JPH11187878A - New potassium channel gene derived from plants belonging to genus nicotiana - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a new gene derived from a plant of genus Nicotiana having a function of giving a salt stress resistance to plants, having a specific DNA sequence and comprising a DNA encoding proteins having potassium channel activity. SOLUTION: This new potassium channel gene derived from genus Nicotiana has a DNA sequence described in the formula or a sequence in which one or some DNA are substituted, deleted, inserted or added and encoding a protein practically having potassium channel activity, has a function affording activity for water stress resistance to a plant, and capable of improving the salt stress resistance of the plants by controlling an expression of this gene. This DNA is obtained by extracting an entire RNA from a chloroplast system of Nicotiana paniculata, separating mRNA and preparing a cDNA library using an mRNA as a template, and screening this in a differential screening method.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to a DNA having an activity resistant to salt stress. More specifically, Nicotian, which has an activity to resist salt stress using genetic engineering technology
It relates to DNA from the genus a.

[0002]

2. Description of the Related Art Plants are constantly under stress even in a normal growing environment. For such stress,
Although various things such as salt, drying, high temperature, low temperature, strong light and air pollution are included, the most problematic from the viewpoint of agricultural production is salt damage due to salt damage and drought. Salt damage has become a problem not only in areas with high salinity, but also in agricultural lands where there was no problem with irrigation. At present, it is said that more than 10% of the total arable land has been damaged by some kind of salt. There is also a view that to increase food production to catch up with population growth, agricultural production on land that is currently considered as uncultivated soil due to salt damage, etc., will be required.

[0003] Therefore, finding a plant that is resistant to such a salt stress is very important in view of a food crisis that may occur in the future.

[0004] We screened plants of the genus Nicotiana for salt and drought stress tolerance (Plan
t Physiology (1995), 108, 106), Nicotiana excelsi
or and Nicotiana paniculata were selected as salt stress-tolerant species (Journal of Breeding Science, Vol. 46, Supplement No. 2, 188)
page). Its degree of tolerance is about half the salt concentration of seawater (25
Even if the solution is irrigated with a 0 mM NaCl) solution, it can grow. From studies on so-called salt-tolerant plants that have been conducted in Japan and overseas, when salt-tolerant plants are moved from non-stress conditions to salt stress conditions, the expression of new genes is induced, and the products of these genes are produced. It is known to be helpful in salt stress tolerance.

There is also a report that a salt stress tolerance gene has been isolated from a plant of the genus Nicotiana (Nelson et al. (1).
992) Plant Molecular Biology, vol. 19, 577-588, Yu
n etal. (1996) Plant Physiology, vol. 111, 1219-12
25) However, these reports did not relate to the presence of the potassium channel gene in Nicotiana plants, and there was no report that the potassium channel gene was isolated.

It has been known that a potassium channel gene exists in plants. A potassium channel is also called a potassium ion channel, and is a channel that exists in a biological membrane and through which potassium ions pass. The potassium channel genes include Arabidopsis thaliana and potato (Solanum).
tuberosum), maize (Zea maize), broad bean (Vicia fava), rye (Secale cereale), barley (Hordeum vulgare), plantain (Plantago major),
Japanese pine (Mesembryanthemum crystallinum) and the like are known, but the potassium channel gene is not known or isolated for plants of the genus Nicotiana.
On the other hand, it is not known that potassium channels are channels induced by salt stress, and there is no report that potassium channels derived from the above-mentioned various plants are related to salt stress. WO93 / 22422 discloses a potassium channel gene (KAT1) and a potassium transporter gene (TRK1, TRK) derived from Arabidopsis thaliana.
There are descriptions of yeasts deficient in K2), potassium channel genes using them, and methods for screening specific inhibitors thereof. However, there is no description in the above publication about salt stress tolerance, nor is there any description about transformed plants. Moreover, the gene of the present invention described below and KAT1 are DN
There is only about 56% homology at the A level and about 50% at the amino acid level.

[0007]

As described above, it is an urgent task to find a plant that is resistant to salt stress. For example, there is a method by controlling the salt content of a plant. Generally, it is considered that a plant having salt stress tolerance can be obtained by reducing the salt content of a plant, that is, by enhancing the ability to eliminate salt. Therefore,
An object of the present invention is to find a gene having resistance to salt stress. More specifically, an object of the present invention is to find a gene having resistance to salt stress by genetic manipulation or the like, and to improve the salt stress tolerance of a plant using the gene.

[0008]

Means for Solving the Problems The present inventors have proposed Nicoti.
NicotianaPa, a salt stress-tolerant species of ana plants
We found a novel potassium channel gene induced by salt stress from niculata and isolated the gene. From the above facts, it is considered that the potassium channel gene encoded by the gene obtained this time has a function of imparting water stress tolerance to plants. Thus, by controlling the expression of this gene, it is possible to improve the salt stress tolerance of plants.

The present invention has been made in view of the above circumstances, and proposes a method for introducing a plant-derived salt stress tolerance gene into a plant to regulate the salt content of the plant. The purpose was to obtain plants.

[0010] More specifically, the present invention relates to (1) a potassium channel gene derived from a plant of the genus Nicotiana. (2) SEQ ID NO: 1 or one or several DNAs in the sequence are substituted, deleted, inserted or added,
DNA encoding a protein having substantially potassium channel activity. (3) The DNA sequence described in SEQ ID NO: 1.

