WO2007086402A1

WO2007086402A1 - Plant stomatal regulatory factor

Info

Publication number: WO2007086402A1
Application number: PCT/JP2007/051049
Authority: WO
Inventors: Tatuo Kakimoto; Ryoko Kajita; Kenta Hara
Original assignee: Osaka University
Priority date: 2006-01-25
Filing date: 2007-01-24
Publication date: 2007-08-02

Abstract

It is intended to provide a plant stomatal regulatory factor. The plant stomatal regulatory factor of the invention comprises an amino acid sequence selected from the group consisting of the following (1) to (3) or an amino acid sequence derived therefrom and has a function to inhibit the plant stomatal formation density. (1) An amino acid sequence represented by SEQ ID NO: 2 or 4 in the sequence listing. (2) An amino acid sequence encoded by an orthologous gene of at least one of At1g34245 gene of Arabidopsis thaliana and At2g20875 gene of Arabidopsis thaliana. (3) An amino acid sequence in which one or several amino acids have been substituted, deleted or added in the amino acid sequence of (1) or (2).

Description

Specification

Plant stomatal regulator

Technical field

[0001] The present invention relates to a plant stomatal regulator.

Background art

[0002] There are pores in the epidermis of all higher terrestrial plants. These plants open and close the pores according to the situation, and adjust the amount of oxygen and carbon dioxide necessary for respiration and carbon dioxide assimilation and the amount of water in the plant body. The pores are two lens-like guard cells that open and close due to inflation pressure. The number and density of pores vary depending on intrinsic (genetic) factors and extrinsic (environmental) factors. For example, it is generally known that the pores are present at high density on the back side of the leaf, and the density of the pores decreases under a growing environment where the carbon dioxide concentration in the atmosphere is increased. However, in any case, the pores are uniformly distributed without concentrating on one leaf. This is because the pattern formation control system that creates pores uniformly in the plant epidermis is working.

[0003] Regarding the pore pattern formation control system, the following mechanism is known. In other words, secretory signal molecules secreted from stomatogenic stem cells (meristemoids) are processed by SDDI protease, which is recognized by TMM receptor kinase and ER ECTA family receptor kinase family (See Patent Document 1 and Non-Patent Documents 1 and 2). However, it is not clear what the secretory signal molecule is. Therefore, understanding the stomatal pattern formation control system that regulates the balance between stomatal density and distribution requires the elucidation of the secretory signal.

[0004] On the other hand, regulation of the density of leaf stomata and epidermal cells has great significance in the fields of agriculture and forestry. For example, increasing the stomatal density or epidermal cell density can be expected to increase the carbon dioxide assimilation rate, increase tl of the accumulated amount of sugar and protein, that is, increase the biomass (for example, Patent Document 1). In addition, for example, improvement in drying resistance can be expected by reducing the pore density.

Patent Document 1: Japanese Translation of Special Publication 2002-527070 Non-Patent Document 1: Schluter U, Muschak M, Berger D, Altmann T. Pheoto synthetic performance of an Arabidopsis mutant with elevated stomatal density (sadl— 1) under different light regimes. J Exp B ot. 2003 Feb; 54 (383 ): 867- 74.PMID: 12554730

Non-Patent Document 2: Shpak ED, McAbee JM, Pillitteri LJ, Torii KU.Sto matal patterning and differentiation by synergistic interactions of receptor kinases. Science. 2005 Jul 8; 309 (5732): 290-3 PMID:

16002616

Disclosure of the invention

Problems to be solved by the invention

[0005] Accordingly, an object of the present invention is to provide a plant stomatal regulator.

Means for solving the problem

[0006] In order to achieve the above object, the stomatal regulator of the plant of the present invention comprises an amino acid sequence selected from the group force consisting of the following (1) to (3) or an amino acid sequence derived therefrom, and the plant It has a function of suppressing the pore formation density.

(1) The amino acid sequence of SEQ ID NO: 2 or 4 in the sequence listing.

(2) Amino acid sequence encoded by at least one orthologous gene of Arabidopsis Atlg34245 gene and Arabidopsis At2g20875 gene.

(3) An amino acid sequence in which one or several amino acids are substituted, deleted or added in the amino acid sequence of (1) or (2).

