JPH11187879A - New inps gene derived from plant belonging to genus nicrotiana - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a new gene derived from genus Nicotiana having a function of giving a salt stress resistance to plants, and having a specific DNA sequence and encoding proteins having inositol-1-phosphate synthetase activity. SOLUTION: This new INPS 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 the inositol-1-phosphate synthetase(INPS) activity, having 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 an mRNA and preparing a cDNA library using the mRNA as a template, and screening this in a differential screening method.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a DNA having an activity of resisting water stress such as salt stress or drought stress.
About. More specifically, the present invention relates to DNA derived from the genus Nicotiana having an activity of resistance to water stress such as salt stress or drought stress using genetic engineering technology.

[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] Drying stress is caused by drought caused by irregular weather. Every few years in the United States, droughts have reduced agricultural output. Therefore, finding a plant that is resistant to such salt stress is very important in view of a possible 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 indicate that INPS in Nicotiana plants
There was no report that the INPS gene was isolated, not regarding the presence of the gene.

It has been known that an INPS gene exists in a plant. INPS (Inositol monophosphate synthase) converts glucose-6-phosphate (D-glucose 6-phosphate) to inositol-1-phosphate (1L-myo-inositol 1-phosphate).
Have the effect of synthesizing The above-mentioned INPS gene is known from Arabidopsis thaliana, pine blossom (Mesembryanthemum crystallinum) and the like, and reported by Ishitani et al. (Ishitani et al. (1996)
Plant Journal, vol. 9, 537-548) states that INPS gene is induced by salt stress in halophyte pine birch. On the other hand, it is not induced in Arabidopsis thaliana, a non-halophyte. In Matsubaiku, inositol synthesized by INPS is finally converted into a sugar alcohol that functions as a compatible solute called pinitol via a pinitol synthesis system. However, the INPS that exists in Matsubagic
Since Arabidopsis lacks the pinitol synthesis system located further downstream, it is considered that INPS itself is not induced by salt stress because pinitol is not produced from inositol. Based on this report, this time Nicotiana pani
Looking at the phenomenon observed in culata, as described below, salt stress produces INPS, indicating that Nicotiana pan
In iculata, it is highly likely that INPS has contributed to salt stress tolerance. Nicotiana paniculata is also considered to be lacking the pinitol synthesis system downstream of INPS. Therefore, it is thought that increasing the production of inositol can improve the resistance to salt stress. However, the INPS gene was not known or isolated for Nicotiana plants.

[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 water content of a plant. Generally, it is considered that a plant having water stress tolerance can be obtained by increasing the water content of a plant, that is, by increasing the water holding capacity. 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 water stress resistance 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 the ana genus
Novel INPS induced by salt stress from niculata
The gene was found and the gene was isolated. From the above facts, the INPS gene encoded by the gene obtained this time is
It is considered that plants have a function of imparting water stress tolerance. Thus, by controlling the expression of this gene, it is possible to improve the salt stress tolerance of plants. Further, since cultivated plants are constantly exposed to the risk of being subjected to water stress, it is useful to quickly recognize the stress state of the plant for a subsequent countermeasure. Therefore,
By monitoring the expression status of this gene, the stress status of the plant can be known.

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

[0010] More specifically, the present invention relates to (1) an INPS 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,
And a DN encoding a protein having substantially INPS activity
A. (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. “Having substantially INPS activity” means having substantially INPS activity against salt stress.

Hereinafter, the present invention will be described in more detail. The present invention is characterized by introducing an Nicotiana-derived INPS 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 newly produced under the environment was taken out, and the gene of the present invention was derived by analyzing the sequence. INP derived from Nicotiana used in the present invention
The S gene may be introduced into the plant in the sense direction,
Further, it may be introduced in the antisense direction. In the present invention, for the purpose of increasing the water holding capacity of the plant under water stress conditions, it is preferable to introduce the plant in the sense direction, but depending on the nature of the stress, the effect is reduced by suppressing the metabolic change of the plant. In some cases, 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 the sense or antisense INPS gene into a plant to be transformed does not require a special method, and may be any method usually used for transforming a plant. Any method can be used. For example, a transformation method using a gene gun, an electroporation method, a leaf disk method using Agrobacterium, and the like can be mentioned, and preferably a leaf disk method using Agrobacterium is used. This will be described below.

