WO2006057306A1 - Poaceous plant with enhanced stress tolerance and/or productivity and method of creating the same - Google Patents

Abstract

A rice plant, or progeny thereof, enhanced in various stress tolerances, which stably retains a gene encoding enzyme associated with polyamine metabolism; a method of creating the plant; and a method of creating a callus of the plant. Further, there is provided a rice plant, or progeny thereof, enhanced in productivity and trait, which stably retains a gene encoding enzyme associated with polyamine metabolism. Still further, there are provided a method of creating the plant, and a method of creating a callus of the plant.

Description

 Specification

 Gramineae plants with improved stress tolerance and Z or productivity, and methods for producing them

 Technical field

 [0001] The present invention relates to a Gramineae plant having improved stress tolerance, lodging resistance and productivity! / More particularly, improved salt stress tolerance, low temperature stress tolerance, or improved The present invention relates to a gramineous plant having productivity or traits such as ears, pods, seeds, pods, rice, fruits, tillers, and spikelets.

 [0002] The present invention also relates to a method for producing the gramineous plant.

 [0003] Further, the present invention relates to a method for producing a useful substance (eg starch, protein) or a derivative thereof (eg biodegradable plastic).

 Background art

 [0004] Plants live by adapting to various environmental stresses such as temperature and salt in their habitats. However, for example, in the case of temperature stress, if a plant encounters an environment where the upper limit or lower limit of the optimum growth temperature is exceeded, it undergoes high temperature stress or low temperature stress, and the physiological function of cells is gradually or suddenly impaired, causing damage. Until now, in order to use wild plants adapted to various temperature environments for food crops and craft crops, efforts have been made to expand the temperature adaptability of crops by breeding means such as selection and cross breeding. However, especially in Japan, the climate varies significantly depending on the region that extends from north to south, and the changes in the four seasons are significant. Therefore, depending on the region and season, crops are exposed to a temperature environment that is inappropriate for growth. For example, rice originating from the tropics can be cultivated in cool areas such as Tohoku and Hokkaido as a result of cultivar improvement since the Meiji era and is now cultivated as a key crop in these areas. Even if there is an abnormally low temperature in early summer, it is still a problem that it suffers from cold damage and a significant decrease in sales. In recent years, crops have been severely damaged by abnormal weather, which is thought to be caused by global warming and the El Nino phenomenon.

[0005] About 10% of the land area of salt stress is said to be a salt damage area. Expansion of salt soil is a serious problem in agriculture, especially in dry land such as Africa

[0006] Drought stress and water stress are important stresses for plants. When temperature is not a limiting factor, it is greatly affected by rainfall and its distribution. In particular, in semi-arid areas, which are the main crop cultivation areas, the growth and yield of crops are significantly affected by drought stress and water stress.

 [0007] In addition, the food crisis is a problem on a global scale, and it is necessary to improve plant productivity or traits (production potential, hereinafter simply referred to as “productivity” in the background section). Is an important issue.

 [0008] In order to enhance various environmental stress tolerances, cross breeding, breeding using recent genetic engineering techniques, methods using the action of plant hormones and plant regulators, and the like are performed.

 [0009] So far, environmental stress-tolerant plants have been created using genetic engineering techniques. The genes used to improve low-temperature stress tolerance include fatty acid desaturase genes (ω-3 desaturase gene, glycerol 3-phosphate acyltransferase gene, stearoyl-ACP desaturase gene). In addition, pyruvate phosphate dikinase gene involved in photosynthesis, genes encoding proteins with cryoprotective activity (COR15, COR85, kinl) have been reported.

 [0010] Examples of genes used to improve tolerance to salt stress and drought 'water stress include glycine betaine synthase genes (choline monooxygenase gene, betaine aldehyde dehydrogenase gene), proline synthase Genes (1 pyrroline 5-force rubonic acid synthetase) have been reported.

 [0011] However, many of the plants transformed with these genes have not yet been sufficiently effective to be industrially usable, and have not yet been put into practical use.

[0012] On the other hand, in order to improve the productivity of plants, cross breeding, breeding using recent genetic engineering techniques, methods utilizing the action of plant hormones and plant regulators, and the like have been carried out. In particular, attempts have been made to improve productivity by cross breeding centering on rice, and many high-yielding varieties with dramatically improved productivity have been developed so far. Breeding with genetic engineering technology improves productivity using genes of key enzymes related to photosynthesis and carbohydrate metabolism Has been done.

 [0013] For example, fructose-derived 1,6-bisphosphatase Z cedeheptulose derived from cyanobacteria

 -Expression of 1,7-bisphosphatase gene in tobacco chloroplasts enhances photosynthesis and increases the content of photosynthetic metabolic intermediates (hexose, sucrose, starch) and promotes growth. Productivity improved (Patent Document 1). In addition, sucrose phosphate synthase (SPS), a carbohydrate metabolism, is thought to affect not only carbon partitioning but also production capacity, and SPS genes were introduced into tomatoes, potatoes and tobacco. It was confirmed that the introduction of SPS gene in tomato tends to increase the dry matter weight of the above-ground part during vegetative growth (Non-patent Document 1). In potatoes, it was confirmed that the dry matter weight of the above-ground part (leaves and stems) and tubers increased! / Non-patent document (Non-patent document 2; Non-patent document 3). However, many of the plants transformed with these genes have not yet been sufficiently effective to be industrially usable, and are currently being put into practical use. It is.

 [0014] Polyamine is a general term for aliphatic hydrocarbons having two or more primary amino groups, and is a natural product that exists universally in the living body. More than 20 types of polyamines have been found. Typical polyamines include putrescine, spermidine and spermine. The main physiological functions of polyamines are as follows: (1) Stabilization and structural changes of nucleic acids by interaction with nucleic acids (2) Promotion of various nucleic acid synthesis systems (3) Activation of protein synthesis systems (4) Cell membranes Stabilization of materials and enhancement of membrane permeability of substances are known. The role of polyamines in plants has been reported to promote nucleic acid and protein biosynthesis and cell protection during cell growth and division, but recently, the relationship between polyamines and environmental stress tolerance has also attracted attention.

[0015] In recent years, the relationship of polyamines to various environmental stresses has been reported (Patent Document 2). Low temperature stress (Non-patent document 4, Non-patent document 5, Non-patent document 6), salt stress (Non-patent document 7: Plant Physiol, 91, 500-504, 1984), acid stress (non-patent document 8), osmotic stress (Non-patent document 9), pathogen infection stress (Non-patent document 10), herbicide stress (Non-patent document 11), etc. have been reported. This is an estimation of the involvement of polyamines, and the gene-level involvement of polyamine metabolism-related enzyme genes that encode polyamine metabolism-related enzymes and environmental stress tolerance is sufficient. It is obscene to be investigated by. [0016] Polyamine metabolism-related enzymes involved in plant polyamine biosynthesis include arginine decarboxylase (ADC), ornithine decarboxylase (ODC), S-adenosylmethionine decarboxylase (SAMDC), spermidine Synthase (SPDS), spermine synthase (SPMS) and the like are known. Some of the polyamine metabolism-related genes encoding these polyamine metabolism-related enzymes have already been isolated. ADC gene is Enmbata (Non-patent document 12), Tomato (Non-patent document 13), Arabidopsis (Non-patent document 14), Endu (Non-patent document 15), ODC gene is Datura (Non-patent document 16), The SAM DC gene is isolated from potato (Non-patent document 17), spinach (Non-patent document 18), tobacco, and SPDS gene from Arabidopsis (Non-patent document 19).

 [0017] Furthermore, a relationship to polyamine morphogenesis has been reported. In Arabidopsis mutants with reduced arginine decarboxylase (ADC) activity, it has been reported that root growth was altered (Non-patent Document 20). The gene level of polyamine metabolism-related enzyme genes and productivity has been thoroughly investigated.

 [0018] Furthermore, when the potato-derived S-adenosylmethionine decarboxylase gene was introduced into potato in the sense direction, no transformant was obtained, and when introduced in the antisense direction, abnormal growth was observed. (Non-patent document 21), the ability of each plant to disclose abnormal growth (non-patent document 22) when arginine decarboxylase gene derived from empak was introduced into tobacco. Has not been investigated at all. Patent Document 1: Patent No. 3357909

 Patent Document 2: WO02Z23974

 Non-patent literature l: Plant Physiol, 101, 535, 1993, Planta, 196, 327, 1995

 Non-Patent Document 2: Jpn. J. Crop Sci "65, 102, 1996

 Non-Patent Document 3: Jpn. J. Crop Sci, 65, 143, 1996

 Non-Patent Document 4: J. Japan So Hortic. Sci., 68, 780-787, 1999

 Non-Patent Document 5: J. Japan Soc. Hortic. Sci., 68, 967-973, 1999

 Non-Patent Document 6: Plant Physiol. 124, 431-439, 2000

Non-Patent Document 7: Plant Physiol, 91, 500-504, 1984 Non-Patent Document 8: Plant Cell Physiol, 38 (10), 156-1166, 1997

 Non-Patent Document 9: Plant Physiol. 75, 102-109, 1984

 Non-Patent Document 10: New PhytoL, 135, 467-473, 1997

 Non-Patent Document ll: Plant Cell Physiol, 39 (9), 987-992, 1998

 Non-Patent Document 12: Mol. Gen. Genet., 224, 431-436, 1990

 Non-Patent Document 13: Plant Physiol, 103, 829-834, 1993

 Non-Patent Document 14: Plant Physiol., Ill, 1077-1083, 1996

 Non-Patent Document 15: Plant Mol. Biol, 28, 997-1009, 1995

 Non-Patent Document 16: Biocem. J., 314, 241-248, 1996

 Non-Patent Document 17: Plant Mol. Biol, 26, 327-338, 1994

 Non-Patent Document 18: Plant Physiol., 107, 1461-1462, 1995

 Non-Patent Document 19: Plant Cell Physiol, 39 (1), 73-79, 1998

 Non-Patent Document 20: The Plant Journal, 13 (2), 231-239, 1998

 Non-Patent Document 21: The Plant Journal, 9 (2), 147-158, 1996

 Non-Patent Document 22: The Plant Journal, 11 (3), 465-473, 1997

 Disclosure of the invention

 Problems to be solved by the invention

[0019] An object of the present invention is to produce a grass plant with improved productivity by artificially controlling the expression of genes related to polyamine metabolism and changing polyamine levels.

