WO2006098423A1

WO2006098423A1 - Polynucleotides imparting environmental stress-tolerance to plants

Info

Publication number: WO2006098423A1
Application number: PCT/JP2006/305328
Authority: WO
Inventors: Hirokazu Kobayashi
Original assignee: Shizuoka Prefectural Government
Priority date: 2005-03-14
Filing date: 2006-03-13
Publication date: 2006-09-21
Also published as: JPWO2006098423A1; JP5152845B2

Abstract

It is intended to provide a polynucleotide containing a polynucleotide comprising a base sequence represented by SEQ ID NO:3, 7, 9 or 11; a vector containing this polynucleotide; a transgenic plant having this vector transferred thereinto; and so on. The above-described polynucleotide containing a polynucleotide comprising a base sequence represented by SEQ ID NO:3, 7, 9 or 11 and so on can impart salt tolerance to plants. Therefore, the transgenic plant as described above and so on are useful in greening soil with salt accumulation, cultivating soil with salt accumulation, etc.

Description

Specification

Polynucleotide technology for imparting environmental stress tolerance to plants,

The present invention relates to a polynucleotide that imparts salt stress tolerance to a plant, and a transforming product into which the polynucleotide is introduced. Background art

There are various environmental stresses on plants, such as salt, drying, high temperature, low temperature, and air pollution, but the most problematic from the viewpoint of agricultural production is salt and drought. -The barren arid region on the earth occupies up to 1 Z 3 on land, and is increasing by 60,000 km ² Z years (area combined with Kyushu and Shikoku). In addition, the tropical rainforest is decreasing by 130,000 km 2 / year (the combined area of Hokkaido and Kyushu). In many farmland, the accumulation of soil salinity (mainly salty sodium) due to water evaporation has become a problem, and the total amount of salt accumulation soil distributed on the earth is 95,500,000 km ² . This is comparable to the land area of the United States.

On the other hand, the number of starved deaths on the earth is 28 (including 21 children) per minute, and the population of starvation is 15,000,000,000. More than a few people in the world are not satisfied with hunger, and the global food drought is obvious. Furthermore, since the world population is on the rise, a “world food crisis” is expected around 2010.

In addition, since global warming has become serious in recent years, it is highly necessary to increase the amount of carbon dioxide in the atmosphere by living organisms from the viewpoint of environmental problems. It is strongly desired.

On the other hand, various researches have been conducted on salt-tolerant plants. For example, it is a kind of salt-tolerant photosynthetic growth tolerance pst (photoautotrophic salt tolerance) in which photosynthetic growth (autotrophic growth) is more salt-tolerant than wild strains. There are reports about pst2 '(eg Tsugane, K., Kobayashi, K., Niwa, Υ., Ohba, Υ., Wada, Κ., and Kobayashi, Η., A recessive Arabidopsis mutant that grows photoautotrophically under salt stress shows enhanced active oxygen detoxification. See Plant Cell., 11, 1195-1206 (1999)). Disclosure of the invention

In the above situation, various researches have been conducted on plants having salt tolerance, but at present, the development of plants having sufficient salt tolerance has not been successful. Thus, development of transformed plants having sufficient salt tolerance is strongly desired.

As a result of intensive studies to solve the above-mentioned problems, the present inventors have found that a polynucleotide having a specific sequence can impart salt tolerance to a plant, and a transformed plant into which the polynucleotide has been introduced has salt tolerance. The present invention was completed. That is, the present invention provides the following polynucleotides, vectors containing the polynucleotides, plant cells transformed with the polynucleotides, plants transformed with the polynucleotides, and the like. To do.

[1] The polynucleotide according to any one of the following (a) to (f).

(a) a polynucleotide containing a polynucleotide comprising the nucleotide sequence set forth in SEQ ID NO: 3, 7, 9, or 11;

(b) a polynucleotide containing a polynucleotide encoding a protein consisting of the amino acid sequence set forth in SEQ ID NO: 1, 6, 8, or 10;

(c) the amino acid sequence set forth in SEQ ID NO: 1, 6, .8 or 10 consisting of an amino acid sequence in which one or more amino acids are deleted, substituted, inserted and Z or added; and A polynucleotide comprising a polynucleotide encoding a protein having a function of enhancing plant salt tolerance;

(d) encodes a protein having a homology of 80% or more with a protein consisting of the amino acid sequence set forth in SEQ ID NO: 1, 6, 8 or 10 and having a function of enhancing plant salt tolerance A polynucleotide containing a polynucleotide that:

(e) has a function of hybridizing with a polynucleotide comprising a nucleotide sequence complementary to the nucleotide sequence set forth in SEQ ID NO: 3, 7, 9 or 11 under stringent conditions and increasing the salt tolerance of plants A polynucleotide comprising a polynucleotide encoding the protein; and (f) encodes a protein having a homology of 80% or more with the polynucleotide comprising the nucleotide sequence set forth in SEQ ID NO: 3, 7, 9, or 11 and having a function of enhancing plant salt tolerance A polynucleotide containing a polynucleotide.

[2] The polynucleotide according to any one of the following (a) to (f).

(a) a polynucleotide comprising the base sequence described in SEQ ID NO: 4;

(b) a polynucleotide encoding a protein consisting of the amino acid sequence of SEQ ID NO: 2;

(c) The amino acid sequence shown in SEQ ID NO: 2, consisting of an amino acid sequence in which one or several amino acids are deleted, substituted, inserted, or attached, and has a function of increasing the salt tolerance of plants. A polynucleotide encoding a protein having;

(d) A polynucleotide encoding a protein having 80% or more homology with the protein consisting of the amino acid sequence set forth in SEQ ID NO: 2 and having a function of enhancing plant salt tolerance.

(e) a polynucleotide that encodes a protein that hybridizes with a polynucleotide comprising a nucleotide sequence complementary to the nucleotide sequence set forth in SEQ ID NO: 4 under stringent conditions and has a function of enhancing plant salt tolerance; Eiji

(f) a polynucleotide encoding a protein having a homology of 80% or more with the polynucleotide comprising the nucleotide sequence of SEQ ID NO: 4 and having a function of enhancing plant salt tolerance.

[3] The polynucleotide according to any one of the following (a) to (f).

(a) a polynucleotide containing a polynucleotide comprising the nucleotide sequence set forth in SEQ ID NO: 26;

(b) a polynucleotide containing a polynucleotide encoding a protein consisting of the amino acid sequence set forth in SEQ ID NO: 25;

(c) The amino acid sequence described in SEQ ID NO: 25 comprises an amino acid sequence in which one or more amino acids are deleted, substituted, inserted and / or added, and enhances salt tolerance of plants. A polynucleotide comprising a polynucleotide encoding a functional protein;

(d) a protein comprising the amino acid sequence set forth in SEQ ID NO: 25 and at least 80% A polynucleotide comprising a polynucleotide encoding a protein having homology and a function of enhancing plant salt tolerance;

(E) SEQ ID NO: 2 6 Porinukure Ochido and Sutorinjiwento conditions consisting of a nucleotide sequence complementary to the nucleotide sequence hybridizes described, and encodes a protein having high mel function salt ¹ production plant A polynucleotide containing a polynucleotide; and ¹ (f) having a homology of 80% or more with a polynucleotide comprising the nucleotide sequence set forth in SEQ ID NO: 26, and a function of increasing plant salt tolerance A polynucleotide comprising a polynucleotide encoding a protein.

[4] A polynucleotide comprising a polynucleotide comprising any one of the polynucleotides described in [1] above and any one of the polynucleotides described in [2] or [3] above.

[5] The polynucleotide of [4] above, comprising a polynucleotide having the base sequence of SEQ ID NO: 5.

[6] The polynucleotide according to any one of the following (a) to (f).

