CN113227383B

CN113227383B - Role of HAK gene in regulating and controlling plant traits

Info

Publication number: CN113227383B
Application number: CN202080003873.1A
Authority: CN
Inventors: 不公告发明人
Original assignee: Shandong Shunfeng Biotechnology Co Ltd
Current assignee: Shandong Shunfeng Biotechnology Co Ltd
Priority date: 2019-12-30
Filing date: 2020-12-29
Publication date: 2022-11-22
Anticipated expiration: 2040-12-29
Also published as: WO2021136255A1; CN113227383A; CN113122566B; CN113122566A

Abstract

Use of a substance for improving a trait in a plant or for preparing a formulation or composition for modulating the shape of a plant, wherein the trait in the plant comprises one or more traits selected from the group consisting of: (ii) (i) root length, root branching and/or root weight; (ii) plant height; and (iii) salt tolerance; wherein the substance is selected from the group consisting of: (a) a HAK protein; (b) a nucleic acid sequence encoding a HAK protein; (c) promoters of the HAK protein and its coding nucleic acid sequences; or a combination thereof. The HAK protein or the mutant protein thereof and the corresponding method can realize the regulation and control of properties such as lodging resistance, biomass resistance, salt resistance and the like, and have important scientific value for cultivating new varieties of plants with high quality, high yield and stress tolerance.

Description

Role of HAK gene in regulation and control of plant traits

Technical Field

The invention relates to the field of biology, in particular to the field of plant biology, and specifically relates to an effect of a HAK gene in regulation and control of plant traits.

Background

The prior art reports that the KUP/HAK/KT family is the potassium transport family which is first discovered, has the largest number and most abundant functions in the biology world and is widely present in a plurality of species. It mediates the potassium absorption of plant in low potassium stress, and participates in the physiological process of plant cell enlargement, root auxin transport and the like, the expression of the family gene is regulated and controlled by the factors of plant growth and development regulating factors, environmental stimulation and the like, and participates in the growth and development and stress response mechanism of the plant. KUP (K + uptake permenase) is firstly found in Escherichia coli and is responsible for coding Escherichia coli potassium absorption permease; HAK (high-affinity K +) is a KUP homologous gene identified in Schwanniomyces. KT (K + transporter) is a transporter with a potassium transport function found in arabidopsis thaliana.

Salt stress is one of the major abiotic stresses of plants, and causes changes in a plurality of metabolic pathways of the plants, thereby generating complex physiological level changes, mainly manifested as ion imbalance, water deficiency and oxidative toxicity, and further causing reduction of plant photosynthesis, increase of energy consumption, acceleration of senescence, limitation of growth, reduction of yield and quality. One of the main ways plants increase their own salt stress resistance is to regulate potassium (K) and sodium (Na) transport in vivo through xylem parenchyma. The tolerance to salt stress is realized by the adaptation to the toxic action of Na + and the maintenance of K + nutrition, and the plant with higher K +/Na + ratio has stronger tolerance to salt stress.

Research reports that KUP/HAK/KT family genes are also induced by high-salt adversity stress, however, at present, the genes exerting salt resistance in a plurality of HAK subtypes and the mechanism thereof are not clear.

Therefore, there is a strong need in the art to resolve the mechanism of action of the relevant genes and to develop a method capable of enhancing the tolerance of plants to high salt stress.

Disclosure of Invention

The invention aims to provide a method for enhancing the tolerance of plants to high-salt stress by regulating and controlling HAK genes. Further provided is a method for enhancing salt resistance of a plant by mediating regulation of Na +.

Another objective of the invention is to provide the use of HAK gene and its mutant in enhancing plant salt tolerance, wherein the salt tolerance of plants can be significantly enhanced by over-expressing the gene or mutating to obtain mutant protein with better activity.

In a first aspect of the invention there is provided the use of a substance for improving a trait in a plant or for the preparation of a formulation or composition for modulating a trait in a plant, wherein the trait in the plant comprises one or more traits selected from the group consisting of: (ii) (i) root length, root branching and/or root weight; (ii) plant height; and (iii) salt tolerance;

wherein the substance is selected from the group consisting of: (a) a HAK protein; (b) a nucleic acid sequence encoding a HAK protein; (c) promoters of the HAK protein and its coding nucleic acid sequences; or a combination thereof.

In another preferred embodiment, the HAK protein includes a wild-type HAK protein and a mutant HAK protein.

In another preferred embodiment, the HAK protein mediates Na in plants ⁺ The function of transport.

In another preferred embodiment, the amino acid sequence of the wild-type HAK protein is shown in SEQ ID NO. 2.

In another preferred embodiment, the sequence of the HAK protein mutant has at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity compared to the sequence from which it is derived.

In another preferred embodiment, the sequence from which it is derived is the amino acid sequence of a wild-type HAK protein.

In another preferred embodiment, the use further comprises regulating Na in plants ⁺ Ion transport, embodied in one or any of the following i-iv:

i. regulating the accumulation of Na + ions of the plant roots;ii. Promote Na in the middle ⁺ Transfer to and unloading from the xylem; and iii, reducing the Na +/K + ratio of the roots and the overground parts.

In another preferred embodiment, the plant includes angiosperms and gymnosperms.

In another preferred embodiment, the gymnosperm is selected from the group consisting of: cycadaceae (Cycadaceae), podocarpaeaceae (podocarpaeceae), araucaceae (araucaceae), pinaceae (Pinaceae), cedaceae, cypress, cephalotaxaceae, taxaceae, ephedra, gnetaceae, monotype, welchidaceae, or combinations thereof.

In another preferred embodiment, the plant includes a monocotyledon and a dicotyledon.

In another preferred embodiment, the plant includes herbaceous plants and woody plants.

In another preferred embodiment, the herbaceous plant is selected from the group consisting of: solanaceae, poaceae, leguminous plants, or combinations thereof.

In another preferred embodiment, the woody plant is selected from the group consisting of: actinidiaceae, rosaceae, moraceae, or their combination.

In another preferred embodiment, the plant is selected from the group consisting of: cruciferous plants, gramineae, leguminous plants, solanaceae, actinidiaceae, malvaceae, paeoniaceae, rosaceae, liliaceae, or combinations thereof.

In another preferred embodiment, the plant is selected from the group consisting of: arabidopsis, rice, soybean, tomato, corn, sorghum, quinoa, millet, potato, tobacco, wheat, sorghum, rape, spinach, lettuce, chinese cabbage, cucumber, garland chrysanthemum, water spinach, celery, lettuce or a combination thereof.

In another preferred embodiment, the HAK gene is derived from: gymnosperms or angiosperms.

In another preferred example, the HAK gene is derived from a monocotyledon or a dicotyledon.

In another preferred embodiment, the HAK gene is derived from herbaceous plants and woody plants.

In another preferred example, the HAK gene is derived from a solanaceae plant, a gramineae plant, a leguminous plant, or a combination thereof.

In another preferred example, the HAK gene is derived from actinidiaceae, rosaceae, moraceae, or a combination thereof.

In another preferred example, the HAK gene is derived from a crucifer, a gramineae, a leguminous plant, a solanaceae, a actinidiaceae, a malvaceae, a paeoniaceae, a rosaceae, a liliaceae, or a combination thereof.

In another preferred example, the HAK gene is derived from arabidopsis thaliana, rice, soybean, tomato, corn, sorghum, quinoa, millet, potato, tobacco, wheat, sorghum, canola, spinach, lettuce, chinese cabbage, cucumber, garland chrysanthemum, water spinach, celery, lettuce or a combination thereof.

In another preferred embodiment, the nucleic acid sequence encoding the HAK protein is a nucleic acid sequence of a gene selected from the group consisting of: slHAK20, osHAK4, osHAK17, zmHAK4, sbHAK9, sbHAK26, grHAK8, gmHAK12, ptHAK21, mdHAK5, or a combination thereof.

In another preferred embodiment, the nucleic acid sequence encoding the HAK protein is selected from the group consisting of: slHAK20 gene from tomato, osHAK4 gene or OsHAK17 gene from rice.

In another preferred embodiment, the amino acid sequence of the HAK protein is selected from the group consisting of:

(i) A polypeptide having an amino acid sequence shown in

SEQ ID NO

2, 4 or 6;

(ii) 2, 4 or 6 through one or more (such as 1-10) amino acid residue substitution, deletion or addition, and (i) derived polypeptide with the function of regulating plant traits; or

(iii) The amino acid sequence has at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence as set forth in

SEQ ID NO

2, 4, or 6, and has the ability to mediate Na in plants ⁺ A functional polypeptide of transport.

In another preferred embodiment, the nucleotide sequence encoding the HAK protein is selected from any one of the following groups or a combination thereof:

(a) A polynucleotide encoding a polypeptide as set forth in

SEQ ID NO

2, 4 or 6;

(b) 1, 3 or 5 as shown in SEQ ID NO; or

(c) A sequence having substitution, deletion or addition (e.g., 1 to 10) of one or more bases as compared with the nucleotide sequence represented by

SEQ ID NO

1, 3 or 5;

(d) A nucleotide sequence having at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% sequence identity to the nucleotide sequence set forth in SEQ ID NO. 1, 3 or 5;

(e) A nucleotide sequence in which 1 to 60 (preferably 1 to 30, more preferably 1 to 10) nucleotides are truncated or added at the 5 'end and/or 3' end of the nucleotide sequence shown in SEQ ID NO. 1, 3 or 5;

(f) A polynucleotide sequence complementary to the nucleotide sequence of any one of (a) - (e).

In another preferred embodiment, the HAK protein mutant is a SlHAK20 protein mutant.

In another preferred example, the nucleic acid sequence encoding a mutant HAK protein is a mutant SlHAK20 nucleic acid sequence.

In another preferred embodiment, the mutant form of the protein comprises substitution, deletion or addition of one or more amino acids.

In another preferred embodiment, the amino acid sequence of the HAK protein mutant includes:

(i) 2, 4 or 6, by deletion of 10 to 600 (preferably 100 to 500, more preferably 400 to 500, more preferably 450 to 480, more preferably 460 to 480, more preferably 470 to 480, more preferably 472 or 473) amino acid residues at the C-terminus; and

(ii) Optionally, an unequal number of amino acid fragments of random sequence added by cellular repair mechanisms are located C-terminal to the sequence of amino acids.

In another preferred embodiment, the amino acid fragments with variable numbers and random sequences comprise 1 to 100 amino acids, preferably 1 to 50 amino acids, preferably 1 to 30 amino acids, preferably 5 to 25 amino acids, preferably 10 to 20 amino acids.

In another preferred embodiment, the deletion is preferably a continuous deletion of amino acids in the C-stretch.

In another preferred embodiment, the number of deleted amino acids is more than half the number of amino acids in the amino acid sequence shown in

SEQ ID NO

2, 4 or 6.

In another preferred embodiment, the HAK protein mutant has at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to the amino acid sequence set forth in SEQ ID NO. 2, 4 or 6.

In another preferred embodiment, the mutant protein at least substantially retains the biological function of the sequence from which it is derived.

In another preferred embodiment, the sequence of the HAK protein mutant is selected from the group consisting of:

(i) A polypeptide having an amino acid sequence shown in

SEQ ID NO

12 or 14;

(ii) 12 or 14 through one or more (such as 1-10) amino acid residues, and has the plant character regulating function and is derived from the polypeptide (i); or

(iii) The polypeptide with the function of regulating Na + transport in plants, wherein the homology of the amino acid sequence and the amino acid sequence shown in SEQ ID NO. 12 or 14 is more than or equal to 80 percent (preferably more than or equal to 90 percent, more preferably more than or equal to 95 percent or more than or equal to 98 percent).

In another preferred embodiment, the nucleotide sequence encoding the mutant HAK protein is selected from the group consisting of:

(a) A polynucleotide encoding a polypeptide as set forth in

SEQ ID NO

12 or 14;

(b) A nucleotide sequence as shown in SEQ ID NO. 11 or 13; or

(c) A sequence having substitution, deletion or addition (e.g., 1 to 10) of one or more bases as compared with the nucleotide sequence shown in SEQ ID NO. 11 or 13;

(d) A nucleotide sequence having homology of 80% or more (preferably 90% or more, more preferably 95% or 98% or more) with the nucleotide sequence represented by SEQ ID NO. 11 or 13;

(e) A nucleotide sequence in which 1 to 60 (preferably 1 to 30, more preferably 1 to 10) nucleotides are truncated or added at the 5 'end and/or the 3' end of the nucleotide sequence shown in SEQ ID NO. 11 or 13;

In another preferred embodiment, the nucleic acid sequence encoding the mutant is obtained by natural mutagenesis or gene editing techniques.

In another preferred example, the gene editing technology comprises TALEN, ZIN, CRISPR.

In another preferred embodiment, the composition comprises an agricultural composition.

In another preferred embodiment, the composition further comprises an agronomically acceptable carrier.

In another preferred embodiment, the composition comprises the substance in an amount of (a) HAK protein; (b) a nucleic acid sequence encoding a HAK protein; (c) promoters of the HAK protein and its coding nucleic acid sequences; or a combination thereof, from 0.0001 to 99wt%, preferably from 0.1 to 90wt%, more preferably from 1 to 80wt%, based on the total weight of the composition.

In another preferred embodiment, the composition or formulation is in a dosage form selected from the group consisting of: a solution, an emulsion, a suspension, a powder, a foam, a paste, a granule, an aerosol, or a combination thereof.

In another preferred embodiment, the promoter comprises a small molecule compound that promotes expression of the HAK gene or its encoded protein.

In another preferred embodiment, the accelerator is selected from the group consisting of: a small molecule compound, a nucleic acid molecule, or a combination thereof.

In a second aspect of the present invention, there is provided a method of improving a trait in a plant comprising the steps of: modulating the expression level and/or activity of a HAK protein or a nucleic acid sequence encoding the same in said plant, thereby improving the trait of the plant.

In another preferred embodiment, the trait improvement of the plant is selected from one or more of the group consisting of:

(i) Increase root length and/or root branching;

(ii) The plant height is increased; and

(iii) And the salt tolerance is enhanced.

In another preferred embodiment, the improvement of the trait further comprises: (iv) promoting Na + ion transport in plants.

In another preferred example, said modulating the expression level and/or activity of a HAK protein or a nucleic acid encoding the same in said plant comprises:

(a) Enhancing the expression amount and/or activity of an endogenous HAK protein or a nucleic acid sequence encoding the same in said plant;

(b) Introducing a foreign HAK protein or a nucleic acid sequence encoding the same, or a mutant HAK protein or a nucleic acid sequence encoding the same, into the plant, thereby allowing the plant to have the expression level and/or activity of the foreign HAK protein or the nucleic acid sequence encoding the same, or the mutant HAK protein or the nucleic acid sequence encoding the same; or

(c) An enhancer for administering HAK proteins and nucleic acid sequences encoding the same.

In another preferred embodiment, the ratio (E1/E0) of the activity E1 of the HAK protein mutant or the nucleic acid sequence encoding the mutant in the plant to the background activity E0 of the same wild-type HAK protein or the nucleic acid sequence encoding the mutant in the plant of the same type (E1/E0) is not less than 1.5 times or not more than 2 times or not, preferably not less than 5 times or not, more preferably not less than 10 times.

In another preferred example, the method comprises the steps of:

(i) Providing a plant or plant cell; and

(ii) Introducing into said plant or plant cell a HAK protein or a nucleic acid encoding it, or a promoter thereof, or a mutant HAK protein or a nucleic acid encoding it, thereby improving the trait of the plant.

In another preferred example, the method comprises the steps of:

(i) Providing a plant or plant cell; and

(ii) Introducing gene editing tool, contacting with nucleotide sequence in plant, mutating to obtain HAK protein mutant nucleotide sequence, and improving plant properties.

In another preferred embodiment, the method for introducing includes: agrobacterium transformation, particle gun, microinjection, electroporation, ultrasound, and polyethylene glycol (PEG) mediated methods.

In a third aspect of the invention, there is provided a mutein having an amino acid sequence selected from the group consisting of:

(i) An amino acid sequence formed by deleting 10 to 600 (preferably 100 to 500, more preferably 400 to 500, more preferably 450 to 480, for example, 472, 473) amino acid residues at the C terminal based on the amino acid sequence shown in SEQ ID NO. 2; and

(ii) Optionally, an unequal number of amino acid fragments of random sequence are added by cellular repair mechanisms at the C-terminus of the sequence of amino acids.

In another preferred embodiment, the deletion is preferably a continuous deletion of amino acids in the C stretch.

In another preferred embodiment, the number of deleted amino acids is more than half of the number of amino acids in the amino acid sequence shown in SEQ ID NO. 2.

In one embodiment, the amino acid sequence of the mutein is an amino acid sequence formed by the continuous deletion of 473 amino acid residues in the C-terminus on the basis of the amino acid sequence shown in SEQ ID NO. 2. Optionally, 1-100 amino acids, preferably 1-50 amino acids, preferably 1-30 amino acids, preferably 5-25 amino acids, preferably 10-20 amino acids are added at the C-terminal of the mutant protein.

In another preferred embodiment, the amino acid sequence of the mutein is selected from the group consisting of:

(i) A polypeptide having an amino acid sequence shown in

SEQ ID NO

12 or 14;

(ii) 12 or 14 through one or more (such as 1-10) amino acid residue substitution, deletion or addition, and has the plant character regulating function; or

(iii) (ii) the amino acid sequence has homology of 80% or more (preferably 90% or more, more preferably 95% or 98% or more) with the amino acid sequence shown in

SEQ ID NO

12 or 14, and has at least substantially the same activity as the polypeptide of (i).

In a fourth aspect of the invention, there is provided an isolated polynucleotide encoding a mutein according to the third aspect of the invention.

In another preferred embodiment, the nucleotide sequence of said polynucleotide is selected from the group consisting of:

(a) A polynucleotide encoding a polypeptide as set forth in

SEQ ID NO

12 or 14;

(b) A nucleotide sequence shown as SEQ ID NO. 11 or 13; or

(e) A nucleotide sequence in which 1 to 60 (preferably 1 to 30, more preferably 1 to 10) nucleotides are truncated or added at the 5 'end and/or 3' end of the nucleotide sequence shown in SEQ ID NO. 11 or 13;

In a fifth aspect of the invention, there is provided a vector comprising a polynucleotide according to the fourth aspect of the invention.

In a sixth aspect of the invention, there is provided a host cell comprising a vector or genome according to the fifth aspect of the invention into which has been integrated a polynucleotide according to the fourth aspect of the invention.

In another preferred embodiment, the host cell includes eukaryotic cells and prokaryotic cells.

In a seventh aspect of the invention, there is provided a genetically engineered plant cell, tissue or organ comprising a mutant HAK protein according to the third aspect of the invention or a nucleic acid sequence encoding the same.

In an eighth aspect of the invention, there is provided a method of producing a genetically engineered plant cell, plant tissue or organ according to the seventh aspect of the invention, comprising the method steps of: regulating the expression level and/or activity of the HAK gene or the protein coded by the HAK gene in the plant, thereby obtaining the genetically engineered plant tissue or plant cell.

In another preferred example, the method comprises the steps of:

(1) Providing a plant cell, plant tissue or organ; and

(2) Introducing a means for gene editing into contact with the nucleotide sequence in the plant, and mutating to obtain a nucleotide sequence encoding a mutant HAK protein according to the third aspect of the present invention.