 [0020] Another object of the present invention is to improve the yield and productivity of gramineous plants and to produce more useful substances.

[0021] Furthermore, an object of the present invention is to provide a technique for enhancing practical plant productivity or traits.

[0022] Gramineae plants (including rice, corn, wheat, rye, barley, embata, shiba), for example, rice is a tropical plant, so it is susceptible to low-temperature stress damage (cold damage) and is extremely resistant to low-temperature stress. It is an important issue. If the resistance to low temperature stress of grasses can be increased, it will be a problem especially in cold regions (for example, Hokkaido and Tohoku regions). The decline in the yield of cereals (rice, wheat, barley, corn, etc.) due to cold damage can be reduced, and a stable supply of cereals becomes possible. Furthermore, if the tolerance to salt stress can be increased, grasses can be cultivated even in areas where cultivation is difficult due to high salt damage.

 [0023] An object of the present invention is to artificially control the expression of genes related to polyamine metabolism and to change the polyamine level to produce a variety of gramineous plants with improved stress tolerance. To do.

 [0024] Another object of the present invention is to improve the yield and productivity of gramineous plants and to produce more useful substances.

 Means for solving the problem

 As a result of diligent efforts to achieve the above object, the present inventors have isolated a polyamine metabolism-related enzyme gene involved in polyamine biosynthesis and introduced the gene into a grass family plant to overexpress it. Thus, it was found that by manipulating polyamine metabolism and changing polyamine concentration, productivity or trait parameters were improved regardless of the cultivation environment, in other words, with or without environmental stress. Furthermore, it was revealed that plants with improved various stress tolerance parameters can be obtained by changing the polyamine concentration.

 [0026] For example, it is shown that when the amount of polyamine is increased during the tillering period when ears or pods are formed, the number of spikes, the number of pods, the above-ground weight, the above-ground dry weight, and the culm yield are significantly increased. It was. Furthermore, gramineous plants have the ability to reduce fertility or sterility if various stresses such as low temperature, drying, salt, herbicides, pests, etc. are applied during heading, pollen formation or booting. During this period, we found that the increased amount of polyamines acted to improve the fertility against various environmental stresses (inhibition of sterility).

 [0027] The present invention provides the following inventions.

 1. a nucleic acid sequence that regulates the amount of polyamines under the control of a promoter that can function in plants; stably maintains a nucleic acid sequence, and is less productive than a control plant that does not have the nucleic acid sequence, regardless of the cultivation environment. Gramineae plants and their progeny with improved traits and with improved Z or at least one stress tolerance.

17. Useful substances obtained from the grasses and their descendants according to Item 1. 18. A step of stably maintaining a nucleic acid sequence that regulates the amount of polyamine under the control of a promoter that can function in a plant, and transforming cells of a plant that has the nucleic acid sequence! , A method for producing a Gramineae plant having improved productivity or traits irrespective of the cultivation environment and Z or at least one stress tolerance improved compared to a control plant not having the nucleic acid sequence .

 20. An expression vector comprising a nucleic acid sequence that regulates the amount of polyamine under the control of a promoter capable of functioning in a plant, comprising the step of transforming cells of the ヽ plant that has the nucleic acid sequence! A method for producing a Gramineae plant having improved productivity or traits irrespective of the cultivation environment and Z or at least one stress tolerance improved as compared with a comparative control plant not having the nucleic acid sequence.

 33. The following steps:

 (1) An expression vector comprising a nucleic acid sequence that regulates the amount of polyamine under the control of a promoter capable of functioning in a plant, transforming a cell of a culm plant having the nucleic acid sequence,

(2) From the transformed cell, regenerate a plant body having the nucleic acid sequence!

 (3) Collecting seeds by pollination of the plant, and

 (4) Plant physical strength obtained by cultivating the seed Examining the nucleic acid sequence in the seed obtained by pollination and selecting a homozygote of the nucleic acid sequence

Which is homozygous for the nucleic acid sequence, has improved productivity or traits independent of the cultivation environment compared to a comparative control plant not having the nucleic acid sequence, and Z or at least one of A method to create a grass family with improved stress tolerance.

The invention's effect

According to the present invention, productivity of gramineous plants can be improved, and improvement in quality, value, productivity, yield, etc. of plant organs and tissues can be expected. Specifically, when the organ is produced as an agricultural crop by increasing the number and size of spikelets, pods, tillers, seeds, pods, rice, berries, and spikelets, quality, productivity An increase in yield is expected. In particular, polyamines act during the tilling and booting periods, and the yield of grasses (especially rice) increases as the number of spikes, pods, and seeds per pod increases. I can expect that. [0029] The gramineous plant with improved stress tolerance according to the present invention is used not only in an area subjected to environmental stress, but also used (growth) to cope with environmental stress that is not subject to environmental stress and cannot be predicted even in the area. ) Power that can be used It may be used exclusively in areas subject to environmental stress.

 [0030] According to the present invention, various environmental stress tolerances of gramineous plants can be improved, and avoidance of damages caused by various environmental stresses encountered during the growth process of gramineous plants and suppression of growth can be reduced. Stabilization of cultivation, improvement of productivity, and expansion of cultivation areas can be expected.

 [0031] In particular, since it is resistant to stress during the booting stage and improves the fertility, it is possible to avoid a reduction in the yield.

 [0032] Furthermore, the increased amount of polyamines acts on the ears, pods and tillers during the tillering period, so that the number of spikes and pods increases and the yield is increased. , Low lodging resistance and improved yield loss due to hurricanes.

 Brief Description of Drawings

 [0033] FIG. 1 is a diagram showing the structure of an expression construct containing a polyamine metabolism-related enzyme gene.

FIG. 2 is a diagram showing the results of Western blotting of rice introduced with a polyamine metabolism-related enzyme gene.

 FIG. 3 is a diagram showing a comparison of salt stress tolerance (growth) between rice and a wild strain into which a polyamine metabolism-related enzyme gene has been introduced.

 FIG. 4 is a diagram showing a comparison of salt stress tolerance (grass form) between rice and a wild strain introduced with a polyamine metabolism-related enzyme gene.

 FIG. 5 is a diagram showing a comparison of low temperature stress tolerance (growth) between rice and a wild strain introduced with a polyamine metabolism-related enzyme gene.

 FIG. 6 is a photograph showing a comparison of low temperature stress tolerance (grass form) between rice and a wild strain introduced with a polyamine metabolism-related enzyme gene.

 BEST MODE FOR CARRYING OUT THE INVENTION

[0034] Polyamine is a basic substance that contains a large amount of amine in the molecule. There are putrescine containing two molecules of amine, spermidine containing three molecules of amine, spermine containing four molecules of amine, and the like. In plants, ADC, ODC, SAMDC, SPDS, and SAMDC, SPMS, etc. for putrescine and SAMDC have been found as enzymes related to polyamine metabolism involved in polyamine biosynthesis. Polyamine metabolism-related enzyme genes encoding these polyamine metabolism-related enzymes have already been isolated in several plants. For example, polyamine metabolism-related enzymes involved in plant polyamine biosynthesis include arginine decarboxylase (ADC), ornithine decarboxylase (ODC), S-adenosylmethionine decarboxylase (SAMDC), and spermidine synthesis. Enzymes (SPDS), spermine synthase (SPMS), etc. are known. Several polyamine metabolism-related genes encoding these polyamine metabolism-related enzymes have already been isolated from plants. ADC gene is embata (Mol. Gen. Genet., 224, 431-436, 1 990), tomato (Plant Physiol, 103, 829-834, 1993), Arabidopsis (Plant Physiol, 11 1, 1077-1083, 1996) Endo (Plant Mol. Biol, 28, 997-1009, 1995), ODC gene is Datura (Biocem. J., 314, 241-248, 1996), SAMDC gene is potato (Plant Mol. Biol, 26, 327-338, 1994), spinach (Plant Physiol, 107, 1461-1462, 1995), tobacco, SPDS genes from Arabidopsis (Plant cell Physiol, 39 (1), 73-79, 1998), etc. It has been isolated. Furthermore, some polyamine metabolism-related enzyme genes have been tried to be introduced into plants. The obtained transformations, products are productive, and improvements in traits have been reported. .

 [0035] If a grass plant is stressed at the time of ear formation (blossoming season), it will become sterile even if there is no stress at other times, and the yield will be greatly reduced. The present inventor has found that polyamine metabolism-related enzyme genes (SPDS, SAMDC, ADC, etc.) are effective against the stress at the booting stage and can improve fertility (prevent sterility).

 [0036] Gramineae plants are composed of a tissue called panicles and pods that grows their root strength, and when subjected to the action of an increased amount of polyamines during the formation of these spikes or pods (split period). The present inventors have found that the number of spikes Z is increased, resulting in an improvement in lodging resistance due to an increase in yield or a decrease in plant height (height).

[0037] The amount of polyamine depends on the tillering period (time involved in the number of pods, number of ears, number of tillers) Polymorphine compared to plants before transformation (for example, wild strains) for all buds during the 'early panicle formation period (period related to pollen formation, ear formation, fertility, Z sterility) The amount (especially the amount of spermidine and the amount of spermine) increases by about 1.1 to 3 times (preferably about 1.3 to 2.5 times). As the amount of polyamine increases, the number of pods, ears and tillers is increased by increasing the number of pods, ears and tillers during the tillering period. Inhibition of pollen formation and spike formation due to stress during the young panicle formation period can avoid deterioration of fertility and sterility, and increase yield and reduction of height (improvement of lodging resistance). Therefore, in the present invention, the amount of polyamine is increased at the tillering stage, the booting stage, and the young panicle formation stage at 1.1% compared with the plant before transformation (for example, a wild strain) in all pods. A transgenic plant that regulates genes, promoters, enzymes, etc. to increase by about 3 times (preferably about 1.3 to 2.5 times), or can realize such polyamine content. Can be selected.

 [0038] In the present invention, "stress" refers to any stress that is also subject to environmental forces, such as high temperature, low temperature, low pH, low oxygen, oxidation, salt, osmotic pressure, drying, water, flooding, cadmium, copper, ozone, Air pollution, ultraviolet rays, strong light, low light, pathogens, pathogens, pests, herbicides, etc.

