JPH10229883A - Improvement of plant water potential maintenance function and water channel protein gene used for the same - Google Patents

Abstract

(57) [Problem] To produce a plant resistant to water stress without wasting energy stored in photosynthesis. SOLUTION: A plant cell membrane-localized water channel protein gene is introduced into a plant to improve the water potential maintaining function of the plant, thereby obtaining a water stress-tolerant plant.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a technique for improving a plant water potential maintenance function for obtaining a plant resistant to water stress such as salt stress or drought stress.

[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 water stress due to salt damage and drying. Salt damage has been 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. Some believe that increasing food production to keep up with population growth will require agricultural production on land that is now considered to be uncultivated due to salt damage.

[0003] Drying stress is caused by drought caused by irregular weather. Every few years in the United States, drought causes agricultural output to decline.

[0004] Therefore, finding a plant that is resistant to such water stress is very important in view of a possible food crisis that may occur in the future.

[0005] On the other hand, a water channel protein (hereinafter sometimes referred to as WCH protein) is known as a protein which is considered to be involved in water movement of plants.

[0006] The WCH proteins of such plants include:
There are a cell membrane-localized WCH protein and a vacuolar membrane-localized WCH protein, and there have already been some reports on the genes of these proteins.

[0007] As an example of the report of the gene of the cell membrane-localized WCH protein, cloning of the cDNA of the pea-derived inducible WCH protein (Plant Molecular Bio
logy 15, 11-26 (1990)), Cloning of the cDNA of the dry-inducible WCH protein from Arabidopsis thaliana (Plant
Cell Physiology 33, 217-224), those derived from Arabidopsis thaliana (The Plant Journal 6, 187-199 (1994)), those derived from tomato (Plant Molecular Biology 24, 539-543 (199
4)), introducing an antisense gene into Arabidopsis,
Proof of function as water channel (The Plant J
ounal 7, 87-95 (1995)), derived from Matsubagiku (The P
lant Cell 7, 1129-1142 (1995)), and corn, rice, kohlraby, soybean, barley, and the like, which are registered in gene databases and the like.

[0008] As an example of a report on the gene of the vacuolar membrane-localized WCH protein, a gene derived from tobacco (Nucleic Acids Res.
erch 18, 7449 (1990)), from kidney bean (The P
lantCell 2, 525-532 (1990)), those derived from Arabidopsis thaliana (Plant Physiology 99, 561-570 (1992)), and the like. ,
White clover, purple coconut palm and the like.

[0009]

As described above, it is an urgent task to find a plant that is resistant to water stress. For example, there is a method by adjusting the amount of water in a plant body. Generally, it is considered that a plant having water stress tolerance can be obtained by increasing the water content in a plant, that is, by increasing the water holding capacity.

[0010] As a method for obtaining a water stress-tolerant plant by such a method, a gene for an enzyme that synthesizes trehalose is introduced into a plant, and synthesized and accumulated in the plant. A method for obtaining a plant having water stress tolerance by controlling the water content of a plant has been disclosed (WO 96).
/ 00789)).

[0011] However, this method is disadvantageous in terms of energy because fixed carbon must be consumed by photosynthesis, and unexpected effects on product quality and metabolic system due to accumulation of saccharides may occur. There are various problems such as

On the other hand, it is expected that the above-mentioned WCH protein will be involved in water movement of plants, but the idea of using this to improve the water potential maintenance function of plants has been proposed. Not.

[0013]

The present invention has been made in view of the above circumstances, and provides a method for improving a water potential maintenance function of a plant by introducing a plant cell membrane-localized water channel protein gene into a plant. The purpose of the present invention is to obtain a water stress-tolerant plant. Further, the present invention provides a novel Nicotiana excelsiol (Nicotiana e) as a water channel protein gene used for such a method for improving water potential of plants.
xcelsior) -derived plant cell membrane-localized water channel protein. Less than,
The present invention will be described in detail.

The present invention relates to a plant cell membrane-localized WCH
It is characterized by introducing a protein gene and improving the function of maintaining the water potential of the transgenic plant into which the protein gene has been introduced.

Here, the water potential is a unit representing the chemical potential of water, and can be expressed by the equation "water potential = turgor pressure-osmotic pressure". Moisture transfer is
It occurs from high to low water potential. Physiologically, a high water potential in a plant means that there are many available water molecules in a cell. In other words, if the decrease in intracellular water potential under water stress conditions is large, it can be interpreted that the stress has a large effect on plant cells.