In another preferred example, the method comprises the steps of:

(1) Providing a plant cell, plant tissue or organ; and

(2) Introducing a vector comprising the HAK protein mutant of the third aspect of the invention or a nucleic acid sequence encoding the mutant.

In a ninth aspect of the present invention, there is provided a method of preparing a transgenic plant or a gene-editing plant, comprising the steps of:

regenerating a plant cell, tissue or organ according to the seventh aspect of the present invention into a plant body, thereby obtaining a transgenic or gene-edited plant.

In a tenth aspect of the invention, there is provided a method of making a mutein of the third aspect of the invention, comprising the steps of:

culturing the host cell of the sixth aspect of the invention under conditions suitable for expression, thereby expressing the fusion protein; and/or, isolating the fusion protein.

In an eleventh aspect of the present invention, there is provided a method for detecting salt tolerance in a plant, which comprises detecting the expression activity and/or expression amount of a HAK protein or a nucleic acid encoding the same or a mutant thereof.

In another preferred embodiment, the HAK protein is selected from the group consisting of: slHAK20, osHAK4, osHAK17, zmHAK4, sbHAK9, sbHAK26, grHAK8, gmHAK12, ptHAK21, mdHAK5, or any combination thereof.

It is to be understood that within the scope of the present invention, the above-described features of the present invention and those specifically described below (e.g., in the examples) may be combined with each other to form new or preferred embodiments. Not to be reiterated herein, but to the extent of space.

Drawings

Fig. 1 shows the transcription levels of SlHAK20 in different tomato plants. Wherein SlEF1a is used as an internal reference.

FIG. 2 shows the protein expression levels of SlHAK20-YFP in different tomato plants.

FIG. 3 shows the results of the salt tolerance test of transgenic tomato plants.

Wherein, a shows the plant growth state under 175mM NaCl salt stress condition; b shows the growth status of the plants after two weeks of recovery.

FIG. 4 shows the results of survival analysis of transgenic tomato plants.

FIG. 5 shows Na + and K + contents of the mutant roots compared to the wild type roots with time under salt stress conditions.

FIG. 6 shows Na +/K + of the mutant versus wild type roots over time under salt stress conditions.

FIG. 7 shows Na + and K + content of mutants over time compared to wild type xylem under salt stress conditions.

FIG. 8 shows Na + and K + contents of the above-ground parts of the mutant as compared with the wild type under salt stress conditions with time.

FIG. 9 shows Na +/K + in the above-ground portion of the mutant as compared to the wild type over time under salt stress conditions.

FIG. 10 shows the change in root length and number of root branches of the mutant compared to the wild type after salt treatment.

FIG. 11 shows the plant height of the mutant after salt treatment compared with the wild type.

FIG. 12 shows Na +/K + of roots and aerial parts of mutant type more wild type under salt stress conditions.

Wherein, a shows the Na +/K + of the root of the mutant type compared with the wild type; b shows Na +/K + of the aerial parts of the mutant type compared to the wild type.

The sequence listing in this application is as follows:

Detailed Description

The present inventors have extensively and intensively studied and, after extensive screening, have for the first time unexpectedly found that a HAK gene of a plant (such as tomato) or a protein encoded by the gene or a promoter thereof can be used for controlling plant traits, including: root length, root weight, root branching, plant height, salt tolerance. Research has shown that overexpression of the HAK protein (transporter) or its encoding gene in tomato increases the salt resistance of plants. The HAK protein and the encoding gene can be used for regulating and controlling the Na + transport of plants, and under the condition of salt stress, the HAK protein can be used for regulating and controlling the loading and unloading of Na + in the plants so as to improve the salt tolerance of the plants. Specifically, the HAK protein enhances Na + transport to and from the xylem in plants, while facilitating Na + transport from the root to other tissues or the outside world.

Furthermore, the present inventors have also surprisingly found that when the amino acid sequence of the HAK protein is subjected to C-terminal deletion by gene editing techniques, under salt stress, root length and root branching can be increased, plant height of a plant can be increased, and salt tolerance of the plant can be enhanced. The improvement of the root can enhance the lodging resistance of the plant, promote the absorption of nutrient substances and enhance the environmental adaptability. And the increase of the plant height can increase the biomass and/or the yield of the plant.

The research also finds that the deletion of 6 bases at the downstream 48bp of the HAK gene promoter and the replacement of the base G to A at 3093bp cause the reduction of the salt tolerance of the plant.

On the basis of this, the present invention has been completed.

HAK protein and encoding nucleic acid sequence

As used herein, the terms "HAK protein", "HAK polypeptide", "HAK transporter" are used interchangeably and refer to a class of ion transporters that are present on different organelle membranes, such as the plasma membrane, the vacuolar membrane, and the thylakoid membrane.

As used herein, the terms "mutant HAK protein", and "mutant HAK protein" are used interchangeably and refer to a mutant obtained by mutation of the amino acid sequence of a wild-type HAK protein.

As used herein, the terms "HAK gene of the present invention", "HAK gene", "nucleic acid sequence encoding HAK protein", which are used interchangeably under appropriate conditions, all refer to DNA sequences, all refer to HAK genes or variants thereof derived from plants (e.g., tomato, wheat, etc.).

In a preferred embodiment, the HAK gene of the invention is SlHAK20 gene, the nucleotide sequence of which is shown in SEQ ID NO. 1, and the coded amino acid sequence of which is shown in SEQ ID NO. 2.

The invention also includes nucleic acids having at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence homology to a preferred gene sequence of the invention (SEQ ID NO: 1), which nucleic acids are also effective in modulating an agronomic trait in a plant, such as tomato.

"homology" or "identity" refers to the match of sequences between two polypeptides or between two nucleic acids. When a position in both of the sequences being compared is occupied by the same base or amino acid monomer subunit (e.g., a position in each of two DNA molecules is occupied by adenine, or a position in each of two polypeptides is occupied by lysine), then the molecules are identical at that position. The "percent identity" between two sequences is a function of the number of positions compared at the first by the number of matching positions shared by the two sequences x 100. For example, if 6 of 10 positions of two sequences match, then the two sequences have 60% identity. Typically, the comparison is made when it is difficult to Align the two sequences to produce maximum identity, such alignment can be conveniently performed by using, for example, a computer program such as the Align program (DNAstar, inc.) Needleman et al (1970) j.mol.biol.j.mol.mol.biol.j.mol.biol.j.mol.j.mol.biol.48: 443-453. The algorithm of e.meyers and w.miller (comput.appl biosci.,4, 11-17 (1988)) which has been incorporated into the ALIGN program (version 2.0) can also be used to determine percent identity between two amino acid sequences using a PAM120 weight residue table (weight residue table), a gap length penalty of 12, and a gap penalty of 4. The method is realized. Furthermore, percent identity between two amino acid sequences can be determined using the Needleman and Wunsch (J MoI biol.48:444-453 (1970)) algorithms that have been incorporated into the GAP program of the GCG software package (available at www.gcg.com), using either the Blossum 62 matrix or the PAM250 matrix, and GAP weights (GAP weights) of 16, 14, 12, 10, 8, 6, or 4, and length weights of 1, 2, 3, 4, 5, or 6. In this context, variants of the genes can be obtained by insertion or deletion of regulatory regions, random or site-directed mutagenesis, and the like.

In the present invention, the nucleotide sequence of SEQ ID NO. 1 may be substituted, deleted or added with one or more nucleotides to generate a derivative sequence of SEQ ID NO. 1, and due to the degeneracy of codons, even if the homology with SEQ ID NO. 1 is low, the amino acid sequence shown as SEQ ID NO. 2 can be basically encoded. In addition, the meaning of "a nucleotide sequence in SEQ ID NO. 1 is substituted, deleted or added with at least one nucleotide-derived sequence" also includes a nucleotide sequence that hybridizes to the nucleotide sequence shown in SEQ ID NO. 1 under moderate stringency conditions, more preferably under high stringency conditions. These variants include (but are not limited to): deletion, insertion and/or substitution of several (usually 1 to 90, preferably 1 to 60, more preferably 1 to 20, most preferably 1 to 10) nucleotides, and addition of several (usually less than 60, preferably less than 30, more preferably less than 10, most preferably less than 5) nucleotides at the 5 'and/or 3' end.

It is to be understood that although the genes provided in the examples of the present invention are derived from tomato, the gene sequences of HAK derived from other similar plants (especially plants belonging to the same family or genus as tomato) and having a certain homology (conservation) with the sequence of the present invention (preferably, the sequence shown in SEQ ID NO: 1) are also included in the scope of the present invention, as long as the sequence can be easily isolated from other plants by those skilled in the art based on the information provided in the present application after reading the present application.

The polynucleotide or nucleic acid sequence of the present invention may be in the form of DNA or RNA. The DNA forms include: DNA, genomic DNA or synthetic DNA, DNA may be single-stranded or double-stranded. The DNA may be the coding strand or the non-coding strand. The sequence of the coding region encoding the mature polypeptide may be identical to the sequence of the coding region shown in SEQ ID NO. 1 or may be a degenerate variant.

Polynucleotides encoding a mature polypeptide include coding sequences that encode only a mature polypeptide; the coding sequence for the mature polypeptide and various additional coding sequences; the coding sequence (and optionally additional coding sequences) for the mature polypeptide as well as non-coding sequences.

The term "polynucleotide encoding a polypeptide" may include a polynucleotide encoding the polypeptide, and may also include additional coding and/or non-coding sequences. The invention also relates to variants of the above polynucleotides which encode fragments, analogs and derivatives of the polyglycosides or polypeptides having the same amino acid sequence as the invention. The variant of the polynucleotide may be a naturally occurring allelic variant or a non-naturally occurring variant. These nucleotide variants include substitution variants, deletion variants and insertion variants. As is known in the art, an allelic variant is a substitution form of a polynucleotide, which may be a substitution, deletion, or insertion of one or more nucleotides, without substantially changing the function of the encoded polypeptide.

The present invention also relates to polynucleotides which hybridize to the sequences described above and which have at least 50%, preferably at least 70%, and more preferably at least 80% identity between the two sequences. The present invention particularly relates to polynucleotides which hybridize under stringent conditions to the polynucleotides of the invention. In the present invention, "stringent conditions" mean: (1) Hybridization and elution at lower ionic strength and higher temperature, e.g., 0.2 XSSC, 0.1% SDS,60 ℃; or (2) adding denaturant during hybridization, such as 50% (v/v) methylphthalamide, 0.1% calf serum/0.1% Ficoll,42 deg.C, etc.; or (3) hybridization only when the identity between two sequences is at least 90% or more, preferably 95% or more.

It is to be understood that although the HAK gene of the invention is preferably from tomato, other genes from other plants that have some homology and function in close proximity to the tomato HAK gene are also within the contemplation of the invention. Methods and means for aligning sequence identity are also well known in the art, for example BLAST.

For example, in another preferred embodiment of the present invention, the HAK gene of the present invention may also be OsHAK4 gene derived from rice, the nucleotide sequence of which is shown in SEQ ID NO. 3, and the amino acid sequence of the encoded protein of which is shown in SEQ ID NO. 4. In another preferred embodiment of the present invention, the HAK gene of the present invention may also be OsHAK17 derived from rice, the nucleotide sequence of which is shown in SEQ ID NO. 5, and the amino acid sequence of the encoded protein of which is shown in SEQ ID NO. 6. Further comprises ZmHAK4 from corn; sbHAK9, sbHAK26 derived from sorghum; grHAK8 from cotton; gmHAK12 derived from soybean, and the like.

The full-length sequence of the HAK nucleotide or a fragment thereof of the present invention can be obtained by a PCR amplification method, a recombination method, or an artificial synthesis method. For PCR amplification, primers can be designed based on the nucleotide sequences disclosed herein, particularly open reading frame sequences, and amplified using commercially available DNA libraries or cDNA libraries prepared by conventional methods known to those skilled in the art as templates to obtain the sequences. When the sequence is long, it is often necessary to perform two or more PCR amplifications, and then splice together the amplified fragments in the correct order. Once the sequence of interest has been obtained, it can be obtained in large quantities by recombinant methods. Usually, it is cloned into a vector, transferred into a cell, and then isolated from the propagated host cell by a conventional method to obtain the relevant sequence.

In addition, the sequence can be synthesized by artificial synthesis, especially when the fragment length is short. Typically, long fragments are obtained by first synthesizing a plurality of small fragments and then ligating them together. At present, DNA sequences encoding the proteins of the present invention (or fragments or derivatives thereof) have been obtained completely by chemical synthesis. The DNA sequence may then be introduced into various existing DNA molecules (or vectors, for example) and cells known in the art. Furthermore, mutations can also be introduced into the protein sequences of the invention by chemical synthesis.

As used herein, the terms "mutein of the invention", "SlHAK20 mutein", "protein encoded by mutant of SlHAK20 gene" and "polypeptide of the invention" are used interchangeably and all refer to a mutein of the invention formed after deletion of the C-terminus of the protein encoded by the SlHAK20 gene.

In another preferred embodiment, a typical amino acid sequence of the SlHAK20 mutein of the invention is shown in SEQ ID NO 12.

MDRQTGRPDLTAAAAAQSDAAVTVAVHDGETINNDQKDHSRWETLVLAYKTLGVVFGGLVTSPLYVYPSMPLKSPTEDDYLGIYSIMFWTLSLIGVVKYATIALQADDQGEGGTFALYSLLCRNINIGILSSKSASLNSSHSYVNQSKKPSRLGKFCERSLIARRVLLFIAMLGMCMLIGDGILTPAISVLSAMGGLRARFSSVSKSLVEGLSAIILIVLFLLQKFGTSRVSFLLSPIMGHGLLPLLSLGYTVS*(SEQ ID NO:12)

In another preferred embodiment, a typical amino acid sequence of the SlHAK20 mutein of the invention is shown in SEQ ID No. 14.

MDRQTGRPDLTAAAAAQSDAAVTVAVHDGETINNDQKDHSRWETLVLAYKTLGVVFGGLVTSPLYVYPSMPLKSPTEDDYLGIYSIMFWTLSLIGVVKYATIALQADDQGEGGTFALYSLLCRNINIGILSSKSASLNSSHSYVNQSKKPSRLGKFCERSLIARRVLLFIAMLGMCMLIGDGILTPAISVLSAMGGLRARFSSVSKSLVEGLSAIILIVLFLLQKFGTSRVSFLLSPIMGCMDSYHSSHWDIQYHKALSEHI*(SEQ ID NO:14)

Researches show that under the condition of salt stress, the HAK protein can regulate and control the loading and unloading of Na + in plants so as to improve the salt tolerance of the plants.

Specifically, the HAK protein enhances Na + transport to and from the xylem in plants, while facilitating Na + transport from the root to other tissues or the outside world.

The invention relates to a SlHAK20 protein for improving plant traits and a mutant protein thereof, wherein in a preferred embodiment of the invention, the amino acid sequence of the SlHAK20 protein is shown as SEQ ID NO. 2; the amino acid sequence of the SlHAK20 mutant protein is shown in

SEQ ID NO

12 or 14. The polypeptide of the invention can effectively regulate and control the character of plants (such as rice).

The invention also includes polypeptides or proteins having at least 50% or more (at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) homology to the sequences set forth in

SEQ ID NOs

2, 12 or 14 of the invention, and having the same or similar function.

The "same or similar functions" mainly refer to: the root length, the root branching, the plant height, the salt tolerance and other properties of the plant are improved.

The polypeptide of the present invention may be a recombinant polypeptide, a natural polypeptide, or a synthetic polypeptide. The polypeptides of the invention can be naturally purified products, or chemically synthesized products, or using recombinant technology from prokaryotic or eukaryotic hosts (e.g., bacteria, yeast, higher plant, insect and mammalian cells). Depending on the host used in the recombinant production protocol, the polypeptides of the invention may be glycosylated or may be non-glycosylated. The polypeptides of the invention may or may not also include an initial methionine residue.

The invention also includes SlHAK20 mutein fragments and analogs having SlHAK20 mutein activity. As used herein, the terms "fragment" and "analog" refer to a polypeptide that retains substantially the same biological function or activity of a native SlHAK20 protein of the invention.

The polypeptide fragment, derivative or analogue of the invention may be: (i) Polypeptides in which one or more conserved or non-conserved amino acid residues (preferably conserved amino acid residues) are substituted, and such substituted amino acid residues may or may not be encoded by the genetic code; or (ii) a polypeptide having a substituent group in one or more amino acid residues; or (iii) a polypeptide formed by fusing the mature polypeptide with another compound, such as a compound that increases the half-life of the polypeptide, e.g., polyethylene glycol; or (iv) a polypeptide formed by fusing an additional amino acid sequence to the polypeptide sequence (e.g., a leader or secretory sequence or a sequence used to purify the polypeptide or a proprotein sequence, or a fusion protein). Such fragments, derivatives and analogs are within the scope of those skilled in the art as defined herein.

In the present invention, the polypeptide variant is an amino acid sequence shown in SEQ ID NO. 2, 12 or 14, a derivative sequence obtained by several (usually 1-60, preferably 1-30, more preferably 1-20, and most preferably 1-10) substitutions, deletions or additions of at least one amino acid, and one or several (usually less than 20, preferably less than 10, and more preferably less than 5) amino acids added to the C-terminus and/or N-terminus. For example, in the protein, when the performance similar or similar amino acid substitution, usually does not change the protein function, C terminal and/or \ terminal addition of one or several amino acids usually does not change the protein function. These conservative changes are best made by making substitutions according to table 1.

TABLE 1

The invention also includes analogs of the claimed proteins. The differences between these analogues and the sequences of

SEQ ID NO

2, 12 or 14 of the present invention may be differences in amino acid sequence, differences in modified form which do not affect the sequence, or both. Analogs of these proteins include natural or induced genetic variants. Induced variants can be obtained by various techniques, such as random mutagenesis by irradiation or exposure to mutagens, site-directed mutagenesis, or other well-known biological techniques. Analogs also include analogs having residues other than the natural L-amino acids (e.g., D-amino acids), as well as analogs having non-naturally occurring or synthetic amino acids (e.g., beta, gamma-amino acids). It is to be understood that the proteins of the present invention are not limited to the representative proteins exemplified above.

Modified (generally without altering primary structure) forms include: chemically derivatized forms of the protein in vivo or in vitro, said modifications being capable of maintaining or enhancing or partially inhibiting the transport function of the protein; the modification comprises chemical modification of amino acid side chains, chemical modification of peptide chain end groups, such as chemical modification of sulfydryl, chemical modification of amino, chemical modification of carboxyl, chemical modification of disulfide bonds and other modifications; such chemical modifications include phosphorylation modifications (e.g., phosphotyrosine, phosphoserine, phosphothreonine), glycosylation modifications (mediated by glycosylases, e.g., N-glycosylation, O-glycosylation), lipid acylation modifications (e.g., acetylation, palmitoylation), and the like.

Of particular note, in the present invention, there is also provided a loss-of-function transgenic plant, said loss-of-function comprising one or more of the following characteristics: (i) root length and root branching are reduced compared to wild type; (ii) reduced plant height relative to wild type; and (iii) reduced salt tolerance compared to wild type.