 [0039] Rice is classified into wild rice and cultivated rice in the genus Gramineae, and cultivated rice is Asian rice (Oryz a sativa L.) and African rice (Oryza. Grabbelima L.), and the other species are wild rice. It is. Rice is used for food and livestock feed, and starch is used as an industrial raw material.

 [0040] In the present invention, "productive and improved traits" means all organs (tissues) of plants, such as ears, pods, seeds, pods, rice, berries, and tillers. , Spikelet size, total weight, quantity, etc., growth period shortened (improvement of productivity), traits related to the organ (tissue) (for example, rice, corn, etc. Increase in the number and size of pods, rice, corn, etc., increase in the number and size of seeds (endosperm), change in shape, coloring (for example, change in balance between pigments and increase in pigment production), etc. ) Is improved. These improvements are based on the action of polyamines during periods specific to gramineous plants such as the tillering stage and the booting stage.

[0041] According to one preferred embodiment, the plant obtained by the present invention has an increased polyamine expression level regardless of the cultivation environment (for example, environmental stress), and can improve productivity. The

 [0042] As used herein, the term "Gramineae plant" means cultivated rice, wild rice, corn, wheat, rye, barley, embatta, and shiba.

 [0043] Examples of the nucleic acid sequence that regulates the amount of polyamine include exogenous polyamine metabolism-related enzyme genes or suppressors of endogenous polyamine metabolism-related enzyme genes. An exogenous polyamine metabolism-related enzyme gene can increase the amount of polyamine in the plant, and an endogenous polyamine metabolism-related enzyme gene suppressor can decrease and increase the amount of polyamine in the plant.

 In the present invention, “a control plant having a nucleic acid sequence that regulates the amount of the polyamine” refers to the nucleic acid sequence (for example, an exogenous polyamine metabolism-related enzyme gene or an endogenous polyamine). It means any plant before the introduction of the metabolic factor enzyme inhibitor). Therefore, in addition to so-called wild species, cultivars established by normal mating, natural or artificial mutants thereof, and transgenic plants into which exogenous genes other than polyamine metabolism-related enzyme genes have been introduced. Include.

 [0045] The "polyamine" referred to in the present invention is a general natural product universally present in living organisms, and is an aliphatic hydrocarbon compound having two or more primary amino groups. For example, 1,3-diaminopropane, putrescine, cadaverine, cardine, spermidine, homospermidine, aminopropylcadaverine, theremin, spermine, thermospermine, canabalmine, amaminopentylnorspermidine, N, N-bis (amaminopropyl) Examples include cadaverine, homospermine, force dodopentamine, homo force dodopentamine, force dodohexamine, and homo force dodohexamine.

 Polyamine metabolism

In the present invention, the “polyamine metabolism-related enzyme gene” is a gene encoding an amino acid of an enzyme involved in polyamine biosynthesis in plants. For example, arginine is representative of putrescine, which is a typical polyamine. Decarboxylase (ADC) gene and orthine decarboxylase (ODC) gene, spermidine, S-adenosylmethionine decarboxylase (SAMDC) gene and spermidine synthase (SPDS) gene, spermine About S-adenosylmethionine decarboxylase (SAMDC) gene and spermine synthase Elementary (SPMS) gene is involved, and is considered to be rate-limiting! /.

[0046] Arginine decarboxylase (ADC: arginine decarboxylase EC4.1.1.19.) Is an enzyme that catalyzes the reaction of L-arginine power to produce agmatine and diacid-carbon. Orthine decarboxylase (ODC: ornithine decarboxylase EC4.1.1.17) is an enzyme that catalyzes the reaction of producing putrescine and carbon dioxide from L-orthine. S-adenosylmethionine decarboxylase (SAMDC: S-adenosylmethionine decarboxylase EC4.1.1.50.) Catalyzes the reaction to produce S-adenosylmethionine force adenosylmethylthiopropylamine and diacid-carbon. It is an enzyme. Spermidine synthase (SPDS) is an enzyme that catalyzes the reaction of producing spermidine and methylthioadenosine from putrescine and adenosylmethylthiopropylamine.

[0047] These genes may be derived from any source, but can be isolated from various plants, for example. Specifically, dicotyledonous plants such as cucurbitaceae; solanaceae; Brassicaceae such as Arabidopsis; legumes such as Alf alfa and cowpy (Vigna unguiculata); These include those whose group power has also been selected, or monocotyledonous plants such as rice, wheat, barley, corn and the like. Preferably, cucurbitaceae plants, more preferably Kurodanekabotya!

[0048] The plant tissue for isolating the plant-derived polyamine metabolism-related enzyme gene of the present invention is in the form of seed or in the growth process. Growing plants can be isolated in whole or in part. The site that can be isolated is not particularly limited, but preferably ears, inflorescences, strawberries, berries, seeds, grains, grains, strawberries, rice, whole strawberries, strawberries, flowers, ovary, fruits, Leaves, stems, roots, etc. More preferably, it is a site showing stress resistance

[0049] Preferable examples of polyamine metabolism-related enzyme genes used in the present invention include supermidine synthase gene, S-adenosylmethionine decarboxylase gene, and alginine decarboxylase gene. be able to. In particular,

 A DNA having a base sequence represented by base numbers 77 to 1060 in the base sequence represented by SEQ ID NO: 1,

• The base sequence shown in SEQ ID NO: 3 has the base sequence shown in base numbers 456 to 1547 DNA,

 A DNA having a base sequence represented by base numbers 541 to 2661 in the base sequence represented by SEQ ID NO: 5;

 Is mentioned. In addition,

 Examples include DNA that has a nucleotide sequence that can be hybridized under stringent conditions with any of the above sequences and that encodes a polypeptide having a polyamine metabolism-related enzyme activity equivalent to that sequence. . Furthermore,

 -In any of the above sequences, a DNA encoding a polypeptide having a base sequence ability in which one or more bases are deleted, substituted, inserted or added and having a polyamine metabolism-related enzyme activity equivalent to that sequence. Can be mentioned.

[0050] Here, "stringent conditions" refers only to a base sequence encoding a polypeptide having a polyamine metabolism-related enzyme activity equivalent to the polyamine metabolism-related enzyme encoded by the specific polyamine metabolism-related enzyme gene sequence. Forms a hybrid with the specific sequence (, so-called specific hybrid), and a base sequence encoding a polypeptide having no equivalent activity forms a hybrid with the specific sequence (, so-called non-specific hybrid) Shina! Means a condition. Those skilled in the art can easily select such conditions by changing the temperature during the hybridization reaction and washing, the salt concentration of the hybridization reaction solution and the washing solution, and the like. Specifically, 6 X SSC (0.9 M NaCl, 0.09 M trisodium citrate) or 6 X SSPE (3 M NaCl, 0.2 M NaH PO, 20 mM EDTA-2

 twenty four

Na, pH 7.4) Noble at 42 ⁰ C, then 0.5 X SSC at 42 ⁰ C. This is one of the stringent conditions of the present invention. This is not a limitation.

[0051] The "base sequence in which one or more bases have been deleted, substituted, inserted or added" means that one or more amino acids are generally substituted in the amino acid sequence of a protein having physiological activity. It will be widely recognized by those skilled in the art that even when deleted, inserted or added, the physiological activity may be maintained. Such a modification is added to the present invention, and a gene encoding a polyamine metabolism-related enzyme is also included in the scope of the present invention. For example, polyA tails and untranslated regions at the 5 ′ and 3 ′ ends may be “deleted”. Alternatively, it may be deleted in such a range that amino acids are deleted. In addition, the base may be “substituted” within a range in which no frame shift occurs. Further, it may be “added with a basic strength” in such a range that an amino acid is added. However, even with such modifications, it is necessary to have polyamine metabolism-related enzyme activity. Preferably, “a gene in which one or several bases have been deleted, replaced or added” is preferred.

 Such modified DNA can be substituted, deleted, inserted, or added at a specific site by, for example, site-directed mutagenesis (Nucleic Acid Research, Vol. 10, No. 20, 6487-6500, 1982). Thus, it is obtained by modifying the base sequence of the DNA of the present invention. Gramineae plants and their progeny with improved stress tolerance

In the present invention, “stress” includes high temperature, low temperature, low pH, low oxygen, oxidation, salt, osmotic pressure, drying, water, flooding, cadmium, copper, ozone, air pollution, ultraviolet light, strong light as described above. Examples of such stress include environmental light such as low light, pathogens, pathogens, pests and herbicides. In this context, “high temperature stress” refers to the stress that a plant experiences when it encounters an environment that exceeds the upper limit of plant growth temperature. Physiological function is impaired and injury is caused. “Low-temperature stress” refers to the stress that plants receive when they encounter an environment that exceeds the lower limit of plant growth temperature. The physiology of the vesicle is impaired and injury is caused. `` Salt stress '' is the stress that plants receive when they encounter an environment that exceeds the upper limit of the appropriate salt concentration for plant growth. Excess salt flows into cells in plants that have received salt stress. As a result, the physiology of the cells is gradually or suddenly impaired, causing injury. “Dry stress” refers to the stress that a plant experiences when the plant encounters an environment that exceeds the lower limit of the water concentration suitable for growth of the plant. Function is impaired and injury is caused. “Water stress” refers to the stress that a plant experiences when it encounters an environment that exceeds the lower limit of the water concentration suitable for growth of the plant. Function is impaired and injury is caused. “Low light stress” refers to the stress that plants receive when they encounter an environment that exceeds the lower limit of the optimal light intensity for plant growth. In the plant that receives it, the physiological function of the cell is gradually or suddenly damaged, causing injury. “Pathogenic stress” refers to the stress that a plant receives as a result of encountering or infecting the plant with a pathogenic fungus. A plant that has undergone pathogenic stress gradually or suddenly loses its cellular physiology and causes injury. “Pest stress” refers to the stress that a plant receives when the plant encounters the pest. The plant that receives the pest stress gradually or suddenly loses its physiological function and causes injury.