The plant cell membrane localized WC used in the present invention
As described above, genes encoding the H protein include pea, Arabidopsis, tomato, pinewood,
Corn, rice, kohlraby, soybean, various things such as those derived from barley are known, in the present invention, it is also possible to use these known genes,
However, the present invention is not limited thereto, and a plant cell membrane localized WCH
The gene is not particularly limited as long as it encodes a protein.

As a method for obtaining such a plant cell membrane-localized WCH protein gene, for example, a method for obtaining one derived from Nicotiana excelsior is described in Examples below. Further, as a method for obtaining a substance derived from another plant, it can be obtained by referring to the following literature. Pea (Pisum sativum) (Plant Molecular Biolog
y 15, 11-26 (1990)) Arabidopsis thaliana (Plant Cell
Physiology 33, 217-224 (1992), Plant Molecular Bi
ology 23, 1187-1198 (10993), The PlantJournal 6, 1
87-199 (1994)) Tomato (Plant Molecular Biology 24, 539-543 (199
4)) Matsugiku (The Plant Cell 7, 1129-1142 (1995))

The gene encoding the plant WCH protein used in the present invention may be introduced into a plant body in the sense direction or in the antisense direction.
In the present invention, for the purpose of increasing the function of maintaining the water potential of a plant under water stress conditions, it is preferable to introduce it in the sense direction.

In the present invention, the gene to be introduced when the gene encoding the plant WCH protein is introduced in the sense direction is not particularly limited.
For example, Nicotiana excelsiol
Those derived from sior) are preferred.

As the expression promoter used in the present invention, those having strong transcriptional power and expressed in all cells, (35S,
19S, nos, etc.), those that respond to light (such as rbc), those that respond to temperature (such as hsp), those that respond to hormones, those that respond to tissue, etc. If there is no particular limitation,
Particularly preferred is a strong one such as the 35S promoter.

In the present invention, the method of introducing the WCH protein gene in the sense or antisense direction into a plant to be transformed does not require a special method, and may be any method usually used for transforming plants. Any method can be used. As an example, a case where a leaf disk method using Agrobacterium sp. Is used will be described.

W in the sense or antisense direction
The CH protein 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. Selection of the transformed plants can be carried out by adding an appropriate antibiotic to the medium for forming callus and determining whether or not the medium has resistance. The transformation method using Agrobacterium can be applied not only to dicotyledonous plants but also to monocotyledonous plants (WO 94/00977).

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, tobacco and the like. Among them, soybean and corn are preferred.

The water potential can be measured by a method of attaching a detection portion of a water potential meter to a leaf tissue of a plant, or a method of punching a leaf disk from the plant and placing it on a measuring device. The former method, which is non-destructive and can measure the water potential in the plant over time, is preferred. The method of measuring the water potential includes a method of measuring the dew point in a space where the plant is closed, and a method of measuring the freezing point of the squeezed liquid from the plant,
The former method, which is non-destructive and reflects the entire tissue, is preferred. The water potential of the plant is affected by the volume and salt concentration of the solution to be irrigated, the lapse of time after irrigation, and the environmental temperature, humidity, sunshine, etc., which affect transpiration. Therefore, it is necessary to measure the water potential under stress conditions over time in an artificial weather device with constant environmental conditions. The tissue to be measured is preferably performed on leaf tissue that actively performs physiological activities such as photosynthesis.

[0025]

【Example】

Embodiment 1 FIG. mRNA extraction Nicotiana tabacum Green leaf tissue and Nicotiana excelsior
Extraction of total RNA from green leaf tissue was performed according to the method of Ostrem et al.
strem et al., Plant Physiology 84, 1270-1275 (198
7)), but the following points were improved upon implementation. 1) The ground plant was shaken on ice for 1 hour together with an extraction buffer and phenol. 2) The supernatant after centrifugation was shaken with chloroform for 1 hour on ice.

Purification of poly (A) ⁺ RNA is performed using QuickPrep mRNA Purification Kit (QuickPr
ep mRNA purification Kit (Pharmacia))
Performed according to the manufacturer's instructions.

