KR20010041943A - Genes encoding MLO proteins and conferring fungal resistance upon plants - Google Patents

Abstract

The present invention relates to genes encoding proteins that regulate plant resistance to fungal pathogens. The invention also relates to transformed plants that are resistant to fungal pathogens and methods of making plants that are resistant to fungal pathogens. The invention also relates to a method for isolating additional genes encoding additional proteins that modulate the plant's resistance to fungal pathogens.

Description

Genes encoding MLO proteins and conferring fungal resistance upon plants

The present invention relates to a nucleotide sequence encoding a protein that modulates a plant's resistance to fungal disease. The invention also relates to plants which are resistant to fungal diseases and to plants which are resistant to fungal diseases.

Fungal diseases cause about $ 9.1 billion in damage to crops in the United States each year and are caused by a wide variety of biologically diverse pathogens. Different methods are commonly used to control them. Resistance develops agriculturally important strains, providing varying levels of resistance to a narrow range of pathogen isolates or populations, or to a broader range of pathogen isolates or populations. However, this involves a long and labor-intensive process that introduces desirable properties into commercial plant stocks by genetic hybridization, and due to the risk of pests evolving to overcome natural plant tolerance, it is necessary to improve new resistance to commercial plant stocks. Requires constant effort Alternatively, fungal diseases were controlled by applying chemical fungicides. Such methods typically control efficiently, but allow for the development of resistant pathogens and adversely affect the environment. In addition, in certain crops such as barley and wheat, control of fungal pathogens by chemical fungicides is difficult or impractical. Recent techniques have made the mechanism of resistance partly understood, making the interaction between plants and their pathogens better understood at the molecular level. While much of this molecular characterization is performed in the model plant Arabidopsis, resistance mechanisms have begun to elucidate in economically important crops.

Powdery powdery mildew is a major disease affecting most plant species and has been widely studied. They are characterized by white to gray spots or patches on plant tissue, which correspond to fungal mycelium and occlusion follicles. Powdery powdery mildew is caused by several fungi of the genus Erysiphales. For example, Erysiphe graminis causes powdery powdery grains of cereals and grasses. While powdery white powder is difficult to control in most crops, barley plant stocks are available that are resistant to isolates of mostly known pathogens. Mutations at the Mlo position, alone, have been found to be involved in the resistance phenotype. The mechanism of mlo resistance has been found in part; At the site of contact with the pathogen, it entails the formation of enormous cell wall juxtapositions, called dendritic cells, which contain mainly carloose but contain carbohydrates, phenols and proteins. In mlo plants, cell wall juxtaposition provides resistance by preventing the invasion of pathogens.

Unfortunately, the only powerful way to control this powdery powder is limited to barley. In view of the problems caused by fungal diseases in agriculture, especially powdery powdery mildew, the need for new and effective methods for controlling this type of pathogen in other crops is not realized, which is economically important for farmers and the environment. It is also desired.

The present invention focuses on the need for novel disease control strategies by applying genetic engineering techniques. In particular, the present invention relates to control strategies for powdered white powder, preferably for distributed white powder in economically important crops. The present invention relates to an isolated DNA molecule encoding Mlo protein that confers plant resistance to fungal pathogens. In particular, the present invention relates to an Mlo protein comprising a conserved amino acid sequence first discovered by the inventors of the present invention, and to an isolated DNA molecule encoding such an Mlo protein. The invention also relates to a vector for expressing a DNA molecule of the invention in a plant. The invention also relates to a transformed plant comprising one of the DNA molecules of the invention. The present invention also relates to agricultural products having improved phytosanitary properties, including transformed plants that are resistant to fungal pathogens by expressing one of the DNA molecules of the invention. The invention also relates to modification of the expression of a protein encoded by an endogenous copy of a gene corresponding to one of the DNA molecules of the invention in a transformed plant or by endogenous copy of a gene corresponding to one of the DNA molecules of the invention. A method of making a plant resistant to fungal diseases by modifying the activity or stability of the encoded protein. Such transformed plants are preferably pathogens for infecting epithelial cells of surviving plants, more preferably the causative agent of fungi from the neck of Eripapales, preferably known as powdery powdery mildew, preferably powdery powdery mildew. It is resistant to fungi from the genus Erysipe, and more preferably the plants are resistant to erythrope gramminis. The present invention also relates to a method for encoding a protein having a function identical or similar to a DNA molecule of the present invention or for separating a DNA molecule encoding a conserved amino acid sequence provided herein. Thus, the present invention provides a novel and effective method for controlling fungal diseases in economically important crops, potentially reducing the amount of chemicals applied to the crop and reducing the risk of the emergence of pathogens resistant to the control agents. to provide.

Therefore, the present invention,

Provided is a DNA molecule, preferably a cDNA molecule, encoding a Mlo protein comprising at least one amino acid sequence identical or substantially similar to the amino acid sequence set forth in SEQ ID NO: 1 or 2, which confers resistance to a fungal pathogen to the plant. . In a preferred embodiment, the DNA molecule is preferably not derived from barley and is derived from a dicotyledonous or plant group consisting of wheat, corn, rice, oats, rye, sorghum, sorghum, millet, milo, and palmaceae. In a preferred embodiment, the DNA molecules of the invention are identical or substantially similar to one of the nucleotide sequences set forth in SEQ ID NO: 3, SEQ ID NO: 5 or SEQ ID NO: 7, or identical or substantially similar to the Mlo protein set forth in SEQ ID NO: 4, SEQ ID NO: 6 or SEQ ID NO: 8 Encode the Mlo protein. In a more preferred embodiment, a DNA molecule comprising the nucleotide sequence set forth in SEQ ID NO: 3, SEQ ID NO: 5 or SEQ ID NO: 7 is derived from wheat. In another preferred embodiment, a DNA molecule of the invention is the same as or substantially similar to one of the nucleotide sequences set forth in SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, or SEQ ID NO: 17, or SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16 Or an Mlo protein identical or substantially similar to the Mlo protein encoded by one of the nucleotide sequences set forth in SEQ ID NO: 18. In a more preferred embodiment, the DNA molecule comprising the nucleotide sequence set forth in SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, or SEQ ID NO: 17 is derived from Arabidopsis thaliana. In another preferred embodiment, the above-mentioned DNA molecules are modified such that the activity of the endogenous protein is lost. In one particular aspect of the invention, the amino acid sequence of the protein corresponding to said DNA modification is changed in accordance with one, all, or a combination thereof:

-Trp (163) to Arg

Frame transition after Pro (396)

Frame switching after Trp 160

Met (1) is Ile

Gly (227) to Asp

-Mep (1) goes to Val

Arg (11) to Trp

-Phe (183), Thr (184) deleted

Val (31) to Glu

-Ser (32) to Phe

Leu (271) to His

In a further preferred embodiment, the fungal pathogen preferably infects surviving epithelial cells, and more preferably the fungal pathogen is of the genus Erysipales, preferably of the genus Erysipe, also known as powdery powdery mildew. Derived from, more preferably the fungal pathogen is eryrecipe graminis.

In a further aspect, an isolated DNA molecule comprises a Mlo protein comprising at least one amino acid sequence that is identical or substantially similar to the amino acid sequence described in SEQ ID NO: 1, or SEQ ID NO: 2, to the isolated molecule as described above. Antisense to the DNA molecule encoding, i.e., the same or substantially similar to the cDNA molecule, in particular the DNA molecule set forth in SEQ ID NO: 3, 5, 7, 9, 11, 13, 15 or 17, and SEQ ID NO: 4, 6, 8, 10, Antisense for DNA molecules encoding Mlo proteins identical or substantially similar to the Mlo proteins described in 12, 14, 16 or 18.

The present invention further provides

A protein is provided comprising at least one amino acid sequence identical or substantially similar to the amino acid sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2, wherein the protein is an MLO protein and confers resistance to fungal pathogens to the plant. The protein is preferably not liberated from barley and is derived from a dicotyledonous or plant group consisting of wheat, corn, rice, oats, rye, sorghum, sugar cane, millet, mylo, and palm family. In a preferred embodiment, the protein of the invention is encoded by a nucleotide sequence identical or substantially similar to one of the nucleotide sequences set forth in SEQ ID NO: 3, SEQ ID NO: 5 or SEQ ID NO: 7, or with one of the Mlo proteins set forth in SEQ ID NO: 4, SEQ ID NO: 6 or SEQ ID NO: 8 Same or substantially similar. In a more preferred embodiment, the protein is derived from wheat. In another preferred embodiment, the protein of the invention is encoded by or is encoded by a nucleotide sequence identical to or substantially similar to one of the nucleotide sequences set forth in SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, or SEQ ID NO: 17; , Identical to or substantially similar to one of the Mlo proteins set forth in SEQ ID NO: 16 or SEQ ID NO: 18. In a more preferred embodiment, the protein is a. It is derived from Talina. In another preferred embodiment, the fungal pathogen preferably infects surviving epithelial cells, more preferably the fungal pathogen is from the genus Erysipales, preferably of the genus Erysipe, also known as powdery powdery mildew. Derived, more preferably, the fungal pathogen is eryrecipe graminis. In a further aspect, the invention also encompasses mutated or truncated forms of the protein encoded by one of the aforementioned DNA molecules.

The present invention further provides

Provided is an expression cassette comprising one of the aforementioned DNA molecules, eg, one of the cDNA molecules described above, operably linked to a termination signal and a promoter capable of expressing the DNA molecule in a plant. In a preferred embodiment, the expression cassettes of the invention are heterologous. In a further preferred embodiment, the promoter and the termination signal are eukaryotic. In a further preferred embodiment, the promoter and termination signal are heterogeneous with respect to the cryptographic region.

The present invention further provides

Provided are vectors comprising one of the expression cassettes described above. In a preferred embodiment, the vector is used for transformation of an expression cassette in a plant. In another preferred embodiment, the vectors of the present invention are used to amplify one of the DNA molecules described above.

The present invention further provides

Provided is a cell comprising an expression cassette or portion thereof comprising an isolated DNA molecule of the invention capable of expressing a DNA molecule in an expression cassette in a cell. In a preferred embodiment, the DNA molecule is not derived from barley. In another preferred embodiment, said cell is a plant cell. In a further preferred embodiment, the expression cassette is stably integrated in the genome of the cell or contained in a self-replicating vector and maintained in the cell as an extrachromosomal molecule.

The present invention further provides

Provided are plants comprising an expression cassette or portion thereof comprising an isolated DNA molecule of the invention. In a preferred embodiment, the DNA molecule is not derived from barley. In another preferred embodiment, the DNA molecules contained in the expression cassette can be expressed in a plant. In another preferred embodiment, the DNA molecule is stably integrated in the plant genome or included in a self-replicating vector and maintained in the cell as an extrachromosomal molecule. In another preferred embodiment, the plant is resistant to fungal pathogens, preferably fungal pathogens that infect surviving epithelial cells, more preferably the fungal pathogen is known as erythropoiesis. From the neck, preferably of the genus erycife and more preferably the fungal pathogen is eryrecipe graminis.

The present invention also relates to seeds for such plants, optionally treated (eg primed or coated), packaged, for example in bags with instructions for use.

The present invention further provides

Provided are agricultural products comprising plants comprising the isolated DNA molecules of the invention. In a preferred embodiment, the agricultural product is used, for example, as feed, food, or grasses and does not contain fungal toxin produced by fungal pathogens such as, for example, aflatoxin. Therefore, the agricultural product of the present invention has improved phytosanitary properties.

The present invention further provides

a) expressing in the plant an RNA transcript encoded by one of the DNA molecules described above in a "sense" orientation; or

b) expressing in the plant an RNA transcript encoded by one of the DNA molecules described above in an "antisense" orientation; or

c) expressing in the plant a ribozyme capable of specifically cleaving a messenger RNA transcript encoded by an endogenous gene corresponding to one of the above DNA molecules; or

d) expressing in the plant an aptamer specifically directed to an endogenous protein encoded by a gene corresponding to one of the above DNA molecules; or

e) expressing in the plant a mutated or truncated form of one of the above DNA molecules so that it can act as a dominant-negative mutant; or

f) modifying one or more chromosomal copies of a gene corresponding to one of the above DNA molecules by homologous recombination in a plant; or

g) a method of making a plant resistant to fungal pathogens comprising modifying one or more chromosomal copies of a regulatory factor of a gene corresponding to one of the above DNA molecules by homologous recombination in the plant.

The present invention further provides

Optionally obtained (eg primed or coated), packaged, for example, obtained by one of the aforementioned methods comprising seeds for such plants, contained in a bag with instructions for use, for example. Provide plants. In another preferred embodiment, the plant obtained is resistant to fungal pathogens, preferably fungal pathogens that infect surviving epithelial cells, more preferably the fungal pathogens are known as erythropoiesis. It is derived from the family Pace neck, preferably of the genus erycife and more preferably the fungal pathogen is erycife graminis.

The present invention further provides

Provided are agricultural products having improved phytosanitary properties obtained by any of the methods described above.

The present invention further provides

a) complementary to a degenerate oligonucleotide encoding at least 6 of the amino acids of SEQ ID NO: 1 and to a sequence encoding at least 6 of the amino acids of SEQ ID NO: 2 under conditions that the DNA extracted from the plant and the degenerate oligonucleotide hybridize Mixing a degenerate oligonucleotide with DNA extracted from a plant; And

b) amplifying a DNA fragment of said plant DNA comprising at its left and right ends a nucleotide sequence that can be annealed to said degenerate oligonucleotide in step a); And

c) obtaining a full length cDNA clone comprising the DNA fragment of step b).

The present invention further provides

Provided are methods for producing mutated copies of nucleotide sequences of the invention by "in vitro recombination" or "DNA shuffling". Mutant copies of the nucleotide sequences of the present invention are used to confer improved resistance to fungal pathogens. In a preferred embodiment, mutant copies of the nucleotide sequences of the invention are used to confer resistance to a wide range of pathogens. One such method is described below:

A method of mutating a DNA molecule according to the invention wherein the DNA molecule is cleaved into double stranded random fragments of the desired size,

a) adding to at least one single-chain or double-stranded oligonucleotide comprising a region homologous to the double-stranded template polynucleotide and a heterologous region to the resulting double-strand random fragment;

b) denaturing the resulting mixture of double stranded random fragments and oligonucleotides with single chain fragments;

c) incubating the polymerase with the resulting population of single-chain fragments and the polymerase to form mutated double-stranded polynucleotides under conditions where the single-chain fragments are annealed in the homologous region to form pairs of annealed fragments, wherein Homology regions are sufficient for a pair of members to prime replication of another member); And

d) repeating the second and third steps in two or more additional cycles, where the mixture produced in the second step of one additional cycle comprises the mutated double-stranded polynucleotides from the third step of the previous cycle; Forming the more mutated double-stranded polynucleotide.

Justice

An "isolated DNA molecule" is a nucleotide sequence that, by human hands, is separate from its natural environment and is therefore not a natural product. The isolated nucleotide sequence may be present in purified form or may be present in a non-natural environment such as, for example, a transformed host cell.

A “protein” as defined herein is a complete protein encoded by the corresponding nucleotide sequence or part of a protein encoded by a portion of the corresponding nucleotide sequence.

An "isolated protein" is a protein that is encoded by an isolated nucleotide sequence and therefore is not a natural product. An isolated protein may be present in purified form or may be present in a non-natural environment, such as, for example, a transformed host cell, wherein the protein is not normally expressed or in a homogeneous non-transformed host cell. It is expressed in different forms or in different amounts.

Plants that are "resistant to fungal pathogens" inhibit or limit the ability of fungal pathogens to proliferate on plants, thereby exhibiting fewer or less symptoms of fungal infections caused by fungi. As a result, plants grow better, yield higher and produce more seeds.

"Protein conferring resistance to fungal pathogens to plants" means that the protein is involved in the regulation of plant genetic pathways involved in the plant's resistance to fungal pathogens. The protein may be a positive modulator that enhances the plant's resistance to fungal pathogens, or may be a negative modulator that inhibits the plant's resistance to fungal pathogens. A particular example of a protein that confers resistance to fungal pathogens to plants is the Mlo protein.

"Mlo protein" refers to the group of proteins (Mlo protein) in the present invention that have substantially similar functions in the disease resistance pathway and also share some structural homology. The structural homology may be, for example, sharing one or more regions where members of the class are conserved.

In the broadest sense, as used herein for a nucleotide sequence, the term "substantially similar" refers to a nucleotide sequence corresponding to the reference nucleotide sequence, where the corresponding sequence has a substantially identical structure and the reference nucleotide sequence ( For example, only changes in amino acids that do not affect polypeptide function). Reasonably, substantially similar nucleotide sequences are those that encode a polypeptide encoded by a reference nucleotide sequence. The percent identity between the substantially similar nucleotide sequence and the reference nucleotide sequence is suitably 80% or more, more preferably 85% or more, preferably 90% or more, more preferably 95% or more, even more preferably 99% or more to be.

As used herein for a protein, the term "substantially similar" refers to a protein corresponding to a reference protein, such as an amino acid, that does not affect polypeptide function, having substantially the same structure and function as the reference protein. It means a protein with only changes. When used with respect to a protein or amino acid sequence, the percent identity between a substantially similar protein or amino acid sequence thereof and the reference protein or amino acid sequence thereof is suitably 80% or greater, more preferably 85% or greater, preferably 90% or greater, More preferably at least 95%, even more preferably at least 99%. Percent sequence identity is determined using a computer program based on a dynamic programming algorithm. Preferred computer programs within the scope of the present invention include a BLAST (Basic Local Alignment Search Tool) search program designed to search all available sequence databases, whether protein or DNA to be queried. A version of the search tool BLAST 2.0 (Gapped BLAST) is publicly available on the Internet (currently http://www.ncbi.nlm.nih.gov/BLAST/). This uses a heuristic algorithm that examines local alignments as opposed to generic alignments, and thus can detect relationships between sequences that share only discrete regions. The score assigned in the BLAST search has a well-defined statistical interpretation. The program is preferably performed by selecting a variable set for the error value.

The term "gene" refers to a coding sequence and related regulatory sequences, wherein the coding sequence is transcribed into RNA, such as mRNA, rRNA, tRNA, snRNA, sense RNA, or antisense RNA. Examples of regulatory sequences are promoter sequences, 5 'and 3' untranslated sequences and termination sequences. Additional factors that may be present are, for example, introns.

"Expression" refers to the transcription and / or translation of an endogenous or transgene in a plant. For antisense constructs, for example, expression may refer to antisense DNA only transcription.

As used herein, an "expression cassette" refers to a DNA sequence capable of directing the expression of a particular nucleotide sequence in a suitable host cell, including a promoter operably linked to the nucleotide sequence of interest operably linked to the termination signal. Means. It also includes sequences necessary for proper translation of nucleotide sequences. The coding region typically encodes the protein of interest but may also encode non-ready RNA that inhibits the expression of the functional RNA of interest, for example antisense RNA or specific genes in the sense or antisense direction, such as antisense RNA. An expression cassette comprising a nucleotide sequence of interest may be chimeric, meaning that one or more of its components are heterologous to one or more of the other components. The expression cassette may also be one that is natural but obtained in recombinant form useful for heterologous expression. Typically, however, the expression cassette is heterologous to the host, ie the specific DNA sequence of the expression cassette is not naturally present in the host cell and must be introduced by transformation during the progenitor of the host cell or host cell. The expression of the nucleotide sequence in the expression cassette may be under the control of an inducible promoter that initiates transcription only when the constitutive promoter or host cell is exposed to some specific external stimulus. In multicellular organisms such as plants, the promoter may also be specific for a particular tissue or organ or stage of development.

As used herein, “heterologous” means “of different natural or synthetic origin” or refers to a non-natural state. For example, when the host cell is transformed with nucleic acid sequences from other organisms, in particular from other species, the gene is heterologous to the offspring of the host cell and also of the host cell comprising the gene. The transforming nucleic acid may comprise a heterologous promoter, a heterologous coding sequence, or a heterologous termination sequence. Alternatively, the transforming nucleic acid may be completely heterologous or may include possible combinations of heterologous and endogenous nucleic acid sequences. Similarly, heterogeneity refers to the same natural, native cell type, or nucleotide sequence derived from and inserted into a cell type, but such nucleotide sequences may be non-natural, eg, different copy numbers, or control of different regulatory factors. Is under.

The term "promoter" refers to a DNA sequence that initiates the transcription of an associated DNA sequence. Promoter regions may also include factors that act as regulators of gene expression such as activating factors, enhancers and / or refreshers.

As used herein, “synthetic nucleotide sequence” means a nucleotide sequence that includes structural features not present in the native sequence. For example, synthetic sequences are mentioned that mimic the G + C content of dicotyledonous and / or monocotyledonous genes and very similarly to conventional codon distributions.

Regulatory DNA sequence means that the regulatory DNA sequence is "operably linked" or "associated with" the DNA sequence coding for the RNA or protein when two sequences are located so as to affect the expression of the coding DNA sequence. .

"Regulatory factor" refers to a sequence involved in conferring expression of a nucleotide sequence. Regulatory factors include promoters and termination signals operably linked to the nucleotide sequence of interest. They also typically encompass sequences necessary for proper translation of nucleotide sequences.

"Plant" refers to any plant or part of a plant and in particular seed plants of the developmental stage. Also included are cuts, cell or tissue cultures and seeds. The term "plant tissue" as used in connection with the present invention includes whole plants, plant organs, plant seeds, protoplasts, callus, cell cultures, and groups of plant cells organized into structural and / or functional units. This is not restrictive.

"Plant cell" refers to the structural and physiological units of a plant, including protoplasts and cell walls. Plant cells may be in the form of isolated single cells or cultured cells, or may be part of a unit organized in higher stages, such as, for example, plant tissue, or may be plant organs.

As used herein, “transformation” refers to the introduction of nucleic acid into a cell. In particular, it refers to the stable integration of DNA molecules into the genome of the organism of interest.

A "selective marker" is conferred by a gene whose expression in plant cells provides a selective advantage for the cell. The selective advantage possessed by cells transformed with the selectable marker gene may be due to their viability in the presence of negative selection agents such as antibiotics or herbicides, as compared to the growth of non-transformed cells. Compared to non-transformed cells, the selective benefits possessed by the transformed cells may also be due to their improved or new ability to use compounds added as nutrients, growth factors or energy sources. Selectable marker genes also refer to genes or gene combinations whose expression in plants gives both negative and positive selective advantages to cells.

A "selective marker" is conferred by a gene whose expression does not provide a selective advantage for the transformed cells, but which allows the cells transformed by their expression to be distinguished from non-transformed cells.

〈Simple description of sequence in sequence list〉

SEQ ID NO: 1 conserved amino acid sequence 1

SEQ ID NO: 2 conserved amino acid sequence 2

SEQ ID NO: 3 nucleotide sequence of wheat Mlo protein TrMlo1

SEQ NO: 4: protein sequence of TrMlo1

SEQ ID NO: 5 nucleotide sequence of the wheat Mlo protein TrMlo2.

SEQ ID NO: 6 protein sequence of TrMlo2.

SEQ ID NO: 7 nucleotide sequence of the wheat Mlo protein TrMlo3.

SEQ ID NO: 8 protein sequence of TrMlo3.

SEQ ID NO: 9 nucleotide sequence of the Arabidopsis Mlo protein CIB10259

SEQ NO: 10 protein sequence of CIB10259.

SEQ ID NO: 11 nucleotide sequence of the Arabidopsis Mlo protein CIB10295

SEQ ID NO: 12 protein sequence of CIB10295.

SEQ NO: 13: nucleotide sequence of the Arabidopsis Mlo protein CIB10296

SEQ ID NO: 14 protein sequence of CIB10296.

SEQ NO: 15 nucleotide sequence of Arabidopsis Mlo protein F19850

SEQ ID NO: 16: Protein sequence of F19850

SEQ NO: 17 nucleotide sequence of Arabidopsis Mlo protein U95973

SEQ ID NO: 18 protein sequence of U95973.

SEQ ID NO: 19 oligonucleotide MLO-1

SEQ ID NO: 20 oligonucleotide MLO-3

SEQ ID NO: 21 oligonucleotide MLO-5

SEQ ID NO: 22 oligonucleotide MLO-7

SEQ ID NO: 23 oligonucleotide MLO-10

SEQ ID NO: 24 oligonucleotide MLO-15

SEQ ID NO: 25 oligonucleotide MLO-26

SEQ ID NO: 26 oligonucleotide MLO-GSP1

SEQ ID NO: 27 oligonucleotide MLO-GSP2

SEQ ID NO: 28 oligonucleotide ST27

SEQ ID NO: 29 oligonucleotide N37544-1

SEQ ID NO: 30 oligonucleotide N37544-2

SEQ ID NO: 31 oligonucleotide T22146-1

SEQ ID NO: 32 oligonucleotide T22146-2

SEQ ID NO: 33 oligonucleotide H76041-1

SEQ ID NO: 34 oligonucleotide H76041-2

SEQ ID NO: 35 oligonucleotide SAS-1

SEQ ID NO: 36 oligonucleotide SAS-2

SEQ ID NO: 37 oligonucleotide SAS-3

SEQ ID NO: 38 oligonucleotide SAS-4

SEQ ID NO: 39 oligonucleotide SAS-5

SEQ ID NO: 40 oligonucleotide SAS-6

SEQ ID NO: 41 oligonucleotide SAS-7

SEQ ID NO: 42 oligonucleotide SAS-8

<submission>

Deposited material Accession number Deposit date pCIB 10259 NRRL B-21945 3/10/98 pCIB 10295 NRRL B-21946 3/10/98 pCIB 10296 NRRL B-21947 3/10/98 TrMlo1 NRRL B-21948 3/10/98 TrMlo1-5 NRRL B-21949 3/10/98 TrMlo2 NRRL B-21950 3/10/98 TrMlo2-5 NRRL B-21951 3/10/98 TrMlo3 NRRL B-21952 3/10/98 TrMlo3-5 NRRL B-21953 3/10/98

All deposits were deposited at the Northern Regional Research Center at 1604 Peoria Northern University Street 1815, Illinois, USA.

The present invention relates to DNA molecules encoding Mlo proteins that confer resistance to fungal pathogens on plants. The inventors of the present invention first identified the amino acid sequence conserved in the Mlo protein. The conserved amino acid sequence of the present invention comprises three Mlo proteins derived from wheat and a. It is conserved between three Mlo proteins derived from Tagliana. These amino acids are also conserved in two expected Arabidopsis Mlo proteins. The first conserved amino acid sequence, set forth in SEQ ID NO: 1, comprises 13 amino acids. The fourth amino acid in SEQ ID NO: 1 is one of L, V or I, its fifth amino acid is one of V or L and its seventh amino acid is F or L. The thirteenth amino acid in SEQ ID NO: 1 is not I and is preferably one of T, S or A. The second conserved amino acid sequence, set forth in SEQ ID NO: 2, comprises 14 amino acids. The first amino acid of SEQ ID NO: 2 is not M and is preferably one of I, V, S or G. Its third amino acid is one of F, L or V. Its sixth amino acid is Y or N, its seventh amino acid is A or V, its eighth amino acid is L or I, and its tenth amino acid is T or S. The present invention encompasses an isolated Mlo protein comprising one or more conserved amino acid sequences described above and an isolated DNA molecule encoding such an Mlo protein. The invention also encompasses an isolated Mlo protein comprising both the conserved sequences set forth in SEQ ID NO: 1 and SEQ ID NO: 2. In a preferred embodiment, the isolated DNA molecule encoding the Mlo protein of the invention is a cDNA molecule.