IN in the sense direction or the antisense direction
The PS gene 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 the host into which the INPS 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 introduced into a vector such as a plasmid vector, a phage vector, a retrovirus vector, etc., and the cells are transformed, or after in vitro packaging, transfected into host cells (transfection).
Thus, a transformed cell stably retaining the reporter gene 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. pGEX-5X-1) or an expression vector derived from SV-40 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 whose water content 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 as a method for measuring the water 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, the aerial part of the plant body is harvested, the fresh weight is measured, and these plants are dried at 60 ° C. for several days, and then the dry weight is measured. A method of measuring the water content based on the ratio between the raw weight and the dry weight may be used.

[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 <Nicotiana paniculata IN
Isolation of PS cDNA> A stressed Nicotiana paniculata green leaf cDNA library was prepared by using poly (A) + RNA extracted and purified from the stress-treated plant in Example 2 as a template using 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 produced was carried out according to Amersham's manual. 32 used for screening
The P-labeled probe was prepared as follows. Example 2
Was dissolved in 30 μl of sterile water, 1 μl of primer (Takara Shuzo) was added, and the mixture was placed at 65 ° C. for 10 minutes. Then cool to room temperature and add RNse inhibitor
5 μl of 5X buffer (Takara Shuzo) attached to MMLV reverse transcriptase
l, 5 μl of dATP / dGTP / dTTP solution (10 mM each), 32P-dCTP
1 μl (10 μCi) (Amersham) and 6 μl of distilled water were added, mixed well, and MMLV reverse transcriptase (Takara Shuzo) was added.
μl was added. 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 N
aCl, 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, cDN shown in Sequence Listing 1
A, NpINPS1 was obtained.

[Example 4] <Analysis of change in INPS transcript accumulation amount due to salt stress> N. panicul treated with salt stress according to the method described in Example 1
From ata, total RNA was extracted and purified over time, and the expression of the relevant gene was examined by RNA gel blotting. Total RNA was extracted using the method described in Example 2 and 0% after the start of the stress treatment.
Hour, 8 hours, 1st, 3rd day. Total RNA extracted
Was purified by LiCl treatment and electrophoresed on formaldehyde gel according to the method of Vernon et al. (Vern
on and Bohnert, EMBO Journal 11: 2077-2085 (199
2)). RNA was used in an amount of 5 μg per lane. After electrophoresis,
RNA was transferred to a nylon membrane (HybondN +, manufactured by Amersham) and hybridization was performed according to the manufacturer's instructions. The probe labeled with 32P-dCTP is RediprimeT
This was performed using an M DNA labeling system (manufactured by Amersham) according to the manufacturer's instructions. After completion of the reaction, free 32
P-dCTP was removed using a spin column and used for hybridization. As a hybridization solution, a solution containing 0.25 M Na2HPO4 (pH 7.2) and 7% SDS was used at 65 ° C. overnight. Washing after completion of hybridization is performed with 300 mM NaCl, 30 mM trisodium citrate,
Twice at 65 ° C for 20 minutes using 0.1% SDS, followed by 75 mM
NaCl, 7.5 mM trisodium citrate, 0.1% SDS
Twice at 20 ° C. for 20 minutes. After the washing, the nylon film was dried and brought into close contact with an imaging plate (Fuji Photo Film Co., Ltd.) and exposed. Thereafter, the imaging plate was analyzed by a bioimage analyzer BAS100 (Fuji Photo Film Co., Ltd.) to image the distribution of radiation energy remaining on the nylon film. as a result,
An expression pattern as shown in FIG. 1 was obtained.

In FIG. 1, the higher the amount of RNA production, the darker the image. The comparison between the control and stress on the first and third days shows that the amount of RNA production under stress is greater than that under the control. Higher RNA production means higher gene product production.

[0040]

From the above facts, it is considered that INPS 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. Further, since cultivated plants are constantly exposed to the risk of being subjected to water stress, it is useful to quickly recognize the stress state of the plant for a subsequent countermeasure. Therefore, by monitoring the expression status of this gene, the stress status on the plant can be known.