Here, the term "salt stress" means that the concentration of sodium chloride in the soil increases, which is disadvantageous for the growth of plants. "Salt stress tolerance" means resistance to the above-mentioned salt stress. "Substantially having potassium channel activity" means having substantially potassium channel activity against salt stress.

Hereinafter, the present invention will be described in more detail. The present invention is characterized by introducing a Nicotiana-derived potassium channel gene into a plant in order to control the water content of the plant. First, a plant was exposed to a salt stress environment, a gene (mRNA) newly produced under the environment was extracted, and the sequence was analyzed to derive the gene of the present invention. The potassium channel gene derived from the genus Nicotiana used in the present invention may be introduced into a plant in a sense direction or may be introduced in an antisense direction. In the present invention, for the purpose of enhancing the ability of plants to eliminate salt under salt stress conditions, it is preferable to introduce them in the antisense direction. In such a case, it is preferable to introduce in the antisense direction.

On the other hand, when the gene is introduced in the antisense direction, the closer the species from which a transformed plant is to be produced and the species from which the gene to be introduced is derived, the more preferable it is, and particularly preferably the same species. In addition, when introducing in the antisense direction, it is not always necessary to introduce the whole, and even when a part is used, a sufficient effect may be exhibited. Therefore, when a gene is introduced in the antisense direction in the present invention, at least a part of the gene needs to be introduced. Examples of the expression promoter used in the present invention include those which have strong transcriptional power and are expressed in all cells (such as 35S, 19S, nos), those which respond to light (such as rbc), those which respond to temperature (such as hsp), There is no particular limitation as long as it is conventionally known, such as those that respond to hormones and those that respond tissue-specifically. Particularly preferred are strong ones such as the 35S promoter.

In the present invention, a method for introducing a potassium channel gene in the sense or antisense direction into a plant to be transformed does not need to be a special method, and may be a method usually used for transforming a plant. If so, any method can be used. For example,
Transformation method using a gene gun, electroporation, leaf disk method using Agrobacterium sp., And the like, but preferably the leaf disk method using Agrobacterium sp. This will be described below.

[0015] A potassium channel gene in the sense or antisense direction is inserted into an appropriate plant expression vector, and this vector is introduced into Agrobacterium. Next, after immersing the leaf disk collected from the sterile leaves of the plant to be transformed in the culture solution of the above-described Agrobacterium, callus was formed, and only the transformed plants were selected. A transformed plant can be obtained.

When transforming into Agrobacterium, another host may be transformed if necessary, and then the desired vector may be introduced into Agrobacterium. Examples of the other host include bacteria (Escherichia, Bacillus), yeast (Saccharomyces, Pichia, etc.), animal cells, insect cells, etc., and preferably Escherichia coli (DH5, HB101, etc.). However, the present invention is not limited to these.

The detection of a host into which a potassium channel gene has been introduced is achieved by introducing a vector into which a reporter gene has been introduced into a host cell (Agrobacterium). The reporter gene is stably maintained by introducing the reporter gene into a vector such as a plasmid vector, a phage vector, and a retrovirus vector, and transforming the cell, or transfecting (transfecting) the host cell after in vitro packaging. A transformed cell is prepared.

The plasmid vector used here is not particularly limited as long as it can be replicated and maintained in the host cell, and any phage vector may be used as long as it can be propagated in the host cell. PUC119, λgt10, λgt11, pBlues
Examples include cript, λZAP II, λZAP XR, and the like.

A method for incorporating cDNA into a plasmid is described in, for example, "Maniatis, T. et al., Molecular Cloning, A Laboratory Manual (Molecular Clonin).
g, A Laboratory Manual, second edition), Cold Spri
ng Harbor Laboratory, 1.53 (1989) ". As a method of incorporating cDNA into a phage vector, Hyunh, TV et al. (Hyunh, TV, DNA
Cloning, a practical approach, 1, 49 (1985)). For convenience, a commercially available ligation kit (for example, Takara Shuzo) can be used. The thus obtained recombinant plasmid or phage vector is
Prokaryotic cells (eg, E. coli HB101, DH5 or MC1061 / P3
Etc.) and / or eukaryotic cells (J774.1, PU5-1.8, RAW264.
7. Introduce into ST2) various suitable host cells.

A method for introducing a plasmid into a host cell is described in "Maniatis, T. et al., Molecular Cloning, A Laboratory Manual (Molecular Cloning, A La
boratory Manual, second edition), Cold Spring Harb
or Laboratory, 1.74 (1989) ", calcium chloride method or calcium chloride / rubidium chloride method, electroporation method, electroinjection method, PE
Examples thereof include a method using a chemical treatment such as G, a method using a gene gun, and the like. Examples of a method for introducing a phage vector into a host cell include a method in which phage DNA is packaged in vitro and then introduced into the grown host cell. In vitro packaging is
It can be easily performed by using a commercially available in vitro packaging kit (for example, manufactured by Stratagene, Amersham, etc.).