The invention's effect

[0007] The present inventors paid attention to the fact that there is no known gene that increases the number of cells per individual due to suppression of gene function in plants, and conducted extensive research to identify the gene. As a result, two Arabidopsis genes Atlg34245 and At2g20875, which encode a secretory peptide and increase the number of epidermal cells and stomata of leaves by destroying the gene, were identified and reached the present invention.

[0008] The plant stomatal regulator of the present invention has a function of suppressing plant pore-forming density. Therefore, for example, preferably, the formation density of pores can be adjusted using the pore regulating factor of the present invention. That is, for example, if the expression of the stomatal regulator of the present invention is inhibited, for example, preferably the number of epidermal cells and the number of pores in the leaves without affecting the growth, size, shape and reproduction of the plant are increased. be able to. In addition, for example, if the expression of the stomatal regulator of the present invention is promoted, the leaf stomatal density can be preferably reduced.

[0009] As described above, by increasing the pore density and epidermal cell density, for example, an increase in the carbon assimilation rate and an increase in the amount of sugar and protein accumulated can be expected, and by reducing the pore density, drought resistance can be expected. Therefore, according to the present invention, various water environments and CO

2 Plants can be modified to accommodate the environment.

Brief Description of Drawings

[0010] [FIG. 1] Panels A, B, C and D in FIG. 1 are microscopic photographs of the leaves of the wild strain, Atlg34245 disruption mutant, At2g20875 disruption mutant and double disruption, respectively. An example is shown.

FIG. 2 is an example of a phylogenetic tree of the Arabidopsis Atlg34245 gene family.

[FIG. 3] FIG. 3 shows the first leaf (primary leaf) and cotyledon in the At2g20875 expression vector transformation ^ ¾ (X), the Atlg34245 expression vector transformation strain (參), and the vector transformation strain (〇). It is an example of the scatter diagram of the pore density.

[FIG. 4] FIG. 4 shows an example of the results of Northern blotting in which At2g20875 expression vector transformation and expression of At2g20875 mRNA in the vector transformation were confirmed. 18S rRNA is an internal standard.

[FIG. 5] FIG. 5 shows an example of photographs taken on Day 20 of a control strain into which only a vector was introduced and an overexpression strain of Atlg34245.

BEST MODE FOR CARRYING OUT THE INVENTION

[0011] The stomatal regulator of the present invention preferably further has, for example, a function of suppressing epidermal cell density.

[0012] The plant production method of the present invention is a plant production method including a step of promoting or suppressing the expression of the stomatal regulator of the present invention.

[0013] The plant breeding method of the present invention comprises the nucleotide base sequence according to any one of (4) to (7) below: Alternatively, a plant breeding method comprising a step of detecting the presence of a gene of the stomatal regulatory factor of the present invention or a substitution, deletion or addition in the gene using a polynucleotide having a complementary sequence ability or a part thereof. .

(4) A nucleotide base sequence from the 158th to 517th positions of SEQ ID NO: 1 or SEQ ID NO: 1 in the sequence listing, or from the 102nd to 413th positions of SEQ ID NO: 3 or SEQ ID NO: 3.

(5) The nucleotide base sequence of at least one orthologous gene of Arabidopsis Atlg34245 gene and Arabidopsis At2g20875 gene or mRNA thereof.

(6) A nucleotide base sequence homologous to the nucleotide base sequence of (4) or (5) above, wherein at least one nucleotide is substituted, deleted or added.

(7) A nucleotide base sequence that is hybridized under stringent conditions with a complementary sequence of the nucleotide base sequence of (4) or (5) above.