Embodiment 2 FIG. Nicotiana Tabakam (Nicotiana
tabacum). Isolation of Cell Membrane-Localized WCH Protein Gene from Tabacum) Primers for isolating the cDNA encoding the WCH protein were prepared using
llinum) -derived WCH protein cDNA base sequence (T
he Plant Cell 7, 1129-1142 (1995))
It was prepared using an NA synthesizer (manufactured by Applied Biosystems). The sequence is described below. Primer 1 GAAGATCTATGATCTTTGCCCTTGTTTACTGC Primer 2 GTCAGATCTAGCACGCGACTTGAATGGAATAGCC

The synthesis of the template cDNA used for the reverse transcription replication chain reaction (RT-PCR) is performed by using the StrataScript ^™ RT-PCR + kit (StrataScript ^™ RT-PCR + Kit).
(STRATAGENE)))
Poly (A) ⁺ RNA extracted and purified from (Nicotiana tabacum)
Using 50 ng of oligo dT primers as primers, according to the manufacturer's instructions.

The RT-PCR reaction uses the above-mentioned cDN as a template.
A, The primers 1 and 2 were used as primers for 20 each.
pmol, dATP, dGTP, dCTP, and dTTP were each 200 μmol,
1X PCR buffer (Takara Shuzo) and 2.5 U AmpliTaq DNA polymerase (Takara Shuzo)
In a total volume of 100 μl. The reaction is performed at 91 ° C. for 1 minute, 42 ° C. for 1 minute, 2 minutes
The cycle of 72 ° C. was repeated 32 times. 1% PCR product
After separation on an agarose gel and staining with ethidium bromide, a product was detected at about 550 bp.

The PCR fragment was excised from the gel and subcloned into the pCRII plasmid using a TA cloning kit (TA Cloning Kit (Invitrogen)) according to the manufacturer's instructions.

Determination of the nucleotide sequence of the obtained PCR fragment
Using a DNA sequencer model 373A (manufactured by Applied Biosystems), the reaction was performed using a tack die terminator cycle sequencing kit (Taq D
This was performed using ye Terminator Cycle Sequencing Kit (manufactured by Applied Biosystems) according to the manufacturer's guide. In this example, a partial cDNA fragment of the cell membrane-localized WCH protein gene having the nucleotide sequence shown in SEQ ID NO: 1 was obtained.

Example 3 Nicotiana excelsiol (N
Isolation of WCH protein gene localized in cell membrane of N. icotiana excelsior) The production of N. excelsior green leaf cDNA library was carried out in Example 2 by N. excelsior. Excelsior (N.exc
elsior) Using a poly (A) ⁺ RNA extracted and purified from green leaves as a template, a Time Saver cDNA Synthetic Kit (TimeS
aver cDNA Synthesis Kit (Pharmacia)) and cloning vector using λZAPII. XLI-Blue was used as a host cell.

Screening of the cDNA library was performed using the PCR fragment obtained in Example 2 as a probe.
To prepare the probe, add 50 ng of the above PCR fragment as a template, 32 P-dCTP containing a radioisotope for labeling to the 0.74 MBq reaction solution, and prepare a Ready-Pri
me Labelling System Kit (Amersham))
Performed according to the manufacturer's instructions.

The screening was performed using Hybond N + (Hybond N +).
+ (Amersham)) for 16 hours under standard conditions as described in the manufacturer's instructions. Washing was performed using 300 mM NaCl, 30 mM trisodium citrate 0.1% SDS using 65%
Twice at 20 ° C. for 20 minutes, and then twice at 65 ° C. for 20 minutes with 75 mM NaCl 2, 7.5 mM trisodium citrate 0.1% SDS. Positive plaques were isolated and subcloned in vitro into pBluescript as described by the manufacturer of the λZAPII cloning vector.

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 a tack dye terminator cycle sequencing kit (Taq Dye Terminator).
Cycle Sequencing Kit (manufactured by Applied Biosystems)) according to the manufacturer's instructions. The gene sequence showed that three independent WCH genes were obtained. NeMip1
(SEQ ID NO: 2), NeMip2 (SEQ ID NO: 3) and NeMip3 (SEQ ID NO: 4). The amino acid sequences of the water channel proteins encoded by them are shown in SEQ ID NOS: 5 to 7, respectively.

Embodiment 4 FIG. Construction of Transgene Gene construction for introduction into Nicotiana tabacum was performed using the cDNA obtained in Example 3. NeMip2 (SEQ ID NO: 3) inserted into pBluescript was purified by digestion with restriction enzymes EcoRV and SacI.
This fragment was ligated with SmaI of pBI121 plasmid (CloneTech).
PNe10 construct gene inserted into the site generated by SacI digestion
I got

Example 5 Nicotiana tabacam
Transformation of Nicotiana tabacum (cv. SR1) was carried out according to the method of Horsch et al.
l., Science 277, 1229-1231, (1985)). PNe10 prepared in Example 4 was introduced into Agrobacterium tumefacience. Using these fungi, Nicotiana tabacum was transformed according to the method of Horsch et al. The regenerated plants were grown in a closed greenhouse, and the seeds produced by self-pollination were collected.