In a preferred embodiment, the loss-of-function transgenic plant of the present invention comprises HAK mutein having an amino acid sequence as shown in SEQ ID NO. 8 and a gene mutation sequence as shown in SEQ ID NO. 7.

MDRQTRA*(SEQ ID NO:8)

In another preferred embodiment, the loss-of-function transgenic plant of the present invention comprises HAK mutein having the amino acid sequence shown in SEQ ID NO. 10 and the gene mutation sequence shown in SEQ ID NO. 9.

MDRQTGRPDLTAAAAAQSDAAVSGGA*(SEQ ID NO:10)

Expression vector

The invention also relates to a vector comprising the polynucleotide of the invention, as well as a host cell comprising the vector of the invention or the coding sequence of the mutein of the invention, and a method for producing the SlHAK20 mutein of the invention by recombinant techniques.

The polynucleotide sequences of the present invention may be used to express or produce recombinant muteins by conventional recombinant DNA techniques. Generally, the following steps are provided:

(1) Transforming or transducing a suitable host cell with a polynucleotide (or variant) of the invention encoding a mutein of the invention, or with a recombinant expression vector containing the polynucleotide;

(2) A host cell cultured in a suitable medium;

(3) Isolating and purifying the protein from the culture medium or the cells.

The invention also provides a recombinant vector comprising the gene of the invention. In a preferred embodiment, the promoter downstream of the recombinant vector comprises a multiple cloning site or at least one cleavage site. When the target gene is required to be expressed, the target gene is connected into a suitable multiple cloning site or enzyme cutting site, so that the target gene is operably connected with the promoter. As another preferred mode, the recombinant vector comprises (in the 5 'to 3' direction): a promoter, a gene of interest, and a terminator. If desired, the recombinant vector may further comprise an element selected from the group consisting of: a 3' polyadenylation signal; an untranslated nucleic acid sequence; transport and targeting nucleic acid sequences; resistance selection markers (dihydrofolate reductase, neomycin resistance, hygromycin resistance, green fluorescent protein, etc.); an enhancer; or operator.

In the present invention, the polynucleotide sequence encoding the mutein may be inserted into a recombinant expression vector. The term "recombinant expression vector" refers to a bacterial plasmid, bacteriophage, yeast plasmid, plant cell virus, mammalian cell virus such as adenovirus, retrovirus, or other vectors well known in the art. Any plasmid or vector can be used as long as it can replicate and is stable in the host. An important feature of expression vectors is that they generally contain an origin of replication, a promoter, a marker gene and translation control elements.

Methods well known to those skilled in the art can be used to construct expression vectors containing the DNA sequences encoding the muteins of the present invention and appropriate transcription/translation control signals. These methods include in vitro recombinant DNA techniques, DNA synthesis techniques, in vivo recombinant techniques, and the like. The DNA sequence may be operably linked to a suitable promoter in an expression vector to direct mRNA synthesis. Representative examples of such promoters are: lac or trp promoter of E.coli; a lambda phage PL promoter; eukaryotic promoters include CMV immediate early promoter, HSV thymidine kinase promoter, early and late SV40 promoter, LTRs from retrovirus, and other known promoters which control the expression of genes in prokaryotic or eukaryotic cells or viruses. The expression vector also includes a ribosome binding site for translation initiation and a transcription terminator.

The inclusion of the gene, expression cassette or vector of the invention may be used to transform an appropriate host cell to allow the host to express the protein. The host cell may be a prokaryotic cell, such as E.coli, streptomyces, agrobacterium; or lower eukaryotic cells, such as yeast cells; or higher eukaryotic cells, such as plant cells. It will be clear to one of ordinary skill in the art how to select an appropriate vector and host cell. Transformation of a host cell with recombinant DNA may be carried out using conventional techniques well known to those skilled in the art. When the host is a prokaryote (e.g., escherichia coli), caCl may be used ₂ The treatment can also be carried out by electroporation. When the host is a eukaryote, the following DNA transfection methods may be used: calcium phosphate coprecipitation, conventional mechanical methods (e.g., microinjection, electroporation, liposome encapsulation, etc.). Agrobacterium may also be used to transform plantsExamples of the transformation method include a leaf disk method, a young embryo transformation method, and a flower bud soaking method. The transformed plant cells, tissues or organs can be regenerated into plants by conventional methods to obtain transgenic plants.

When the polynucleotide of the present invention is expressed in higher eukaryotic cells, transcription will be enhanced if an enhancer sequence is inserted into the vector. Enhancers are cis-acting elements of DNA, usually about 10 to 300 base pairs, that act on a promoter to increase transcription of a gene. Examples include the SV40 enhancer, which is 100 to 270 bp on the late side of the replication origin, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers, among others.

It will be clear to one of ordinary skill in the art how to select appropriate vectors, promoters, enhancers and host cells.

The obtained transformant can be cultured by a conventional method to express the polypeptide encoded by the gene of the present invention. The medium used in the culture may be selected from various conventional media depending on the host cell used. The culturing is performed under conditions suitable for the growth of the host cell. After the host cells have been grown to an appropriate cell density, the selected promoter is induced by suitable means (e.g., temperature shift or chemical induction) and the cells are cultured for an additional period of time.

The recombinant polypeptide in the above method may be expressed intracellularly or on the cell membrane, or secreted extracellularly. If necessary, the recombinant protein can be isolated and purified by various separation methods using its physical, chemical and other properties. These methods are well known to those skilled in the art. Examples of such methods include, but are not limited to: conventional renaturation treatment, treatment with a protein precipitant (such as salt precipitation), centrifugation, cell lysis by osmosis, sonication, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high Performance Liquid Chromatography (HPLC), and other various liquid chromatography techniques, and combinations thereof.

Plant traits

Plant traits involved in the present invention include root length, root weight and root branching, plant height, salt tolerance, na + ion transport in plants, yield or biomass. Application of the present invention can significantly improve one or more of the traits described above in plants. Furthermore, the application of the present invention may improve other traits related to the above traits, such as increase in root length or root branching, and improve biological traits of plants such as lodging resistance and drought resistance, and other traits related to the implementation of the present invention are included in the scope of the present invention.

The terms "aerial parts" and "parts above the ground" are used interchangeably and refer to parts of a plant exposed above the ground surface, including, for example, the stems, leaves, flowers and fruits of the plant.

The survival rate of the plants obtained by applying the present invention is improved by at least 1-fold, preferably at least 2-fold, preferably at least 3-fold, more preferably at least 4-fold under the saline conditions compared to the wild type plants.

The plant height or biomass of the plants obtained by applying the present invention is increased by at least about 0.1-fold, preferably at least 0.2-fold, more preferably at least 0.5-fold, and most preferably at least 1-fold under the salt condition compared to the wild-type plants.

The main advantages of the invention include:

1) The invention firstly discloses the regulation mechanism of HAK genes, particularly SlHAK20 and homologous genes thereof on Na + ion transport. The over-expression HAK gene can enhance the salt resistance of plants, and the research result has important scientific value and social significance for cultivating novel salt-resistant crop varieties, enlarging the crop planting area, increasing the crop yield and improving the income of farmers.

2) The invention also obtains the hak mutant which is beneficial to phenotype improvement, can realize the regulation and control of the properties such as lodging resistance, biomass resistance, salt resistance and the like by regulating and controlling the endogenous gene of the plant, and has important scientific value for cultivating new varieties of plants with high quality, high yield and stress tolerance.

The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Experimental procedures without specific conditions noted in the following examples, generally followed by conventional conditions, such as Sambrook et al, molecular cloning: the conditions described in the Laboratory Manual (New York: cold Spring Harbor Laboratory Press, 1989), or according to the manufacturer's recommendations. Unless otherwise indicated, percentages and parts are percentages and parts by weight.

Example 1: effect of overexpression of HAK20 (SEQ ID NO: 2) in tomato on salt resistance in plants

In the experiment, a transgenic method is adopted to construct an overexpression vector of HAK20 to transfect tomato callus so as to obtain three groups of transgenic positive plants, namely, hap1OE-1, hap1OE-2 and Hap1OE-3.

1. Vector construction

The upstream and downstream primers of the cloning primer of SlHAK20 CDS (coding sequence, CDS) with the stop codon removed are respectively synthesized with the linker sequences (from the target vector) of CAAGAGACAGGATCGAATTC and ACCTCCGACCCGGGTGCACTAGT at the 5' end. Using PCR amplification, the corresponding linker CDS-containing fragment was cloned from the total cDNA product obtained by total RNA inversion of the wild type variety TS-21. Further, the SlHAK20 CDS is shuttled into the target vector by recombination with the homologous sequence of the target vector pCAMBIA1300-YFP catalyzed by recombinase through specific site recombination.

2. Genetic transformation

Is realized by utilizing agrobacterium-mediated callus infection. The agrobacterium carrying the target vector is incubated with the detoxified tomato cotyledons for 20min for infection, after the agrobacterium is abandoned, the tomato cotyledons are placed in a co-culture medium (components 4.3g/L MS salt (519), 0.2mg/L IAA,2mg/L ZT,30g/L sucrose,7.4g/L agar, pH 5.5-5.6) for dark culture for 2-3 days. The co-cultured tomato cotyledons were transferred to a selection medium containing 6mg/L of Hygromycin and 300mg/L of Timentin antibiotic (the composition was the same as the co-culture medium) for 7-10 days of selection subculture each generation until tissue culture seedlings were differentiated. The tissue culture seedlings are cut down and inserted into a rooting culture medium (components are 4.3g/L MS salt (524), 20g/L sucrose,300mg/L Timentin,7.4g/L Agar, pH is 5.7-5.8) for rooting, and the seedlings can be acclimatized and transplanted after the root systems develop to be strong.

3. Transformed plant identification and culture

Taking the rooted tissue culture seedling leaves, extracting total DNA, amplifying YFP gene through PCR, carrying out agarose gel electrophoresis, if a target strip is a positive seedling, if no target strip is used as a negative control, transplanting the positive seedling and the positive seedling into peat soil for culture, and propagating seeds for subsequent tests.

4. Molecular detection

The transcript level of SlHAK20 in over-expression strains Hap1OE-1, hap1OE-2 and Hap1OE-3 is detected by real-time quantitative PCR (qRT-PCR). After extracting total RNA of over-expressed strains Hap1OE-1, hap1OE-2 and Hap1OE-3 by an RNA extraction Kit, obtaining cDNA by reverse transcription with a reverse transcription Kit (iScriptTM cDNA Synthesis Kit, BIO-RAD), taking an upstream primer 5-. Finally, the relative level of SlHAK20 transcript was calculated using the transcript of the housekeeping gene SlEF1a (the upstream and downstream primers were 5 'GACAGGCGTTCAGGTAAG GA-3' and 5 'GGGTATTCAGCAAAGGTCTC 3', respectively) as an internal reference.

Detection of protein levels was achieved by Western Blot. Total protein in the over-expressed strains Hap1OE-1, hap1OE-2 and Hap1OE-3 was extracted with 1% SDS protein extract, 20. Mu.g of each total protein was run on SDS-PAGE gels, after which the proteins were transferred to Nitrocellulose membranes (0.45. Mu.m HATF, nitrocellulose membrane, merck Millipore), 5% skim milk powder was blocked for 1h, 1.

5. Salt resistance test

And disinfecting the seeds of the positive seedlings and the negative seedlings by using 10% sodium hypochlorite for 20min, ending in a 1/4MS culture medium, horizontally placing for germination, transferring to the 1/4MS culture medium, and vertically culturing for about 10 days. The seedlings were transplanted into soil or liquid culture boxes (1/4 MS liquid medium), and after 18 days of further culture 175mM NaCl was added to the culture or used to irrigate the tomatoes cultured in the soil. After 2-3 weeks of salt treatment, rehydration is carried out, i.e. tomatoes are cultured and irrigated by using culture solution or water without NaCl, and the survival rates of different transgenic materials are counted after 2 weeks.

6. Results and analysis of the experiments

(1) Level of overexpression

As shown in FIG. 1 and FIG. 2, the transcription level and protein expression level of plants of Hap1OE-1, hap1OE-2 and Hap1OE-3 are higher than those of wild type (TS-670), and the transcription level and protein expression level of plants of Hap1OE-1 are slightly higher than those of plants of Hap1OE-2, and both are better than those of Hap1OE-3.

(2) Transgenic tomato plant salt resistance detection

As shown in FIG. 3, the transgenic and wild tomato plants show wilting after being treated with 175mM NaCl, and the wilting status of the wild plants is significantly higher than that of the wild plants, wherein the status of the Hap1OE-1 transgenic plants is slightly better than that of the other two groups. After rehydration, the transgenic plant can gradually recover the normal growth state, and the wild plant has no sign of reversion.

(3) Survival analysis

As shown in FIG. 4, the survival rate of transgenic tomato plants after salt treatment is significantly better than that of wild type. The survival rate of the transgenic plant is positively correlated with the transcription level and the protein expression level of the SlHAK20 gene.

7. Conclusion of the experiment

The overexpression of SlHAK20 can obviously enhance the salt resistance of plants and improve the adaptability of the plants to the adverse environment.

The research result is helpful for cultivating more new salt-resistant plant varieties, and has important scientific value and social significance for expanding the plant planting area, increasing the utilization rate of saline-alkali soil, increasing the yield of agricultural products and improving the income of farmers.

Example 2: regulating and controlling effect of HAK20 in tomato on Na +

In this example, the slhak20 gene in wild-type tomato TS-21 variety was knocked out by gene editing, and two mutants, slhak20-1 and slhak20-2, were generated.

1. Construction of CRISPR editing tools

A GATTG and an AAAC.. C linker were added to upstream and downstream primers of Single guide RNA1 (sgRNA 1) 5 'CATGGATCGACAACCGG A3' and sgRNA2 (5 'TCAGATGCAGCTGTTACAG 3') in the vicinity of ATG of SlHAK20 genome, respectively, and synthesized (Thermo Fisher Scientific), and then diluted to a final concentration of 10. Mu.M, and 10. Mu.L of each was mixed. Annealing in a PCR instrument (BioRA D) to form double chains, directly carrying out enzyme digestion on the double chains and Bsa I-HF (New England Biolabs) of a target vector pCAMBIA1300-Cas9, carrying out T4 chaining for 2h, carrying out kanamycin screening to obtain a correct recombinant binary vector after a reaction product is transformed into escherichia coli DH5 alpha competence, carrying out colony PCR identification by using M13F and sgRNA downstream primers, carrying out Sanger sequencing on a PCR product, and transforming the tomato by using an agrobacterium-mediated callus floral dip transformation method for subsequent experiments.

2. Genetic transformation

Is realized by utilizing agrobacterium-mediated callus infection. The agrobacterium carrying the target vector is incubated with detoxified tomato cotyledons for 20min for infection, after the agrobacterium is discarded, the tomato cotyledons are placed in a co-culture medium (components 4.3g/L MS salt (519), 1mg/L IAA,0.3mg/L ZT,30g/L sucrose,7.4g/L agar, pH 5.5-5.6) for dark culture for 2-3 days. The co-cultured tomato cotyledons were transferred to a selection medium containing 6mg/L of Hygromycin and 300mg/L of Timentin antibiotic (the composition was the same as the co-culture medium) for 7-10 days of selection subculture each generation until tissue culture seedlings were differentiated. The tissue culture seedlings are cut down and inserted into a rooting culture medium (components are 4.3g/L MS salt (524), 20g/L sucrose,300mg/L Timentin,7.4g/L Agar, pH is 5.7-5.8) for rooting, and the seedlings can be acclimatized and transplanted after the root systems develop to be strong.

3. Plant culture and mutant screening

Taking the rooted tissue culture seedling leaves, extracting total DNA, carrying out PCR amplification through an upstream primer at about 300bp of the upstream of the sgRNA and a downstream primer at 300bp of the downstream of the sgRNA to obtain a target band, carrying out Sanger sequencing on a target segment, comparing a sequencing result with a reference genome sequence of SlHAK20 to obtain a gene editing result, transplanting the homozygous positive seedlings into peat soil for culture, and carrying out expanded propagation on seeds for subsequent tests.

4. Sodium and potassium ion content determination

And (3) measuring the content of sodium ions and potassium ions of roots and aerial parts: 19 day old tomato seedlings were cultured in 1/4MS liquid medium and after treatment with 1/4MS containing 50mM NaCl for 0, 1, 2, 7, 14 days or for the indicated time, aerial parts and roots, respectively, were taken (roots needed to be washed 3 times with deionized water and blotted dry with absorbent paper). Oven drying at 75 deg.C for more than 24h, adding 1mL of concentrated nitric acid containing internal standard indium (In) into 1mg (accurately weighing and recording weight) dry sample, digesting at 115 deg.C for 4h, adding 9.2mL of deionized water, diluting, and mixing. The diluted samples were examined by ICP-MS (NexION 350D.

And (3) measuring the content of sodium and potassium ions in the xylem flow: culturing 19-day-old tomato seedlings in 1/4MS liquid medium, treating with 1/4MS containing 50mM NaCl for 0, 1, 2 or a designated time, removing cotyledons and their upper parts with a sharp blade, and collecting the remaining xylem flow. Adding 5% into 20 μ L xylem, diluting with In-containing concentrated nitric acid, detecting by ICP-MS (NexION 350D, perkinelmer), and calculating sodium and potassium ion content.

5. Results of the experiment

As shown in FIG. 5, under the salt stress condition, the Na + content of the mutant slhak20-1 and slhak20-2 is obviously increased and the K + content is equivalent compared with that of the wild type roots along with the prolonging of time.

As shown in FIG. 6, under salt stress conditions, the Na +/K + of the mutant slhak20-1 and slhak20-2 was gradually increased compared to wild-type roots with time.

As shown in FIG. 7, under the salt stress condition, the Na + content of the mutant slhak20-1 and slhak20-2 is obviously reduced, and the K + content is equivalent.

As shown in FIG. 8, under the salt stress condition, the Na + content of the mutant slhak20-1 and slhak20-2 is significantly increased and the K + content is equivalent compared with that of the wild-type above-ground part with the time being prolonged.

As shown in FIG. 9, the mutant has increased Na +/K + over wild-type aerial part with time under the salt stress condition.

6. Conclusion of the experiment

The function of the SlHAK20 gene is closely related to the transport of Na + ions in a plant body, and the SlHAK20 gene can regulate the accumulation of Na + ions at the root of the plant, promote the transport of Na + from the root to xylem and unload Na + from xylem to root parts, thereby reducing the Na +/K + ratio of the root and the above-ground parts and enhancing the salt resistance of the plant.

Example 3: HAK20 mutation improves effects on plant root development, plant height and salt resistance

In this experimental example, mutants of the slhak20 gene, slhak20-3 and slhak20-4, in tomato TS-21 variety were generated by a gene editing method. The amino acid sequences of slhak20-3 and slhak20-4 are shown in SEQ ID NO. 12 and SEQ ID NO. 14, respectively.