 [0053] In the present invention, "the grass family with improved stress tolerance" and "the grass family with improved stress tolerance" refers to the introduction of an exogenous polyamine metabolism-related enzyme gene. This refers to a grass family that has been given or improved stress tolerance (resistance) compared to before introduction. For example, by introducing polyamine metabolism-related enzyme genes into plants, low temperature stress resistance (resistance) ゝ salt stress resistance (resistance) ゝ herbicide stress resistance (resistance), resistance to air pollution stress (resistance) Resistance to pathogenic stress (resistance), resistance to drought stress (resistance), or resistance to osmotic stress (resistance), yield of useful substance or rice, number of rice, etc. Examples of such a plant include, but are not limited to, plants that are improved compared to plants.

[0054] Specifically, "a gramineous plant with improved salt stress tolerance" refers to a rice plant that has been able to avoid or reduce the growth suppression and injury caused by salt stress encountered during the growth process of gramineous plants. It is a family plant. “Gramineae plants with improved resistance to low-temperature stress” are grass plants that have been able to avoid or reduce the growth suppression and injury caused by low-temperature stress encountered during the growth process of gramineous plants. “Gramineae plants with improved resistance to pathogenic stress” refers to grasses that have been able to avoid or reduce the growth inhibition and damage caused by pathogenic stress encountered in the growth process of gramineous plants. “Gramineae plant with improved resistance to pest stress” refers to a gramineous plant that has been able to avoid or reduce the growth inhibition and damage caused by pest stress encountered during the growth process of the gramineous plant. This can be expected to stabilize cultivation, improve productivity and yield, expand cultivation area and area. In addition, the productivity and yield of Gramineae plants and rice can be expected to improve the productivity of various useful substances (starch, protein, etc.) obtained from Gramineae plants. it can.

 [0055] Particularly in the case of gramineous plants, the ability to become sterile when stressed during the booting stage. The transgenic gynecaceous plant of the present invention is particularly resistant to stress during the booting stage. It can effectively prevent the gramineous plants from becoming sterile.

 [0056] In the present specification, the "blossoming period" is a certain period before the heading period of a gramineous plant (for example, from a few days to about 2 to 3 weeks) and is important for the ears to grow. Say the right time. In general, if stress such as low temperature stress is applied during the booting period, the ear (corn), grain (wheat, barley) or grain (rice) cannot be harvested. “Tillering period” is the time when ears, pods and tillers are formed, and is an important period for determining the number of spikes, pods and tillers. Increasing the number of spikes and pods improves the lodging resistance through a decrease in the height (plant height) of gramineous plants that directly leads to an increase in yield.

 Ti who was self and its

 In the present invention, “productivity” includes all organs (tissues) of the gramineous plants as described above, for example, ears, pods, tillers, seeds, pods, rice, fruits, spikelets, etc. Examples of the traits of the organ (tissue) include quantity, growth period, shape, color, property, and characteristics.

In the present invention, “a gramineous plant with improved productivity” and “a gramineous plant with improved productivity” mean an exogenous polyamine metabolism-related enzyme gene or an endogenous polyamine metabolism-related enzyme gene. This refers to a grass family plant that has been imparted or improved or suppressed in quantity, growth period, shape, coloration, properties, and characteristics, which are traits related to productivity compared to before introduction. For example, by introducing a polyamine metabolism-related enzyme gene or the suppressor into a plant, traits related to ears, pods, tillers, seeds, pods, rice, fruits, and spikelets may be converted into the exogenous polyamine metabolism-related enzymes. Examples thereof include, but are not limited to, V, which has a gene or the repressing factor, and improved gramineous plants compared to gramineous plants.

 [0058] The amount of polyamines in the grass family is high due to the presence of high concentrations (eg, about 1 to 3 times that of the wild strain) in the ears, pods and tillers during the tillering period. Activating tillering increases calories, ears and tillers.

[0059] This improves the productivity, yield, yield and quality of gramineous plants (organs' tissues, etc.) Expected to improve merchantability. In grasses, reducing the amount of polyamines in the edible part (rice, corn, wheat, barley, etc.) may positively affect quality and other characteristics. Therefore, it is possible to increase the yield while improving the quality by expressing a suppressor in these parts and expressing a polyamine-related enzyme gene in the other parts.

 [0060] The plant of the present invention is not limited to the whole gramineous plant (whole culm), but its callus, seeds, all plant tissues, leaves, stems, ears, buds, tubers, roots, tuberous roots, pods, Includes flowers, petals, ovary, fruit, pods, ovules, fibers, cocoons, rice, etc. Furthermore, the progeny are also included in the plant of the present invention. The important parts of Gramineae plants are ears, buds, tillers, seeds, pods, rice, berries, and spikelets.

 [0061] In the present invention, "a useful substance obtained from a gramineous plant and its progeny" means that productivity has been improved as compared to that before introduction by introducing an exogenous polyamine metabolism-related enzyme gene. Examples of useful substances produced in grasses and their descendants include, for example, amino acids, fats and oils, starches, proteins, phenols, hydrocarbons, cellulose, natural rubber, pigments, enzymes, antibodies, vaccines, pharmaceuticals And biodegradable plastics.

 [0062] Gramineous plant power provides a large amount of carbohydrates such as starch, which can be used as a raw material for producing biodegradable plastics. Biodegradable plastics include polyhydroxy propylate, polyhydroxy valerate, poly 13-hydroxybutyric acid, poly force prolatatone, polybutylene succinate, polybutylene adipate, polyethylene succinate, poly (D, L, DL) lactic acid (polylactide) ), Polyglycolic acid (polyglycoside), cellulose acetate, chitosan Z cellulose Z starch, modified starch, etc., or a binary copolymer or ternary copolymer thereof. These biodegradable plastics are known and can be produced using known fermentation methods, chemical synthesis methods, and the like.

 [0063] The plant of the present invention is a plant that does not have the exogenous polyamine metabolism-related enzyme gene.

An exogenous polyamine metabolism-related enzyme gene has been introduced and stably maintained by genetic engineering techniques. Here, “stablely maintained” means that the polyamine metabolism-related enzyme gene is expressed at least in the present plant into which the polyamine-metabolism-related enzyme gene has been introduced, which is sufficient to improve stress tolerance. It is retained in the plant cell for a period of time. Therefore, in reality, the polyamine metabolism-related enzyme gene is U, preferably integrated into the plant chromosomes. The polyamine metabolism-related enzyme gene is more preferably inherited stably in the next generation.

 [0064] Here, "exogenous" means that the plant is not naturally present and is introduced from the outside. Therefore, the “exogenous polyamine metabolism-related enzyme gene” of the present invention may be a polyamine metabolism-related enzyme gene of the same kind as the host plant (ie, derived from the host plant) introduced from the outside by genetic manipulation. . Considering the identity of codon usage, the use of host-derived polyamine metabolism-related enzyme genes is also preferred.

 [0065] An exogenous polyamine metabolism-related enzyme gene may be introduced into a plant by any genetic engineering technique. For example, protoplast fusion with a heterologous plant cell having a polyamine metabolism-related enzyme gene, polyamine metabolism-related enzyme gene Infection with a plant virus having a viral genome genetically engineered to express or transformation of host plant cells with an expression vector containing a polyamine metabolism-related enzyme gene.

 [0066] Preferably, the plant of the present invention is an expression vector containing an exogenous polyamine metabolism-related enzyme gene under the control of a promoter that can function in the plant, and has the exogenous polyamine metabolism-related enzyme gene. Transgenic plants obtained by transforming cells of non-plants.

 [0067] Examples of promoters that can function in plants include the cauliflower mosaic virus (CaMV) 35S promoter, nopaline synthase gene (NOS) promoter, and octopine synthase gene (OCS) promoter that are constitutively expressed in plant cells. , Phenylalanine ammonia lyase (PAL) gene promoter, canolecon synthase (CHS) gene promoter, and the like. Furthermore, the well-known plant promoter which is not limited to these is also mentioned.

[0068] Not only a promoter that is constitutively expressed throughout the organ, such as the 35S promoter, but also an organ- or tissue-specific promoter can be used to express a target gene only in a specific organ or tissue. Only one organ or tissue can improve stress tolerance. For example, by using polyamine metabolism-related enzyme genes and promoters that specifically act on flower organs (for example, W099 / 43818, JP-A-11-178572, JP-A-2000-316582) The flower size can be improved. In addition, seed The number and size of seeds, ovaries, and fruits can be improved by using a promoter that specifically acts on fruits and ovaries (eg, Plant Mol. Biol, 11, 651-662, 1 988). . Furthermore, by using polyamine metabolism-related enzyme genes and promoters that can cause transcription only when plants encounter low temperatures (eg, BN115 promoter: Plant physiol., 106, 917-928, 199 9), It can control the body's polyamine metabolism and improve cold stress resistance. Furthermore, by using a polyamine metabolism-related enzyme gene and a promoter that can cause transcription only when the plant encounters drought (eg, Atmyb2 promoter: The Plant Cell, 5, 1529-1539, 1993), It can control the body's polyamine metabolism and improve drought stress resistance.

 [0069] If an organ- or tissue-specific promoter is used, the target gene can be expressed only in a specific organ or tissue. For example, by using a polyamine metabolism-related enzyme gene and a promoter that specifically acts on the seed, stress tolerance can be improved only by the seed. If a time-specific promoter is used, the target gene can be expressed only at a specific time, and stress tolerance can be improved only at a specific time. For example, by using a polyamine metabolism-related enzyme gene and a promoter that works during vegetative growth, stress tolerance can be improved only during vegetative growth.

 [0070] If a time-specific promoter is used, a target gene can be expressed only at a specific time, and productivity can be improved only at a specific time. For example, by using a polyamine metabolism-related enzyme gene and a promoter that works during the vegetative growth period, productivity can be improved only during the vegetative growth period.

 [0071] In the expression vector of the present invention, the exogenous polyamine metabolism-related enzyme gene is arranged downstream of the promoter so that its transcription is controlled by a promoter that can function in plants. It is preferable that a transcription termination signal (terminator region) capable of functioning in plants is further added downstream of the polyamine metabolism-related enzyme gene. For example, the terminator NOS (nopaline synthase) gene and the like can be mentioned.

[0072] The expression vector of the present invention may further contain a cis-regulatory element such as an enhancer sequence. In addition, the expression vector is a marker gene for selection of transformants such as drug resistance gene markers, such as neomycin phosphotransferase II (NPTI It may further include I) a gene, a phosphinothricin acetyl transferase (PAT) gene, a glyphosate resistance gene, and the like. Under conditions where selective pressure is not applied, the incorporated gene may drop out, so if the herbicide-tolerant gene coexists on the vector, the herbicide can be used during cultivation. There is also an advantage that a condition under selective pressure can always be realized.

