WO2008047834A1 - Procédé de production d'une nouvelle souche mutante utile de bactérie et/ou de plante - Google Patents

Procédé de production d'une nouvelle souche mutante utile de bactérie et/ou de plante Download PDF

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WO2008047834A1
WO2008047834A1 PCT/JP2007/070253 JP2007070253W WO2008047834A1 WO 2008047834 A1 WO2008047834 A1 WO 2008047834A1 JP 2007070253 W JP2007070253 W JP 2007070253W WO 2008047834 A1 WO2008047834 A1 WO 2008047834A1
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mutant
plant
strain
progeny
singlet oxygen
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Japanese (ja)
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Kohji Hasunuma
Yusuke Yoshida
Fuminori Satou
Hiroaki Harata
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Yokohama City University
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/01—Preparation of mutants without inserting foreign genetic material therein; Screening processes therefor
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H1/00—Processes for modifying genotypes ; Plants characterised by associated natural traits
    • A01H1/12—Processes for modifying agronomic input traits, e.g. crop yield
    • A01H1/122—Processes for modifying agronomic input traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance

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  • the present invention relates to a method for obtaining a novel useful mutant of fungi and / or plants, and a fungal and plant mutant obtained by the method.
  • Patent Document 1 Japanese Unexamined Patent Publication No. 2006-296359
  • Mv taken up into cells produces reduced Mv instead of producing reduced ferredoxin due to the reducing power generated in PSI (photosystem I) as a result of photosynthesis.
  • Reduced Mv generates electrons and superoxides in triplet oxygen.
  • Superoxide is reduced to hydrogen peroxide by the action of superoxide dismutase (SOD).
  • SOD superoxide dismutase
  • reduced ferredoxin turns 2 monodehydroascorbic acid into ascorbic acid.
  • the ascorbic acid and ascorbate peroxidase (Apx) are coupled to convert hydrogen peroxide into water and oxygen. Due to the reducing power generated in PSI (Photosystem I), superoxide is generated through the Mailer reaction even under normal conditions. This superoxide is converted to hydrogen peroxide by SOD, and further, ascorbic acid and Apx are conjugated, making hydrogen peroxide water and oxygen, and detoxified.
  • Arabidopsis thaliana yeast AtNDK-1 using yeast two-hybrid method Arabidopsis catalase, AtCat-l, AtCat-2, AtCat-3 form a complex with AtNDK-1, AtNDK-1 Strains overexpressing -1 have become resistant to Mv and hydrogen peroxide (Fukamatsu, Tsuji, Yabe, N. and Hasunuma,. 2003, Arabidopsis NDKl isa component of ROS signaling by interacting with three catalases Plant Cell Physi ol. 44, 982-989.). In plants, singlet oxygen is eliminated in the same way as akapankabi.
  • AtNDK-lP93S mutant protein has phosphorylation by ATP of about 1% compared to wild type.
  • AtCat Clearly increases the binding of singlet oxygen and has a higher mobility in native gel electrophoresis than AtCat of the strain overexpressing wild-type AtNDK-1.
  • the mobility of AtCat is slower than that of the wild strain, and it can be judged that there is less singlet oxygen binding.
  • an object of the present invention was to develop a new method for isolating strains resistant to singlet / oxygen.
  • the present inventors irradiate fungi or plants with ultraviolet rays to induce mutations, then add riboflavin to the medium, irradiate with sunlight, and grow under conditions filled with singlet oxygen.
  • the present inventors have found that the following mutant strains are frequently included in the mutant strains, and have completed the present invention.
  • the gist of the present invention is as follows.
  • nucleoside diphosphate kinase activity is 1.3 times or more higher than that of a strain into which no mutation has been introduced! /, (1) to (8)! /, Or a mutant according to any one of the above Or a mutant plant or its progeny.
  • the nucleoside diphosphate kinase has a higher phosphorylation activity in legumes, gramineous and sweet potatoes compared to a strain into which no mutation has been introduced.
  • the mutant or mutant plant or progeny thereof according to (9) is the mutant or mutant plant or progeny thereof according to (9) .
  • nucleoside diphosphate kinase and catalase are higher in legumes and gramineae than in strains not introduced with mutations! /, (9) mutant strain or mutation described in (9) Plant or its progeny.
  • ultraviolet irradiation is to irradiate the 3 ⁇ 10 mole 'm- 2' sec- 1 UV 1-5 minutes fungi or plants (24) The method according.
  • composition for producing a fungus or plant in which a useful mutation is induced comprising a singlet oxygen generation photosensitizer.
  • FIG. 2 shows a scheme for generating and erasing active oxygen.
  • Molecular oxygen 0 is triplet oxygen in the ground state.