(a) a polynucleotide containing a polynucleotide comprising the base sequence set forth in SEQ ID NO: 28;

() A polynucleotide containing a polynucleotide encoding a protein comprising the amino acid sequence set forth in SEQ ID NO: 27;

(c) The amino acid sequence described in SEQ ID NO: 27 comprises an amino acid sequence in which one or more amino acids are deleted, substituted, inserted and Z or added, and the salt tolerance of the plant is increased. A polynucleotide comprising a polynucleotide encoding a protein having an enhancing function;

(d) a polynucleotide comprising a polynucleotide encoding a protein having a homology of 80% or more with the protein comprising the amino acid sequence of SEQ ID NO: 27 and having a function of enhancing plant salt tolerance ;

(e) A protein that hybridizes under stringent conditions with a polynucleotide comprising a nucleotide sequence complementary to the nucleotide sequence set forth in SEQ ID NO: 28 and that has the function of increasing plant salt tolerance. A polynucleotide containing a polynucleotide Leotide;

(f) a polynucleotide comprising a polynucleotide encoding a protein having a function of increasing the salt tolerance of a plant and having a homology of 80% or more with the polynucleotide comprising the nucleotide sequence of SEQ ID NO: 28.

[7] The polynucleotide according to any one of [1] to [6], which is DNA.

[8] A modified solid comprising the polynucleotide according to any one of [1] to [7] above,

[9]. The polynucleotide according to any one of [1] to [7] above, or the above

[8] A transformed cell carrying the vector according to [8].

[10] A transformed object incorporating the polynucleotide according to any one of [1] to [7] above or the vector according to [8] above.

[11] A transformed plant which is a descendant or clone of the transformed object according to [0]. By using the polynucleotide of the present invention, salt tolerance can be imparted to a plant, and therefore, a transformed cell having a salt tolerance can be obtained. Since the transformant or object of the present invention thus obtained can be grown even in salt-accumulated soil, it can contribute to the greening of the salt-accumulated soil. In addition, a salt-tolerant transformed plant obtained by incorporating a vector containing the polynucleotide of the present invention into an edible plant has the advantage that it can be cultivated in a place where it is difficult to grow normally due to salt damage. Brief Description of Drawings

FIG. 1 shows the bHLH19 gene genome sequence.

Fig. 2 shows the RT-PCR product (qualitative experiment) of the bHLH19 gene.

Figure 3 shows a possible mechanism for the generation of bHLH transcription factor mRNAs of different sizes.

FIG. 4 shows the results of RT-PCR for the N-terminal intron. 'Figure 5 shows alternative splicing of intron 1.

FIG. 6 is a diagram showing introduction of the bHLH19 gene into a binary vector. FIG. 7 shows growth from germination under salt stress conditions.

FIG. 8 is a diagram showing the results of RT-PCT for the C-terminal intron. BEST MODE FOR CARRYING OUT THE INVENTION

Hereinafter, the present invention will be described in detail based on the embodiments.

The present inventors have found that Arabidopsis Genome Initiative: Analysis of the genome sequence of the flowering plant Arabidopsis tha liana. Nature, 408, 796—815 (2000) In the case of the salt-tolerant photosynthetic growth mutant gun st (photoautotrophic salt tolerance) Pst2 (e.g. Tsugane, K., Kobayashi,-K., Niwa, Y., Ohba, Υ., Wada, K., and Kobayashi, H., A recessive Arabidopsis mutant that grows photoautotrophica 丄丄 y Under Cell stress shows enhanced active oxygen detoxification. Plant Cell., 11, 1195-1206 (1999)), the genes expressed under non-stress conditions were analyzed using DNA chips (oligo microarray), and At2g227 We found that the 60 (bHLH19) gene was highly expressed specifically in the wild strain. In addition, surprisingly, we found that alternative splicing occurs at the At2g22760 gene due to salt stress, resulting in a new transcript (RNA). The coding sequence of the cDNA of the new transcript found in the present invention is shown in SEQ ID NOs: 3, 4, 7, 9, and 11. The new cDNA found in the present invention also includes a nucleotide sequence (SEQ ID NO: 5) containing both the coding sequence represented by SEQ ID NO: 3 and the coding sequence represented by SEQ ID NO: 4. included.

The amino acid 酉 S rows of the proteins encoded by these cDNAs of the present invention are shown in SEQ ID NOs: 1, 2, 6, 8, and 10. In Examples described later, a vector containing the cDNA of SEQ ID NO: 5 was prepared, and a transformed plant transformed with this vector was prepared. A transformed plant transformed with a vector containing the cDNA of this new transcript of the present invention is a normal transcript in the At2 _g 22760 gene. More highly salt-tolerant than plants into which a vector containing a cDNA, ie, a vector containing a DNA having the base sequence (SEQ ID NO: 13) encoding normal bHLH19 (SEQ ID NO: 12) is introduced. I found.

Based on these findings, the present invention provides a polynucleotide encoding a protein that imparts salt stress tolerance, a vector containing the polynucleotide, and a transformed cell into which the vector or polynucleotide has been introduced. Pi transformation A plant body is provided.

1. Polynucleotide of the present invention

The polynucleotide of the present invention is preferably DN A, and specific examples thereof include cDNA and synthetic DNA. The biological species from which DNA is derived is not particularly limited, but is preferably a plant. Plants include, for example, Arabidopsis, rice, corn, wheat, barley, soybean, tomato, straw, tobacco, rapeseed, potato, sugar beet, sugarcane, sunflower, shiba, trees (poplar, eucalyptus, etc.) However, it is not limited to this.

Specific examples of the polynucleotide of the present invention include, for example,

[1] The polynucleotide according to any one of the following (a) to (f) is exemplified. (a) a polynucleotide containing a polynucleotide comprising the nucleotide sequence set forth in SEQ ID NO: 3, 7, 9, or 11,

(b) a polynucleotide comprising a polynucleotide encoding a protein consisting of the amino acid sequence set forth in SEQ ID NO: 1, 6, 8 or 10;

(c) In the amino acid sequence set forth in SEQ ID NO: 1, 6, 8 or 10, consisting of an amino acid sequence in which one or more amino acids are deleted, substituted, inserted or added, and a plant A polynucleotide comprising a polynucleotide encoding a protein having a function of increasing the salt tolerance of

(d) a protein encoding a protein having a homology of 80% or more with a protein comprising the amino acid sequence set forth in SEQ ID NO: 1, 6, 8 or 10 and having a function of enhancing plant salt tolerance. A polynucleotide containing nucleotides,

.. (e) Is the base sequence complementary to the base sequence described in SEQ ID NO: 3, 7, 9 or 11 Or a polynucleotide containing a polynucleotide encoding a protein or quality having a function of increasing the salt tolerance of a plant and hybridizing under stringent conditions, or

(f) encodes a protein having a homology of 80% or more with the polynucleotide comprising the nucleotide sequence set forth in SEQ ID NO: 3, 7, 9 or 11 and having a function of enhancing salt tolerance of plants. A polynucleotide containing a polynucleotide to be treated.

