WO2010093015A1 - Gene associated with deep-water tolerance and use thereof - Google Patents

Abstract

Disclosed is the isolation/identification of a gene associated with deep-water tolerance.  Also disclosed is a use of the gene. A polynucleotide encoding a protein comprising the amino acid sequence depicted in SEQ ID NO:4 is isolated as a gene associated with deep-water tolerance.  The use of the gene enables the production of a plant which can exhibit deep-water tolerance readily.

Description

Submerged response-related genes and their use

The present invention relates to isolation and identification of a gene relating to a response (flooding response) to an increase in water depth of a plant, and a method for improving the flooding tolerance of a plant using the gene.

自然 Natural disasters such as drought and flooding due to heavy rains make the food production such as rice and other cereals unstable and reduce the yield. In recent years, there has been an increasing need to stabilize food production by breeding crops that are resistant to such natural disasters.

Rice (0ryza sativa) is considered to be a semi-aqueous plant, depending on the need for water and the system for water, upland rice (lowland rice) and floating rice (deep water rice) (deepwater rice or floating rice) Can be classified. Land rice and water rice are cultivated in high altitudes or shallow water areas (about 10 cm deep), but when the water level rises due to the rainy season or heavy rain in these cultivated areas, rice is submerged, and this type of rice dies due to oxygen deficiency. Resulting in. On the other hand, floating rice can take a strategy of avoiding flooding by extending the internodes of the stem according to the water level. Floating rice is able to survive against flooding by increasing water depth, i.e. dramatically extending internodes by flooding and deploying leaves on the water surface to avoid oxygen deficiency and restore gas exchange . In some types of floating rice, a significant internode stretch of 20 to 25 cm per day occurs during the flooding, but what was about 1 m in the air can be extended to a maximum of 8 m by flooding. It is known to do (Non-Patent Document 1). Such dramatic and rapid growth in response to flooding is one of the unique biological adaptations to harsh environments.

Molecular genetic approaches such as cDNA and microarrays have been carried out to elucidate the mechanism for flood response. Moreover, some genes involved in the submergence response have been clarified by classical experiments on the submergence response (Non-Patent Documents 2 and 3). In addition, physiological studies on floating rice suggest that ethylene, abscisic acid and gibberellin are involved in the flooding response (Non-Patent Document 4).

However, the genes involved in such submergence responses have not been isolated and identified so far. The information about genes revealed by the prior art is not consistent with each other, and the genes that have been implicated have not yet been located.

On the other hand, by using such a gene, it is possible to breed a flood-resistant plant that survives without dying due to lack of oxygen even in the rainy season or heavy rain. In addition, plants that can increase the yield as soft biomass such as rice straw can be bred using the gene.

An object of the present invention is to isolate and identify a gene related to the submergence response and to use the gene.

The present inventors tried to identify genes involved in QTL analysis combined with positional cloning in order to isolate and identify the genes involved in the submergence response in rice and to understand the molecular mechanism of submergence response. . As a result of enormous experimentation and analysis, the present inventors succeeded in isolating and identifying a submergence response-related gene for the first time, and further introducing the identified gene into a non-floating rice species to obtain a submergence response capability. A converted plant was obtained. According to the present invention, the following means are provided based on these findings.

According to the present invention, the following polynucleotides (a) to (f) are provided. The polynucleotide of the present invention may be derived from rice.
(A) A polynucleotide encoding a protein consisting of the amino acid sequence represented by SEQ ID NO: 4.
(B) a protein having an amino acid sequence in which one or more amino acids are substituted, deleted, added, and / or inserted in the amino acid sequence represented by SEQ ID NO: 4, and having internode elongation activity of stems during submergence Encoding polynucleotide.
(C) A polynucleotide encoding a protein having an amino acid sequence having 70% or more identity with the amino acid sequence represented by SEQ ID NO: 4 and having stem internode elongation activity during submergence.
(D) A polynucleotide comprising the base sequence represented by SEQ ID NO: 3.
(E) a polynucleotide that hybridizes with a complementary strand of a polynucleotide comprising the base sequence represented by SEQ ID NO: 3 under stringent conditions and encodes a protein having internode elongation activity of a stem when submerged.
(F) A polynucleotide encoding a protein having 70% or more identity with the base sequence represented by SEQ ID NO: 3 and having internode elongation activity of stems during submergence.

According to the present invention, there are also provided a vector comprising the polynucleotide, a plant cell into which the vector has been introduced, a transformed plant comprising the plant cell, a progeny or clone of the plant, and a propagation material for the plant. .

Moreover, according to the present invention, there is also provided a method for producing a transformed plant comprising the steps of introducing the polynucleotide of the present invention into a plant cell and regenerating the plant from the plant cell.

Furthermore, according to the present invention, there is provided a production method for useful crops, comprising the step of cultivating the transformed plant of the present invention and the step of harvesting the transformed plant. . In the production method, the cultivation step may include a step S that induces a hand completion response in the transformed plant body.