 [0073] Furthermore, to facilitate large-scale preparation and purification, the expression vector has an origin of replication that enables autonomous replication in E. coli and a selectable marker gene in E. coli (eg, ampicillin resistance gene, tetracycline resistance Gene etc.). The expression vector of the present invention is simply constructed by inserting the above-mentioned polyamine metabolism-related enzyme gene expression cassette and, if necessary, a selection gene into the cloning site of a pUC or pBR E. coli vector. can do.

 [0074] When an exogenous polyamine metabolism-related enzyme gene is introduced by using an infection by Agrobacterium rhizogenes, Agrobacterium tumefaciens In general, the polyamine metabolism-related enzyme gene expression cassette can be inserted into the T DNA region (region transferred to the plant chromosome) on the Ri plasmid. At present, the standard method of transformation by agro-batterium method uses a noinary vector system. The functions required for T-DNA transfer are supplied independently by both T-DNA itself and the Ti (or Ri) plasmid, and each component can be split on separate vectors. The binary plasmid has 25 bp border sequences at both ends necessary for T-DNA excision and integration, and the plant hormone gene that causes crown gall (or hairy root) has been removed, and there is room for insertion of foreign genes at the same time. Is given. As such binary vectors, for example, ρΒΙΙΟΙ and PBI121 (manufactured by Clontech) are commercially available. The Vir region, which acts on T-DNA integration, acts on trans on another Ti (or Ri) plasmid called a helper plasmid.

[0075] Various conventionally known methods can be used for plant transformation. For example, plant cell power protoplasts are isolated by cell wall degrading enzyme treatment such as cellulase or hemicellulase, and the expression cassette of the protoplasts and the above polyamine metabolism-related enzyme genes is isolated. A method in which polyethylene glycol is placed in a suspension with an expression vector containing a phospholipid and the expression vector is incorporated into a protoplast in an endocytic manner (PEG method), lipid membrane vesicles such as phosphatidylcholine Putting an expression vector into the cell by sonication, etc., fusing the vesicle and protoplast in the presence of PEG (ribosome method), fusing in the same process using a minicell, suspension of protoplast and expression vector For example, a method of applying an electric pulse to the protoplast to incorporate the vector in the external solution into the protoplast (electrovolation method) can be mentioned. However, these methods are complicated in that they require a culture technique for redifferentiating protoplasts into plants. As a means of gene transfer into an intact cell with a cell wall, a microinjection method in which a micropipette is inserted into the cell and vector DNA in the pipette is injected into the cell by hydraulic pressure or gas pressure, and DNA is coated There are a direct introduction method such as a particle gun method in which fine gold particles are accelerated using explosive explosion or gas pressure and introduced into the cell, and a method using infection by agrobacterium. Microinstruction has the disadvantages that it requires skill in operation and the number of cells that can be handled is small. Therefore, considering the simplicity of operation, it is preferable to transform plants by the agrobacterium method and the particle gun method. The particle gun method is further useful in that the gene can be directly introduced into the apical meristem of the plant being cultivated. In addition, in the agrobacterium method, a plant virus, for example, a genomic virus DNA such as tomato golden mosaic virus (TGMV), is inserted into a binary vector at the same time between border sequences. By simply inoculating cells at any part of the plant with a bacterial suspension using a syringe or the like, viral infection spreads throughout the plant, and at the same time, the target gene is introduced into the entire plant. These methods are well known in the art, and a method suitable for the plant to be transformed can be appropriately selected by those skilled in the art.

 [0076] The transformed product produced by the method described above is subjected to Southern analysis or Northern analysis for gene expression analysis of polyamine metabolism-related enzyme genes, polyamine content analysis, stress tolerance evaluation, and productivity evaluation. It can be carried out.

[0077] For example, polyamine is quantified by sampling a sample of 0.05-: Lg and adding a 5% aqueous perchloric acid solution to extract the polyamine. The quantification of the extracted polyamine After labeling with benzo louis, etc., it can be analyzed by internal standard method using high performance liquid chromatography (HPLC) connected with fluorescence or uv detector.

[0078] For example, as an evaluation of productivity, a T1 seed or a T2 seed obtained by self-pollination of a transgeneic gramineous plant or a transgeneic gramineous plant is appropriately grown. Planted or sown in culture medium or growing soil, grown at 20-30 ° C under long-day conditions (day Z night: 16 hours Z8 hours day length), number and size of spikelets and pods (eg, parting), It can be evaluated by examining the shape. For inflorescences, fruits, seeds, grains, grapes, and rice, use T1 seeds or T2 seeds obtained by self-pollination of Transgeneic Gramineae plants or Transgenic Gramineae plants in an appropriate growth medium or growth Planted or sown in soil and grown at 20-30 ° C under long-day conditions (day Z night: 16 hours Z8 hours day length), number of inflorescences, fruits, seeds, grains, straw, rice It can be evaluated by examining the size, shape, etc.

[0079] For example, low-temperature stress tolerance can be evaluated by examining growth conditions, low-temperature injury, etc. after growth at 25-30 ° C after low-temperature treatment at 0-20 ° C for 1-10 days. Low temperature stress tolerance can be evaluated by growing gramineous plants at 10 ° C to 18 ° C for the entire period and examining their growth status and body weight (yield). High temperature stress tolerance can be evaluated by examining growth conditions and high temperature injury after growing at 25-30 ° C after low temperature treatment at 35-50 ° C for 1-: LO days. High temperature stress tolerance can be evaluated by growing grasses at 35 ° C to 45 ° C for the entire period and examining their growth status and fresh body weight (yield). Resistance to salt stress can be evaluated by growing at 25-30 ° C in a medium containing 10-300 mM NaCl and examining the growth status for salt stress disorder. Salt stress tolerance can be evaluated by growing grasses in culture soil containing 10-150 mM NaCl for the entire period and examining the growth status and body weight (yield). The resistance to drought and water stress can be evaluated by examining the growth situation and the degree of damage after stopping the water supply. Dry water stress can be evaluated by growing grasses in culture soil with limited irrigation for the entire period and examining the growth status and body weight (yield). Pathogenic stress can be assessed by inoculating rice with rice blast fungus or rice leaf blight fungus and examining the disease spots, susceptibility response, growth status and degree of damage after inoculation. Pest stress can be evaluated by examining the extent of food damage and growth conditions by releasing rice, rice, rice leafhopper and kokuzo to rice and rice. [0080] In the present invention, the method for improving (improving) productivity is a comparative control by stably maintaining a nucleic acid sequence that regulates the amount of polyamine under the control of a promoter capable of functioning in a plant. Yield or useful substance (eg starch, fat, protein, cellulose, etc.) such as inflorescence, fruit, seed, grain, straw, rice (eg rice, corn, wheat, barley), etc. A method in which (content) increases. Preferably, the taste of the edible substance is not deteriorated by the method, and is equal to or higher than that of the control plant.

 [0081] The gramineous plant to be transformed of the present invention is not particularly limited, and examples thereof include monocotyledonous plants. Examples include cultivated rice, wild rice, corn, wheat, rye, wheat, and embata. Rice and corn are preferable.

 [0082] The present invention can be preferably implemented for, for example, rice.

 [0083] Gramineous plant power provides sugars such as starch, which can be used as a raw material for producing biodegradable plastics. Biodegradable plastics include polyhydroxy propylate, polyhydroxy valerate, poly 13-hydroxybutyric acid, poly force prolatatone, polybutylene succinate, polybutylene adipate, polyethylene succinate, poly (D, L, DL) lactic acid (borilactide) And polyglycolic acid (polyglycolide), cellulose acetate, chitosan Z cellulose Z starch, modified starch and the like, and binary copolymers and ternary copolymers thereof. These biodegradable plastics are known and can be produced using known fermentation methods, chemical synthesis methods, and the like.

 Example

 [0084] The present invention will be described more specifically with reference to the following examples. However, these are merely examples and do not limit the scope of the present invention.

[0085] It should be noted that in the following examples, an example of using rice as a gramineous plant is described. With respect to other gramineous plants such as corn, wheat, and barley, various environmental stress tolerances and yields are similarly increased. To do.

 ^ l: Cloning of plant-derived polyamine metabolism-related enzyme genes

In accordance with the description in Example 2 of WO02Z23974, a full-length spridine synthase gene (FSPD1; SEQ ID NO: 1, 2), S adenosylmethionine decarboxylase gene (FSAM24; SEQ ID NO: 3, 4) ), The arginine decarboxylase gene (FAD C76; SEQ ID NO: 5, 6) was obtained.

 Example 2: Production and analysis of transgenic rice

 (1) Production of expression construct

 The polyamine metabolism-related gene FSPD1 shown in SEQ ID NO: 1 was cleaved with Xhol so as to include all open reading frames from the base sequence of FSPD1, and purified by the glass milk method. Next, pGEM-7Zf (Promega) was cleaved with Xhol, and the FSPD1 fragment was subcloned in the sense and antisense directions, respectively. The FSPD1 fragment was excised again with the restriction enzymes Xbal and Kpnl of the pGEM-7Zf multicloning site and subcloned into the binary vector pBI101-Hm2 linked to the 35S promoter. These plasmids were named PBI35S—FSPD1 +/—. The structure of the expression construct is shown in Fig. 1. The transformed E. coli JM109 was named Escherichia coli JM109 / pBI35S—FSPD1 + Z—.

 [0086] The polyamine metabolism-related gene FSAM24 shown in SEQ ID NO: 3 was cleaved with Notl so as to include all open reading frames from the base sequence of FSAM24, and each was blunt-ended. These fragments were ligated with a blunt-ended 35S promoter and subcloned into the binary vector pBI101-Hm2 in the sense and antisense directions. These plasmids were named PBI35S—FSAM24 + Z—. The structure of the expression construct (sense direction only) is shown in FIG. The transformed E. coli JM109 was named Escheri chia coli JM109 / pBI35S— FSAM24 + / —.