The vector can be prepared simply by ligating a desired gene to a vector for recombination (plasmid DNA and bacterial phage DNA) available in the art in a conventional manner. As the vector to be used, specifically, as a plasmid derived from E. coli, for example, pBR322, pBR325, pUC12, pUC13, and the like,
As yeast-derived plasmids, for example, pSH19, pSH15 and the like, as Bacillus subtilis-derived plasmids, for example, pUB110, pT
P5, pC194 and the like are exemplified, but not limited thereto.
As phage, bacteriophages such as λ phage, and animal and insect viruses such as retrovirus, vaccinia virus, and nucleopolyhedrovirus [pVL
1393 (manufactured by Invitrogen)] and the like, but not limited thereto.

For the purpose of producing a desired protein, an expression vector is particularly useful. The expression vector is not particularly limited as long as it has a function of expressing a desired gene and producing a desired protein in various host cells of prokaryotic cells and / or eukaryotic cells. pQE vector, pGEX-5X-1), or SV-4
A 0-derived expression vector is preferred.

When a bacterium, particularly Escherichia coli, is used as a host cell before transformation into Agrobacterium, the expression vector generally comprises at least a promoter-operator region, an initiation codon, a desired gene, a stop codon and a replicable unit. Is done. When Agrobacterium is used as a host cell, it is generally preferable that the expression vector contains at least a promoter, an initiation codon, a desired gene, and a termination codon. It may also contain a DNA encoding a signal peptide, an enhancer sequence, 5 ′ and 3 ′ untranslated regions of the desired gene, a splicing junction, a polyadenylation site, a selectable marker region or a replicable unit as appropriate. .
Further, it may contain a gene amplification gene (marker) which is generally used depending on the purpose.

In the vector used for the gene of the present invention, a suitable initiation codon is methionine codon (AT
G) is exemplified. Examples of the stop codon include common stop codons (eg, TAG, TGA, etc.).

[0025] A replicable unit refers to its entire DN in a host cell.
A DNA capable of replicating the A sequence, which includes a natural plasmid, an artificially modified plasmid (a DNA fragment prepared from the natural plasmid), and a synthetic plasmid. Suitable plasmids include E. coli. In E. coli, plasmid pBR322 or its artificial modification (obtained by treating pBR322 with appropriate restriction enzymes)
DNA yeast), yeast 2μ plasmid or yeast chromosomal DNA in yeast, and plasmid pRSVneo ATCC 37198 and plasmid pSV2dhfr ATCC 37 in mammalian cells.
145, plasmid pdBPV-MMTneo ATCC 37224, plasmid
pSV2neo ATCC 37149, plasmid pME18S and the like.

As the enhancer sequence, polyadenylation site and splicing junction site, those commonly used by those skilled in the art, for example, those derived from SV40 can be used. Gene amplification genes include dihydrofolate reductase (DHFR) gene, thymidine kinase gene, neomycin resistance gene, glutamate synthase gene, adenosine deaminase gene, ornithine decarboxylase gene, hygromycin-B-phosphotransferase gene,
Aspartate transcarbamylase gene and the like can be exemplified.

An expression vector can be prepared by linking at least the above-mentioned promoter, start codon, desired gene, stop codon, and terminator region to an appropriate replicable unit in a continuous and circular manner. At this time, digestion with restriction enzymes and T4 DNA
Suitable methods such as ligation using ligase
DNA fragments (eg, linkers, other restriction sites, etc.) can be used.

The selection of transformed plants can be carried out by adding an appropriate antibiotic to a medium for forming callus and determining whether or not the medium has resistance. In this case, a commonly used selection marker can be used in a conventional manner. For example, an antibiotic resistance gene such as tetracycline, ampicillin, or kanamycin or neomycin is exemplified. At this time, it is preferable to introduce into the vector, in addition to the potassium channel gene, an antibiotic resistance gene added to the medium.

The test for confirming the production of the desired protein from the host cell prepared as described above may be performed as follows.
That is, a reporter gene may be introduced into the host cell prepared as described above at a desired gene site of the vector, a protein serving as a reporter may be produced from the host cell, and the protein may be detected.

Here, reporter genes used for the desired gene site include chloramphenicol acetyltransferase (CAT) and β-glucuronidase (GU
S), luciferase gene, β-galactosidase, green fluorescein protein (GFP), aequorin, β-lactamase and the like.

The transformation method using Agrobacterium can be applied not only to dicotyledonous plants but also to monocotyledonous plants (WO94 / 00977).

The plant transformed by the present invention, that is, the plant in which the salt content of the plant is controlled by the present invention is not particularly limited, but includes soybean, corn, potato, tomato, rice and tobacco. Is mentioned. Various methods can be considered for measuring the salt content of the transformed plant in the present invention. As one method, for example, a plant grown to a predetermined size is grown for a certain period of time under a stressed environment using an artificial weather machine or the like. Then, there is a method in which the aerial part of the plant is harvested and the salt content in the living body is measured by HPLC.

[0033]

EXAMPLES [Example 1] <Growth of plant material> Nicotiana paniculata was seeded on a culture soil in a greenhouse and allowed to germinate. When about four true leaves had been grown, they were transplanted to a black vinyl pot of about 10 cm in diameter filled with a mixture of vermiculite and hydroball (1: 1 by volume). After that, it was transferred into an artificial weather machine (12 hours day length, 23 ° C., relative humidity 70%) and irrigated with 100 mL of 1/4 diluted Hoagland solution per day. Salt stress treated plants contain 1/4 containing 250 mM sodium chloride.
The diluted Hoagland solution was irrigated with 100 mL per day.