It is a figure which shows the outline of a flooding response. It is a figure which shows the preparation protocol of near-isogenic lines NIL-12 and NIL-12B. It is a figure which shows the flooding response in T65, C9285, and NIL-12. The upper row schematically shows the genotype of each line, and the lower row shows an example of cultivation of each plant body in air and under submerged conditions. It is a figure which shows the measurement result of the degree of internode elongation at the time of flooding response in various systems. It is a figure which shows the result of the positional cloning of QTL on the 12th chromosome of Bhadua and C9285. It is a figure which shows the expression pattern of 18 putative genes in the candidate area | region in C9285BAC. It is a figure which shows the expression analysis result of SK1, SK2, and ACS5. It is a figure which shows the function acquisition analysis result of SK1 and SK2. It is a figure which shows the measurement result of the number of internode elongation in the overexpression body of SK1 and SK2. It is a figure which shows the tissue specific expression analysis result of SK1, SK2, and ACS5. It is a figure which shows the genome structure of SK1 and SK2. It is a figure which shows the phylogenetic tree of SK1 and SK2 based on the comparison with the AP2 / ERF gene derived from a typical or typical plant. It is a figure which shows the intracellular localization of SK1 protein and SK2 protein. It is a figure which shows the measurement result of the transactivation activity in yeast. It is a figure which shows the relationship between a plant hormone and the induction | guidance | derivation of SK1 and SK2. The numbers in the figure indicate the number of days of hormone treatment. It is a figure which shows the measurement result of the ethylene density | concentration in a plant body under the flooding conditions in T65 and C9285. It is a figure which shows the expression pattern of the ethylene related gene under the flooding conditions in T65 and C9285. It is a figure which shows internode elongation promotion by ethylene under non-flooding conditions. It is a figure which shows internode elongation suppression under the flooding conditions to which the ethylene inhibitor was added. It is a figure which shows the phylogenetic tree and structure of EIN3-like protein in rice. It is a figure which shows the alignment with the primer for confirmation of the binding site of EIN3, known EBS, etc. FIG. 2 shows sequence specific binding of OsEIN3 protein to SK1 and SK2. It is a figure which shows the competition test result by the mutant of the promoter region of SK1 and SK2. It is a figure which shows the submergence treatment time and the density | concentration change of GA1 in a plant body. It is a figure which shows the measurement result of internode elongation under GA process. It is a figure which shows the measurement result of internode elongation in NIL-12 etc.

The present invention relates to genes involved in submergence response and their use. The present invention is based on the fact that the present inventors succeeded in isolating and identifying a gene that triggers a submergence response for the first time as follows.

That is, a near-isogenic line (NIL-12) was created for QTL seated on the long arm of chromosome 12, which is one of QTLs likely to have genes related to the submergence response (FIG. 2). That is, NIL-12 having a genetic background of T65 and having a C9285 gene fragment at the end of chromosome 12 was prepared. NIL-12 was found to respond by internode elongation to increased water depth.

Furthermore, based on these results, two different mapping populations, NIL12 hetero-isogenic line 16000 derived from T65 / C9285 hybrids (FIG. 2) and NIL- derived from T65 / Bhadua hybrids. A 12B hetero near-isogenic line 12000 strain (FIG. 2) was prepared, and the QTL was subjected to positional cloning. Candidate regions for QTL are duplicated in two independent populations. In order to identify QTL, a BAC library covering this duplicated candidate region was further constructed. As a result of nucleotide sequence analysis, additional regions were added in C9285. A region was found. The total 67.5 kb DNA fragment found was predicted to have 18 ORFs (FIG. 5).

Of the 18 ORFs, two genes, Snorkel1 (SK1) and Snorkel2 (SK2), were not expressed in the air, but were strongly expressed in deep water conditions (FIGS. 6 and 7). Therefore, in order to confirm that SK1 and SK2 induce a submergence response, three subclones carrying one or both of the two genes (SK1, SK2) are selected from the sub-library of the BAC library. These were used to transform T65, a non-floating rice species, using these. Similarly, a BAC library was prepared for Bhadua species, and T65 was transformed with SK1 and SK2.

As a result of evaluating submergence responses to various transformants, it was found that both SK1 and SK2 are involved in submergence responses (FIGS. 7, 8, and 9). It was also found that SK2 gene is essential and strongly contributes to submergence response, and SK1 acts weakly.

According to the present invention, a transformed plant having flood resistance or improved can be obtained by modifying a plant using a gene newly isolated and identified by the present inventors. The genes found by the present inventors are particularly useful in the agricultural field, the energy field using biomass as a raw material, and the chemical industry field. For example, it is possible to obtain plants such as rice that can survive and be harvested against flooding during the rainy season or during heavy rain. Moreover, since a plant with stems and leaves extended is obtained, a plant with a high yield can be obtained when the stems and leaves are used as useful parts (for food or as biomass).

When a plant is created using a gene isolated and identified by the present inventors, it is preferable to use transformation. The period required for transformation is extremely short compared to gene transfer by mating, and it is possible to impart or improve submergence tolerance without changing other traits. By utilizing the gene isolated by the present inventors, the flood resistance can be easily improved. In addition, since the genome synteny (gene homology) is well preserved in cereals, the gene isolated by the present inventors can be expected to be applied to grain breeding such as wheat, barley and corn. Furthermore, since the gene isolated by the present inventors is an ethylene-responsive transcription factor and widely distributed in plants, it is considered that introduction of the gene can improve the water-resistant response in all plants.

The genes found by the present inventors are considered to be used for plants including other gramineous plants (for example, wheat, barley, corn, sugarcane, sorghum, etc.) other than rice. Useful in the chemical industry.

(Polynucleotide)
The polynucleotide of the present invention encodes a protein that is a nuclear localization ethylene-responsive transcription factor and induces internode elongation of stems as a submergence response (the protein of the present invention, hereinafter also referred to as the present protein). Hereinafter, the present protein will be described.

(protein)
The protein has a nuclear localization signal (NLS). The nuclear localization signal is usually composed mainly of basic amino acids. Examples of the NLS of this protein include, but are not limited to, SEQ ID NO: 5 and SEQ ID NO: 6 (both are deduced sequences). Moreover, this protein can have an ERF domain which is a DNA binding domain. According to the phylogenetic analysis of certain ERF domains (AP2 / ERF domains) (SEQ ID NO: 7 and SEQ ID NO: 8) encompassed by this protein, these amino acid sequences are similar to OsSUB1 protein and OsERF I know that. SEQ ID NOs: 5 and 7 are NLS (presumed) and ERF in the amino acid sequence represented by SEQ ID NO: 2, and SEQ ID NOs: 6 and 8 are NLS (presumed) and NLS in the amino acid sequence represented by SEQ ID NO: 4. ERF.

This protein can have ethylene-responsive transcription factor activity on the C-terminal side. More specifically, it preferably has ethylene-responsive transcription factor activity on the C-terminal side of the ERF domain retained by the protein. The ethylene-responsive transcription factor activity can be detected, for example, by the method disclosed in the examples in the subsequent stage.