  • NDK histidine kinase including NDP kinase
  • SOD superoxide dismutase
  • Hydrogen peroxide, H 0 and 0. Mv produces reduced ⁇ in the living body instead of generating reduced ferredoxin under light irradiation, and catalytically generates 0'- (superoxide). Hydrogen peroxide, ⁇ 0 becomes ⁇ 0 and 0 by catalase. 0 '— reacts with NO to produce ON 00—, peroxynitrite. It has strong oxidative activity, oxidizes C, 5-Methyto C in DNA, deaminates, and translocates to U and T. Na nitropulsid is a NO generator. NO is also a kind of active oxygen, and these active oxygen molecular species are collectively referred to as Reactive 0 xygen Species (ROS).
  • ROS Reactive 0 xygen Species
  • H 0 produces the hydroxy radical ⁇ ⁇ —.
  • Riboflavin catalytically generates molecular oxygen (0 2 ) to singlet oxygen (0) in a living body (cell) under light irradiation.
  • the present invention provides a method for obtaining useful mutant strains of fungi and / or plants.
  • the present invention also provides fungal and plant mutants having properties and traits superior to those of strains into which no mutation has been introduced (for example, wild strains).
  • FIG. 1 Distribution of solar energy received by leaves in the photosynthesis process (Amended from the 1997 Annual Report of Carnegie Institute, USA). Leaves that have received sunlight use about 10% of their energy for the carbon dioxide immobilization reaction. Solar energy excites chlorophyll, generating triplet chlorophyll. Triplet chlorophyll passes its energy to triplet oxygen, producing singlet oxygen. Singlet oxygen reacts with the chloroplast thylakoid membrane, etc., causing ion outflow, etc., causing a decrease in its function.
  • Fig.2 Geneation of active oxygen containing heavy oxygen and their elimination.
  • triplet oxygen in the ground state receives strong light in the presence of a dye such as riboflavin, it receives the energy and is activated to singlet oxygen.
  • Singlet oxygen is trapped by the Cat-1 / NDK-l complex in red mold. Further, it has not been proved that it can receive electrons from NAD (P) H and become superoxide. Electrons with high energy levels are taken into triplet oxygen and become superoxide.
  • the superoxide is converted to hydrogen peroxide by superoxide dismutase (SOD). In red mold, the hydrogen peroxide is reduced to water and oxygen by three types of power tarase, Cat-1, Cat-2, and Cat_3.
  • FIG. 6 Comparison of wild potato and R8 mutants. The wild strain and the R8 mutant were similar! /, But there were two good mutants isolated.
  • FIG. 7 Comparison of wild sweet potato and R40 mutant. There are significant differences between the wild type and the R40 mutant. Two good mutants were isolated.
  • FIG. 8 Comparison of wild sweet potato and R80 mutant. There are significant differences between the wild type and the R80 mutant. Two good mutants were isolated.
  • FIG.15 Harvest of sweet potato. This shows the F80-4-20 mutant strain that showed good vine growth in the mutant strain. F80-5-20 in the photo is an error of F80-4-20.
  • FIG. 16 shows F0-0-4, a wild rice strain with relatively good growth.
  • FIG. 17 shows a rice mutant F80-1-1-1.
  • F80-1-1-2 and F80-1-1-3 mutant strains were further isolated from this strain. All the mutants grow very well and the harvest is very good.
  • FIG. 18 Number of tillers / number of heading of mutants produced from wheat seeds treated with methyl viologen (Chinese spring).
  • FIG.21 Harvesting wheat (Chinese spring). The ears of the R4_3_3, R4-4-2, and R8-1-1 mutants that showed a large ear weight among the mutants are shown. For comparison, the ears of wild strains, R0-l_5, R0_2_3, R0_3_3, and R 0-4-3 are also shown.
  • FIG. 22 Number of spikes and weight of mutants produced from Nambu wheat seeds treated with Methyl viologen.
  • FIG. 23 Generation and elimination process of active oxygen containing singlet oxygen.
  • Singlet oxygen is generated when intense light is irradiated in the presence of a photosensitizer such as riboflavin. At present, it is unclear how this singlet oxygen will be detoxified.
  • catalase binds two molecules of singlet oxygen in this patent. The NDK power SNADH is carried there and two electrons are donated. And it produces two molecules of superoxide.
  • FIG. 24 Conidia for hydrogen peroxide (H 0) using wild-type strain, cat-l RIP , ndk-l p72H , cat-l; ndk-ll, cat-l; ndk-l-2 mutant Sensitivity to germination of (conidia).
  • the ndk-l p72H mutant is highly sensitive to hydrogen peroxide.
  • the cat_l RIP mutant is highly sensitive.
  • these double mutants are insensitive, as seen in cat_l; ndk-ll and cat_2; ndk-l-2. This is probably because CAT-2 is overexpressed in double mutants. It is.