In a further embodiment, the DNA molecule encoding the Mlo protein comprising one or more conserved amino acid sequences is not derived from barley. In other embodiments, such DNA molecules are derived from dicotyledonous plants or wheat, corn, rice, oats, rye, sorghum, sugar cane, millet, milo, or palm family. In a preferred embodiment, the DNA molecules of the invention are identical or substantially similar to the DNA molecules set forth in SEQ ID NO: 3, 5 or 7 and SEQ ID NOs: 9, 11, 13, 15 or 17 or in SEQ ID NO: 4, 6 or 8 or SEQ ID NOs: 10, 12, It encodes an Mlo protein that is the same or substantially similar to one of the Mlo proteins described in 14, 16 or 18. The DNA molecule set forth in SEQ ID NO: 3, 5 or 7 is derived from wheat and encodes the Mlo protein set forth in SEQ ID NO: 4, 6 or 8, respectively. Separation of such DNA molecules is further described in Example 1. The DNA molecules set forth in SEQ ID NO: 9, 11, 13, 15 or 17 are derived from arabidopsis and encode the Mlo proteins set forth in SEQ ID NO: 10, 12, 14, 16 or 18, respectively. Separation of such DNA molecules is further described in Example 2. The DNA molecule of SEQ ID NO: 3 encoding wheat Mlo protein, designated TrMlo1, has been deposited as strains TrMlo1 and TrMlo1-5 under accession numbers NRRL B-21948 and NRRL B-21949, respectively. The DNA molecules of SEQ ID NO: 5 encoding wheat Mlo protein, designated TrMlo2, have been deposited as strains TrMlo2 and TrMlo2-5 under accession numbers NRRL B-21950 and NRRL B-21951, respectively. The DNA molecules of SEQ ID NO: 7 encoding wheat Mlo protein designated TrMlo3 have been deposited with strains TrMlo3 and TrMlo3-5 under accession numbers NRRL B-21952 and NRRL B-21953, respectively. TrMlo1 and TrMlo3 contain the full length cDNAs of the corresponding Mlo genes and also include portions of the corresponding 5 ′ and 3 ′ non-toxic regions. TrMlo2 is the longest cDNA clone of the corresponding gene recovered. It includes the entire coding region except for the first methionine (initiation codon) as deduced from the comparison with TrMlo1 and TrMlo3. TrMlo2 also includes a portion of the 3 'untranslated region of the corresponding gene.

The DNA molecule of SEQ ID NO: 9 encoding Arabidopsis Mlo protein designated CIB10259 has been deposited as strain pCIB10259 under accession number NRRL B-21945. The DNA molecule of SEQ ID NO: 11 encoding Arabidopsis Mlo protein named CIB10295 has been deposited as strain pCIB10295 under accession number NRRL B-21946. The DNA molecule of SEQ ID NO: 13 encoding arabidopsis Mlo protein designated CIB10296 has been deposited as strain pCIB10296 under accession number NRRL B-21947. CIB10259, CIB10295 and CIB10296 contain the full length cDNA of the corresponding Mlo gene and also include some of the corresponding 5 ′ and 3 ′ unread regions. Nucleotide sequences encoding F19850 and U95973, members of the Arabidopsis Mlo protein class, are obtained from Genbank. However, the predicted amino acid sequence for both clones has been determined and found to be inconsistent with the predicted amino acid sequence in the Genbank entry. It is therefore evident that the Mlo protein determined by the inventors of the present invention is novel. The newly predicted proteins both contain the conserved amino acid sequences set forth in SEQ ID NOs: 1 and 2 and thus are encompassed by the present invention as well as isolated cDNAs encoding them.

The Mlo protein encoded by the DNA molecule of the present invention is known as the fungal pathogen that infects the plant, the fungal pathogen that infects epithelial cells of a surviving plant, more reasonably known as powdery white powder (Agrios G. (1988). ) Plant Pathology, Third Edition, Academic Press Inc., in particular p271), confers resistance to fungal pathogens from the throat of Eripalace. Preferably, the Mlo protein encoded by the DNA molecule of the present invention confers resistance to the plant from the genus erythrope, and more preferably the fungal pathogen is erythrope graminis.

The invention also encompasses recombinant vectors comprising one of the DNA molecules of the invention. Among these vectors, such DNA molecules are preferably contained in an expression cassette containing a regulatory factor for expression of the DNA molecule in a host cell capable of expressing these DNA molecules. Such regulatory factors are typically promoters and termination signals and preferably also include factors that allow for efficient translation of the protein encoded by the DNA molecule of the invention. In a preferred embodiment, the expression cassette is heterologous. Such vectors are used to transform an expression cassette comprising one of the DNA molecules of the invention into a host cell. In a preferred embodiment, the expression cassette is stably integrated into the DNA of such a host cell. In another preferred embodiment, the expression cassette is included in a vector, which can be replicated in the host cell and remains as an extrachromosomal molecule in the host cell. In a further preferred embodiment, such extrachromosomal replication molecules are used to amplify the DNA molecules of the invention in host cells. In a preferred embodiment, such host cells are bacteria, in particular E. coli. It is a microorganism such as collie. In another preferred embodiment, the host cell is a eukaryotic cell such as, for example, a yeast cell, an insect cell, or a plant cell.

In a further embodiment, the DNA molecules of the invention are modified by incorporating random mutations by techniques known as in vitro recombination or DNA shuffling. Such techniques are described herein by reference: Stemmer et al., Nature 370: 389-391 (1994) and US Pat. No. 5,605,793. Millions of mutant copies of the nucleotide sequence are produced based on the original nucleotide sequence described herein and variants with improved properties, such as increased resistance to fungal pathogens or resistance to a broader range of pathogens, are recovered. The method encompasses forming a double stranded polynucleotide mutated from a double stranded polynucleotide template that has been cleaved into a double stranded random fragment of the desired size, comprising the nucleotide sequence of the present invention, and the resulting double stranded random fragment population. Adding to the at least one single-chain or double-stranded oligonucleotide comprising a region identical to the double-chain polynucleotide template and a region heterologous; Denaturing the resulting mixture of double stranded random fragments and oligonucleotides with single chain fragments; Incubating the resulting population of single-chain fragments with a polymerase to form mutated double-stranded polynucleotides under conditions in which the single-chain fragments form pairs of annealed fragments in the same region, wherein the same region is a pair of Sufficient for a member to prime another replica); And repeating the second and third steps for at least two additional cycles, wherein the mixture produced in the second step of the further cycle comprises the mutated double-stranded polynucleotide from the third step of the preceding cycle, further Cycles form additional mutated double-stranded polynucleotides). In a preferred embodiment, the single species concentration of the double stranded random fragment in the population of double stranded random fragments is less than 1% by weight of the total DNA weight. In a further preferred embodiment, the double-stranded polynucleotide template comprises at least about 100 polynucleotides. In another embodiment, the double stranded random fragment is about 5 bp to 5 kb in size. In a further aspect, the fourth step of the method includes repeating the second and third steps for at least ten cycles.

The invention also encompasses cells comprising the DNA molecules of the invention, wherein the DNA molecules are not in a natural cellular environment. In a preferred embodiment, such cells are plant cells. In another preferred embodiment, the DNA molecules of the present invention can be expressed in such cells and are included in expression cassettes that allow them to be expressed in such cells. In a preferred embodiment, the expression cassette is stably integrated into the DNA of such a host cell. In another preferred embodiment, the expression cassette is included in a vector that can be replicated in a cell and remains as an extrachromosomal molecule in the cell.

The present invention also encompasses plants comprising the plant cells described above. In a further aspect, the DNA molecules of the present invention may be expressed in said plants, and the transformed plants confer resistance to fungal pathogens by expression of one or a portion of the DNA molecules of the present invention in the transformed plants. do. In a preferred embodiment, the fungal pathogen infects viable epithelial cells, and more preferably the fungal pathogen is from the genus Erysipales, preferably of the genus Erysipe, also known as powdery powdery mildew. And more preferably said fungal pathogen is eryrecipe graminis. The present invention therefore also encompasses transformed plants that have become resistant to fungal pathogens by expression of one or a portion of the DNA molecules of the invention.

Plants transformed according to the invention may be monocots or dicotyledons, corn, wheat, barley, rye, sweet potatoes, soybeans, peas, chicory, lettuce, cabbage, cauliflower, broccoli, turnips, radish, spinach , Asparagus, onion, garlic, pepper, celery, squash, pumpkin, hemp, zucchini pumpkin, apple, pear, quince, melon, plum, cherry, peach, nectarine, apricot, strawberry, grape, raspberry, blackberry, pineapple , Avocado, Papaya, Mango, Banana, Soybean, Tomato, Sorghum, Sugar Cane, Beet, Sunflower, Rapeseed, Clover, Tobacco, Carrot, Cotton, Alfalfa, Rice, Potato, Eggplant, Cucumber, Arabilopesis Talina, And wood plants such as conifers and hardwoods, in particular corn, wheat, or sugar beet.

Once the desired nucleotide sequence is transformed into a particular plant species, it can be propagated to the species using conventional breeding techniques or transferred to other variants of the same species, especially commercial ones. For their expression in transformed plants, DNA molecules need to be modified and optimized. It is known in the art that all organisms have specific preferences for codon use, and the nucleotide sequences contained in the DNA molecules of the present invention can thereby be changed to match specific plant preferences while maintaining the encoded amino acids. . In addition, high expression in plants is best achieved from coding sequences having a GC content of at least 35%, preferably at least 45%. Nucleotide sequences with a low GC content can be poorly expressed due to the presence of ATTTA motifs that can destabilize the signal and AATAAA motifs that can lead to inappropriate polyadenylation. While preferred gene sequences can be appropriately expressed in both monocotyledonous and dicotyledonous species, the sequences can be modified to take account of specific codon preferences and as these preferences have been found to differ in GC content preference for monocotyledonous or dicotyledonous plants: Murray et al. Nucl. Acids Res. 17: 477-498 (1989). In addition, the nucleotide sequence is searched for the presence of an inappropriate splice site that causes signal interruption. All changes required to be made within the nucleotide sequence as described above are made using site directed mutations, known techniques of PCR, and the methods described in published patent applications EP 0 385 962, EP 0 359 472 and WO 93/07278. This is done using synthetic gene construction.

For efficient translational initiation, it is necessary to modify the sequence adjacent to the starting methionine. For example, they can be modified by including sequences known to be effective in plants. Yoshi presented a standard sequence suitable for plants (NAR 15: 6643-6653 (1987)) and Clontech presented additional standard sequence translation initiation factors (1993/1994 catalog, 210 if). These standard sequences are suitable for use with the nucleotide sequences of the invention. Of the constructs comprising the nucleotide sequence, the sequence up to and including ATG (with the second amino acid unmodified), or alternatively the nucleotide sequence up to and including GTC following ATG (transgenic genes) Together with the possibility of modification of the second amino acid of).

DNA molecules in the transformed plant are driven by promoters found to be functional in the plant. The choice of promoter varies according to the temporal and spatial requirements for expression and depending on the target species. For the protection of plants against the pathogens of the leaves, expression in the leaves is preferred; For protection of plants against Isaac pathogens, expression in inflorescences (eg, inflorescences, cones, corn genus, etc.) is preferred; For the protection of plants against root pathogens, expression in the roots is preferred; For protection of seedlings against soil-originating pathogens, expression in roots and / or seedlings is preferred. In many cases, however, protection against one or more plant pathogens is sought, and therefore expression in multiple tissues is justified. Numerous promoters from dicotyledons have been found to be operative in monocotyledonous plants and vice versa, but ideally the dicotyledonous promoter is selected for expression in dicotyledonous plants, and the monocotyledonous promoter is for expression in monocotyledonous plants Is selected. However, there is no limitation on the origin of the selected promoter; It is sufficient if they are functional in expressing DNA molecules in the cells of interest.

Preferred promoters that are expressed continuously are those derived from the Agrobacterium opin synthase gene, for example the nos promoter, or a dual promoter from the Agrobacterium Ti plasmid (Velten et al. (1984) EMBO J. 3: 2723-2730), or viral promoters operable in plants, such as the CaMV 35S and 19S promoters, and promoters from genes encoding actin or ubiquitin (Ni et al. (1995) Plant J. 7: 661-676 There is). The DNA molecules of the invention can also be expressed under the control of chemically regulated promoters. This allows proteins that confer fungal disease to be synthesized only when the crop plants are treated with induction chemicals. Preferred techniques for chemical induction of gene expression are described in detail in published patent application EP 0 332 104 and US Pat. No. 5,614,395. A preferred promoter for chemical induction is the tobacco PR-1a promoter.

A preferred category of promoter is one that is wound inducible. Numerous promoters are described that are expressed at the wound site and also at the site of phytopathogen infection. Ideally, such a promoter should be only active locally at the site of infection, in which the proteins controlling the fungal disease accumulate in the cells that require their synthesis and kill offending insect pests. Preferred promoters of this kind are those described in Stanford et al. Mol. Gen. Genet. 215: 200-208 (1989), Xu et al. Plant Molec. Biol. 22: 573-588 (1993), Logemann et al. Plant Cell 1: 151-158 (1989), Rohrmeier & Lehle, Plant Molec. Biol. 22: 783-792 (1993), Firek et al. Plant Molec. Biol. 22: 129-142 (1993), and Warner et al. Plant J. 3: 191-201 (1993).

Preferred tissue specific expression patterns include green tissue specific, root specific, stem specific, and flower specific. Suitable promoters for expression in green tissues include many that regulate genes involved in photosynthesis, many of which have been cloned from both monocots and dicots. Preferred promoters are corn PEPC promoters from the phosphoenol carboxylase gene (Hudspeth & Grula, Plant Molec. Biol. 12: 579-589 (1989)). Preferred promoters for root specific expression are de Framond [FEBS 290: 103-106 (1991); EP 0 452 269) and further preferred root-specific promoters are from the T-1 gene provided by the present invention. Preferred stem specific promoters are described in US Pat. No. 5,625,136 which directs the expression of the maize trpA gene.

Preferred embodiments of the invention are transformed plants which express the DNA molecule in a root-specific manner. A further preferred embodiment is a transformed plant which expresses the DNA molecule in a wound-induced or pathogen infection-induced manner.

In addition to the selection of suitable promoters, constructs for protein expression in plants require the attachment of a transcription terminator downstream of the heterologous nucleotide sequence. Several such terminators are available and are known in the art (eg, tm1 from CaMV, E9 from rbcS). Available terminators known to function in plants can be used in connection with the present invention.

Numerous other sequences can be incorporated into the expression cassettes for the DNA molecules of the invention. These include sequences found to enhance expression such as intron sequences (eg from Adh1 and bronze1) and viral leader sequences (eg from TMV, MCMV and AMV).

It may be desirable to target expression of DNA molecules with different cell localization in plants. In some cases, localization in the cytoplasm may be justified, while in other cases localization in some blast organs may be desirable. Subcellular localization of the transgene encoded enzyme can be performed using techniques known in the art. Typically, DNA that encodes a target peptide from a known organ-targeted gene product is engineered and fused upstream of the nucleotide sequence. Numerous such target sequences are known for chloroplasts and their function in heterologous construction is known.

Suitable vectors for plant transformation are described herein. For Agrobacterium-mediated transformation, a binary vector or a vector comprising one or more T-DNA border sequences is suitable and any vector for direct gene transfer is suitable and of interest. Linear DNA containing only offerings may be preferred. For direct gene transfer, transformation or co-transformation with a single DNA species can be used (Schocher et al. Biotechnology 4: 1093-1096 (1986)). For both direct gene transfer and Agrobacterium-mediated delivery, transformation is usually (but not required) antibiotics (ganamycin, hygromycin or metatrexate) or herbicides (glufosinate, glyphosate or proto Or a selectable marker capable of conferring selective benefit to the transformed cells, such as a phospho-mannose isomerase gene. However, the selection of selectable markers is not important for the present invention.

In another preferred embodiment, the DNA molecules of the invention are directly transformed into the chromatin genome. Chromosome transformation techniques are described in US Pat. Nos. 5,451,513, 5,545,817, and 5,545,818, PCT Patent Application WO95 / 16783, and McBride et al (1994) Proc. Natl. Acad. Sci. USA 91, 7301-7305. The basic technique of chloroplast transformation involves the area of the cloned chromatin DNA flanking selectable marker with the DNA molecule of interest into a suitable target tissue, for example, a biocarb or protoplast transformation (e.g. calcium chloride or PEG mediated transformation). To be introduced using 1 to 1.5 kb flanking regions, designated targeting sequences, facilitate homologous recombination with the chromatin genome to replace or modify specific regions of the Plastom. Initially, point mutations in the chloroplast 16S rRNA and rps12 genes that confer resistance to spectinomycin and / or streptomycin are used as selectable markers for transformation (Svab, Z., Hajdukiewicz, P., and Maliga, P. (1990) Proc. Natl. Acad. Sci. USA 87, 8526-8530; Staub, JM, and Maliga, P. (1992) Plant Cell 4, 39-45). This gives a homoplasmic transformant that is stable at approximately one frequency per bombardment on 100 target leaves. The presence of cloning sites between these markers allows constructing a chromatin targeting vector for introduction of foreign genes (Staub, J.M., and Maliga, P. (1993) EMBO J. 12, 601-606, incorporated by reference). A substantial increase in the frequency of transformation was with the bacterial aadA gene encoding spectinomycin-detoxification enzyme aminoglycoside-3'-adenyltransferase, a selectable marker of the recessive rRNA or r-protein antibiotic resistance gene. Obtained by replacement (Svab, Z., and Maliga, P. (1993) Proc. Natl. Acad. Sci. USA 90, 913-917). Previously, these markers have been used successfully for high-frequency transformation of the chromatin genome of green alga Chlamydomonas lyhardti (Goldschmidt-Clermont, M. (1991) Nucl. Acids Res. 19, 4083-4089). ). Other selectable markers useful for chromatographic transformation are known in the art and are encompassed within the scope of the present invention. Typically, approximately 15-20 cell division cycles are required after transformation to reach homoplasmid status. Chromosome expression, in which genes are inserted by homologous recombination into the thousands of copies of all circular chromosomal genomes present in each plant cell, facilitates 10% of the total soluble plant protein using numerous copy number benefits on the nuclear-expressed genes. Allow for expression levels that may be exceeded.

The invention also provides for agricultural products comprising transformed plants that are resistant to fungal pathogens by expression of one of the DNA molecules of the invention or resistant to fungal pathogens by one of the methods described herein. Comprehensive Since such plants are resistant to fungal pathogens, the growth of pathogens on these is inhibited. Therefore, such plants and agricultural products derived therefrom are less likely to contain fungal toxins, which are naturally produced by numerous fungal pathogens and can be very toxic to humans and animals. Therefore, such agricultural products have better phytosanitary properties. In a preferred embodiment, such agricultural products are used for feed, silage or food.

A further object of the present invention is to provide a method of making a plant resistant to fungal pathogens. The Mlo protein encoded by the DNA molecule of the present invention confers resistance to fungal pathogens in plants and it is a preferred object of the present invention to modify the expression of such proteins in their natural host environment. Such modification of expression, stability, or activity in plants of proteins encoded by the DNA molecules of the present invention increases the plant's resistance to fungal pathogens. In a preferred embodiment, the protein encoded by the DNA molecule of the present invention is a negative regulator of plant resistance to fungal pathogens, which inhibits the genetic pathway in plants involved in the plant's resistance to fungal pathogens. Thus, a preferred object of the present invention is to reduce the expression of Mlo protein encoded by a DNA molecule in a natural host environment or to reduce the stability or activity of such a protein in a natural host environment.

"Sense" suppression

In a preferred embodiment, reduced expression of the protein encoded by the DNA molecule of the invention is obtained by "sense inhibition" (eg, Jorgensen et al. (1996) Plant Mol. Biol. 31, 957-973). In this case, all or part of the DNA molecule of the present invention is included in the expression cassette, which is introduced into a host cell, preferably a plant cell, into which the DNA molecule can be expressed. The DNA molecule is inserted into the expression cassette in a "sense orientation", which means that the 5 'end of the DNA molecule is adjacent to the promoter in the expression cassette and a coding strand of the DNA molecule can be obtained. In a preferred embodiment, the DNA molecules are all translatable and the genetic information contained in some of the DNA molecules are all translated into proteins. In another preferred embodiment, the DNA molecules are partially translatable and short peptides are translated. In a preferred embodiment, this is accomplished by inserting at least one immature stop codon into the DNA molecule, which terminates the translation. In another more preferred embodiment, the DNA molecule is translated but no translation product is produced. This is typically accomplished by removing the start codon of the protein encoded by the DNA molecule, eg "ATG". In another preferred embodiment, an expression cassette comprising a DNA molecule or part thereof is stably integrated into the genome of a host cell. In another preferred embodiment, expression cassettes comprising DNA molecules or portions thereof are included in extrachromosomal replication molecules. In transformed plants containing one of the above expression cassettes, the expression of the gene corresponding to the DNA molecule contained in the expression cassette is reduced or abolished, thereby reducing or eliminating the level of the protein in the transformed plant. As a result, the transformed plant is resistant to fungal pathogens.

"Antisense" suppression

In another preferred embodiment, inhibition of expression of the protein encoded by the DNA molecule of the invention is obtained by "antisense" inhibition. All or part of the DNA molecule of the present invention is inserted into an expression cassette into which a DNA molecule is introduced into a host cell, preferably a plant cell, into which the DNA molecule can be expressed. The DNA molecule is inserted into the expression cassette in an "antisense orientation", which means that the 3 'end of the DNA molecule is adjacent to the promoter in the expression cassette and that non-coding strands of the DNA molecule can be transcribed. . In a preferred embodiment, the expression cassette comprising the DNA molecule or portion thereof is stably integrated into the genome of the host cell. In another preferred embodiment, expression cassettes comprising DNA molecules or portions thereof are included in extrachromosomal replication molecules. Several documents describing the method are cited for further explanation (Green, PJ et al., Ann. Rev. Biochem. 55: 569-597 (1986); van der Krol, AR et al, Antisense Nuc.Acids & Proteins, pp. 125-141 (1991); Abel, PP et al., Proc. Natl. Acad. Sci. USA 86: 6949-6952 (1989); Ecker, JR et al., Proc. Natl. Acad. Sci. USA 83: 5372-5376 (Aug. 1986)).

Homologous recombination

In another preferred embodiment, one or more genomic copies corresponding to the DNA molecules of the invention are modified by homologous recombination in the genome of the plant as further described in the following literature: Paszkowski et al., EMBO Journal 7: 4021 -26 (1988). This technique takes advantage of the properties of homologous sequences that recognize each other and exchange nucleotide sequences to each other by homologous recombination by processes known in the art. Homologous recombination may occur between the chromosomal copy of the nucleotide sequence in the cell and the inflow copy of the nucleotide sequence introduced into the cell by transformation. Thus specific modifications are precisely introduced into the chromosomal copy of the nucleotide sequence. In one embodiment, the regulatory factors of the genes encoding proteins of the invention are modified. Regulatory factors present are replaced by different regulatory factors to inhibit the expression of, or to mutate or eliminate, the expression of the protein. In other embodiments, the coding region of a protein is modified by mutation or by removing some of the coding sequence of the entire coding sequence. Expression of the mutated protein can also confer plants with increased resistance to fungal pathogens. In another preferred embodiment, a mutation is introduced in the chromosomal copy of the DNA molecule by transforming the cell with a chimeric oligonucleotide consisting of a contact stretch of RNA and DNA residues in a double helix structure with a double hairpin cap on the end. A further feature of the oligonucleotides is the presence of 2'-0-methylation at the RNA residue. RNA / DNA sequences are designed to align with the sequence of chromosomal copies of the DNA molecules of the invention and to contain the desired nucleotide change. This technique is further described in US Pat. No. 5,501,967.

Ribozyme

In a further embodiment, the RNA encoding the protein of the invention is cleaved with catalytic RNA, or ribozyme specific for such RNA. Ribozymes are expressed in transformed plants and reduce the amount of RNA encoding for the proteins of the invention in plant cells, thus slowing down the amount of protein accumulated in the cells and increasing plant resistance to fungal pathogens. Let's do it. This method is further described in US Pat. No. 4,987,071.

Dominant-negative mutants

In another preferred embodiment, the activity of the protein encoded by the nucleotide sequence of the invention is altered. This is achieved by the loss of the activity of endogenous proteins due to the expression of the dominant negative mutants of the protein in transformed plants.

Aptamers

In a further aspect, the activity of a protein encoded by the DNA molecule of the present invention is inhibited by the expression of a nucleic acid ligand in a transgenic plant, called aptamer, that specifically binds to the protein. Aftamers are preferentially obtained by the SELEX (Systematic Evolution of Ligands by Exponential Enrichment) method. In the SELEX method, a candidate mixture of single-stranded nucleic acids with regions of randomized sequences is contacted with the protein and nucleic acids with increased affinity for the target are separated from the rest of the candidate mixture. Amplified fractionated nucleic acids yield a ligand rich mixture. After several repetitions, nucleic acids with optimal affinity for the protein are obtained and used for expression in the transformed plant. The method is further described in US Pat. No. 5,270,163.

Isolation of Nucleotide Sequences Conserved Sequences

The conserved sequence contained in the Mlo protein encoded by the DNA molecule of the present invention is used for isolation of other DNA molecules encoding such sequences. In a preferred embodiment, a mixture of degenerate oligonucleotides containing at least one of the possible oligonucleotides encoding the sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2 is produced. A mixture of oligonucleotides encoding the sequence set forth in SEQ ID NO: 1 and oligonucleotides complementary to the sequence encoding the sequence set forth in SEQ ID NO: 2 is used for PCR amplification reactions with the selected template DNA. Mixtures of degenerate oligonucleotides are known in the art and the degree of degeneracy varies as needed. In a preferred embodiment, the template DNA is a complete DNA sample from a plant, wherein the DNA sample is obtained by methods known in the art. Amplified fragments generated from the above PCR reactions are isolated by methods known in the art and used to select cDNA libraries, as known in the art, or to isolate corresponding full length cDNAs using the RACE protocol. The method represents a novel and useful method of isolating new genes that confer plants with resistance to fungal pathogens.

The invention is further illustrated with reference to the following detailed examples. These examples are provided for illustrative purposes and are not limiting unless otherwise specified.

