac tgacgttatt tatgagatgg gtttttatga ttagagtccc 4020 gcaattatac atttaatacg cgatagaaaa caaaat atag cgcgcaacct aggataaatt 4080 gtgtcatcta atcgcgcgcg tgttactaga tetetagaaa gcttcgtacg ttaattaatt 4140 cgaatccgga gcggccgcag ggetageate gatggtaccg agetegagac tatacaggcc 4200 aaattegetc ttagccgtac aatattaetc accggtgcga tgccccccat cgtaggtgaa 4260 ggtggaaatt aatgatccat caggcccaca cttgagacca acagctacca gtttcctcaa 4320 gggtccacca aaaacgtaag cgcttacgta catggtcgat aagaaaaggc aatttgtaga 4380 tgttaacatc caacgtcgct ttcagggatc ccgaattcca agcttggaat tcgggatcct 4440 acaggccaaa ttegetetta gccgtacaat attactcacc ggtgcgatgc cccccatcgt 4500 aggtgaaggt ggaaattaat gatecatett gagaccacag gcccacaaca gctaccagtt 4560 tcctcaaggg tccaccaaaa acgtaagcgc ttacgtacat ggtcgataag aaaaggcaat 4620 ttgtagatgt taacatecaa cgtcgctttc agggatcccg aattecaage ttggaattcg 4680 ggatectaca ggccaaattc gctcttagcc gtacaatatt actcaccggt gcgatccccc 4740 gaaggtggaa catcgtaggt attaatgatc catcttgaga ccacaggccc acaacageta 4800 caagggtcca ccagtttcct ccaaaaacgt aagcgcttac gtacatggtc gataagaaaa 4860 ggcaatttgt agatgttaac atccaacgtc gctttcaggg to tecegaatt ccaagcttgg 4920 gctgcaggtc aatcccattg cttttgaagc agetcaacat tgatctcttt ctcgagggag 4980 tcagtgcgca atttttcaaa agacgtgacg taagtatccg agtcagtttt tatttttcta 5040 ctaatttggt cgtttatttc ggcgtgtagg acatggcaac cgggcctgaa tttcgcgggt 5100 attctgtttc tattecaact ttttcttgat ccgcagccat taacgacttt tgaatagata 5160 cgctgacacg ccaagcctcg ctagtcaaaa gtgtaccaaa caaegettta cageaagaac 5220 ggaatgcgcg tgacgctcgc ggtgacgcca tttcgccttt tcagaaatgg ataaatagee 5280 ttatatette ttgettecta ccaaattacc aatacattac actageatet gaatttcata 5340 accaatctcg atacaccaaa tcgagatctg cagggatccc cgatcatgca aaaactcatt 5400 aactcagtgc aaaactatgc ctggggcagc aaaacggcgt tgactgaact ttatggtatg 5460 gaaaatccgt ccagccagcc gatggccgag ctgtggatgg gcgcacatcc gaaaagcagt 5520 tcacgagtgc agaatgccgc cggagatatc gtttcactgc gtgatgtgat tgagagtgat 5580 aaatcgactc tgctcggaga ggccgttgcc aaacgctttg gcgaactgcc tttcctgttc 5640 aaagtattat gcgcagcaca gccactctcc attcaggttc atccaaacaa acacaattct 5700 ttgccaaaga gaaatcggtt aaatgccgca ggtatcccga tggatgccgc cgagc gtaac 5760 tataaagatc ctaaccacaa gccggagctg gtttttgcgc tgacgccttt ccttgcgatg 5820 aacgcgtttc gtgaattttc cgagattgtc tccctactcc agccggtcgc aggtgcacat 5880 ccggcgattg ctcacttttt acaacagcct gatgccgaac gtttaagcga actgttcgcc 5940 agcctgttga atatgcaggg tgaagaaaaa tcccgcgcgc tggcgatttt aaaatcggcc 6000 ctcgatagcc agcagggtga accgtggcaa acgattcgtt taatttctga attttacccg 6060 gaagacagcg gtctgttctc cccgctattg ctgaatgtgg tgaaattgaa ccctggcgaa 6120 gcgatgttcc tgttcgctga aacaccgcac gcttacctgc aaggcgtggc gctggaagtg 6180 atggcaaact ccgataacgt gctgcgtgcg ggtctgacgc ctaaatacat tgatattccg 6240 gaactggttg ccaatgtgaa attcgaagcc aaaccggcta accagttgtt gacccagccg 6300 gtgaaacaag gtgcagaact ggacttcccg attccagtgg atgattttgc cttctcgctg 6360 gtgataaaga catgacctta aaccaccatt agccagcaga gtgccgccat tttgttctgc 6420 gtcgaaggcg atgcaacgtt gtggaaaggt tctcagcagt tacagcttaa accgggtgaa 6480 ttgccgccaa tcagcgttta gtgactgtca cgaatcaccg aaggccacgg ccgtttagcg 6540 acaagctgta cgtgtttaca agagcttact gaaaaaatta acatctcttg ctaagctggg 6600 agctcgtcga cggatcgaat tcctgcagat cgttcaaaca tttggcaata aagtttctta 