[0041]

[Sequence list]

SEQ ID NO: 1 Sequence length: 1950 Sequence type: nucleic acid Number of strands: double-stranded Topology: linear Sequence type: cDNA to mRNA Origin Organism: Nicotiana paniculata Nature: INPS cDNA (NpINPS1 ) sequence of feature 92-1703 CDS 1 CTTAGCCTTAAAGTATACTCATAGCGTGTTCTTCTCTTTGTTTGTTACAAAACCTCTTTC 60 61 CCCTTCGATACTTTAGAAAAGTTTCCTCAAAATGTTTATTGAGAATTTTAAGGTTGAGAG 120 MetPheIleGluAsnPheLysValGluSer 121 CCCCAACGTTAAGTACACCGAAAGTGAAATTCACTCTGTCTATGATTATCAAACCACTGA 180 ProAsnValLysTyrThrGluSerGluIleHisSerValTyrAspTyrGlnThrThrGlu 181 GTTAGTTCATGATGAGAAAAATGGGACATATCAATGGACCGTCAAGCCTAAGACTGTCAA 240 LeuValHisAspGluLysAsnGlyThrTyrGlnTrpThrValLysProLysThrValLys 241 ATATGAGTTCAAGACTGATGTTCATGTTCCCAAATTAGGGGTTATGCTTGTTGGATGGGG 300 TyrGluPheLysThrAspValHisValProLysLeuGlyValMetLeuValGlyTrpGly 301 TGGAAACAATGGTTCAACCTTGACCGGTGGTGTTATTGCTAACAGAGAAGGAATTTCATG 360 GlyAsnAsnGlySerThrLeuThrGlyGlyValIleAlaAsnArgGluGlyIleSerTrp 361 GGCCACCAAAGATAAGGTGCAACAAGCCAATTACTTTGGCTCTCT TACTCAGGCTTCTAC 420 AlaThrLysAspLysValGlnGlnAlaAsnTyrPheGlySerLeuThrGlnAlaSerThr 421 TATTCGAGTTGGGTCTTTCAATGGAGAAGAGATCTATGCTCCATTTAAAAGCCTCCTTCC 480 IleArgValGlySerPheAsnGlyGluGluIleTyrAlaProPheLysSerLeuLeuPro 481 AATGGTCAATCCAGATGACGTAGTGTTTGGAGGATGGGACATCAGCGACATGAATTTAGC 540 MetValAsnProAspAspValValPheGlyGlyTrpAspIleSerAspMetAsnLeuAla 541 AGATGCCATGGCCAGGGCTAAGGTATTTGATATTGATCTACAAAAGCAGTTGAgGCCCTA 600 AspAlaMetAlaArgAlaLysValPheAspIleAspLeuGlnLysGlnLeuArgProTyr 601 CATGGAATCTATGGTCCCACTCCCTGGTATCTATGACCCTGATTTCATTGCTGCTAACCA 660 MetGluSerMetValProLeuProGlyIleTyrAspProAspPheIleAlaAlaAsnGln 661 AGGGTCACGTGCCAACAACGTGATCAAAGGAACCAAGAAAGAACAAATTGATCAAATCAT 720 GlySerArgAlaAsnAsnValIleLysGlyThrLysLysGluGlnIleAspGlnIleIle 721 TAAGGATATTAGGGAGTTTAAGGAAAAGAACAAAGTGGACAAGGTGGTAGTATTGTGGAC 780 LysAspIleArgGluPheLysGluLysAsnLysValAspLysValValValLeuTrpThr 781 TGCTAACACTGAAAGATACAGTAATGTGGTTGTTGGACTTAATGACACTATGGAAAACCT 840 AlaAsnThrGluArgTyrSerAsnValValValGlyLeuAsnAspThrMetGluAsnLeu 841 CTT TGCTTCTGTGGACAGAAATGAAGCTGAAATATCTCCTTCCACTTTGTATGCTATTGC 900 PheAlaSerValAspArgAsnGluAlaGluIleSerProSerThrLeuTyrAlaIleAla 901 CTGCATTCTTGAAAATGTGCCTTTTATTAATGGAAGCCCCCAGAACACCTTTGTCCCAGG 960 CysIleLeuGluAsnValProPheIleAsnGlySerProGlnAsnThrPheValProGly 