In the present invention, the “plant” is not particularly limited, but is preferably a higher terrestrial plant having pores, and is used for, for example, food, feed, ornamental, environmental, experimental, and resource use. Includes plants. The “plant” in the present invention is, for example, a seed plant, which may be a gymnosperm plant, an angiosperm plant, a monocotyledonous plant, or a dicotyledonous plant. Furthermore, the “plant” in the present invention may be a woody plant or a plant plant. Examples of plants for food and feed include monocotyledonous plants such as wheat, barley, sorghum, millet, rye, triticale, corn, rice (rice), oats and sugar cane, rapeseed, pepper, Arabidopsis, cabbage or canola Brassicaceae, Soybean, Alfalfa, Endo, Kidney Bean or Legume, etc., Potato, Tobacco, Tomato, Eggplant or Pepper, etc. Includes dicotyledonous plants such as cucurbitaceae and celeryaceae such as carrots. Ornamental plants include roses such as roses, rhododendrons such as rhododendrons and azaleas; Genus, spinach, etc., orchids such as orchids, gladiolus, freesia containing Aya, crocuses, etc. Includes cucumbers such as figs. Environmental and resource plants include various trees and experiments Examples of plants for use include Arabidopsis thaliana, rice, corn, Miyakogusa and the like.

In the present invention, “the nucleotide base sequence of SEQ ID NO: 1 or 3 in the sequence listing” is an mRNA (cDNA) sequence encoded by the Arabidopsis gene Atlg34245 and At2g20875, respectively, Technology Information Center) Nucleotide nucleotide sequences with Nucleotide database registration numbers NM-103147 and NM-127657, respectively. In the present invention, the “mRNA sequence” includes a non-translated sequence and a coding sequence (CDS; Coding Sequence), one of them, or a partial force sequence thereof. For example, a sequence obtained by removing the stop codon from CDS is also included. “The sequence from the 158th to the 517th sequence of SEQ ID NO: 1” refers to the sequence obtained by removing the stop codon from the CDS of the Atlg34245 gene. “Sequence” refers to a sequence obtained by removing the stop codon from the CDS of the At2g20875 gene.

In the present invention, the “amino acid sequence of SEQ ID NO: 2 or 4 in the sequence listing” is the amino acid sequence encoded by the Arabidopsis gene Atlg34245 and At2g20875, respectively, and NCBI (US Biotechnology Information) Center) The amino acid sequences whose protein database registration numbers are NP-564442 and NP-179684, respectively.

[0017] In the present invention, the Arabidopsis Atlg34245 gene and At2g20875 gene refer to genes whose NCBI (National Center for Biotechnology Information) Nucleotide database registration numbers are NC-003070 and NC-003071, respectively.

[0018] In the present invention, "orthologous gene" refers to a corresponding gene in a different species. The orthologous gene or protein is also simply referred to as an ortholog. Orthologues in different species corresponding to a certain protein can be determined using, for example, homology with the amino acid sequence or nucleotide base sequence of the protein as an index. That is, the gene showing the highest homology among the genes of the different species can be determined as the orthologous gene. Here, homology means identity or similarity between two or more sequences. In the present invention, proteins include peptides, oligopeptides, and polypeptides.

[0019] The Arabidopsis Atlg34245 gene and the Arabidopsis At2g in (2) above Examples of orthologous genes of 20875 include rice (Orvza sativa) OSJNBa0036B21.12 and soybean (Glycine max) (BU550776jf). The phylogeny of this figure was created by analyzing the amino acid sequence of the protein encoded by the described gene using analysis software (ClustalW), as shown in the phylogenetic tree of the figure. The genes may include Sarasuko, maize BI43667 gene, and rice (rice) OSJNBb0034I13.14 gene.

Here, the Arabidopsis thaliana At4gl4723 gene and At3g22820 gene shown in FIG. 2 can reduce the stomatal density by overexpression. Therefore, the stomatal regulator of the present invention, in other embodiments, includes the cDNA sequences of At4gl4723 and At3g22820 (NM—001036564 (SEQ ID NO: 5) and NM—113181 (SEQ ID NO: 7)), a part thereof, a complementary sequence thereof, Or an amino acid sequence encoded by the At4gl4723 and At3g22820 genes (NP — 001031641 (SEQ ID NO: 6) and NP — 188921 (SEQ ID NO: 8)), a part thereof, or a portion thereof. Includes proteins consisting of these homologous sequences.

[0021] Gene A and gene B in Fig. 2 are unregistered genes of Arabidopsis thaliana and have the sequences of SEQ ID NOs: 9 and 10, respectively.