6660 agattgaatc ctgttgccgg tcttgcgatg attatcatat aatttctgtt gaattacgtt 6720 aagcatgtaa taattaacat gtaatgcatg acgttattta tgagatgggt ttttatgatt 6780 agagtcccgc aattatacat ttaatacgcg atagaaaaca aaatatagcg cgcaaactag 6840 gataaattat cgcgcgcggt gtcatctatg ttactagatc tctagaacta gtggatctgc 6900 tagccctgca ggaaatttac cggtgcccgg gcggccagca tggccgtatc cgcaatgtgt 6960 tattaagttg tctaagcgtc aatttgttta tatcctgcca caccacaata ccagccagcc 7020 aacagctccc cgaccggcag ctcggcacaa aatcaccact cgatacaggc agcccatcag 7080 aattaattct catgtttgac agcttatcat cgactgcacg gtgcaccaat gcttctggcg 7140 tcaggcagcc atcggaagct gtggtatggc tgtgcaggtc gtaaatcact gcataattcg 7200 tgtcgctcaa ggcgcactcc cgttctggat aatgtttttt gcgccgacat cataacggtt 7260 ctggcaaata ttctgaaatg agctgttgac aattaatcat cggctcgtat aatgtgtgga 7320 attgtgagcg gataacaatt tcacacagga aacagaccat gagggaagcg gtgatcgccg 7380 tcaactatca aagtatcgac gaggtagttg gcgtcatcga gcgccatctc gaaccgacgt 7440 t gctggccgt acatttgtac ggctccgcag tggatggcgg cctgaagcca cacagtgata 7500 ttgatttgct ggttacggtg accgtaaggc ttgatgaaac gctttgatca aacgcggcga 7560 acgacctttt ggaaacttcg gcttcccctg gagagagcga gattctccgc gctgtagaag 7620 tcaccattgt tgtgcacgac gacatcattc cgtggcgtta tccagctaag cgcgaactgc 7680 aatttggaga atggcagcgc aatgacattc ttgcaggtat cttcgagcca gccacgatcg 7740 acattgatct ggctatcttg ctgacaaaag caagagaaca tagcgttgcc ttggtaggtc 7800 cagcggcgga ggaactcttt gatccggttc ctgaacagga tctatttgag gcgctaaatg 7860 aaaccttaac gctatggaac tcgccgcccg actgggctgg cgatgagcga aatgtagtgc 7920 ttacgttgtc ccgcatttgg tacagcgcag taaccggcaa aatcgcgccg aaggatgtcg 7980 ctgccgactg ggcaatggag cgcctgccgg gcccgtcata cccagtatca cttgaagcta 8040 tcttggacaa ggcaggctta gaagatcgct tggcctcgcg cgcagatcag ttggaagaat 8100 ttgttcacta cgtgaaaggc gagatcacca aggtagtcgg caaataaagc tctagtggat 8160 ccccgaggaa tcggcgtgac ggtcgcaaac catccggccc ggtacaaatc ggcgcggcgc 8220 tgggtgatga cctggtggag aagttgaagg ccgcgcaggc cgcccagcgg caacgcatcg 8280 aggcaga agc acgccccggt gaatcgtggc aagcggccgc tgatcgaatc cgcaaagaat 8340 cccggcaacc gccggcagcc ggtgcgccgt cgattaggaa gccgcccaag ggcgacgagc 8400 aaccagattt tttcgttccg atgctctatg acgtgggcac ccgcgatagt cgcagcatca 8460 tggacgtggc cgttttccgt ctgtcgaagc gtgaccgacg agctggcgag gtgatccgct 8520 acgagcttcc agacgggcac gtagaggttt cagcagggcc ggccggcatg gccagtgtgt 8580 gggattacga cctggtactg atggcggttt cccatctaac cgaatccatg aaccga acc 8640 gggaagggaa gggagacaag cccggccgcg tgttccgtcc acacgttgcg gacgtactca 8700 agttctgccg gcgagccgat ggcggaaagc agaaagacga cctggtagaa acctgcattc 8760 ggttaaacac cacgcacgtt gccatgcagc gtacgaagaa ggccaagaac ggccgcctgg 8820 tgacggtatc cgagggtgaa gccttgatta gccgctacaa gatcgtaaag agcgaaaccg 8880 ggcggccgga gtacatcgag atcgagctag ctgattggat gtaccgcgag atcacagaag 8940 gcaagaaccc ggacgtgctg acggttcacc ccgattactt tttgatcgat cccggcatcg 9000 gccgttttct ctaccgcctg gcacgccgcg ccgcaggcaa ggcagaagcc agatggttgt 9060 tcaagacgat ctacgaacgc agtggcagcg ccggagagtt caagaagttc tgtttcaccg 9120 tgcgcaagct gatcg ggtca aatgacctgc cggagtacga tttgaaggag gaggcggggc 9180 aggctggccc gatcctagtc atgcgctacc gcaacctgat cgagggcgaa gcatccgccg 9240 gttcctaatg tacggagcag atgctagggc aaattgccct agcaggggaa aaaggtcgaa 9300 aaggtctctt tcctgtggat agcacgtaca ttgggaaccc