Other specific examples of the polynucleotide of the present invention include, for example, the polynucleotide described in any one of the following (a) to (f): (a) a polynucleotide comprising the base sequence described in SEQ ID NO: 4;

(b) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO: 2,

(c) the amino acid sequence of SEQ ID NO: 2, comprising an amino acid sequence in which one or more amino acids have been deleted, substituted, inserted and / or attached, and has a function of enhancing plant salt tolerance (D) encoding a protein having a homology of 80% or more with the protein comprising the amino acid sequence described in SEQ ID NO: 2 and having a function of enhancing plant salt tolerance Polynucleotides,

(e) encodes a protein that hybridizes under stringent conditions with a polynucleotide consisting of a base sequence complementary to the base sequence set forth in SEQ ID NO: 4 and has a function of increasing plant I ¹ tolerance. Polynucleotide, or

(f) a polynucleotide encoding a protein having a homology of 80% or more with the polynucleotide comprising the nucleotide sequence of SEQ ID NO: 4 and a function of enhancing plant salt tolerance;

Other specific examples of the polynucleotide of the present invention include, for example,

[3] The polynucleotide according to any one of the following (a) to (f) can be mentioned. '

(a) a polynucleotide containing a polynucleotide comprising the base sequence set forth in SEQ ID NO: 26 or 28;

(b) a protein comprising the amino acid sequence set forth in SEQ ID NO: 25 or 27 A polynucleotide comprising a polynucleotide to be isolated;

(c) In the amino acid sequence set forth in SEQ ID NO: 25 or 27, it consists of an amino acid sequence in which one or more amino acids are deleted, substituted, inserted and Z or added, and is salt-tolerant in plants. A polynucleotide containing a polynucleotide encoding a protein having a function of enhancing the activity;

(d) containing a polynucleotide encoding a protein having a homology of 80% or more with the protein consisting of the amino acid sequence described in SEQ ID NO: 25 or 27 and having a function of enhancing plant salt tolerance A polynucleotide to:

(e) a protein that hybridizes with a polynucleotide comprising a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 26 or 28 under stringent conditions and has a function of enhancing plant salt tolerance A polynucleotide containing the polynucleotide to be treated;

(f) containing a polynucleotide encoding a protein having a homology of 80% or more with the polynucleotide comprising the nucleotide sequence set forth in SEQ ID NO: 26 or 28 and having a function of enhancing plant salt tolerance Polynucleotide.

The polynucleotide of the present invention includes a polynucleotide comprising a polynucleotide comprising any one of the polynucleotides exemplified in the above [1] and any one of the polynucleotides exemplified in the above [2]. And a polynucleotide containing a polynucleotide having the base sequence set forth in SEQ ID NO: 5 is preferred.

For proteins with a certain amino acid sequence, one or more amino acids are deleted, substituted, inserted and protein with Z or added amino acid sequence. It is already known to maintain its biological activity. (For example, Mark, DF et al., Proc. Natl. Acad. Sci. USA 81, 5662-5666 (1984), Zoller, M. J "and Smith, M., Nucleic Acids Research 10, 6487-6500 (1982 ), Dalbadie- McFarland, G. et al "Proc. Natl. Acad. Sci. USA 79, 6409-6413 (1982), Bowie et al., Science 247, 1306-1310 (1990), etc.). Accordingly, in the amino acid sequence set forth in SEQ ID NO: 1; 6, 8, or 10, it comprises an amino acid sequence in which one or more amino acids are deleted, substituted, inserted, and / or attached, and A polynucleotide containing a polynucleotide encoding a protein having a function of enhancing salt tolerance is also within the scope of the present invention.

A polynucleotide encoding a protein having an amino acid sequence in which one or several amino acids have been deleted, substituted, inserted and / or added to a certain amino acid sequence is obtained by site-directed mutagenesis (eg, Gotoh, T. et al., Gene 152, 271-275 (1995), Zoller, MJ, and Smith, M., Methods Enzymol. 100, 468-500

(1983), Kramer, W. et al., Ucleic "Acids Res. 12, 9441-9456 (1984), Kramer W, and Fritz HJ, Methods. Enzymol. 154, 350-367 (1987), Kunkel, TA, Proc. Natl. Acad. Sci. USA. 82, 488-492 (1985), Kunkel, Methods Enzymol. 85, 2763-2766 (1988), etc.), methods using amber mutation (eg, Gapped duple method) , Ucleic Acids Res. 12, 9441-9456

(See 1984), etc.).

In addition, PCR using a pair of primers (for example, Ho SN et al., Gene) having a sequence introduced with the desired mutation (deletion, addition, substitution and Z or insertion) at the 5 'end of each. 77, 51 (1989), etc.) can also introduce mutations into polynucleotides.

A polynucleotide that encodes a partial fragment of a protein that is a type of deletion mutant has a sequence that matches the base sequence at the 5th and end of the region that encodes the partial fragment to be prepared in the polynucleotide that encodes the protein. It can be obtained by performing PCR using an oligonucleotide having a nucleotide sequence complementary to the nucleotide sequence at the 3 'end as a primer and using a polynucleotide encoding the protein as a cocoon.

The number of amino acids to be deleted, added, substituted, or inserted is not particularly limited,

Any number of 1 or more is acceptable, but it is preferable that the number be sufficient to allow deletion, addition, substitution, or insertion by the above-mentioned site-directed mutagenesis method or PCR. In general, it is about 1 to 50, preferably 1 to 30, more preferably 1 to 20, more preferably 1 to 10, particularly preferably 1 to several (example 6).

In addition, in order for the protein of the present invention to have a function of increasing plant salt tolerance, The homology with the amino acid sequence described in column numbers 1, 2, 6, 8 or 10 is BLAST (see, for example, Altzshul SF et al "J. Mol. Biol. 215, 403 (1990), etc.) and FASTA (See Pearson WR, Methods in Enzymology 183, 63 (1990), etc.) When using the default (initial setting) parameters in an analysis program such as, at least 80% or more, preferably 90% or more More preferably, it is 93% or more, more preferably 95% or more, particularly preferably 97% or more, and most preferably 98% or more.

“Polynucleotide hybridizing under stringent conditions” refers to a colony 'hybrid using a polynucleotide consisting of a base sequence described in 3, 4, 7, 9, or 11 as a probe. This refers to polynucleotides obtained by using the dialysis method, plaque hybridization method, or Southern plot hyper-precipitation method, and more specifically, DNA derived from colonies or plaques. After the hybridization at 65 ° C in the presence of 0.7 to 1.0.mo 1 ZL of Na C 1 using a filter with immobilized SSC (Saline-sodium citrate) solution (The composition of 1-fold concentration of SSC solution consists of 15 Ommo1 / L sodium chloride, 15mmO1 / L sodium citrate) Clean the filter at 65 ° C It is possible to increase the polynucleotide that can be identified by.

Molecular hybridization, Sambrook J. et al., Molecular Cloning: A Laboratory Manual, Third Edition, Cold Spring Harbor Laboratory Press (2001) (hereinafter abbreviated as Molecular 'Cloning 3rd Edition), Ausbel FM et al., Current Protocols m Molecular Biology, Supplement 1-38, dohn Wiley and Sons (1987-1997), Glover DM and Hames BD, DNA Cloning 1: Core Techniques, A practical Approach, Second Edition, Oxford University Press (1995) Can be carried out according to the method described in. . In the hybridization reaction of the present invention, various stringent conditions can be used. The more severe the conditions, the higher the complementarity required for duplex formation.

“Low stringent conditions” suitable for the purposes of the present invention include, for example, 5 XSSC, 5 X Denhardt's solution, 0.5% SDS, 50% formamide, hybridization at 32 ° C, followed by 2XSSC, 0.1% SSC, washing at room temperature. “Medium stringent conditions” are, for example, 5 XSSC, 5 X Denhardt's solution, 0.5% SDS, 50% formamide, 42. Hybridization under C conditions, followed by 0.2XSSC, 0.1% SDS, 37 ° C washing. “High stringency conditions” include, for example, 5 XSSC, 5 X Denhanol solution, 0.5% SDS, 50% formamide, hybridization at 42 ° C, followed by 0.1 XSSC, 0.1% Wash with SDS at 65 ° C. Under these conditions, the higher the temperature, the higher the DNA with homology can be expected to be obtained efficiently. However, there are several factors that influence the stringency of hybridization, such as temperature, probe concentration, probe length, ionic strength, time, and salt concentration. It is possible to achieve the same stringency by appropriately selecting.