As an embodiment of the present protein, there are proteins having the amino acid sequences represented by SEQ ID NO: 2 and SEQ ID NO: 4. These proteins were isolated from C9285, a floating rice cultivar native to Bangladesh. As other aspects of the present protein, as described later, a protein having an amino acid sequence highly identical to the amino acid sequence represented by SEQ ID NO: 2 or 4, or 1 or 2 in the amino acid sequence represented by SEQ ID NO: 2 or 4 A protein having an amino acid sequence in which a plurality of amino acids are substituted, deleted, added, and / or inserted, and is encoded by a base sequence that hybridizes under stringent conditions to a complementary strand of the base sequence represented by SEQ ID NO: 1 or 3. And a protein having an amino acid sequence.

Examples of the present polynucleotide include a polynucleotide encoding the amino acid sequence represented by SEQ ID NO: 2 or SEQ ID NO: 4. Each of these polynucleotides can be used alone. In that case, a polynucleotide encoding the amino acid sequence represented by SEQ ID NO: 4 can be preferably used. It is more preferable to use both of these as the present polynucleotide. Examples of the polynucleotide encoding the amino acid sequence represented by SEQ ID NO: 2 and SEQ ID NO: 4 include polynucleotides having the base sequences represented by SEQ ID NO: 1 and SEQ ID NO: 3, respectively. Such polynucleotides can be prepared by known techniques. For example, total mRNA is prepared from a rice tissue extract, a primer is designed based on the nucleotide sequence represented by SEQ ID NO: 1 or SEQ ID NO: 3, and PCR method such as RACE method is performed to perform the full length of the above SEQ ID NO: cDNA can be obtained. Alternatively, a cDNA library can be prepared from a rice tissue extract, a probe can be designed based on the above base sequence, and obtained by a hybridization method or the like. Further, it may be artificially synthesized based on the base sequence described in the above SEQ ID NO.

This polynucleotide is not limited to those encoding the amino acid sequences represented by SEQ ID NOs: 2 and 4, but encodes a protein exhibiting an activity of extending between stem nodes during submergence (submergence responsive activity) If it is. Such a polynucleotide may be prepared from nature or artificially prepared as long as it has the above-mentioned submergence responsive activity. For example, orthologs, homologues, and artificially introduced mutations of the above SEQ ID NO. The submergence responsive activity can be evaluated by at least one of the methods disclosed in the Examples. For example, a transformed plant can be prepared and measured by measuring internode elongation of the plant during submergence or by measuring internode elongation of the plant by exposure to ethylene or ethephon in the air.

This polynucleotide has a high degree of identity with these sequences by performing a homology search against the plant genome database using the amino acid sequence of SEQ ID NO: 2 or 4 or the base sequence of SEQ ID NOs: 1, 3. It may be. For example, based on information on the amino acid sequences of NLS and ERF or the base sequences encoding them from grasses or other plants such as wild and cultivated varieties such as rice that exhibit internode elongation of stems as a submergence response In addition, it may encode an amino acid sequence extracted by homology search using a plant genome database such as a rice genome database.

The polynucleotide preferably encodes an amino acid sequence having high identity with the amino acid sequence represented by SEQ ID NO: 2 or 4, and high identity is at least 40% or more, preferably 60% or more, and more preferably Means 80% or more, more preferably 90% or more, more preferably at least 95% or more, more preferably at least 97% or more (eg, 98 to 99%) of sequence identity. The identity of the amino acid sequence and the base sequence is, for example, the algorithm BLAST (Proc. Natl. Acad. Sci. USA 87: 2264-2268, 1990, Proc. Natl. Acad. Sci. USA 90: 5873-5877 by Karlin and Altschul. , 1993). Programs called BLASTN and BLASTX based on the BLAST algorithm have been developed (Altschul SF, et al: J Mol Biol 215: 403, 1990). When analyzing a base sequence using BLASTN, parameters are set to, for example, score = 100 and wordlength = 12. In addition, when an amino acid sequence is analyzed using BLASTX, the parameters are, for example, score = 50 and wordlength = 3. When using BLAST and Gapped BLAST programs, use the default parameters of each program. Specific techniques for these analysis methods are known (http://www.ncbi.nlm.nih.gov.).

The present polynucleotide may encode an amino acid sequence in which one or more amino acids are substituted, deleted, added, and / or inserted in the amino acid sequence represented by SEQ ID NO: 2 or 4. It can be prepared by introducing a mutation site-specifically or randomly by applying a gene modification method known to those skilled in the art, such as a mutation introduction method by PCR or a cassette mutation method. Alternatively, it is also possible to synthesize a sequence in which a mutation is introduced into the base sequence described in the above SEQ ID No. using a commercially available nucleic acid synthesizer.

Note that degenerate mutants that do not involve amino acid mutations are also included in the polynucleotide.

The present polynucleotide may be one that hybridizes under stringent conditions to a complementary strand of the base sequence represented by SEQ ID NO: 1 or 3. Here, stringent hybridization conditions can be appropriately selected by those skilled in the art. For example, 25% formamide, 50% formamide under more severe conditions, 4 × SSC, 50 mM HEPES® (pH 7.0), 10 × Denhardt's solution, hybridization solution containing 20 μg / ml denatured salmon sperm DNA at 42 ° C. After overnight prehybridization, add a labeled probe and incubate at 42 ° C. overnight. The cleaning solution and temperature conditions for the subsequent cleaning are about `` 1xSSC, 0.1% SDS, 37 ° C '', more severe conditions are about `` 0.5xSSC, 0.1% SDS, 42 ° C '', and more severe conditions are `` 0.2xSSC , 0.1% SDS, 65 ° C. ”. Thus, isolation of a polynucleotide having high homology with the probe sequence can be expected as the conditions for washing for hybridization become more severe. However, combinations of the above SSC, SDS, and temperature conditions are exemplary, and those skilled in the art will understand the above or other factors that determine the stringency of hybridization (eg, probe concentration, probe length, hybridization reaction). It is possible to achieve the same stringency as above by appropriately combining the time and the like. Such a polynucleotide is usually at least 40% or more, preferably 60% or more, more preferably 80% or more, more preferably 90% or more, more preferably at least 95% with the base sequence represented by SEQ ID NO: 1 or 3. More preferably, the sequence identity is at least 97% or more (for example, 98 to 99%).