Example 2 <Extraction of mRNA> Extraction of total RNA from green leaf tissue of Nicotiana paniculata was performed by Ostre.
m et al.'s method (Ostrem etal., Plant Physiology 84, 1270-
1275 (1987)), but the following points were improved upon implementation. 1) The ground plant was shaken on ice for 1 hour with extraction buffer and phenol. 2) The supernatant after centrifugation was shaken together with chloroform on ice. For purification of poly (A) +-RNA, use QuickPrep mRNA puri
This was performed using a fication Kit (Pharmacia) according to the manufacturer's instructions.

Example 3 <Isolation of Nicotiana paniculata Potassium Channel cDNA> A stressed Nicotiana paniculata green leaf cDNA library was prepared by extracting and purifying poly (A) from the stress-treated plant in Example 2. + RNA as template, ZAP-cDNA
Synthesis Kit (Stratagene) and Uni-ZAP XR (Stratagene) were used as cloning vectors. XLI-Blue was used as a host cell. The screening of the cDNA library was performed by a differential screening method. The detailed procedure is described below.

Lytic plaques for screening were performed according to Stratagene's instructions. For the plaque lift from the agar medium where plaques appeared, a nylon membrane Hybond N + (manufactured by Amersham) was used. Plaque lift was performed twice from one agar medium. The denaturation of the nylon membrane for screening thus prepared was performed by Amersham.
I went according to the company's guide. Used for screening
The 32P-labeled probe was prepared as follows. 50-100 ng of poly (A) + RNA obtained in Example 2 was dissolved in 30 μl of sterilized water, 1 μl of primer (Takara Shuzo) was added, and the mixture was placed at 65 ° C. for 10 minutes. After cooling to room temperature, add 1 μl of RNase inhibitor and 5X buffer (Takara Shuzo) attached to MMLV reverse transcriptase.
5 μl, 5 μl of dATP / dGTP / dTTP solution (10 mM each), 32P-d
1 μl (10 μCi) of CTP (Amersham) and 6 μl of distilled water
After mixing well, MMLV reverse transcriptase (Takara Shuzo)
Was added in an amount of 1 μl. After reacting this reaction solution at 37 ° C. for 1 hour, free 32 P-dCTP was removed using a spin column.
Probes were prepared for poly (A) + RNA prepared from a stress-treated plant and a non-stressed control plant, respectively. Hybridization was performed using one set of the nylon membranes prepared using the control probe and another set using the stress probe.
Hybridization was performed for 16 hours under standard conditions as described in the manufacturer's instructions. Wash with 300 mM
NaCl, 30 mM trisodium citrate, 0.1% SDS
C. Twice for 20 minutes at 75 ° C., followed by 75 mM NaCl, 7.5 mM trisod
This was performed twice using ium citrate, 0.1% SDS at 65 ° C. for 20 minutes. Compare the results of the control and stress treatment plots,
Positive clones showing a strong signal were obtained in the stress-treated section. The inserted cDNA fragment contained in the obtained clone was Stra
Subcloning into the pBluescript plasmid was performed according to tagene's instructions.

The nucleotide sequence of the obtained positive clone was determined as follows:
Using a DNA sequencer Model 373A (manufactured by Applied Biosystems), the reaction was performed using Taq Dye Terminator Cyc
This was performed using a le Sequencing Kit (manufactured by Applied Biosystems) according to the manufacturer's instructions. Analysis of the obtained base sequence was performed by GENETYX-MAC ver.8 (software development). As a result, Sequence Listing 1 cDNA, NPKT
Got one.

[0038]

From the above facts, it is considered that the potassium channel encoded by the gene obtained this time has a function of imparting water stress tolerance to plants. Thus, by controlling the expression of this gene, it is possible to improve the salt stress tolerance of plants.

[0039]

[Sequence list]