The polynucleotide is hybridized with a polynucleotide comprising a nucleotide sequence complementary to the nucleotide sequence set forth in SEQ ID NO: 3, 4, 7, 9 or 11 under stringent conditions, and has a function of increasing plant salt tolerance. To be a polynucleotide that encodes a protein, it is described in SEQ ID NO: 3, 4, 7, 9 or 11 when calculated using the default (initial setting) parameters in an analysis program such as BLAST or FAS TA. At least 80% or more, preferably 90% or more, more preferably 93% or more, more preferably 95% or more, particularly preferably 97% or more, and most preferably 98% or more. It is desirable to have a polynucleotide.

Examples of the nucleic acid of the polynucleotide of the present invention include DNA and RNA. The polynucleotide of the present invention may be single-stranded or double-stranded, and both are included.

The polynucleotide of the present invention is, for example, a suitable cell having DNA encoding the protein of the present invention (for example, a protein having the amino acid sequence described in SEQ ID NO: 1, 2, 6, 8 or 10). Alternatively, cDNA can be synthesized based on mRNA prepared from tissues and inserted into a vector such as I ZAP. This can be obtained by developing and developing and screening using a probe prepared based on the polynucleotide of the present invention. In addition, a primer specific to a polynucleotide encoding the protein of the present invention (for example, a polynucleotide having the base sequence described in SEQ ID NO: 3, 4, 7, 9, or 11) is prepared, and this is used. It can also be obtained by performing PCR, and cDNA is cloned by PCR using sequence information (MJ McPherson et al., PCR, A practical Approach, Oxford University Press (1991)). You can also. For such cDNA cloning, for example, the RACE (Rapid Amplification of cDNA ends) method is used.

The base sequence of the obtained cDNA is DNA sequencer (Applied Biosystems 3 7 7 etc.) manufactured by Applied Biosystems, Applied Biosystems, Amersham 'Falmasia' Biotech, and LI-COR ) And the dideoxy method (Sanger F. et al., Proc. Natl. Acad. Sci. USA 74, 5463 (1977)).

2. Recombinant vector for plant transformation of the present invention

The recombinant vector of the present invention can be prepared by inserting the above DNA of the present invention into an appropriate vector. As a vector, a pBluescript type vector, a pBI type vector, a pUC type vector and the like can be used.

Binary vector vectors such as pBluescript vectors and pBI vectors are preferred because the target DNA can be introduced into plants via agrobacterium. Examples of the pBluescript vector include pBluescript SK (+), pBluescript SK, pBluescript II KS (+), pBluescript II KS, pBluescript II SK (ten), and pBluescript II SK (-). Examples of pBI vectors include pBI121, pBI101, pBI101.2, pBI101.3, pBI221 and the like. A pUC vector is preferable in that DNA can be directly introduced into a plant. 'The vector contains a promoter for constitutive gene expression in plant cells (eg, the cauliflower mosaic virus (CaMV) 35S promoter). Or a promoter that is inducibly activated by an external stimulus (eg, drying, irradiation with ultraviolet rays, salt stress). Such promoters include, for example, the Arabidopsis rabl6 gene promoter induced by drying (Nundy et al., Proc. Natl. Acad. Sci. USA 87, 1406 (1990)), UV irradiation. The promoter of the parsley chalcone synthase gene induced by lysine (Sclmlze-Lefert et al "EMBO J. 8, 651 (1989)) and the promoter induced by salt stress (Shinozaki, K. and Yamaguchi-Shinozaki, K , Curr. Opin. Plant Biol. 3, 217-223 (2000)).

Furthermore, a promoter, an enhancer, a terminator, a poly A addition signal, and the like may be linked to the vector of the present invention as necessary.

The promoter does not have to be derived from a plant as long as it can function in plant cells. Specific examples include CaMV35S promoter, promoter of nopaline synthase gene (Pnos), ubiquitin promoter derived from maize, actin promoter derived from rice, and PR protein promoter derived from tobacco. Furthermore, there is a promoter that is activated inductively by the aforementioned external stimulus.

An example of an enhancer is an enhancer region containing an upstream sequence in the CaMV35S promoter.

The terminator may be any sequence that can terminate the transcription of the gene transcribed by the above-mentioned promoter. Specific examples include nopaline synthase gene terminator (Tnos) and CaMV poly A terminator.

In order to insert the DNA of the present invention into a vector, first, the purified DNA is cleaved with an appropriate restriction enzyme, and inserted into a restriction enzyme site or a multicloning site of an appropriate vector DNA. Used.

3. Transformed plant cell and transformed plant body of the present invention ''

The transformed plant cell of the present invention can be obtained by introducing the recombinant vector of the present invention into a plant cell. Recombinant vectors can be introduced into plant cells by methods known in the art, such as those introduced via viruses or bacteria that infect plants (I. Potrylkus, Annu. Rev. Plant Physiol. Plant Mol. Biol. 42, 205 (1991))

For example, direct introduction of DNA. Specifically, the agro-pacteria method, particle gun method, PEG method, electoral position method, etc. can be used. These methods can be appropriately determined depending on, for example, the type of host plant to be transformed.

Cauliflower mosaic virus, gemini virus, tobacco mosaic virus, prom mosaic virus, etc. can be used as viruses that infect plants. .

Transfer of the vector to the agglomerate can be done by the Elect Mouth Position method.

Examples of methods for directly introducing exogenous DNA into plant cells include the microinjection method, the electoral position method, and the like. One known method such as the Takenoregan method, the polyethylene Daricol method, the fusion method, and the high-speed paristic penetration method (see L Potrykus, Annu. Rev. Plant Physiol. Plant Mol. Biol. 42, 205 (1991)). ) The electroporation method is preferably applied, for example, to plant cells in which protoplast culture is stable and easy and can be easily regenerated. In addition, since the particle gun method is not limited to the host, it is preferably applied to, for example, plant cells that are difficult to infect agrobacterium and plant cells that are difficult to prepare protoplasts. In addition, when the electopore position method is performed in this way, as a vector constituting the recombinant vector, for example, pUC18, pUC19, pBR322, pBR325, pBluescript and the like are preferable. Since many monocotyledonous plants are not susceptible to infection by agrobacterium, the indirect introduction method using agrobacterium, which is widely used as a DNA introduction method, cannot be used. Is effective.

Next, T1 DNA is introduced into the plant from agrobacterium, etc. into which the vector of the present invention has been introduced, and the plant is transformed. For example, after agrobacterium having introduced a vector as described above was allowed to coexist for several minutes with callus or tissue pieces of plant cells, in a medium such as 2N6-AS or N6CO, 25-28 ° C for about 3 days Incubate. Here, seed plants having different levels of difficulty in co-cultivation are used. In particular, monocotyledonous crops that have been difficult to transform until now, such as rice, wheat, barley, corn, and buckwheat, and trees (such as poplar and eucalyptus) can also be targeted.

After co-cultivation with the above agrobacterium, calli or tissue pieces are selectively cultured in a medium containing an appropriate antibiotic. For example, when a hygromycin resistance gene is introduced into a vector as a selectable marker, hygromycin (10 to: 100

IX g / mL) and 2N6-CH or N6Se medium containing cefotaxime (25 g / mL) or carpenicillin (500 / ig / inL) for agropactericum removal for 1-3 weeks By performing selective culture, transformed callus bodies can be selectively obtained. The callus body obtained selectively is induced to re-differentiate using an appropriate re-differentiation medium such as N6S3-CH or MSre to obtain re-differentiated individuals. As described above, a DNA fragment can be introduced into a plant and transformed using the vector of the present invention. Recombinant individuals can be selected by examining the expression of an antibiotic resistance gene (eg, a hygromycin gene) using a marker gene. In addition, chromosomal DNAs of the obtained transformed plant cells and transformed plant bodies are respectively prepared, for example, by PCR or Southern blotting using a primer or probe specific to the target DNA sequence. If the expression of DNA is confirmed, the desired transformed cells and transformed plants are obtained.