The present polynucleotide includes genomic DNA, cDNA, and chemically synthesized DNA, and may be a DNA / RNA hybrid or a DNA / RNA chimera. Moreover, the base of RNA may be provided, and the base and sugar chain which chemically modified the natural base may be provided.

This polynucleotide does not ask its origin. The polynucleotide is preferably derived from a plant. As described in the Examples, the present polynucleotide is isolated from rice (Poaceae), but it is considered to exist as long as it exhibits a similar flooding response. The present polynucleotide is preferably derived from a monocotyledonous plant, more preferably from a grass family.

(Expression vector)
The expression vector of the present invention is an expression vector that retains the present polynucleotide. The vector of the present invention is used as a vector for expressing the present polynucleotide in plant cells in order to produce the present protein and also to produce transformed plants. Such a vector is not particularly limited as long as it contains a promoter sequence that can be transcribed in plant cells and a terminator sequence containing a polyadenylation site necessary for stabilization of the transcript. For example, plasmids “pBI121”, “pBI221” ”,“ PBI101 ”(both manufactured by Clontech). The vector used for the transformation of plant cells is not particularly limited as long as the inserted gene can be expressed in the cells. For example, using a vector having a promoter (eg, cauliflower mosaic virus 35S promoter) for constitutive gene expression in plant cells or a vector having a promoter that is inducibly activated by external stimuli Is also possible. As used herein, “plant cells” include various forms of plant cells, such as suspension culture cells, protoplasts, leaf sections, and callus.

The vector of the present invention may contain a promoter for constitutively or inducibly expressing the protein of the present invention. Examples of promoters for constant expression include cauliflower mosaic virus 35S promoter (Odell et al. 1985 Nature 313: 810), rice actin promoter (Zhang et al.1991 Plant Cell 3: 1155), maize Ubiquitin promoter (Cornejo et al. 1993 Plant Mol.Biol. 23: 567) and the like.

In addition, promoters for inducible expression are known to be expressed by external factors such as infection and invasion of filamentous fungi / bacteria / viruses, low temperature, high temperature, drying, ultraviolet irradiation, spraying of specific compounds, etc. Promoters and the like. Examples of such promoters include rice chitinase gene promoter (Xu et al. 1996 Plant Mol. Biol. 30: 387) and tobacco PR protein gene promoter (Ohshima et al. 1990 Plan Cell 2:95), Rice “lip19” promoter (Aguan et al. 1993 Mol.GenGenet. 240: 1), rice “hsp80” and “hsp72” promoters (Van Breusegem et al. 1994 Planta 193: 57), Arabidopsis "Rab16" gene promoter (Nundy et al. 1990 Proc.Natl.Acad.Sci.USA 87: 1406), parsley chalcone synthase gene promoter (Schulze-Lefert et al. 1989 EMBO J. 8: 651) And the promoter of corn alcohol dehydrogenase gene (Walker et al. 1987 Proc.Natl.Acad.Sci.USA 84: 6624). According to the present invention, a host cell into which such an expression vector has been introduced is also provided.

(Transformed cells)
The transformed cell of the present invention is a cell into which the vector of the present invention has been introduced. The cells into which the vector of the present invention is introduced include plant cells for producing transformed plants, in addition to cells such as E. coli, yeast, animal and plant cells and insect cells used for production of recombinant proteins. There is no restriction | limiting in particular as a plant cell, For example, cells, such as Arabidopsis thaliana, rice, corn, potato, and tobacco, are mentioned. The plant cells of the present invention include cultured cells as well as cells in the plant body. Also included are protoplasts, shoot primordia, multi-buds, and hairy roots. For introduction of a vector into a plant cell, various methods known to those skilled in the art such as polyethylene glycol method, electroporation (electroporation), Agrobacterium-mediated method, and particle gun method can be used.

(Transformed plant)
The transformed plant of the present invention contains a plant cell into which the vector of the present invention has been introduced. A transformed plant can be obtained by regenerating a plant from a plant cell transformed by introducing the vector of the present invention. Transformed plant cells can be prepared by a known method as described above. For example, gene transfer into protoplasts using polyethylene glycol (Datta, SK (1995) In Gene Transfer To Plants (Potrykus I and Spangenberg Eds.) Pp66- 74), gene transfer to protoplasts by electric pulse (Toki et al. (1992) Plant Physiol. 100, 1503-1507), gene transfer directly to cells by particle gun method (Christou et al. (1991) Bio / technology, 9: 957-962.) And gene transfer via Agrobacterium (Hiei et al. (1994) Plant J. 6: 271-282.). In addition, regeneration of plant bodies from converted plant cells can be performed by methods known to those skilled in the art depending on the type of plant cells (see Toki et al. (1995) Plant Physiol. 100: 1503-1507). ). For example, the method of Fujimura et al. (Plant Tissue Culture Lett. 2:74 (1995)) can be cited for rice, and the method of Shillito et al. (Bio / Technology 7: 581 (1989)) or Gorden-Kamm for maize. (Plant Cell 2: 603 (1990)), for potatoes the method of Visser et al. (Theor.Appl.Genet 78: 594 (1989)), for tobacco, Nagata and Takebe (Planta 99 : 12 (1971)), and for Arabidopsis, the method of Akama et al. (Plant Cell Reports 12: 7-11 (1992)) is used.

If a transformed plant into which the present polynucleotide is introduced into the genome is obtained, offspring can be obtained from the plant by sexual reproduction or asexual reproduction. It is also possible to obtain a propagation material (for example, seeds, fruits, cuttings, tubers, tuberous roots, strains, callus, protoplasts, etc.) from the plant body, its descendants or clones, and mass-produce the plant body based on them. Is possible. The present invention includes a plant cell into which the DNA of the present invention has been introduced, a plant containing the cell, progeny and clones of the plant, and propagation material of the plant, its progeny and clones.

The plant body produced in this way has been given or improved the ability to respond to submergence, and has improved resistance to submergence. Therefore, it is a plant body that can be expected to be harvested even in submersion and can be expected to increase the yield of the foliage part.