Example 6 Measurement of Change in Biological Water Potential Under Stress Conditions Transformant seeds obtained in Experimental Example 5 are germinated on an MS medium containing kanamycin (50 μg / ml) to produce about two true leaves. It was grown until. Plants that were not whitened at this time were transplanted into flower pots in a closed greenhouse. The leaves were grown until about four true leaves were formed, and then transplanted to an equal mixture of vermiculite and hydroball. In this state, 50 ml of a 1/4 concentration Hoagland solution (SIGMA) was watered per day and grown for 2 weeks. And plant the artificial weather machine (temperature
23 ℃, humidity 70%, 12 hours light / 12 hours dark)
After growing for 5 days and training in the environment inside the artificial weather machine, stress treatment was performed. 250 mM for plants in salt stress treatment
100 ml of a 1/4 concentration Hoagland solution containing NaCl was watered on the first day, and 50 ml from the second day. During the experiment, the intracellular water potential in the leaf tissue was measured over time using a water potential meter manufactured by WESKCOR. As a result, as shown in FIG. 1, it was clarified that the transformant into which pNe10 was introduced maintained a higher water potential than the non-transformant under the conditions of salt stress and drought stress.

[0039]

Industrial Applicability The present invention is a method for improving the water potential maintaining function of a plant by introducing a plant cell membrane-localized water channel protein gene into the plant. There are no disadvantages such as energy disadvantages or unexpected effects on product quality or metabolic system due to accumulation of saccharides,
By improving the water potential maintenance function of a plant, a plant having water stress tolerance can be produced.

[0040]

[Sequence list]

SEQ ID NO: 1 Sequence length: 587 Sequence type: Number of DNA strands: Double strand Topology: Linear Origin Organism: Nicotiana tabacum Nature: Cell membrane localized water channel protein cDNA sequence GAAGATCTAT GATCTTTGCC CTTGTTTACT GCACTGCTGG TATCTCAGGA GGACACATTA 60 ACCCAGCAGT GACATTTGGT CTGTTTTTGG CAAGAAAGTT GTCCTTAACA AGGGCTCTGT 120 TCTACATGGT GATGCAGTGC CTTGGTGCAA TCTGTGGTGC TGGTGTGGTT AAAGGTTTCA 180 TGGTGGGTCC ATACCAGAGA CTTGGTGGTG GGGCCAACAT GGTTCAACCT GGCTACACAA 240 AAGGTGATGG ACTTGGTGCT GAGATTATTG GGACCTTTGT CCTAGTTTAC ACTGTTTTCT 300 CTGCCACTGA TGCCAAGAGA AATGCTAGAG ATTCTCATGT CCCTATTTTG GCACCTCTTC 360 CTATTGGATT CGCGGTGTTC TTGGTTCATT TGGCCACCAT CCCAATCACC GGAACCGGTA 420 TCAACCCCGC CAGGAGCCTT GGAGCTGCTA TCATCTTCAA CCAAGACCAG GCATGGGATG 480 ATCACTGGAT CTTCTGGGTT GGACCATTCA TTGGAGCTGC ACTTGCTGCA GTTTACCACC 540 AGATAATCAT CAGGGCTATT CCATTCAAGT CGCGTGCTAG ATCTGAC 587