Example 1

Cloning and Sequencing of the Mlo Gene from Wheat

The Mlo gene from wheat is cloned using the reverse transcription-PCR method. RNA is prepared from the leaves of wheat cultivar UC703 and used in reverse transcription programs using the Stratagene RT-PCR kit. The resulting cDNA is used in a PCR reaction using the following primers:

MLO-26 5 'TTC CAG CAC CGG CAC AAG AA 3' (SEQ ID NO: 25)

MLO-10 5 'AAG AAC TGC CTG AAG AAG GC 3' (SEQ ID NO: 23)

MLO-7 5 'CAG AAA CTT GTC TCA TCC CTG G 3' (SEQ ID NO: 22)

MLO-5 5 'ACA GAG ACC ACC TCC TTG GAA 3' (SEQ ID NO: 21)

MLO-15 5 'CAC CAC CTT CAT GAT GCT CA 3' (SEQ ID NO: 24)

PCR is performed using the primer pairs specified below, and the reaction amplifies fragments of the specified size:

MLO-26 and MLO-10 503 bp

MLO-26 and MLO-7 1481 bp

MLO-5 and MLO-15 650 bp

The fragment is cloned into pCR2.1 or pCR2.1-TOPO (Invitrogen). Plasmid DNA is prepared from the transformants and DNA sequenced. Sequencing revealed that there are three different cDNAs that are very similar to each other. These wheat Mlo genes are named TrMlo1, TrMlo2 and TrMlo3.

Additional wheat Mlo clones are isolated by screening wheat cDNA libraries constructed in lambda-ZAPII vectors. To screen the library, a mass incision is performed to convert the library to a BlueScript-based collection of cDNA clones. They are grown separately in 80,000 independent clones and pools of plasmid DNA prepared. PCR reactions are performed on pooled DNAs using oligonucleotide primers MLO-5 and MLO-15 (see above). These pools produce bands with an expected size of 650 base pairs. Subsequently, fractionation is achieved by continuous incubation of bacterial clones at low density, then plasmid DNA is prepared in each step and PCR is performed for each subpool using primers MLO-5 and MLO-15. After several fractionation a single clone containing the insert with the Mlo sequence is isolated. Sequencing two of these clones reveals that they contain the same insert. It is identical to the sequence of the second clone with the sequence of two other clones, but with an insert with 40 additional bases at the 5 'end.

Cloning of the rest of the wheat Mlo is performed by random amplification of the cDNA ends (RACE). RACE reactions are performed using the Marathon cDNA Amplification Kit (Clontech) on wheat UC703 poly-A + RNA. Poly-A + RNA is prepared from whole wheat RNA using an oligo-dT cellulose column (Gibco BRL). Oligonucleotides used in the RACE reaction are as follows:

MLO-GSP1 5'TGG ACC TCT TCA TGT TCG ATC CCA TCT G3 '(SEQ ID NO: 26)

MLO-GSP2 5'CCT GAC GCT GTT CCA GAA TGC GTT TCA3 '(SEQ ID NO: 27)

Amplification using the primers MLO-GSP1 and 5 ′ adapter primers provided in the kit resulted in DNA fragments of approximately 1300 nucleotides. Amplification with primers MLO-GSP2 and 3 ′ adapters result in DNA fragments of approximately 600 nucleotides. The fragment was cloned into pCR2.1-TOPO and named TrMlo1-5, TrMlo2-5 and TrMlo3-5, which comprise the 5 'ends of the wheat Mlo genes TrMlo1, TrMlo2 and TrMlo3, respectively. Plasmid DNA is prepared from the clones and plasmid inserts are sequenced.

Example 2

Cloning of Mlo cDNA from Arabidopsis

The program TBLASTN was used to compare database entry and Mlo protein sequences to find the number of entries with similarities to Mlo. Of these, full-length cDNAs corresponding to three Arabidopsis EST entries were cloned and assigned the access numbers H76041, N37544, and T22146. For each EST, oligonucleotides are designed to amplify the sequences corresponding to the EST from the arabidopsis cDNA library constructed in plasmid pFL61 (Minet et al. (1992) Gene Nov 16; 121 (2): 393 -396). Oligonucleotides used are as follows:

N37544-1 5'AAG ATC AAG ATG AGG ACG TGG AAG TCG TGG 3 '(SEQ ID NO: 29)

N37544-1 5'AGG CTG AAC CAC TGG GGC GCC TCT CAC CAC 3 '(SEQ ID NO: 30)

T22146-1 5'CAA GTA TAT GAT GCG CGC TCT AGA GGA TGA 3 '(SEQ ID NO: 31)

T22146-2 5'AGG TTT CAC CAC TAA GTC TCC TTC AAT GGC 3 '(SEQ ID NO: 32)

H76041-1 5 'GAT CAT TCA AGA CTT AGG CTC ACT CAT GAG 3' (SEQ ID NO: 33)

H76041-2 5 'AAC AGC AAG GAA GAT TAC AAA TGA TGC CCA 3' (SEQ ID NO: 34)

Amplify -500 base pair fragments from DNA prepared from the cDNA library with primers N37544-1 and N37544-2, and -250 base pair fragments with primers T22146-1 and T22146-2. Amplify two fragments of approximately 350 and approximately 300 base pairs with primers H76041-1 and H76041-2. Approximately 300 base fragments are of the size expected from the EST sequence and then used to diagnose the presence of cDNA corresponding to H76041 EST in the cDNA library. DNA from the library. Coli is transformed and the clones consist of approximately 20,000 clones each. DNA from each pool is screened by PCR using different primer pairs, and then the positive pools are fractionated into smaller numbers of clones. Isolation of each positive clone is completed through this process, or in some cases by colony hybridization using the EST sequence as a probe once the pool size reaches about 200 clones. For ESTs N37544 and T22146, clones corresponding to EST were successfully isolated and the insert was sequenced. The cDNA corresponding to EST N37544 is designated CIB10295 in plasmid pCIB10295 and the plasmid containing cDNA corresponding to EST T22146 (CIB10296) is designated pCIB10296. For these two ESTs, the corresponding genomic sequences are available in some form of Arabidopsis BAC clone sequence recently deposited with Genebank. Clearly, as measured by GenBank, the protein sequences predicted to be translated from these genomic sequences do not correspond to the sequences measured by direct sequencing of the cDNAs. Therefore, the amino acid sequences of the genes corresponding to ESTs N37544 and T22146 are clearly distinguished from GenBank entries and are only revealed through cloning and sequencing of the cDNA clones. Clones isolated using primers for H76041 EST did not contain the gene for H76041, but were found to contain members of the new Mlo gene family as inserts. The insert was fully sequenced and members of the Mlo gene family were designated CIB10259 in plasmid pCIB10259.

Example 3

Construction of vector for expression of Mlo gene in wheat

Two vectors for "antisense" expression of barley Mlo gene are constructed in wheat. PCR is performed using barley cDNA and primer pairs MLO-5 and MLO-7 (see (1) above), and the result of the reaction is amplified 1124 bp fragment, which is cloned into pGEM-T (Promega). The fragment is cleaved from pGEM-T using the enzymes SacII and NotI. The 1124 base pair fragment is cloned into pBlueScrib-SK (+). The insert is now cleaved into BamHI and SacI restriction sites and cloned into a BamHI-SacI-digested pCIB9806 (patent application 08 / 838,219) in the opposite orientation to the maize ubiquitin promoter. The plasmid is designated pCK01. To construct vectors for "antisense" expression of the entire Mlo gene in wheat, primer pairs MLO-1 (5 'ATG TCG GAC AAA AAA GGG GT 3' (SEQ ID NO: 19) and MLO-10 (see (1) above)) were used. PCR was performed, and the reaction amplified the 635 bp fragment which was cloned into pCR2.1 (Invitrogen) The fragment was cut from pCR2.1 into an EcoRI fragment and inserted into pGEM-9Zf (−) (Promega). Cleavage of 320 nucleotide fragments from SacI site to MLO-10 site in Mlo with SacI and BstXI digest pCK01 with SacI and BstXI and insert 320 base fragments Mlo in monocot plant expression vector To complete the construction of the gene, 210 nucleotide SacI fragments are cleaved from the pGEM-9Zf (-) derivative, which cuts the 5 'end of the Mlo coding sequence from the primer site MLO-1 to the native SacI site in the Mlo gene. PCK01 oil The sieve is digested with SacI and 210 base fragments are inserted .. For orientation of the 210 base fragments in the newly-built vector, clones are analyzed by PCR using primers MLO-1 and MLO-10. Only clones whose fragments were inserted in antisense orientation with respect to the ubiquitin promoter produced 530 base pair products corresponding to the 5 'end of the Mlo coding sequence The resulting plasmid was the entire Mlo coding sequence in an "antisense" culture for the ubiquitin promoter. And designated pCK02.

To construct a vector for expression of the Mlo gene in a "sense" orientation, plasmid pCK02 is digested with BamHI to separate the Mlo coding sequence as an insert. Relink the BamHI fragment to the pCK02 base vector. Colonies with Mlo coding sequences in reverse orientation for pCK02 were identified by SacI digestion, resulting in 1.8 kb fragments of such clones, as opposed to 210 base fragments in clones of the same configuration as pcK02. Clones with Mlo coding sequence in the "sense" orientation for the corn ubiquitin promoter are selected and designated pCK03.

Example 4

Construction of a Vector for Expression of Mlo Gene in Arabidopsis

In combination with pCK02 (for the Barley Mlo gene), Mlo clones of pCIB10259, pCIB10295 and pCIB10296 are used in PCR reactions to generate bands with full length gene sequences flanked by BamHI restriction sites. The sequence of primers used is as follows:

SAS-1: 5'GGA TTA AGA TCT AAT GGC 3 '(SEQ ID NO: 35 for pCIB10259)

SAS-2: 5'CAA AGA TCT TCA TTT CTT AAA AG 3 '(SEQ ID NO: 36 for pCIB10295)

SAS-3: 5'GCG GAT CCA TGT CGG ACA AAA AAG G 3 '(SEQ ID NO: 37 for Barley Mlo)

SAS-4: 5'TGCG GAT CCT CAT CCC TGG CTG AAG G 3 '(SEQ ID NO: 38 for Barley Mlo)

SAS-5: 5'GGA TCC ACC ATG GCC ACA AGA TG 3 '(SEQ ID NO: 39 for pCIB10259)

SAS-6: 5'GGA TCC TTA GTC AAT ATC ATT AGC 3 '(SEQ ID NO: 40 for pCIB10259)

SAS-7: 5'GCG GAT CCA TGG GTC ACG GAG GAG AAG 3 '(SEQ ID NO: 41 for pCIB10269)

SAS-8: 5'GCG GAT CCT CAG TTG TTA TGA TCA GGA 3 '(SEQ ID NO: 42 for pCIB10296)

The band was cloned into pCR2.1-TOPO and the insert was sequenced from the resulting plasmid to confirm that there were no mutations introduced by PCR. The plasmid was digested with BamHI and the insert was purified to produce BamHI-degraded pPEH28 (Norris et al. (1993) Plant Molecular, a shuttle vector containing a copy of the Arabidopsis ubiquitin gene promoter UBQ3 downstream of the BamHI site. Biology 21: 895-906). Clones containing Mlo sequences fused to UBQ3 are identified, and restriction analysis is performed to identify clones with inserts in the "sense" and "antisense" orientations for UBQ3. For each Mlo gene, clones with inserts in the "sense" orientation and clones with the inserts in the "antisense" orientation are digested with XbaI and the insert is purified and cloned into XbaI-digested pCIB200. This positions the UBQ3-Mlo gene fusion between T-DNA boundaries.

Example 5

Wheat Transformation and Expression Identification

The wheat is transformed by particle foaming of immature embryos as described in detail in patent application WO 94/13822. Seedlings are resuscitated on Basta containing medium and analyzed by PCR. To diagnose the presence of the Mlo transgene by PCR, the following primers are used:

MLO-3: 5 'ATG CTA CCA CAC GCA GAT CG 3'

ST27: 5 'ACT TCT GCA GGT CGA CTC TA 3'

Primer MLO-3 corresponds to the region of the Mlo transgene, while primer ST27 lies in the corn ubiquitin promoter sequence. Both Mlo gene and ubiquitin promoter primers are used during PCR to eliminate error positives resulting from the use of two Mlo primers, which can be primed from chromosomal copies of the Mlo gene present in wheat. Plants identified as containing Mlo transgenes are analyzed by RNA gel blot to determine if they contain modified levels of chromosomal-encoded wheat Mlo mRNAs. Poly-A + RNAs are prepared from each transformed plant line and blotted onto Hybond-N + filters. The blot is probed with 530 base fragments corresponding to the 5 'end of the Mlo gene. This region was absent from the pCK01 clone; Thus, no hybridization to the antisense RNA expressed from the transgene in the transformed plant strain containing pCK01 occurs. For pCK02 transformed plant strains where the transgene contains the 5 'terminal fragment, the probe is hybridized to two bands of different sizes. The ˜2.5 kb mRNA corresponding to the antisense transgene is distinguished from the 2.0 kb mRNA derived from the wheat chromosome-encoded Mlo gene. Multiple 2.0 kb mRNAs are monitored as a measure of gene suppression efficiency achieved by each plant strain.

Example 6

Disease Testing of Transformed Wheat Plants

Transformed and untransformed UC703 (control) wheat plant stocks are grown in greenhouses until they are two weeks old. The plants were transferred to a Percival growth chamber (cycles of 8 hours dark, 16 ° C., and 16 hours clear room, 20 ° C.), and eryrecipe graminis f species. Inoculate with a sufficient amount of spores with Tritici. The degree of sporulation of fungi is assessed 2 weeks after inoculation. Evaluate plants as 1 (little or no mycelial growth, no visible spore formation), 2 (some mycelium growth and sporulation, but less than control plants) or 3 (hyphae growth and sporulation comparable to control) do. Transformed wheat plant lines expressing the Mlo- construct show increased resistance to pathogens. The results obtained with the antisense barley Mlo constructs are shown below.

Screening of siblings from plant lines R1 and R2 transformed for disease resistance

Clan members (T2 seeds) of Mlo antisense transformants were transplanted to E. coli. Inoculate with graminis and assess for disease resistance.

R1 R2 UC703 (Control) Transplanted seeds 24 24 24 Germinated 14 24 21 Disease score 1 4 2 0 Disease score 2 0 0 0 Disease score 3 10 22 21

The small percentage of R1 and R2 plants that are resistant may be due to the fact that a still dividing T2 population for the transgene is tested.

Example 7

Analysis of Arabidopsis strains expressing the Mlo gene

A derivative of pCIB200 containing the Mlo gene is used to transform Arabidopsis ecotype Ws-O by vacuum infiltration (Bechtold, N., Ellis, J. and Pelletier, G. (1993) CR Acad. Sci. Paris 316, 1194-1199). Transformants are identified by screening offspring by kanamycin screening. For Mlo transformed plant strains, plants expressing Mlo are identified by RNA gel blot analysis. For the Mlo gene, transformants are analyzed for changes in steady-state levels of mRNA accumulation by RNA gel blot analysis. Transformants exhibiting either the sense or antisense inhibition of the target genes were identified as the phytopathogenic fungi Erysiphe cichoracearum and Peronospora parasitica, and the bacterial pathogen Pseudomonas syringae pv. . Assay for changes in response to tomatoes. The leaves of the transformed plants are examined both macroscopically and microscopically using trypan blue staining to test for the presence of necrosis.

For erycife inoculation, spores are applied abundantly to Arabidopsis rosete and the plants are kept at 25 ° C. in a Percival growth chamber. The degree of fungal sporulation is assessed 10 days after inoculation.

Example 8

Use of similar regions between Mlo sequences to isolate additional Mlo gene family members

The alignment of amino acid sequences expected to be encoded by the Mlo gene revealed numerous short regions with high amino similarity between all gene products. Degenerate primers are designed for these regions and PCR reactions with these primers are performed according to PCR reagent supplier recommendations. The amplified fragment is used as a probe to isolate the full length cDNA or genomic clone of the new Mlo gene. The amino acid sequences conserved between the Mlo protein (bold) of the invention and the degenerate oligonucleotides used for the isolation of additional Mlo genes are shown below:

Example 9

Modification of coding sequences and contiguous sequences

The DNA molecules described herein can be modified for expression in a transformed plant host to perform and optimize or down-regulate their expression. The following problems may be encountered and modifications of these DNA molecules can be performed using techniques known in the art.

(1) codon use

Preferred codon usage for some plants differs from the preferred codon usage for some other plant species. Typically, plant evolution tends to strongly favor nucleotides C and G at the third base position of the monocotyledonous plant, while dinucleotides often use nucleotides A or T at this position. By modifying the gene to use the desired codons for the particular target transformed species, numerous problems described below for GC / AT content and inadequate splicing can be overcome.

(2) GC / AT content

Plant genes typically have a GC content of at least 35%. DNA molecules rich in A and T nucleotides can cause several problems in plants. First, the motif of ATTTA is believed to cause message destabilization and is found at the 3 'end of numerous short-lived mRNAs. Second, the presence of polyadenylation signals such as AATAAA at inappropriate locations in the message is believed to cause immature cleavage of transcription. Monocots can also recognize AT-rich sequences as splice sequences (see below).

(3) sequences adjacent to the starting methionine

Ribosomes are believed to examine the first available ATG that attaches to the 5 'end of messenger RNA to initiate translation. Nevertheless, there is a preference for certain nucleotides adjacent to ATG and it is believed that the expression of the DNA molecules of the invention can be improved by including new conserved sequence translation initiators in ATG. Clontech (1993/1994 catalog, p. 210) shows that E. Sequences as conserved nucleotide translational initiators for the expression of the Coli uidA gene are shown. In addition, Joshi (NAR 15: 6643-6653 (1987)) compared the numerous plant sequences adjacent to ATG and suggested conserved base sequences. If difficulties are encountered in the expression of DNA molecules in plants, inclusion of one of these sequences in the starting ATG can improve translation. In such cases the last three nucleotides of the conserved sequence may not be suitable for inclusion in the modified sequence due to modification of the second AA residue. Preferred sequences adjacent to the starting methionine may also differ between different plant species. Fourteen corn genes in the GenBank database were examined to obtain the following results:

Location of initiation ATG ahead of 14 maize genes

-10 -9 -8 -7 -6 -5 -4 -3 -2 -1

C 3 8 4 6 2 5 6 0 10 7

T 3 0 3 4 3 2 1 1 1 0

A 2 3 1 4 3 2 3 7 2 3

G 6 3 6 0 6 5 4 6 1 5

This analysis can be performed for the desired plant species into which the nucleotide sequence is incorporated and the sequence adjacent to the ATG can be modified to incorporate the desired nucleotide.

(4) Removal of nonconforming splice sites

The DNA molecules of the present invention may also be recognized as 5 'or 3' splice sites in plants and may contain motifs that can be degraded, resulting in interrupted or deleted messages. These sites can be removed using techniques known in the art.

(5) generation of dominant-negative mutants

In addition, the DNA molecules of the present invention may also include molecules modified in such a way that the activity of the protein encoded by the nucleotide sequence of the present invention is changed. This is accomplished by expressing the dominant negative mutant of the protein in the transformed plant, thereby losing the activity of the endogenous protein. The positions of the mutations in the Mlo nucleotide sequence that give rise to such dominant-negative mutations are listed below. The single mutations or combinations of different mutations listed below can be introduced.

Location of Mutations in Wheat Mlo Gene (nt Number) Mutation number Description of the mutation (Aa) Location of mutation in wheat Mlo protein of SEQ ID NOs: 4, 6 and 8 TrMlo1 SEQ ID NO: 3 TrMlo2 Sequence 5 TrMlo3 SEQ ID NO: 7 One T is A Trp (163) goes to Arg 662 487 684 2 fruition Frame transition after Pro 306 1366-1367 1191-1192 1388-1389 3 fruition Frame Switching After Trp (160) 656-666 481-491 678-688 4 G is A Met (1) is Ile 178 3 200 5 G is A Gly (227) to Asp 855 680 877 6 A is G Met (1) goes to Val 176 One 198 7 A is T Arg (11) to Trp 206 31 228 8 fruition Phe (183), Thr (184) Deletion 721-726 546-551 743-748 9 T is A Val (31) to Glu 267 92 289 10 C is T Ser (32) to Phe 270 95 292 11 T is A Leu (271) is His 987 812 1009

Techniques for modifying coding sequences and contiguous sequences are known in the art. If the initial expression of the DNA molecule of the present invention is low and it is considered appropriate to modify the sequence as described above, synthetic genes can be constructed according to methods known in the art. For example, they are described in published patent applications EP 0 385 962, EP 0 359 472 and WO 93/07278. In most cases it is desirable to assay expression of the gene constructs using a temporary assay protocol (which is known in the art) prior to delivering them to the transformed plants.

Example 10

Construction of Plant Transformation Vectors

Numerous transformation vectors are available for plant transformation, and the molecules of the invention can be used in conjunction with such vectors. The choice of vector for use depends on the desired transformation technique and the type of target for transformation. For certain target species, different antibiotic or herbicide selection markers may be preferred. Selection markers commonly used for transformation include nptII genes that confer resistance to kanamycin, paromomycin, geneticin and related antibiotics (Vieira and Messing, 1982, Gene 19: 259-268; Bevan et al. , 1983, Nature 304: 184-187), a bacterial aadA gene (Goldschmidt-Clermont, 1991, Nucl.Acids Res) that encodes an aminoglycoside 3′-adenylyltransferase and confers resistance to streptomycin or spectinomycin. 19: 4083-4089), hph gene conferring resistance to antibiotic hygromycin (Blochlinger and Diggelmann, 1984, Mol. Cell. Biol. 4: 2929-2931), and dhfr gene conferring resistance to methotrexate. (Bourouis and Jarry, 1983, EMBO J. 2: 1099-1104). Other markers that may be used include the phosphinothricin acetyltransferase gene (White et al., 1990, Nucl. Acids Res. 18: 1062; Spencer et al. 1990, Theor, which confers resistance to the herbicide phosphinothricin). Appl. Genet. 79: 625-631), mutant EPSP synthase genes encoding glyphosate resistance (Hinchee et al., 1988, Bio / Technology 6: 915-922), imidazolion or sulfonylureas Mutant acetolactate synthase (ALS) gene to confer resistance (Lee et al., 1988, EMBO J.7: 1241-1248), and mutant psbA gene to confer resistance to atrazine (Smeda et al., 1993, Plant Physiol. 103: 911-917), or mutant protoporpynogen oxidase genes as described in US Pat. No. 5,767,373. Selection markers are also used that allow for positive selection, such as the phosphomannose isomerase gene, as described in US Pat. No. 5,767,378. Identification of transformed cells also confers phenotypic definite characteristics to chloramphenicol acetyl transferase (CAT), β-glucuronidase (GUS), luciferase, and green fluorescent protein (GFP) or transformed cells. It can be carried out through the expression of selectable marker genes, such as genes encoding other proteins.

(1) Construction of a vector suitable for Agrobacterium transformation

Numerous vectors for transformation using Agrobacterium tumefaciens are available. These typically include one or more T-DNA border sequences and include vectors such as pBIN19 (Bevan, Nucl. Acids Res. (1984)) and pXYZ. The construction of two representative vectors is described below.

Construction of pCIB200 and pCIB2001

Dual vectors pCIB200 and pCIB2001 are used for the construction of recombinant vectors for use against Agrobacterium and are constructed in the following manner. pTJS75 (Schmidhauser & Helinski, J Bacteriol. 164: 446-455 (1985)) is digested with NarI to cleave tetracycline-resistant genes to construct pTJS75kan, and then insert AccI fragments from pUC4K containing NPTII ( Vieira & Messing, Gene 19: 259-268 (1982): Bevan et al., Nature 304: 184-187 (1983); McBride et al., Plant Molecular Biology 14: 266-276 (1990)). XhoI linker is bound to the EcoRV fragment of pCIB7 (Rothstein et al., Gene 53: 153-161 (1987)) containing left and right T-DNA boundaries, plant selective nos / nptII chimeric gene and pUC polylinker, PCIB200 is constructed by cloning XhoI-digested fragments into SaII-digested pTJS75kan (see EP 0 332 104, Example 19). pCIB200 contains the following unique polylinker restriction sites: EcoRI, SstI, KpnI, BgIII, XbaI, and SalI. pCIB2001 is a derivative of pCIB200 constructed by insertion into a polylinker of additional restriction sites. Specific restriction sites in the polylinker of pCIB2001 are EcoRI, SstI, KpnI, BglII, XbaI, SalI, MluI BclI, AvrlL, ApaI, HpaI, and StuI. In addition to containing these unique restriction sites, pCIB2001 also contains plant and bacterial kanamycin selectivity, Agrobacterium-mediated left and right T-DNA boundaries, E. coli. RK2-derived trfA function for migration between collie and other hosts, and also OriT and OriV functions from RK2. The pCIB2001 polylinkers are suitable for cloning plant expression cassettes containing their own regulatory signals.

Construction of pCIB10 and Hygromycin Selective Derivatives thereof

The double vector pCIB10 contains genes encoding kanamycin resistance for selection in plants, T-DNA right and left border sequences and includes sequences from a broad host-range plasmid pRK252. Allows replication in both Collie and Agrobacterium. Its construction is described in the following literature: Rothstein et al. Gene 53: '153-161 (1987). Several derivatives of pCIB10 have been constructed that contain genes for hygromycin B phosphotransferase described in the following literature: Gritz et al. Gene 25: 179-188 (1983). These derivatives can select plants transformed only for hygromycin (pCIB743), or for hygromycin and kanamycin (pCIB715, pCIB717).

(2) Construction of vectors suitable for non-Agrobacterium transformation

Transformations that do not use Agrobacterium tumefaciens do not require T-DNA sequences in the selected transfection vector such that a vector lacking these sequences contains a T-DNA sequence, such as one of those described above. Can be used besides. Transformation techniques that do not depend on Agrobacterium include particle foaming, protoplast uptake (eg PEG and electroporation) and transformation via microinjection. The choice of vector depends largely on the preferred choice of species to be transformed. The construction of some representative vectors will now be described.

Construction of pCIB3064

pCIB3064 is a pUC-derived vector suitable for direct gene transfer techniques with selection by the herbicide Basta (or phosphinothricin). Plasmid pCIB246 is E. coli. The CaMV 35S promoter and CaMV 35S transcription terminator operably fused with the Coli GUS gene are described in published PCT patent application WO 93/07278. The 35S promoter of the vector contains two ATG sequences 5 'at the start site. These sites are mutated in a manner that removes ATGs using standard PCR techniques and produces restriction sites SspI and PvulI. The new restriction site is 96 and 37 bp from the unique SalI site and 101 and 42 bp from the actual initiation site. The resulting derivative of pCIB246 is designated as pCIB3025. The plasmid pCIB3060 is constructed by digesting the GUS gene with SalI and SacI, cleaving from pCIB3025, and smoothing the ends to reconnect. Obtain plasmid pJIT82 from the John Innes Centre, Norswich and cut the 400 bp SamI fragment containing the bar gene from Streptomyces viridochromogenes and insert it into the HpaI site of pCIB3060 (Thompson et al. EMBO). J 6: 2519-2523 (1987). Thus pCIB3064 comprising a bar gene, a gene for ampicillin resistance (for selection in E. coli) and a polylinker with specific sites SphI, PstI, HindIII, and BamHI, under the control of the CaMV 35S promoter and terminator for herbicide selection To construct. The vectors are suitable for cloning plant expression cassettes containing their own regulatory signals.