If a transformed m 3 object is obtained, offspring can be obtained from the plant body by sexual reproduction or asexual reproduction, and clones can also be obtained by known methods. In addition, further progeny or clones can be obtained from the plant body, its progeny or clone.

The plant transformed by introducing the vector or polynucleotide of the present invention is not particularly limited, and examples thereof include crops and ornamental plants. Examples of crops include rice, corn, wheat, barley, soybeans, tomatoes, mandarin oranges, apples, strawberries, strawberries, tobacco, coffee trees, chiya, rapeseed, potatoes, sugar beets, sugar cane, sunflower seeds, and rubber trees. Ornamental plants include chrysanthemum, carnation, para, cyclamen, Cattleya, petunia, tulips, gerberas and so on.

In the present invention, “increasing salt tolerance” means, for example, a plant cell or plant,

When compared to non-transformed plant cells or plants, the total weight of callus or plant, root elongation, leaf It means that the growth under salt stress is improved, such as the force with larger total area or number of sheets, or the suppression of whitening of Z and leaves (foliage, true leaves, etc.). -Salt stress conditions such as the salt used for salt stress, its concentration, and growing period can be appropriately set according to the type of plant used. Examples of salts used for salt stress include salt potassium and salt sodium, with sodium chloride being preferred. The salt concentration is, for example, 0.1 to 2 M, preferably about 0.05 to 1 M, more preferably 0.1 to 0.5 M, and particularly preferably 0.2 to 0.3 M. . The period for applying salt stress is, for example, 1 hour to 10 weeks, preferably 2 hours to 8 weeks, more preferably 1 day to 4 weeks, and particularly preferably 1 week to 3 weeks.

In the present invention, unless otherwise specified, detailed experimental procedures are described in Molecular _Cloning _3rd Edition, _Augbel F M. et al., Current Protocols in Molecular Biology, Supplement _1-38 , John Wiley and Sons (1987.1997), Glover DM and Hames BD, DNA Cloning 1: Core Techniques, A practical Approach, Second Edition, Oxford University Press (1995), etc. This can be done by the method described in the instruction manual of the commercially available kit. Example

Hereinafter, the present invention will be described more specifically based on examples. The present invention is not limited to these examples.

First, the materials and experimental methods used in the present invention will be described. -

1. Plant material

In this study, we used the Arabidopsis wild-type strain Columbia (Col) and our laboratory. Preservation · Used Arabidopsis thaliana. 2-1-6 transgenic plants were created in our laboratory. Under the control of the photosynthetic gene RBCS-3B promoter, the β-dalcronidase (GUS) gene (uidA) and the chlorophyll-binding protein gene CAB1 are under the control of the promoter. A wild-type strain using the agro-pacterium method has a construct having a luciferase gene in the RBCS-3B promoter and a hygromycin resistance gene (iph) under the control of the RBCS-3B promoter and a bialaphos resistance gene Cbar under the control of the CAB1 promoter. _P st2 (63) derived from the 2-1-6 line was used as the mutant plant introduced into Col. 2. Growth conditions

Using an artificial weather machine (Biotron NC220, Nksystem), they were grown for 3 weeks under continuous white light conditions of 22 ° C, humidity 40-60%, and illuminance 3,000-5,000 lux. When salt stress was applied, the cells were transferred to a medium supplemented with 0.2 M NaCl and cultured under the same conditions. 3. Seed sterilization and sowing

Seeds were dispensed into a 1.5-mL tube, 1 mL of 70% ethanol was added, and the mixture was shaken for 1 minute. Centrifuge, discard the supernatant, add 1 mL of sterile solution (0.25% hypochlorous acid, 0.1% Tween 20) and shake for 10-15 minutes. Centrifuge lightly in a clean bench, discard the supernatant, and rinse with 1 mL ultrapure water. Rinse was repeated 5 times, and 1 mL of ultrapure water was added, wrapped in aluminum foil, and springed at 4 ° C for about 1 week. The sample was centrifuged briefly in a clean bench, the supernatant was discarded, and it was suspended in 1 mL of 0.3% agarose solution that had been autoclaved (121 ° C, 20 minutes). A filter paper that had been autoclaved (121 ° C, 20 minutes) was seeded on a petri dish that had been spread on the medium.

4. Preparation of fungal medium

(1) Mineral medium

The following composition is included. _{5.0 mM KNO 3, 2.5 mM K} 2 HP0 4 -H 3 P0 4 (H 5.5), 50 mM Fe- EDTA, 2.0 mM MgS0 4. 7H 2 0, microelements [70 mM H 3 B0 4, 14 mM MnCl 2 · 4H ₂ 0, 0.5 mM CuS0 ₄ 5¾0, 1 mM ZnS0 ₄ 7H ₂ 0, 0.2 mM Na ₂ Mo0 ₄ 2¾0, lO mM NaCl, 0.01 mM CoCl ₂ · 6¾0], 0.1 mM Ca (N0 ₃ ) ₂ , 0.3% Gelrite ₀ medium solution is prepared, then autoclaved (121 ° C, 20 minutes) and cooled to about 50 ° C Then dispensed into petri dishes. However, Ca (N0 ₃ ) ₂ was autoclaved separately (121 ° C, 20 minutes) and added before dispensing. When 200 mM NaCl was added, 11.69 g NaCl / 1 L was added to the above medium composition.

(2) Murashige Skoog medium (MS medium)

The following composition is contained in 1L of ultrapure water (prepared to pH 6.3 with 2N KOH). After the medium solution was prepared, it was autoclaved (121 ° C, 20 minutes), cooled to about 50 ° C, and dispensed into a shear.

Mixed salt for Murashige Skoog medium 4.3 g

Sucrose 20 g

1000 X vitamin stock 1 mL

2.4 m pyndoxine hydrochloride

Gelrite

(3) 1 ^ Medium

The following composition is contained in 1L of ultrapure water. The medium solution was adjusted and autoclaved (121 ° C, 20 minutes). In the case of a solid medium, 1.5% Bacto-agax was added to the medium solution, and after autoclaving, the solution was cooled to about 50 ° C and then dispensed into a petri dish.

Bacto-Peptone 10 g

Bacto- Yeast extract 5 g

NaCl 10 g

5. Oligo microarray

(1) RNA purification 'fluorescent labeling

RNA 20 purified using RNeasy Plant Mini Kit (Qiagen) was fluorescently labeled using Agilent Fluorescent Direct Label Kit (Agilent Technologies).

(2) Hybridization ぉ yopi data processing

An Agilent Araoiaopsis 1 icroarray Kit (Agilent Technologies) was used and followed the standard y-mouth protocol. Data acquisition is performed using the Agilent DNA Microarray Scanner. (Agilent Technologies) was used, and Feature Extraction (Agilent Technologies) was used for the numerical value of Signanore. Excel (Microsoft) was used for the subsequent analysis.

6. R A C E

Using the SMART ™ RACE cDNA Amplification Kit (Clontech), primer design, cDNA preparation and RACE PCR were performed according to standard protocols.

(1) TA cloning

The RACE PGR product was ligated and transformed using the pGEM-T (Promega) vector with Compitent High JM109 (Toyobo) as the host E. coli according to the standard protocol of the pGEM-T Vector System.