SEQ ID NO: 2 Sequence length: 1078 Sequence type: nucleic acid Number of strands: double-stranded Topology: linear Sequence type: cDNA to mRNA Origin Organism: Nicotiana excelsior Nature: cell membrane localized water Channel protein cDNA (N
EMip1) sequence of feature 34..894 CDS sequence AGAAAACTTA GTTGAGTGAC TGAAGAAACA AAAATGGCAG AGAACAAGGA AGAAGATGTT 60 AAGCTAGGAG CAAACAAGTA CAGAGAAACT CAACCCTTGG GCACAGCAGC TCAAACAGAC 120 AAGGACTATA AGGAGCCACC ACCAGCTCCT TTGTTTGAGC CAGGGGAGTT GTCGTCATGG 180 TCTTTTTACA GAGCTGGAAT TGCAGAATTC ATGGCTACTT TCTTGTTCTT GTACATCACT 240 ATCTTGACTG TTATGGGTCT CAAAAGGTCA GATAGTTTGT GTTCTTCTGT TGGTATTCAA 300 GGAGTTGCTT GGGCTTTTGG TGGCATGATC TTTGCTCTTG TCTACTGCAC TGCTGGTATC 360 TCAGGAGGAC ACATTAACCC AGCAGTGACA TTTGGTCTGT TCTTGGCAAG AAAGTTGTCT 420 TTAACAAGGG CTCTGTTCTA CATGGTGATG CAGTGCCTTG GTGCAATCTG TGGTGCTGGT 480 GTTGTTAAAG GTTTTATGGT GGGTCCATAT CAGAGACTTG GTGGTGGGGC CAACGTGGTT 540 CAACCTGGCT ACACCAAAGG TGATGGACTT GGTGCTGAGA TTATTGGCAC CTTTGTCCTT 600 GTCTACACTG TTTTCTCTGC CACTGATGCC AAGAGAAATG CTAGAGATTC TCATGTTCCT 660 ATTTTGGCAC CTCTTCCTAT TGGATTCGCG GTGTTCTTGG TTCATTTGGC CACCATCCCT 720 ATCACTGGAA CCGGCATCAA CCCCGCCCGG AGCCTTGGAG CTGCTATCAT CTTCAACCAC 780 GACCAGGCAT GGGATGATCA CTGGATCTTC TGGGTTGGAC CATTC ATTGG AGCTGCTCTC 840 GCTGCAGTTT ACCACCAGAT AATCATCAGA GCCATTCCAT TCAAGACCAA ATCTTAAATT 900 ATCTTGAAGA GTCCTCCTTT TATGTTTCTT CCCCTGCTTA TGTACCCTTT TTCCCCTTTC TG TGGTGCTGTTATATCATGTG ATTTTCGTAATGATGATGAATGATGATGAATGATGATGAAAA

SEQ ID NO: 3 Sequence length: 1125 Sequence type: nucleic acid Number of strands: double-stranded Topology: linear Sequence type: cDNA to mRNA Origin Organism: Nicotiana excelsior Nature: Cell membrane localized water Channel protein cDNA (N
EMip2) sequence of feature 66..926 CDS sequence GTTTTGTACT ATCCCACTTA GCACAAAAAA GAGAGAAAAA CAAGCTAAGT TTAGTGAGTG 60 TTCAAATGGC AGAAAACAAA GAAGAAGATG TTAAGCTTGG AGCTAACAAA TTCAGAGAAA 120 CACAACCATT AGGAACAGCT GCTCAAACAG ACAAAGATTA CAAAGAACCA CCACCAGCTC 180 CATTGTTTGA ACCAGGGGAG TTATCATCAT GGTCTTTTTA CAGAGCTGGA ATTGCAGAAT 240 TTATGGCTAC TTTCTTGTTT TTGTACATCA CTATCTTGAC TGTTATGGGT CTTAAAAGAT 300 CTGATAGTCT GTGTAGTTCA GTTGGTATTC AAGGTGTTGC TTGGGCTTTT GGTGGTATGA 360 TCTTTGCTTT GGTTTACTGC ACTGCTGGTA TCTCAGGAGG ACACATCAAC CCAGCTGTGA 420 CCTTTGGATT GTTCTTGGCA AGGAAACTGT CCTTAACCAG GGCTATTTTC TACATGGTGA 480 TGCAATGCCT TGGTGCAATT TGTGGTGCTG GTGTTGTGAA AGGATTCATG GTTGGTCCAT 540 ACCAGAGACT TGGTGGTGGT GCTAATGTTG TTAACCCTGG TTACACCAAA GGTGATGGCC 600 TTGGTGCTGA AATTATTGGC ACTTTTGTCC TTGTTTACAC TGTTTTCTCT GCTACTGATG 660 CTAAGAGAAA TGCCAGAGAC TCACATGTTC CTATTTTGGC ACCACTTCCC ATCGGATTCG 720 CGGTGTTCTT GGTTCATTTG GCCACCATTC CCATCACCGG AACTGGCATC AACCCCGCTA 780 GGAGTCTTGG AGCTGCGATC ATCTACAACG ACGAACATGC ATGGG ACGAC CACTGGATCT 840 TTTGGGTTGG ACCATTCATT GGAGCTGCAC TTGCTGCAGT TTACCATCAA ATAATCATCA 900 GAGCTATTCC ATTCCACAAG TCTTCTTAAG TTTGCTCAAG TAGCCTCATC TTTCTGAATT 960 TTCAAGACTT TGTACCTTTT TTTTGGGTTA AATTTTAATC CTCTCCTTCT GATGTATTTG 1020 TAATGTGGAT TTTCTTGTGT TCAAATGTCT ATTATGTGTG TAAATTATCA GGTTTTGTTG 1080 TACTGTACCC TATTAATGAA GGTTCTTTAT TTTATCAAAA AAAAA 1125