Construction of pSOG19 and pSOG35

pSOG35 is a selective marker that confers resistance to methotrexate. It is a transformation vector using the colli dihydrofolate reductase (DHFR) gene. PCR is used to amplify the 35S promoter (-800 bp) from pSOG10, intron 6 from the corn Adh1 gene (approximately 550 bp) and 18 bp of the GUS non-read leader sequence. this. The 250 bp fragment encoding the collie dihydrofolate reductase type II gene was also amplified by PCR and these two PCR fragments were SacI from pBI221 (Clontech) containing the backbone of the pUC19 vector and the nopaline synthase terminator. Concatenate with the PstI fragment Linkage of these fragments constructs pSOG19 containing an intro 6 sequence, a GUS leader, a DHFR gene and a 35S promoter fused with a nopaline synthase terminator. The vector pSOG35 was constructed by replacing the GUS leader in pSOG19 with the leader sequence from maize bleaching virus (MCMV). pSOG19 and pSOG35 contain the pUC gene for ampicillin resistance and have HindIII, SphI, PstI and EcoRI sites that can be used for cloning of foreign sequences.

Example 11

Requirements for the Construction of Plant Expression Cassettes

The gene sequence intended for expression in the transformed plant is first linked directly upstream of the suitable promoter and suitable transcription terminator in the expression cassette.

Promoter Selection

The choice of promoter used in the expression cassette determines the spatial and temporal expression patterns of the transgene in the transformed plant. The selected promoter expresses the transgene in a specific cell type (e.g., epithelial cells, leafy cells, root cortical cells of leaves) or in certain tissues or organs (roots, leaves or flowers) and this selection results in biosynthesis of the DNA molecules of the invention. Reflect the intended location of the. In addition, selected promoters can express genes under light-induced or other temporarily regulated promoters. A further alternative is that the selected promoter is chemically regulated. This provides the ability to induce the expression of nucleotide sequences only if they are induced at the desired time by treatment with chemical inducers.

Warrior Terminator

Various transcription terminators are available for use in expression cascades. They are involved in the transcription termination of transgenic genes and their correct polyadenylation. Suitable transcription terminators are those known to work in plants, including CaMV 35S terminator, tml terminator, nopalin synthase terminator, and pea rbcS E9 terminator. They can be used in both monocotyledonous and dicotyledonous plants.

Sequences for enhancing or regulating expression

Numerous sequences have been identified that enhance gene expression within a transcription unit and they can be used in conjunction with the genes of the invention to enhance expression of the genes in the transformed plant.

Various intron sequences have been found to enhance expression, especially in monocots. For example, introns of the maize Adh1 gene have been found to significantly enhance the expression of wild-type genes under its homologous promoter upon introduction into maize cells. Intron 1 has been shown to be particularly effective and enhance expression in fusion constructs with the chloramphenicol acetyltransferase gene (Callis et al., Genes Develop 1: 1183-1200 (1987)). In the same experimental system, introns from the corn bronze 1 gene have a similar effect in enhancing expression (Callis et al., Supra). Intron sequences are typically incorporated into plant transformation vectors, typically within a non-translating leader.

Numerous non-translating leader sequences derived from viruses are also known to enhance expression, which are particularly effective in dicotyledonous cells. Leader sequences from tobacco mosaic virus (TMV, 'Ω-sequence'), corn white spotted virus (MCMV), and alfalfa mosaic virus (AMV) have been found to be effective in enhancing expression (eg Gallie et al. Nucl.Acids Res. 15: 8693-8711 (1987); Skuzeski et al., Plant Molec. Biol. 15, 65-79 (1990)).

Targeting Intracellular Gene Products

Various mechanisms for targeting gene products are known to exist in plants and the sequences regulating the functionalization of these mechanisms are characterized in some detail. For example, targeting gene products with chloroplasts is regulated by signal sequences present at the amino terminus of various proteins and degraded during transport to chloroplasts to become mature proteins (eg, Comai et al. J. Biol. Chem 263: 15104-15109 (1988). Fusion of these signal sequences to heterologous gene products is effective for introducing heterologous products into chloroplasts (van den Broeck et al. Nature 313: 358-363 (1985)). DNA encoding a suitable signal sequence can be isolated from the 5 'end of the cDNAs encoding RUBISCO protein, CAB protein, EPSP synthase enzyme, GS2 protein and numerous other proteins known to be transferred to chloroplasts.

Other gene products are transferred to other organs such as mitochondria and peroxysomes (eg, Unger et al. Plant Molec. Biol. 13: 411-418 (1989)). CDNAs encoding these products can also be engineered to target heterologous gene products to these organs. Examples of such sequences are nuclear-encoded ATPases and aspartate amino transferases specific for mitochondria. Targeting to cellular protein bodies is described in Rogers et al. (Pro. Natl. Acad. Sci. USA 82: 6512-6516 (1985)).

Also characterized are sequences that target gene products to other cell compartments. Amino terminal sequences are involved in targeting by extracellular secretion from ER, apoplasts, and allureon cells (Koehler & Ho, Plant Cell 2: 769-783 (1990)). In addition, amino terminal sequences associated with carboxy terminal sequences are involved in vacuole targeting of gene products (Shinshi et al. Plant Molec. Biol. 14: 357-368 (1990)).

The appropriate targeting sequence described above may be fused to the transforming gene sequence of interest to direct the transformation gene product to the organ or cell compartment. In the case of chloroplast targeting, for example, chloroplast signal sequences from the RUBISCO gene, CAB gene, EPSP synthase gene, or GS2 gene are fused into the frame of the amino terminal ATG of the transgene. The signal sequence selected should comprise a known cleavage site and the constructed fusion should take into account the amino acids following the cleavage site required for cleavage. In some cases this requirement can be met by adding a few amino acids between the cleavage site and the transgene ATG, or alternatively replacing some amino acids in the transgene sequence. Fusions constructed for transport to chloroplasts can be tested for chloroplast uptake efficacy by in vitro detoxification of in vitro transcribed constructs followed by in vitro chloroplast acquisition using techniques described in the following literature: Bartlett et al. In: Edelmann et al. (Eds.) Methods in Chloroplast Molecular Biology, Elsevier. pp 1081-1091 (1982); Wasmann et al. Mol. Gen. Genet. 205: 446-453 (1986)). These construction techniques are known in the art and can be equally applied to mitochondria and peroxysomes. The choice of targeting may be required for pesticidal toxins. It may in some cases be a mitochondria or a peroxysome, but typically may be cytoplasmic or chloroplast. Expression of nucleotide sequences may also require targeting to ER, apoplasts or vacuoles.

The cell targeting mechanisms described above can be used not only in relation to cognate promoters, but also in relation to heterologous promoters to perform specific cell targeting purposes under the transcriptional control of promoters having expression patterns different from those of the promoters that induce targeting signals. have.

Example 12

Example of Expression Cassette Construction

The present invention includes expression of DNA molecules under the control of a promoter that can be expressed in a plant, regardless of the origin of the promoter. The invention also encompasses the use of plant-expressing promoters in connection with additional sequences needed or selected for expression of a DNA molecule. Such sequences include transcription terminators, exogenous sequences that enhance expression (eg, introns [eg, Adh intron 1], viral sequences [eg, TMV-Ω]), and gene products to specific organ and cell compartments. Include but are not limited to sequences that enable them.

Persistent Expression: CaMV 35S Promoter

The construction of the plasmid pCGN1761 is described in published patent application EP 0 392 225. pCGN1761 includes a 'double' 35S promoter and a tml transcription terminator with a distinct EcoRI site between the promoter and terminator and has a pUC-type backbone. A derivative of pCGN1761 with a modified polylinker comprising NotI and XhoI in addition to the EcoRI site present is constructed. The derivative is designated pCGN1761ENX. pCGN1761ENX is useful for cloning cDNA sequences or gene sequences (including microbial ORF sequences) in polylinkers for their expression purposes under the control of a 35S promoter in transformed plants. The entire 35S promoter-gene sequence-tml terminator cassette of such construct was terminated with HindIII, SphI, SalI, and XbaI at the 5 ′ site for the promoter for delivery to the transformation vector as described above in Example 35. Cleavage at the 3 ′ site of XbaI, BamHI and BglI for the factor. In addition, the dual 35S promoter fragments were subjected to 5 ′ cleavage at the HindIII, SphI, SalI, XbaI, or PstI sites, and 3 ′ cleavage at any polylinker restriction site (EcoRI, NotI or XhoI) to replace other promoters. Can be removed.

Modification of pCGN1761ENX by Optimization of the Site of Initiation

For the constructs described in this section, modifications can be made around the cloning site by introducing sequences that can enhance translation. Since these genes may not contain sequences adjacent to their starting methionine, which may be suitable for initiating translation in plants, this is particularly useful when genes derived from microorganisms are introduced into plant expression cassettes. When cloning genes derived from microorganisms from their ATG into plant expression cassettes, it may be useful to modify their insertion sites to optimize their expression. Modifications of pCGN1761ENX are described as an example for incorporating one of several optimized sequences for plant expression (eg Joshi, supra).

Expression under chemically controllable promoters

This section describes replacing the dual 35S promoter in pCGN1761ENX with one of the selected promoters: As an example, a chemically regulated PR-1a promoter is described. The promoter of choice is preferably cleaved by restriction enzymes from its source, but may be PCR-amplified using primers that otherwise include suitable termination restriction sites. When performing PCR-amplification, the promoter should be resequenced and examined for amplification errors after cloning of the amplified promoter in the target vector. The chemically controllable tobacco PR-1a promoter is cleaved from plasmid pCIB1004 (see EP 0 332 104, Example 21 for construction) and transferred to plasmid pCGN1761ENX. pCIB1004 is cleaved with NcoI and the 3 'overhang of the resulting linearized fragments is smoothed by treatment with T4 DNA polymerase. The fragment is then cleaved with HindIII and the resulting PR-1a promoter containing fragments are gel purified and cloned into pCGN176ENX from which the 35S promoter has been removed. It is cleaved with XhoI and smoothed with T4 polymerase, then cleaved with HindIII and the huge vector-terminator containing fragments with the pCIB1004 promoter fragment cloned are isolated. This constructs a pCGN1761ENX derivative having a PR-1a promoter and a tml terminator and an insertion polylinker with unique EcoRI and NotI sites. DNA molecules of the invention can be inserted into the vector and the fusion product (ie, promoter-gene-terminator) can then be delivered to a selected transformation vector, including those described herein.

Persistent Expression: Actin Promoter

Several isoforms of actin are known to be expressed in most cell types, with the result that the actin promoter is well selected for the persistent promoter. In particular, promoters from the rice Act1 gene have been cloned and characterized (McElroy et al. Plant Cell 2: 163-171 (1990)). The 1.3 kb fragment of this promoter was found to contain all of the regulatory components required for expression in rice protoplasts. In addition, numerous expression vectors based on the Act1 promoter have been specially constructed for use in monocots (McElroy et al. Mol. Gen. Genet. 231: 150-160 (1991)). These include Act1-intron 1, Adh1 5 'flanking sequences and sequences from Adh1-intron 1 (from corn alcohol dehydrogenase gene) and CaMV 35S promoters. The vector with the highest expression is a fusion of 35S with Act1 intron or Act1 5 'flanking sequence with Act1 intron. Optimization of the sequence around the starting ATG (GUS reporter gene) also enhances expression. McElroy et al. The promoter expression cassette described in (Mol. Gen. Genet 231: 150-160 (1991)) can be readily modified for the expression of the DNA molecules of the invention and is particularly suitable for use in monocot plant hosts. Promoter containing fragments can be removed from the McElroy construct and used to replace the double 35S promoter in pCGN1761ENX, which can then be used for insertion or for specific gene sequences. Other reports have shown that the Rice Act1 promoter with the first intron also directs high expression in cultured barley cells (Chibbar et al. Plant Cell Rep. 12: 506-509 (1993)).

Sustained Expression: Ubiquitin Promoter

Ubiquitin is another gene product known to accumulate in numerous cell types and its promoter has been cloned from several species for use in transformed plants [eg sunflower-Binet et al. Plant Science 79: 87-94 (1991), corn-Christensen et al. Plant Molec. Biol. 12: 619-632 (1989). Corn ubiquitin promoters have been developed in transformed monocotyledonous systems and their sequences and vectors constructed for monocotyledonous transformation are described in patent publication EP 0 342 926. See also Taylor et al. Plant Cell Rep. 12: 491-495 (1993) describes a vector containing the corn ubiquitin promoter and a first intron (pAHC25) and its high activity in cell suspensions of numerous monocotyledonous plants when introduced via micropropulsion foaming. Ubiquitin promoters are particularly suitable for the expression of the DNA molecules of the invention in transformed plants, in particular monocots, Suitable vectors include derivatives of pAHC25 or suitable ubiquitin promoters and / or intron sequences as described herein. One of the transformation vectors introduced and modified.

Root Specific Expression

A preferred expression pattern for the nucleotide sequence of the invention is root expression. Root expression is particularly useful for the control of soil-based fungal pathogens. Suitable root promoters are described in de Framond; FEBS 290: 103-106 (1991) and also published patent application EP 0 452 269. The promoter is transferred to a suitable vector, such as pCGN1761ENX for nucleotide sequence insertion, followed by transfer of the entire promoter-gene-terminator cassette to the transformation vector of interest.

Wound-inducible promoter

Wound inducible promoters are particularly suitable for the expression of the DNA molecules of the invention because they are typically active at wound induction as well as at the site of phytopathogenic infection. Numerous such promoters are described in Xu et al. Plant Molec. Biol. 22: 573-588 (1993), Logemann et al. Plant Cell 1: 151-158 (1989), Rohrmeler & Lehle, Plant Molec. Biol. 22: 783-792 (1993), Firek et al. Plant Molec. Biol. 22: 129-142 (1993), Warner et al. Plant J. 3: 191-201 (1993), all of which are suitable for use in the present invention. Rosemann (Logemann et al.) (See above) describes the 5 'upstream sequence of the dicotyledonous potato wun1 gene. Su (Xu et al.) (See above) found that the wound inducible promoter (pin2) from dicotyledonous potatoes is active in monocotyledonous rice. In addition, Rohrmeier & Lehle (see above) describes the cloning of maize Wip1 cDNA that is wound-induced and can be used to isolate cognate promoters using standard techniques. Similarly, Firek et al. (See above) and Warner et al. (See above) are wound-induced from monocotyledonous Asparagus officinalis expressed at local wounds and site of pathogen invasion. Describe the gene. Using cloning techniques known in the art, these promoters can be delivered to suitable vectors, fused to the DNA molecules of the invention and then used to express these genes at the site of phytopathogen infection.

Preferred expression of pith

Patent application WO 93/07278 (Ciba-Geigy) describes the isolation of maize trpA gene, which is preferentially expressed in mesenchymal cells. Extended gene sequences and promoters are provided from the transcriptional initiation site to -1726. Using standard molecular biological techniques, the promoter or portion thereof can be delivered to a vector such as pCGN1761, where it can be used to induce the expression of the DNA molecule of the invention in a heavy-figure fashion, replacing the 35S promoter. Indeed, fragments containing the sheath-preferred promoter or a portion thereof can be delivered to a vector and modified for use in the transformed plant.

Pollen-specific Expression

Patent application WO 93/07278 (Ciba-Geigy) also describes the isolation of corn calcium-dependent protein kinase (CDPK) genes expressed in pollen cells. The gene sequence and promoter extend up to 1400 bp from the transcription initiation. Using standard molecular biological techniques, the promoter or portion thereof can be delivered to a vector such as pCGN1761, which can be used to express the DNA molecule of the invention in place of the 35S promoter. In fact, fragments containing pollen-specific promoters or portions thereof can be delivered to vectors and modified for use in transformed plants.

Leaf-specific expression

Corn genes encoding phosphoenol-carboxylase (PEPC) are described in Hudspeth & Grula, Plant Molec Biol 12: 579-589 (1989). Standard molecular biology techniques can be used to express genes in a leaf-specific manner in transformed plants using promoters for these genes.

Expression by Chloroplast Targeting

Chen & Jagendorf, J. Biol. Chem. 268: 2363-2367 (1993) describe the successful use of chloroplast transporting peptides for heterologous transgene introduction. The peptide used is a carrier peptide from the rbcS gene from Nicotiana plumbaginifolia (Poulsen et al. Mol. Gen. Genet. 205: 193-200 (1986)). DNA sequences encoding the transport peptides using the restriction enzymes DraI and SphI, or Tsp5091 and SphI, can be cleaved from plasmid prbcS-8B (Poulsen et al., Supra) and manipulated for use in one of the constructs described above. . The DraI-SphI fragment extends from -58 for the initiating rbcS ATG to the first amino acid (also methionine) of the mature peptide immediately after the induction cleavage site, while the Tsp5091-SphI fragment is at -8 for the initiating rbcS ATG Extends to the first amino acid. Thus, these fragments can insert the DNA molecules of the invention into the correct fusion downstream of the carrier peptide and generate the transcriptional fusion with a non-ready leader of selected promoters (eg, 35S, PR-1a, actin, ubiquitin, etc.). Can be appropriately inserted into the polylinker of the selected expression cassette. This kind of construction is common in the art. For example, while the DraI terminus is already smoothed, the 5 'Tsp5091 site can be blunted by T4 polymerase treatment or otherwise bound to a linker or adapter sequence to facilitate fusion of the selected promoter. The 3 ′ SphI site can be maintained intact or otherwise facilitated insertion into a selected vector in such a way that it can be bound to an adapter of the linker sequence so that a restriction site suitable for subsequent insertion of the DNA molecule of the invention can be used. Ideally the ATG of the SphI site is maintained and contains the first ATG of the DNA molecule of the invention. Chen & Jagendorf (see above) provides a conserved base sequence for cleavage that is ideal for chloroplast introduction, in which case it is preferred that methionine is present at the first position of the mature protein. The next position is more varied and the amino acids may not be so important. In some cases, fusion constructs are described in Bartlett et al (In: Edelmann et al. (Eds.) Methods in Chloroplast Molecular Biology, Elsevier. Pp. 1081-1091 (1982) and Wasmann et al. (Mol. Gen. The in vitro transduction efficiency can be assessed using the method described in Genet. 205: 446-453 (1986). Typically, the best method is to construct a fusion using the DNA molecule of the invention without modification at the amino terminus. And it is clear that such a fusion is not introduced into the chloroplast with high efficiency [in this case, the established literature (Chen & Jahendorf, supra; Wasman et al., Supra; Ko & Ko, J. Biol. Chem. 267: 13910-13916 (1992)).

Similar manipulations can be made with other GS2 chloroplast transporting peptides that encode sequences from other sources (monolitic and dicotyledonous) and other genes. Similar processes can also be targeted to other subcellular compartments, such as mitochondria.

〈110〉 Novartis AG

〈120〉 Genes encoding MLO proteins and conferring fungal resistance upon plants

<130> S-30431 / A

〈150〉 US 09/042763

<151> 1998-03-17

〈160〉 42

〈170〉 KOPATIN 1.5

〈210〉 1

<211> 13

<212> PRT

〈213〉 Artificial Sequence

〈220〉

<223> Conserved amino acid sequence

<400> 1

Glu Leu Met Xaa Xaa Gly Xaa Ile Ser Leu Leu Leu Xaa

1 5 10

〈210〉 2

<211> 14

<212> PRT

〈213〉 Artificial Sequence

〈220〉

<223> conserved amino acid sequence

<400> 2

Xaa Thr Xaa Pro Leu Xaa Xaa Xaa Val Xaa Gln Met Gly Ser

1 5 10

〈210〉 3

〈211〉 1868

<212> DNA

213 Triticum sp.

〈220〉

<221> CDS

222 (176) .. (1777)

〈220〉

〈221〉 misc_feature

222 (365) .. (403)

223 location of the amino acid sequence set forth in SEQ ID No: 1

〈220〉

〈221〉 misc_feature

222 (1352) .. (1393)