In addition to being resistant to flooding due to natural disasters, the plant produced in this way is not only flooded artificially or exposed to ethylene in the air, intentionally generating a flooding response, resulting in stem nodes. The space can be extended. For this reason, a plant body with a high plant height or a large number of stems and leaves can be easily obtained.

(Production method)
The method for producing useful crops of the present invention comprises a step of cultivating the transformed plant of the present invention and a step of harvesting the transformed plant. According to the production method of the present invention, a crop can survive even during a rainy season or flooding due to heavy rain, so that the crop can be reliably harvested. Moreover, the cultivation process may include the step of inducing a flooding response in the transformed plant body. The submergence response can be induced artificially by flooding the transformant or exposing it to ethylene. By carrying out such a treatment, it is possible to induce a submergence response in the transformed plant body and extend the internodes of the stem. As a result, it becomes possible to harvest plants having a high plant height or a large number of foliage.

In addition, what is necessary is just to supply water to a plant body so that it may become the water level of the grade which can induce a flooding response in the flooding process for inducing a flooding response. Preferably, the water level is 50% or more of the height of the plant, more preferably 70% or more, still more preferably 80% or more, still more preferably 90% or more, most Preferably, it is enough to be completely submerged. In addition, when the submergence response is induced by exposure to ethylene, exposure to a concentration or amount of ethylene that can induce the submergence response may be performed. The exposure to ethylene can be supplied as ethylene gas, for example, in a gas atmosphere where plants can survive, such as in the air. Appropriate ethylene concentration and exposure time in the gas atmosphere can be determined in advance.

Hereinafter, the present invention will be specifically described with reference to examples, but these do not limit the present invention. Hereinafter, the material and method of the present invention will be described.

(1) Evaluation of plant material and submergence response W0120 (O. ru The Indian wild rice species W0120 (O. rufipogon; perennial), (Morishima et al. 1962), Bangladesh C9285 and Bhadua (both O. sativa and ssp. Indeca species), which are cultivated floating rice cultivated in Hokkaido, were used as floating rice species in the following experiments.

Wild rice W0106 (O. rufipogon; one year) native to India and cultivated species Taichung 65 (T65; O. sativa ssp. Japonica) are used for rice that does not have floating rice characteristics (hereinafter, non-floating rice species or controls) Used for crossing). Furthermore, O. glumaepatula (W2199) was used as a cultivated species. These lines, C9285, W0120, W0106 and O. glumaepatula were all obtained from the National Institute of Genetics (Japan), and T65 was cultivated at Nagoya University.

Plants were germinated by treatment in water in a petri dish at 30 ° C. for 72 hours, and then transplanted to pots having a diameter of 10 cm and a height of 13 cm. At the 10th leaf stage, in order to evaluate the internode elongation length (TIL) of the stem, the plant body was submerged to 70% of its height in a 3000 L tank for 1 week (see FIG. 1).

(2) Construction of cultivated and wild species BAC libraries BAC libraries were constructed from young leaves of plants in accordance with a conventional method. That is, partial digestion of DNA with HindIII, size fractionation of large DNA by pulse field gel electrophoresis (CHEF, Bio-Rad Laboratories, Hercules, California, USA), vector (pIndigo BAC-5, EPICENTRE Biotechnologies, Madison, Wisconsin, USA) and transformation into E. coli (DH10B strain). Positive BAC clones were screened by PCR from the pooled DNA for each of a sufficient number of BAC libraries holding total DNA equivalent to at least 6 times the amount of rice genomic DNA. For these BAC libraries, positive clones were screened by Southern hybridization from a high-density BAC filter.

(3) Preparation of quasi-isogenic lines and pyramiding lines A quasi-equipment with a gene fragment corresponding to QTL on chromosomes 1, 3 and 12 of C9285, a floating rice species, against the genetic background of T65 Isogenic lines (NIL-1, NIL-3, and NIL-12) were produced respectively. As shown in FIG. 2, floating rice cultivars C9285 and Bhadua were crossed with non-floating rice species T65, respectively. For these hybrids, NIL-12 (BC ₄ F ₁ population) was obtained by 4 backcrosses using T65 as a recurrent parent and marker-based selection (MAS). Furthermore, self-breeding was performed to obtain progeny NIL-12 and NIL-12B (BC ₄ F ₂ population) of near-isogenic lines. Similarly, BC ₄ F ₂ populations were obtained for NIL-1 and NIL-3. The pyramiding lines NIL1 + 3, NIL3 + 12, NIL1 + 12 and NIL1 + 3 + 12 were obtained by crossing near isogenic lines NILs with each other.

(4) SK1 gene and SK2 gene cloning T65 and C9285 and 16000 individuals (BC ₄ F ₂₎ hetero systems NIL-12 from hybrids of (BC ₄ F _2), T65 and Bhadua the (BC ₄ F ₂₎ 12000 individuals of the heterozygous line NIL-12B (BC ₄ F ₂ ) obtained from the hybrid were used for the positional cloning of SK1 and SK2.

Evaluation of SK1 and SK2 phenotypes (measurement of internode elongation length) under submerged conditions was performed using F3 and F4 individuals. Phenotypes were compared for DNA fragments within the candidate regions of C9285, Bhadua and T65. The full length genomic DNAs of SK1 and SK2 including the promoter region were introduced into the binary vector pBI-Hm12. These DNA fragments were introduced into T65, which is a japonica species, by the Agrobacterium method. As a control, an empty vector without these DNA fragments was introduced into T65.

(5) RNA isolation and semi-quantitative RT-PCR
Total RNA was prepared by the method of Samblook et al. (Molecular Cloning A Laboratory Manual Cold Spring Harbor, 1989) and the first strand of cDNA was prepared using 2 μg of the Omniscript Reverse Transcription Kit (Qiagen, California, USA). Synthesized from total RNA. Semi-quantitative RT-PCR was performed by the method of Kaneko et al. (Kaneko et al., 2003) using gene specific primers.