961 CCTCATTGATTTGGCCATCAAGAAGAACACATTGATTGGTGGTGATGACTTTAAGAGTGG 1020 LeuIleAspLeuAlaIleLysLysAsnThrLeuIleGlyGlyAspAspPheLysSerGly 1021 TCAAACCAAAATGAAGTCAGTGCTGGTTGATTTCCTTGTTGGAGCTGGTATTAAGCCAAC 1080 GlnThrLysMetLysSerValLeuValAspPheLeuValGlyAlaGlyIleLysProThr 1081 ATCAATTGTGAGCTACAACCATTTGGGTAACAATGATGGAATGAATCTGTCTGCCCcTCA 1140 SerIleValSerTyrAsnHisLeuGlyAsnAsnAspGlyMetAsnLeuSerAlaProGln 1141 AACTTTCCGGTCAAAGGAGATCTCGAAAAGTAATGTTGTTGATGACATGGTTTCAAGCAA 1200 ThrPheArgSerLysGluIleSerLysSerAsnValValAspAspMetValSerSerAsn 1201 TGCCATCCTTTATGAGCCTGGAGAGCACCCTGACCATGTTGTTGTGATTAAGTATGTGCC 1260 AlaIleLeuTyrGluProGlyGluHisProAspHisValValValIleLysTyrValPro 1261 ATATGTGGGAGACAGCAAGAgGGCAATGGATGAGTACACATCTGAGATTTTCATGGGGGG 1320 TyrValGlyAspSerLy sArgAlaMetAspGluTyrThrSerGluIlePheMetGlyGly 1321 AAAGAACACCATTGTTTTGCACAATACTTGTGAgGATTCACTTTTAGCTGCTCCAATTAT 1380 LysAsnThrIleValLeuHisAsnThrCysGluAspSerLeuLeuAlaAlaProIleIle 1381 ATTGGATTTGGTCCTTCTTGCTGAACTCAGTACCCGCATTCAGCTCAAAGCTGAAGGAGA 1440 LeuAspLeuValLeuLeuAlaGluLeuSerThrArgIleGlnLeuLysAlaGluGlyGlu 1441 gGGTaAGTTCCACTCCTTCCACCCCGTGGcTACTATCcTCAGCTACCTTACCAAgGcTCc 1500 GlyLysPheHisSerPheHisProValAlaThrIleLeuSerTyrLeuThrLysAlaPro 1501 TCTGGTACCACCAGGTACACCAGTGGTGAATGCACTCTCAAAGCAGAGGGCAATGCTTGA 1560 LeuValProProGlyThrProValValAsnAlaLeuSerLysGlnArgAlaMetLeuGlu 1561 GAACATATTGAGGGCTTGTGTTGGACTTGCACCAGAGAACAACATGATTCTGGAATACAA 1620 AsnIleLeuArgAlaCysValGlyLeuAlaProGluAsnAsnMetIleLeuGluTyrLys 1621 ATGAAGATCCATTTCCCTAAGAAGGGAAAGGACTCAAGAAGCTGTACGGCAGAAGAACCA 1680 *** ArgSerIleSerLeuArgArgGluArgThrGlnGluAlaValArgGlnLysAsnHis 1681 TAATGGTCTTTCTTTTCTTTAGTATTTGTAATCTTCATCTTTCATTAGTACTATGATTAG 1740 AsnGlyLeuSerPheLeu *** 1741 CTAAATGTCGGCAACCCTTCGAGGTATAGCTGTTTTGTAATGGCAGGCCAAGTTGAATGT 1800 1801 CATTATAATTTCTTTGTTGTCTTTGTATGTTAGCCTTCTATTTACAAGGTTGTATCCGTG 1860 1861 TTGCTCTTTCTCATTTAGAGCTAGCAACGCTTTTCTCTAATGGAAATACAATGTGTAGTC 1920 1921 CTTTAGACGTTAAAAAAAAAAAAAAAAAAA 1950

[0043]

[Brief description of the drawings]

FIG. 1 is a diagram showing RNA gel blot analysis of the INPS gene.

Claims (3)

[Claims] 1. An INPS 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 substantially having INPS activity. 3. The DNA sequence according to SEQ ID NO: 1.