(2) Colony PCR

Colony PCR was performed to confirm whether cloning was successful. Using TaKaRa Ex Taq ™ Hot Start Version (Takara) with DNA polymerase, 94 ° C 5 min → (94 ° C 15 sec, 55 ° C 15 sec, 72 ° C 30 sec) X 35 cycle → 72 ° C 5 Based on the temperature conditions of the samples, _c- agarose gel electrophoresis was performed by PCR according to the size of the primer and target product, and the size of the product was confirmed.

(3) Plasmid recovery

The transformed Escherichia coli was cultured in an LB liquid medium containing 50 mg / mL ampicillin at about 150 rpm at 37 ° C for 14 hours. The culture was transferred to a 1.5 mL tube and centrifuged at 6,000 rpm for 4 minutes at 4 ° C. The supernatant was removed, and the pellet was suspended by pipetting with 200 Solution I. Add 300 xL of Solution Π and gently invert 2 to 3 times and let stand on ice for 10 minutes. 300 iL of Solution was added and gently inverted 2 to 3 times and allowed to stand on ice for 10 minutes. Centrifugation was performed at 15,000 rpm at 4 ° C for 10 minutes, and the supernatant was collected. 1 μΐ: 1 mg / mL RNase A was added and allowed to stand at 37 ° C for 20 minutes or longer. 200 chloroform was added, and the mixture was vigorously stirred and centrifuged at 15,000 rpm at 4 ° C for 10 minutes, and the upper layer (aqueous layer) was collected (performed twice). Stirred vigorously adding an equal volume of isopropanol, 15,000 rpm 4 ° C for 10 minutes centrifugation, the Peretsuto remove the supernatant was centrifuged washed _c 15,000 rpm 4 ° _C 10 minutes with 70% ethanol 600 x, the supernatant was removed Dry up. Dissolve in 10-20 μΐ or ultrapure water to make a plasmid solution. Solution I (in 1L of ultrapure water)

Solutionll (Adjustment at the time of use: Prepare the target amount with ultrapure water)

2NNaOH (Adjustment when using) 1/5 volume

10% SDS 1/10 times

SolutionlE (in 1 L of ultrapure water)

Potassium acetate 29.44 g

Acetic acid 11.5 mL

After adjustment, it was autoclaved (121 ° C, 20 minutes). (4) Restriction enzyme treatment

The recovered plasmid solution was digested with a restriction enzyme that can confirm the correct construct. After the treatment, agarose gel electrophoresis was performed.

(5) DNA sequence

Using the collected plasmid as a template, use DYEnamic ™ Terminator Cycle Sequence Kit (Amercham Biosciences) ¾, anti-L, solution 20 (10 μmol of template 1 μΐ ^ each primer, 8 L premix, 20 μΙ with ultrapure water, Reaction conditions were 94 ° C for 20 seconds → (95 ° C for 20 seconds, 50 ° C for 15 seconds, 60 ° C for 1 minute) X 30 cycle, PCR was performed using Autoseq G-50 (Amersham Biosciences) The sample was purified according to the label standard protocol, and run and analyzed with ABI PRISM ™ 310 Genetic Analyzer (ABI).

7. Real-time RT—PCR

(1) R A purified opi DNase I treatment

Plant plants (about Ol g) were frozen in liquid nitrogen, and the ruptured mortar was transferred to a new tube. 1 mL Isogen ί Nippon which has been incubated at 50 ° C in advance NJ) was added, and violently portexed. Incubated at 50 ° C for 10 minutes, and then allowed to stand at room temperature for 5 minutes. 0.2 mL black mouth form was added, vortexed, and allowed to stand at room temperature for 2 minutes. The mixture was centrifuged at 15,000 rpm (15,000 Xg) for 15 minutes (4 ° C), and the upper layer (aqueous layer) was collected and transferred to a new tube. 0.2 mL of black mouth form was added, mixed vigorously for 1 minute, and centrifuged at 12,000 rpm (10,000 Xg) for 5 minutes (4 ° C) to recover the upper layer (aqueous layer). This black mouth form treatment was repeated until there was no protein in the intermediate layer. 0.5 mL isopropanol was added to the upper layer (aqueous layer), and lightly mixed for 5 minutes. The mixture was centrifuged at 15,000 rpm (15,000 X ^) for 15 minutes (4 ° C), the supernatant was discarded, and the precipitate was air-dried. The precipitate was dissolved in 0.1 mL RNase-free water and mixed with 10 μ∑ 3Μ sodium acetate. The plate was allowed to stand on ice for 20 minutes, and centrifuged at 15,000 rpm (15,000 Xg) for 15 minutes (4 ° C). The supernatant was collected and added with 0.25 mL ethanol and incubated at 70 ° C: 1 hour. The mixture was centrifuged at 15,000 rpm (15,000 X ^) for 10 minutes (4 ° C), the supernatant was discarded, and 1 mL 70% ethanol was added to rinse. The mixture was centrifuged at 15,000 rpm (15,000 Xg) for 5 minutes (4 ° C), the supernatant was discarded, and the precipitate was air-dried. The precipitate was dissolved in 20 μ RNase-free water. RNase-free Deoxyribonuclease I (Takara) was used and reacted at 37 ° C for 30 minutes according to standard protocol. The black mouth form treatment was repeated until the protein in the intermediate layer disappeared, and total RNA was recovered by ethanol precipitation.

(2) Preparation of template for cDNA synthesis and calibration curve

cDNA was prepared using 1st Strand cDNA Synthesis Kit for RT-PCR (AMV) (Roche) according to the standard protocol. The template for the standard curve was Superscript ™ One-Step RT-PCR with Platinum Taq (Life Technologies), swimming with agarose genole, cutting out the gel, and Quantum Prep ™ Freeze 'N Squeeze DNA Gel Extraction Spin Column (Bio -Rad).

(3) Measurement

A 22-25mer primer was designed to sandwich the intron in order to confirm the contamination of the genomic DNA with high specificity to the gene to be measured. The reagent was LightCycler-FastStart DNA Master SYBR Green I (Roche), and real-time RT-PCR was performed using Light-Cycler (Roche) according to the standard protocol. The measured values were corrected using the actin 2 gene (^ Τ2) as an internal standard. 8. Plant transformation

(1) Construction of binary vector

Add restriction enzyme αΐ site to forward primer (Primer (ix) (bHLH19-Nf Fw “5'-ATT ATT TCT AGA CGA TCG TTT CCC ATG GAT GAA GAT TTT TTC TTA CCC GAT TTC TCA CTA G-3 '( SEQ ID NO: 14))) Reverse primer with Sa site (Primer (X) (HLH19-Cr Rv “5'-ACT AAA ACT CGA GAG AAG ACC TAA ACC ATT GCG AGT CTC AGG-3 '(SEQ ID NO: 15)))) A long fragment containing the normal start codon and stop codon was amplified with a combination of primers (ix) and (X) Stress condition sample Salt-foil was amplified as a template, and Normal-foil was amplified as a template for non-stressed samples.

Each insert was first subcloned into the EcoRI site of the pBluescriptn SK (+) (Nibonbon gene) vector, and confirmed by colony PCR, plasmid recovery, restriction enzyme treatment, and DNA sequencing. Thereafter, each of the above plasmids pSMAB701 vector was treated with the restriction enzyme SacVXbal, electrophoresed with agarose gel, the target band was excised from the gel, and Quantum Prep ™ Freeze'N Squeeze DNA Gel Extraction Spin Columns (BIO -RAD). Ligation Pack (Nibonbon Gene) was used for ligation according to the standard protocol, and transformation was performed using Compitent high JM109 (Toyobo) according to the standard protocol. The insert inserted into the GUS coding region that was cleaved at the XbaVSacl site on the pSMAB701 betater (Figure 6). Colonies that grew on LB solid medium containing 100 mg / mL of spectinomycin, a drug marker contained in pSMAB701, were recovered as transformation candidates. After selection by colony PCR, plasmid collection, restriction enzyme treatment, and DNA sequencing were performed, and final confirmation of binary vector construction was performed.