  • FIG. 25 Sensitivity of wild strain, cat-l RIP ndk-l p72H cat-l; ndk-ll cat-l; ndk-l-2 mutant strain to riboflavin; concentration of riboflavin in agar medium is 0 200 400 800 Varying M and the concentration, conidia (conidia) of the above strain is sprinkled for about 200, and singlet oxygen is generated under light irradiation (lOO ⁇ mol / n ⁇ / sec). The ndk-l p72H mutant and the double mutant with cat-l RIP are sensitive to singlet oxygen.
  • FIG. 26 Binding ability of [ 32 P] NADH to histag NDK-1 and histag NDK-2. NDK-1 with histag and histag NDK-l p72H (72nd Pro becomes His in mutant NDK-1).
  • FIG. 28 Sensitivity of wild-type strain, ndk-l p72H mutant strain, and sod- ⁇ mutant strain to singlet oxygen.
  • FIG. 31 Harvesting characteristics of self-bred second generation of high yielding mutants. R4-2-4, R4-5-8 The representative characteristics of high-yielding mutants are shown. Using the leaves of this strain, biochemical characteristics were examined.
  • FIG. 32 SDS-PAGE of wild-type and high-yield mutants, R4-2-4 and R4-5-8 proteins Comparison.
  • the crude extract from leaves was fractionated into soluble, chloroplast and mitochondrial fractions, and 100 ⁇ g was separated by SDS-PAGE and stained with CBB. Due to its molecular weight, there is an increase in the amount of bands that are considered to be catalase and NDK.
  • the catalase band is not very stable even at -80 ° C storage.
  • FIG. 33 Comparison of catalase activity of wild type and high yield mutants, R4-2-4 and R4-5-8.
  • the crude extract from leaves was fractionated into soluble, chloroplast and mitochondrial fractions, and 100 ⁇ g was separated by native gel electrophoresis and stained for activity. There is a clear difference in activity between the wild and high-yield mutants in each fraction.
  • the catalase activity band is not very stable even at -80 ° C storage.
  • FIG. 34 Comparison of catalase activity in the crude extract of R4-5-8 and wild-type and high-yield mutants. Crude extract proteins 6.25, 12.5, 25, 50, and 100 g were separated by native gel electrophoresis and subjected to activity staining. There is a clear difference in activity between wild-type and high-yield mutants. The catalase activity band is not very stable even at -80 ° C storage.
  • FIG. 35 Phosphorylation of crude extract from leaves of wild-type and high-yield mutant R4-2-4. Crude extract 6.25, 12.5, 25, 50, 100 g of ( ⁇ - 32 P) ATP at 0 ° C for 10 seconds after phosphorylation labeling (in this condition, histidine kinase is selectively phosphorylated) SDS-PAGE gels were separated by electrophoresis and autoradiography was performed. Changes were observed in phosphorylation of 15-18 kDa NDK isomer and 37-70 kDa histidine kinase-like protein. NDK isomer is also a histidine kinase.
  • FIG. 36 Phosphorylation of crude extract from leaves of wild-type and high-yield mutant R4-5-8. Crude extract 6.25, 12.5, 25, 50, 100 g of ( ⁇ - 32 P) ATP at 0 ° C for 10 seconds after phosphorylation labeling (in this condition, histidine kinase is selectively phosphorylated) SDS-PAGE gels were separated by electrophoresis and autoradiography was performed. Changes were observed in phosphorylation of 15-18 kDa NDK isomer and 37-70 kDa histidine kinase-like protein.
  • FIG. 37 The crude extract from the leaves of wild-type and high-yield mutant R4-5-8 was further fractionated into soluble, chloroplast and mitochondrial fractions, and their phosphorylation was observed. 100 g of protein in each fraction was phosphorylated with ( ⁇ - 32 P) ATP at 0 ° C for 10 seconds (in this condition histidine kinase was selected Is phosphorylated. ) And then separated by SDS-PAGE gel electrophoresis and autoradiography was performed (right). Similarly, 100 g of protein in each fraction was separated by SDS-PAGE gel electrophoresis and stained with CBB (left).
  • FIG. 38 Morphogenesis of Alaska Endu wild strain and two high-yield mutants HIYl, HIY2 Details are described in the text.
  • Flower development, Stem deveropment (A) 180-day growth of wild and high-yield mutants.
  • D Internode length and number of wild and high yield mutants.
  • FIG. 40 Resistance to Mv (paraquat) and riboflavin (details are explained in the text) of wild strains of Alaska Endu and two high-yield mutants HIYl and HIY2 to paraquat; resistance to riboflavin; (II) Resistance to riboflavin;
  • FIG. 41 Four antioxidant enzymes were measured using the extract of the leaves of Alaska Endu wild strain and two high-yield mutants HIYl and HIY2. The upper part is an extract, and the supernatant per chloroplast and mitochondrial fraction (soluble fraction) was used, and the activity per leaf weight (g) was compared.