[0022] In the present invention, whether or not a factor has a function of suppressing pore formation density is measured by, for example, whether or not the number of pores per unit area of the leaf increases or decreases when the factor gene is disrupted. It can be said that if it breaks and the density increases, it has a suppression function. Similarly, in the present invention, whether a factor has a function of suppressing epidermal cell density depends on whether, for example, the number of epidermal cells per leaf unit area increases or decreases when the gene of the factor is destroyed. It can be measured, and if it breaks and the density increases, it can be said that there is a suppression function.

[0023] The stomatal regulatory factor of the present invention is suitable using, for example, the polynucleotide of SEQ ID NO: 1 or 3 in the sequence listing, preferably using the stomatal regulatory factor expression vector of the present invention described later. It can be obtained by expressing in cells.

[0024] The stomatal regulator of the present invention! , And (1) to (3) an amino group in which a group force is also selected An amino acid sequence derived from an acid sequence refers to an amino acid sequence after being processed by proteolysis in a plant. Examples of the processing include cleavage of a signal sequence and protein degradation by SDD1 protease and its ortholog.

[0025] The amino acid sequence of the stomatal regulator of the present invention includes (1) the amino acid sequence of SEQ ID NO: 2 or 4 in the sequence listing, and (2) the atologue gas of at least one of the Arabidopsis Atlg34245 gene and the Arabidopsis At2g20875 gene. In addition to the amino acid sequence encoded by the gene, it further comprises an amino acid sequence that is homologous to the amino acid sequences of (1) and (2) above.

[0026] In the present invention, the term "homologous" in the amino acid sequence of proteins means that they are sufficiently similar to maintain their functions. For example, even if there are differences in amino acid sequences due to substitutions, deletions, or attachments, they can be said to be “homologous” if they do not affect the function of the protein. In this case, the number of amino acid residues to be substituted, deleted or added is, for example, 1 to 40, 1 to 20, 1 to 15, 1 to: LO, 1 to 5, 1 to 3, One or two or one. Similarly, in the present invention, a nucleotide base sequence that is “homologous” means that it is sufficiently similar to maintain its function. For example, even if there are differences due to point mutations, deletions or additions in the nucleotide base sequence, they can be said to be “homologous” if they do not affect the function of the gene. As the number of different bases, for example, 1 to 60, 1 to 40, 1 to 20, 1 to 15, 1 to: LO, 1 to 5, 1 to 4, 1 to 3, One or two or one. In addition, when hybridizing with two polynucleotide force stringent conditions, both can be said to be homologous. In the present invention, the stringent conditions are not particularly limited. For example, in a solution containing 6 X SSC, 0.5% SDS, 5 X Denhardt, 0.01% denatured salmon sperm nucleic acid [Tm -25 ° C], and other conditions for overnight incubation. The Tm can be obtained by the following equation, for example.

Tm = 81. 5- 16. 6 (log [Na ⁺ ]) +0.41 (% G + C)-(600 / N)

Ten

In the above formula, N is the chain length of the polynucleotide, and% G + C is the content of guanine and cytosine residues in the polynucleotide. [0027] The stomatal regulator of the present invention can be used, for example, by promoting expression in plant cells in order to reduce the stomatal density. The stomatal regulator of the present invention can also be used as a target for destruction or inhibition in the plant genome, for example, to increase stomatal density or cell epidermal density.

[0028] One embodiment of the use of the stomatal regulator of the present invention includes the method for producing the plant of the present invention including the step of promoting or suppressing the expression of the stomatal regulator of the present invention. Here, plants produced by the production method of the plant of the present invention include plant individuals, their progeny and clones, and propagation materials (for example, seeds, fruits, cuttings, tubers, tuberous roots, strains, Callus, protoplast, etc.). Such a plant production method of the present invention can be used, for example, for production of a plant having an increased pore density or epidermal cell density or a plant having a decreased pore density.

[0029] The step of promoting or suppressing the expression of the stomatal regulator of the present invention is not particularly limited, and examples thereof include a method for producing a transgenic plant and a method for molecular breeding.

[0030] The transgenic plant in which the expression of the stomatal regulator of the present invention is promoted or suppressed will be described. In the present invention, the term “transgenic plant” refers to a plant cell, plant tissue, or plant individual that retains a foreign gene that has been incorporated into the genome of the plant by gene transfer. As well as propagation materials (eg, seeds, fruits, cuttings, tubers, tuberous roots, strains, callus, protoplasts, etc.) obtained from these forces. In the transgenic plant of the present invention, the stomatal regulator of the present invention whose expression is promoted or suppressed may be one or two or more when the plant has two or more types.