1. Construction of CRISPR editing tools

A Single guide RNA 5'GGTCTCCTATCATGGGTGCATGG 3' upstream primer and a Single guide RNA downstream 3093bp from SlHAK20 genome ATG were designed, and GATTG and AAAC.. C linkers were added to the primers and synthesized (Thermo Fisher Scientific), and the primers were diluted to a final concentration of 10. Mu.M, and 10. Mu.L of each was mixed. Annealing in a PCR instrument (BioRAD) to form double chains, directly carrying out enzyme digestion on the double chains and Bsa I-HF (New England Biolabs) of a target vector pCAMBIA1300-Cas9, carrying out T4 chaining for 2h, carrying out kanamycin screening to obtain a correct recombinant binary vector after a reaction product is transformed into escherichia coli DH5 alpha competence, carrying out colony PCR identification by using M13F and sgRNA downstream primers, carrying out Sanger sequencing on a PCR product, and transforming the tomato by using an agrobacterium-mediated callus floral dip transformation method for subsequent experiments.

2. Genetic transformation

Is realized by utilizing agrobacterium-mediated callus infection. The agrobacterium carrying the target vector is incubated with the detoxified tomato cotyledons for 20min for infection, after the agrobacterium is abandoned, the tomato cotyledons are placed in a co-culture medium (components 4.3g/L MS salt (519), 1mg/L IAA,0.3mg/L ZT,30g/L sucrose,7.4g/L agar, pH 5.5-5.6) for dark culture for 2-3 days. The co-cultured tomato cotyledons were transferred to a selection medium containing 6mg/L of Hygromycin and 300mg/L of Timentin antibiotic (the composition was the same as the co-culture medium) for 7-10 days of selection subculture each generation until tissue culture seedlings were differentiated. The tissue culture seedlings are cut down and inserted into a rooting culture medium (components of 4.3g/L MS salt (524), 20g/L sucrose,300mg/L Timentin,7.4g/L Agar, pH 5.7-5.8) for rooting, and the seedlings can be acclimatized and transplanted after the root systems develop to be strong.

3. Plant culture and mutant screening

Taking the rooted tissue culture seedling leaves, extracting total DNA, carrying out PCR amplification through an upstream primer at about 300bp of the sgRNA upstream and a downstream primer at 300bp of the sgRNA downstream to obtain a target strip, carrying out Sanger sequencing on the target strip, comparing the sequencing result with a reference genome sequence of SlHAK20 to obtain a gene editing result, transplanting the homozygous positive seedlings into peat soil, and culturing and expanding the propagated seeds for subsequent tests.

4. Identification of phenotype (root development, plant height, sodium and potassium ion content)

And (3) disinfecting the gene editing homozygote and the corresponding wild seeds for 20min by using 10% sodium hypochlorite, then finally placing the seeds in a 1/4MS culture medium horizontally, transferring the seeds after germination, vertically placing the seeds in a 1/4MS culture medium containing 200mM NaCl, and culturing for 1 week or 1 month, and then counting the characters such as fresh weight and the like. The control group was cultured in 1/4MS medium without NaCl for 10 days while standing upright.

Or transferring the germinated seedlings to a 1/4MS culture medium for about 10 days in a vertical culture. The seedlings were transplanted into soil blocks or liquid culture boxes (1/4 MS liquid medium), and after further 18 days of culture, 200mM NaCl was added to the culture solution or the tomatoes cultured in the soil blocks were watered with water containing 200mM NaCl. After 2-3 weeks of salt treatment, rehydration is carried out, namely the characters such as plant height and the like are counted after 2 weeks of culture solution or water culture without NaCl and tomato irrigation. The control group was cultured with NaCl-free 1/4MS medium or water-irrigated soil blocks for 30 days and then the plant heights were counted.

5. Determination of sodium and potassium content

6. Results and analysis of the experiments

(1) SlHAK20 mutation can affect the development of plant roots

As shown in FIG. 10, the mutant slhak20-3, slhak20-4 had increased root length and increased number of root branches compared to wild-type after salt treatment.

(2) Influence of SlHAK20 mutation on plant height and biomass

As shown in FIG. 11, the mutant slhak20-3, slhak20-4 increased in plant height compared to the wild type after salt treatment.

(3) Influence of SlHAK20 mutation on sodium ion and potassium ion content of plants

As shown in FIG. 12a, in salt stress conditions, na +/K + of mutant slhak20-3, slhak20-4 was significantly reduced compared to wild type roots.

As shown in FIG. 12b, under salt stress conditions, the Na +/K + of mutant slhak20-3 and slhak20-4 was significantly reduced compared to wild-type aerial parts.

7. Conclusion of the experiment

The experimental result shows that the root length and the plant height of the SlHAK20 large-fragment deletion mutant are obviously superior to those of a wild type under salt stress, so that the biomass of the plant can be increased, and even the yield of the plant is isophenotypic; the Na +/K + ratio of plant tissues can be reduced, so that the salt resistance of plants is enhanced.

As can be seen from the analysis of the amino acid sequences of slhak20-3 and slhak20-4 (SEQ ID NO:12 and SEQ ID NO: 14), the N-terminal positions 1-240 of these two mutants are completely identical; the amino acid sequence of slhak20-3 further includes the sequence HGLLPLLSLGYTVS at the C-terminus; the amino acid sequence of slh ak20-4 also includes C-terminal CMDSYHSSHWDIQYHKALSEHI sequence. This is because the nucleic acid sequence after the coding amino acids 1 to 240 undergoes frame shift mutation and terminates prematurely during gene editing, resulting in the amino acid sequence after positions 1 to 240 not being identical to the wild type.

Both SlHAK20-3 and SlHAK20-4 can improve salt resistance of plants, and the same amino acid region (1-240 sites at the N terminal) of the SlHAK20 and the SlHAK has a deletion of 473 amino acids at the C terminal compared with the wild type SlHAK 20. In other words, the SlHAK20 protein with 473 amino acids deleted from the C-terminal can be considered to have an important effect on enhancing the salt resistance of plants.

We over-express the amino acid sequences of slhak20-3 and slhak20-4 in rice and maize, which can also enhance the salt resistance of plants.

The research result has application value for improving the characters, can realize the regulation and control of the characters such as salt resistance and the like by improving the HAK gene of the plant, and has important scientific value for cultivating new stress-tolerant plant varieties.

All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes or modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the appended claims of the present application.

Sequence listing

<110> Shandong Shunfeng Biotechnology Co., ltd

<120> role of HAK Gene in regulating plant traits

<130> JH-CNP202152

<160> 14

<170> PatentIn version 3.5

<210> 1

<211> 5685

<212> DNA

<213> tomato (Solanum lycopersicum)

<400> 1

atggatcgac aaaccggacg gcctgacttg acggcggcgg cggcggcggc ggcgcagtca 60

gatgcagctg ttacagtggc ggtgcatgac ggcgaaacca ttaataatga tcaaaaggtt 120

caatggttca tctcattcca cgctctctgt atacttatct ctttgttttg tggttttttt 180

ccccttaaaa atggaacttt gtgctctgtt ttgtagtatt ttgatttctc tctgtgttta 240

acttccgtcg tatgtttaca ttcatttagt cggagtgcct acaaactact gctctctagt 300

tttagtttat gtgatacagt tggaatttcg tgattcaaaa gctttaaatt ttgattatga 360

atccagacat agaaggcttt tgagtttttt tttgaaatga tatacacttt tctctgtttt 420

gattttattt tttttgaact tgacacggag tttaagaaat ttaataaaga ttttagaatt 480

gcatggactt tttttttatg accgtagtgt gttggtcagc tttacgcgca cctcaattaa 540

tttcatgcga tacctgtcac gtcgagcaaa aggtatcaaa taactctgtc catcgaggct 600

cgaagagaat gttagaatcg cctagtgctt tttgtctgta ctgagtttga attgaatggt 660

gtttaaacta aagatacgtg gaatgtatca aactgttttc aatgttttgg gttttaaata 720

cgtcatgaaa agttgaaact gaagtgttgc aaaaaagagt aaaaaaaata gatttttttt 780

ttcaaagtat gacaacaaat tgaaatagag gtagtatttg gaaaactacg tcgaaattag 840

tataaatcta tgttgctcgt actcttccaa gatatcaacc ggtgtgtgtc ggatcctaaa 900

gatagtgtat ttttgaagga tccgacacga atgcggcatt gtttttgggg agtccaccaa 960

cataatataa atcacgataa ctaatataaa atgtttaaaa gacatatgaa taaagtttgg 1020

tcgaagaaaa acgtggttga ctctactgtt ccacgtatat tgtgatttga ggtgaaattg 1080

ttgatgaaga tggtattttc ttaatcccat tccttcatta tatatagtgt atctttcaat 1140

tgaggtgaag ttgtggatca tgaagtagtt gttgatgatg atggttgact ctacgaaata 1200

cttttgcgtt caaagttaag ttgtctctac taagatgttc tttgacattt gtttttactc 1260

ggaaactata tttgtttagg accatagccg atgggagacg ttggtacttg catacaagac 1320

cttaggagtt gtttttgggg gtcttgtcac atctccactc tatgtatatc catcaatgcc 1380

attaaagtct ccaactgagg atgattattt ggggatatac agcataatgt tttggactct 1440

cagtctaatt ggtgttgtca aatatgctac catagctctc caagctgatg atcagggtga 1500

aggtattgct gatcactctc tctttgtact tttgctactt acgacaacat tttcccattg 1560

aaaaatggga attgtttcat atggaaaaaa aaaggaaact tctcgctatt tcagtgagaa 1620

tctgtttccc accaaacatt ttcccagtgg agctggttca gtggggaatg agtggaaaaa 1680

tcatatgtta tagcacaaat ttcccactta tttgcaggtg gaaaaaacct ttatttctag 1740

tagtgaggtg actcgagttt taagaaactg tctgtttgta ttcatactaa aagatttatc 1800

ttttggagaa tgttgaaggt gttaatgaag gatgtttata tataatgttg agtcaattct 1860

taattttctt tatattttgg ttgaaattcc ttttgaaaga cacttcagag tttcttcatt 1920

tctcttttga tgtgatcatt atcgaaattt cagttctaga acatggtctt ttgtaggtgg 1980

aacctttgct ctttattcat tactttgtag aaatataaat attggtattc tttcttccaa 2040

gagtgccagt ttgaattcaa gccactccta tgttaaccaa tcgaaaaagc caagtaggtt 2100

gggtaaattt tgtgagagga gtttgattgc cagaagggta ctgcttttca tcgcaatgtt 2160

gggtatgtgc atgcttattg gcgatggcat acttacacct gccatctcag gtatgttagc 2220

ttgggttggg ccttttagtt tacttttagc aaccttttga gtgcttgtca gttttccctc 2280

tttcattttc agtttcatta attgtccctt ctgtctgtca gtgttatctg caatgggtgg 2340

cttacgagca cgattctcct cggtcagtaa atgtaagtat gacagcatac agtaaggtac 2400

atattgctca ttgctttctt agtcgagctc gttgcatggg gcttgcctag tgcggcttac 2460

cgctcctgtg tggtttgcag gctattaccc cgaagcaggt ttaccctatg cccaccctaa 2520

gggtagcagc tgtggtttcc cttgtcataa ataaaaaatt gtctttgaac ggagaggcct 2580

ttatgatttt gagggtaatt gggaagtggt cgaagaacaa ttttgattct tgtaatttct 2640

caggttttaa ggtattgtgc ttgttaaaag actatctttc acagattcat atattgatgt 2700

tacatgagtc cgattctgga agctaattga aataacttga atccttgttc tagtaattat 2760

ttgaagtaat taagtaatga ttgaacactt taaggtcaat aagtcatatt aacgaaaagg 2820

agcagtgcag tttctgaaag ttaaagtctt cttttatgac aggttgcaag tcctttttaa 2880

acactagatg acgcgaaaca tgttaattgt cccatttatg taaacacatc tctccctctc 2940

gatattcaca atttctctgc ttgccacttg cagcgctggt cgaaggtctc tcagcaatca 3000

ttctcattgt tctattcctc ctgcagaaat ttggtacttc acgagtgagc ttcctcttgt 3060

ctcctatcat gggtgcatgg actcttacca ctcctctcgt tgggatatac agtatcataa 3120

agcattatcc gagcatattt aaggcgatat caccccatta cattgccctc ttcttcttaa 3180

gaaatggaaa acaaggatgg atatatcttg gtggtactgt cctatgcatt acaggttagc 3240

ctcgttgttg gtgtggtctg caaaagttgt tcattgtgat taagatctaa atccatttcc 3300

tcatgtttgg agttctttta cattctttcc tattactttg caattaatat tggggaaaat 3360

ttggaccaat tatcatgtgt gcaatgtcta ccgatggcgg gaaaagctta aacctggtat 3420

cttgtttcat atccagagaa actcgaaagg tgatgaatgg aggtggaaaa tattataaag 3480

aatatggaat gcccctctgg tcaatacttt tatccaaaaa tgataacaat tgttctggtt 3540

tgagcgaaaa aacatggagg ctgttctaga ttatttagct cggggagttc caacgtcgac 3600

catggtgtat ttctcgctaa gttgtcagat tcctcaccct tctatttttg tgtagtgggg 3660

ggtttgagga ttagagaaaa agacgggatg aaagaggata aggggtggac agtacctttc 3720

accgaaacat attaggccgc tagagttatt ttctggccaa gatctgtttc acttccaccc 3780

aatatctgct taacagtaca atcctttcat atttttaggt tctgaagcaa tgtttgctga 3840

tcttggtcat ttcaacagga gttcaattcg ggtaaaaata cttgctatgt cttggaattt 3900

ctcttagtct cttcttcttg cttgttttat atgatttacg ggatcttgtt ttctggcaga 3960

tagcatttct ctatactata tatccatcat tggtgctgac atatgcggga cagacagcat 4020

atctgattag aaatcccgat gaccactttg atggatttta caagtttata ccctctgctg 4080

tgtactggcc aatttttgtt atagccacat tagctgctat agtagcaagt cagtcgctga 4140

tttcagccac cttttcggtt atcaagcaat cagttgtgtt ggattatttt cctcgggtga 4200

aggtagttca cacatcctcc agcaaagaag gcgaggttta ctcaccagaa gttaactaca 4260

tcctcatgat tctctgtgtt gcagtcatac tagtcttcgg agatggacaa gacatcggaa 4320

atgccttcgg taagatgccc taaacccttt gcattttatc ttccatattt agggactatc 4380

ttaactgaat tgatcccttt atgaaagtta tctttcactt gactcctctt cctaggatgg 4440

tggttgttaa catcgactac tagcattttc tttacctaat ttactactac ttggtgatga 4500

cttgcttact ttaaggggta actagtgtaa tgaattgagc taaacaaatt gtggattgga 4560

attttctcat tctgtttgct cgttgttttc agattttgtc tcgtcttgtt tagcattatc 4620

caataagata tttcactagt gataacgcac atatatgtgg tgcaaatcaa gttcaacaaa 4680

attgtcttgt cttgtttagc atagttaaaa taaaattaca tatatgtatg atcttgtatg 4740

tatatattaa ggtataactt gcattttttc ttgcaacggt tcaactcttt cgtattgtca 4800

gcatagtaaa tattgcatta actaattaaa atggattctc taacaggtgt tgttgttagc 4860

atagttatgc ttataacgac catattgttg acattggtta tgatcattat ttggagaact 4920

ccaccagctc tggtcgggct ctacttcgtt gtattttttg tgatggaaag tgtatatgtt 4980

agtgcaatct tcacaaaaat tcccgaaggt ggttggattc cttttgccat ttctctcatt 5040

cttgctttca ttatgtttgg ttggttctac ggtaggcaga gaaaacttga gtatgaattg 5100

acacacaaga tagactcaga gagactaagg acactgctaa tcgatccagg tcttcagaga 5160

gttcctggac tctgcttctt ctacacaaat atccaagatg gtttaacccc aattttaggt 5220

cattacataa agaacatgag atctcttcac aaagttactg tatttacaac tcttcgttac 5280

ttgctggttc ctaaagtagc acctggtgaa agaatcgttg ttagtaaact aggtctcaga 5340

ggggtttata gatgtgtgat tcgttatggt tatgcagata agcttagtct tgaaggagat 5400

gatttagtta atcaagtcat ccaaagttta cgatctcatg tgctccactg ctccaattca 5460

ttggaggttg acactgaagt ttccgagttg gatgaggcaa agcttgctgg agtagtacat 5520

attcgtggaa agacgaggtt ttatattggt aaggactgtg gatggtttga tagaacaatg 5580

cttgctttct atgaagtttt gcatagcaat tgcagatctg cattgcctgc tatgggtgta 5640

ccattacctc aacgtatcga ggttggaatg ctctacgcgg cgtaa 5685

<210> 2

<211> 713

<212> PRT

<213> tomato (Solanum lycopersicum)

<400> 2

Met Asp Arg Gln Thr Gly Arg Pro Asp Leu Thr Ala Ala Ala Ala Ala

1 5 10 15

Gln Ser Asp Ala Ala Val Thr Val Ala Val His Asp Gly Glu Thr Ile

20 25 30

Asn Asn Asp Gln Lys Asp His Ser Arg Trp Glu Thr Leu Val Leu Ala

35 40 45

Tyr Lys Thr Leu Gly Val Val Phe Gly Gly Leu Val Thr Ser Pro Leu

50 55 60

Tyr Val Tyr Pro Ser Met Pro Leu Lys Ser Pro Thr Glu Asp Asp Tyr

65 70 75 80

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

85 90 95

Val Lys Tyr Ala Thr Ile Ala Leu Gln Ala Asp Asp Gln Gly Glu Gly

100 105 110

Gly Thr Phe Ala Leu Tyr Ser Leu Leu Cys Arg Asn Ile Asn Ile Gly

115 120 125

Ile Leu Ser Ser Lys Ser Ala Ser Leu Asn Ser Ser His Ser Tyr Val

130 135 140

Asn Gln Ser Lys Lys Pro Ser Arg Leu Gly Lys Phe Cys Glu Arg Ser

145 150 155 160

Leu Ile Ala Arg Arg Val Leu Leu Phe Ile Ala Met Leu Gly Met Cys

165 170 175

Met Leu Ile Gly Asp Gly Ile Leu Thr Pro Ala Ile Ser Val Leu Ser

180 185 190

Ala Met Gly Gly Leu Arg Ala Arg Phe Ser Ser Val Ser Lys Ser Leu

195 200 205

Val Glu Gly Leu Ser Ala Ile Ile Leu Ile Val Leu Phe Leu Leu Gln

210 215 220

Lys Phe Gly Thr Ser Arg Val Ser Phe Leu Leu Ser Pro Ile Met Gly

225 230 235 240

Ala Trp Thr Leu Thr Thr Pro Leu Ile Gly Ile Tyr Ser Ile Ile Lys

245 250 255

His Tyr Pro Ser Ile Phe Lys Ala Ile Ser Pro His Tyr Ile Ala Leu

260 265 270

Phe Phe Leu Arg Asn Gly Lys Gln Gly Trp Ile Tyr Leu Gly Gly Thr

275 280 285

Val Leu Cys Ile Thr Gly Ser Glu Ala Met Phe Ala Asp Leu Gly His

290 295 300

Phe Asn Arg Ser Ser Ile Arg Ile Ala Phe Leu Tyr Thr Ile Tyr Pro

305 310 315 320

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

325 330 335

Pro Asp Asp His Phe Asp Gly Phe Tyr Lys Phe Ile Pro Ser Ala Val

340 345 350

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

355 360 365

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

370 375 380

Leu Asp Tyr Phe Pro Arg Val Lys Val Val His Thr Ser Ser Ser Lys

385 390 395 400

Glu Gly Glu Val Tyr Ser Pro Glu Val Asn Tyr Ile Leu Met Ile Leu

405 410 415

Cys Val Ala Val Ile Leu Val Phe Gly Asp Gly Gln Asp Ile Gly Asn

420 425 430

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

435 440 445

Leu Thr Leu Val Met Ile Ile Ile Trp Arg Thr Pro Pro Ala Leu Val

450 455 460

Gly Leu Tyr Phe Val Val Phe Phe Val Met Glu Ser Val Tyr Val Ser

465 470 475 480

Ala Ile Phe Thr Lys Ile Pro Glu Gly Gly Trp Ile Pro Phe Ala Ile

485 490 495

Ser Leu Ile Leu Ala Phe Ile Met Phe Gly Trp Phe Tyr Gly Arg Gln

500 505 510

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

515 520 525

Arg Thr Leu Leu Ile Asp Pro Gly Leu Gln Arg Val Pro Gly Leu Cys

530 535 540

Phe Phe Tyr Thr Asn Ile Gln Asp Gly Leu Thr Pro Ile Leu Gly His

545 550 555 560

Tyr Ile Lys Asn Met Arg Ser Leu His Lys Val Thr Val Phe Thr Thr

565 570 575

Leu Arg Tyr Leu Leu Val Pro Lys Val Ala Pro Gly Glu Arg Ile Val

580 585 590

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

595 600 605

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

610 615 620

Val Ile Gln Ser Leu Arg Ser His Val Leu His Cys Ser Asn Ser Leu

625 630 635 640

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

645 650 655

Val Val His Ile Arg Gly Lys Thr Arg Phe Tyr Ile Gly Lys Asp Cys

660 665 670

Gly Trp Phe Asp Arg Thr Met Leu Ala Phe Tyr Glu Val Leu His Ser

675 680 685

Asn Cys Arg Ser Ala Leu Pro Ala Met Gly Val Pro Leu Pro Gln Arg

690 695 700

Ile Glu Val Gly Met Leu Tyr Ala Ala

705 710

<210> 3

<211> 3536

<212> DNA

<213> Rice (Oryza sativa)