  • SOD Superoxide dismutase
  • AP X Ascorbate peroxidase
  • C Catalase
  • NDK Nucleoside diphosphate kinase
  • bottom unmodified gel electrophoresis Then, SOD, APX, and CAT were stained with activity. For NDK activity, a dialyzed soluble fraction was used.
  • E) and (F) were obtained by tracing (C) and (D). Details are explained in the text Yes.
  • Fig.42 Four antioxidant enzymes were measured using leaves of Alaska Endu wild strain and two high-yield mutants HIYl and HIY2. The upper part is an extract, and the supernatant per chloroplast and mitochondrial fraction (soluble fraction) was used, and the activity per leaf weight (g) was compared.
  • C After binding ( 32 P) NADH, unmodified gel electrophoresis was performed. Its autoradiography Ichiriki to give al (32 P) NADH binding signal. Another gel was used for CAT activity staining. The green region showing CAT activity, the red region showing radio activity, and the blue region of the control experiment were cut out to extract proteins.
  • the phosphoryl transfer reaction of ND ⁇ was measured using ( ⁇ _ 32 ⁇ ).
  • FIG. 43 Protein content in each cell fraction of wild type and high yield mutants. Protein content mg / g Leaf fresh weight, soluble fraction (Cs frac), chloroplast fraction (CI frac), mitochondrial-peroxisome fraction (MP frac).
  • FIG. 44 Separation of proteins in each cell fraction of wild-type and high-yield mutants by SDS-PAGE electrophoresis. Each cell fraction protein, 100 ⁇ g, was isolated. Soluble fraction (Cs f rac), chloroplast fraction (CI frac), mitochondrial-peroxisome fraction (MP frac).
  • FIG. 45 Enzyme activity (unit, mmol / min / mg protein) of active oxygen metabolism enzyme in each cell fraction of wild type and high yield mutant. Soluble fraction (Cs), chloroplast fraction (C1), mitochondria-peroxisome fraction (MP).
  • FIG. 46 Enzyme activity (unit, mmol / min / mg protein) of active oxygen metabolism enzyme in each cell fraction of wild type and high yield mutant. Soluble fraction (Cs), chloroplast fraction (C1), mitochondria-peroxisome fraction (MP).
  • FIG. 47 Protein phosphorylation activity in each cell fraction of wild-type strain and high-yield mutant. Soluble fraction (Cs), chloroplast fraction (Cl), mitochondrial-peroxisome fraction (MP).
  • FIG. 48 Characteristics of conidia formation rhythm exhibited by red mold sod-1 mutant
  • the wild strains, sod-l and band (bd), are the forces S and sod_l; bd that form bands at 22.1, 20.3, and 22.1 time periods, respectively.
  • FIG. 49 Knockout mutation of rhythm of akapankabi, the role of frq 1Q in rhythm formation ij.
  • the present invention provides a mutant bacterium, a mutant plant, or a progeny thereof that is resistant to singlet oxygen.
  • Tolerance to singlet oxygen is the result of culturing mutant bacteria or mutant plants or their progeny under conditions where singlet oxygen is generated, examining their growth state, and comparing with strains that have not been mutated (for example, wild strains). It can be confirmed by doing.
  • Examples of the culture under conditions where singlet oxygen is generated include culture under light irradiation in a medium containing a singlet oxygen-generating photosensitizer.
  • Singlet oxygen generation photosensitizers include riboflavin (7,8-dimethyl-10-D-ribitylisoaloxazine), porphyrin, methylene blue, rose bengal, FMN (flavin mononucleotide), FAD (flavin adenine) Forces that can exemplify dinucleotide) and the like, but are not limited thereto.
  • Riboflavin can be permeated into cells even when applied to the leaf surface. Riboflavin is excited by light irradiation and passes its energy to triplet oxygen, producing singlet oxygen.
  • the irradiating light should be sunlight (direct sunlight), but heat is cut off in a 12 cm water tank, ventilated with a draft, and two 400 W incandescent lamps are used for 1500 mole ⁇ m— 2 'sec— 1 . These numerical values may be changed as appropriate.
  • culture under conditions where singlet oxygen is generated in a medium containing 10 to 80 M riboflavin, fungi or plants under solar irradiation of 1000 to 2000 01 0 16 '01— ⁇ sec— 1 Can be mentioned.
  • the mutant bacterium or mutant plant of the present invention or its progeny is compared with a strain into which no mutation has been introduced, in a medium containing 10 M riboflavin in lOOO mole 'm— ⁇ sec— 1. It grows well under sunlight.
  • the mutant bacterium or mutant plant of the present invention or its progeny is white light exceeding lOO mole 'm-sec- 1 in a medium containing 100 M riboflavin as compared to a strain into which no mutation has been introduced. It may be one that grows well under irradiation.
  • the mutant bacterium or mutant plant of the present invention or progeny thereof may further be resistant to active oxygen other than singlet oxygen.