<223> location of the amino acid sequence set forth in SEQ ID No: 2

<400> 3

cacgcccaca cttcgccaac acacaacgta cctgcgtacg tacgctttcc atttcctttc 60

ttgctccggc cggccggcca cgtagaatag atacccggcc aggtaggtac ctcgttggct 120

cagacgaccg gcggctgggt ctccggacaa ggaaagaggt tgcgctcggg gaccg 175

atg gcg gac gac gac gag tac ccc cca gcg agg acg ctg ccg gag acg 223

Met Ala Asp Asp Asp Glu Tyr Pro Pro Ala Arg Thr Leu Pro Glu Thr

1 5 10 15

ccg tcc tgg gcg gtg gcc ctc gtc ttc gcc gtc atg atc atc gtg tcc 271

Pro Ser Trp Ala Val Ala Leu Val Phe Ala Val Met Ile Ile Val Ser

20 25 30

gtc ctc ctg gag cac gcg ctc cat aag ctc ggc cat tgg ttc cac aag 319

Val Leu Leu Glu His Ala Leu His Lys Leu Gly His Trp Phe His Lys

35 40 45

cgg cac aag aac gcg ctg gcg gag gcg ctg gag aag atc aag gcg gag 367

Arg His Lys Asn Ala Leu Ala Glu Ala Leu Glu Lys Ile Lys Ala Glu

50 55 60

ctc atg ctg gtg ggc ttc atc tcg ctg ctg ctc gcc gtg acg cag gac 415

Leu Met Leu Val Gly Phe Ile Ser Leu Leu Leu Ala Val Thr Gln Asp

65 70 75 80

ccc atc tcc ggg ata tgc atc tcc gag aag gcc gcc agc atc atg cgg 463

Pro Ile Ser Gly Ile Cys Ile Ser Glu Lys Ala Ala Ser Ile Met Arg

85 90 95

ccc tgc aag ctg ccc cct ggc tcc gtc aag agc aag tac aaa gac tac 511

Pro Cys Lys Leu Pro Pro Gly Ser Val Lys Ser Lys Tyr Lys Asp Tyr

100 105 110

tac tgc gcc aaa cag ggc aag gtg tcg ctc atg tcc acg ggc agc ttg 559

Tyr Cys Ala Lys Gln Gly Lys Val Ser Leu Met Ser Thr Gly Ser Leu

115 120 125

cac cag ctg cac ata ttc atc ttc gtg ctc gcc gtc ttc cat gtc acc 607

His Gln Leu His Ile Phe Ile Phe Val Leu Ala Val Phe His Val Thr

130 135 140

tac agc gtc atc atc atg gct cta agc cgt ctc aaa atg aga acc tgg 655

Tyr Ser Val Ile Ile Met Ala Leu Ser Arg Leu Lys Met Arg Thr Trp

145 150 155 160

aag aaa tgg gag aca gag acc gcc tcc ctg gaa tac cag ttc gca aat 703

Lys Lys Trp Glu Thr Glu Thr Ala Ser Leu Glu Tyr Gln Phe Ala Asn

165 170 175

gat cct gcg cgg ttc cgc ttc acg cac cag acg tcg ttc gtg aag cgg 751

Asp Pro Ala Arg Phe Arg Phe Thr His Gln Thr Ser Phe Val Lys Arg

180 185 190

cac ctg ggc ctc tcc agc acc ccc ggc gtc aga tgg gtg gtg gcc ttc 799

His Leu Gly Leu Ser Ser Thr Pro Gly Val Arg Trp Val Val Ala Phe

195 200 205

ttc agg cag ttc ttc agg tcg gtc acc aag gtg gac tac ctc acc ttg 847

Phe Arg Gln Phe Phe Arg Ser Val Thr Lys Val Asp Tyr Leu Thr Leu

210 215 220

agg gca ggc ttc atc aac gcg cat ttg tcg cat aac agc aag ttc gac 895

Arg Ala Gly Phe Ile Asn Ala His Leu Ser His Asn Ser Lys Phe Asp

225 230 235 240

ttc cac aag tac atc aag agg tcc atg gag gac gac ttc aaa gtc gtc 943

Phe His Lys Tyr Ile Lys Arg Ser Met Glu Asp Asp Phe Lys Val Val

245 250 255

gtt ggc atc agc ctc ccg ctg tgg tgt gtg gcg atc ctc acc ctc ttc 991

Val Gly Ile Ser Leu Pro Leu Trp Cys Val Ala Ile Leu Thr Leu Phe

260 265 270

ctt gac att gac ggg atc ggc acg ctc acc tgg att tct ttc atc cct 1039

Leu Asp Ile Asp Gly Ile Gly Thr Leu Thr Trp Ile Ser Phe Ile Pro

275 280 285

ctc gtc atc ctc ttg tgt gtt gga acc aag ctg gag atg atc atc atg 1087

Leu Val Ile Leu Leu Cys Val Gly Thr Lys Leu Glu Met Ile Ile Met

290 295 300

gag atg gcc ctg gag atc cag gac cgg gcg agc gtc atc aag ggg gcg 1135

Glu Met Ala Leu Glu Ile Gln Asp Arg Ala Ser Val Ile Lys Gly Ala

305 310 315 320

ccc gtg gtt gag ccc agc aac aag ttc ttc tgg ttc cac cgc ccc gac 1183

Pro Val Val Glu Pro Ser Asn Lys Phe Phe Trp Phe His Arg Pro Asp

325 330 335

tgg gtc ctc ttc ttc ata cac ctg acg cta ttc cag aac gcg ttt cag 1231

Trp Val Leu Phe Phe Ile His Leu Thr Leu Phe Gln Asn Ala Phe Gln

340 345 350

atg gca cat ttc gtg tgg aca gtg gcc acg ccc ggc ttg aag aaa tgc 1279

Met Ala His Phe Val Trp Thr Val Ala Thr Pro Gly Leu Lys Lys Cys

355 360 365

ttc cat atg cac atc ggg ctg agc atc atg aag gtc gtg ctg ggg ctg 1327

Phe His Met His Ile Gly Leu Ser Ile Met Lys Val Val Leu Gly Leu

370 375 380

gct ctt cag ttc ctc tgc agc tat atc acc ttc ccg ctc tac gcg ctc 1375

Ala Leu Gln Phe Leu Cys Ser Tyr Ile Thr Phe Pro Leu Tyr Ala Leu

385 390 395 400

gtc aca cag atg gga tca aac atg aag agg tcc atc ttc gac gag cag 1423

Val Thr Gln Met Gly Ser Asn Met Lys Arg Ser Ile Phe Asp Glu Gln

405 410 415

acg gcc aag gcg ctg aca aac tgg cgg aac acg gcc aag gag aag aag 1471

Thr Ala Lys Ala Leu Thr Asn Trp Arg Asn Thr Ala Lys Glu Lys Lys

420 425 430

aag gtc cga gac acg gac atg ctg atg gcg cag atg atc ggc gac gcg 1519

Lys Val Arg Asp Thr Asp Met Leu Met Ala Gln Met Ile Gly Asp Ala

435 440 445

acg ccc agc cga ggg gcg tcg ccc atg cct agc cgg ggc tcg tcg cca 1567

Thr Pro Ser Arg Gly Ala Ser Pro Met Pro Ser Arg Gly Ser Ser Pro

450 455 460

gtg cac ctg ctt cac aag ggc atg gga cgg tcc gac gat ccc cag agc 1615

Val His Leu Leu His Lys Gly Met Gly Arg Ser Asp Asp Pro Gln Ser

465 470 475 480

acg cca acc tcg cca agg gcc atg gag gag gct agg gac atg tac ccg 1663

Thr Pro Thr Ser Pro Arg Ala Met Glu Glu Ala Arg Asp Met Tyr Pro

485 490 495

gtt gtg gtg gcg cat cca gtg cac aga cta aat cct gct gac agg aga 1711

Val Val Val Ala His Pro Val His Arg Leu Asn Pro Ala Asp Arg Arg

500 505 510

agg tcg gtc tcg tcg tcg gca ctc gat gtc gac att ccc agc gca gat 1759

Arg Ser Val Ser Ser Ser Ala Leu Asp Val Asp Ile Pro Ser Ala Asp

515 520 525

ttt tcc ttc agc cag gga tga gacaagtttc tgtattgatg ttagtccaat 1810

Phe Ser Phe Ser Gln Gly

530

gtatagccaa cataggatgt catgattcgt acaataagaa atacaaattt ttactgag 1868

〈210〉 4

<211> 534

<212> PRT

213 Triticum sp.

<400> 4

Met Ala Asp Asp Asp Glu Tyr Pro Pro Ala Arg Thr Leu Pro Glu Thr

1 5 10 15

Pro Ser Trp Ala Val Ala Leu Val Phe Ala Val Met Ile Ile Val Ser

20 25 30

Val Leu Leu Glu His Ala Leu His Lys Leu Gly His Trp Phe His Lys

35 40 45

Arg His Lys Asn Ala Leu Ala Glu Ala Leu Glu Lys Ile Lys Ala Glu

50 55 60

Leu Met Leu Val Gly Phe Ile Ser Leu Leu Leu Ala Val Thr Gln Asp

65 70 75 80

Pro Ile Ser Gly Ile Cys Ile Ser Glu Lys Ala Ala Ser Ile Met Arg

85 90 95

Pro Cys Lys Leu Pro Pro Gly Ser Val Lys Ser Lys Tyr Lys Asp Tyr

100 105 110

Tyr Cys Ala Lys Gln Gly Lys Val Ser Leu Met Ser Thr Gly Ser Leu

115 120 125

His Gln Leu His Ile Phe Ile Phe Val Leu Ala Val Phe His Val Thr

130 135 140

Tyr Ser Val Ile Ile Met Ala Leu Ser Arg Leu Lys Met Arg Thr Trp

145 150 155 160

Lys Lys Trp Glu Thr Glu Thr Ala Ser Leu Glu Tyr Gln Phe Ala Asn

165 170 175

Asp Pro Ala Arg Phe Arg Phe Thr His Gln Thr Ser Phe Val Lys Arg

180 185 190

His Leu Gly Leu Ser Ser Thr Pro Gly Val Arg Trp Val Val Ala Phe

195 200 205

Phe Arg Gln Phe Phe Arg Ser Val Thr Lys Val Asp Tyr Leu Thr Leu

210 215 220

Arg Ala Gly Phe Ile Asn Ala His Leu Ser His Asn Ser Lys Phe Asp

225 230 235 240

Phe His Lys Tyr Ile Lys Arg Ser Met Glu Asp Asp Phe Lys Val Val

245 250 255

Val Gly Ile Ser Leu Pro Leu Trp Cys Val Ala Ile Leu Thr Leu Phe

260 265 270

Leu Asp Ile Asp Gly Ile Gly Thr Leu Thr Trp Ile Ser Phe Ile Pro

275 280 285

Leu Val Ile Leu Leu Cys Val Gly Thr Lys Leu Glu Met Ile Ile Met

290 295 300

Glu Met Ala Leu Glu Ile Gln Asp Arg Ala Ser Val Ile Lys Gly Ala

305 310 315 320

Pro Val Val Glu Pro Ser Asn Lys Phe Phe Trp Phe His Arg Pro Asp

325 330 335

Trp Val Leu Phe Phe Ile His Leu Thr Leu Phe Gln Asn Ala Phe Gln

340 345 350

Met Ala His Phe Val Trp Thr Val Ala Thr Pro Gly Leu Lys Lys Cys

355 360 365

Phe His Met His Ile Gly Leu Ser Ile Met Lys Val Val Leu Gly Leu

370 375 380

Ala Leu Gln Phe Leu Cys Ser Tyr Ile Thr Phe Pro Leu Tyr Ala Leu

385 390 395 400

Val Thr Gln Met Gly Ser Asn Met Lys Arg Ser Ile Phe Asp Glu Gln

405 410 415

Thr Ala Lys Ala Leu Thr Asn Trp Arg Asn Thr Ala Lys Glu Lys Lys

420 425 430

Lys Val Arg Asp Thr Asp Met Leu Met Ala Gln Met Ile Gly Asp Ala

435 440 445

Thr Pro Ser Arg Gly Ala Ser Pro Met Pro Ser Arg Gly Ser Ser Pro

450 455 460

Val His Leu Leu His Lys Gly Met Gly Arg Ser Asp Asp Pro Gln Ser

465 470 475 480

Thr Pro Thr Ser Pro Arg Ala Met Glu Glu Ala Arg Asp Met Tyr Pro

485 490 495

Val Val Val Ala His Pro Val His Arg Leu Asn Pro Ala Asp Arg Arg

500 505 510

Arg Ser Val Ser Ser Ser Ala Leu Asp Val Asp Ile Pro Ser Ala Asp

515 520 525

Phe Ser Phe Ser Gln Gly

530

<210> 5

<211> 1693

<212> DNA

213 Triticum sp.

〈220〉

<221> CDS

222 (1) .. (1602)

〈220〉

〈221〉 misc_feature

222 (190) .. (228)

223 location of the amino acid sequence set forth in SEQ ID No: 1

〈220〉

〈221〉 misc_feature

222 (1177) .. (1218)

<223> location of the amino acid sequence set forth in SEQ ID No: 2

<400> 5

atg gcg gag gac tac gag tac ccc ccg gcg cgg acg ctg ccg gag acg 48

Met Ala Glu Asp Tyr Glu Tyr Pro Pro Ala Arg Thr Leu Pro Glu Thr

1 5 10 15

ccg tcc tgg gcg gtg gcg ctc gtc ttc gcc gtc atg atc atc gtg tcc 96

Pro Ser Trp Ala Val Ala Leu Val Phe Ala Val Met Ile Ile Val Ser

20 25 30

gtc ctc ctg gag cac gcg ctc cac aag ctc ggc cat tgg ttc cac aag 144

Val Leu Leu Glu His Ala Leu His Lys Leu Gly His Trp Phe His Lys

35 40 45

cgg cac aag aac gcg ctg gcg gag gcg ctg gag aag atc aaa gcg gag 192

Arg His Lys Asn Ala Leu Ala Glu Ala Leu Glu Lys Ile Lys Ala Glu

50 55 60

ctg atg ctg gtg ggg ttc atc tcg ctg ctg ctc gcc gtg acg cag gac 240

Leu Met Leu Val Gly Phe Ile Ser Leu Leu Leu Ala Val Thr Gln Asp

65 70 75 80

cca atc tcc ggg ata tgc atc tcc gag aag gcc gcc agc atc atg cgg 288

Pro Ile Ser Gly Ile Cys Ile Ser Glu Lys Ala Ala Ser Ile Met Arg

85 90 95

ccc tgc agc ctg ccc cct ggt tcc gtc aag agc aag tac aaa gac tac 336

Pro Cys Ser Leu Pro Pro Gly Ser Val Lys Ser Lys Tyr Lys Asp Tyr

100 105 110

tac tgc gcc aaa aag ggc aag gtg tcg cta atg tcc acg ggc agc ttg 384

Tyr Cys Ala Lys Lys Gly Lys Val Ser Leu Met Ser Thr Gly Ser Leu

115 120 125

cac cag ctc cac atg ttc atc ttc gtg ctc gcc gtc ttc cat gtc acc 432

His Gln Leu His Met Phe Ile Phe Val Leu Ala Val Phe His Val Thr

130 135 140

tac agc gtc atc atc atg gct cta agc cgt ctc aaa atg agg aca tgg 480

Tyr Ser Val Ile Ile Met Ala Leu Ser Arg Leu Lys Met Arg Thr Trp

145 150 155 160

aag aaa tgg gag aca gag acc gyc tcc ttg gaa tac cag ttc gca aat 528

Lys Lys Trp Glu Thr Glu Thr Ala Ser Leu Glu Tyr Gln Phe Ala Asn

165 170 175

gat cct gcg cgg ttc cgc ttc acg cac cag acg tcg ttc gtg aag cgt 576

Asp Pro Ala Arg Phe Arg Phe Thr His Gln Thr Ser Phe Val Lys Arg

180 185 190

cac ctg ggc ctc tcc agc acc ccc ggc atc aga tgg gtg gtg gcc ttc 624

His Leu Gly Leu Ser Ser Thr Pro Gly Ile Arg Trp Val Val Ala Phe

195 200 205

ttc agg cag ttc ttc agg tcg gtc acc aag gtg gac tac ctc acc ctg 672

Phe Arg Gln Phe Phe Arg Ser Val Thr Lys Val Asp Tyr Leu Thr Leu

210 215 220

agg gca ggc ttc atc aac gcg cat ttg tcg cat aac agc aag ttc gac 720

Arg Ala Gly Phe Ile Asn Ala His Leu Ser His Asn Ser Lys Phe Asp

225 230 235 240

ttc cac aag tac atc aag agg tcc atg gag gac gac ttc aaa gtc gtc 768

Phe His Lys Tyr Ile Lys Arg Ser Met Glu Asp Asp Phe Lys Val Val

245 250 255

gtt ggc atc agc ctc ccg ctg tgg tgt gtg gcg atc ctc acc ctc ttc 816

Val Gly Ile Ser Leu Pro Leu Trp Cys Val Ala Ile Leu Thr Leu Phe

260 265 270

ctt gat att gac ggg atc ggc acg ctc acc tgg att tct ttc atc cct 864

Leu Asp Ile Asp Gly Ile Gly Thr Leu Thr Trp Ile Ser Phe Ile Pro

275 280 285

ctc gtc atc ctc ttg tgt gtt gga acc aag ctg gag atg atc atc atg 912

Leu Val Ile Leu Leu Cys Val Gly Thr Lys Leu Glu Met Ile Ile Met

290 295 300

gag atg gcc ctg gag atc cag gac cgg gcg agc gtc atc aag ggg gcg 960

Glu Met Ala Leu Glu Ile Gln Asp Arg Ala Ser Val Ile Lys Gly Ala

305 310 315 320

ccc gtg gtt gag ccc agc aac aag ttc ttc tgg ttc cac cgc ccc gac 1008

Pro Val Val Glu Pro Ser Asn Lys Phe Phe Trp Phe His Arg Pro Asp

325 330 335

tgg gtc ctc ttc ttc ata cac ctg acg ctg ttc cag aat gcg ttt cag 1056

Trp Val Leu Phe Phe Ile His Leu Thr Leu Phe Gln Asn Ala Phe Gln

340 345 350

atg gca cat ttc gtc tgg aca gtg gcc acg ccc ggc ttg aag aaa tgc 1104

Met Ala His Phe Val Trp Thr Val Ala Thr Pro Gly Leu Lys Lys Cys

355 360 365

ttc cat atg cac atc ggt ctg agc atc atg aag gtc gtg ctg ggg ctg 1152

Phe His Met His Ile Gly Leu Ser Ile Met Lys Val Val Leu Gly Leu

370 375 380

gct ctt cag ttc ctc tgc agc tat atc acc ttc ccc ctc tac gcg ctc 1200

Ala Leu Gln Phe Leu Cys Ser Tyr Ile Thr Phe Pro Leu Tyr Ala Leu

385 390 395 400

gtc aca cag atg gga tcg aac atg aag agg tcc atc ttc gac gag cag 1248

Val Thr Gln Met Gly Ser Asn Met Lys Arg Ser Ile Phe Asp Glu Gln

405 410 415

acg gcc aag gcg ctg acc aac tgg cgg aac acg gcc aag gag aag aag 1296

Thr Ala Lys Ala Leu Thr Asn Trp Arg Asn Thr Ala Lys Glu Lys Lys

420 425 430

aag gtc cga gac acg gac atg ctg atg gcg cag atg atc ggc gac gcg 1344

Lys Val Arg Asp Thr Asp Met Leu Met Ala Gln Met Ile Gly Asp Ala

435 440 445

acg ccc agc cga ggc acg tcg ccg atg cct agc cgg gct tcg tca ccg 1392

Thr Pro Ser Arg Gly Thr Ser Pro Met Pro Ser Arg Ala Ser Ser Pro

450 455 460

gtg cac ctg ctt cac aag ggc atg gga cgg tcc gac gat ccc cag agc 1440

Val His Leu Leu His Lys Gly Met Gly Arg Ser Asp Asp Pro Gln Ser

465 470 475 480

gcg ccg acc tcg cca agg acc atg gag gag gct agg gac atg tac ccg 1488

Ala Pro Thr Ser Pro Arg Thr Met Glu Glu Ala Arg Asp Met Tyr Pro

485 490 495

gtt gtg gtg gcg cat ccc gtg cac aga cta aat cct gct gac agg cgg 1536

Val Val Val Ala His Pro Val His Arg Leu Asn Pro Ala Asp Arg Arg

500 505 510

agg tcg gtc tct tcg tcg gca ctc gat gcc gac atc ccc agc gca gat 1584

Arg Ser Val Ser Ser Ser Ala Leu Asp Ala Asp Ile Pro Ser Ala Asp

515 520 525

ttt tcc ttc agc cag gga tgagacaa gtttctgtat tgatgttagt ccaatgtata 1640

Phe Ser Phe Ser Gln Gly

530

gccaacatag gatgtcatga ttcgtacaat aagaaataca aatttttact gag 1693

〈210〉 6

<211> 534

<212> PRT

213 Triticum sp.

<400> 6

Met Ala Glu Asp Tyr Glu Tyr Pro Pro Ala Arg Thr Leu Pro Glu Thr

1 5 10 15

Pro Ser Trp Ala Val Ala Leu Val Phe Ala Val Met Ile Ile Val Ser

20 25 30

Val Leu Leu Glu His Ala Leu His Lys Leu Gly His Trp Phe His Lys

35 40 45

Arg His Lys Asn Ala Leu Ala Glu Ala Leu Glu Lys Ile Lys Ala Glu

50 55 60

Leu Met Leu Val Gly Phe Ile Ser Leu Leu Leu Ala Val Thr Gln Asp

65 70 75 80

Pro Ile Ser Gly Ile Cys Ile Ser Glu Lys Ala Ala Ser Ile Met Arg

85 90 95

Pro Cys Ser Leu Pro Pro Gly Ser Val Lys Ser Lys Tyr Lys Asp Tyr

100 105 110

Tyr Cys Ala Lys Lys Gly Lys Val Ser Leu Met Ser Thr Gly Ser Leu

115 120 125

His Gln Leu His Met Phe Ile Phe Val Leu Ala Val Phe His Val Thr

130 135 140

Tyr Ser Val Ile Ile Met Ala Leu Ser Arg Leu Lys Met Arg Thr Trp

145 150 155 160

Lys Lys Trp Glu Thr Glu Thr Ala Ser Leu Glu Tyr Gln Phe Ala Asn

165 170 175

Asp Pro Ala Arg Phe Arg Phe Thr His Gln Thr Ser Phe Val Lys Arg

180 185 190

His Leu Gly Leu Ser Ser Thr Pro Gly Ile Arg Trp Val Val Ala Phe

195 200 205

Phe Arg Gln Phe Phe Arg Ser Val Thr Lys Val Asp Tyr Leu Thr Leu

210 215 220

Arg Ala Gly Phe Ile Asn Ala His Leu Ser His Asn Ser Lys Phe Asp

225 230 235 240

Phe His Lys Tyr Ile Lys Arg Ser Met Glu Asp Asp Phe Lys Val Val

245 250 255

Val Gly Ile Ser Leu Pro Leu Trp Cys Val Ala Ile Leu Thr Leu Phe

260 265 270

Leu Asp Ile Asp Gly Ile Gly Thr Leu Thr Trp Ile Ser Phe Ile Pro

275 280 285

Leu Val Ile Leu Leu Cys Val Gly Thr Lys Leu Glu Met Ile Ile Met

290 295 300

Glu Met Ala Leu Glu Ile Gln Asp Arg Ala Ser Val Ile Lys Gly Ala

305 310 315 320

Pro Val Val Glu Pro Ser Asn Lys Phe Phe Trp Phe His Arg Pro Asp

325 330 335

Trp Val Leu Phe Phe Ile His Leu Thr Leu Phe Gln Asn Ala Phe Gln

340 345 350

Met Ala His Phe Val Trp Thr Val Ala Thr Pro Gly Leu Lys Lys Cys

355 360 365

Phe His Met His Ile Gly Leu Ser Ile Met Lys Val Val Leu Gly Leu

370 375 380

Ala Leu Gln Phe Leu Cys Ser Tyr Ile Thr Phe Pro Leu Tyr Ala Leu

385 390 395 400

Val Thr Gln Met Gly Ser Asn Met Lys Arg Ser Ile Phe Asp Glu Gln

405 410 415

Thr Ala Lys Ala Leu Thr Asn Trp Arg Asn Thr Ala Lys Glu Lys Lys

420 425 430

Lys Val Arg Asp Thr Asp Met Leu Met Ala Gln Met Ile Gly Asp Ala

435 440 445

Thr Pro Ser Arg Gly Thr Ser Pro Met Pro Ser Arg Ala Ser Ser Pro

450 455 460

Val His Leu Leu His Lys Gly Met Gly Arg Ser Asp Asp Pro Gln Ser

465 470 475 480

Ala Pro Thr Ser Pro Arg Thr Met Glu Glu Ala Arg Asp Met Tyr Pro

485 490 495

Val Val Val Ala His Pro Val His Arg Leu Asn Pro Ala Asp Arg Arg

500 505 510

Arg Ser Val Ser Ser Ser Ala Leu Asp Ala Asp Ile Pro Ser Ala Asp

515 520 525

Phe Ser Phe Ser Gln Gly

530

〈210〉 7

<211> 1886

<212> DNA

213 Triticum sp.

〈220〉

<221> CDS

222 (198) .. (1799)

〈220〉

〈221〉 misc_feature

222 (387) .. (425)

223 location of the amino acid sequence set forth in SEQ ID No: 1

〈220〉

〈221〉 misc_feature

222 (1374) .. (1415)

<223> location of the amino acid sequence set forth in SEQ ID No: 2

<400> 7

gcagcaacaa gctagacata cctgcgtgcg tacgtacgtt ttcgttttcc tttcttgctc 60

cggccggccg gccggccacg tagaatagat acctgcccag gtacgtacct cgttggctca 120

gacgatcggc ggttggactt gggtgcgcgc cctgccctgc tccggccaag gaaagaggtt 180

gcgctaaaga cgggcgg atg gca aag gac gac ggg tac ccc ccg gcg cgg 230

Met Ala Lys Asp Asp Gly Tyr Pro Pro Ala Arg

1 5 10

acg ctg ccg gag acg ccg tcc tgg gcg gtg gcg ctg gtc ttc gcc gtc 278

Thr Leu Pro Glu Thr Pro Ser Trp Ala Val Ala Leu Val Phe Ala Val

15 20 25

atg atc atc gtc tcc gtc ctc ctg gag cac gcg ctc cac aag ctc ggc 326

Met Ile Ile Val Ser Val Leu Leu Glu His Ala Leu His Lys Leu Gly

30 35 40

cat tgg ttc cac aag cgg cac aag aac gcg ctg gcg gag gcg ctg gag 374

His Trp Phe His Lys Arg His Lys Asn Ala Leu Ala Glu Ala Leu Glu

45 50 55

aag atg aag gcg gag ctg atg ctg gtg gga ttc atc tcg ctg ctg ctc 422

Lys Met Lys Ala Glu Leu Met Leu Val Gly Phe Ile Ser Leu Leu Leu

60 65 70 75

gcc gtc acg cag gac cca atc tcc ggg ata tgc atc tcc cag aag gcc 470

Ala Val Thr Gln Asp Pro Ile Ser Gly Ile Cys Ile Ser Gln Lys Ala

80 85 90

gcc agc atc atg cgc ccc tgc aag gtg gaa ccc ggt tcc gtc aag agc 518

Ala Ser Ile Met Arg Pro Cys Lys Val Glu Pro Gly Ser Val Lys Ser

95 100 105

aag tac aag gac tac tac tgc gcc aaa gag ggc aag gtg gcg ctc atg 566

Lys Tyr Lys Asp Tyr Tyr Cys Ala Lys Glu Gly Lys Val Ala Leu Met

110 115 120

tcc acg ggc agc ctg cac cag ctc cac ata ttc atc ttc gtg cta gcc 614

Ser Thr Gly Ser Leu His Gln Leu His Ile Phe Ile Phe Val Leu Ala

125 130 135

gtc ttc cat gtc acc tac agc gtc atc atc atg gct cta agc cgt ctc 662

Val Phe His Val Thr Tyr Ser Val Ile Met Ala Leu Ser Arg Leu

140 145 150 155

aag atg aga aca tgg aag aaa tgg gag aca gaa acc gcc tcc ttg gaa 710

Lys Met Arg Thr Trp Lys Lys Trp Glu Thr Glu Thr Ala Ser Leu Glu

160 165 170

tac cag ttc gca aat gat cct gcg cgg ttc cgc ttc acg cac cag acg 758

Tyr Gln Phe Ala Asn Asp Pro Ala Arg Phe Arg Phe Thr His Gln Thr

175 180 185

tcg ttc gtg aag cgg cac ctg ggc ctg tcc agc acc ccc ggc gtc aga 806

Ser Phe Val Lys Arg His Leu Gly Leu Ser Ser Thr Pro Gly Val Arg

190 195 200

tgg gtg gtg gcc ttc ttc agg cag ttc ttc agg tcg gtc acc aag gtg 854

Trp Val Val Ala Phe Phe Arg Gln Phe Phe Arg Ser Val Thr Lys Val

205 210 215

gac tac ctc acc ttg agg gca ggc ttc atc aac gcg cac ttg tcg cag 902

Asp Tyr Leu Thr Leu Arg Ala Gly Phe Ile Asn Ala His Leu Ser Gln

220 225 230 235

aac agc aag ttc gac ttc cac aag tac atc aag agg tcc atg gag gac 950

Asn Ser Lys Phe Asp Phe His Lys Tyr Ile Lys Arg Ser Met Glu Asp

240 245 250

gac ttc aaa gtc gtc gtt ggc atc agc ctc ccg ctg tgg gct gtg gcg 998

Asp Phe Lys Val Val Val Gly Ile Ser Leu Pro Leu Trp Ala Val Ala

255 260 265

atc ctc acc ctc ttc ctt gat atc gac ggg atc ggc aca ctc acc tgg 1046

Ile Leu Thr Leu Phe Leu Asp Ile Asp Gly Ile Gly Thr Leu Thr Trp

270 275 280

gtt tct ttc atc cct ctc atc atc ctc ttg tgt gtt gga acc aag cta 1094

Val Ser Phe Ile Pro Leu Ile Ile Leu Leu Cys Val Gly Thr Lys Leu

285 290 295

gag atg atc atc atg ggg atg gcc ctg gag atc cag gac cgg tcg agc 1142

Glu Met Ile Ile Met Gly Met Ala Leu Glu Ile Gln Asp Arg Ser Ser

300 305 310 315

gtc atc aag ggg gca ccc gtg gtc gag ccc agc aac aag ttc ttc tgg 1190

Val Ile Lys Gly Ala Pro Val Val Glu Pro Ser Asn Lys Phe Phe Trp

320 325 330

ttc cac cgc ccc gac tgg gtc ctc ttc ttc ata cac ctg acg ctg ttc 1238

Phe His Arg Pro Asp Trp Val Leu Phe Ile His Leu Thr Leu Phe

335 340 345

cag aac gcg ttt cag atg gca cat ttc gtg tgg aca gtg gcc acg ccc 1286

Gln Asn Ala Phe Gln Met Ala His Phe Val Trp Thr Val Ala Thr Pro

350 355 360

ggc ttg aag gac tgc ttc cat atg aac atc ggg ctg agc atc atg aag 1334

Gly Leu Lys Asp Cys Phe His Met Asn Ile Gly Leu Ser Ile Met Lys

365 370 375

gtc gtg ctg ggg ctg gct ctc cag ttc ctg tgc agc tac atc acc ttc 1382

Val Val Leu Gly Leu Ala Leu Gln Phe Leu Cys Ser Tyr Ile Thr Phe

380 385 390 395

ccc ctc tac gcg cta gtc aca cag atg gga tca aac atg aag agg tcc 1430

Pro Leu Tyr Ala Leu Val Thr Gln Met Gly Ser Asn Met Lys Arg Ser

400 405 410

atc ttc gac gag cag aca gcc aag gcg ctg acc aac tgg cgg aac acg 1478

Ile Phe Asp Glu Gln Thr Ala Lys Ala Leu Thr Asn Trp Arg Asn Thr

415 420 425

gcc aag gag aag aag aag gtc cga gac acg gac atg ctg atg gcg cag 1526

Ala Lys Glu Lys Lys Lys Val Arg Asp Thr Asp Met Leu Met Ala Gln

430 435 440

atg atc ggc gac gca aca ccc agc cga ggc acg tcc ccg atg cct agc 1574

Met Ile Gly Asp Ala Thr Pro Ser Arg Gly Thr Ser Pro Met Pro Ser

445 450 455

cgg ggc tca tcg ccg gtg cac ctg ctt cag aag ggc atg gga cgg tct 1622

Arg Gly Ser Ser Pro Val His Leu Leu Gln Lys Gly Met Gly Arg Ser

460 465 470 475

gac gat ccc cag agc gca ccg acc tcg cca agg acc atg gag gag gct 1670

Asp Asp Pro Gln Ser Ala Pro Thr Ser Pro Arg Thr Met Glu Glu Ala

480 485 490

agg gac atg tac ccg gtt gtg gtg gcg cat cct gta cac aga cta aat 1718

Arg Asp Met Tyr Pro Val Val Val Ala His Pro Val His Arg Leu Asn

495 500 505

cct gct gac agg cgg agg tcg gtc tct tca tca gcc ctc gat gcc gac 1766

Pro Ala Asp Arg Arg Arg Ser Val Ser Ser Ser Ala Leu Asp Ala Asp

510 515 520

atc ccc agc gca gat ttt tcc ttc agc cag gga t gagacaagtt 1810

Ile Pro Ser Ala Asp Phe Ser Phe Ser Gln Gly

525 530

tctgtattga tgttagtcca atgtatagcc aacataggat gtgatgattc gtacaataag 1870

aaatacaatt ttttac 1886

〈210〉 8

<211> 534

<212> PRT

213 Triticum sp.

<400> 8

Met Ala Lys Asp Asp Gly Tyr Pro Pro Ala Arg Thr Leu Pro Glu Thr

1 5 10 15

Pro Ser Trp Ala Val Ala Leu Val Phe Ala Val Met Ile Ile Val Ser

20 25 30

Val Leu Leu Glu His Ala Leu His Lys Leu Gly His Trp Phe His Lys

35 40 45

Arg His Lys Asn Ala Leu Ala Glu Ala Leu Glu Lys Met Lys Ala Glu

50 55 60

Leu Met Leu Val Gly Phe Ile Ser Leu Leu Leu Ala Val Thr Gln Asp

65 70 75 80

Pro Ile Ser Gly Ile Cys Ile Ser Gln Lys Ala Ala Ser Ile Met Arg

85 90 95

Pro Cys Lys Val Glu Pro Gly Ser Val Lys Ser Lys Tyr Lys Asp Tyr

100 105 110

Tyr Cys Ala Lys Glu Gly Lys Val Ala Leu Met Ser Thr Gly Ser Leu

115 120 125

His Gln Leu His Ile Phe Ile Phe Val Leu Ala Val Phe His Val Thr

130 135 140

Tyr Ser Val Ile Ile Met Ala Leu Ser Arg Leu Lys Met Arg Thr Trp

145 150 155 160

Lys Lys Trp Glu Thr Glu Thr Ala Ser Leu Glu Tyr Gln Phe Ala Asn

165 170 175

Asp Pro Ala Arg Phe Arg Phe Thr His Gln Thr Ser Phe Val Lys Arg

180 185 190

His Leu Gly Leu Ser Ser Thr Pro Gly Val Arg Trp Val Val Ala Phe

195 200 205

Phe Arg Gln Phe Phe Arg Ser Val Thr Lys Val Asp Tyr Leu Thr Leu

210 215 220

Arg Ala Gly Phe Ile Asn Ala His Leu Ser Gln Asn Ser Lys Phe Asp

225 230 235 240

Phe His Lys Tyr Ile Lys Arg Ser Met Glu Asp Asp Phe Lys Val Val

245 250 255

Val Gly Ile Ser Leu Pro Leu Trp Ala Val Ala Ile Leu Thr Leu Phe

260 265 270

Leu Asp Ile Asp Gly Ile Gly Thr Leu Thr Trp Val Ser Phe Ile Pro

275 280 285

Leu Ile Ile Leu Leu Cys Val Gly Thr Lys Leu Glu Met Ile Ile Met

290 295 300

Gly Met Ala Leu Glu Ile Gln Asp Arg Ser Ser Val Ile Lys Gly Ala

305 310 315 320

Pro Val Val Glu Pro Ser Asn Lys Phe Phe Trp Phe His Arg Pro Asp

325 330 335

Trp Val Leu Phe Phe Ile His Leu Thr Leu Phe Gln Asn Ala Phe Gln

340 345 350

Met Ala His Phe Val Trp Thr Val Ala Thr Pro Gly Leu Lys Asp Cys

355 360 365

Phe His Met Asn Ile Gly Leu Ser Ile Met Lys Val Val Leu Gly Leu

370 375 380

Ala Leu Gln Phe Leu Cys Ser Tyr Ile Thr Phe Pro Leu Tyr Ala Leu

385 390 395 400

Val Thr Gln Met Gly Ser Asn Met Lys Arg Ser Ile Phe Asp Glu Gln

405 410 415

Thr Ala Lys Ala Leu Thr Asn Trp Arg Asn Thr Ala Lys Glu Lys Lys

420 425 430

Lys Val Arg Asp Thr Asp Met Leu Met Ala Gln Met Ile Gly Asp Ala

435 440 445

Thr Pro Ser Arg Gly Thr Ser Pro Met Pro Ser Arg Gly Ser Ser Pro

450 455 460

Val His Leu Leu Gln Lys Gly Met Gly Arg Ser Asp Asp Pro Gln Ser

465 470 475 480

Ala Pro Thr Ser Pro Arg Thr Met Glu Glu Ala Arg Asp Met Tyr Pro

485 490 495

Val Val Val Ala His Pro Val His Arg Leu Asn Pro Ala Asp Arg Arg

500 505 510

Arg Ser Val Ser Ser Ser Ala Leu Asp Ala Asp Ile Pro Ser Ala Asp

515 520 525

Phe Ser Phe Ser Gln Gly

530

〈210〉 9

<211> 2197

<212> DNA

〈213〉 Arabidopsis thaliana

〈220〉

<221> CDS

222 (331) .. (2037)