SEQ ID NO: 1 Sequence length: 2700 Sequence type: number of nucleic acid chains: double-stranded Topology: linear Sequence type: other nucleic acids (DN including 5 ′ and 3 ′ regions
A) Origin Organism: Nicotiana paniculata Nature: Potassium channel cDNA (NPKT1) Sequence characteristics 3-1703 CDS AAC ATG ATA ATT GAA GAT AGC CAA ATT AAA GAC CAA CAT GTG CAA GAT 4 8 Met Ile Ile Glu Asp Ser Gln Ile Lys Asp Gln His Val Gln Asp 1 5 10 15 AAT AGC CAT GGG AGT AGC AAT AAT TCG GGT ACT AAT TCA GAA GAA CTC 96 Asn Ser His Gly Ser Ser Asn Asn Ser Gly Thr Asn Ser Glu Glu Leu 20 25 30 AGT TTT CGT AAC CTG TCA AAG CTC ATT CTA CCT CCT CTT GGT TCC AAT 14 4 Ser Phe Arg Asn Leu Ser Lys Leu Ile Leu Pro Pro Leu Gly Ser Asn 35 40 45 GGT TAC AAC CAG AAT CAA ACT CAG CAG AAA GGC AAG ATC ATC ACC CCT 19 2 Gly Tyr Asn Gln Asn Gln Thr Gln Gln Lys Gly Lys Ile Ile Thr Pro 50 55 60 ATG GAT TCA AGA TAC AGG TGT TGG GAG ACG CTA ATG GTA GTA ATG GTG 24 0 Met Asp Ser Arg Tyr Arg Cys Trp Glu Thr Leu Met Val Val Met Val 65 70 75 GCG TAT TCT GTA TGG GTA TGT CCA TTT GAG ATA GCA TTC ATG CAC TCT 28 8 Ala Tyr Ser Val Trp Val Cys Pro Phe Glu Ile Ala Phe Met His Ser 80 85 90 95 AAC CCA AAC AGA GCG C TC TAC TTG GCA GAC AAC GTT GTT GAT CTC TTC 33 6 Asn Pro Asn Arg Ala Leu Tyr Leu Ala Asp Asn Val Val Asp Leu Phe 100 105 110 TTT GCT GTT GAT ATC ATC TTG ACA TTC TTT GTT GCC TAT ATT GAT ACC 38 4 Phe Ala Val Asp Ile Ile Leu Thr Phe Phe Val Ala Tyr Ile Asp Thr 115 120 125 ACG ACT CAG CTT CTC GTA CGT GAC AGG AGA AGA ATT GCC ACA AGA TAT 43 2 Thr Thr Gln Leu Leu Val Arg Asp Arg Arg Arg Ile Ala Thr Arg Tyr 130 135 140 ATA TCT ACA TGG TTT ATG ATG GAT GTT GCC TCA ACC ATA CCA TTC GAC 48 0 Ile Ser Thr Trp Phe Met Met Asp Val Ala Ser Thr Ile Pro Phe Asp 145 150 155 CTT CTT GCC TTG ATT TTC ACT GGT AAA CAC CAA ATT GGT GTT TCT TAC 52 8 Leu Leu Ala Leu Ile Phe Thr Gly Lys His Gln Ile Gly Val Ser Tyr 160 165 170 175 TCA GTT CTA GGA ATG CTC AGA TTT TGG CGT CTT CGC AGG GTT AAA CAG 57 6 Ser Val Leu Gly Met Leu Arg Phe Trp Arg Leu Arg Arg Val Lys Gln 180 185 190 TTT TTT ACC AGA CTT GAA AAG GAC ATG AGA TTC AGC TAT TTT TGG GTC 62 4 Phe Phe Thr Arg Leu Glu Lys Asp Met Arg Phe Ser Tyr Phe Trp Val 195 200 205 AG A TGT GCC AGG CTT CTA TTT GTA ACA CTA TTG ACA GTG CAC TGT GCT 67 2 Arg Cys Ala Arg Leu Leu Phe Val Thr Leu Leu Thr Val His Cys Ala 210 215 220 GGA TGC CTC TAC TAT TTG CTA GCT GAT AGA TAT CCA CAC CAA GGA GAT 72 0 Gly Cys Leu Tyr Tyr Leu Leu Ala Asp Arg Tyr Pro His Gln Gly Asp 225 230 235 ACT TGG TTA GGA GCT ATG AAT CCA AAT TAC AAG GAG ACA AGT CTT CTT 76 8 Thr Trp Leu Gly Ala Met Asn Pro Asn Tyr Lys Glu Thr Ser Leu Leu 240 245 250 255 ATT AGG TAC ATT GCA GCT TTG TAT TGG TCC ATT ACT ACC ATG ACA ACA 81 6 Ile Arg Tyr Ile Ala Ala Leu