[0031] The method for producing a transgenic plant in which the expression of the stomatal regulator of the present invention is promoted is not particularly limited. For example, first, as a first step, the aforementioned ( Introducing a foreign gene into which a polynucleotide having the sequence of 4) to (7) is transcribed in a transcribable manner into a plant, and as a second step, selecting a host cell into which the foreign gene has been introduced and redifferentiating it into an individual. The method containing is mentioned. Examples of methods for introducing foreign genes into plants include the particle gun method, the polyethylene glycol method (PEG method), A direct physical method, such as the electroporation method, which directly punctures a part of a plant cell and introduces a gene into the plant cell from this hole, such as the agrobacterium method, virus Examples include indirect and biological methods that utilize the gene transfer ability of microorganisms and viruses, such as the vector method. The expression vector used for introduction into the plant is not particularly limited, but can be expressed in a plant cell, for example, a plasmid having a T-DNA region used in the agrobacterium method, or a virus vector. Tobacco Mosaic Virus (TMV), Bromo Mosaic Virus (BM)

V) and vectors produced by modifying plant RNA viruses such as cucumber mosaic virus (CMV).

[0032] The method for producing the transgenic plant in which the expression of the stomatal regulator of the present invention is suppressed is not particularly limited. For example, the Atlg34245 gene and the Z or At 2g20875 gene, or the gene for these orthologous genes. A method of inhibiting the synthesis of a complete protein by introducing a mutation such as substitution 'deletion' or addition to the sequence, a method of inhibiting transcription of the gene by inhibiting a transcription promoting factor or activating a transcription repressing factor, RNA Examples thereof include a method of inhibiting protein synthesis using interference and a method of overexpressing a dominant negative mutant of the gene product. As a method for introducing mutation into the gene sequence, for example, a transgenic plant library in which T DNA or transposon is inserted into a host plant is prepared, or an existing mutant library is screened, Screening for mutants in which the T-DNA or transposon is inserted at the locus to be detected, or by using the tilling (targeting induced local lesions m genomes) method ~ How to jung is mentioned. Examples of the RNA interference method include methods using antisense RNA, short inhibitory RNA (siRNA), microRNA (miRNA) and the like.

[0033] Examples of the method of molecular breeding a plant in which the expression of the stomatal regulator of the present invention is promoted or suppressed include a reverse genetic method that does not perform genetic recombination such as a tilling method. In such a molecular breeding method, the polynucleotides (4) to (7) and a part thereof may include the presence or absence of the gene of the stomatal regulatory factor of the present invention, substitution, deletion, addition, etc. It can be used as a primer or a probe in screening for detecting mutations.

[0034] The present invention will be further described with reference to the following examples, but the present invention is not limited thereto.

Example 1

[0035] Phenotypic analysis of Atlg34245 strain, At2 _g 20875 strain, and double strain

These double disruption strains were prepared from the Atlg34245 disruption mutant (SALK-047918; Arabidopsis BBiological Resource Center) and the At2g20875 disruption mutant (SALK-137549; the same as above). Specifically, seeds were obtained by self-pollination of a plant (F1) of a generation in which the Atlg34245 disruption mutant and the At2g20875 disruption mutant were crossed. Next, plant power DNA cultivated with these seed strengths was obtained, and those in which both genes were disrupted as homozygotes were selected as double disruption strains.

[0036] Next, for the obtained mutant, the density of epidermal cells (individual Zmm ² ) on the back side of the dorsal axis of the first leaf was measured 20 days after sowing. The results are shown in Table 1 below, and examples of microscopic photographs of wild strains, Atlg34245 disruption mutants, At2g20875 disruption mutants and double disruption strains are shown in panels A, B, C and D in Fig. 1, respectively. Show. As shown in the table and figure, the porosity density increased when either Atlg34245 or At2g20875 gene was disrupted (panels B and C in Fig. 1). In particular, in the Atlg34245 disruption mutant, epidermal cells other than the pores increased more markedly than the increase in pore density tl (Panel C in Fig. 1). Furthermore, in the case of the double disrupted strains of Atlg34245 and At2g20875, both stomatal density and epidermal cell density were greatly increased (Panel D in FIG. 1). It should be noted that the pore density decreased compared to the overexpressing strains of Atlg34245 and At2g20875 (see Example 2).