SEQ ID NO: 4 Sequence length: 1132 Sequence type: nucleic acid Number of strands: double-stranded Topology: linear Sequence type: cDNA to mRNA Origin Organism: Nicotiana excelsior Nature: Cell membrane localized water Channel protein cDNA (N
EMip3) sequence of feature 46..903 CDS sequence CCGTCCTCAA TTTGAGTAAA TTTTCCTGTG AATTGAGAAA ACAAAATGGA GAACAAAGAA 60 GAAGATGTAA GATTAGGAGC AAACAAATAT TCAGAGAGGC AAGCGATTGG GACGGCAGCG 120 CAGAGTGACA AGGATTATAC AGAGCCACCA CCAGCGCCAC TGTTTGAGGC AGGAGAATTG 180 ACGTCTTGGT CATTCTATCG TGCTGGAATT GCTGAGTTCA TGGCCACTTT CCTCTTCCTT 240 TACATCACTA TCTTAACAGT GATGGGTGTT TCAAAATCTG AATCCAAATG CTCCACTGTT 300 GGTATTCAAG GCATTGCTTG GGCTTTTGGG GGCATGATTT TTGCCCTTGT TTACTGTACT 360 GCTGGCATTT CTGGAGGACA CATAAATCCA GCTGTGACCT TTGGGCTGTT CTTGGCAAGG 420 AAATTGTCGT TGACAAGGGC AGTGTTCTAC ATGGTGATGC AGTGTCTTGG TGCTATATGT 480 GGTGCTGGTG TGGTCAAAGG GTTCGGCAAA ACTCTTTATC AAACAAAGGG TGGTGGTGCC 540 AATGTTGTAA ATCATGGTTA CACAAAGGGA TCAGGTCTTG GTGCTGAGAT CGTTGGCACT 600 TTTGTTCTTG TTTACACTGT TTTCTCTGCC ACAGATGCTA AGCGTAGTGC TAGGGATTCA 660 CATGTCCCTA TTTTGGCACC ATTGCCTATT GGATTTGCGG TGTTCTTGGT ACACTTGGCC 720 ACAATTCCAA TCACTGGAAC TGGTATTAAC CCAGCTAGGA GTCTTGGCGC AGCCATCATC 780 TACAACCAAG ACCACGCATG GGATGACCAC TGGATTTTCT GGGTT GGACC TTTCATTGGA 840 GCAGCACTAG CAGCTCTGTA CCAACAGGTG GTGATCAGGG CAATCCCATT TAAGTCCAAG 900 TGAAATCTCT TTTAACCACA GATTTCTCAA GCTTAATCAT CATCATCTGG TCAAAGATCA 960 AAAGCCTAAT TTCTGCCCAT TTCCCCTTTG CATTAGATCC TCTTTTTCTT TTGGTTTTGT 1020 TTTCTATAAC TATGTATTTG TAAGTCTCGT AAGTGGGAGA ATGTATCTGA TGGGGGCATA 1080 TTTATTATCT ATAAAAAGGA TCTTCGCTTC TCAAAAAAAA AAAAAAAAAA AA 1132