〈220〉

〈221〉 misc_feature

222 (589) .. (627)

223 location of the amino acid sequence set forth in SEQ ID No: 1

〈220〉

〈221〉 misc_feature

222 (1603) .. (1644)

<223> location of the amino acid sequence set forth in SEQ ID No: 2

<400> 9

agtaatttag ctgttcttct acccctctga tctctcacag gggatcaaat agttttgata 60

catagagcca caacagtgac attagtgtgt tgttgactac tgtaagggtt gggttttgaa 120

aagagacatg aaggagtgtt attaggttga ttgtcttcaa gtacctccag tgtcaaacaa 180

acattgacga ttgattctct tcccataatt tattgttatg cattacatat cacagtaaac 240

ggactttcaa gtcaacaccg catttatttg ccctcttcat tgtttcacgt acgtaatcaa 300

ggaccaaggg attttgttct tttggctacc atg gcc aca aga tgc ttt tgg tgt 354

Met Ala Thr Arg Cys Phe Trp Cys

1 5

tgg acc act ttg ctc ttc tgc tct cag ctg ctt acc ggc ttt gcc cga 402

Trp Thr Thr Leu Leu Phe Cys Ser Gln Leu Leu Thr Gly Phe Ala Arg

10 15 20

gct tcc tct gca ggc ggc gcc aaa gag aaa gga ctc tcc caa act ccc 450

Ala Ser Ser Ala Gly Gly Ala Lys Glu Lys Gly Leu Ser Gln Thr Pro

25 30 35 40

acc tgg gcc gtt gcc ctc gtc tgt acc ttt ttc att ctt gtc tcc gtc 498

Thr Trp Ala Val Ala Leu Val Cys Thr Phe Phe Ile Leu Val Ser Val

45 50 55

ctt ctc gag aag gct ctt cac aga gtt gcc acg tgg ttg tgg gag aaa 546

Leu Leu Glu Lys Ala Leu His Arg Val Ala Thr Trp Leu Trp Glu Lys

60 65 70

cat aag aac tct ctg ctt gaa gcc ttg gaa aaa ata aag gcc gag ctg 594

His Lys Asn Ser Leu Leu Glu Ala Leu Glu Lys Ile Lys Ala Glu Leu

75 80 85

atg att cta gga ttc att tcc ttg ttg cta acc ttc gga gag cag tac 642

Met Ile Leu Gly Phe Ile Ser Leu Leu Leu Thr Phe Gly Glu Gln Tyr

90 95 100

att ctc aag att tgt att cct gaa aag gct gca gcc tct atg tta cct 690

Ile Leu Lys Ile Cys Ile Pro Glu Lys Ala Ala Ala Ser Met Leu Pro

105 110 115 120

tgt cca gct cct tct act cat gac caa gac aag acc cac cgc aga cgt 738

Cys Pro Ala Pro Ser Thr His Asp Gln Asp Lys Thr His Arg Arg Arg

125 130 135

cta gct gct gct acg acc tct tcc cgc tgc gat gag ggt cat gaa cca 786

Leu Ala Ala Ala Thr Thr Ser Ser Arg Cys Asp Glu Gly His Glu Pro

140 145 150

ctc ata cct gcc acg ggt ttg cac cag cta cac att cta ttg ttc ttc 834

Leu Ile Pro Ala Thr Gly Leu His Gln Leu His Ile Leu Leu Phe Phe

155 160 165

atg gct gcc ttt cat atc ctc tac agt ttc atc acc atg atg ctt ggc 882

Met Ala Ala Phe His Ile Leu Tyr Ser Phe Ile Thr Met Met Leu Gly

170 175 180

aga ctc aag atc cgt ggc tgg aaa aag tgg gag cag gag aca tgt tct 930

Arg Leu Lys Ile Arg Gly Trp Lys Lys Trp Glu Gln Glu Thr Cys Ser

185 190 195 200

cat gat tac gag ttt tca atc gat cca tca aga ttc aga ctc act cat 978

His Asp Tyr Glu Phe Ser Ile Asp Pro Ser Arg Phe Arg Leu Thr His

205 210 215

gag acg tcc ttt gtt aga caa cat tcc agt ttc tgg aca aaa atc ccc 1026

Glu Thr Ser Phe Val Arg Gln His Ser Ser Phe Trp Thr Lys Ile Pro

220 225 230

ttc ttc ttt tat gct ggg tgc ttc cta cag cag ttt ttc cga tct gtc 1074

Phe Phe Phe Tyr Ala Gly Cys Phe Leu Gln Gln Phe Phe Arg Ser Val

235 240 245

ggg agg act gac tac tta act ctg cgc cat ggc ttc atc gct gcc cat 1122

Gly Arg Thr Asp Tyr Leu Thr Leu Arg His Gly Phe Ile Ala Ala His

250 255 260

tta gct cca gga aga aag ttc gac ttc cag aag tat atc aaa aga tca 1170

Leu Ala Pro Gly Arg Lys Phe Asp Phe Gln Lys Tyr Ile Lys Arg Ser

265 270 275 280

ttg gaa gac gat ttc aag gtg gta gtt gga ata agt cct ctt ttg tgg 1218

Leu Glu Asp Asp Phe Lys Val Val Val Gly Ile Ser Pro Leu Leu Trp

285 290 295

gca tca ttt gta att ttc cta ctt ctg aat gtt aat ggc tgg gaa gca 1266

Ala Ser Phe Val Ile Phe Leu Leu Leu Asn Val Asn Gly Trp Glu Ala

300 305 310

ttg ttt tgg gcg tca atc cta cct gta ctt atc att cta gct gtc agt 1314

Leu Phe Trp Ala Ser Ile Leu Pro Val Leu Ile Ile Leu Ala Val Ser

315 320 325

acg aag ctt caa gcg atc cta aca aga atg gct ctg gga atc acg gag 1362

Thr Lys Leu Gln Ala Ile Leu Thr Arg Met Ala Leu Gly Ile Thr Glu

330 335 340

aga cac gca gtt gtt caa ggg ata cct ctc gtg cat ggt tca gat aag 1410

Arg His Ala Val Val Gln Gly Ile Pro Leu Val His Gly Ser Asp Lys

345 350 355 360

tac ttt tgg ttt aat cgc cct cag ttg cta ctt cat ctt ctt cac ttc 1458

Tyr Phe Trp Phe Asn Arg Pro Gln Leu Leu Leu His Leu Leu His Phe

365 370 375

gcc tta ttt cag aat gct ttc cag cta aca tac ttc ttc tgg gtc tgg 1506

Ala Leu Phe Gln Asn Ala Phe Gln Leu Thr Tyr Phe Phe Trp Val Trp

380 385 390

tat tcc ttt ggg cta aaa tct tgc ttt cac acg gat ttc aaa cta gtc 1554

Tyr Ser Phe Gly Leu Lys Ser Cys Phe His Thr Asp Phe Lys Leu Val

395 400 405

atc gta aaa ctc tct cta ggc gtt gga gct ttg att ttg tgc agc tac 1602

Ile Val Lys Leu Ser Leu Gly Val Gly Ala Leu Ile Leu Cys Ser Tyr

410 415 420

atc aca ctt cct ttg tat gca cta gtt act cag atg ggt tca aac atg 1650

Ile Thr Leu Pro Leu Tyr Ala Leu Val Thr Gln Met Gly Ser Asn Met

425 430 435 440

aag aaa gct gtg ttt gat gag caa atg gca aaa gcg ttg aag aaa tgg 1698

Lys Lys Ala Val Phe Asp Glu Gln Met Ala Lys Ala Leu Lys Lys Trp

445 450 455

cac atg act gtg aag aag aag aaa ggc aaa gcg aga aag cca cca aca 1746

His Met Thr Val Lys Lys Lys Lys Gly Lys Ala Arg Lys Pro Pro Thr

460 465 470

gag acc ctt ggt gtt tct gac act gtc agc acc tct acc tca tcc ttt 1794

Glu Thr Leu Gly Val Ser Asp Thr Val Ser Thr Ser Thr Ser Ser Phe

475 480 485

cac gcc tct gga gcc act cta ctc cgc tcc aag acc act ggt cac tcg 1842

His Ala Ser Gly Ala Thr Leu Leu Arg Ser Lys Thr Thr Gly His Ser

490 495 500

aca gcc tct tat atg agt aat ttc gag gac caa agc atg tct gat ctt 1890

Thr Ala Ser Tyr Met Ser Asn Phe Glu Asp Gln Ser Met Ser Asp Leu

505 510 515 520

gaa gct gag cca tta tcc cct gaa cca ata gag ggg cac act ctc gtc 1938

Glu Ala Glu Pro Leu Ser Pro Glu Pro Ile Glu Gly His Thr Leu Val

525 530 535

agg gtt ggt gat cag aac aca gag ata gaa tat act gga gat att agt 1986

Arg Val Gly Asp Gln Asn Thr Glu Ile Glu Tyr Thr Gly Asp Ile Ser

540 545 550

cct gga aac caa ttc tcc ttt gtg aag aac gtt cct gct aat gat att 2034

Pro Gly Asn Gln Phe Ser Phe Val Lys Asn Val Pro Ala Asn Asp Ile

555 560 565

gac taa tattcaaaat gaatgcagaa caaatccatc atccggtctt tattttctat 2090

Asp

tacatgtatg ccaacaattg cttcgccaag tgttaccaac taggttttct gtataaggct 2150

gtattttaga gctaaaaaaa aaaaaaaaaa aaaaaaacta aattact 2197

<210> 10

<211> 569

<212> PRT

〈213〉 Arabidopsis thaliana

<400> 10

Met Ala Thr Arg Cys Phe Trp Cys Trp Thr Thr Leu Leu Phe Cys Ser

1 5 10 15

Gln Leu Leu Thr Gly Phe Ala Arg Ala Ser Ser Ala Gly Gly Ala Lys

20 25 30

Glu Lys Gly Leu Ser Gln Thr Pro Thr Trp Ala Val Ala Leu Val Cys

35 40 45

Thr Phe Phe Ile Leu Val Ser Val Leu Leu Glu Lys Ala Leu His Arg

50 55 60

Val Ala Thr Trp Leu Trp Glu Lys His Lys Asn Ser Leu Leu Glu Ala

65 70 75 80

Leu Glu Lys Ile Lys Ala Glu Leu Met Ile Leu Gly Phe Ile Ser Leu

85 90 95

Leu Leu Thr Phe Gly Glu Gln Tyr Ile Leu Lys Ile Cys Ile Pro Glu

100 105 110

Lys Ala Ala Ala Ser Met Leu Pro Cys Pro Ala Pro Ser Thr His Asp

115 120 125

Gln Asp Lys Thr His Arg Arg Arg Leu Ala Ala Ala Thr Thr Ser Ser

130 135 140

Arg Cys Asp Glu Gly His Glu Pro Leu Ile Pro Ala Thr Gly Leu His

145 150 155 160

Gln Leu His Ile Leu Leu Phe Phe Met Ala Ala Phe His Ile Leu Tyr

165 170 175

Ser Phe Ile Thr Met Met Leu Gly Arg Leu Lys Ile Arg Gly Trp Lys

180 185 190

Lys Trp Glu Gln Glu Thr Cys Ser His Asp Tyr Glu Phe Ser Ile Asp

195 200 205

Pro Ser Arg Phe Arg Leu Thr His Glu Thr Ser Phe Val Arg Gln His

210 215 220

Ser Ser Phe Trp Thr Lys Ile Pro Phe Phe Phe Tyr Ala Gly Cys Phe

225 230 235 240

Leu Gln Gln Phe Phe Arg Ser Val Gly Arg Thr Asp Tyr Leu Thr Leu

245 250 255

Arg His Gly Phe Ile Ala Ala His Leu Ala Pro Gly Arg Lys Phe Asp

260 265 270

Phe Gln Lys Tyr Ile Lys Arg Ser Leu Glu Asp Asp Phe Lys Val Val

275 280 285

Val Gly Ile Ser Pro Leu Leu Trp Ala Ser Phe Val Ile Phe Leu Leu

290 295 300

Leu Asn Val Asn Gly Trp Glu Ala Leu Phe Trp Ala Ser Ile Leu Pro

305 310 315 320

Val Leu Ile Ile Leu Ala Val Ser Thr Lys Leu Gln Ala Ile Leu Thr

325 330 335

Arg Met Ala Leu Gly Ile Thr Glu Arg His Ala Val Val Gln Gly Ile

340 345 350

Pro Leu Val His Gly Ser Asp Lys Tyr Phe Trp Phe Asn Arg Pro Gln

355 360 365

Leu Leu Leu His Leu Leu His Phe Ala Leu Phe Gln Asn Ala Phe Gln

370 375 380

Leu Thr Tyr Phe Phe Trp Val Trp Tyr Ser Phe Gly Leu Lys Ser Cys

385 390 395 400

Phe His Thr Asp Phe Lys Leu Val Ile Val Lys Leu Ser Leu Gly Val

405 410 415

Gly Ala Leu Ile Leu Cys Ser Tyr Ile Thr Leu Pro Leu Tyr Ala Leu

420 425 430

Val Thr Gln Met Gly Ser Asn Met Lys Lys Ala Val Phe Asp Glu Gln

435 440 445

Met Ala Lys Ala Leu Lys Lys Trp His Met Thr Val Lys Lys Lys Lys

450 455 460

Gly Lys Ala Arg Lys Pro Pro Thr Glu Thr Leu Gly Val Ser Asp Thr

465 470 475 480

Val Ser Thr Ser Thr Ser Ser Phe His Ala Ser Gly Ala Thr Leu Leu

485 490 495

Arg Ser Lys Thr Thr Gly His Ser Thr Ala Ser Tyr Met Ser Asn Phe

500 505 510

Glu Asp Gln Ser Met Ser Asp Leu Glu Ala Glu Pro Leu Ser Pro Glu

515 520 525

Pro Ile Glu Gly His Thr Leu Val Arg Val Gly Asp Gln Asn Thr Glu

530 535 540

Ile Glu Tyr Thr Gly Asp Ile Ser Pro Gly Asn Gln Phe Ser Phe Val

545 550 555 560

Lys Asn Val Pro Ala Asn Asp Ile Asp

565

<210> 11

<211> 1935

<212> DNA

〈213〉 Arabidopsis thaliana

〈220〉

<221> CDS

222 (89) .. (1807)

〈220〉

〈221〉 misc_feature

222 (269) .. (307)

223 location of the amino acid sequence set forth in SEQ ID No: 1

〈220〉

〈221〉 misc_feature

222 (1370) .. (1411)