(6) Plasmid construction and plant transformation To produce transformants overexpressing SK1 and SK2, SK1 and SK2 cDNAs were amplified and introduced into pCR4 Blunt-TOPO (Invitrogen, California, USA). did. Analysis of the base sequence of this plasmid confirmed that SK1 and SK2 were correctly introduced. The plasmid was subjected to restriction enzyme treatment, and the DNA fragment was introduced into the pBI101 binary vector having ACTIN1 promoter and nos terminator. This binary vector was introduced into Agrobacterium strain EHA101 (A. tumefaciens strain EHA101 (Hood et al., 1986)) by electroporation. Rice (Hiei, Y., Ohta, S., Komari, T. & Kumashiro, T. Efficient transformation of rice (Oryza sativa L.) mediated by Agrobacterium and sequence analysis of the boundaries of the T-DNA. J. 6, 271-282 (1994).). Briefly, the Agrobacterium EHA101 strain into which the DNA fragment was introduced was introduced into T65 callus, a cultivated rice plant, by electroporation, and the transformed plant was placed in a medium containing 50 mg / l hygromycin. Selected. The hygromycin-resistant plant was transplanted to soil and cultivated at 30 ° C. for 16 hours in light conditions / 8 hours in dark conditions.

(7) Construction of plasmid for expressing GFP fusion protein In order to produce a GFP fusion protein, SK1 and SK2 cDNAs were amplified by PCR using primers based on the sequences. In order to express a fusion protein in which GFP was linked to the N-terminus of the SK1 gene or SK2 gene under the control of the ACTIN1 promoter, the PCR product was introduced into pCR4 Blunt-TOPO (Invitrogen) and then into the pUC119 vector.

(8) Microscopic observation Gold particles are coated with DNA constructs encoding each fusion protein of GFP-SK1 and GFP-SK2, and onion epidermis cells using PDS-1000 / He biolistic system (Bio-Rad, California, USA) Introduced particles with shock waves. Onion epidermal cells were cultured in a dark room at 22 ° C., and after 24 hours, the cell layer was placed on a slide glass. This sample was used to stain the nucleus and evaluate the intracellular localization of SK1 and SK2 derivatives at 2 μg / μL of 4 ′, 6-diamidino-2-phenylindole n-hydrate (DAPI; Dojindo, Kumamoto , Japan) Immerse in the solution. The stained sample was observed with a confocal microscanning laser microscope (FV500; Olympus, Tokyo, Japan).

(9) Measurement of transactivation activity GAL-4 matchmaker 2-hybrid system 3 (Clontech) was used for measurement of transactivation activity. Constructs pGBKT7-BD-SK1 (or SK2) -Full, pGBKT7-BD-SK1 (or SK2) -NT, pGBKT7-BD-SK1 (or SK2) -AP2 / ERF and pGBKT7-BD-SK1 (or SK2) -CT To construct SK1, SK1 and SK2, the full length (Full), N-terminal side (NT), AP2 / ERF domain and C-terminal (CT) were amplified using PCR. The PCR product confirmed by the analysis was treated with EcoRI and PstI, introduced into the pGBKT7 vector, and ligated with the GAL-4-binding domain. All constructs were introduced into the yeast strain AH109. Liquid cultures of the respective yeasts were sequentially diluted to OD600 = 0.6, and 2 μl of each diluted solution was inoculated into tryptophan-histidine negative SD (Synthetic Dropout) medium.

(10) Quantification of plant hormones Plant hormones (auxin (IAA), brassinosteroid (BR), cytokinin (CK), gibberellin (GA), abscisic acid (ABA)) are about 100 mg (raw weight) of rice stem. Extracted from. These plant hormones are prepared by liquid chromatography-mass spectroscopy system chromatography (UPLC / Quattro Premier XE; Waters, Massachusetts, USA) and ODS column (Acquity) according to the method described in the literature (Hirano et al. 2008). -UPLC BEH-C ₁₈ , 1.7 μm, 2.1 x 100 mm, Waters). The ethylene content was measured using a gas chromatograph (Hitachi, 263-30). The stem base including nodes and internodes was harvested from the flooding after 1 day, placed in a 6 ml glass vial, and held for 1 hour. The amount of ethylene in 1 ml of gas collected from the vial was measured.

(11) Treatment with plant hormones and inhibition of plant hormones For analysis of plant hormone responses, plants were cultivated in air for 1 month. Thereafter, for treatment with plant hormones, the plants were transferred to water containing 10 ppm ethylene, 10 μM GA, 10 nM BR, 20 μM IAA, 1 μM CK and 100 μM ABA, respectively.

(12) Electrophoretic gel mobility shift assay To produce OsEIL1 protein, its full-length DNA (Mao et al. 2006) was amplified by PCR, and the pET-32a (+) vector (Novagen, Madison, Wisconsin, USA) Introduced to XbaI and BglII sites. Probes of the SK1 and SK2 promoter fragments were labeled by introducing [ ³² P] dATP Klenow fragment into the 5 ′ overhang site. The DNA binding reaction was performed for 30 minutes at 4 ° C. with a binding buffer containing 0.5 ng of ³² P-labeled probe and a bacterially produced fusion protein (12.5 mM Tris-HCl, 60 mM NaCl, 0.25 mM DTT, 12.5% Electrophoresis was performed on a 13% acrylamide gel at 200 V for 2 hours in glycerin, 1 mM EDTA, 0.05% NP-40, and 2 0.25 × Tris-borate-EDTA buffer.

The competition test was performed by adding an unlabeled competitive oligonucleotide to the binding reaction and then adding a labeled oligonucleotide.

(Identification of SK1 and SK2 genes)
The present inventors have already found three main QTLs present on chromosomes 1, 3, and 12, respectively, using a hybrid of T65 and C9298. (Non-Patent Documents 2 and 3). Among these QTLs, QTL on chromosome 12 is considered to be most involved in the water depth increasing response (Non-Patent Documents 2 and 3). Based on these documents, in order to evaluate the influence of the strongest QTL on chromosome 12, a near-isogenic line (NIL) having a genetic background of T65 and having C9285 and Bhadua gene fragments at the end of chromosome 12. -12) was produced by the above method. In addition to the produced NIL lines, the flooding response was evaluated for floating rice species C9285 and non-floating rice species C65. In addition to the produced NIL-12, the submergence response for T65 and C9285 was evaluated. The results are shown in FIGS.