<400> 3

atgcactcaa gtttcaacct cccttctcca acttgcaatg tcttcatctc acacggtgac 60

agtctccatg gatgttgaag ccggacagaa aaacaaagat gtaagtgaga gtgattaacc 120

gtagcgctat cattacatct catcagtcgt acttgttcat cactatcttc atgatcagat 180

catctcctac gtaataccta gattggatac aaatccttgg ttaaccactg ctaattaact 240

tcatgatggg gttaaccact gttaaactaa cttcaagaag gaactcagga aggtagatga 300

ttggtaagag caggagtgct ggttcccaaa tgtttgggtt tcattttttt tataaaaaaa 360

atacgcaaaa ggaacagtgt atttttgtat taggataaat aaggtttaca actaaagaac 420

caaaataaga gagctaagcc tattctcgag acatgaatgt ggctaccaga atatttgggt 480

tgtggacagt aaaaatcagt aaaattaacc atgtagggat gtattggttg agtggttgat 540

actgtttgct gttctcccct agtttcttct gaaaaaaagt tttggacatt ggctaacaaa 600

gtgtatatta tgtatgaatg aatgattatg ctgatgtgta gttaagtgag tttgttgatc 660

tgtgcttgat gaaagcacat gtaatctggt gctgacatta gtaaaccttg cattcacaaa 720

gatacaaatg tttgattcta ttttctacat tatttaccct gctgttattg ttgcagaaga 780

aggggatcag ccaagatctg atcctggctt acaagactct tggtgttgtt ttcggtggcc 840

ttgttacctc accactctac gtctacccat ccatgaattt gacgaatcct actgaagaag 900

actacctggg aatatacagc atcatgttct ggaccctcac tctcattggc gtagtcaagt 960

acatatgcat cgctctcaac gctgacgacc acggcgaagg ttggatcaaa tgccccaatg 1020

ttcaattggt gaacaatctg tagctacaat ttctaacttg atatgatcac tgtgtgtttt 1080

gcaggtggca catttgccat gtattctctg ctctgtcagc acgcaaacat tgggatcctt 1140

ccatccaaga agatctacac tgaagaagag aacctgatct cgaatcagcc tgtcgtagct 1200

ggaaggcctg gaagactgag aaggttcatc gaaagcagca taatcgccag gaggctgctg 1260

ctgctcacag ccatcctagg catgtgcatg ctcatcggcg atggcatcct cactcctgca 1320

atctcagtct tgtcagcgat tgatggactc agaggcccgt tcccatccgt cagtaaacgt 1380

aaggcacact gactttgcag aacataacat atctgatcag tgatcaatgt gataaatcat 1440

ataccgatgt tgttttctga taaaaaacaa ttgtcgtgca gctgctgtgg agggcctgtc 1500

ggccgcgatt ctcgttgggc tgttcttgct gcagaagtac ggcacgtcga aggtgagctt 1560

catgttctcg ccgatcatgg cggcgtggac gttcgccacc ccggtcatcg gcgtgtacag 1620

catctggcgc tactaccctg gcatcttcaa agccatgtcg ccgcattaca tcgtcagatt 1680

cttcatgacg aaccagacca gaggctggca gctactcggc ggcaccgtcc tctgcatcac 1740

aggcacgtag tacatgttct tgtctgatca tgtcgaaacg tctcactgaa tttgattttg 1800

ttttgagata tatggcgatg cgtggatgca ggtgccgagg cgatgttcgc ggatcttggt 1860

cacttcagca agaggtccat ccagatcgcg ttcatgtcga gcatataccc ttcgctggtg 1920

ctgacgtacg ccgggcagac ggcgtacctg atcaacaacg tcgacgactt cagcgacggg 1980

ttctacaagt tcgtgccgcg gccggtgtac tggccgatgt tcatcatcgc gacgctggcg 2040

gcgatcgtgg cgagccagtc gctgatctcg gccaccttct ccgtcatcaa gcagtcggtg 2100

gtgctggact acttcccgcg ggtgaaggtg gtgcacacgt ccaaggacaa ggaagggggg 2160

tgtactcgcc ggagaccaac tacatgctga tgctgctgtg cgtgggcgtc atcctcggct 2220

tcggcgacgg caaggacatc ggcaacgcgt tcggggtggt ggtgatcctc gtgatgctca 2280

tcaccaccat cctgctcacg ctggtgatgc tcatcatctg gggcacccac gtcgtgctgg 2340

tggcgctcta cttagtgccg ttcctgctcc tcgaggccac atacgtgagc gccgtgtgca 2400

ccaagatcct ccgcggaggg tgggtgccgt tcgcggtgtc ggtggcgctg gcggcggtca 2460

tgttcgggtg gtactacggc cggcagcgga agacggagta cgaggcggcg aacaaggtga 2520

cgctggagcg gctcggcgag ctgctgtccg gccccggctt gcgccgcgtc ccgggcctct 2580

gcttcttcta cagcaacagg caggacggcg ggtggctcac cccggtgctc gcgcactaca 2640

tcaggaacat gcggtcgctg cacgaggtga ccgtgttcct cacgctccgg tacctgctgg 2700

tggcgaaggt ggacggcaag gacagggtgc aggccgtgcg ccggctgggg ccagcggggg 2760

tgtacggctg cacgatccag tacgggtacg ccgacgcgat cgacttcgag gaggacgaca 2820

tcgccgggca ggtggtgggg gcgctgcggg agcgggtcgt cgacggggag gaggaggggg 2880

agcgggtgga ggcggcgagg gcggccgggg tggtgcacgt gagggggaag atgaggttcc 2940

acgtcgggaa ggacacgagg ctgttcgacc gggtgctgct cgggttctac gagttgctgc 3000

acggcgcgtg ccgctccgcg ctgccggcgc tcgggatccc gctgcagcag cgcgtcgaga 3060

tcggcatgct ctacaaggcc taacggcgcc gaacgccacg cacgcgcccc acgaatccgt 3120

acgtacgtac gtggccatta attaccatta gcagtataac ccctattatt agtgtggata 3180

ttatgggtac cccataccca tacggcatgg ttatccgacc agtcataggg gataacttat 3240

atctatagaa tatgtaatag atcatgactc gggtattacg tttccttata tattacggaa 3300

cagcctaaag tcctggtttg ataatattgt aaagtagatt taggaaaccg ataccgtatt 3360

ggttaaggtt tctatcttgt aatcctgccc cccaccctat ataaggtggg caggaggccc 3420

tctagggggc atgagacaac ctgatcgtca gatctataat acacccggcg gattcaaatc 3480

cccaaacagg agtagggtat tacctctcat tgagagggcc cgaacctgtc taaatc 3536

<210> 4

<211> 697

<212> PRT

<213> Rice (Oryza sativa)

<400> 4

Met Ser Ser Ser His Thr Val Thr Val Ser Met Asp Val Glu Ala Gly

1 5 10 15

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

20 25 30

Tyr Lys Thr Leu Gly Val Val Phe Gly Gly Leu Val Thr Ser Pro Leu

35 40 45

Tyr Val Tyr Pro Ser Met Asn Leu Thr Asn Pro Thr Glu Glu Asp Tyr

50 55 60

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

65 70 75 80

Val Lys Tyr Ile Cys Ile Ala Leu Asn Ala Asp Asp His Gly Glu Gly

85 90 95

Gly Thr Phe Ala Met Tyr Ser Leu Leu Cys Gln His Ala Asn Ile Gly

100 105 110

Ile Leu Pro Ser Lys Lys Ile Tyr Thr Glu Glu Glu Asn Leu Ile Ser

115 120 125

Asn Gln Pro Val Val Ala Gly Arg Pro Gly Arg Leu Arg Arg Phe Ile

130 135 140

Glu Ser Ser Ile Ile Ala Arg Arg Leu Leu Leu Leu Thr Ala Ile Leu

145 150 155 160

Gly Met Cys Met Leu Ile Gly Asp Gly Ile Leu Thr Pro Ala Ile Ser

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

Ile Met Ala Ala Trp Thr Phe Ala Thr Pro Val Ile Gly Val Tyr Ser

225 230 235 240

Ile Trp Arg Tyr Tyr Pro Gly Ile Phe Lys Ala Met Ser Pro His Tyr

245 250 255

Ile Val Arg Phe Phe Met Thr Asn Gln Thr Arg Gly Trp Gln Leu Leu

260 265 270

Gly Gly Thr Val Leu Cys Ile Thr Gly Ala Glu Ala Met Phe Ala Asp

275 280 285

Leu Gly His Phe Ser Lys Arg Ser Ile Gln Ile Ala Phe Met Ser Ser

290 295 300

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

305 310 315 320

Ile Asn Asn Val Asp Asp Phe Ser Asp Gly Phe Tyr Lys Phe Val Pro

325 330 335

Arg Pro Val Tyr Trp Pro Met Phe Ile Ile Ala Thr Leu Ala Ala Ile

340 345 350

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

355 360 365

Ser Val Val Leu Asp Tyr Phe Pro Arg Val Lys Val Val His Thr Ser

370 375 380

Lys Asp Lys Glu Gly Glu Val Tyr Ser Pro Glu Thr Asn Tyr Met Leu

385 390 395 400

Met Leu Leu Cys Val Gly Val Ile Leu Gly Phe Gly Asp Gly Lys Asp

405 410 415

Ile Gly Asn Ala Phe Gly Val Val Val Ile Leu Val Met Leu Ile Thr

420 425 430

Thr Ile Leu Leu Thr Leu Val Met Leu Ile Ile Trp Gly Thr His Val

435 440 445

Val Leu Val Ala Leu Tyr Leu Val Pro Phe Leu Leu Leu Glu Ala Thr

450 455 460

Tyr Val Ser Ala Val Cys Thr Lys Ile Leu Arg Gly Gly Trp Val Pro

465 470 475 480

Phe Ala Val Ser Val Ala Leu Ala Ala Val Met Phe Gly Trp Tyr Tyr

485 490 495

Gly Arg Gln Arg Lys Thr Glu Tyr Glu Ala Ala Asn Lys Val Thr Leu

500 505 510

Glu Arg Leu Gly Glu Leu Leu Ser Gly Pro Gly Leu Arg Arg Val Pro

515 520 525

Gly Leu Cys Phe Phe Tyr Ser Asn Arg Gln Asp Gly Gly Trp Leu Thr

530 535 540

Pro Val Leu Ala His Tyr Ile Arg Asn Met Arg Ser Leu His Glu Val

545 550 555 560

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

565 570 575

Lys Asp Arg Val Gln Ala Val Arg Arg Leu Gly Pro Ala Gly Val Tyr

580 585 590

Gly Cys Thr Ile Gln Tyr Gly Tyr Ala Asp Ala Ile Asp Phe Glu Glu

595 600 605

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

610 615 620

Asp Gly Glu Glu Glu Gly Glu Arg Val Glu Ala Ala Arg Ala Ala Gly

625 630 635 640

Val Val His Val Arg Gly Lys Met Arg Phe His Val Gly Lys Asp Thr

645 650 655

Arg Leu Phe Asp Arg Val Leu Leu Gly Phe Tyr Glu Leu Leu His Gly

660 665 670

Ala Cys Arg Ser Ala Leu Pro Ala Leu Gly Ile Pro Leu Gln Gln Arg

675 680 685

Val Glu Ile Gly Met Leu Tyr Lys Ala

690 695

<210> 5

<211> 5285

<212> DNA

<213> Rice (Oryza sativa)