<223> location of the amino acid sequence set forth in SEQ ID No: 2

<400> 11

cagtgtgagt aatttagtaa aaagacaaga tctctggtct ggaattagaa gaatcttatt 60

tgggtttttt tcttaggatt aagctcta atg gca gat caa gta aaa gag cgg 112

Met Ala Asp Gln Val Lys Glu Arg

1 5

act tta gag gag acc tct acg tgg gca gtt gct gtt gtt tgc ttt gtc 160

Thr Leu Glu Glu Thr Ser Thr Trp Ala Val Ala Val Val Cys Phe Val

10 15 20

tta ctc ttt att tcg att gtc ctc gaa cat tct att cac aaa att gga 208

Leu Leu Phe Ile Ser Ile Val Leu Glu His Ser Ile His Lys Ile Gly

25 30 35 40

acc tgg ttt aaa aag aag cac aag cag gct ctt ttt gaa gct ctt gaa 256

Thr Trp Phe Lys Lys Lys His Lys Gln Ala Leu Phe Glu Ala Leu Glu

45 50 55

aag gtc aaa gca gag ctt atg ctg ttg gga ttc ata tca cta cta ctc 304

Lys Val Lys Ala Glu Leu Met Leu Leu Gly Phe Ile Ser Leu Leu Leu

60 65 70

aca att gga caa aca cca atc tca aat atc tgc atc tcc cag aaa gtt 352

Thr Ile Gly Gln Thr Pro Ile Ser Asn Ile Cys Ile Ser Gln Lys Val

75 80 85

gcg tca aca atg cac cct tgc agt gct gct gaa gaa gct aaa aaa tac 400

Ala Ser Thr Met His Pro Cys Ser Ala Ala Glu Glu Ala Lys Lys Tyr

90 95 100

ggc aag aaa gac gcc gga aag aaa gat gat gga gat gga gat aaa ccc 448

Gly Lys Lys Asp Ala Gly Lys Lys Asp Asp Gly Asp Gly Asp Lys Pro

105 110 115 120

ggt cga aga ctt ctt ctt gag tta gct gaa tct tat atc cat aga aga 496

Gly Arg Arg Leu Leu Leu Glu Leu Ala Glu Ser Tyr Ile His Arg Arg

125 130 135

agt tta gcc acc aaa ggc tat gac aaa tgt gca gag aag ggg aaa gtg 544

Ser Leu Ala Thr Lys Gly Tyr Asp Lys Cys Ala Glu Lys Gly Lys Val

140 145 150

gct ttt gta tct gct tat gga atc cac cag ctg cat ata ttc atc ttc 592

Ala Phe Val Ser Ala Tyr Gly Ile His Gln Leu His Ile Phe Ile Phe

155 160 165

gtg ctc gcg gtt gtt cat gtt gtt tac tgc att gtt act tat gct ttc 640

Val Leu Ala Val Val His Val Val Tyr Cys Ile Val Thr Tyr Ala Phe

170 175 180

gga aag atc aag atg agg acg tgg aag tcg tgg gag gaa gag aca aag 688

Gly Lys Ile Lys Met Arg Thr Trp Lys Ser Trp Glu Glu Glu Thr Lys

185 190 195 200

aca ata gag tat cag tat tcc aac gat cct gag agg ttc agg ttt gcg 736

Thr Ile Glu Tyr Gln Tyr Ser Asn Asp Pro Glu Arg Phe Arg Phe Ala

205 210 215

agg gac aca tct ttt ggg aga aga cat ctc aat ttc tgg agc aag acg 784

Arg Asp Thr Ser Phe Gly Arg Arg His Leu Asn Phe Trp Ser Lys Thr

220 225 230

aga gtc aca cta tgg att gtt tgt ttt ttt aga cag ttc ttt gga tct 832

Arg Val Thr Leu Trp Ile Val Cys Phe Phe Arg Gln Phe Phe Gly Ser

235 240 245

gtc acc aaa gtt gat tac tta gca cta aga cat ggt ttc atc atg gcg 880

Val Thr Lys Val Asp Tyr Leu Ala Leu Arg His Gly Phe Ile Met Ala

250 255 260

cat ttt gct ccc ggt aac gaa tca aga ttc gat ttc cgc aag tat att 928

His Phe Ala Pro Gly Asn Glu Ser Arg Phe Asp Phe Arg Lys Tyr Ile

265 270 275 280

cag aga tca tta gag aaa gac ttc aaa acc gtt gtt gaa atc agt ccg 976

Gln Arg Ser Leu Glu Lys Asp Phe Lys Thr Val Val Glu Ile Ser Pro

285 290 295

gtt atc tgg ttt gtc gct gtg cta ttc ctc ttg acc aat tca tat gga 1024

Val Ile Trp Phe Val Ala Val Leu Phe Leu Leu Thr Asn Ser Tyr Gly

300 305 310

tta cgt tct tac ctc tgg tta cca ttc att cca cta gtc gta att cta 1072

Leu Arg Ser Tyr Leu Trp Leu Pro Phe Ile Pro Leu Val Val Ile Leu

315 320 325

ata gtt gga aca aag ctt gaa gtc ata ata aca aaa ttg ggt cta aga 1120

Ile Val Gly Thr Lys Leu Glu Val Ile Ile Thr Lys Leu Gly Leu Arg

330 335 340

atc caa gag aaa ggt gat gtg gtg aga ggc gcc cca gtg gtt cag cct 1168

Ile Gln Glu Lys Gly Asp Val Val Arg Gly Ala Pro Val Val Gln Pro

345 350 355 360

ggt gat gac ctc ttc tgg ttt ggc aag cca cgc ttc att ctt ttc ctt 1216

Gly Asp Asp Leu Phe Trp Phe Gly Lys Pro Arg Phe Ile Leu Phe Leu

365 370 375

att cac ttg gtc ctt ttt acg aat gca ttt caa ctt gca ttc ttt gcc 1264

Ile His Leu Val Leu Phe Thr Asn Ala Phe Gln Leu Ala Phe Phe Ala

380 385 390

tgg agt acg tat gaa ttc aat ctc aat aat tgt ttc cat gaa agc act 1312

Trp Ser Thr Tyr Glu Phe Asn Leu Asn Asn Cys Phe His Glu Ser Thr

395 400 405

gca gat gtg gtc att aga ctt gta gtt gga gct gtt gtg cag ata ctt 1360

Ala Asp Val Val Ile Arg Leu Val Val Gly Ala Val Val Gln Ile Leu

410 415 420

tgc agc tat gtg act ctt cca ctc tat gca ctt gtt act cag atg ggt 1408

Cys Ser Tyr Val Thr Leu Pro Leu Tyr Ala Leu Val Thr Gln Met Gly

425 430 435 440

agt aaa atg aag cca aca gta ttc aac gat aga gta gcc acg gca tta 1456

Ser Lys Met Lys Pro Thr Val Phe Asn Asp Arg Val Ala Thr Ala Leu

445 450 455

aag aag tgg cat cac act gca aag aac gag acg aaa cac gga aga cac 1504

Lys Lys Trp His His Thr Ala Lys Asn Glu Thr Lys His Gly Arg His

460 465 470

tcg gga tcc aat aca cct ttc tct agc cgt cca act aca cca aca cat 1552

Ser Gly Ser Asn Thr Pro Phe Ser Ser Arg Pro Thr Thr Pro Thr His

475 480 485

ggc tca tct cca atc cat ctc ctt cac aat ttc aat aac cgg agc gtt 1600

Gly Ser Ser Pro Ile His Leu Leu His Asn Phe Asn Asn Arg Ser Val

490 495 500

gaa aat tac cca agt tct cct tct cct aga tac tct ggt cat ggt cat 1648

Glu Asn Tyr Pro Ser Ser Pro Ser Pro Arg Tyr Ser Gly His Gly His

505 510 515 520

cat gaa cac caa ttt tgg gat cct gag tct caa cac caa gaa gct gaa 1696

His Glu His Gln Phe Trp Asp Pro Glu Ser Gln His Gln Glu Ala Glu

525 530 535

act tcc aca cat cat tct ctt gcg cat gaa agc tca gaa cct gtt ctt 1744

Thr Ser Thr His His Ser Leu Ala His Glu Ser Ser Glu Pro Val Leu

540 545 550

gca tct gtg gaa ctt cct cct ata agg act agc aaa agc tta aga gat 1792

Ala Ser Val Glu Leu Pro Pro Ile Arg Thr Ser Lys Ser Leu Arg Asp

555 560 565

ttt tct ttt aag aaa tga tgattcttgt ttgctatatt tgatttcgta 1840

Phe Ser Phe Lys Lys

570

cagtgggaat tttgtcatat gaaaataatt tcttgtacat tactagtttg ataagaaata 1900

accatatcta tatggataca aaaaaaaaaa aaaaa 1935

<210> 12

<211> 573

<212> PRT

〈213〉 Arabidopsis thaliana

<400> 12

Met Ala Asp Gln Val Lys Glu Arg Thr Leu Glu Glu Thr Ser Thr Trp

1 5 10 15

Ala Val Ala Val Val Cys Phe Val Leu Leu Phe Ile Ser Ile Val Leu

20 25 30

Glu His Ser Ile His Lys Ile Gly Thr Trp Phe Lys Lys Lys His Lys

35 40 45

Gln Ala Leu Phe Glu Ala Leu Glu Lys Val Lys Ala Glu Leu Met Leu

50 55 60

Leu Gly Phe Ile Ser Leu Leu Leu Thr Ile Gly Gln Thr Pro Ile Ser

65 70 75 80

Asn Ile Cys Ile Ser Gln Lys Val Ala Ser Thr Met His Pro Cys Ser

85 90 95

Ala Ala Glu Glu Ala Lys Lys Tyr Gly Lys Lys Asp Ala Gly Lys Lys

100 105 110

Asp Asp Gly Asp Gly Asp Lys Pro Gly Arg Arg Leu Leu Leu Glu Leu

115 120 125

Ala Glu Ser Tyr Ile His Arg Arg Ser Leu Ala Thr Lys Gly Tyr Asp

130 135 140

Lys Cys Ala Glu Lys Gly Lys Val Ala Phe Val Ser Ala Tyr Gly Ile

145 150 155 160

His Gln Leu His Ile Phe Ile Phe Val Leu Ala Val Val His Val Val

165 170 175

Tyr Cys Ile Val Thr Tyr Ala Phe Gly Lys Ile Lys Met Arg Thr Trp

180 185 190

Lys Ser Trp Glu Glu Glu Thr Lys Thr Ile Glu Tyr Gln Tyr Ser Asn

195 200 205

Asp Pro Glu Arg Phe Arg Phe Ala Arg Asp Thr Ser Phe Gly Arg Arg

210 215 220

His Leu Asn Phe Trp Ser Lys Thr Arg Val Thr Leu Trp Ile Val Cys

225 230 235 240

Phe Phe Arg Gln Phe Phe Gly Ser Val Thr Lys Val Asp Tyr Leu Ala

245 250 255

Leu Arg His Gly Phe Ile Met Ala His Phe Ala Pro Gly Asn Glu Ser

260 265 270

Arg Phe Asp Phe Arg Lys Tyr Ile Gln Arg Ser Leu Glu Lys Asp Phe

275 280 285

Lys Thr Val Val Glu Ile Ser Pro Val Ile Trp Phe Val Ala Val Leu

290 295 300

Phe Leu Leu Thr Asn Ser Tyr Gly Leu Arg Ser Tyr Leu Trp Leu Pro

305 310 315 320

Phe Ile Pro Leu Val Val Ile Leu Ile Val Gly Thr Lys Leu Glu Val

325 330 335

Ile Ile Thr Lys Leu Gly Leu Arg Ile Gln Glu Lys Gly Asp Val Val

340 345 350

Arg Gly Ala Pro Val Val Gln Pro Gly Asp Asp Leu Phe Trp Phe Gly

355 360 365

Lys Pro Arg Phe Ile Leu Phe Leu Ile His Leu Val Leu Phe Thr Asn

370 375 380

Ala Phe Gln Leu Ala Phe Phe Ala Trp Ser Thr Tyr Glu Phe Asn Leu

385 390 395 400

Asn Asn Cys Phe His Glu Ser Thr Ala Asp Val Val Ile Arg Leu Val

405 410 415

Val Gly Ala Val Val Gln Ile Leu Cys Ser Tyr Val Thr Leu Pro Leu

420 425 430

Tyr Ala Leu Val Thr Gln Met Gly Ser Lys Met Lys Pro Thr Val Phe

435 440 445

Asn Asp Arg Val Ala Thr Ala Leu Lys Lys Trp His His Thr Ala Lys

450 455 460

Asn Glu Thr Lys His Gly Arg His Ser Gly Ser Asn Thr Pro Phe Ser

465 470 475 480

Ser Arg Pro Thr Thr Pro Thr His Gly Ser Ser Pro Ile His Leu Leu

485 490 495

His Asn Phe Asn Asn Arg Ser Val Glu Asn Tyr Pro Ser Ser Pro Ser

500 505 510

Pro Arg Tyr Ser Gly His Gly His His Glu His Gln Phe Trp Asp Pro

515 520 525

Glu Ser Gln His Gln Glu Ala Glu Thr Ser Thr His His Ser Leu Ala

530 535 540

His Glu Ser Ser Glu Pro Val Leu Ala Ser Val Glu Leu Pro Pro Ile

545 550 555 560

Arg Thr Ser Lys Ser Leu Arg Asp Phe Ser Phe Lys Lys

565 570

〈210〉 13

<211> 1811

<212> DNA

〈213〉 Arabidopsis thaliana

〈220〉

<221> CDS

222 (56) .. (1633)

〈220〉

〈221〉 misc_feature

222 (236) .. (274)

223 location of the amino acid sequence set forth in SEQ ID No: 1

〈220〉

〈221〉 misc_feature

222 (1328) .. (1369)

<223> location of the amino acid sequence set forth in SEQ ID No: 2

〈400〉 13

gttcccagat tcatctttac ttattgtcta aattctctct ggtgtgagaa gtaaa 55

atg ggt cac gga gga gaa ggg atg tcg ctt gaa ttc act ccg acg tgg 103

Met Gly His Gly Gly Glu Gly Met Ser Leu Glu Phe Thr Pro Thr Trp

1 5 10 15

gtc gtc gcc gga gtt tgt acg gtc atc gtc gcg att tca ctg gcg gtg 151

Val Val Ala Gly Val Cys Thr Val Ile Val Ala Ile Ser Leu Ala Val

20 25 30

gag cgt ttg ctt cac tat ttc ggt act gtt ctt aag aag aag aag caa 199

Glu Arg Leu Leu His Tyr Phe Gly Thr Val Leu Lys Lys Lys Lys Gln

35 40 45

aaa ccc ctt tac gaa gcc ctt caa aag gtt aaa gaa gag ctg atg ttg 247

Lys Pro Leu Tyr Glu Ala Leu Gln Lys Val Lys Glu Glu Leu Met Leu

50 55 60

tta ggg ttt ata tcg ctg tta ctg acg gta ttc caa ggg ctc att tcc 295

Leu Gly Phe Ile Ser Leu Leu Leu Thr Val Phe Gln Gly Leu Ile Ser

65 70 75 80

aaa ttc tgt gtg aaa gaa aat gtg ctt atg cat atg ctt cca tgt tct 343

Lys Phe Cys Val Lys Glu Asn Val Leu Met His Met Leu Pro Cys Ser

85 90 95

ctc gat tca aga cga gaa gct ggg gca agt gaa cat aaa aac gtt aca 391

Leu Asp Ser Arg Arg Glu Ala Gly Ala Ser Glu His Lys Asn Val Thr

100 105 110

gca aaa gaa cat ttt cag act ttt tta cct att gtt gga acc act agg 439

Ala Lys Glu His Phe Gln Thr Phe Leu Pro Ile Val Gly Thr Thr Arg

115 120 125

cgt cta ctt gct gaa cat gct gct gtg caa gtt ggt tac tgt agc gaa 487

Arg Leu Leu Ala Glu His Ala Ala Val Gln Val Gly Tyr Cys Ser Glu

130 135 140

aag ggt aaa gta cca ttg ctt tcg ctt gag gca ttg cac cat cta cat 535

Lys Gly Lys Val Pro Leu Leu Ser Leu Glu Ala Leu His His Leu His

145 150 155 160

att ttc atc ttc gtc ctc gcc ata tcc cat gtg aca ttc tgt gtc ctt 583

Ile Phe Ile Phe Val Leu Ala Ile Ser His Val Thr Phe Cys Val Leu

165 170 175

acc gtg att ttt gga agc aca agg att cac caa tgg aag aaa tgg gag 631

Thr Val Ile Phe Gly Ser Thr Arg Ile His Gln Trp Lys Lys Trp Glu

180 185 190

gat tcg atc gca gat gag aag ttt gac ccc gaa aca gct ctc agg aaa 679

Asp Ser Ile Ala Asp Glu Lys Phe Asp Pro Glu Thr Ala Leu Arg Lys

195 200 205

aga agg gtc act cat gta cac aac cat gct ttt att aaa gag cat ttt 727

Arg Arg Val Thr His Val His Asn His Ala Phe Ile Lys Glu His Phe

210 215 220

ctt ggt att ggc aaa gat tca gtc atc ctc gga tgg acg caa tcc ttt 775

Leu Gly Ile Gly Lys Asp Ser Val Ile Leu Gly Trp Thr Gln Ser Phe

225 230 235 240

ctc aag caa ttc tat gat tct gtg acg aaa tca gat tac gtg act tta 823

Leu Lys Gln Phe Tyr Asp Ser Val Thr Lys Ser Asp Tyr Val Thr Leu

245 250 255

cgt ctt ggt ttc att atg aca cat tgt aag gga aac ccc aag ctt aat 871

Arg Leu Gly Phe Ile Met Thr His Cys Lys Gly Asn Pro Lys Leu Asn

260 265 270

ttc cac aag tat atg atg cgc gct yta gag gat gat ttc aaa caa gtt 919

Phe His Lys Tyr Met Met Arg Ala Xaa Glu Asp Asp Phe Lys Gln Val

275 280 285

gtt ggt att agt tgg tat ctt tgg atc ttt gtc gtc atc ttt ttg ctg 967

Val Gly Ile Ser Trp Tyr Leu Trp Ile Phe Val Val Ile Phe Leu Leu

290 295 300

cta aat gtt aac gga tgg cac aca tat ttc tgg ata gca ttt att ccc 1015

Leu Asn Val Asn Gly Trp His Thr Tyr Phe Trp Ile Ala Phe Ile Pro

305 310 315 320

ttt gct ttg ctt ctt gct gtg gga aca aag ttg gag cat gtg att gca 1063

Phe Ala Leu Leu Leu Ala Val Gly Thr Lys Leu Glu His Val Ile Ala

325 330 335

cag tta gct cat gaa gtt gca gag aaa cat gta gcc att gaa gga gac 1111

Gln Leu Ala His Glu Val Ala Glu Lys His Val Ala Ile Glu Gly Asp

340 345 350

tta gtg gtg aaa ccc tca gat gag cat ttc tgg ttc agc aaa cct caa 1159

Leu Val Val Lys Pro Ser Asp Glu His Phe Trp Phe Ser Lys Pro Gln

355 360 365

att gtt ctc tac ttg atc cat ttt atc ctc ttc cag aat gct ttt gag 1207

Ile Val Leu Tyr Leu Ile His Phe Ile Leu Phe Gln Asn Ala Phe Glu

370 375 380

att gcg ttt ttc ttt tgg att tgg gtt aca tac ggc ttc gac tcg tgc 1255

Ile Ala Phe Phe Phe Trp Ile Trp Val Thr Tyr Gly Phe Asp Ser Cys

385 390 395 400

att atg gga cag gtg aga tac att gtt cca aga ttg gtt atc ggg gtc 1303

Ile Met Gly Gln Val Arg Tyr Ile Val Pro Arg Leu Val Ile Gly Val

405 410 415

ttc att caa gtg ctt tgc agt tac agt aca ctg cct ctt tac gcc atc 1351

Phe Ile Gln Val Leu Cys Ser Tyr Ser Thr Leu Pro Leu Tyr Ala Ile

420 425 430

gtc tca cag atg gga agt agc ttc aag aaa gct ata ttc gag gag aat 1399

Val Ser Gln Met Gly Ser Ser Phe Lys Lys Ala Ile Phe Glu Glu Asn

435 440 445

gtg cag gtt ggt ctt gtt ggt tgg gca cag aaa gtg aaa caa aag aga 1447

Val Gln Val Gly Leu Val Gly Trp Ala Gln Lys Val Lys Gln Lys Arg

450 455 460

gac cta aaa gct gca gct agt aat gga aac gaa gga agc tct cag gct 1495

Asp Leu Lys Ala Ala Ala Ser Asn Gly Asn Glu Gly Ser Ser Gln Ala

465 470 475 480

ggt cct ggt cct gat tct ggt tct ggt tct gct cct gct gct ggt cct 1543

Gly Pro Gly Pro Asp Ser Gly Ser Gly Ser Ala Pro Ala Ala Gly Pro

485 490 495

ggt gca ggt ttt gca gga att cag ctc agc aga gta aca aga aac aac 1591

Gly Ala Gly Phe Ala Gly Ile Gln Leu Ser Arg Val Thr Arg Asn Asn

500 505 510

gca ggg gac aca aac aat gag att aca cct gat cat aac aac tgagcag 1640

Ala Gly Asp Thr Asn Asn Glu Ile Thr Pro Asp His Asn Asn

515 520 525

agatattatc ttttccattt agaggatcat catcagattt tagcttcaag gtccggtttt 1700

gtggtttata cataagttat agtgacttga tttttttgtt ttgttacaaa gttaccatct 1760

ttggattaga attgggaaat tgaatctgtt tgtaaaaaaa aaaaaaaaaa a 1811

〈210〉 14

<211> 526

<212> PRT

〈213〉 Arabidopsis thaliana

<400> 14

Met Gly His Gly Gly Glu Gly Met Ser Leu Glu Phe Thr Pro Thr Trp

1 5 10 15

Val Val Ala Gly Val Cys Thr Val Ile Val Ala Ile Ser Leu Ala Val

20 25 30

Glu Arg Leu Leu His Tyr Phe Gly Thr Val Leu Lys Lys Lys Lys Gln

35 40 45

Lys Pro Leu Tyr Glu Ala Leu Gln Lys Val Lys Glu Glu Leu Met Leu

50 55 60

Leu Gly Phe Ile Ser Leu Leu Leu Thr Val Phe Gln Gly Leu Ile Ser

65 70 75 80

Lys Phe Cys Val Lys Glu Asn Val Leu Met His Met Leu Pro Cys Ser

85 90 95

Leu Asp Ser Arg Arg Glu Ala Gly Ala Ser Glu His Lys Asn Val Thr

100 105 110

Ala Lys Glu His Phe Gln Thr Phe Leu Pro Ile Val Gly Thr Thr Arg

115 120 125

Arg Leu Leu Ala Glu His Ala Ala Val Gln Val Gly Tyr Cys Ser Glu

130 135 140

Lys Gly Lys Val Pro Leu Leu Ser Leu Glu Ala Leu His His Leu His

145 150 155 160

Ile Phe Ile Phe Val Leu Ala Ile Ser His Val Thr Phe Cys Val Leu

165 170 175

Thr Val Ile Phe Gly Ser Thr Arg Ile His Gln Trp Lys Lys Trp Glu

180 185 190

Asp Ser Ile Ala Asp Glu Lys Phe Asp Pro Glu Thr Ala Leu Arg Lys

195 200 205

Arg Arg Val Thr His Val His Asn His Ala Phe Ile Lys Glu His Phe

210 215 220

Leu Gly Ile Gly Lys Asp Ser Val Ile Leu Gly Trp Thr Gln Ser Phe

225 230 235 240

Leu Lys Gln Phe Tyr Asp Ser Val Thr Lys Ser Asp Tyr Val Thr Leu

245 250 255

Arg Leu Gly Phe Ile Met Thr His Cys Lys Gly Asn Pro Lys Leu Asn

260 265 270

Phe His Lys Tyr Met Met Arg Ala Xaa Glu Asp Asp Phe Lys Gln Val

275 280 285

Val Gly Ile Ser Trp Tyr Leu Trp Ile Phe Val Val Ile Phe Leu Leu

290 295 300

Leu Asn Val Asn Gly Trp His Thr Tyr Phe Trp Ile Ala Phe Ile Pro

305 310 315 320

Phe Ala Leu Leu Leu Ala Val Gly Thr Lys Leu Glu His Val Ile Ala

325 330 335

Gln Leu Ala His Glu Val Ala Glu Lys His Val Ala Ile Glu Gly Asp

340 345 350

Leu Val Val Lys Pro Ser Asp Glu His Phe Trp Phe Ser Lys Pro Gln

355 360 365

Ile Val Leu Tyr Leu Ile His Phe Ile Leu Phe Gln Asn Ala Phe Glu

370 375 380

Ile Ala Phe Phe Phe Trp Ile Trp Val Thr Tyr Gly Phe Asp Ser Cys

385 390 395 400

Ile Met Gly Gln Val Arg Tyr Ile Val Pro Arg Leu Val Ile Gly Val

405 410 415

Phe Ile Gln Val Leu Cys Ser Tyr Ser Thr Leu Pro Leu Tyr Ala Ile

420 425 430

Val Ser Gln Met Gly Ser Ser Phe Lys Lys Ala Ile Phe Glu Glu Asn

435 440 445

Val Gln Val Gly Leu Val Gly Trp Ala Gln Lys Val Lys Gln Lys Arg

450 455 460

Asp Leu Lys Ala Ala Ala Ser Asn Gly Asn Glu Gly Ser Ser Gln Ala

465 470 475 480

Gly Pro Gly Pro Asp Ser Gly Ser Gly Ser Ala Pro Ala Ala Gly Pro

485 490 495

Gly Ala Gly Phe Ala Gly Ile Gln Leu Ser Arg Val Thr Arg Asn Asn

500 505 510

Ala Gly Asp Thr Asn Asn Glu Ile Thr Pro Asp His Asn Asn

515 520 525

<210> 15

<211> 1782

<212> DNA

〈213〉 Arabidopsis thaliana

〈220〉

<221> CDS

222 (1) .. (1779)

〈220〉

〈221〉 misc_feature

222 (274) .. (313)

223 location of the amino acid sequence set forth in SEQ ID No: 1

〈220〉

〈221〉 misc_feature

222 (1327) .. (1370)