(2) Transformation into agrobacterium [Transformation into agrobacterium (GV3101) was performed by electroporation. GV3101 40 μΐ and 10-fold diluted plasmid solution are Moved to a cuvette for cuvoroporation. The condition of the gene pulser was 2.5 kV, 25 μΡ, 200 Ω. The bacterial solution in the cuvette was transferred to 1 mL of LB liquid medium and cultured at 30 ° C for 20 minutes at about 150 rpm. Colonies that have been grown at 28 ° C in an LB solid medium containing 100 g / mL of spectinomycin, 25 g / mL of gentamicin, and 50 μ ^ τήΐ of rifampicin, which are selective markers for GV3101 and pSMAB701 Was recovered as a transformation candidate strain.

The transformed agrobacterium was inoculated into 1.5 mL of LB liquid medium containing 100 g / mL spectinomycin, 25 g / mL gentamicin, and 50 g / mL rifampicin. The culture was performed at 28 ° C and about 170 rpm for about 20 hours. Subsequently, plasmid recovery and restriction enzyme treatment were performed for confirmation. For transformation, transformation was performed using the two long chain patterns described above.

(3) Transformation into wild Arabidopsis

Agrobatatarum (GV3101) introduced with a binary vector was inoculated into 1.5 mL of LB liquid medium containing 100 g / mL spectinomycin, 25 g / mL gentamicin, and 50 g / mL rifampicin. Incubation was performed at 28 ° C and about 170 rpm for about 20 hours. As the main culture, the preculture was transferred 1/1000 times to 200 mL of liquid medium with the same composition, and cultured at 28 ° C and about 170 rpm for about 22 hours. Bacteria collected by centrifugation were suspended in about 80 mL of infiltration medium. The suspension was transferred to an appropriately sized container, and several mL of infiltration medium was further added to suspend it.

The Arabidopsis wild line Col grown for about 2 weeks in MS medium was transferred to Mouth Wool and further grown for about 2 weeks, and the strain from the time when the buds just before flowering appeared was used. The cells were directly immersed in the suspension for 3 minutes to infect and transformed.

infiltration medium

Mixed salt for MurasHge Skoog medium 4.3 g

1000 X vitamin stock ²

sucrose 100 _&

1 mg / mL benzylamino purine μΐ ^

silicon L-77 ⁴⁰⁰ ^

2 L with ultra pure water. '' Example 1

Oligo Microarray pst2 mutant lines and wild-type guns were grown in low K + medium (mineral medium) and high K ⁺ medium (MS medium) for 3 weeks, and R ^ A was prepared from the leaves. Arabidopsis thaliana nuclear gene chip (14,880 gene 60mer synthetic ori Gonucleide (Agilent Technologies) was used for analysis. As an index, the P value (significance probability) of the significant difference is 0.01 or less as a result of removing the test noise by the standard deviation. The putative bHLH transcription factor (bHLH19) was highly expressed in a lineage-specific manner in low K + medium that was close to the growth conditions in the field (Table 1). This high expression was also confirmed in real-time RT-PCR (Figure 2). Table 1. Genes with increased or decreased expression in ^ compared to wild lines psi2Z wild lines> 3

Low K + medium (mineral medium) putative bHLH transcription factor 14.37

anter-specific proline-rich-like protein (APG-like) 6

High K + medium (MS medium)

233

633 psi2Z wild strain <0.333

Low K ⁺ medium (mineral medium)

High K + medium (MS medium)

putative receptor protein kinase 0 putative protein 0 putative protein 0 ethylene responsive element binding factor-like 0 Example 2

R A C E

The possibility of either salt stress-specific transcription initiation site or salt stress-selective splicing was considered (Fig. 3). For 5 'RACE PCR, primers are used to ensure amplification of the end region. One upstream and one downstream of the inlet that is thought to change pricing (Primer (i) (pst-p6-r-fukuya Rv '5'-CTT GAA AGA AGA CCT AAA CCA TTG CG-3' (sequence Number: 1 6) "))), (ii) (pst-p3-r-fukuya Rv"5'-TTC ATT GCT CTT TCA GCT CTC CTT CCC G-3 '(SEQ ID NO: 17) "), 3, side In the RACE PCR, one upstream of the intron (primer (iii) (pst-pl-f-fukuya Fw "5'-CTC ATG GCA CAA GAT CTC CGG TTC TTG C-3 '(SEQ ID NO: 1 8) ”) Designed Total RNA was recovered from plants that had been treated with salt stress for 12 hours, and RACE was performed with each primer and a primer complementary to the sequence added to the terminal during reverse transcription. In addition to the above primers, and three additional primers (primer (iv) (pst-555-r-fukuya Fw "5'-AGT CGA AAT CAA TGT CTA CTA G-3 '(SEQ ID NO: 1 9)")) ( v) (pst-1776-f-fukuya Fw `` 5'-CAA AGC TA T TTC CGA TAA CGA CC-3 '(SEQ ID NO: 20) "), (vi) (pst-1140-r-fukuya Rv (5'-GTT GTT CTT GGA GTT GTT TC-3 * (SEQ ID NO: 2 1)))) The analysis was carried out as a result of determining the nucleotide sequence, and both the plant that was treated with salt stress for 12 hours and the plant under non-stress conditions were expected to be the transcription start site. The end was found to be A approximately HObp upstream from the predicted start codon (Figure 1), and the RACE product was sequenced against 57, and the intron status was examined. There are five patterns, each of which is a normal type where normal splicing occurs, and three patterns which are thought to be induced by salt stress (salt-induced types 2, 3, 4), and salt induction where no splicing occurs Type 1 was present (Figure 5).

Normal type is 551 to 688 from the transcription start point

Salt-induced type 4 is from 551 bases to 660 from the transcription start point

Salt-induced type 3 is from 551 bases to 643 from the transcription start point

Salt-induced type 2 excludes 618 bases from 551 to 618 from the transcription start point, and salt-derived type 1 does not exclude bases 551 to 688.

As a result of sequencing 57 samples in total for 5 side RACE and 3 side RACE, 28 samples for normal type, 2 samples for salt induced type 4, 15 samples for salt induced type 3, 7 samples for salt induced type 2 There were 5 samples of salt-derived type 1 (Fig. 5). The cloned salt-induced type 3 and normal full-length cDNAs were salt-f ll. (SF) and Normal-foil (NP) were used for future experiments. Example 3

Real time R T— P C R

We tried to observe the stress treatment time, changes in RNA content and splicing patterns in each part of the plant (leaves, roots). Samples were grown in MS medium for 3 weeks, conditioned for 16 hours in mineral liquid medium, then transferred to medium containing 200 mM NaCl, and the roots and leaves were separated after 0, 1, 2, 5, 10, 24 hours. The recovered RNA was used. Primer (vii) (at2g22760-f Fw "5'-TGT TCT CAT GGC ACAAGA TCT CC-3 '(SEQ ID NO: 2 2)"), (viii) (at2g22760-r Rv "5'-AAG AAT CGT TAG CTT GTC CGC C-3 '(SEQ ID NO: 2 3) "). RT-PCR and gel amplification products were observed • When salt stress was observed for 2 hours, splicing occurred in both roots and leaves. The amount of RA with the size of the pattern that was not reached the maximum, and then the intron gradually shifted to a shorter pattern and returned to the normal splicing pattern. After 24 hours, spurious splicing occurred again (Fig. 4). Example 4