) o

[0037] As shown in panel B of Fig. 1, in the Atlg34245 disruption mutant, the pattern formation control in which the pores are increased and the pores are evenly distributed apart from each other is normal. On the other hand, in the conventionally known mutants of TMM and SDD1, the pores are adjacent and formed in an uneven distribution as the pore density increases. The efficiency of air and gas reaching the inside of the leaves is better when the pore formation distribution is uniform. In this respect, the regulation of pore formation by controlling the Atlg 34245 gene is the suppression of conventional TMM, SDD1, etc. It is superior to the pore formation control by.

[0038] [Table 1]

(1)

Example 2

[0039] Preparation of expression vectors for Atig34245 and At2g20875

First, a binary vector pTKO 16 having a cauliflower mosaic virus (CaMV) -derived 35S promoter in the T-DNA region was prepared as described in the literature (WO02Z07 2818). That is, an omega sequence, which is a translation promoting sequence, is ligated downstream of the 35SRNA gene promoter that is engineered so that the enhancer of the cauliflower mosaic virus 35SRNA gene is duplicated twice, and a multicloning site and a transcription termination sequence are downstream of it. The cassette was prepared and the pGPTV—Bar (Becker R et al., Plant

Molecular biology 20, 1195-1197, 1992) was ligated with the longer fragment of two fragments obtained by restriction enzyme treatment with Hindlll and EcoRI to obtain pTK016. Atlg34245 and At2g 20875 CDS (see SEQ ID NOS: 1 and 3 in the sequence listing) were linked to this multi-cleaning site of pTK016 to allow expression, and expression vectors pTK016—Atlg34245 and pTK016—At2g 20875 were prepared respectively .

[0040] Phenotypic analysis of eddy surplus strains

The expression vectors PTK016-Atlg34245 and pTK016-At2g20875 and the control vector pTK016 were transfected into callus of Arabidopsis thaliana by a conventional agrobacterium method to obtain a transformed strain. The agrobacterium method followed the method of Akama et al. (Akama, K. et al. 1992 Plant Cell Rep. 12, 7-11). After culturing the obtained transformant for 15 days, the pore density (individual Zmm ² ) of the pores behind the cotyledons and the first leaves (primary leaves) was measured. The results are shown in Fig. 3. As shown in the figure, the stomatal density of A tlg34245 and At2g20875 transformants tended to be low in both the cotyledons and the first leaves. When this result was Student t-tested, in both cases of Atlg34245 and At2g20875, the effect of suppressing the stomatal density due to overexpression was significant at p <0.01.

[0041] Further, for pTK016-At2g20875 transformation, it was confirmed that the decrease in stomatal density correlated with the expression level of At2g20875 gene. In other words, total RNA was prepared from the above-ground part of the 15 day individual sample (excluding cotyledons and first leaves), and the mRNA expression of At2g2-8075 gene was confirmed by Northern blotting. The pore density was measured. The results are shown in Fig. 4 and Table 2 below. Figure 4 shows total RNA prepared from pTK016-A t2 _g 20875 transformed sample numbers 5, 8, 21, 9, 16, and 18 and total RNA prepared from control strain sample numbers 1, 2, and 3. The results of Northern blotting for V2 and At2g20875 mRNA and 18S rRNA are shown below. The 18S rRNA is an internal standard. As shown in the figure, the pore density of the samples Nos. 5, 8, and 21 in which the expression level of At2g20875 mRNA was particularly high was significantly reduced as shown in Table 2 below. On the other hand, No. 9, 16, and 18 samples with low expression levels of At2g20875 mRNA showed high pore density close to that of the control strain (FIG. 4 and Table 2 below). Thus, it was confirmed that the degree of decrease in the pore density depends on the expression level of At2g20875.