<223> location of the amino acid sequence set forth in SEQ ID No: 2

<400> 15

atg gga atc atc gac ggt tct ttg ctt cgg cgg ttg att tgt ctc tgt 48

Met Gly Ile Ile Asp Gly Ser Leu Leu Arg Arg Leu Ile Cys Leu Cys

1 5 10 15

ctc tgg tgt ctt ctc ggt gga gga gtg acg gtg gtt acg gcg gag gat 96

Leu Trp Cys Leu Leu Gly Gly Gly Val Thr Val Val Thr Ala Glu Asp

20 25 30

gag aag aaa gtg gta cat aaa cag ctt aat caa act ccg act tgg gct 144

Glu Lys Lys Val Val His Lys Gln Leu Asn Gln Thr Pro Thr Trp Ala

35 40 45

gtt gct gct gtt tgt act ttc ttc atc gtt gtt tct gtt ctt ctt gaa 192

Val Ala Ala Val Cys Thr Phe Phe Ile Val Val Ser Val Leu Leu Glu

50 55 60

aaa ctt ctt cac aaa gtt gga aag gtt cta tgg gat cgg cac aag aca 240

Lys Leu Leu His Lys Val Gly Lys Val Leu Trp Asp Arg His Lys Thr

65 70 75 80

gct ctt ctt gac gct ttg gag aag atc aaa gca gag ctg atg gtt ctt 288

Ala Leu Leu Asp Ala Leu Glu Lys Ile Lys Ala Glu Leu Met Val Leu

85 90 95

gga ttc atc tct ttg ctt ctg aca ttt gga caa acc tac att ttg gat 336

Gly Phe Ile Ser Leu Leu Leu Thr Phe Gly Gln Thr Tyr Ile Leu Asp

100 105 110

att tgt atc cct tca cat gtt gct cgt acg atg ctc ccg tgt cct gct 384

Ile Cys Ile Pro Ser His Val Ala Arg Thr Met Leu Pro Cys Pro Ala

115 120 125

cct aac ttg aaa aag gag gat gat gac aat ggt gaa agt cac agg aga 432

Pro Asn Leu Lys Lys Glu Asp Asp Asp Asn Gly Glu Ser His Arg Arg

130 135 140

ctc ttg tcg ttt gag cac aga ttt tta tct gga ggt gaa gca tct ccc 480

Leu Leu Ser Phe Glu His Arg Phe Leu Ser Gly Gly Glu Ala Ser Pro

145 150 155 160

act aaa tgc acg aag gag ggt tat gta gag ctt atc tct gcc gag gca 528

Thr Lys Cys Thr Lys Glu Gly Tyr Val Glu Leu Ile Ser Ala Glu Ala

165 170 175

ctc cat cag ttg cac atc ctt ata ttc ttc tta gcc att ttc cac gtt 576

Leu His Gln Leu His Ile Leu Ile Phe Phe Leu Ala Ile Phe His Val

180 185 190

ctt tac agc ttc tta act atg atg ctt gga agg ttg aag att cgc gga 624

Leu Tyr Ser Phe Leu Thr Met Met Leu Gly Arg Leu Lys Ile Arg Gly

195 200 205

tgg aag cat tgg gag aat gag aca tca tcc cat aat tac gag ttt tca 672

Trp Lys His Trp Glu Asn Glu Thr Ser Ser His Asn Tyr Glu Phe Ser

210 215 220

aca gac act tcc aga ttc agg cta act cat gaa aca tct ttt gtg aga 720

Thr Asp Thr Ser Arg Phe Arg Leu Thr His Glu Thr Ser Phe Val Arg

225 230 235 240

gcg cac acc agt ttc tgg acc cgg att cca ttc ttt ttc tat gtt gga 768

Ala His Thr Ser Phe Trp Thr Arg Ile Pro Phe Phe Phe Tyr Val Gly

245 250 255

tgc ttt ttc aga cag ttt ttc aga tcc gtt ggg aga act gac tat ttg 816

Cys Phe Phe Arg Gln Phe Phe Arg Ser Val Gly Arg Thr Asp Tyr Leu

260 265 270

aca ttg aga aat ggt ttc atc gct gtt cat tta gct cca gga agt caa 864

Thr Leu Arg Asn Gly Phe Ile Ala Val His Leu Ala Pro Gly Ser Gln

275 280 285

ttt aac ttc caa aaa tac att aaa aga tcg ttg gag gat gat ttc aag 912

Phe Asn Phe Gln Lys Tyr Ile Lys Arg Ser Leu Glu Asp Asp Phe Lys

290 295 300

gta gtc gtt gga gtc agc cct gtc ttg tgg gga tct ttt gtg cta ttc 960

Val Val Val Gly Val Ser Pro Val Leu Trp Gly Ser Phe Val Leu Phe

305 310 315 320

ctc ctc cta aat att gac ggt gag tat atg atg ttc atc ggc act gca 1008

Leu Leu Leu Asn Ile Asp Gly Glu Tyr Met Met Phe Ile Gly Thr Ala

325 330 335

ata ccg gtt att atc att tta gct gta ggg aca aag ctt caa gcg att 1056

Ile Pro Val Ile Ile Ile Leu Ala Val Gly Thr Lys Leu Gln Ala Ile

340 345 350

atg aca agg atg gct ctt ggt atc aca gat aga cat gcg gta gtt caa 1104

Met Thr Arg Met Ala Leu Gly Ile Thr Asp Arg His Ala Val Val Gln

355 360 365

gga atg ccg ctt gta caa ggc aac gat gag tat ttc tgg ttc ggt cgt 1152

Gly Met Pro Leu Val Gln Gly Asn Asp Glu Tyr Phe Trp Phe Gly Arg

370 375 380

ccc cat ttg att ctc cat ctc atg cat ttc gcc ttg ttt cag aac gca 1200

Pro His Leu Ile Leu His Leu Met His Phe Ala Leu Phe Gln Asn Ala

385 390 395 400

ttt cag atc act tat ttc ttc tgg ata tgg tat tcc ttt gga tca gat 1248

Phe Gln Ile Thr Tyr Phe Phe Trp Ile Trp Tyr Ser Phe Gly Ser Asp

405 410 415

tct tgc tac cat cct aat ttc aag att gca ctt gta aaa gta gcg att 1296

Ser Cys Tyr His Pro Asn Phe Lys Ile Ala Leu Val Lys Val Ala Ile

420 425 430

gct tta gga gta ttg tgt ctt tgc agc tac atc aca ctt cct ctt tac 1344

Ala Leu Gly Val Leu Cys Leu Cys Ser Tyr Ile Thr Leu Pro Leu Tyr

435 440 445

gca ctc gta act cag atg ggt tct cgg atg aaa aaa tcg gta ttc gat 1392

Ala Leu Val Thr Gln Met Gly Ser Arg Met Lys Lys Ser Val Phe Asp

450 455 460

gaa caa acg tca aaa gca ctc aag aaa tgg aga atg gca gtg aag aag 1440

Glu Gln Thr Ser Lys Ala Leu Lys Lys Trp Arg Met Ala Val Lys Lys

465 470 475 480

aag aaa ggt gtg aaa gcc act act aag aga cta ggt gga gat gga agt 1488

Lys Lys Gly Val Lys Ala Thr Thr Lys Arg Leu Gly Gly Asp Gly Ser

485 490 495

gcg agc cct acg gca tcg aca gtt agg tct act tcg tct gta cgt tca 1536

Ala Ser Pro Thr Ala Ser Thr Val Arg Ser Thr Ser Ser Val Arg Ser

500 505 510

ttg cag cgt tac aaa acc aca cca cat tcg atg aga tac gaa gga ctt 1584

Leu Gln Arg Tyr Lys Thr Thr Pro His Ser Met Arg Tyr Glu Gly Leu

515 520 525

gac cct gaa aca tcg gat ctc gac aca gat aat gaa gct ttg act cct 1632

Asp Pro Glu Thr Ser Asp Leu Asp Thr Asp Asn Glu Ala Leu Thr Pro

530 535 540

ccc aaa tct cct cca agc ttc gag ctt gtt gtg aaa gtt gaa cca aat 1680

Pro Lys Ser Pro Pro Ser Phe Glu Leu Val Val Lys Val Glu Pro Asn

545 550 555 560

aag acc aat acc ggt gag act agc cgt gac act gaa act gat tct aaa 1728

Lys Thr Asn Thr Gly Glu Thr Ser Arg Asp Thr Glu Thr Asp Ser Lys

565 570 575

gag ttc tct ttc gtc aag cct gct ccg agt aat gaa tca tct caa gac 1776

Glu Phe Ser Phe Val Lys Pro Ala Pro Ser Asn Glu Ser Ser Gln Asp

580 585 590

cgg t ga 1782

Arg

<210> 16

<211> 593

<212> PRT

〈213〉 Arabidopsis thaliana

<400> 16

Met Gly Ile Ile Asp Gly Ser Leu Leu Arg Arg Leu Ile Cys Leu Cys

1 5 10 15

Leu Trp Cys Leu Leu Gly Gly Gly Val Thr Val Val Thr Ala Glu Asp

20 25 30

Glu Lys Lys Val Val His Lys Gln Leu Asn Gln Thr Pro Thr Trp Ala

35 40 45

Val Ala Ala Val Cys Thr Phe Phe Ile Val Val Ser Val Leu Leu Glu

50 55 60

Lys Leu Leu His Lys Val Gly Lys Val Leu Trp Asp Arg His Lys Thr

65 70 75 80

Ala Leu Leu Asp Ala Leu Glu Lys Ile Lys Ala Glu Leu Met Val Leu

85 90 95

Gly Phe Ile Ser Leu Leu Leu Thr Phe Gly Gln Thr Tyr Ile Leu Asp

100 105 110

Ile Cys Ile Pro Ser His Val Ala Arg Thr Met Leu Pro Cys Pro Ala

115 120 125

Pro Asn Leu Lys Lys Glu Asp Asp Asp Asn Gly Glu Ser His Arg Arg

130 135 140

Leu Leu Ser Phe Glu His Arg Phe Leu Ser Gly Gly Glu Ala Ser Pro

145 150 155 160

Thr Lys Cys Thr Lys Glu Gly Tyr Val Glu Leu Ile Ser Ala Glu Ala

165 170 175

Leu His Gln Leu His Ile Leu Ile Phe Phe Leu Ala Ile Phe His Val

180 185 190

Leu Tyr Ser Phe Leu Thr Met Met Leu Gly Arg Leu Lys Ile Arg Gly

195 200 205

Trp Lys His Trp Glu Asn Glu Thr Ser Ser His Asn Tyr Glu Phe Ser

210 215 220

Thr Asp Thr Ser Arg Phe Arg Leu Thr His Glu Thr Ser Phe Val Arg

225 230 235 240

Ala His Thr Ser Phe Trp Thr Arg Ile Pro Phe Phe Phe Tyr Val Gly

245 250 255

Cys Phe Phe Arg Gln Phe Phe Arg Ser Val Gly Arg Thr Asp Tyr Leu

260 265 270

Thr Leu Arg Asn Gly Phe Ile Ala Val His Leu Ala Pro Gly Ser Gln

275 280 285

Phe Asn Phe Gln Lys Tyr Ile Lys Arg Ser Leu Glu Asp Asp Phe Lys

290 295 300

Val Val Val Gly Val Ser Pro Val Leu Trp Gly Ser Phe Val Leu Phe

305 310 315 320

Leu Leu Leu Asn Ile Asp Gly Glu Tyr Met Met Phe Ile Gly Thr Ala

325 330 335

Ile Pro Val Ile Ile Ile Leu Ala Val Gly Thr Lys Leu Gln Ala Ile

340 345 350

Met Thr Arg Met Ala Leu Gly Ile Thr Asp Arg His Ala Val Val Gln

355 360 365

Gly Met Pro Leu Val Gln Gly Asn Asp Glu Tyr Phe Trp Phe Gly Arg

370 375 380

Pro His Leu Ile Leu His Leu Met His Phe Ala Leu Phe Gln Asn Ala

385 390 395 400

Phe Gln Ile Thr Tyr Phe Phe Trp Ile Trp Tyr Ser Phe Gly Ser Asp

405 410 415

Ser Cys Tyr His Pro Asn Phe Lys Ile Ala Leu Val Lys Val Ala Ile

420 425 430

Ala Leu Gly Val Leu Cys Leu Cys Ser Tyr Ile Thr Leu Pro Leu Tyr

435 440 445

Ala Leu Val Thr Gln Met Gly Ser Arg Met Lys Lys Ser Val Phe Asp

450 455 460

Glu Gln Thr Ser Lys Ala Leu Lys Lys Trp Arg Met Ala Val Lys Lys

465 470 475 480

Lys Lys Gly Val Lys Ala Thr Thr Lys Arg Leu Gly Gly Asp Gly Ser

485 490 495

Ala Ser Pro Thr Ala Ser Thr Val Arg Ser Thr Ser Ser Val Arg Ser

500 505 510

Leu Gln Arg Tyr Lys Thr Thr Pro His Ser Met Arg Tyr Glu Gly Leu

515 520 525

Asp Pro Glu Thr Ser Asp Leu Asp Thr Asp Asn Glu Ala Leu Thr Pro

530 535 540

Pro Lys Ser Pro Pro Ser Phe Glu Leu Val Val Lys Val Glu Pro Asn

545 550 555 560

Lys Thr Asn Thr Gly Glu Thr Ser Arg Asp Thr Glu Thr Asp Ser Lys

565 570 575

Glu Phe Ser Phe Val Lys Pro Ala Pro Ser Asn Glu Ser Ser Gln Asp

580 585 590

Arg

〈210〉 17

<211> 1629

<212> DNA

〈213〉 Arabidopsis thaliana

〈220〉

<221> CDS

222 (1) .. (1626)

〈220〉

〈221〉 misc_feature

222 (184) .. (223)

223 location of the amino acid sequence set forth in SEQ ID No: 1

〈220〉

〈221〉 misc_feature

222 (1141) .. (1183)

<223> location of the amino acid sequence set forth in SEQ ID No: 2

〈400〉 17

atg gag cat atg atg aaa gaa gga agg tct ctt gca gag acg ccg act 48

Met Glu His Met Met Lys Glu Gly Arg Ser Leu Ala Glu Thr Pro Thr

1 5 10 15

tac tct gtt gct tcg gtt gtt act gtt ttg gtc ttt gtt tgc ttt ctc 96

Tyr Ser Val Ala Ser Val Val Thr Val Leu Val Phe Val Cys Phe Leu

20 25 30

gtt gaa cgc gcc att tac aga ttt gga aag tgg tta aag aag act aga 144

Val Glu Arg Ala Ile Tyr Arg Phe Gly Lys Trp Leu Lys Lys Thr Arg

35 40 45

aga aag gca ctt ttt act tca ctt gag aaa atg aaa gag gag ttg atg 192

Arg Lys Ala Leu Phe Thr Ser Leu Glu Lys Met Lys Glu Glu Leu Met

50 55 60

ttg ctg gga ctt ata tca ctt ctg ttg tca caa agc gcg aga tgg att 240

Leu Leu Gly Leu Ile Ser Leu Leu Leu Ser Gln Ser Ala Arg Trp Ile

65 70 75 80

tca gaa atc tgt gtt aac tct tcc ctt ttc aat agt aaa ttc tac att 288

Ser Glu Ile Cys Val Asn Ser Ser Leu Phe Asn Ser Lys Phe Tyr Ile

85 90 95

tgc tct gaa gag gac tat gga atc cat aag aaa gtt ctt ctg gaa cac 336

Cys Ser Glu Glu Asp Tyr Gly Ile His Lys Lys Val Leu Leu Glu His

100 105 110

acc tct tct aca aac cag agc tcc tta cct cat cat gga ata cat gaa 384

Thr Ser Ser Thr Asn Gln Ser Ser Leu Pro His His Gly Ile His Glu

115 120 125

gcc tct cat caa tgt ggt cat ggc cgt gaa cca ttt gtg tcg tat gag 432

Ala Ser His Gln Cys Gly His Gly Arg Glu Pro Phe Val Ser Tyr Glu

130 135 140

gga ctc gag caa ctc cta aga ttc tta ttc gtc ctg ggt atc act cat 480

Gly Leu Glu Gln Leu Leu Arg Phe Leu Phe Val Leu Gly Ile Thr His

145 150 155 160

gtt cta tac agt ggc att gcc att ggt tta gcc atg agc aag att tac 528

Val Leu Tyr Ser Gly Ile Ala Ile Gly Leu Ala Met Ser Lys Ile Tyr

165 170 175

agt tgg aga aaa tgg gaa gcc caa gcg atc ata atg gct gaa tca gat 576

Ser Trp Arg Lys Trp Glu Ala Gln Ala Ile Ile Met Ala Glu Ser Asp

180 185 190

atc cac ctt tgt ttc ctg cgg caa ttt aga ggc tcc ata cga aag tct 624

Ile His Leu Cys Phe Leu Arg Gln Phe Arg Gly Ser Ile Arg Lys Ser

195 200 205

gac tac ttc gca ctt cgg tta ggt ttc ctc act aaa cat aat ttg cca 672

Asp Tyr Phe Ala Leu Arg Leu Gly Phe Leu Thr Lys His Asn Leu Pro

210 215 220

ttt aca tac aac ttc cat atg tat atg gta cgg acg atg gaa gat gag 720

Phe Thr Tyr Asn Phe His Met Tyr Met Val Arg Thr Met Glu Asp Glu

225 230 235 240

ttt cat ggc att gtt gga att agc tgg cca ctt tgg gtt tac gct ata 768

Phe His Gly Ile Val Gly Ile Ser Trp Pro Leu Trp Val Tyr Ala Ile

245 250 255

gta tgc atc tgc ata aat gtt cat ggc ctg aat atg tac ttt tgg ata 816

Val Cys Ile Cys Ile Asn Val His Gly Leu Asn Met Tyr Phe Trp Ile

260 265 270

tca ttc gtt cct gcc att ctt gtc atg ttg gtt gga acc aaa ctt gag 864

Ser Phe Val Pro Ala Ile Leu Val Met Leu Val Gly Thr Lys Leu Glu

275 280 285

cat gtt gtc tcc aag ctt gct ctc gag gtt aag gag cag cag aca ggc 912

His Val Val Ser Lys Leu Ala Leu Glu Val Lys Glu Gln Gln Thr Gly

290 295 300

aca tct aat ggg gct caa gtc aaa cca cgt gat ggg ctc ttc tgg ttt 960

Thr Ser Asn Gly Ala Gln Val Lys Pro Arg Asp Gly Leu Phe Trp Phe

305 310 315 320

ggg aaa cca gaa att ctg cta cgg ttg ata caa ttt atc att ttt cag 1008

Gly Lys Pro Glu Ile Leu Leu Arg Leu Ile Gln Phe Ile Ile Phe Gln

325 330 335

aat gca ttt gaa atg gca aca ttc atc tgg ttc ttg tgg gga atc aag 1056

Asn Ala Phe Glu Met Ala Thr Phe Ile Trp Phe Leu Trp Gly Ile Lys

340 345 350

gaa aga tct tgc ttc atg aag aac cat gtg atg ata tca agc cgg cta 1104

Glu Arg Ser Cys Phe Met Lys Asn His Val Met Ile Ser Ser Arg Leu

355 360 365

att tct ggg gtt ctc gtt cag ttc tgg tgt agt tat ggc act gtg cct 1152

Ile Ser Gly Val Leu Val Gln Phe Trp Cys Ser Tyr Gly Thr Val Pro

370 375 380

ctc aat gta atc gtt act cag atg gga tct cgg cat aag aaa gct gtg 1200

Leu Asn Val Ile Val Thr Gln Met Gly Ser Arg His Lys Lys Ala Val

385 390 395 400

ata gca gag agc gta aga gac tca ctt cac agt tgg tgc aag aga gtg 1248

Ile Ala Glu Ser Val Arg Asp Ser Leu His Ser Trp Cys Lys Arg Val

405 410 415

aaa gag agg tct aag cac acg aga tca gtg tgt tcc ctt gac aca gca 1296

Lys Glu Arg Ser Lys His Thr Arg Ser Val Cys Ser Leu Asp Thr Ala

420 425 430

aca ata gac gag aga gac gag atg aca gtg ggg aca ttg tct agg agc 1344

Thr Ile Asp Glu Arg Asp Glu Met Thr Val Gly Thr Leu Ser Arg Ser

435 440 445

tca tcg atg act tca ctg aat cag att acc ata aac tcc ata gac caa 1392

Ser Ser Met Thr Ser Leu Asn Gln Ile Thr Ile Asn Ser Ile Asp Gln

450 455 460

gca gag tct ata ttc gga gca gca gct tca tcc agc agt cct caa gat 1440

Ala Glu Ser Ile Phe Gly Ala Ala Ala Ser Ser Ser Ser Pro Gln Asp

465 470 475 480

gga tac acg tcg agg gtg gaa gaa tat ctg tct gaa aca tac aat aac 1488

Gly Tyr Thr Ser Arg Val Glu Glu Tyr Leu Ser Glu Thr Tyr Asn Asn

485 490 495

atc ggt tcg ata ccg cct tta aac gat gag att gag att gag att gaa 1536

Ile Gly Ser Ile Pro Pro Leu Asn Asp Glu Ile Glu Ile Glu Ile Glu

500 505 510

ggt gaa gaa gat aat gga ggg aga gga agt ggg agt gat gag aat aac 1584

Gly Glu Glu Asp Asn Gly Gly Arg Gly Ser Gly Ser Asp Glu Asn Asn

515 520 525

ggt gat gct gga gaa aca ctt ctt gag ttg ttt agg agg act tga 1629

Gly Asp Ala Gly Glu Thr Leu Leu Glu Leu Phe Arg Arg Thr

530 535 540

〈210〉 18

<211> 542

<212> PRT

〈213〉 Arabidopsis thaliana

〈400〉 18

Met Glu His Met Met Lys Glu Gly Arg Ser Leu Ala Glu Thr Pro Thr

1 5 10 15

Tyr Ser Val Ala Ser Val Val Thr Val Leu Val Phe Val Cys Phe Leu

20 25 30

Val Glu Arg Ala Ile Tyr Arg Phe Gly Lys Trp Leu Lys Lys Thr Arg

35 40 45

Arg Lys Ala Leu Phe Thr Ser Leu Glu Lys Met Lys Glu Glu Leu Met

50 55 60

Leu Leu Gly Leu Ile Ser Leu Leu Leu Ser Gln Ser Ala Arg Trp Ile

65 70 75 80

Ser Glu Ile Cys Val Asn Ser Ser Leu Phe Asn Ser Lys Phe Tyr Ile

85 90 95

Cys Ser Glu Glu Asp Tyr Gly Ile His Lys Lys Val Leu Leu Glu His

100 105 110

Thr Ser Ser Thr Asn Gln Ser Ser Leu Pro His His Gly Ile His Glu

115 120 125

Ala Ser His Gln Cys Gly His Gly Arg Glu Pro Phe Val Ser Tyr Glu

130 135 140

Gly Leu Glu Gln Leu Leu Arg Phe Leu Phe Val Leu Gly Ile Thr His

145 150 155 160

Val Leu Tyr Ser Gly Ile Ala Ile Gly Leu Ala Met Ser Lys Ile Tyr

165 170 175

Ser Trp Arg Lys Trp Glu Ala Gln Ala Ile Ile Met Ala Glu Ser Asp

180 185 190

Ile His Leu Cys Phe Leu Arg Gln Phe Arg Gly Ser Ile Arg Lys Ser

195 200 205

Asp Tyr Phe Ala Leu Arg Leu Gly Phe Leu Thr Lys His Asn Leu Pro

210 215 220

Phe Thr Tyr Asn Phe His Met Tyr Met Val Arg Thr Met Glu Asp Glu

225 230 235 240

Phe His Gly Ile Val Gly Ile Ser Trp Pro Leu Trp Val Tyr Ala Ile

245 250 255

Val Cys Ile Cys Ile Asn Val His Gly Leu Asn Met Tyr Phe Trp Ile

260 265 270

Ser Phe Val Pro Ala Ile Leu Val Met Leu Val Gly Thr Lys Leu Glu

275 280 285

His Val Val Ser Lys Leu Ala Leu Glu Val Lys Glu Gln Gln Thr Gly

290 295 300

Thr Ser Asn Gly Ala Gln Val Lys Pro Arg Asp Gly Leu Phe Trp Phe

305 310 315 320

Gly Lys Pro Glu Ile Leu Leu Arg Leu Ile Gln Phe Ile Ile Phe Gln

325 330 335

Asn Ala Phe Glu Met Ala Thr Phe Ile Trp Phe Leu Trp Gly Ile Lys

340 345 350

Glu Arg Ser Cys Phe Met Lys Asn His Val Met Ile Ser Ser Arg Leu

355 360 365

Ile Ser Gly Val Leu Val Gln Phe Trp Cys Ser Tyr Gly Thr Val Pro

370 375 380

Leu Asn Val Ile Val Thr Gln Met Gly Ser Arg His Lys Lys Ala Val

385 390 395 400

Ile Ala Glu Ser Val Arg Asp Ser Leu His Ser Trp Cys Lys Arg Val

405 410 415

Lys Glu Arg Ser Lys His Thr Arg Ser Val Cys Ser Leu Asp Thr Ala

420 425 430

Thr Ile Asp Glu Arg Asp Glu Met Thr Val Gly Thr Leu Ser Arg Ser

435 440 445

Ser Ser Met Thr Ser Leu Asn Gln Ile Thr Ile Asn Ser Ile Asp Gln

450 455 460

Ala Glu Ser Ile Phe Gly Ala Ala Ala Ser Ser Ser Ser Pro Gln Asp

465 470 475 480

Gly Tyr Thr Ser Arg Val Glu Glu Tyr Leu Ser Glu Thr Tyr Asn Asn

485 490 495

Ile Gly Ser Ile Pro Pro Leu Asn Asp Glu Ile Glu Ile Glu Ile Glu

500 505 510

Gly Glu Glu Asp Asn Gly Gly Arg Gly Ser Gly Ser Asp Glu Asn Asn

515 520 525

Gly Asp Ala Gly Glu Thr Leu Leu Glu Leu Phe Arg Arg Thr

530 535 540

〈210〉 19

<211> 20

<212> DNA

〈213〉 Artificial Sequence

〈220〉

<223> Oligonucleotide

〈400〉 19

atgtcggaca aaaaaggggt 20

<210> 20

<211> 20

<212> DNA

〈213〉 Artificial Sequence

〈220〉

<223> Oligonucleotide

<400> 20

atgctaccac acgcagatcg 20

〈210〉 21

<211> 21

<212> DNA

〈213〉 Artificial Sequence

〈220〉

<223> Oligonucleotide

<400> 21

acagagacca cctccttgga a 21

〈210〉 22

<211> 22

<212> DNA

〈213〉 Artificial Sequence

〈220〉

<223> Oligonucleotide

<400> 22

cagaaacttg tctcatccct gg 22

<210> 23

<211> 20

<212> DNA

〈213〉 Artificial Sequence

〈220〉

<223> Oligonucleotide

<400> 23

aagaactgcc tgaagaaggc 20

<210> 24

<211> 20

<212> DNA

〈213〉 Artificial Sequence

〈220〉

<223> Oligonucleotide

<400> 24

caccaccttc atgatgctca 20

<210> 25

<211> 20

<212> DNA

〈213〉 Artificial Sequence

〈220〉

<223> Oligonucleotide

<400> 25

ttccagcacc ggcacaagaa 20

<210> 26

<211> 28

<212> DNA

〈213〉 Artificial Sequence

〈220〉

<223> Oligonucleotide

<400> 26

tggacctctt catgttcgat cccatctg 28

<210> 27

<211> 27

<212> DNA

〈213〉 Artificial Sequence

〈220〉

<223> Oligonucleotide

<400> 27

cctgacgctg ttccagaatg cgtttca 27

<210> 28

<211> 20

<212> DNA

〈213〉 Artificial Sequence

〈220〉

<223> Oligonucleotide

<400> 28

acttctgcag gtcgactcta 20

<210> 29

<211> 30

<212> DNA

〈213〉 Artificial Sequence

〈220〉

<223> Oligonucleotide

<400> 29

aagatcaaga tgaggacgtg gaagtcgtgg 30

<210> 30

<211> 30

<212> DNA

〈213〉 Artificial Sequence

〈220〉

<223> Oligonucleotide

<400> 30

aggctgaacc actggggcgc ctctcaccac 30

<210> 31

<211> 30

<212> DNA

〈213〉 Artificial Sequence

〈220〉

<223> Oligonucleotide

<400> 31

caagtatatg atgcgcgctc tagaggatga 30

<210> 32

<211> 30

<212> DNA

〈213〉 Artificial Sequence

〈220〉

<223> Oligonucleotide

<400> 32

aggtttcacc actaagtctc cttcaatggc 30

<210> 33

<211> 30

<212> DNA

〈213〉 Artificial Sequence

〈220〉

<223> Oligonucleotide

<400> 33

gatcattcaa gacttaggct cactcatgag 30

<210> 34

<211> 30

<212> DNA

〈213〉 Artificial Sequence

〈220〉

<223> Oligonucleotide

<400> 34

aacagcaagg aagattacaa atgatgccca 30

<210> 35

<211> 18

<212> DNA

〈213〉 Artificial Sequence

〈220〉

<223> Oligonucleotide

<400> 35

ggattaagat ctaatggc 18

<210> 36

<211> 23

<212> DNA

〈213〉 Artificial Sequence

〈220〉

<223> Oligonucleotide

<400> 36

caaagatctt catttcttaa aag 23

〈210〉 37

<211> 25

<212> DNA

〈213〉 Artificial Sequence

〈220〉

<223> Oligonucleotide

<400> 37

gcggatccat gtcggacaaa aaagg 25

<210> 38

<211> 25

<212> DNA

〈213〉 Artificial Sequence

〈220〉

<223> Oligonucleotide

<400> 38

gcggatcctc atccctggct gaagg 25

<210> 39

<211> 23

<212> DNA

〈213〉 Artificial Sequence

〈220〉

<223> Oligonucleotide

<400> 39

ggatccacca tggccacaag atg 23

〈210〉 40

<211> 24

<212> DNA

〈213〉 Artificial Sequence

〈220〉

<223> Oligonucleotide

<400> 40

ggatccttag tcaatatcat tagc 24

<210> 41

<211> 27

<212> DNA

〈213〉 Artificial Sequence

〈220〉

<223> Oligonucleotide

<400> 41

gcggatccat gggtcacgga ggagaag 27

<210> 42

<211> 27

<212> DNA

〈213〉 Artificial Sequence

〈220〉

<223> Oligonucleotide

<400> 42

gcggatcctc agttgttatg atcagga 27

Claims (28)

A DNA molecule comprising at least one of the sequences set forth in SEQ ID NO: 1 or SEQ ID NO: 2 and encoding a Mlo protein that confers resistance to fungal pathogens to plants. The Mlo of claim 1, wherein the DNA molecule is identical or substantially similar to one of the nucleotide sequences set forth in SEQ ID NO: 3, SEQ ID NO: 5 or SEQ ID NO: 7, or identical or substantially similar to the Mlo protein set forth in SEQ ID NO: 4, SEQ ID NO: 6 or SEQ ID NO: 8. DNA molecule encoding a protein. The method of claim 1, wherein the DNA molecule is the same as or substantially similar to one of the nucleotide sequences set forth in SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, or SEQ ID NO: 17, or SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, or SEQ ID NO: A DNA molecule encoding a Mlo protein, identical or substantially similar to the encoded Mlo protein described in 18. The DNA molecule of claim 1, wherein the DNA molecule is not derived from barley. The DNA molecule of claim 1, wherein the DNA is modified such that the activity of the endogenous protein is lost. The DNA molecule of claim 5, wherein the amino acid sequence of the protein corresponding to the DNA modification is modified by one, all, or a combination of the following: -Trp (163) to Arg Frame transition after Pro (396) Frame switching after Trp 160 Met (1) is Ile Gly (227) to Asp -Mep (1) goes to Val Arg (11) to Trp -Phe (183), Thr (184) deleted Val (31) to Glu -Ser (32) to Phe Leu (271) to His A DNA molecule that is antisense to the DNA molecule of any one of claims 1 to 6. A protein comprising at least one of the sequences set forth in SEQ ID NO: 1 or SEQ ID NO: 2 and which is a Mlo protein conferring resistance to fungal pathogens to plants. The compound of claim 8, wherein the protein is encoded by a nucleotide sequence identical or substantially similar to one of the sequences set forth in SEQ ID NO: 3, SEQ ID NO: 5 or SEQ ID NO: 7, or is identical to one of the proteins set forth in SEQ ID NO: 4, SEQ ID NO: 6 or SEQ ID NO: 8; Substantially similar protein. The protein of claim 8, wherein the protein is encoded by a nucleotide sequence identical or substantially similar to one of the sequences set forth in SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, or SEQ ID NO: 17, or SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, Sequence A protein identical or substantially similar to one of 16 or a protein set forth in SEQ ID NO: 18. The protein of claim 8, wherein the protein is not derived from barley. An expression cassette comprising the DNA molecule of any one of claims 1 to 7. A vector comprising an expression cassette comprising the DNA molecule of claim 12. A cell into which a DNA molecule in an expression cassette can be expressed, comprising an expression cassette comprising a DNA molecule of any one of claims 1 to 7 or a portion thereof. The cell of claim 14, wherein the DNA molecule is stably integrated in the genome of the cell. The cell according to claim 14 or 15, which is a plant cell. A plant in which a DNA molecule in an expression cassette can be expressed, comprising an expression cassette comprising a DNA molecule of any one of claims 1 to 7 or part thereof. 18. The plant of claim 17, wherein the DNA molecule is stably integrated in the genome of the plant. An agricultural product with improved phytosanitary properties, comprising a plant comprising the isolated DNA molecule of any one of claims 1-7. a) expressing the DNA molecule of any one of claims 1 to 6 in a plant in a "sense" orientation; or b) expressing the DNA molecule of any one of claims 1 to 6 in a plant in an "antisense" orientation; or c) expressing in the plant a ribozyme capable of specifically cleaving a messenger RNA transcript encoded by an endogenous gene corresponding to the DNA molecule of any one of claims 1 to 6; or d) expressing in the plant an aptamer specifically directed to the protein or portion of the protein encoded by the DNA molecule of any one of claims 1 to 6; or e) expressing in the plant a mutated or truncated form of the DNA molecule of any one of claims 1 to 6; or f) resistance to fungal pathogens to plants, comprising modifying at least one of the chromosomal copies of the gene corresponding to the DNA molecule of any one of claims 1 to 6 by homologous recombination in the plant. How to grant. A plant made to be resistant to fungal pathogens by the method of claim 20. The plant of claim 21, wherein the fungal pathogen infects the epithelial cells that survive. 22. The plant of claim 21, wherein the fungal pathogen is from the neck of Erysiphales. The plant of claim 21, wherein the fungal pathogen is of genus Erysiphe. The plant of claim 21, wherein the fungal pathogen is Erysiphe graminis. Agricultural products having improved phytosanitary properties obtained using the method of claim 20. a) a degenerate oligonucleotide complementary to a degenerate oligonucleotide encoding at least 6 of the amino acids of SEQ ID NO: 1 and a sequence encoding at least 6 of the amino acids of SEQ ID NO: 2 under conditions that the degenerate oligonucleotide hybridizes with DNA Mixing the nucleotides with DNA extracted from the plant; And b) amplifying a DNA fragment of plant DNA comprising at its left and right ends a nucleotide sequence that can be annealed to the degenerate oligonucleotide in step a); And c) obtaining a full length cDNA clone comprising the DNA fragment of step b). a) adding at least one single-chain or double-stranded oligonucleotide comprising a region homologous to the double-stranded template polynucleotide and a heterologous region to the population of resulting double-stranded random fragments; b) denaturing the resulting mixture of double stranded random fragments and oligonucleotides with single chain fragments; c) incubating the resulting population of single-chain fragments with the polymerase under conditions such that one member of the pair anneals the single-chain fragment in a region of homology sufficient to prime the replication of the other to form a pair of annealed fragments. Treating to form a mutated double stranded polynucleotide; And d) the second and third agents such that the mixture produced in the second stage of the further cycle comprises a mutated double-stranded polynucleotide originating from the third stage of the previous cycle, such that the further cycle forms a more mutated double-stranded polynucleotide. A method of mutating the DNA molecule of claim 1, wherein the DNA molecule is cleaved into two-stranded-random fragments of the desired size, comprising repeating three steps at least two additional cycles.