Tyr Trp Ser Ile Thr Thr Met Thr Thr 260 265 270 GTT GGC TAT GGT GAT CTC CAT GCT GTC AAC ACT TTG GAA ATG GTC TTC 86 4 Val Gly Tyr Gly Asp Leu His Ala Val Asn Thr Leu Glu Met Val Phe 275 280 285 ATC ATT TTC TAC ATG CTC TTT AAT CTT GGC CTC ACT GCT TAT ATT ATT 91 2 Ile Ile Phe Tyr Met Leu Phe Asn Leu Gly Leu Thr Ala Tyr Ile Ile 290 295 300 GGT AAT ATG ACC AAT TTA GTC GTT GAA GGA ACT CGT CGT ACC ATG GAA 96 0 Gly Asn Met Thr Asn Leu Val Val Glu Gly Thr Arg Arg Thr Met Glu 305 310 315 TTC AGA AAT AGC ATC GAA GCA GCA TCA AAT TTC GTG TGC CGA AAT AGG 100 8 Phe Arg Asn Ser Ile Glu Ala Ala Ser Asn Phe Val Cys Arg Asn Arg 320 325 330 335 TTG CCT CCA AGA TTG AAA GAG CAA ATA TTG GCC TAT ATG TGT TTA AGA 105 6 Leu Pro Pro Arg Leu Lys Glu Gln Ile Leu Ala Tyr Met Cys Leu Arg 340 345 350 TTC AGG GCA GAG AGC CTA AAC CAG CAG CAA TTG ATT GAA CAA CTT CCC 110 4 Phe Arg Ala Glu Ser Leu Asn Gln Gln Gln Leu Ile Glu Gln Leu Pro 355 360 365 AAG ACA ATC TGC AAA AGC ATT AGG CAC CAT TTG TTT TTA CCA ACA GTG 115 2 Lys Thr Ile Cys Lys Ser Ile Arg His His Leu Phe Leu Pro Thr Val 370 375 380 GAG AAG GTT TAT CTT TTC AAG GGT GTC TCA AGG GAA ATT CTG TTA CTT 120 0 Glu Lys Val Tyr Leu Phe Lys Gly Val Ser Arg Glu Ile Leu Leu Leu 385 390 395 TTA GTT GCA GAT ATG AAG GCT GAG TAC ATA CCC CCA AGA GAG GAT GTA 124 8 Leu Val Ala Asp Met Lys Ala Glu Tyr Ile Pro Pro Arg Glu Asp Val 400 405 410 415 ATA ATG CAG AAC GAA TCG CCA GAT GAG GTA TAC ATC ATA GTG TCA GGG 129 6 Ile Met Gln Asn Glu Ser Pro A sp Glu Val Tyr Ile Ile Val Ser Gly 420 425 430 GAG GTG GAA ATG ATT GAG TGT GAG ATG GAA AAT GAG CAG GTT TGT TGG 134 4 Glu Val Glu Met Ile Glu Cys Glu Met Glu Asn Glu Gln Val Cys Trp 435 440 445 ACA TTC AAA TCT GGA GAT ATG TTA GGA GAA GTT GGG GCA TTT TGT TGT 139 2 Thr Phe Lys Ser Gly Asp Met Leu Gly Glu Val Gly Ala Phe Cys Cys 450 455 460 AGG CCT CAG AGC TAC ACG TAT CGA ACC AAG ACA CTT TCA CAA CTC TTG 144 0 Arg Pro Gln Ser Tyr Thr Tyr Arg Thr Lys Thr Leu Ser Gln Leu Leu 465 470 475 AAG ATA AGA ACA ACC TCT TTG ATT GAA GCA ATG AAA ACT AGA CAA GAA 148 8 Lys Ile Arg Thr Thr Ser Leu Ile Glu Ala Met Lys Thr Arg Gln Glu 480 485 490 495 GAT AAT CTC ATA ATG ATC AAG AAC TTC CTT CAG CAT CAC AAG AAG CTC 153 6 Asp Asn Leu Ile Met Ile Lys Asn Phe Leu Gln His His Lys Lys Leu 500 505 510 AGG GAT TTA AAG CTT GGA GAT TTA TTT CAT GAA GTT CGG GCA GAA AAT 158 4 Arg Asp Leu Lys Leu Gly Asp Leu Phe His Glu Val Arg Ala Glu Asn 515 520 525 GGT GAT CCA AAC ATG TCT GTT AAT TTG CTA ACT GTT GCT AGTCA GGT 163 2 Gly Asp Pro Asn Met Ser Val Asn Leu Leu Thr Val Ala Ser Thr Gly 530 535 540 AAT GCT GCT TTT CTT GAG GAA CTT CTC AAG GCA AGA TTA GAT CCT GAT 168 0 Asn Ala Ala Phe Leu Glu Glu Leu Leu Lys Ala Arg Leu Asp Pro Asp 545 550 555 ATT GGA GAT GCC CAA GGA