aaagccgtac attgggaacc 9360 ggaacccgta cattgggaac ccaaagccgt acattgggaa ccggtcacac atgtaagtga 9420 agagaaaaaa ctgatataaa ggcgatt tt ccgcctaaaa ctctttaaaa cttattaaaa 9480 ctcttaaaac ccgcctggcc tgtgcataac tgtctggcca gcgcacagcc gaagagctgc 9540 aaaaagcgcc tacccttcgg tcgctgcgct ccctacgccc cgccgcttcg cgtcggccta 9600 tcgcggccgc tggccgctca aaaatggctg gcctacggcc aggcaatcta ccagggcgcg 9660 gccgtcgcca gacaagccgc ctcgaccgcc ggcgctgagg tctgcctcgt gaagaaggtg 9720 ttgctgactc ataccaggcc tgaatcgccc catcatccag ccagaaagtg agggagccac 9780 ggttgatgag agctttgttg taggtggacc agttggtgat tttgaacttt tgctttgcca 9840 cggaacggtc tgcgttgtcg ggaagatgcg tgatctgatc cttcaactca gcaaaagttc 9900 acaaagccgc gatttattca cgtcccgtca agtcagcgta atgctctgcc agtgttacaa 9960 ccaattaacc aattctgatt agaaaaactc atcgagcatc aaatgaaact gcaatttatt 10020 catatcagga ttatcaatac catatttttg aaaaagccgt ttctgtaatg aaggagaaaa 10080 ctcaccgagg cagttccata ggatggcaag atcctggtat cggtctgcga ttccgactcg 10140 atacaaccta tccaacatca ttaatttccc ctcgtcaaaa ataaggttat caagtgagaa 10200 gtgacgactg atcaccatga aatccggtga gaatggcaaa agctctgcat taatgaatcg 10260 gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg 10320 actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa 10380 cacagaatca tacggttatc ggggataacg caggaaagaa catgtgagca aaaggccagc 10440 aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc 10500 ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat 10560 aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc 10620 cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcaatgct 10680 cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg 10740 aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc 10800 cga cggtaagaca cttatcg ccactggcag cagccactgg taacaggatt agcagagcga 10860 cggtgctaca ggtatgtagg gagttcttga agtggtggcc taactacggc tacactagaa 10920 ggacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta 10980 gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc 11040 agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg 11100 acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga 11160 tcttcaccta gatccttttg atccggaatt aattcctgtg gttggcatgc acatacaaat 11220 ggacgaacgg ataaaccttt tcacgccctt ttaaatatcc gattattcta ataaacgctc 11280 ttttctctta ggtttacccg ccaatatatc ctgtcaaaca ctgatagttt aaactgaagg 11340 cgggaaacga caatctgatc atgagcggag aattaaggga gtcacgttat gacccccgcc 11400 gatgacgcgg gacaagccgt tttacgtttg gaactgacag aaccgcaacg ctgcaggaat 11 60t 11461

Claims (11)

CLAIMS Having described the foregoing invention, it is considered as a novelty, and therefore, the content of the following claims is claimed as property:

1 . A method for altering the expression of a viral genome, characterized in that it comprises introducing into a cell a first DNA sequence capable of expressing in this cell an RNA fragment in the sense of that viral genome, and a second DNA sequence capable of expressing in This cell is an anti-sense RNA fragment of said viral genome, wherein the sense RNA fragment and the anti-sense RNA fragment are capable of forming double-stranded RNA.

2 . Eli pétocfo of reivirriicaaón 1, characterized perqué haae that this cell is resistant or tolerant to viruses.