Production of transformed lines

The first generation seeds of each line obtained by transformation were designated as T1 seeds. The T-DNA used for transformation has a region encoding the gene bar and can be selected by the herbicide bialaphos. T1 lines of each transformed line used 16 normal-foil lines and 9 salt-foil lines. A homozygous line of each gun was maintained. After examining the expression level of the introduced gene by real-time PCR, seeds and Cols of each transformed line were sown and grown in a mineral medium supplemented with 75 mM NaCl, and the phenotype was observed. . Example 5 'Phenotype under salt stress conditions

Seeds of each transformed line and wild line Col of Arabidopsis obtained in Example 4 Were seeded and grown in mineral media supplemented with 75 mM NaCl, and their phenotypes were observed. In the growth under salt stress conditions, each line showed higher salt tolerance than the wild line (Fig. 7). In addition, the line that introduced NF gained the most salt tolerance. The line introduced with SF also acquired salt tolerance compared to the wild type. For each transformation line, no difference from the wild line Col was observed in the growth in normal MS medium. This experiment showed that bHLH19 expression is involved in the salt tolerance mechanism. Example 6

Real time R T—P C R

Attempts were made to observe the stress treatment time, changes in the amount of RA in each part of the plant (leaves, roots), and the splicing pattern for the C-terminal intron. Blimmer (xi) (pst-p7-r-fukuya Rv "5'-GAC CTA AAC CAT TGC GAG TCT CAG GTT CCG-3 '(SEQ ID NO: 2 9)"), (xii) (1328-f Fw " Except for using 5'-CTT TGC GAG AAA AGC AAA GGG TGC ATG ATC-3 '(SEQ ID NO: 30))), RT-PCR was performed in the same manner as in Example 3 to observe the amplified product by gel. After 2 hours from salt stress, the amount of RNA in the size of the pattern where splicing does not occur in both roots and leaves is maximized, and then the intron gradually shifts to a shorter pattern and returns to the normal splicing pattern. I went. After 24 hours, spurious splicing occurred again (Fig. 8). Industrial applicability

Since the use of the polynucleotide of the present invention can impart salt tolerance to plants, it is possible to provide transformed cells and transformants having salt tolerance. In addition, salt tolerance often accompanies resistance to environmental stresses, including water drought resistance (drying tolerance), so the greening of barren dry areas contributes to agriculture in these areas. Be expected. Furthermore, in fruit cultivation, irrigation is restricted to increase sugar content, but this is expected to contribute to alleviating the yield loss associated with this. In addition, it is expected to reduce irrigation costs on rooftop greening of buildings and road slopes. In addition, resistance to environmental stresses such as strong light, low temperature, and high temperature 6Z

° 2 Summary ZCS0C / 900Zdf / X3d £ H 860 / 900Z OAV

Claims

The scope of the claims

1. The polynucleotide according to any one of the following (a) to (f).

) A polynucleotide containing a polynucleotide comprising the nucleotide sequence set forth in SEQ ID NO: 3, 7, 9 or 11;

(c) In the amino acid sequence described in SEQ ID NO: 1, 6, 8 or 10, from one or more amino acid deletions, substitutions, insertions Z or added amino acid sequences And a polynucleotide comprising a polynucleotide encoding a protein having a function of enhancing the salt-salt property of a plant;

(d), which encodes a protein having a function of increasing the salt tolerance of plants and having a homology of 80% or more with a protein comprising the amino acid sequence set forth in SEQ ID NO: 1, 6, 8 or 10 A polynucleotide containing a polynucleotide that:

(e) a function of hybridizing with a polynucleotide comprising a nucleotide sequence complementary to the nucleotide sequence set forth in SEQ ID NO: 3, 7, 9 or 11 under stringent conditions and enhancing the salt tolerance of plants A polynucleotide comprising a polynucleotide encoding a protein having:

(f) encodes a protein having a homology of 80% or more with the polynucleotide comprising the nucleotide sequence set forth in SEQ ID NO: 3, 7, 9, or 11 and having a function of enhancing plant salt tolerance A polynucleotide containing a polynucleotide.

2. The polynucleotide according to any one of the following (a) to (f).

(a) a polynucleotide comprising the base sequence set forth in SEQ ID NO: 4;

(c) a protein comprising an amino acid sequence in which one or several amino acids are deleted, substituted, inserted and added or added in the amino acid sequence described in SEQ ID NO: 2 and having a function of enhancing plant salt tolerance A polynucleotide encoding

(d) a protein comprising the amino acid sequence set forth in SEQ ID NO: 2 and at least 80% phase A polynucleotide encoding a protein having the same sex and having a function of enhancing plant salt tolerance.

(e) a polynucleotide encoding a protein that hybridizes with a polynucleotide comprising a nucleotide sequence complementary to the nucleotide sequence set forth in SEQ ID NO: 4 under stringent conditions and has a function of enhancing plant salt tolerance; Eiji

3. The polynucleotide according to any one of the following (a) to (f).

(a) a polynucleotide comprising a polynucleotide comprising the nucleotide sequence set forth in SEQ ID NO: 26;

(c) The amino acid sequence described in SEQ ID NO: 25, which consists of an amino acid sequence in which one or more amino acids are deleted, substituted, inserted and Z or attached, and is salt-tolerant in plants. A polynucleotide comprising a polynucleotide encoding a protein having a function of enhancing sex;

(d) a polynucleotide containing a polynucleotide encoding a protein having a homology of 80% or more with the protein comprising the amino acid sequence of SEQ ID NO: 25 and having a function of enhancing plant salt tolerance ;

(e) encodes a protein that hybridizes with a polynucleotide comprising a nucleotide sequence complementary to the nucleotide sequence set forth in SEQ ID NO: 26 under stringent conditions and has a function of enhancing plant salt tolerance. A polynucleotide containing the polynucleotide; and

(f) a polynucleotide comprising a polynucleotide encoding a protein having a homology of 80% or more with the polynucleotide comprising the nucleotide sequence set forth in SEQ ID NO: 26 and having a function of enhancing plant salt tolerance; nucleotide. '

4. A polynucleotide comprising a polynucleotide comprising any one of the polynucleotides according to claim 1 and any one of the polynucleotides according to claim 2 or 3. Renucleotide.

5. The polynucleotide according to claim 4, comprising a polynucleotide having the base sequence set forth in SEQ ID NO: 5.

6. The polynucleotide according to any one of the following (a) to (f).

(c) The amino acid sequence described in SEQ ID NO: 27 comprises an amino acid sequence in which one or more amino acids are deleted, substituted, inserted and Z or added, and the salt tolerance of the plant is increased. A polynucleotide comprising a polynucleotide encoding a protein having the function of enhancing;

(d) a polynucleotide comprising a polynucleotide that encodes a protein having a function of increasing the salt tolerance of a plant and having a homology of 80% or more with the protein comprising the amino acid sequence of SEQ ID NO: 27;

(e) a polynucleotide that encodes a protein that hybridizes with a polynucleotide comprising a nucleotide sequence complementary to the nucleotide sequence set forth in SEQ ID NO: 28 under stringent conditions and has a function of enhancing plant salt tolerance. A polynucleotide containing nucleotides; and

(f) a polynucleotide comprising a polynucleotide encoding a protein having a homology of 80% or more with the polynucleotide comprising the nucleotide sequence set forth in SEQ ID NO: 28 and having a function of enhancing plant salt tolerance; nucleotide.

7. The polynucleotide according to any one of claims 1 to 6, which is DNA.

8. Recombinant vector comprising the polynucleotide according to any one of claims 1 to 7 <9. The polynucleotide according to any one of claims 1 to 7 or the vector according to claim 8 is retained. Transformed cells.

10. A transformant object into which the polynucleotide according to any one of claims 1 to 7 or the vector according to claim 8 has been introduced.

1 1. A trait that is a descendant or clone of the transformed plant according to claim 10. εε

° Tanran ZCS0C / 900Zdf / X3d £ 860 / 900Z OAV