 [0087] The polyamine metabolism-related gene FADC76 shown in SEQ ID NO: 5 was cleaved with Notl so as to include all open reading frames from the base sequence, and each was blunt-ended. These fragments were subcloned in the sense and antisense directions into a binary vector pBI101-Hm2 ligated with a 35S promoter with blunt ends. These plasmids were named PBI35S— FADC76 + Z—. The structure of the expression construct (sense direction only) is shown in FIG. The transformed E. coli JM109 was named Escherichia coli JM109ZpBI35S— FADC76 + Z—.

 (2) Introduction of plasmid into agrobacterium

E. coli pBI35S-FSPD 1 + Z— obtained in (1), E. coli pBI35S-FSAM24 + /-, Escherichia coli pBI35S-FADC76 +/- and E. coli HB101 strains with helper plasmid pRK2013, 1 mg at 37 ° C in LB medium each containing 50 mgZl of kanamycin, 50 mgZl of Agrovaterium EHA101 The cells were cultured at 37 ° C for 2 days in LB medium containing kanamycin. 1.5 ml of each culture solution was collected in an Eppendorf tube and then washed with LB medium. After suspending these cells in 1 ml of LB medium, mix three types of bacteria 100 1 at a time, spread them on LB medium agar medium, and incubate at 28 ° C to transfer the plasmid to agrobacterium. Three-party joining method). One or two days later, a portion was scraped with a platinum loop and applied to LB agar medium containing 50 mg Zl kanamycin, 20 mg / remyromycin, and 25 mg / l chloramphee-chol. After culturing at 28 ° C for 2 days, a single colony was selected. The resulting transformants were named EHA10lZpBI35S—FSPD1 + Z—, EHA10lZpBI35S—FSAM2 4 + Z—, EHA101 / pBI35S—FADC76 +/—.

(3) Rice transformation

Rice transformation was performed by referring to the method of Hiei Y. et al. (Plant J., 6, 271-282, 1994). Ripe seeds of rice varieties 'Yukihikari' (hereinafter referred to as “Yukihikari” or “wild-type”) are removed from the pods, immersed in 70% ethanol for 5 minutes, and then placed in a sterilized beaker in the same manner ( Disinfection was performed by immersing in 5% sodium hypochlorite, 0.02% Triton X-100) for 20 minutes. Sterilized seeds were washed three times with sterile water in a sterile beaker. After washing, place on a callus induction medium [N6C1 plate: N6 inorganic salt, N6 vitamin, 2mgZl 2, 4—D, 30g / l sucrose, 2g / l gellite, pH5.8], plant incubator , MLR- 350HT) 25 ° C in, bright place ^{(60 mol'm _2 's _1,} 16 h light period Z8 hours dark, were cultured in the following this light conditions and photopic) conditions. About one month later, embryonic callus having the ability to redivide the plant body was selected from the grown tissue. The selected embryonic callus was then transplanted to a new N6C1 plate every month for growth. Agrobataterum infection is caused by transformed agrobataterum strains EHA10lZpBI35S—FSPDl + Z ―, EHA101 / pBIC2—FSPD1 + / — (CaMV35S promoter replaced with peroxidase promoter derived from horseradish rust), EHA101 / pBI35S — FS AM24 + Z—, EHA10lZpBI35S— FADC76 + Z— was cultured on LB agar medium containing 50mgZl kanamycin and 50mgZ lygromycin at 27 ° C Scattered about 2 grains of rice, infection medium (AA: AA mineral salt, amino acid, B5 vitamin, 20g / l sucrose, 2mg / l 2, 4—D, 0.2mg / l force rice, 10mg / l acetosyringone, It was suspended in pH 5.8). This cell suspension was transferred to a 300 ml sterilized beaker containing a sterilized stainless steel net basket. The embryo callus pre-cultured in a beaker is placed in this beaker basket and soaked for 2 minutes. Then, the basket is placed on two sterilized filter papers to remove excess water, and the coculture medium (N6CO plate: N6 inorganic salt) , N6 vitamin, 30 g / \ sucrose, lOgZl glucose, 2 mg / l 2, 4-D, 10 mg / l acetosyringone, 2 g / l gellite, pH 5.8), 3 at 28 ° C in the dark Co-cultured for days. Embryo callus co-cultured for 3 days is transferred to a 300m 1 beaker basket containing a sterile stainless steel basket containing 50ml of sterilization solution (sterilized water plus carbecillin to a final concentration of 500mgZl) I picked up the basket with tweezers and washed it well for several minutes. The embryonic callus was then transferred to a 300 ml sterilized beaker containing the sterilization solution along with the basket and washed again. Repeat the same procedure, remove excess water on sterile filter paper, and select medium (N6SE plate: N6 mineral salt, N6 vitamin, 30g / l sucrose, 2mgZl 2, 4-D, 50mg / l neugromycin. , 500 mg / l carbe-cillin, 2 g / l gellite, ρρ5.8) and cultured at 25 ° C. under dark conditions for about 3 to 4 weeks. Regeneration medium (MSRE plate: MS mineral salt, MS vitamin, 30g / l sucrose, 30g / l sorbitol, 2g / l casamino acid, lmgZl NAA, 2mg / l BAP 250mg / l force Norebenicillin 50 mg / l Neugromycin, 4 g / l Genorelite, pH 5.8) [The transplanted cells were cultured at 25 ° C under dark conditions until redifferentiation. Confirmation and expression analysis of the introduced gene were performed on the obtained transformants. Specifically, for the confirmation of the transgene, the genomic DNA was prepared and then PCR and Southern hybridization were performed. For the expression analysis of the introduced gene, Northern analysis and Western analysis were performed. Western analysis showed that 4 strains were transformed ^^ (RSP— SS— 1— 1, RSP-SS- 1-2, RSP— CS— 3— 1, RSP -CS- 3-2, 3, RSP—CS— 3— 4) We extracted leaves by sampling leaves. The extracted protein was subjected to SDS-PAGE according to a conventional method and then transferred to a membrane by an electroblot method. Western blotting was performed using a peptide antibody of FSPD1 (SPDS—SP3: LCSTEGPPLDFKHP). Fig. Shown in 2. As is clear from the results in Fig. 2, the transformations (RSP— SS— 1— 1, RSP- SS- 1-2, RSP-CS-3- 1, RSP— CS— 3— 2, RSP— CS— In 3-3, RSP-CS-3-4), a band showing immunoreactivity with the FSPD1 peptide antibody was detected, indicating that the target gene was transcribed and translated. Based on the above, it was possible to obtain V, a transformant with the target gene introduced.

(4) Analysis of polyamine

 The transformed rice produced in (3) was selected as a result of PCR (or Southern analysis), Northern analysis, or Western analysis. Polyamine analysis was performed on lines in which a polyamine metabolism-related enzyme gene was reliably introduced and the gene was stably expressed. Cell lines with FSPD1 introduced in the sense direction (+), RSP—SS 1 1, RSP—SS—1, 2, RSP—CS—3—1, RSP—CS—3—2, RSP—CS—3— 3. RSP—CS—3—4 was selected, RSP—SS—1-1, RSP—SS—1-2—has been introduced with CaMV35S promoter as a promoter, RSP—CS—3-1— RSP—CS—3—2, RSP—CS—3—3, and RSP—CS—3—4 are strains into which the horseradish penoleoxidase promoter (C2 promoter) has been introduced. FSPD 1 has been introduced in the antisense direction (1)! Cell lines, RSP-SA-1, RSP-SA-2 have been selected and all are CaMV 35S promoters. FS AM24 is introduced in the sense direction (+) !, Cell Line, RSP—SM—1—1, TSP—SM—1-2, RSP—SM—1-1, RSP—SM—1 -2 is a strain in which the CaMV35S promoter is introduced as a promoter. About 0.3 to 1. Og young leaves were sampled and stored frozen from wild strains (WT) and transformants (TSP) grown at the same time. Add the diluted internal standard solution (1, 6-hexanediamine, internal standard amount = 7.5 or 12 nmol) and 5% aqueous perchloric acid solution (sample body weight: 1 to 5 ml per Og: LOml) to the sampled sample, And thoroughly ground and extracted at room temperature. Grinding liquid,

Centrifugation was carried out at 4 ° C · 35,000 Xg for 20 minutes to collect the supernatant, and this solution was used as a free polyamine solution. To a microtube with a screw cap, 400 1 free polyamine solution, 200 1 saturated aqueous sodium carbonate solution, 200 1 Dansilk mouthride Z acetone solution (lOmgZml) were added and mixed gently. After tightly closing the tube stopper, aluminum It was covered with foil and heated for 1 hour with water nose at 60 ° C, and then dansill candy was performed. After allowing the tube to cool, an aqueous proline solution (lOOmgZml) was added at 200 / zl and mixed. It was covered with aluminum foil and reheated in a water bath for 30 minutes. After standing to cool, nitrogen gas was blown to remove acetone, and 6001 toluene was added and mixed vigorously. After leaving the tube to separate into two phases, the upper toluene layer was fractionated into 3001 microtubes. Toluene was completely removed by blowing nitrogen gas to the collected toluene. The tube was filled with 200 1 methanol to dissolve the dansylated free polyamine. The amount of free polyamines of putrescine, spermidine, and spermine was analyzed by an internal standard method using high performance liquid chromatography connected with a fluorescence detector (excitation wavelength: 365 nm · emission wavelength: 51 Onm). The HPLC column used was / z Bondapak C18 (Waters: 027324, 3.9 X 300 mm, particle size 10 m). The polyamine content in the sample was calculated by obtaining the peak areas of each polyamine and internal standard from the standard solution and the HPLC chart of the sample, respectively. As a result, cell lines in which polyamine metabolism-related enzyme genes were introduced in the sense direction had significantly higher putrescine content, spermidine content, and spermine content than the wild type strain (WT), and the total polyamine content was also higher than the wild type strain (WT). A significant increase was evident. In particular, the increase in spermidine and spermine content was remarkable. Furthermore, cell lines in which polyamine metabolism-related enzyme genes were introduced in the direction of antisense showed increased putrescine content, especially spermidine content and spermine content were significantly reduced compared to the wild strain (WT), and the total polyamine content was also increased in the wild strain (WT ) It became clear that it decreased more significantly. From the above results, it has become clear that it is possible to increase or decrease the content of endogenous polyamines by introducing a polyamine metabolism-related enzyme gene into rice in the sense or antisense direction. These results indicate that polyamine content can be controlled by manipulating polyamine metabolism by introducing polyamine metabolism-related enzyme genes into plants.