SEQ ID NO: 5 Sequence length: 287 Sequence type: number of amino acid chains: single chain Topology: linear Sequence type: protein Origin Organism name: Nicotiana excelsior Properties: cell membrane localized water channel protein (NeMip1) Sequence Met Ala Glu Asn Lys Glu Glu Asp Val Lys Leu Gly Ala Asn Lys 1 5 10 15 Tyr Arg Glu Thr Gln Pro Leu Gly Thr Ala Ala Gln Thr Asp Lys 20 25 30 Asp Tyr Lys Glu Pro Pro Pro Ala Pro Leu Phe Glu Pro Gly Glu 35 40 45 Leu Ser Ser Trp Ser Phe Tyr Arg Ala Gly Ile Ala Glu Phe Met 50 55 60 Ala Thr Phe Leu Phe Leu Tyr Ile Thr Ile Leu Thr Val Met Gly 65 70 75 Leu Lys Arg Ser Asp Ser Leu Cys Ser Ser Val Gly Ile Gln Gly 80 85 90 Val Ala Trp Ala Phe Gly Gly Met Ile Phe Ala Leu Val Tyr Cys 95 100 105 Thr Ala Gly Ile Ser Gly Gly His Ile Asn Pro Ala Val Thr Phe 110 115 120 Gly Leu Phe Leu Ala Arg Lys Leu Ser Leu Thr Arg Ala Leu Phe 125 130 135 Tyr Met Val Met Gln Cys Leu Gly Ala Ile Cys Gly Ala Gly Val 140 145 150 Val Lys Gly Phe Met Val Gly Pro Tyr Gln Arg Leu Gly Gly Gly 155 160 165 Ala Asn Val Val Gln Pro Gly Tyr Thr Lys Gly Asp Gly Leu Gly 170 175 180 Ala Glu Ile Ile Gly Thr Phe Val Leu Val Tyr Thr Val Phe Ser 185 190 195 Ala Thr Asp Ala Lys Arg Asn Ala Arg Asp Ser His Val Pro Ile 200 205 210 Leu Ala Pro Leu Pro Ile Gly Phe Ala Val Phe Leu Val His Leu 215 220 225 Ala Thr Ile Pro Ile Thr Gly Thr Gly Ile Asn Pro Ala Arg Ser 230 235 240 Leu Gly Ala Ala Ile Ile Phe Asn His Asp Gln Ala Trp Asp Asp 245 250 255 His Trp Ile Phe Trp Val Gly Pro Phe Ile Gly Ala Ala Leu Ala 260 265 270 Ala Val Tyr His Gln Ile Ile Ile Arg Ala Ile Pro Phe Lys Thr 275 280 285 Lys Ser

SEQ ID NO: 6 Sequence length: 287 Sequence type: number of amino acid chains: single chain Topology: linear Sequence type: protein Origin Organism name: Nicotiana excelsior Properties: cell membrane localized water channel protein (NeMip2) Sequence Met Ala Glu Asn Lys Glu Glu Asp Val Lys Leu Gly Ala Asn Lys 1 5 10 15 Phe Arg Glu Thr Gln Pro Leu Gly Thr Ala Ala Gln Thr Asp Lys 20 25 30 Asp Tyr Lys Glu Pro Pro Pro Ala Pro Leu Phe Glu Pro Gly Glu 35 40 45 Leu Ser Ser Trp Ser Phe Tyr Arg Ala Gly Ile Ala Glu Phe Met 50 55 60 Ala Thr Phe Leu Phe Leu Tyr Ile Thr Ile Leu Thr Val Met Gly 65 70 75 Leu Lys Arg Ser Asp Ser Leu Cys Ser Ser Val Gly Ile Gln Gly 80 85 90 Val Ala Trp Ala Phe Gly Gly Met Ile Phe Ala Leu Val Tyr Cys 95 100 105 Thr Ala Gly Ile Ser Gly Gly His Ile Asn Pro Ala Val Thr Phe 110 115 120 Gly Leu Phe Leu Ala Arg Lys Leu Ser Leu Thr Arg Ala Ile Phe 125 130 135 Tyr Met Val Met Gln Cys Leu Gly Ala Ile Cys Gly Ala Gly Val 140 145 150 Val Lys Gly Phe Met Val Gly Pro Tyr Gln Arg Leu Gly Gly Gly 155 160 165 Ala Asn Val Val Asn Pro Gly Tyr Thr Lys Gly Asp Gly Leu Gly 170 175 180 Ala Glu Ile Ile Gly Thr Phe Val Leu Val Tyr Thr Val Phe Ser 185 190 195 Ala Thr Asp Ala Lys Arg Asn Ala Arg Asp Ser His Val Pro Ile 200 205 210 Leu Ala Pro Leu Pro Ile Gly Phe Ala Val Phe Leu Val His Leu 215 220 225 Ala Thr Ile Pro Ile Thr Gly Thr Gly Ile Asn Pro Ala Arg Ser 230 235 240 Leu Gly Ala Ala Ile Ile Tyr Asn Asp Glu His Ala Trp Asp Asp 245 250 255 His Trp Ile Phe Trp Val Gly Pro Phe Ile Gly Ala Ala Leu Ala 260 265 270 Ala Val Tyr His Gln Ile Ile Ile Arg Ala Ile Pro Phe His Lys 275 280 285 Ser Ser