<400> 5

atccgcacga gccagagcct cgttccggtc cggctggtct gtacggcccg ctcacataag 60

ctcggaacga acgagaggcg ccgcacgcgc accgccctga cactcgagca ccgcgacgga 120

gcgcctgggc taccggcggc ggcggcggcg gcgagtcggc gacgacgtct tctaccgcgg 180

ggggtcacac gaaggccggc caggccaacc tccgcccgca acgcaacgcc ctagctgcct 240

gccacacggt ctcggcctct ctcgggcggc cgcacgcacg ccacgccacg cccactcgcc 300

tcgcttctcg gccgcgtcgc gcgccgccgc gtggcgtagc ctcccccctc tccaaccttg 360

ttgttcctcg ctcctcctca gtgcggcttc ctgcgtgctc caggccccgt gacaagcgcg 420

gggggaggcg gcccacatgg atctcgaggc ggggtcaatc cgaccccgca gcgacggcga 480

aggcggcggt ccggcggccg gcagggagac cgatgtgagt gtccccgtgg ggaaaacccc 540

cacccttttc tctttcatgt gcagcaatgt tcttttatcg agttcttaag ttcttctaat 600

catatatggt aatttgttgc ggttcaccca atacgtctcg tgattcctgt gcgctttctt 660

gccatggaac tcgctccatt gtctgcaggt ttaaggcctg aacagaacac tcgtgctgtt 720

tattgcgagt ttctgacaag agaataagat atttgcatgg aagaacatgc atcttattac 780

aatattactg tacgaatgat caagcatgtt tatggtttag ttggtcaatg tgatgcagac 840

atgtgctcat gtagagaact agagatggtg tagattttat tttgtatgac atcgttgcgt 900

tcgacttgtc gccccatgct gtagagccga gggtgttgga aattctgact ttggctgctg 960

ctgctgcaaa cgactgatga tagcttcgtg tctcattttt ctctaagctc gtgctcttct 1020

tttcccagcc ttttctggcc ggctttggtg acaactgaga aattaatact gattttgttt 1080

cgtttatttt cgaatactgg atatgtggta gtggtacttc agaatagtcc aaacagatag 1140

atataacgcg aggcatgtgt tgtttgtttc tttggaaaaa tgactaatat attttgtctt 1200

ggtgtggaat aggggaatat tttccttttt cttgtatcct ttttttttgt ggatgataat 1260

ttcacattca ctcaccccct ccccctatct tcaatgcaga acatggtgaa tgtattaata 1320

gtattgtttg ttcagttagc tgcatccttt tgcatagcta tccaaaaact gaaaagtgtt 1380

attgtgaatg aaacacggta gctgcagtgg atatgaagga gctgcatgca agcttgatta 1440

gaatattatt ttgcatttta agtgaagttt ttaaaaagat cttcaggtga ttaagtaacc 1500

agattgtccc cttcctttga atttggatgt atctgatcat tctgctgctg attatttaat 1560

gttttaagat atctgaccat ataagtatca gaagggatgc tcaccccccc ccccccccct 1620

gatgatgatg tcacgaatgc ttgcatcatc aggctctgtt tccacatccc tacaagtatc 1680

caatgtaatc agatgtgaac caagaagacc tgtatgcatt accacgtggt caacgggacc 1740

agtcccacag tacccatcag cataataaat cttaaatttt attatgtaat ttgtgttttg 1800

cttgactgca ttgacttgct atgagtaggt ctgatcagtt gcatttatat gataaatgtg 1860

gttcatcgct cttcttcgtt gtgttccaaa tagaaacttg aagtatcatc tttgtgtttt 1920

acagtttata ttaatgtttt ctctcaactc tgagcaggat agcaatgttt ggaaggatct 1980

attccttgct tacaagactc ttggtgttgt ttttggtggc ctcgtcactt ctcccctcta 2040

cgtttatccc tctatgaact tgtcatctcc cacagaagct gactacctgg ggatatacag 2100

cataatgttt tggactctta ctttaattgg tgtggtcaag tatgtatgca tagctctcaa 2160

tgctgatgat catggtgaag gtaactaacc aattctactg ctacctttcc tctctctatt 2220

cttcattttt gagtggctac ctttccagat aattgttgaa ttaagcaata gcatttggac 2280

aagaaaaata ttcataaaaa aaaggatgat tcattacaaa aaaaaagaag atatgttgat 2340

gaaagatatg tatgtttggg gctatgcatg cctcttcgat ctatagtgtg catagggtgt 2400

gtttttttat cagacatagg gtgtttctca atagtactca ttattcccgt atggaggcag 2460

catgcatcaa tcttttaaca ttcatttagt tttattactg ccttactggg aaaatatctc 2520

tagatttttc tttcttcaga cagcacttga taagctgaat tgtaagtctg atagagatgg 2580

aacatactgt attttctttg gaaggcaatc tttattttat ggataggtct gaaattcttt 2640

tttgcaatgt atcgatatgt attgttgctt aaaagtatac tttctgcagg tggtacattt 2700

gccatgtatt ctttgttgtg taggcatgct gatataggca tccttccttc caaaagggtg 2760

tatgcagaag aagatccact gcttcacagt cagtctgcaa tagccagaag gcctagtagg 2820

ctgggaaagt tctttgaaca gagcataact gcaagaagag ttttgttatt cgtagcagta 2880

cttgggatgt gcatgctcat tggagatggc atacttactc ctgctatttc aggtttgcta 2940

tttataaatt acatgccaat ctaaccattc ttgtgatcat agtgagacct gacagtattt 3000

ctttacccct ttacagtact atcagcaatt gatgggataa gagggccatt tcctactgtt 3060

agcaaacgta aggcctcagg aacattgtag ctaatgaaca atgaaaaatc tttctaatac 3120

taaccttttg acattcatcc tttcttttct tttttgtttc ttccagctgt tgtggaggcc 3180

ctttctgcag caattctcat tggcttattc ttattgcaaa agtatgggac ttcaaaagtg 3240

agctttctgt tttctccaat catggcagca tggactttca ccactccaat tatcggctta 3300

tacagtattg tacattacta tcctggcatc ttcaaagcca tttcaccgta ttatattgtt 3360

catttcttcc tgagaaataa aaggcaaggt tggcaactgc ttggtgggac tgttttatgc 3420

atcacaggta cattccatac cagcattatc aactttgagt attttctttt ctcctaccaa 3480

tgatgatagg tgtaaagttc aacctcctgc acatacacag gtgctgaagc tatgttcgca 3540

gatcttgggc acttcagcaa aaaggctatt caggttagtt tcattgagac gtccatctgc 3600

aatttataca cgatataatt tcttacagtt gatctttttt ttttcagata gcatttctgt 3660

ctagcatata tccttctttg gtgctcactt atgctgggca aacagcatac cttataaaca 3720

atgtcaatga ctttggtgat ggtttctata aatttgttcc tcgtcccgtt tactggccaa 3780

tgtttgtcgt tgcaacacta gcagcaattg ttgcgagcca atccttaata tccgcgacat 3840

tctctgtcat caagcaatca gttgtcctgg attacttccc tcgtgttaaa gtggtgcaca 3900

cgtcgcaaca caaagaaggc gaggtttact ccccagaaat caattacatt ctcatggtac 3960

tgtgtgtcgg tgttatacta ggatttggtg gtggcaaggc tatagggaat gctttcggta 4020

agcaagttct acaaaattct cgacttgtca atacaaaatt attttgtata agaagattca 4080

gtacctcaat gtacattatt tttgcaggtg ttgtggtcat catggtcatg ctcataacta 4140

cagtcctact cacccttgtg atgatcatca tatggagaac accacttgtg cttgctgggc 4200

tgtacttcgt ccccttcttc atcatggaag gggcctatgt cagtgcagtt ttcaccaaga 4260

tccctgaagg aggttggctt cctttcgcgg tttccataac ccttgcaatg atcatgttcg 4320

gctggtacta cggtcggcaa aggaaatttg agtacgagat gacaaacaaa gtgagcttgg 4380

agcacctcgg cgagctcttg gcaaggcctg aggttcagag ggtccctggc ctctgcttct 4440

tctacagcaa catacaggac gggctaactc caatcctcag ccattacatc aagaacatga 4500

gctcactgca cacggtcaca atttttgtca ccctgaggtc cctgctcgtc gccaaagttg 4560

atcaaagcaa aaggatcctg ataaacaggc ttggaccaaa cggggtgtac ggctgcaccg 4620

ttcagtacgg ctacgccgac aacctcagcc tcgagggcgg cgacgacctc gccgcgcagg 4680

tcacgagctg cctgcaatgg cacatccaga tggacactga tggccgccgg tcaccggagg 4740

aagagatggc gcagctggag gcggcgaggt tggccggcgt ggtgcacgtc cggggcaaga 4800

tgaggttcta cgtcggcgag gacgccggct ggtttgataa gatcatgctc ggattctatg 4860

agttcttgca tgggatctgc cggtcggctc tgccggttct tgggatgcct ctgcaacagc 4920

gagttgagat cggcatgctg tacaaggtct gatgagctgt tgatcgattg aatcactagt 4980

gttgtctgtt gccttacgat gttgatcgat tggaactagg aatcagacgg ttagaagtgt 5040

aagatcagga aattgtgtta ctgcgttatg taaactgtat acattatacg tatagaaaaa 5100

ttcagcacgt cgtactgaca tggttcgcat gttgcatctt ctaaaaactg aagaaggaat 5160

cgcgtcaact ttcacaaatt tgaagtctgt atctctcata tttgatcttc aagttcagtt 5220

cagtgcgtac aaatatagct tccaagacga actcaacgtg gaagtgctga aattttgtac 5280

agtgg 5285

<210> 6

<211> 714

<212> PRT

<213> Rice (Oryza sativa)

<400> 6

Met Trp Phe Ile Ala Leu Leu Arg Cys Val Pro Asn Arg Asn Leu Lys

1 5 10 15

Tyr His Leu Cys Val Leu Gln Phe Ile Leu Met Phe Ser Leu Asn Ser

20 25 30

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

35 40 45

Leu Gly Val Val Phe Gly Gly Leu Val Thr Ser Pro Leu Val Tyr Pro

50 55 60

Ser Met Asn Leu Ser Ser Pro Thr Glu Ala Asp Tyr Leu Gly Ile Tyr

65 70 75 80

Ser Ile Met Phe Trp Thr Leu Thr Leu Ile Gly Val Val Lys Tyr Val

85 90 95

Cys Ile Ala Leu Asn Ala Asp Asp His Gly Glu Gly Gly Thr Phe Ala

100 105 110

Met Tyr Ser Leu Leu Cys Arg His Ala Asp Ile Gly Ile Leu Pro Ser

115 120 125

Lys Arg Val Tyr Ala Glu Glu Asp Pro Leu Leu His Ser Gln Ser Ala

130 135 140

Ile Ala Arg Arg Pro Ser Arg Leu Gly Lys Phe Phe Glu Gln Ser Ile

145 150 155 160

Thr Ala Arg Arg Val Leu Leu Phe Val Ala Val Leu Gly Met Cys Met

165 170 175

Leu Ile Gly Asp Gly Ile Leu Thr Pro Ala Ile Ser Val Leu Ser Ala

180 185 190

Ile Asp Gly Ile Arg Gly Pro Phe Pro Thr Val Ser Lys Pro Val Val

195 200 205

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

210 215 220

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

225 230 235 240

Trp Thr Phe Thr Thr Pro Ile Ile Gly Leu Tyr Ser Ile Val His Tyr

245 250 255

Tyr Pro Gly Ile Phe Lys Ala Ile Ser Pro Tyr Tyr Ile Val His Phe

260 265 270

Phe Leu Arg Asn Lys Arg Gln Gly Trp Gln Leu Leu Gly Gly Thr Val

275 280 285

Leu Cys Ile Thr Gly Ala Glu Ala Met Phe Ala Asp Leu Gly His Phe

290 295 300

Ser Lys Lys Ala Ile Gln Ile Ala Phe Leu Ser Ser Ile Tyr Pro Ser

305 310 315 320

Leu Val Leu Thr Tyr Ala Gly Gln Thr Ala Tyr Leu Ile Asn Asn Val

325 330 335

Asn Asp Phe Gly Asp Gly Phe Tyr Lys Phe Val Pro Arg Pro Val Tyr

340 345 350

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

355 360 365

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

370 375 380

Asp Tyr Phe Pro Arg Val Lys Val Val His Thr Ser Gln His Lys Glu

385 390 395 400

Gly Glu Val Tyr Ser Pro Glu Ile Asn Tyr Ile Leu Met Val Leu Cys

405 410 415

Val Gly Val Ile Leu Gly Phe Gly Gly Gly Lys Ala Ile Gly Asn Ala

420 425 430

Phe Gly Val Val Val Ile Met Val Met Leu Ile Thr Thr Val Leu Leu

435 440 445

Thr Leu Val Met Ile Ile Ile Trp Arg Thr Pro Leu Val Leu Ala Gly

450 455 460

Leu Tyr Phe Val Pro Phe Phe Ile Met Glu Gly Ala Tyr Val Ser Ala

465 470 475 480

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

485 490 495

Ile Thr Leu Ala Met Ile Met Phe Gly Trp Tyr Tyr Gly Arg Gln Arg

500 505 510

Lys Phe Glu Tyr Glu Met Thr Asn Lys Val Ser Leu Glu His Leu Gly

515 520 525

Glu Leu Leu Ala Arg Pro Glu Val Gln Arg Val Pro Gly Leu Cys Phe

530 535 540

Phe Tyr Ser Asn Ile Gln Asp Gly Leu Thr Pro Ile Leu Ser His Tyr

545 550 555 560

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

565 570 575

Arg Ser Leu Leu Val Ala Lys Val Asp Gln Ser Lys Arg Ile Leu Ile

580 585 590

Asn Arg Leu Gly Pro Asn Gly Val Tyr Gly Cys Thr Val Gln Tyr Gly

595 600 605

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

610 615 620

Val Thr Ser Cys Leu Gln Trp His Ile Gln Met Asp Thr Asp Gly Arg

625 630 635 640

Arg Ser Pro Glu Glu Glu Met Ala Gln Leu Glu Ala Ala Arg Leu Ala

645 650 655

Gly Val Val His Val Arg Gly Lys Met Arg Phe Tyr Val Gly Glu Asp

660 665 670

Ala Gly Trp Phe Asp Lys Ile Met Leu Gly Phe Tyr Glu Phe Leu His

675 680 685

Gly Ile Cys Arg Ser Ala Leu Pro Val Leu Gly Met Pro Leu Gln Gln

690 695 700

Arg Val Glu Ile Gly Met Leu Tyr Lys Val

705 710

<210> 7

<211> 5686

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 7

atggatcgac aaacccggac ggcctgactt gacggcggcg gcggcggcgg cggcgcagtc 60

agatgcagct gttacagtgg cggtgcatga cggcgaaacc attaataatg atcaaaaggt 120

tcaatggttc atctcattcc acgctctctg tatacttatc tctttgtttt gtggtttttt 180

tccccttaaa aatggaactt tgtgctctgt tttgtagtat tttgatttct ctctgtgttt 240

aacttccgtc gtatgtttac attcatttag tcggagtgcc tacaaactac tgctctctag 300

ttttagttta tgtgatacag ttggaatttc gtgattcaaa agctttaaat tttgattatg 360

aatccagaca tagaaggctt ttgagttttt ttttgaaatg atatacactt ttctctgttt 420

tgattttatt ttttttgaac ttgacacgga gtttaagaaa tttaataaag attttagaat 480

tgcatggact ttttttttat gaccgtagtg tgttggtcag ctttacgcgc acctcaatta 540

atttcatgcg atacctgtca cgtcgagcaa aaggtatcaa ataactctgt ccatcgaggc 600

tcgaagagaa tgttagaatc gcctagtgct ttttgtctgt actgagtttg aattgaatgg 660

tgtttaaact aaagatacgt ggaatgtatc aaactgtttt caatgttttg ggttttaaat 720

acgtcatgaa aagttgaaac tgaagtgttg caaaaaagag taaaaaaaat agattttttt 780

tttcaaagta tgacaacaaa ttgaaataga ggtagtattt ggaaaactac gtcgaaatta 840

gtataaatct atgttgctcg tactcttcca agatatcaac cggtgtgtgt cggatcctaa 900

agatagtgta tttttgaagg atccgacacg aatgcggcat tgtttttggg gagtccacca 960

acataatata aatcacgata actaatataa aatgtttaaa agacatatga ataaagtttg 1020

gtcgaagaaa aacgtggttg actctactgt tccacgtata ttgtgatttg aggtgaaatt 1080

gttgatgaag atggtatttt cttaatccca ttccttcatt atatatagtg tatctttcaa 1140

ttgaggtgaa gttgtggatc atgaagtagt tgttgatgat gatggttgac tctacgaaat 1200

acttttgcgt tcaaagttaa gttgtctcta ctaagatgtt ctttgacatt tgtttttact 1260

cggaaactat atttgtttag gaccatagcc gatgggagac gttggtactt gcatacaaga 1320

ccttaggagt tgtttttggg ggtcttgtca catctccact ctatgtatat ccatcaatgc 1380

cattaaagtc tccaactgag gatgattatt tggggatata cagcataatg ttttggactc 1440

tcagtctaat tggtgttgtc aaatatgcta ccatagctct ccaagctgat gatcagggtg 1500

aaggtattgc tgatcactct ctctttgtac ttttgctact tacgacaaca ttttcccatt 1560

gaaaaatggg aattgtttca tatggaaaaa aaaaggaaac ttctcgctat ttcagtgaga 1620

atctgtttcc caccaaacat tttcccagtg gagctggttc agtggggaat gagtggaaaa 1680

atcatatgtt atagcacaaa tttcccactt atttgcaggt ggaaaaaacc tttatttcta 1740

gtagtgaggt gactcgagtt ttaagaaact gtctgtttgt attcatacta aaagatttat 1800

cttttggaga atgttgaagg tgttaatgaa ggatgtttat atataatgtt gagtcaattc 1860

ttaattttct ttatattttg gttgaaattc cttttgaaag acacttcaga gtttcttcat 1920

ttctcttttg atgtgatcat tatcgaaatt tcagttctag aacatggtct tttgtaggtg 1980

gaacctttgc tctttattca ttactttgta gaaatataaa tattggtatt ctttcttcca 2040

agagtgccag tttgaattca agccactcct atgttaacca atcgaaaaag ccaagtaggt 2100

tgggtaaatt ttgtgagagg agtttgattg ccagaagggt actgcttttc atcgcaatgt 2160

tgggtatgtg catgcttatt ggcgatggca tacttacacc tgccatctca ggtatgttag 2220

cttgggttgg gccttttagt ttacttttag caaccttttg agtgcttgtc agttttccct 2280

ctttcatttt cagtttcatt aattgtccct tctgtctgtc agtgttatct gcaatgggtg 2340

gcttacgagc acgattctcc tcggtcagta aatgtaagta tgacagcata cagtaaggta 2400

catattgctc attgctttct tagtcgagct cgttgcatgg ggcttgccta gtgcggctta 2460

ccgctcctgt gtggtttgca ggctattacc ccgaagcagg tttaccctat gcccacccta 2520

agggtagcag ctgtggtttc ccttgtcata aataaaaaat tgtctttgaa cggagaggcc 2580

tttatgattt tgagggtaat tgggaagtgg tcgaagaaca attttgattc ttgtaatttc 2640

tcaggtttta aggtattgtg cttgttaaaa gactatcttt cacagattca tatattgatg 2700

ttacatgagt ccgattctgg aagctaattg aaataacttg aatccttgtt ctagtaatta 2760

tttgaagtaa ttaagtaatg attgaacact ttaaggtcaa taagtcatat taacgaaaag 2820

gagcagtgca gtttctgaaa gttaaagtct tcttttatga caggttgcaa gtccttttta 2880

aacactagat gacgcgaaac atgttaattg tcccatttat gtaaacacat ctctccctct 2940

cgatattcac aatttctctg cttgccactt gcagcgctgg tcgaaggtct ctcagcaatc 3000

attctcattg ttctattcct cctgcagaaa tttggtactt cacgagtgag cttcctcttg 3060

tctcctatca tgggtgcatg gactcttacc actcctctcg ttgggatata cagtatcata 3120

aagcattatc cgagcatatt taaggcgata tcaccccatt acattgccct cttcttctta 3180

agaaatggaa aacaaggatg gatatatctt ggtggtactg tcctatgcat tacaggttag 3240

cctcgttgtt ggtgtggtct gcaaaagttg ttcattgtga ttaagatcta aatccatttc 3300

ctcatgtttg gagttctttt acattctttc ctattacttt gcaattaata ttggggaaaa 3360

tttggaccaa ttatcatgtg tgcaatgtct accgatggcg ggaaaagctt aaacctggta 3420

tcttgtttca tatccagaga aactcgaaag gtgatgaatg gaggtggaaa atattataaa 3480

gaatatggaa tgcccctctg gtcaatactt ttatccaaaa atgataacaa ttgttctggt 3540

ttgagcgaaa aaacatggag gctgttctag attatttagc tcggggagtt ccaacgtcga 3600

ccatggtgta tttctcgcta agttgtcaga ttcctcaccc ttctattttt gtgtagtggg 3660

gggtttgagg attagagaaa aagacgggat gaaagaggat aaggggtgga cagtaccttt 3720

caccgaaaca tattaggccg ctagagttat tttctggcca agatctgttt cacttccacc 3780

caatatctgc ttaacagtac aatcctttca tatttttagg ttctgaagca atgtttgctg 3840

atcttggtca tttcaacagg agttcaattc gggtaaaaat acttgctatg tcttggaatt 3900

tctcttagtc tcttcttctt gcttgtttta tatgatttac gggatcttgt tttctggcag 3960

atagcatttc tctatactat atatccatca ttggtgctga catatgcggg acagacagca 4020

tatctgatta gaaatcccga tgaccacttt gatggatttt acaagtttat accctctgct 4080

gtgtactggc caatttttgt tatagccaca ttagctgcta tagtagcaag tcagtcgctg 4140

atttcagcca ccttttcggt tatcaagcaa tcagttgtgt tggattattt tcctcgggtg 4200

aaggtagttc acacatcctc cagcaaagaa ggcgaggttt actcaccaga agttaactac 4260

atcctcatga ttctctgtgt tgcagtcata ctagtcttcg gagatggaca agacatcgga 4320

aatgccttcg gtaagatgcc ctaaaccctt tgcattttat cttccatatt tagggactat 4380

cttaactgaa ttgatccctt tatgaaagtt atctttcact tgactcctct tcctaggatg 4440

gtggttgtta acatcgacta ctagcatttt ctttacctaa tttactacta cttggtgatg 4500

acttgcttac tttaaggggt aactagtgta atgaattgag ctaaacaaat tgtggattgg 4560

aattttctca ttctgtttgc tcgttgtttt cagattttgt ctcgtcttgt ttagcattat 4620

ccaataagat atttcactag tgataacgca catatatgtg gtgcaaatca agttcaacaa 4680

aattgtcttg tcttgtttag catagttaaa ataaaattac atatatgtat gatcttgtat 4740

gtatatatta aggtataact tgcatttttt cttgcaacgg ttcaactctt tcgtattgtc 4800

agcatagtaa atattgcatt aactaattaa aatggattct ctaacaggtg ttgttgttag 4860

catagttatg cttataacga ccatattgtt gacattggtt atgatcatta tttggagaac 4920

tccaccagct ctggtcgggc tctacttcgt tgtatttttt gtgatggaaa gtgtatatgt 4980

tagtgcaatc ttcacaaaaa ttcccgaagg tggttggatt ccttttgcca tttctctcat 5040

tcttgctttc attatgtttg gttggttcta cggtaggcag agaaaacttg agtatgaatt 5100

gacacacaag atagactcag agagactaag gacactgcta atcgatccag gtcttcagag 5160

agttcctgga ctctgcttct tctacacaaa tatccaagat ggtttaaccc caattttagg 5220

tcattacata aagaacatga gatctcttca caaagttact gtatttacaa ctcttcgtta 5280

cttgctggtt cctaaagtag cacctggtga aagaatcgtt gttagtaaac taggtctcag 5340

aggggtttat agatgtgtga ttcgttatgg ttatgcagat aagcttagtc ttgaaggaga 5400

tgatttagtt aatcaagtca tccaaagttt acgatctcat gtgctccact gctccaattc 5460

attggaggtt gacactgaag tttccgagtt ggatgaggca aagcttgctg gagtagtaca 5520

tattcgtgga aagacgaggt tttatattgg taaggactgt ggatggtttg atagaacaat 5580

gcttgctttc tatgaagttt tgcatagcaa ttgcagatct gcattgcctg ctatgggtgt 5640

accattacct caacgtatcg aggttggaat gctctacgcg gcgtaa 5686

<210> 8

<211> 7

<212> PRT

<213> Artificial sequence (Artificial sequence)