 ^ Mffi: Productivity evaluation

Transform with wild strain (WT) ^ ^ (RSP— SS— 1— 1, RSP-SS-1-2, RSP— CS-3- 1, RSP-CS- 3- 2, RSP-CS- 3- 3, RSP—CS— 3-4) Seeds were removed one by one with tweezers. To the seeds, 5 ml of a seed sterilizing solution (2.5% antiformin, Tween 20) was added and shaken at 150 rpm for 30 minutes. Discard the seed sterilization solution, This operation was repeated 4 times by adding sterilized water and shaking at 150 rpm for 10 minutes. Seeds of wild type (WT) are growth medium (RGM: MS inorganic salt, MS vitamin, 30g / l sucrose, 8gZ 1 phytowager, pH 5.8), seeds of transformed rice are selective growth medium (RGM: MS inorganic salt MS vitamin, 30 g / l sucrose, 8 g / l phytowager, 50 mgZl kanamycin, 20 mgZl hygromycin, pH 5.8). The cells were grown in a growth chamber set at 26 ° C, low light conditions (4 000 lux), and 16 hours day length (16 hours light period Z8 hours dark period). On the 7th to 10th days after sowing, individuals with the same growth of wild strains and transformed rice were selected and planted in plastic pots filled with commercially available culture soil. After acclimatization, the cultivation test was started in a closed glass room (set temperature daytime Z night: 23-24 ° C Z21-22 ° C, humidity 55%, natural day length). The growth status was examined during the booting, heading, flowering, and maturity periods. As a result, there was no difference in the booting, heading, and flowering periods between wild-type and transformed rice. During maturity, the transformation showed a tendency to be 4 to 7 days later than the wild type. Yield components were examined 4 months after sowing. The results are shown in Table 1. From the results in Table 1, it was confirmed that the productivity and traits of transformed rice plants into which polyamine metabolism-related enzyme genes were introduced can be improved compared to the wild type.

[0088] [Table 1]

[0089] Example 4: Evaluation of various stress tolerance of transformed rice

 (1) Evaluation of salt stress tolerance

The seeds of the wild strain (WT) and the transformed ^^ (RSP-SS-1-1, RSP—SS—1-2) were removed with a pair of tweezers. 5 ml of seed sterilization solution (2.5% antiformin, Tween20) was added to the seeds and shaken at 150 rpm for 30 minutes. The seed sterilization solution was discarded, 10 ml of sterilized water was added, and this operation was repeated 4 times by shaking at 150 rpm for 10 minutes. Wild strain ( WT) seeds contain NaCl (75mM NaCl, lOOmM NaCl) or no growth medium (RGM: MS inorganic salt, MS vitamin, 30gZl sucrose, 8g / l phytofagger, pH5.8), transformed rice (RSP— SS-1-1, RSP-SS-1-2) seeds with or without NaCl (75 mM NaCl, lOOmM NaCl) or selected growth medium (RGM: MS inorganic salt, MS vitamins, 30 g / l sucrose , 8 g / l fiaguar, 50 mg / l kanamycin, 20 mg Λ, igromycin, pH 5.8). Growth tests were conducted in a growth chamber set at 26 ° C, low light conditions (4000 lux), and 16 hours day length (16 hours light period Z8 hours dark period). On the 10th day after sowing, the body weight (total) and shoot length were investigated.

 In the group containing no NaCl (non-stressed group), the wild strain and the transformed rice germinated in the same manner from the second day of sowing. On the other hand, in the salt-stressed area, transformed rice germinated on the third day, whereas in the wild strain, germination was delayed and on the 4th and 5th days, germination occurred. It was confirmed that transformed rice was superior in germination vigor under salt stress conditions compared to the wild type. Fig. 3 shows the results of the growth survey (live weight 'shoot length) on the 10th day after sowing, and Fig. 4 shows the appearance of the 75mM NaCl. Results of Figure 3 In the non-stressed area, there was no difference in the live weight and shoot length between the wild strain and the transformed strain, but in the salt stress area, both the live weight and the shoot length were wild. The growth was significantly larger than that of. As can be seen from the results in FIG. 4, the transformed rice showed significantly better growth of both shoots and roots than the wild type. From the above results, it was shown that transgenic rice introduced with a polyamine metabolism-related enzyme gene has higher tolerance to salt stress than wild-type strains.

(2) Evaluation of low temperature stress tolerance

The seeds of the wild strain (WT) and the transformed ^^ (RSP-SS-1-1, RSP—SS—1-2) were removed with a pair of tweezers. 5 ml of seed sterilization solution (2.5% antiformin, Tween20) was added to the seeds and shaken at 150 rpm for 30 minutes. The seed sterilization solution was discarded, 10 ml of sterilized water was added, and this operation was repeated 4 times by shaking at 150 rpm for 10 minutes. The seeds of the wild strain (WT) are grown in the growth medium (RGM: MS inorganic salt, MS vitamin, 30g / l sucrose, 8g / \ phytowager, ρΗ5.8), and transformed rice (RSP—SS—1—1, RSP— SS-1-2) seeds are selected growth medium (RGM: MS inorganic salt, MS vitamin, 30g / l sucrose, 8g / 1 phytofaga, 50mg / l kanamycin, 20mg / l neugromycin, pH 5.8) Respectively. They were grown for 3 days in a growth chamber set at 26 ° C, low light conditions (4000 lux), 16 hours long (16 hours light period Z 8 hours dark period) and germinated in the same manner. After that, half of the wild and transformed plants were transferred to a growth chamber set at low temperature stress (18 ° C '4000 lux), and the other half were transferred to a growth chamber set at 26 ° C. Continued to grow. On the 10th day after sowing, live weight (total) and shoot length were investigated.

 [0091] Fig. 5 shows the results of the growth survey on the 10th day of sowing (live weight 'shoot length), and Fig. 6 shows the appearance of the grass on the 7th day of sowing. Results of Fig. 5 Forces with no difference in weight and shoot length in wild strains and transformed strains in non-stressed areas Both weight and shoot length in cold stressed areas compared to wild strains in transformed rice The growth was significantly large and excellent. As is clear from the results in Fig. 6, the transformed rice showed significantly better growth in both shoots and roots than the wild type. From the above results, it was shown that the transgenic strain into which the polyamine metabolism-related enzyme gene was introduced was more resistant to low temperature stress than the wild type.

 [0092] In addition, the present inventors have confirmed that the rice produced in the present invention is resistant to various stresses that are known to reduce fruitiness such as low temperature, high temperature, lack of sunshine, and high salt. Therefore, even when various stresses such as low temperature stress, salt stress, lack of sunshine, drought stress, and water stress are applied during the booting period, the fertility does not decrease.

 : Evaluation of rice yield under normal cultivation conditions or salt stress conditions

 RSP-SS-1-1 and RSP-SS-1-2 as described in Example 2 Selected.

[0093] T2 seeds of wild strain (WT, cultivar 'Yukihi forceri') and transformation ^ ^ (RSP -SS-1-1-2, RSP- SS- 1-2-3) one by one I removed the bag with tweezers. The seeds were transferred to a beaker containing water for water absorption treatment. The water-absorbed seeds were sown in deep-bottomed bats packed with commercially available culture soil (Lovely culture soil for rice breeding, manufactured by Sakai Seki). They were covered with Saran wrap and acclimated under humid conditions, and then transferred to a closed glass greenhouse (day / night temperature 25 ° CZ22 ° C, humidity 55%, natural light) to start growing. After 3 weeks, 16 seedlings per cell line (line) were selected. The selected young seedlings were planted in groups of 8 on a 30L large planter packed with 15L of commercially available culture soil (Nawell culture soil for rice breeding, manufactured by Sakai Seki). (Rice planting). Half of the planted planters are untreated (normal cultivation conditions: none), and the power to pack only 15L of the above-mentioned sales culture soil The other half is salt stress (salt stress condition: salt) In addition to commercially available culture soil, 43.8 g of NaCl (50 mM NaCl concentration) is added, and the mixture is thoroughly stirred and mixed. The evaluation test was started with water maintained at a distance of 5.5 cm from the surface and constantly maintained. As the growth status, the tillering period, the booting period, the heading period, the flowering period, the maturation period, etc. were examined. Harvesting was conducted 4 months after the sowing date, and growth characteristics were evaluated. The results are shown in Table 2. Results shown in Table 2 It was shown that the number of spikes, the above-ground weight, and the above-ground dry weight were significantly increased in the transformed strains regardless of the stress treatment compared to the wild type strain. In addition, the yield of salmon in the salt-stressed area was examined for both wild-type strains (WT) and transformed strains (RSP—SS—1-1—2, RSP—SS—l—2-3). The results are shown in Table 3.

 [0094] As a result of examining the polyamine content during the cultivation period, in the transformed rice, the supermidine content and the spermine content were maintained high within the range of 1.5 to 2.0 times that of the wild strain. In transgenic rice overexpressing the supermidine synthase gene (FSPD1), polyamine levels (especially spermidine and spermine) are maintained higher than in wild-type strains, so that growth and growth can be achieved regardless of the presence or absence of stress treatment. It was obvious that it was excellent.

 [0095] With regard to the increase in the number of ears in particular, it is considered that maintaining the high polyamine level promotes the formation of tillering, particularly by increasing cell division activity during the tillering stage. It was confirmed that the yield of rice and rice increased significantly with the increase in the number of spikes in the transgenic rice that showed superior growth and growth compared to the wild type. Furthermore, it was found that the transformed animals had a shorter height and improved lodging resistance. In addition, in transgenic plants, damage caused by pathogenic bacteria and insects was not observed at all during the cultivation test, and as a result, the spermidine and spermine contents were maintained at a high level by overexpressing the spuridine synthase gene. , Showed that pathogen stress and pest stress resistance also improved

[0096] [Table 2] One line of treatment area Number of spikelets (plant) Plant height (cm) Height of back (cm) Above ground weight (g) Above ground dry weight (g) None WT-6 17.00 ± 0.98 106.1 ± 1.2 99.8 ± 0.7 99.0 ± 6.7 55.6 ± 3.5 No RSP-SS + 1-2 22.63 ± 0.99 * 103.9 ± 1.0 96.5 ± 1.1 118.8 ± 4.4 * 65.8 ± 2.7 * No RSP-SS + 2-3 3 24.57 ± 0.97 ** 104.5 ± 1.2 95.3 ± 1.4 127.7 ± 6.3 ** 69.0 ± 2.8 * Salt— WT-6 15.60 ± 1.33 94.7 ± 2.4 91.0 ± 2.8 68.2 ± 11.6 33.4 ± 5.9 Salt— RSP-SS-1 +2 22.29 ± 1.41 * 93.0 ± 1.8 87.5 ± 1.7 89.6 ± 9.6 * 44.0 ± 4.7 ^# salt one RSP - SS tens of 2 - 3 21.33 ± 0.84 * 93.3 ± 2.5 88.4 ± 1.2 86.0 ± 5.1 * 42.2 ± 4.1 *

 : Yes, there is an S difference at the level of 1¾, and there is a significant difference at the level of ': 59ί]

 : Significant difference at 5X level

Claims

The scope of the claims

 [1] A nucleic acid sequence that regulates the amount of polyamine under the control of a promoter that can function in plants is stably maintained, and productivity is higher than a control plant that does not have the nucleic acid sequence, regardless of the cultivation environment. Or a rice plant and its progeny with improved traits and Z or at least one improved stress tolerance.