[0042] [Table 2]

Example 3

[0043] Drought tolerance in eddy-expressing strains of Atlg34245

Next, the drought tolerance of the overexpressing strain of Atlg34245 was confirmed. The pTK016-Atlg34245 transformed T2 generation was used as an overexpressing strain of Atlg34245, and the T2 generation of a vector (pTD016) -introduced strain was used as a control. The drying resistance experiment was performed under the following conditions.

[0044] First, seeds absorbed for about 30 minutes were sterilized with 70% ethanol, washed, and seeded on a GM plate. The composition of the GM plate is MURASHIGE & SKOOG MEDIUM Basal Salt Mixture (DUCHIHEFA BIOCHEMIE No. M0221.0010) 4.3 g, 1% sucrose, 0.05% MES-KOH (pH 5.7), 500 X Vitamin stock solution (500ml, inositol 5g, thiamine hydrochloride lg, pyridoxine hydrochloride 50mg, nicotinic acid 50mg) 2ml, phytagel 3g. A plate with a diameter of 90 mm and a depth of 20 mm was used. After seeding, the plate was stored in a low temperature room for 2 days, and then the GM plate was transferred to a vertically-lighted vertical incubator and incubated at 23 ° C for 8 days. Then replanted to vermulite (50ml), 1Z2MS (2.15g of MURAS HIGE & SKOOG MEDIUM Basal Salt Mixture (same as above) was added to 800ml of deionized water and completely dissolved, adjusted to pH 5.7 with KOH. After that, 25 ml of 1 L) was added, and the plant was placed in a vat and covered with saran wrap, and incubated at 23 ° C under constant light on a room incubator. After this, water was not added to the plant. Moved to a room type incubator and removed Saran Wrap on the second day (Day 0).

[0045] Plants were observed and photographed on Day4, Day7, Dayl0, Dayl5, and Day20. On Day 7, it was almost dry. Also, sample the third leaf of the main leaf on Day 15 and fix it with fixative (90% ethanol, 10% acetic acid) for more than one night. Then, return the fixed leaf to water, and adjust the stomatal density on the back side of the leaf. Observed and measured with a microscope. The results are shown in Table 3 below and FIG. Table 3 below shows the survival rate calculated from the number of individuals alive and dead on Day 20, and Figure 5 shows the state of Day 20 in the control strain into which only the vector was introduced and the overexpressed strain of Atlg34245. An example of a photograph is shown.

[0046] [Table 3] (Table 3)

[0047] As shown in Table 3 above, it was confirmed that the Atlg34245-overexpressing strain significantly increased the survival rate under dry conditions (50%) compared to the control (5.3%). Furthermore, when focusing on the survival rate according to the phenotype (number of stomatal density) of the overexpressing strain, the survival rate increased to 60% in individuals with overexpression of Atlg34245 and the number of pores was about half that of the wild strain, In individuals with few pores, survival increased to 85.7%.

Industrial applicability

[0048] As described above, according to the present invention, since the number of pores and epidermal cells of a plant can be artificially adjusted, for example, an increase in biomass can be expected to improve drought resistance. Therefore, according to the present invention, a plant is modified so that it can cope with various water environments and CO environments.

2

For example, the present invention is useful in the fields of agriculture, forestry, and environmental conservation.

Claims

Claims [1] A stomatal regulator of a plant, comprising the following group powers (1) to (3): a selected amino acid sequence or an amino acid sequence derived therefrom, which suppresses the pore formation density of the plant A stomatal regulator characterized by having a function.

(1) The amino acid sequence of SEQ ID NO: 2 or 4 in the sequence listing.

[2] The stomatal regulator according to claim 1, which further has a function of suppressing epidermal cell density.

[3] A method for producing a plant, comprising the step of promoting or suppressing the expression of a stomatal regulator according to claim 1 or 2.

[4] A plant breeding method according to any one of the following (4) to (7), wherein any one of the nucleotide base sequences or a polynucleotide having a complementary sequence ability thereof or a part thereof is used. A plant breeding method comprising a step of detecting the presence or substitution, deletion or addition of the gene of the stomatal regulatory factor described in 1.

(6) A nucleotide base sequence in which one or several nucleotides are substituted, deleted or added to the nucleotide base sequence of (4) or (5).