AGA ACT CCA CTG CAC ATA GCA GCA TCG AAA 172 8 Ile Gly Asp Ala Gln Gly Arg Thr Pro Leu His Ile Ala Ala Ser Lys 560 560 565 570 570 575 GGG CAC GAA GAG TGT GTA ATG GTT CTA CTT AGA CAT GGA TGT AAC ATA 177 6 Gly His Glu Glu Cys Val Met Val Leu Leu Arg His Gly Cys Asn Ile 580 585 590 CAC CTC CGA GAT GTA AAT GGT AAC ACC GCG TTG TGG GAA GCT ATA GCA 182 4 His Leu Arg Asp Val Asn Gly Asn Thr Ala Leu Trp Glu Ala Ile Ala 595 600 605 GCA AAA CAG CAT CCA ACA TTT CAG ATA TTA TAC CAT TGG GCT TCT GTC 187 2 Ala Lys Gln His Pro Thr Phe Gln Ile Leu Tyr His Trp Ala Ser Val 610 615 620 TCC GAT CCC TAT GTT GCT GGT GAA CTC TTA TGC ACA GCA GCT AAG AGA 192 0 Ser Asp Pro Tyr Val Ala Gly Glu Leu Leu Cys Thr Ala Ala Lys Arg 625 630 635 635 AAT GAA TTA ACA GTG ATG AAA GAA CTC CTA AAA CAT GGA TTA ATA GTT 196 8 Asn Glu Leu Thr Val Met Lys Glu Leu Leu Lys His Gly Leu Ile Val 640 645 650 655 GAC TCA ATA GAT CGC CAC GGA TCA ACT GCT ATC CAT GTT GCA TTG GAA 201 6 Asp Ser Ile Asp Arg His Gly Ser Thr Ala Ile His Val Ala Leu Glu 660 665 670 GAA AAT CAT GAA GAC ATG GTA AAG CTA CTT CTA ATG AAT GGA GCC GAG 206 4 Glu Asn His Glu Asp Met Val Lys Leu Leu Leu Met Asn Gly Ala Glu 675 680 685 ATT AAT GAT AAA TTT AAG CAC AAG CTT TCT TCG ATG AAC CTA AGC GAG 211 2 Ile Asn Asp Lys Phe Lys His Lys Leu Ser Ser Met Asn Leu Ser Glu 690 695 700 ATG CTG CAA AAA CGA GAA GTA GGG CAC AGG GTT ATA GTC CCC GAC ACA 216 0 Met Leu Gln Lys Arg Glu Val Gly His Arg Val Ile Val Pro Asp Thr 705 710 715 ATG GAT GAA GTT GCT CAA AAG TGG CGC GAA CAA GAG CAG AAG TAC AAC 220 8 Met Asp Glu Val Ala Gln Lys Trp Arg Glu Gln Glu Gln Lys Tyr Asn 720 725 730 735 TCG GGA AGT ACT AGG GAT CAA TCG TCT TTT AGA GTT AGC ATA TAC AAA 225 6 Ser Gly Ser Thr Arg Asp Gln Ser Ser Phe Arg Val Ser Ile Tyr Lys 740 745 750 GGC CAT CCT GTG A TC AGA AAA AGA ACT CAT TGC AGT GAA CCT GGG AAA 230 4 Gly His Pro Val Ile Arg Lys Arg Thr His Cys Ser Glu Pro Gly Lys 755 760 765 TTA ATT ATT CTT CCA AAT TCA CTT GCA GAG CTC AAG ATC ATT GCA GGT 235 2 Leu Ile Ile Leu Pro Asn Ser Leu Ala Glu Leu Lys Ile Ile Ala Gly 770 775 780 CAG AAA TTT GGA TTT GAT GCA ACA AAT GCA TTG GTG ACA GAT CAA GAA 240 0 Gln Lys Phe Gly Phe Asp Ala Thr Asn Ala Leu Val Thr Asp Gln Glu 785 790 795 GGA TCA GAA ATT GAC TCT ATT GAA GTG ATA AGA GAT AAT GAT AAG CTA 244 8 Gly Ser Glu Ile Asp Ser Ile Glu Val Ile Arg Asp Asn Asp Lys Leu 800 805 810 815 815 TTT ATA GTT GAA GAT CCA AAA TGC TTG TAG CAATACCTAT TGTGACACTT 249 8 Phe Ile Val Glu Asp Pro Lys Cys Leu * 820 825 AAAAGTGTCT TGTTCACATG CAAAACTTGG ATCTCAGTGA TAATCAACTC CTTCCCCATT 255 8 TGTGTGGCAT GTTGAGCCAC TAAGTTATTA CAGTGGACAA TCTACTGATG GGATCAAAAA 261 8 GTTGATATTA TGTGTGTTGG CCATGAAATG ATATACTTTG GTTGGTGACA ATCTGAAAAA 267 8 AAAAAAAAAA AAAAAAAAAA AAA 270 1