 [2] The grass plant and its progeny according to claim 1, wherein the nucleic acid sequence that regulates the amount of polyamine is an exogenous polyamine metabolism-related enzyme gene.

 [3] An expression vector containing a nucleic acid sequence that regulates the amount of polyamine under the control of a promoter capable of functioning in the plant, wherein the plant having improved productivity or trait or the stress tolerance is the nucleic acid. 2. The gramineous plant and its progeny according to claim 1, which are a transformed plant obtained by transforming a dwarf cocoon plant having a sequence.

 [4] The polyamine metabolism-related enzyme gene is a gene encoding arginine decarboxylase (ADC), a gene encoding ortine decarboxylase (ODC), S-adenosylmethine decarboxylase (SAMDC). The grass plant according to claim 2, which is at least one selected from the group consisting of a gene encoding spermidine synthase (SPDS) and a gene encoding spermine synthase (SPMS). And their descendants.

 [5] The grass plant and its progeny according to claim 4, which is a gene encoding spermidine synthase.

 6. The grass plant and its progeny according to claim 2, wherein the polyamine metabolism-related enzyme gene is a spermidine synthase gene having the following base sequence (a), (b) or (c):

 (a) a base sequence represented by base numbers 77 to 1060 in the base sequence represented by SEQ ID NO: 1 (SPDS, 1328),

 (b) a nucleotide sequence that hybridizes with the nucleotide sequence of (a) above under stringent conditions and encodes a protein having spermidine synthase activity;

(c) In the nucleotide sequence of (a) or (b), it encodes a protein consisting of a nucleotide sequence in which one or more bases are deleted, substituted, inserted or added, and having spermidine synthase activity. Base sequence.

[7] The grass family according to claim 2, wherein the polyamine metabolism-related enzyme gene is an S-adenosylmethionine decarboxylase gene having the following base sequence (a), (b) or (c): Plants and their descendants.

 (a) a base sequence represented by base numbers 456 to 1547 in the base sequence represented by SEQ ID NO: 3 (SAMDC, 1814),

 (b) a nucleotide sequence that hybridizes with the nucleotide sequence of (a) above under stringent conditions and encodes a protein having S-adenosylmethionine decarboxylase activity;

(c) S-adenosylmethionine decarboxylase consisting of a base sequence in which one or more bases are deleted, substituted, inserted or added in the base sequence of (a) or (b) A nucleotide sequence encoding a protein having activity.

 [8] The grass family plant and its progeny according to claim 2, wherein the polyamine metabolism-related enzyme gene is an arginine decarboxylase gene having the following base sequence (a), (b) or (c):

 (a) a base sequence represented by base numbers 541 to 2661 in the base sequence represented by SEQ ID NO: 5 (ADC, 3037),

 (b) a nucleotide sequence that hybridizes with the nucleotide sequence of (a) above under stringent conditions and encodes a protein having an arginine decarboxylase activity;

 (c) A protein having a base sequence in which one or more bases are deleted, substituted, inserted or added in the base sequence of (a) or (b) and having arginine decarboxylase activity. The base sequence to encode.

 [9] The gramineous plant and its progeny according to claim 1, which are gramineous plants with improved salt stress tolerance.

 10. The gramineous plant and its progeny according to claim 1, wherein the gramineous plant has improved low-temperature stress tolerance.

 [11] The gramineous plant and the progeny thereof according to claim 1, which are gramineous plants with improved resistance to pathogenic stress and Z or pest stress.

[12] The organ according to claim 1, wherein the organ whose productivity or trait is improved is at least one selected from the group power of ear, cocoon, seed, cocoon, rice, fruit, tiller and small spike power. Grasses and Its descendants.

 [13] The gramineous plant and its progeny according to claim 1, wherein the productivity or traits are remarkably improved in the tillering stage, the booting stage, and the juvenile stage.

14. The gramineous plant and its progeny according to claim 1, wherein the gramineous plant is rice.

[15] The gramineous plant and its progeny according to claim 1, which are in the form of a panicle, anther, a seed, anther, rice, a fruit, a tiller or a spikelet.

 [16] A spike, anther, a seed, a pod, rice, a fruit, a tiller, or a spikelet from which the gramineous plant according to any one of claims 1 to 15 and its progeny can be obtained.

[17] Useful substances which can also be obtained from the gramineous plant according to any one of claims 1 to 15 and its progeny

[18] a step of stably retaining a nucleic acid sequence that regulates the amount of polyamine under the control of a promoter capable of functioning in a plant, and transforming a cell of a dwarf pod plant with the nucleic acid sequence, A method for producing a Gramineae plant having improved productivity or traits irrespective of the cultivation environment and Z or at least one stress tolerance improved as compared to a control plant not having the nucleic acid sequence.

 [19] The method according to claim 18, wherein the nucleic acid sequence that regulates the amount of polyamine is an exogenous polyamine metabolism-related enzyme gene.

 [20] An expression vector containing a nucleic acid sequence that regulates the amount of polyamine under the control of a promoter that can function in plants, and having the nucleic acid sequence! / Cunning! Including the step of transforming the cells of the plant, the productivity or trait is improved independently of the cultivation environment compared to the control plant not having the nucleic acid sequence, and Z or at least one stress tolerance To produce a grass plant that has improved.

 [21] The method according to claim 20, wherein the nucleic acid sequence that regulates the amount of polyamine is an exogenous polyamine metabolism-related enzyme gene.

 [22] The method according to claim 20, further comprising the step of regenerating a gramineous plant from the transformed cell.

[23] The polyamine metabolism-related enzyme gene is a gene encoding arginine decarboxylase (ADC), a gene encoding ortine decarboxylase (ODC), S-adenosylmethio- And at least one selected from the group consisting of a gene encoding carboxydecarboxylase (SAMDC), a gene encoding spermidine synthase (SPDS), and a gene encoding spermine synthase (SPMS). The method according to 21.

24. The method according to claim 21, wherein the polyamine metabolism-related enzyme gene is a spermidine synthase gene having the following base (a) or (b) or (c):

 (a) a base sequence represented by base numbers 77 to 1060 in the base sequence represented by SEQ ID NO: 1,

(b) a nucleotide sequence that hybridizes with the nucleotide sequence of (a) above under stringent conditions and encodes a protein having spermidine synthase activity;

 (c) In the nucleotide sequence of (a) or (b), it encodes a protein consisting of a nucleotide sequence in which one or more bases are deleted, substituted, inserted or added, and having spermidine synthase activity. Base sequence.

 [25] The method according to claim 21, wherein the polyamine metabolism-related enzyme gene is an S-adenosylmethionine decarboxylase gene having the following base (a) or (b) or (c): .

(a) a base sequence represented by base numbers 456 to 1547 in the base sequence represented by SEQ ID NO: 3,

(b) a nucleotide sequence that hybridizes with the nucleotide sequence of (a) above under stringent conditions and encodes a protein having S-adenosylmethionine decarboxylase activity;

(c) S-adenosylmethionine decarboxylase consisting of a base sequence in which one or more bases are deleted, substituted, inserted or added in the base sequence of (a) or (b) A nucleotide sequence encoding a protein having activity.

 26. The method according to claim 21, wherein the polyamine metabolism-related enzyme gene is an arginine decarboxylase gene having the following base (a) or (b) or (c):

 (a) a base sequence represented by base numbers 541 to 2661 in the base sequence represented by SEQ ID NO: 5,

(b) a nucleotide sequence that hybridizes with the nucleotide sequence of (a) above under stringent conditions and encodes a protein having an arginine decarboxylase activity;

 (c) A protein having a base sequence in which one or more bases are deleted, substituted, inserted or added in the base sequence of (a) or (b) and having arginine decarboxylase activity. The base sequence to encode.

[27] The organs are selected from the group power of ears, pods, tillers, seeds, pods, rice, fruits and spikelets. 21. The method of claim 20, wherein there is at least one.

[28] The method according to claim 20, wherein the plant is a gramineous plant having improved stress tolerance.

[29] The method according to claim 20, wherein the plant is a gramineous plant having improved stress tolerance.

30. The method according to claim 20, wherein the Gramineae plant having improved pathogenic stress resistance and Z or pest stress tolerance is a Gramineae plant having improved salt stress tolerance.

31. The method according to claim 20, wherein the gramineous plant is rice.

 [32] The method according to claim 20, wherein the productivity or character is markedly improved in the tillering stage, the booting stage, and the juvenile stage.

[33] The following steps:

 (1) An expression vector comprising a nucleic acid sequence that regulates the amount of polyamine under the control of a promoter capable of functioning in a plant, transforming a cell of a culm plant having the nucleic acid sequence,

(2) From the transformed cell, regenerate a plant body having the nucleic acid sequence!

 (3) Collecting seeds by pollination of the plant, and

 (4) Plant physical strength obtained by cultivating the seed Examining the nucleic acid sequence in the seed obtained by pollination and selecting a homozygote of the nucleic acid sequence

 Which is homozygous for the nucleic acid sequence, has improved productivity or traits independent of the cultivation environment compared to a comparative control plant not having the nucleic acid sequence, and Z or at least one of A method to create a grass family with improved stress tolerance.