3 . The pétocb of reivirriiracijen 1, characterized perqué the cell is a plant cell. Four . It is characterized by the virus being selected from the group consisting of tospovi-rus, potyvirus, potexvirus, tobamovirus, luteovirus, cucumovirus, bromovirus, closteorvirus, tombusvirus, and furovirus. 5 . The method of i virdicaci? 1, characterize park the DNA sequence comprises a nucleotide sequence derived from a viral coat protein gene, a viral nucleocapsid protein gene, a viral replicase gene, or a viral movement protein gene. 6. The method of reaction 1, characterized because the first DNA sequence and the second DNA sequence are stably integrated into the genome of the cell. 7 The peptide of claim 1, characterized in that the first DNA sequence and the second DNA sequence are comprised in two separate DNA molecules. 8 The plectrum of Revision 1, characterized by introducing at least two pairs of the first and second DNA sequences into a cell. 9. The method of reivipdiradione 8, characterized in that each pair of DNA sequences encodes sense and anti-sense RNA fragments of different species or virus isolates. 10 The method of relying on the first, the first DNA sequence and the second DNA sequence are comprised in a DNA molecule. eleven . The method of re-livening 3Jraci-cr? 10, characterized in that the first DNA sequence and the second DNA sequence are comprised in the same DNA strand of that DNA molecule. 1 . The method of claim 11, characterized in that the sense RNA fragment and the antisense RNA fragment are comprised in an RNA molecule. 13 The method of re-signifying 12, characterized in that the 1 RNA molecule is capable of folding, in such a way that the RNA fragments comprised therein form a double-stranded region. 1 - The method of claim 1, characterized in that the expressed RNA molecule comprises the inverted repeat sequence of SEQ ID NO: 13, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID N0: 25 or SEQ ID NO: 28 fifteen . The method is claimed 24, characterized in that the expression of the RNA molecule is driven by a heterologous promoter, specific to the tissue, regulated by the development, constitutive, or inducible. 16 The method of the reliction, characterized by the expression of the RNA molecule, is driven by a ubiquitin promoter, such as the Ubi3 promoter of Arabidopsis. 17 The method of claiming 15, characterized perqué the expression of the RNA molecule is driven by the RoIC promoter of Agrobacterium. 18 The method of claim 12, characterized in that the DNA molecule comprises a linker between the DNA sequences encoding the sense RNA fragment and the anti-sense DNA fragments. 19 The method of claim 18, characterized in that the linker is defined by the nucleotide sequence of the leader intron of the Actin 2 gene of Arabidopsis. twenty . The method of reivipdication 18, characterized in that the linker is defined by a nucleotide sequence of the viral region flanking the sense or anti-sense RNA fragment. twenty-one . The method of claim 18, characterized in that the linker comprises a cassette for the expression of a functional gene, such as a selectable marker gene. 22 The method of claim 21, characterized in that the linker comprises regulatory sequences, such as introns processing signals. 2. 3 . ^ Method of the claim. 11, characterized in that the RNA fragment in sense and the anti-sense RNA fragment are comprised in two separate RNA molecules. 24 The method of claim 23, characterized in that the first DNA sequence and the second DNA sequence are operatively linked to a bidirectional promoter. 25 The method of claim 11, characterized in that the first DNA sequence and the second DNA sequence are comprised of complementary strands of the DNA molecule. 26 The method of claim 25, characterized in that the first DNA sequence is the DNA strand complementary to the second DNA sequence in said DNA molecule. 27 A DNA construct that alters the expression of a viral gaxma, characterized in that it catches a first sequence of DNA capable of expressing in a cell a fragment of RNA in the sense of the viral genome, and a second DNA sequence capable of expressing in the cell an anti-sense RNA fragment of that viral genome, wherein the RNA fragment in sense and anti-sense RNA fragment are able to form a double-stranded RNA molecule. 28 The DNA construct of claim 27, characterized by the fact that this AEN assay performs a first operand operably linked to the first DNA sequence, and a second promoter operably linked to the second DNA sequence. 29. The DNA construct of claim 27, characterized in that this epinephrine is effec- tionally functionally linked to the first DNA sequence and to the second DNA sequence. 30 A cell that shows an altered expression of A viral germ characterized by a first ACN sequence capable of expressing in this cell an RNA fragment in the sense of that viral genome, and a second DNA sequence able to express in this cell an anti-sense RNA fragment. of that viral genome, wherein the sense RNA fragment and the anti-sense RNA fragment are capable of forming double-stranded RNA. 31 A plant and its derived progeny, which shows an altered expression efe tn win viral, characterized because crrpprn nde would first DNA sequence able to express in the cell a fragment of RNA in the sense of that viral genome, and a second DNA sequence capable of expressing in the cell a fragment of Anti-sense RNA of said viral genome, wherein the sense RNA fragment and the anti-sense RNA fragment are capable of forming double-stranded RNA. 32 The plant efe claim 31, characterized in that this plant is resistant or tolerant to viruses. 33. Seeds derived from the plant of claim 31.