As shown in FIG. 3 and FIG. 4, NIL-12 responded to flooding with internodes and leaf elongation.

Next, based on these results, positional cloning was performed for two populations: 16,000 individuals of the NIL12 line derived from the T65 / C9285 cross and 12000 individuals of the NIL-12B line derived from the T65 / Bhadua cross. Tried. The results are shown in FIG.

As shown in FIG. 5, in the NIL-12B line, QTL on chromosome 12 was mapped to about 88.5 kb between FL-EcoRV and FL-EcoRI. Similarly, in the NIL-12 line, it was mapped to 21.5 kb between the molecular markers DW6-PvuII and DW9-PvuII. The candidate region for this QTL overlapped in two independent mapping populations.

In order to identify this QTL, a C9285 BAC library was constructed according to the above method, and it was found that one BAC clone (C9285_10H05) covers the candidate region. The results are shown in FIG. From the analysis result of the base sequence of the BAC clone, an additional gene region of 46 kb was detected in C9285 but not in T65. The total 67.5 kb DNA fragment had 18 putative ORFs.

Next, expression analysis of these putative ORFs was performed. In the expression analysis, total RNA was extracted by the above method from C9285 submerged for a predetermined time (3 hours, 6 hours, 12 hours) shown in FIG. 6 (A indicates air). Each of these estimated ORFs was extracted. RT-PCR was performed using specific primers. The actin gene was used as a control. As shown in FIG. 6, in C9285, 2 out of 18 ORFs (8: Snorkel1 (SK1) and 12: Snorkel2 (SK2)) are not expressed in the air and are submerged. It was found to be strongly expressed under conditions. The coding region base sequences of the SK1 gene and SK2 gene are shown in SEQ ID NOs: 1 and 3, respectively, and the amino acid sequences of the proteins encoded by these genes are shown in SEQ ID NOs: 2 and 4, respectively. The SK1 gene coding region and its base sequence containing at least part of the promoter region on its 5 ′ side are shown in SEQ ID NO: 9, and the SK2 gene coding region and its base containing at least part of the promoter region on its 5 ′ side. The sequence is shown in SEQ ID NO: 10.

FIG. 7 shows the results of expression analysis for SK1 and SK2. In the expression analysis, total RNA was extracted from C9285 and T65 submerged for a predetermined time (3 hours, 6 hours, 12 hours) as shown in FIG. 7, and RT-PCR was performed using specific primers for these genes. It was performed using. The actin gene was used as a control. As a result, as shown in FIG. 7, in C9285, it was found that SK1 was particularly strongly expressed in the submerged condition among the two genes, Snorkel1 (SK1) and Snorkel2 (SK2).

A BAC library of Bhadua and selected clones covering the desired region was prepared, and it was also confirmed whether Bhadua has SK2 and SK1.

In order to confirm that SK1 and SK2 induce a water depth increasing response, subclone a (containing SK1) and subclone b (containing SK2) were created from the sub library of BAC clone C9285_10H05 (see FIG. 5). . These gene fragments were introduced into non-floating rice species T65 to produce transformed plants by the above method. The submergence response was evaluated for these transformed plants. The results are shown in FIG.

As shown in FIG. 8, the transformed plant carrying SK1 hardly responded to submergence, whereas the transformed plant carrying SK2 showed significant internodal extension to submergence. It was. Transformed plants carrying SK1 and SK2 responded better than plants carrying either.

Also, a transgenic plant having a T65 gene background and overexpressing SK1 or SK2 under the control of the actin promoter was prepared, and internode elongation in air was evaluated. The results are shown in FIG. As shown in FIG. 9, the control (introduced with the empty vector) did not respond in the air. However, in the case of an overexpressing SK1, the first to third internodes were elongated, and SK2 In the overexpressing body, the first to seventh internodes were elongated. From these results, it was found that SK1 and SK2 are both involved in the submergence response, but that the SK2 gene is more effective than SK1 with respect to the submergence response, SK1 is essential for the regulation of submergence response, SK2 was found to act weakly.

Next, tissue-specific expression of SK1 and SK2 was confirmed. Total RNA was extracted from leaf sheath (LS), leaf tip (LB), stem (ST), root (RO) and conical inflorescence (PA), and RT-PCR was performed using specific primers for SK1 and SK2. It was. The results are shown in FIG. As shown in FIG. 10, it was found that these genes are expressed in tissues in which a submergence response occurs, such as leaves (leaf sheaths and leaf tips) and stems including nodes and internodes.

(Functional evaluation 1 of SK1 and SK2)
According to the analysis results of the base sequences of SK1 and SK2, it has a putative nuclear localization signal (NLS) and an AP2 / ERF domain (FIG. 11). According to the phylogenetic analysis of this AP2 / ERF domain, it was speculated that SK1 and SK2 are ethylene-responsive transcription factors (FIG. 12). Moreover, based on the base sequence of this domain, it shows similarities with OsSUB1 protein and various ERFs, but there were no known genes showing similarities with respect to sequences outside this domain.

In order to confirm the intracellular localization of the SK1 protein and SK2 protein, microscopic observation was performed by the above method. The results are shown in FIG. As shown in FIG. 13, the GFP signal was localized in the nucleus.

In addition, using the method for measuring transactivation activity as described above, it is confirmed which protein has transcriptional activity and which part has transcriptional activity using the yeast one-hybrid system. did. The results are shown in FIG. As shown in FIG. 14, it was found that SK1 protein and SK2 protein have strong transcriptional activity at their C-terminus. From the above results, it was found that SK1 and SK2 encode transcription factors.

Next, whether or not the expression of the hormone responses SK1 and SK2 is induced by a plant hormone containing ethylene (IAA, GA, CK, BR, ABA), using the hormone treatment method described above for C9285, a predetermined cultivation After a period of time, total RNA was extracted from the plant body, RT-PCR was performed, and SK1 and SK2 expression analysis was performed. The results are shown in FIG. As shown in FIG. 15, it was found that only ethylene significantly promotes the expression of SK1 and SK2.