  • Tolerance to active oxygen other than singlet oxygen is determined by examining the growth state of the mutant or mutant plant or its progeny after exposure to active oxygen other than singlet oxygen or an active oxygen generator, and introducing the mutation. This can be confirmed by comparison with no strain (eg, wild strain).
  • Examples of exposure to active oxygen other than singlet oxygen or an active oxygen generator include culturing in a medium containing an active oxygen other than singlet oxygen or an active oxygen generator.
  • Examples of active oxygen other than singlet oxygen include superoxide two-one radical, hydroxy radical, peroxynitrite, hydrogen peroxide, nitric oxide, nitrogen dioxide, ozone, lipid peroxide, and the like.
  • Examples of active oxygen generators other than singlet oxygen include, but are not limited to, methyl viologen (Mv), hydrogen peroxide, Na-nitropulsid, and the like. Mv can generate active oxygen in bacterial and plant bodies under light irradiation. Hydrogen peroxide can permeate the plasma membrane of the cell. Therefore, it is said that it enters the cell fairly directly and has the function of secondary information relatively stably. Na Nito Mouth Pulcid dissolves well in water, is unstable as a solution, and enters the medium to produce NO.
  • N 0 becomes NO '(NO radical) and inhibits cytochrome oxidase in the cell, reduces respiration, and promotes the release of Ca ++ from the mitochondria.
  • NO ⁇ binds to and activates guanylate cyclase, increasing cGMP levels.
  • the active oxygen generator is Mv
  • the fungus or plant may be cultured in a medium containing 1 to 80 Mv.
  • a mutant bacterium, a mutant plant, or a progeny thereof that is resistant to active oxygen other than singlet oxygen is, for example, 1 M methylviologen compared to a strain into which no mutation has been introduced. It grows well in a medium containing
  • the type of mutant or plant of the present invention or its progeny is not particularly limited, but other than fungi belonging to ascomycetes (for example, Neurospora, Aspergillus).
  • This technology can be applied to promote the induction of carotenoids of basidiomycetous fungi (eg, shitake, matsutake, enokitake, shimeji, namoko) at low temperatures.
  • basidiomycetous fungi eg, shitake, matsutake, enokitake, shimeji, namoko
  • Aspergillus oryzae which is used for fermentation in ascomycetous fungi, is often similar to Aspergillus oryzae, and its application to fermentation can be expected.
  • Monocotyledonous plants such as ascomycetes and basidiomycetes, oats, corn, wheat, barley, rice (paddy rice and upland rice), dicotyledonous plants such as sweet potato, Alaska endo, broccoli, petunia, rape, soybean, sugar beet
  • Monocotyledonous plants such as ascomycetes and basidiomycetes, oats, corn, wheat, barley, rice (paddy rice and upland rice)
  • dicotyledonous plants such as sweet potato, Alaska endo, broccoli, petunia, rape, soybean, sugar beet
  • mutant bacterium or mutant plant of the present invention or its progeny can exhibit the following traits and / or properties.
  • the number of stems (number of branches and branches) in the grass family and legumes is more than 5 times L.
  • Nucleoside diphosphate kinase activity is 1.3 times higher than that of strains that have not been mutated (for example, wild strains).
  • Nucleoside diphosphate kinase phosphorylation activity is higher in legumes, grasses and sweet potatoes compared to strains that have not been mutated (eg, wild strains).
  • nucleoside diphosphate kinase and catalase are higher in legumes and gramineae than in strains (for example, wild strains) into which no mutation has been introduced.
  • ⁇ Catalase activity is 1.2 times or more higher than that of a strain into which no mutation has been introduced (for example, a wild strain).
  • V ru (flower bud formation time is early, some is late).
  • Nucleoside diphosphate kinase activity is measured by Agarwal, R.P., Robison, B. and Parks, R.E.
  • nucleoside diphosphate kinase The phosphorylation activity of nucleoside diphosphate kinase can be measured by the method described in Oda,. And Hasunuma,. 1994, Light signals are transduced to the phosphorylation of 15 kDa proteins in Neurospor a crassa. When looking at the normal phosphorylation reaction, remove TritonX-100, NADH and riboflavin from the reaction solution in the paper.
  • Catalase activity is reported in Fukamatsu, Tsuji, Yabe, Y. and Hasunuma,. 2003, Arabidops is ND -1 is a component of ROS signaling by interacting with three catalases. Plant Cell Physiol. 44, 982-989 It can be measured by the method described.
  • Crude plant extracts, soluble fractions, chloroplast fractions, and mitochondrial-peroxisome fractions can be prepared as follows.
  • the crude extract was centrifuged at 1,000 rpm (l, 000xg) for 10 minutes, and the precipitate was separated into chloroplast fractions (C hi).