<400> 8

Met Asp Arg Gln Thr Arg Ala

1 5

<210> 9

<211> 5683

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 9

atggatcgac aaaccggacg gcctgacttg acggcggcgg cggcggcggc ggcgcagtca 60

gatgcagctg tcagtggcgg tgcatgacgg cgaaaccatt aataatgatc aaaaggttca 120

atggttcatc tcattccacg ctctctgtat acttatctct ttgttttgtg gtttttttcc 180

ccttaaaaat ggaactttgt gctctgtttt gtagtatttt gatttctctc tgtgtttaac 240

ttccgtcgta tgtttacatt catttagtcg gagtgcctac aaactactgc tctctagttt 300

tagtttatgt gatacagttg gaatttcgtg attcaaaagc tttaaatttt gattatgaat 360

ccagacatag aaggcttttg agtttttttt tgaaatgata tacacttttc tctgttttga 420

ttttattttt tttgaacttg acacggagtt taagaaattt aataaagatt ttagaattgc 480

atggactttt tttttatgac cgtagtgtgt tggtcagctt tacgcgcacc tcaattaatt 540

tcatgcgata cctgtcacgt cgagcaaaag gtatcaaata actctgtcca tcgaggctcg 600

aagagaatgt tagaatcgcc tagtgctttt tgtctgtact gagtttgaat tgaatggtgt 660

ttaaactaaa gatacgtgga atgtatcaaa ctgttttcaa tgttttgggt tttaaatacg 720

tcatgaaaag ttgaaactga agtgttgcaa aaaagagtaa aaaaaataga tttttttttt 780

caaagtatga caacaaattg aaatagaggt agtatttgga aaactacgtc gaaattagta 840

taaatctatg ttgctcgtac tcttccaaga tatcaaccgg tgtgtgtcgg atcctaaaga 900

tagtgtattt ttgaaggatc cgacacgaat gcggcattgt ttttggggag tccaccaaca 960

taatataaat cacgataact aatataaaat gtttaaaaga catatgaata aagtttggtc 1020

gaagaaaaac gtggttgact ctactgttcc acgtatattg tgatttgagg tgaaattgtt 1080

gatgaagatg gtattttctt aatcccattc cttcattata tatagtgtat ctttcaattg 1140

aggtgaagtt gtggatcatg aagtagttgt tgatgatgat ggttgactct acgaaatact 1200

tttgcgttca aagttaagtt gtctctacta agatgttctt tgacatttgt ttttactcgg 1260

aaactatatt tgtttaggac catagccgat gggagacgtt ggtacttgca tacaagacct 1320

taggagttgt ttttgggggt cttgtcacat ctccactcta tgtatatcca tcaatgccat 1380

taaagtctcc aactgaggat gattatttgg ggatatacag cataatgttt tggactctca 1440

gtctaattgg tgttgtcaaa tatgctacca tagctctcca agctgatgat cagggtgaag 1500

gtattgctga tcactctctc tttgtacttt tgctacttac gacaacattt tcccattgaa 1560

aaatgggaat tgtttcatat ggaaaaaaaa aggaaacttc tcgctatttc agtgagaatc 1620

tgtttcccac caaacatttt cccagtggag ctggttcagt ggggaatgag tggaaaaatc 1680

atatgttata gcacaaattt cccacttatt tgcaggtgga aaaaaccttt atttctagta 1740

gtgaggtgac tcgagtttta agaaactgtc tgtttgtatt catactaaaa gatttatctt 1800

ttggagaatg ttgaaggtgt taatgaagga tgtttatata taatgttgag tcaattctta 1860

attttcttta tattttggtt gaaattcctt ttgaaagaca cttcagagtt tcttcatttc 1920

tcttttgatg tgatcattat cgaaatttca gttctagaac atggtctttt gtaggtggaa 1980

cctttgctct ttattcatta ctttgtagaa atataaatat tggtattctt tcttccaaga 2040

gtgccagttt gaattcaagc cactcctatg ttaaccaatc gaaaaagcca agtaggttgg 2100

gtaaattttg tgagaggagt ttgattgcca gaagggtact gcttttcatc gcaatgttgg 2160

gtatgtgcat gcttattggc gatggcatac ttacacctgc catctcaggt atgttagctt 2220

gggttgggcc ttttagttta cttttagcaa ccttttgagt gcttgtcagt tttccctctt 2280

tcattttcag tttcattaat tgtcccttct gtctgtcagt gttatctgca atgggtggct 2340

tacgagcacg attctcctcg gtcagtaaat gtaagtatga cagcatacag taaggtacat 2400

attgctcatt gctttcttag tcgagctcgt tgcatggggc ttgcctagtg cggcttaccg 2460

ctcctgtgtg gtttgcaggc tattaccccg aagcaggttt accctatgcc caccctaagg 2520

gtagcagctg tggtttccct tgtcataaat aaaaaattgt ctttgaacgg agaggccttt 2580

atgattttga gggtaattgg gaagtggtcg aagaacaatt ttgattcttg taatttctca 2640

ggttttaagg tattgtgctt gttaaaagac tatctttcac agattcatat attgatgtta 2700

catgagtccg attctggaag ctaattgaaa taacttgaat ccttgttcta gtaattattt 2760

gaagtaatta agtaatgatt gaacacttta aggtcaataa gtcatattaa cgaaaaggag 2820

cagtgcagtt tctgaaagtt aaagtcttct tttatgacag gttgcaagtc ctttttaaac 2880

actagatgac gcgaaacatg ttaattgtcc catttatgta aacacatctc tccctctcga 2940

tattcacaat ttctctgctt gccacttgca gcgctggtcg aaggtctctc agcaatcatt 3000

ctcattgttc tattcctcct gcagaaattt ggtacttcac gagtgagctt cctcttgtct 3060

cctatcatgg gtgcatggac tcttaccact cctctcgttg ggatatacag tatcataaag 3120

cattatccga gcatatttaa ggcgatatca ccccattaca ttgccctctt cttcttaaga 3180

aatggaaaac aaggatggat atatcttggt ggtactgtcc tatgcattac aggttagcct 3240

cgttgttggt gtggtctgca aaagttgttc attgtgatta agatctaaat ccatttcctc 3300

atgtttggag ttcttttaca ttctttccta ttactttgca attaatattg gggaaaattt 3360

ggaccaatta tcatgtgtgc aatgtctacc gatggcggga aaagcttaaa cctggtatct 3420

tgtttcatat ccagagaaac tcgaaaggtg atgaatggag gtggaaaata ttataaagaa 3480

tatggaatgc ccctctggtc aatactttta tccaaaaatg ataacaattg ttctggtttg 3540

agcgaaaaaa catggaggct gttctagatt atttagctcg gggagttcca acgtcgacca 3600

tggtgtattt ctcgctaagt tgtcagattc ctcacccttc tatttttgtg tagtgggggg 3660

tttgaggatt agagaaaaag acgggatgaa agaggataag gggtggacag tacctttcac 3720

cgaaacatat taggccgcta gagttatttt ctggccaaga tctgtttcac ttccacccaa 3780

tatctgctta acagtacaat cctttcatat ttttaggttc tgaagcaatg tttgctgatc 3840

ttggtcattt caacaggagt tcaattcggg taaaaatact tgctatgtct tggaatttct 3900

cttagtctct tcttcttgct tgttttatat gatttacggg atcttgtttt ctggcagata 3960

gcatttctct atactatata tccatcattg gtgctgacat atgcgggaca gacagcatat 4020

ctgattagaa atcccgatga ccactttgat ggattttaca agtttatacc ctctgctgtg 4080

tactggccaa tttttgttat agccacatta gctgctatag tagcaagtca gtcgctgatt 4140

tcagccacct tttcggttat caagcaatca gttgtgttgg attattttcc tcgggtgaag 4200

gtagttcaca catcctccag caaagaaggc gaggtttact caccagaagt taactacatc 4260

ctcatgattc tctgtgttgc agtcatacta gtcttcggag atggacaaga catcggaaat 4320

gccttcggta agatgcccta aaccctttgc attttatctt ccatatttag ggactatctt 4380

aactgaattg atccctttat gaaagttatc tttcacttga ctcctcttcc taggatggtg 4440

gttgttaaca tcgactacta gcattttctt tacctaattt actactactt ggtgatgact 4500

tgcttacttt aaggggtaac tagtgtaatg aattgagcta aacaaattgt ggattggaat 4560

tttctcattc tgtttgctcg ttgttttcag attttgtctc gtcttgttta gcattatcca 4620

ataagatatt tcactagtga taacgcacat atatgtggtg caaatcaagt tcaacaaaat 4680

tgtcttgtct tgtttagcat agttaaaata aaattacata tatgtatgat cttgtatgta 4740

tatattaagg tataacttgc attttttctt gcaacggttc aactctttcg tattgtcagc 4800

atagtaaata ttgcattaac taattaaaat ggattctcta acaggtgttg ttgttagcat 4860

agttatgctt ataacgacca tattgttgac attggttatg atcattattt ggagaactcc 4920

accagctctg gtcgggctct acttcgttgt attttttgtg atggaaagtg tatatgttag 4980

tgcaatcttc acaaaaattc ccgaaggtgg ttggattcct tttgccattt ctctcattct 5040

tgctttcatt atgtttggtt ggttctacgg taggcagaga aaacttgagt atgaattgac 5100

acacaagata gactcagaga gactaaggac actgctaatc gatccaggtc ttcagagagt 5160

tcctggactc tgcttcttct acacaaatat ccaagatggt ttaaccccaa ttttaggtca 5220

ttacataaag aacatgagat ctcttcacaa agttactgta tttacaactc ttcgttactt 5280

gctggttcct aaagtagcac ctggtgaaag aatcgttgtt agtaaactag gtctcagagg 5340

ggtttataga tgtgtgattc gttatggtta tgcagataag cttagtcttg aaggagatga 5400

tttagttaat caagtcatcc aaagtttacg atctcatgtg ctccactgct ccaattcatt 5460

ggaggttgac actgaagttt ccgagttgga tgaggcaaag cttgctggag tagtacatat 5520

tcgtggaaag acgaggtttt atattggtaa ggactgtgga tggtttgata gaacaatgct 5580

tgctttctat gaagttttgc atagcaattg cagatctgca ttgcctgcta tgggtgtacc 5640

attacctcaa cgtatcgagg ttggaatgct ctacgcggcg taa 5683

<210> 10

<211> 26

<212> PRT

<213> Artificial sequence (Artificial sequence)

<400> 10

Met Asp Arg Gln Thr Gly Arg Pro Asp Leu Thr Ala Ala Ala Ala Ala

1 5 10 15

Gln Ser Asp Ala Ala Val Ser Gly Gly Ala

20 25

<210> 11

<211> 5684

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 11

atggatcgac aaaccggacg gcctgacttg acggcggcgg cggcggcggc ggcgcagtca 60

gatgcagctg ttacagtggc ggtgcatgac ggcgaaacca ttaataatga tcaaaaggtt 120

caatggttca tctcattcca cgctctctgt atacttatct ctttgttttg tggttttttt 180

ccccttaaaa atggaacttt gtgctctgtt ttgtagtatt ttgatttctc tctgtgttta 240

acttccgtcg tatgtttaca ttcatttagt cggagtgcct acaaactact gctctctagt 300

tttagtttat gtgatacagt tggaatttcg tgattcaaaa gctttaaatt ttgattatga 360

atccagacat agaaggcttt tgagtttttt tttgaaatga tatacacttt tctctgtttt 420

gattttattt tttttgaact tgacacggag tttaagaaat ttaataaaga ttttagaatt 480

gcatggactt tttttttatg accgtagtgt gttggtcagc tttacgcgca cctcaattaa 540

tttcatgcga tacctgtcac gtcgagcaaa aggtatcaaa taactctgtc catcgaggct 600

cgaagagaat gttagaatcg cctagtgctt tttgtctgta ctgagtttga attgaatggt 660

gtttaaacta aagatacgtg gaatgtatca aactgttttc aatgttttgg gttttaaata 720

cgtcatgaaa agttgaaact gaagtgttgc aaaaaagagt aaaaaaaata gatttttttt 780

ttcaaagtat gacaacaaat tgaaatagag gtagtatttg gaaaactacg tcgaaattag 840

tataaatcta tgttgctcgt actcttccaa gatatcaacc ggtgtgtgtc ggatcctaaa 900

gatagtgtat ttttgaagga tccgacacga atgcggcatt gtttttgggg agtccaccaa 960

cataatataa atcacgataa ctaatataaa atgtttaaaa gacatatgaa taaagtttgg 1020

tcgaagaaaa acgtggttga ctctactgtt ccacgtatat tgtgatttga ggtgaaattg 1080

ttgatgaaga tggtattttc ttaatcccat tccttcatta tatatagtgt atctttcaat 1140

tgaggtgaag ttgtggatca tgaagtagtt gttgatgatg atggttgact ctacgaaata 1200

cttttgcgtt caaagttaag ttgtctctac taagatgttc tttgacattt gtttttactc 1260

ggaaactata tttgtttagg accatagccg atgggagacg ttggtacttg catacaagac 1320

cttaggagtt gtttttgggg gtcttgtcac atctccactc tatgtatatc catcaatgcc 1380

attaaagtct ccaactgagg atgattattt ggggatatac agcataatgt tttggactct 1440

cagtctaatt ggtgttgtca aatatgctac catagctctc caagctgatg atcagggtga 1500

aggtattgct gatcactctc tctttgtact tttgctactt acgacaacat tttcccattg 1560

aaaaatggga attgtttcat atggaaaaaa aaaggaaact tctcgctatt tcagtgagaa 1620

tctgtttccc accaaacatt ttcccagtgg agctggttca gtggggaatg agtggaaaaa 1680

tcatatgtta tagcacaaat ttcccactta tttgcaggtg gaaaaaacct ttatttctag 1740

tagtgaggtg actcgagttt taagaaactg tctgtttgta ttcatactaa aagatttatc 1800

ttttggagaa tgttgaaggt gttaatgaag gatgtttata tataatgttg agtcaattct 1860

taattttctt tatattttgg ttgaaattcc ttttgaaaga cacttcagag tttcttcatt 1920

tctcttttga tgtgatcatt atcgaaattt cagttctaga acatggtctt ttgtaggtgg 1980

aacctttgct ctttattcat tactttgtag aaatataaat attggtattc tttcttccaa 2040

gagtgccagt ttgaattcaa gccactccta tgttaaccaa tcgaaaaagc caagtaggtt 2100

gggtaaattt tgtgagagga gtttgattgc cagaagggta ctgcttttca tcgcaatgtt 2160

gggtatgtgc atgcttattg gcgatggcat acttacacct gccatctcag gtatgttagc 2220

ttgggttggg ccttttagtt tacttttagc aaccttttga gtgcttgtca gttttccctc 2280

tttcattttc agtttcatta attgtccctt ctgtctgtca gtgttatctg caatgggtgg 2340

cttacgagca cgattctcct cggtcagtaa atgtaagtat gacagcatac agtaaggtac 2400

atattgctca ttgctttctt agtcgagctc gttgcatggg gcttgcctag tgcggcttac 2460

cgctcctgtg tggtttgcag gctattaccc cgaagcaggt ttaccctatg cccaccctaa 2520

gggtagcagc tgtggtttcc cttgtcataa ataaaaaatt gtctttgaac ggagaggcct 2580

ttatgatttt gagggtaatt gggaagtggt cgaagaacaa ttttgattct tgtaatttct 2640

caggttttaa ggtattgtgc ttgttaaaag actatctttc acagattcat atattgatgt 2700

tacatgagtc cgattctgga agctaattga aataacttga atccttgttc tagtaattat 2760

ttgaagtaat taagtaatga ttgaacactt taaggtcaat aagtcatatt aacgaaaagg 2820

agcagtgcag tttctgaaag ttaaagtctt cttttatgac aggttgcaag tcctttttaa 2880

acactagatg acgcgaaaca tgttaattgt cccatttatg taaacacatc tctccctctc 2940

gatattcaca atttctctgc ttgccacttg cagcgctggt cgaaggtctc tcagcaatca 3000

ttctcattgt tctattcctc ctgcagaaat ttggtacttc acgagtgagc ttcctcttgt 3060

ctcctatcat ggggcatgga ctcttaccac tcctctcgtt gggatataca gtatcataaa 3120

gcattatccg agcatattta aggcgatatc accccattac attgccctct tcttcttaag 3180

aaatggaaaa caaggatgga tatatcttgg tggtactgtc ctatgcatta caggttagcc 3240

tcgttgttgg tgtggtctgc aaaagttgtt cattgtgatt aagatctaaa tccatttcct 3300

catgtttgga gttcttttac attctttcct attactttgc aattaatatt ggggaaaatt 3360

tggaccaatt atcatgtgtg caatgtctac cgatggcggg aaaagcttaa acctggtatc 3420

ttgtttcata tccagagaaa ctcgaaaggt gatgaatgga ggtggaaaat attataaaga 3480

atatggaatg cccctctggt caatactttt atccaaaaat gataacaatt gttctggttt 3540

gagcgaaaaa acatggaggc tgttctagat tatttagctc ggggagttcc aacgtcgacc 3600

atggtgtatt tctcgctaag ttgtcagatt cctcaccctt ctatttttgt gtagtggggg 3660

gtttgaggat tagagaaaaa gacgggatga aagaggataa ggggtggaca gtacctttca 3720

ccgaaacata ttaggccgct agagttattt tctggccaag atctgtttca cttccaccca 3780

atatctgctt aacagtacaa tcctttcata tttttaggtt ctgaagcaat gtttgctgat 3840

cttggtcatt tcaacaggag ttcaattcgg gtaaaaatac ttgctatgtc ttggaatttc 3900

tcttagtctc ttcttcttgc ttgttttata tgatttacgg gatcttgttt tctggcagat 3960

agcatttctc tatactatat atccatcatt ggtgctgaca tatgcgggac agacagcata 4020

tctgattaga aatcccgatg accactttga tggattttac aagtttatac cctctgctgt 4080

gtactggcca atttttgtta tagccacatt agctgctata gtagcaagtc agtcgctgat 4140

ttcagccacc ttttcggtta tcaagcaatc agttgtgttg gattattttc ctcgggtgaa 4200

ggtagttcac acatcctcca gcaaagaagg cgaggtttac tcaccagaag ttaactacat 4260

cctcatgatt ctctgtgttg cagtcatact agtcttcgga gatggacaag acatcggaaa 4320

tgccttcggt aagatgccct aaaccctttg cattttatct tccatattta gggactatct 4380

taactgaatt gatcccttta tgaaagttat ctttcacttg actcctcttc ctaggatggt 4440

ggttgttaac atcgactact agcattttct ttacctaatt tactactact tggtgatgac 4500

ttgcttactt taaggggtaa ctagtgtaat gaattgagct aaacaaattg tggattggaa 4560

ttttctcatt ctgtttgctc gttgttttca gattttgtct cgtcttgttt agcattatcc 4620

aataagatat ttcactagtg ataacgcaca tatatgtggt gcaaatcaag ttcaacaaaa 4680

ttgtcttgtc ttgtttagca tagttaaaat aaaattacat atatgtatga tcttgtatgt 4740

atatattaag gtataacttg cattttttct tgcaacggtt caactctttc gtattgtcag 4800

catagtaaat attgcattaa ctaattaaaa tggattctct aacaggtgtt gttgttagca 4860

tagttatgct tataacgacc atattgttga cattggttat gatcattatt tggagaactc 4920

caccagctct ggtcgggctc tacttcgttg tattttttgt gatggaaagt gtatatgtta 4980

gtgcaatctt cacaaaaatt cccgaaggtg gttggattcc ttttgccatt tctctcattc 5040

ttgctttcat tatgtttggt tggttctacg gtaggcagag aaaacttgag tatgaattga 5100

cacacaagat agactcagag agactaagga cactgctaat cgatccaggt cttcagagag 5160

ttcctggact ctgcttcttc tacacaaata tccaagatgg tttaacccca attttaggtc 5220

attacataaa gaacatgaga tctcttcaca aagttactgt atttacaact cttcgttact 5280

tgctggttcc taaagtagca cctggtgaaa gaatcgttgt tagtaaacta ggtctcagag 5340

gggtttatag atgtgtgatt cgttatggtt atgcagataa gcttagtctt gaaggagatg 5400

atttagttaa tcaagtcatc caaagtttac gatctcatgt gctccactgc tccaattcat 5460

tggaggttga cactgaagtt tccgagttgg atgaggcaaa gcttgctgga gtagtacata 5520

ttcgtggaaa gacgaggttt tatattggta aggactgtgg atggtttgat agaacaatgc 5580

ttgctttcta tgaagttttg catagcaatt gcagatctgc attgcctgct atgggtgtac 5640

cattacctca acgtatcgag gttggaatgc tctacgcggc gtaa 5684

<210> 12

<211> 254

<212> PRT

<213> Artificial sequence (Artificial sequence)