Also, for C9285 and T65, the ethylene concentration in the plant body was measured in air and under submerged conditions using a plant hormone quantification method. As shown in FIG. 16, it was found that the ethylene concentration of any plant body was about 2.5 times higher than that in air under flooding conditions. After submerging C9285 and T65 for a predetermined time (6 hours, 12 hours, 24 hours), total RNA was extracted, and expression analysis of ethylene gene (ACS) was performed. The results are shown in FIG. As shown in FIG. 17, it was found that ACS5, which converts SAM to ACC in the rate-limiting step of the ethylene synthesis pathway, was induced by both T65 and C9285 under submerged conditions. From the above, it was found that the synthesis of ethylene induced by activating the ethylene biosynthesis gene ACS5 under submerged conditions induces the expression of SK1 and SK2.

Furthermore, physiological responses to ethylene were investigated for C9285 and T65. That is, the elongation length between nodes when a plant body was exposed to ethylene in the air and ethylene action was inhibited using an ethylene inhibitor was measured. The results are shown in FIGS. As shown in these figures, for C9285, the internode elongation length increased according to the ethylene or etaphone concentration in the air, while the internode elongation length decreased according to the added amount of the inhibitor. In addition, no internode elongation was observed for T65. From the above, it was found that the flooding response occurs through ethylene synthesis.

In order to investigate the relationship between ethylene synthesis and SK1 and SK2, whether or not rice EIN3 (Ethylene Insentive3) (OsEIN3, see FIG. 20) binds to the promoter regions of SK1 and SK2 was examined. As shown in FIG. 21, the SK1 and SK2 promoter regions (−2048 bp, 1797 bp) were divided into 10 and 9 fragments of 200 to 300 bp, respectively, and the above-mentioned electrophoretic mobility shift assay for interaction with OsEIN3 was performed. (EMSA). The results are shown in FIG. As shown in FIG. 22, it was found that OsEIN3 binds to SK1 (-1478 bp to -1196 bp region) and SK2 (-633 bp to -373 bp region). These two regions also had a target core sequence of EINS3 (see FIG. 21). The results of the competition test also supported the interaction between these regions and OsEIN3 (FIG. 23). From the above, it was confirmed that SK1 and SK2 encode ethylene-responsive transcription factors.

(Functional evaluation 2 of SK1 and SK2)
In this example, the mechanism of floating intergrowth in floating rice was investigated. Since plant hormones are essential for plant growth, GC-MS was used to determine the amount of plant hormones before and after flooding for C9285 and T65. The results are shown in FIG. As shown in FIG. 24, a high concentration of active GA was detected in C9285 after flooding. In contrast, T65 did not change significantly before and after flooding. The amount of other hormones was large, suggesting that GA biosynthesis was caused by flooding.

Next, internode elongation was evaluated when GA or GA and GA inhibitor (uniconazole) were administered. In order to treat the plant with GA, a 10 ⁻⁵ M GA1 solution was administered to the root of T65 and NIL-12, and the plant was grown for 7 days and the internodes were measured. In applying the inhibitor, first, a ^10-6 M GA inhibitor was treated under normal cultivation conditions, and then these pretreated plants were submerged with water containing a ^10-6 M GA inhibitor. The length of the internode extended after 7 days was measured. The results are shown in FIG. As shown in FIG. 25, when GA was treated, internodes were elongated for both T65 and C9285, and in the presence of an inhibitor, internode elongation was similarly suppressed. Moreover, as shown in FIG. 26, under submerged conditions, NIL-12 was elongated, but GA-deficient mutants having mutations in GA biosynthetic enzymes did not show a submergence response. From the above, it was found that GA is important for internode elongation during flooding response.

From these results, one model for flood response can be proposed. In other words, under the submerged conditions, ethylene is synthesized by activating the expression of the ethylene biosynthetic enzyme ACS5 gene, and the accumulated ethylene induces the expression of ethylene-responsive transcription factors SK1 and SK2, thereby expressing the submergence response. Is to cause. In addition, as a result of signal transduction by SK1 and SK2 leading to the biosynthetic pathway of GA, it is considered that the GA concentration rises to such an extent that it induces internode elongation directly or indirectly.

Claims (10)

1. The polynucleotide of any one of (a) to (f) below:
   (A) A polynucleotide encoding a protein consisting of the amino acid sequence represented by SEQ ID NO: 4.
   (B) a protein having an amino acid sequence in which one or more amino acids are substituted, deleted, added, and / or inserted in the amino acid sequence represented by SEQ ID NO: 4, and having internode elongation activity of stems during submergence Encoding polynucleotide.
   (C) A polynucleotide encoding a protein having an amino acid sequence having 70% or more identity with the amino acid sequence represented by SEQ ID NO: 4 and having stem internode elongation activity during submergence.
   (D) a polynucleotide comprising the base sequence represented by SEQ ID NO: 2.
   (E) a polynucleotide that hybridizes with a complementary strand of a polynucleotide comprising the base sequence represented by SEQ ID NO: 2 under stringent conditions and encodes a protein having internode elongation activity of a stem when submerged.
   (F) A polynucleotide encoding a protein having 70% or more identity with the base sequence represented by SEQ ID NO: 2 and having internode elongation activity of stems during submergence.
2. The polynucleotide according to claim 1, which is derived from rice.
3. A vector comprising the polynucleotide according to claim 1 or 2.
4. Plant cells into which the vector according to claim 3 has been introduced.
5. A transformed plant comprising the plant cell according to claim 3.
6. A transformed plant that is a descendant or clone of the transformed plant according to claim 5.
7. The propagation material of the transformed plant body according to claim 5 or 6.
8. A method for producing a transformed plant comprising the steps of introducing the polynucleotide according to claim 1 or 2 into a plant cell and regenerating the plant from the plant cell.
9. A method for producing useful crops,
   Cultivating the transformed plant according to claim 5 or 6, and
   Harvesting the transformed plant;
   A production method comprising:
10. The production method according to claim 9, wherein the cultivation step includes a step of inducing a submergence response in the transformed plant body.
    

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