SEQ ID NO: 7 Sequence length: 285 Sequence type: number of amino acid chains: single chain Topology: linear Sequence type: protein Origin Organism name: Nicotiana excelsior Nature: cell membrane localized water channel protein (NeMip3) Sequence Met Glu Asn Lys Glu Glu Asp Val Arg Leu Gly Ala Asn Lys Tyr 1 5 10 15 Ser Glu Arg Gln Ala Ile Gly Thr Ala Ala Gln Ser Asp Lys Asp 20 25 30 Tyr Thr Glu Pro Pro Pro Ala Pro Leu Phe Glu Ala Gly Glu Leu 35 40 45 Thr Ser Trp Ser Phe Tyr Arg Ala Gly Ile Ala Glu Phe Met Ala 50 55 60 Thr Phe Leu Phe Leu Tyr Ile Thr Ile Leu Thr Val Met Gly Val 65 70 75 Ser Lys Ser Glu Ser Lys Cys Ser Thr Val Gly Ile Gln Gly Ile 80 85 90 Ala Trp Ala Phe Gly Gly Met Ile Phe Ala Leu Val Tyr Cys Thr 95 100 105 Ala Gly Ile Ser Gly Gly His Ile Asn Pro Ala Val Thr Phe Gly 110 115 120 Leu Phe Leu Ala Arg Lys Leu Ser Leu Thr Arg Ala Val Phe Tyr 125 130 135 Met Val Met Gln Cys Leu Gly Ala Ile Cys Gly Ala Gly Val Val 140 145 150 Lys Gly Phe Gly Lys Thr Leu Tyr Gln Thr Lys Gly Gly Gly Ala 155 160 165 Asn Val Val Asn His Gly Tyr Thr Lys Gly Ser Gly Leu Gly Ala 170 175 180 Glu Ile Val Gly Thr Phe Val Leu Val Tyr Thr Val Phe Ser Ala 185 190 195 Thr Asp Ala Lys Arg Ser Ala Arg Asp Ser His Val Pro Ile Leu 200 205 210 Ala Pro Leu Pro Ile Gly Phe Ala Val Phe Leu Val His Leu Ala 215 220 225 Thr Ile Pro Ile Thr Gly Thr Gly Ile Asn Pro Ala Arg Ser Leu 230 235 240 Gly Ala Ala Ile Ile Tyr Asn Gln Asp His Ala Trp Asp Asp His 245 250 255 Trp Ile Phe Trp Val Gly Pro Phe Ile Gly Ala Ala Leu Ala Ala 260 265 270 270 Leu Tyr Gln Gln Val Val Ile Arg Ala Ile Pro Phe Lys Ser Lys 275 280 285

[Brief description of the drawings]

FIG. 1 is a graph showing that a transformant into which pNe10 has been introduced maintains a higher water potential than a non-transformant under conditions of salt stress and drought stress.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁶ Identification code FI C12R 1:91) (C12N 5/10 C12R 1:91)

Claims (9)

[Claims] 1. A method for improving a plant water potential maintaining function, comprising introducing a plant cell membrane-localized water channel protein gene into a plant. 2. The method for improving a plant water potential maintenance function according to claim 1, wherein the introduction of the water channel protein gene is in a sense direction. 3. The plant cell membrane-localized water channel protein gene is Nicotiana Excelsiol (Nicotiana).
excelsior). The method for improving a plant water potential maintenance function according to claim 2, wherein the method is derived from excelsior). 4. The nicotiana excelsiol (Nicot)
4. The method for improving a plant water potential maintenance function according to claim 3, wherein the plant cell membrane-localized water channel protein gene derived from iana excelsior) is a gene encoding the amino acid sequence of SEQ ID NO: 5. 5. The method of claim 5, wherein said Nicotiana Excelsior (Nicot)
4. The method for improving a plant water potential maintenance function according to claim 3, wherein the plant cell membrane-localized water channel protein gene derived from iana excelsior) is a gene encoding the amino acid sequence represented by SEQ ID NO: 6. 6. The Nicotiana Excelsior (Nicot)
4. The method for improving a plant water potential maintenance function according to claim 3, wherein the plant cell membrane-localized water channel protein gene derived from iana excelsior) is a gene encoding the amino acid sequence represented by SEQ ID NO: 7. 7. Nicotiana excelsiol (Nicotiana)
excelsior), a gene encoding the amino acid sequence of SEQ ID NO: 5, which is a plant cell membrane-localized water channel protein derived from plant cells. 8. Nicotiana excelsiol (Nicotiana)
excelsior), a gene encoding the amino acid sequence of SEQ ID NO: 6, which is a plant cell membrane-localized water channel protein derived from the plant. 9. Nicotiana Excelsiol (Nicotiana)
excelsior), a gene encoding the amino acid sequence of SEQ ID NO: 7 which is a plant cell membrane-localized water channel protein derived from plant cells.