<400> 12

Met Asp Arg Gln Thr Gly Arg Pro Asp Leu Thr Ala Ala Ala Ala Ala

1 5 10 15

Gln Ser Asp Ala Ala Val Thr Val Ala Val His Asp Gly Glu Thr Ile

20 25 30

Asn Asn Asp Gln Lys Asp His Ser Arg Trp Glu Thr Leu Val Leu Ala

35 40 45

Tyr Lys Thr Leu Gly Val Val Phe Gly Gly Leu Val Thr Ser Pro Leu

50 55 60

Tyr Val Tyr Pro Ser Met Pro Leu Lys Ser Pro Thr Glu Asp Asp Tyr

65 70 75 80

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

85 90 95

Val Lys Tyr Ala Thr Ile Ala Leu Gln Ala Asp Asp Gln Gly Glu Gly

100 105 110

Gly Thr Phe Ala Leu Tyr Ser Leu Leu Cys Arg Asn Ile Asn Ile Gly

115 120 125

Ile Leu Ser Ser Lys Ser Ala Ser Leu Asn Ser Ser His Ser Tyr Val

130 135 140

Asn Gln Ser Lys Lys Pro Ser Arg Leu Gly Lys Phe Cys Glu Arg Ser

145 150 155 160

Leu Ile Ala Arg Arg Val Leu Leu Phe Ile Ala Met Leu Gly Met Cys

165 170 175

Met Leu Ile Gly Asp Gly Ile Leu Thr Pro Ala Ile Ser Val Leu Ser

180 185 190

Ala Met Gly Gly Leu Arg Ala Arg Phe Ser Ser Val Ser Lys Ser Leu

195 200 205

Val Glu Gly Leu Ser Ala Ile Ile Leu Ile Val Leu Phe Leu Leu Gln

210 215 220

Lys Phe Gly Thr Ser Arg Val Ser Phe Leu Leu Ser Pro Ile Met Gly

225 230 235 240

His Gly Leu Leu Pro Leu Leu Ser Leu Gly Tyr Thr Val Ser

245 250

<210> 13

<211> 5686

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 13

atggatcgac aaaccggacg gcctgacttg acggcggcgg cggcggcggc ggcgcagtca 60

gatgcagctg ttacagtggc ggtgcatgac ggcgaaacca ttaataatga tcaaaaggtt 120

caatggttca tctcattcca cgctctctgt atacttatct ctttgttttg tggttttttt 180

ccccttaaaa atggaacttt gtgctctgtt ttgtagtatt ttgatttctc tctgtgttta 240

acttccgtcg tatgtttaca ttcatttagt cggagtgcct acaaactact gctctctagt 300

tttagtttat gtgatacagt tggaatttcg tgattcaaaa gctttaaatt ttgattatga 360

atccagacat agaaggcttt tgagtttttt tttgaaatga tatacacttt tctctgtttt 420

gattttattt tttttgaact tgacacggag tttaagaaat ttaataaaga ttttagaatt 480

gcatggactt tttttttatg accgtagtgt gttggtcagc tttacgcgca cctcaattaa 540

tttcatgcga tacctgtcac gtcgagcaaa aggtatcaaa taactctgtc catcgaggct 600

cgaagagaat gttagaatcg cctagtgctt tttgtctgta ctgagtttga attgaatggt 660

gtttaaacta aagatacgtg gaatgtatca aactgttttc aatgttttgg gttttaaata 720

cgtcatgaaa agttgaaact gaagtgttgc aaaaaagagt aaaaaaaata gatttttttt 780

ttcaaagtat gacaacaaat tgaaatagag gtagtatttg gaaaactacg tcgaaattag 840

tataaatcta tgttgctcgt actcttccaa gatatcaacc ggtgtgtgtc ggatcctaaa 900

gatagtgtat ttttgaagga tccgacacga atgcggcatt gtttttgggg agtccaccaa 960

cataatataa atcacgataa ctaatataaa atgtttaaaa gacatatgaa taaagtttgg 1020

tcgaagaaaa acgtggttga ctctactgtt ccacgtatat tgtgatttga ggtgaaattg 1080

ttgatgaaga tggtattttc ttaatcccat tccttcatta tatatagtgt atctttcaat 1140

tgaggtgaag ttgtggatca tgaagtagtt gttgatgatg atggttgact ctacgaaata 1200

cttttgcgtt caaagttaag ttgtctctac taagatgttc tttgacattt gtttttactc 1260

ggaaactata tttgtttagg accatagccg atgggagacg ttggtacttg catacaagac 1320

cttaggagtt gtttttgggg gtcttgtcac atctccactc tatgtatatc catcaatgcc 1380

attaaagtct ccaactgagg atgattattt ggggatatac agcataatgt tttggactct 1440

cagtctaatt ggtgttgtca aatatgctac catagctctc caagctgatg atcagggtga 1500

aggtattgct gatcactctc tctttgtact tttgctactt acgacaacat tttcccattg 1560

aaaaatggga attgtttcat atggaaaaaa aaaggaaact tctcgctatt tcagtgagaa 1620

tctgtttccc accaaacatt ttcccagtgg agctggttca gtggggaatg agtggaaaaa 1680

tcatatgtta tagcacaaat ttcccactta tttgcaggtg gaaaaaacct ttatttctag 1740

tagtgaggtg actcgagttt taagaaactg tctgtttgta ttcatactaa aagatttatc 1800

ttttggagaa tgttgaaggt gttaatgaag gatgtttata tataatgttg agtcaattct 1860

taattttctt tatattttgg ttgaaattcc ttttgaaaga cacttcagag tttcttcatt 1920

tctcttttga tgtgatcatt atcgaaattt cagttctaga acatggtctt ttgtaggtgg 1980

aacctttgct ctttattcat tactttgtag aaatataaat attggtattc tttcttccaa 2040

gagtgccagt ttgaattcaa gccactccta tgttaaccaa tcgaaaaagc caagtaggtt 2100

gggtaaattt tgtgagagga gtttgattgc cagaagggta ctgcttttca tcgcaatgtt 2160

gggtatgtgc atgcttattg gcgatggcat acttacacct gccatctcag gtatgttagc 2220

ttgggttggg ccttttagtt tacttttagc aaccttttga gtgcttgtca gttttccctc 2280

tttcattttc agtttcatta attgtccctt ctgtctgtca gtgttatctg caatgggtgg 2340

cttacgagca cgattctcct cggtcagtaa atgtaagtat gacagcatac agtaaggtac 2400

atattgctca ttgctttctt agtcgagctc gttgcatggg gcttgcctag tgcggcttac 2460

cgctcctgtg tggtttgcag gctattaccc cgaagcaggt ttaccctatg cccaccctaa 2520

gggtagcagc tgtggtttcc cttgtcataa ataaaaaatt gtctttgaac ggagaggcct 2580

ttatgatttt gagggtaatt gggaagtggt cgaagaacaa ttttgattct tgtaatttct 2640

caggttttaa ggtattgtgc ttgttaaaag actatctttc acagattcat atattgatgt 2700

tacatgagtc cgattctgga agctaattga aataacttga atccttgttc tagtaattat 2760

ttgaagtaat taagtaatga ttgaacactt taaggtcaat aagtcatatt aacgaaaagg 2820

agcagtgcag tttctgaaag ttaaagtctt cttttatgac aggttgcaag tcctttttaa 2880

acactagatg acgcgaaaca tgttaattgt cccatttatg taaacacatc tctccctctc 2940

gatattcaca atttctctgc ttgccacttg cagcgctggt cgaaggtctc tcagcaatca 3000

ttctcattgt tctattcctc ctgcagaaat ttggtacttc acgagtgagc ttcctcttgt 3060

ctcctatcat gggttgcatg gactcttacc actcctctcg ttgggatata cagtatcata 3120

aagcattatc cgagcatatt taaggcgata tcaccccatt acattgccct cttcttctta 3180

agaaatggaa aacaaggatg gatatatctt ggtggtactg tcctatgcat tacaggttag 3240

cctcgttgtt ggtgtggtct gcaaaagttg ttcattgtga ttaagatcta aatccatttc 3300

ctcatgtttg gagttctttt acattctttc ctattacttt gcaattaata ttggggaaaa 3360

tttggaccaa ttatcatgtg tgcaatgtct accgatggcg ggaaaagctt aaacctggta 3420

tcttgtttca tatccagaga aactcgaaag gtgatgaatg gaggtggaaa atattataaa 3480

gaatatggaa tgcccctctg gtcaatactt ttatccaaaa atgataacaa ttgttctggt 3540

ttgagcgaaa aaacatggag gctgttctag attatttagc tcggggagtt ccaacgtcga 3600

ccatggtgta tttctcgcta agttgtcaga ttcctcaccc ttctattttt gtgtagtggg 3660

gggtttgagg attagagaaa aagacgggat gaaagaggat aaggggtgga cagtaccttt 3720

caccgaaaca tattaggccg ctagagttat tttctggcca agatctgttt cacttccacc 3780

caatatctgc ttaacagtac aatcctttca tatttttagg ttctgaagca atgtttgctg 3840

atcttggtca tttcaacagg agttcaattc gggtaaaaat acttgctatg tcttggaatt 3900

tctcttagtc tcttcttctt gcttgtttta tatgatttac gggatcttgt tttctggcag 3960

atagcatttc tctatactat atatccatca ttggtgctga catatgcggg acagacagca 4020

tatctgatta gaaatcccga tgaccacttt gatggatttt acaagtttat accctctgct 4080

gtgtactggc caatttttgt tatagccaca ttagctgcta tagtagcaag tcagtcgctg 4140

atttcagcca ccttttcggt tatcaagcaa tcagttgtgt tggattattt tcctcgggtg 4200

aaggtagttc acacatcctc cagcaaagaa ggcgaggttt actcaccaga agttaactac 4260

atcctcatga ttctctgtgt tgcagtcata ctagtcttcg gagatggaca agacatcgga 4320

aatgccttcg gtaagatgcc ctaaaccctt tgcattttat cttccatatt tagggactat 4380

cttaactgaa ttgatccctt tatgaaagtt atctttcact tgactcctct tcctaggatg 4440

gtggttgtta acatcgacta ctagcatttt ctttacctaa tttactacta cttggtgatg 4500

acttgcttac tttaaggggt aactagtgta atgaattgag ctaaacaaat tgtggattgg 4560

aattttctca ttctgtttgc tcgttgtttt cagattttgt ctcgtcttgt ttagcattat 4620

ccaataagat atttcactag tgataacgca catatatgtg gtgcaaatca agttcaacaa 4680

aattgtcttg tcttgtttag catagttaaa ataaaattac atatatgtat gatcttgtat 4740

gtatatatta aggtataact tgcatttttt cttgcaacgg ttcaactctt tcgtattgtc 4800

agcatagtaa atattgcatt aactaattaa aatggattct ctaacaggtg ttgttgttag 4860

catagttatg cttataacga ccatattgtt gacattggtt atgatcatta tttggagaac 4920

tccaccagct ctggtcgggc tctacttcgt tgtatttttt gtgatggaaa gtgtatatgt 4980

tagtgcaatc ttcacaaaaa ttcccgaagg tggttggatt ccttttgcca tttctctcat 5040

tcttgctttc attatgtttg gttggttcta cggtaggcag agaaaacttg agtatgaatt 5100

gacacacaag atagactcag agagactaag gacactgcta atcgatccag gtcttcagag 5160

agttcctgga ctctgcttct tctacacaaa tatccaagat ggtttaaccc caattttagg 5220

tcattacata aagaacatga gatctcttca caaagttact gtatttacaa ctcttcgtta 5280

cttgctggtt cctaaagtag cacctggtga aagaatcgtt gttagtaaac taggtctcag 5340

aggggtttat agatgtgtga ttcgttatgg ttatgcagat aagcttagtc ttgaaggaga 5400

tgatttagtt aatcaagtca tccaaagttt acgatctcat gtgctccact gctccaattc 5460

attggaggtt gacactgaag tttccgagtt ggatgaggca aagcttgctg gagtagtaca 5520

tattcgtgga aagacgaggt tttatattgg taaggactgt ggatggtttg atagaacaat 5580

gcttgctttc tatgaagttt tgcatagcaa ttgcagatct gcattgcctg ctatgggtgt 5640

accattacct caacgtatcg aggttggaat gctctacgcg gcgtaa 5686

<210> 14

<211> 262

<212> PRT

<213> Artificial sequence (Artificial sequence)

<400> 14

Met Asp Arg Gln Thr Gly Arg Pro Asp Leu Thr Ala Ala Ala Ala Ala

1 5 10 15

Gln Ser Asp Ala Ala Val Thr Val Ala Val His Asp Gly Glu Thr Ile

20 25 30

Asn Asn Asp Gln Lys Asp His Ser Arg Trp Glu Thr Leu Val Leu Ala

35 40 45

Tyr Lys Thr Leu Gly Val Val Phe Gly Gly Leu Val Thr Ser Pro Leu

50 55 60

Tyr Val Tyr Pro Ser Met Pro Leu Lys Ser Pro Thr Glu Asp Asp Tyr

65 70 75 80

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

85 90 95

Val Lys Tyr Ala Thr Ile Ala Leu Gln Ala Asp Asp Gln Gly Glu Gly

100 105 110

Gly Thr Phe Ala Leu Tyr Ser Leu Leu Cys Arg Asn Ile Asn Ile Gly

115 120 125

Ile Leu Ser Ser Lys Ser Ala Ser Leu Asn Ser Ser His Ser Tyr Val

130 135 140

Asn Gln Ser Lys Lys Pro Ser Arg Leu Gly Lys Phe Cys Glu Arg Ser

145 150 155 160

Leu Ile Ala Arg Arg Val Leu Leu Phe Ile Ala Met Leu Gly Met Cys

165 170 175

Met Leu Ile Gly Asp Gly Ile Leu Thr Pro Ala Ile Ser Val Leu Ser

180 185 190

Ala Met Gly Gly Leu Arg Ala Arg Phe Ser Ser Val Ser Lys Ser Leu

195 200 205

Val Glu Gly Leu Ser Ala Ile Ile Leu Ile Val Leu Phe Leu Leu Gln

210 215 220

Lys Phe Gly Thr Ser Arg Val Ser Phe Leu Leu Ser Pro Ile Met Gly

225 230 235 240

Cys Met Asp Ser Tyr His Ser Ser His Trp Asp Ile Gln Tyr His Lys

245 250 255

Ala Leu Ser Glu His Ile

260

Claims

Use of a HAK20 protein or HAK20 protein promoter for improving a trait in a plant under salt stress or for preparing a formulation or composition for modulating a trait in a plant under salt stress, wherein the trait in the plant is salt tolerance;

the amino acid sequence of the HAK20 protein is shown as SEQ ID NO. 2;

the HAK20 protein is derived from tomato;

the plant is tomato.
2. A method for improving a plant trait under salt stress comprising the steps of: up-regulating the expression level and/or activity of the HAK20 protein or a nucleic acid sequence encoding the same in the plant, thereby improving the trait of the plant; the trait of the plant is salt tolerance;

the amino acid sequence of the HAK20 protein is shown as SEQ ID NO. 2;

the HAK20 protein is derived from tomato;

the plant is tomato.
3. The mutein is characterized in that the amino acid sequence of the mutein is shown in SEQ ID NO 12 or SEQ ID NO 14.
4. An isolated polynucleotide encoding the mutein of claim 3.
5. A vector comprising the polynucleotide of claim 4.
6. A host cell comprising the vector or genome of claim 5 having the polynucleotide of claim 4 integrated therein; the host cell is of a non-plant variety.
7. A method of making a transgenic plant or gene-editing plant comprising the steps of:

regenerating a plant cell, plant tissue or organ comprising the mutein of claim 3 or a nucleic acid sequence encoding the mutein into a plant body, thereby obtaining a transgenic or gene-edited plant.
8. A method of making the mutein of claim 3 comprising the steps of:

culturing the host cell of claim 6 under conditions suitable for expression, thereby expressing the mutein; and isolating the mutein.
9. Use of the mutein according to claim 3 for improving a trait of a plant under salt stress or for preparing a formulation or composition that modulates a trait of a plant under salt stress, wherein the trait of the plant is salt tolerance; the plant is tomato, rice, or corn.
10. Use of the mutein according to claim 3 for improving a trait of a plant under salt stress or for preparing a formulation or composition that modulates a trait of a plant under salt stress, wherein the trait of the plant comprises one or more traits selected from the group consisting of: (ii) (i) root length, root branching and/or root weight; (ii) plant height; and (iii) salt tolerance; the plant is tomato.
11. A method for improving a plant trait under salt stress comprising the steps of: (ii) (i) providing a plant or plant cell; and (ii) introducing the mutein of claim 3 or a nucleic acid encoding therefor into said plant or plant cell, thereby improving the trait of the plant; the trait of the plant is salt tolerance; the plant is tomato, rice, or corn.
12. A method of improving a trait in a plant under salt stress comprising the steps of: (i) providing a plant or plant cell; and (ii) introducing the mutein of claim 3 or a nucleic acid encoding therefor into said plant or plant cell, thereby improving the trait of the plant; the trait of the plant comprises one or more traits selected from the group consisting of: (ii) (i) root length, root branching and/or root weight; (ii) plant height; and (iii) salt tolerance; the plant is tomato.