SEQ ID NO: 2 Sequence length: 825 Sequence type: amino acid Topology: linear Sequence type: protein Origin Organism: Nicotiana paniculata Nature: potassium channel amino acid Met Ile Ile Glu Asp Ser Gln Ile Lys
Asp Gln His Val Gln Asp Asn 15
10 15 Ser His Gly Ser Ser Asn Asn Ser Gly
Thr Asn Ser Glu Glu Leu Ser 20 25
30 Phe Arg Asn Leu Ser Lys Leu Ile Leu
Pro Pro Leu Gly Ser Asn Gly 35 40
45 Tyr Asn Gln Asn Gln Thr Gln Gln Lys
Gly Lys Ile Ile Thr Pro Met 50 55
60 Asp Ser Arg Tyr Arg Cys Trp Glu Thr
Leu Met Val Val Met Val Ala 65 70
7580 Tyr Ser Val Trp Val Cys Pro Phe Glu
Ile Ala Phe Met His Ser Asn 85
90 95 Pro Asn Arg Ala Leu Tyr Leu Ala Asp
Asn Val Val Asp Leu Phe Phe 100 105
110 Ala Val Asp Ile Ile Leu Thr Phe Phe
Val Ala Tyr Ile Asp Thr Thr Thr 120
125 Thr Gln Leu Leu Val Arg Asp Arg Arg
Arg Ile Ala Thr Arg Tyr Ile 130 135
140 Ser Thr Trp Phe Met Met Asp Val Ala
Ser Thr Ile Pro Phe Asp Leu 145 150
155 160 Leu Ala Leu Ile Phe Thr Gly Lys His
Gln Ile Gly Val Ser Tyr Ser 165
170 175 Val Leu Gly Met Leu Arg Phe Trp Arg
Leu Arg Arg Val Lys Gln Phe 180 185
190 Phe Thr Arg Leu Glu Lys Asp Met Arg
Phe Ser Tyr Phe Trp Val Arg 195 200
205 Cys Ala Arg Leu Leu Phe Val Thr Leu
Leu Thr Val His Cys Ala Gly 210 215
220 Cys Leu Tyr Tyr Leu Leu Ala Asp Arg
Tyr Pro His Gln Gly Asp Thr 225 230
235 240 Trp Leu Gly Ala Met Asn Pro Asn Tyr
Lys Glu Thr Ser Leu Leu Ile 245
250 255 Arg Tyr Ile Ala Ala Leu Tyr Trp Ser
Ile Thr Thr Met Thr Thr Val 260 260
270 Gly Tyr Gly Asp Leu His Ala Val Asn
Thr Leu Glu Met Val Phe Ile 275 280
285 Ile Phe Tyr Met Leu Phe Asn Leu Gly
Leu Thr Ala Tyr Ile Ile Gly 290 295
300 Asn Met Thr Asn Leu Val Val Glu Gly
Thr Arg Arg Thr Met Glu Phe 305 310
315 320 Arg Asn Ser Ile Glu Ala Ala Ser Asn
Phe Val Cys Arg Asn Arg Leu 325
330 335 Pro Pro Arg Leu Lys Glu Gln Ile Leu
Ala Tyr Met Cys Leu Arg Phe 340 345
350 Arg Ala Glu Ser Leu Asn Gln Gln Gln
Leu Ile Glu Gln Leu Pro Lys 355 360
365 Thr Ile Cys Lys Ser Ile Arg His His
Leu Phe Leu Pro Thr Val Glu 370 375
380 Lys Val Tyr Leu Phe Lys Gly Val Ser
Arg Glu Ile Leu Leu Leu Leu 385 390
395 400 Val Ala Asp Met Lys Ala Glu Tyr Ile
Pro Pro Arg Glu Asp Val Ile 405
410 415 Met Gln Asn Glu Ser Pro Asp Glu Val
Tyr Ile Ile Val Ser Gly Glu 420 425
430 Val Glu Met Ile Glu Cys Glu Met Glu
Asn Glu Gln Val Cys Trp Thr 435 440
445 Phe Lys Ser Gly Asp Met Leu Gly Glu
Val Gly Ala Phe Cys Cys Arg 450 455
460 Pro Gln Ser Tyr Thr Tyr Arg Thr Lys
Thr Leu Ser Gln Leu Leu Lys 465 470
475 480 Ile Arg Thr Thr Ser Leu Ile Glu Ala
Met Lys Thr Arg Gln Glu Asp 485
490 495 Asn Leu Ile Met Ile Lys Asn Phe Leu
Gln His His Lys Lys Leu Arg 500 505
510 Asp Leu Lys Leu Gly Asp Leu Phe His
Glu Val Arg Ala Glu Asn Gly 515 520
525 Asp Pro Asn Met Ser Val Asn Leu Leu
Thr Val Ala Ser Thr Gly Asn 530 535
540 Ala Ala Phe Leu Glu Glu Leu Leu Lys
Ala Arg Leu Asp Pro Asp Ile 545 550
555 560 Gly Asp Ala Gln Gly Arg Thr Pro Leu
His Ile Ala Ala Ser Lys Gly 565
570 575 His Glu Glu Cys Val Met Val Leu Leu
Arg His Gly Cys Asn Ile His 580 585
590 Leu Arg Asp Val Asn Gly Asn Thr Ala
Leu Trp Glu Ala Ile Ala Ala 595 600
605 Lys Gln His Pro Thr Phe Gln Ile Leu
Tyr His Trp Ala Ser Val Ser 610 615
620 Asp Pro Tyr Val Ala Gly Glu Leu Leu
Cys Thr Ala Ala Lys Arg Asn 625 630
635 640 Glu Leu Thr Val Met Lys Glu Leu Leu
Lys His Gly Leu Ile Val Asp 645
650 655 Ser Ile Asp Arg His Gly Ser Thr Ala
Ile His Val Ala Leu Glu Glu 660 665
670 Asn His Glu Asp Met Val Lys Leu Leu
Leu Met Asn Gly Ala Glu Ile 675 680
685 Asn Asp Lys Phe Lys His Lys Leu Ser
Ser Met Asn Leu Ser Glu Met 690 695
700 Leu Gln Lys Arg Glu Val Gly His Arg
Val Ile Val Pro Asp Thr Met 705 710
715 720 Asp Glu Val Ala Gln Lys Trp Arg Glu
Gln Glu Gln Lys Tyr Asn Ser 725
730 735 Gly Ser Thr Arg Asp Gln Ser Ser Phe
Arg Val Ser Ile Tyr Lys Gly 740 745
750 His Pro Val Ile Arg Lys Arg Thr His
Cys Ser Glu Pro Gly Lys Leu 755 760
765 Ile Ile Leu Pro Asn Ser Leu Ala Glu
Leu Lys Ile Ile Ala Gly Gln 770 775
780 Lys Phe Gly Phe Asp Ala Thr Asn Ala
Leu Val Thr Asp Gln Glu Gly 785 790
795 800 Ser Glu Ile Asp Ser Ile Glu Val Ile
Arg Asp Asn Asp Lys Leu Phe 805
810 815 Ile Val Glu Asp Pro Lys Cys Leu * 820 825

Claims (3)

[Claims] 1. A potassium channel gene derived from a plant of the genus Nicotiana. 2. A DNA encoding SEQ ID NO: 1 or a DNA encoding a protein having one or several DNA substitutions, deletions, insertions or additions in said sequence and having substantially potassium channel activity. 3. The DNA sequence according to SEQ ID NO: 1.