  • the supernatant was further centrifuged at 20,000 rpm (2,000 ⁇ g) for 20 minutes, and the supernatant was used as a soluble fraction.
  • the precipitate was suspended in Re-suspension buffer (20 mM Tricine-NaOH, pH 7.8, 5 mM MgCl, 0.01% Dithiothreitol) and centrifuged at l, 000 rpm (l, 000xg) for 10 minutes to obtain chloroplasts.
  • Re-suspension buffer (20 mM Tricine-NaOH, pH 7.8, 5 mM MgCl, 0.01% Dithiothreitol
  • the supernatant was centrifuged at 20,000 ⁇ g for 10 minutes to obtain mitochondrial and peroxisome fractions as precipitates.
  • the precipitate was suspended in Re-suspension buffer, aliquoted, and stored at -80 ° C. Crude extracts, soluble fractions, chloroplast fractions, and mitochondrial-peroxisome fractions of grasses can be prepared according to this method.
  • the supernatant was further precipitated at 10,000 rpm (10,000 ⁇ g) at 10 minutes, and used as a mitochondrial and peroxisome fraction.
  • the supernatant was used as a soluble fraction. These were aliquoted, wrapped in oil and stored at -80 ° C.
  • a crude extract of legumes, a soluble fraction, a chloroplast fraction, and a mitochondrial-peroxisome fraction can be prepared according to this method.
  • the fungal crude extract and soluble fraction can be prepared by the method described in the article of Oda,. And Hasunuma,. 1994, Light signals are transduced to the phosphorylation of 15 kDa proteins in Neurospora crassa. .
  • the 37 ⁇ 70 kDa histidine kinase-like phosphorylated protein is a soluble fraction, a chloroplast fraction, a legume plant soluble fraction, and a chloroplast fraction of the grass family prepared by the above method. Observed in the painting. Phosphorylation can be detected by the same method as NDK phosphorylation.
  • the green density of a plant can be measured, for example, with a commercially available chlorophyll meter (SPD-502, oni ca Minolta).
  • the present invention generates singlet oxygen after mutagenesis treatment of fungi or plants.
  • a method for producing a mutant bacterium or a mutant plant comprising culturing under conditions and selecting a grown strain.
  • a mutant bacterium or a mutant plant having resistance to singlet oxygen or its progeny can be produced. Mutant bacteria or mutant plants or their progeny that are resistant to singlet oxygen are as described above.
  • Examples of mutagenesis treatment include exposure to active oxygen or an active oxygen generator, and ultraviolet irradiation.
  • Active oxygen including singlet oxygen
  • active oxygen generator including singlet oxygen generator
  • UV rays with 3 ⁇ 10 mole ⁇ m- 2 'sec- 1 of light intensity, may be irradiated for 1 to 5 minutes.
  • the mutagenesis treatment is preferably performed on fungal conidia (from which mycelia have been removed), plant germination seeds, plant seedlings, organs containing plant growth points (eg, stems, axillary buds, etc.).
  • Examples of the culture under conditions where singlet oxygen is generated include culture under light irradiation in a medium containing a singlet oxygen-generating photosensitizer.
  • Singlet oxygen generating photosensitizers include riboflavin (7,8-dimethyl-10-D-ribitylisoaloxazine), porphyrin, methylene blue, rose bengal, FMN (flavin mononucleotide), FAD (flavin adenine dinucleotide) ) And the like are not limited to these. Riboflavin can be permeated into cells even when applied to the leaf surface. Riboflavin is excited by light irradiation and passes its energy to triplet oxygen, producing singlet oxygen.
  • the irradiating light should be sunlight (direct sunlight), but heat is cut off in a 12 cm water tank, ventilated with a draft, and two 400 W incandescent lamps are used for 1500 mole ⁇ m— 2 'sec— 1 You may obtain the amount of light, and these numbers and materials may be changed as appropriate.
  • fungi or plants are cultivated in a medium containing 10-80 M riboflavin under 1000-2000 01 0 16 • m-sec- 1 sunlight irradiation. Culturing can be mentioned.
  • the types of fungi and plants to induce mutation by the method of the present invention are not particularly limited, but fungi belonging to ascomycetous fungi (eg, Neurospora, Aspergillus)
  • this technology can be applied to promote the induction of basidiomycetous induction at low temperatures and carotenoid production of basidiomycetous fungi (eg, shitake, matsutake, enoki mushrooms, shimeji, namako, etc.).
  • Aspergillus fungi are used for fermentation. (Aspergillus oryzae) has many similarities to akapankabi, and its application to fermentation can be expected.
  • Monocotyledonous plants such as ascomycetes and basidiomycetes, oats, corn, wheat, barley, rice (paddy rice and upland rice), twins such as sweet potato, Alaska endou, broccoli, petunia, rape, soybean, sugar beet
  • twins such as sweet potato, Alaska endou, broccoli, petunia, rape, soybean, sugar beet
  • the ability to exemplify leaf plants is not limited to these.
  • the fungus or plant subjected to the mutagenesis treatment may be a wild strain, a mutant strain or a progeny thereof.
  • Mutant strains that undergo mutagenesis can include mutants that are resistant to active oxygen other than singlet oxygen, mutant plants, and their progeny. Mutants, mutant plants, and their progeny that are resistant to active oxygen other than singlet oxygen have been exposed to fungi and plants (either wild or mutant) to active oxygen or active oxygen generators other than singlet oxygen. It can be prepared by selecting a strain that has grown later (see the above-mentioned and JP-A-2006-296359). Exposure to an active oxygen other than singlet oxygen or an active oxygen generator is, for example, culture of wild strains of fungi or plants in a medium containing! -80 M methylviologen.
  • the fungal and plant mutants obtained by subjecting the fungus or plant to mutagenesis and then culturing under conditions where singlet oxygen is generated and selecting the grown strain are expressed by the expression level of carotenoid, Properties such as vine length, potato weight, number of tillers, heading, ear weight, etc., or traits may differ from a strain that has not been mutated (eg, a wild strain). For example, compared to fungi or plants that have not been mutated (for example, wild strains), increased carotenoid expression, increased length, increased potato weight, increased number of tillers Examples thereof include, but are not limited to, changes in properties or traits such as an increase in the number of headings and an increase in the weight of the ears.
  • the method of the present invention is effective in producing a plant mutant having an improved yield (ie, resulting in increased production).
  • the method for producing a useful mutant strain of the present invention preferably includes exposing a fungus or a plant to active oxygen other than singlet oxygen or an active oxygen generator.
  • the active oxygen other than singlet oxygen and the active oxygen generator are as described above.
  • Fungi or plants may be cultured.
  • the active oxygen generator is Mv
  • fungi or plants may be cultured in a medium containing! ⁇ 80 ⁇ .
  • active oxygen generators eg wild strains
  • improved resistance to cold injury improved resistance to high temperature injury, improved resistance to oxidative stress injury
  • active oxygen Increase in scavenging ability, decrease in ability to generate active oxygen, change in circadian rhythm, change in characteristics related to temperature and / or photoperiodicity, change in time to form buds, change in time to bloom (early bloom, Late bloom), cancellation of self-incompatibility, change of knots attached to flowers, change in thickness of pods, change in number of fruits, change in size of fruits, change in shape of fruits, change in length of floral pattern Changes in the size of plant organs (leaves and stems), internode lengths, changes in the number of buds and their associated nodes, changes in stem thickness, changes in the size of pods, Changes in the timing of cocoon formation, changes in leaf size, changes in the speed of growth, changes in green intensity, Change in length, a decrease in the degree of oxidation of fat, an increase in antioxidant content
  • Broccoli 1 Increase in the speed of growth, change in flowering time (late blooming), cancellation of self-incompatibility, cold resistance
  • Sweet potato Increase in rapid growth, increase in yield (2-3 times that of wild type)
  • Petunia Increased leaf size, deep green, partial release of self-incompatibility
  • Treatment exposed to active oxygen generators such as methylviologen first treatment, this treatment induces mutations, And mutants resistant to active oxygen are selected
  • culture treatment under singlet oxygen generation conditions such as culture in a medium containing a singlet oxygen generation photosensitizer such as riboflavin.
  • second treatment mutants that are resistant to singlet oxygen are selected by this treatment
  • mutants that are more useful than mutants obtained by a single treatment can be obtained. The possibility increases.
  • the present invention provides fungal and plant mutants resistant to singlet oxygen and their progeny, their cells, tissues and seeds. Such cells, tissues, and seeds can be produced from a fungal or plant mutant having resistance to singlet oxygen and its progeny by a known method.
  • the present invention also provides a method for producing a strain exhibiting a desired property and / or trait by self-fertilization or mating of a mutant strain of a plant resistant to singlet oxygen or its progeny, and a plant strain produced by the method. And their progeny, their cells, tissues and seeds. Methods for self-fertilization or mating of plants are known, and methods for producing cells, tissues and seeds from plants are also known, and these methods can be used.
  • Desirable properties and traits include increased yield, increased carotenoid expression, increased catalase activity and expression, increased NDK activity and increased expression, histidine kinase-like protein phosphorus Increased oxidative activity, increased vine length, increased potato weight, increased number of tillers, increased heading (riboflavin after mutagenesis such as UV irradiation) Examples and the like of the mutant strain obtained by culturing in a medium containing a singlet oxygen-generating photosensitizer such as, but not limited thereto.
  • the strain obtained by self-fertilization or mating of the mutant strain or its progeny includes improved resistance to low temperature damage, improved resistance to high temperature damage, improved resistance to oxidative stress damage, increased active oxygen scavenging ability, reduced active oxygen generation capacity, Cancellation of self-incompatibility, change of circadian rhythm, change of characteristics related to temperature and / or photoperiodism, change of time to form buds, change of time to bloom (early bloom, late bloom), flower Changes in knots, changes in pod thickness, changes in number of fruits, changes in size of fruits, changes in shape of fruits, changes in length of floral pattern, size of plant organs (leaves and stems) Change of length, length of internode, change of number of buds and its attachment Changes in knots, stem thickness, changes in pod lump size,
  • the present invention provides a composition for producing a useful mutation-induced fungus or plant comprising a singlet oxygen generation photosensitizer.
  • a “useful mutation” is a mutation that results in the desired properties and traits as described above.
  • Examples of the singlet oxygen generation photosensitizer include riboflavin, porphyrin, FMN, FAD, methylene blue, rose bengal and the like, but are not limited to these.
  • composition of the present invention it is possible to produce fungi or plants in which useful mutations have been induced.
  • composition of the present invention may further contain a solvent, a medium component, a light source, and the like.
  • Examples of the medium that can be used in the present invention include, but are not limited to, Murashige and Skoog medium and Hoagland medium for plants, and Fries medium and Vogel medium for fungi.
  • Acapankavi wild strains 74-OR23-lA (FGSC # 987), Fungal Genetics Stock Center (Kansas City, USA)
  • nucleoside diphosphate kinase mutants ndk-l P72H (Ogura, ⁇ ⁇ , Yoshida, ⁇ ⁇ , Yabe, N. and Hasunuma,. (2001)
  • a point mutation in nucleoside diphosphate kinase results in a deficient light response for perithecial polarity in Neurospora crassa. J. Biol. Chem. 276, 21228-21234)
  • the conidia that are sex spores were prepared.
  • This mutagenized strain was expressed as riboflavin (0 , 10, 20, 40, 80 M) in a colony-forming medium (sorbose medium (XI Fries salt medium, levrose 0.5 g, glucose 0.5 g, sonolevose 10 g, agar 20 g, 1M OH, 2 ml / 1) 05 Uniformly in cells / plates! /, Etc.
  • sorbose medium XI Fries salt medium, levrose 0.5 g, glucose 0.5 g, sonolevose 10 g, agar 20 g, 1M OH, 2 ml / 1
  • the plates were exposed to sunlight (23 ° C) in the greenhouse (noon; 107 0 ⁇ mole 'm— 2 ⁇ sec— 1 , sunny) 2 After one day, the formed colonies were isolated with Pasteur pipettes and transferred to liquid medium containing 80 M riboflavin (Fries salt medium, 15 g sucrose / 1), again in the greenhouse under sunlight (noon; l lOO mole 'n ⁇ ' sec- 1, exposure to fine weather), later and assayed for its growth situation three days. Many of the isolates under these conditions does not grow. grown in the wild-type strain Approximately 80% of the isolated strains the hyphae was not etc. does O coercive accumulation of white tool carotenoids. Ndk-l P?
  • Figure 23 shows the known events for the active oxygen elimination process.
  • Singlet oxygen is super It was hypothesized that the conversion to oxide was hypothesized by a complex of catalase and NDK. Resistance to active oxygen has already been investigated using wild akapankabi, ndk-l P72H and sod-1 mutants. Both ndk-l P72H and sod-1 mutants have similar properties and have been established to be sensitive to the superoxide generator Methyl viologen (Mv) and hydrogen peroxide (Yoshida, Y., Ogura, Y. and ⁇ asunuma,. 2006, Interaction of nucleoside diphosphate kinase and catalase for stress and light responses in Neurospora crassa. FEBS Lett.
  • Methyl viologen Mv
  • hydrogen peroxide Yoshida, Y., Ogura, Y. and ⁇ asunuma,. 2006, Interaction of nucleoside diphosphate kinas
  • [0057] was prepared wild strain, Cat_l RIP mutants, n dk-l P72H mutant, double mutant 2 strain ndk-l P72H and cat-l RIP mutants. That is, the wild strain 74-OR23_lA (74A; FGSC # 987) was obtained from Fungal enetics Stock and enter (FSC; University of Kansas Medical, enter, Kansas City, KA, USA). The ndk-l P72H mutant, an NDK-1 point mutant, was isolated from the mutation present in the wc-1 mutant (FGSC # 3628) (Oda,. And Hasunuma,. 1997, Gene tic analysis of signal transduction).
  • ndk-l P72H mutant was sensitive to singlet oxygen, superoxide and hydrogen peroxide. It has lost its ability to reduce reactions that are common to these three stages. Therefore, it was suggested that the ndk-l P 72H mutation acts as a carrier for NADH or NADPH necessary for the reduction reaction (electron donation reaction)!
  • oligonucleotide primers (NEF; 5'-ACGGATCCTGTCCAACCA-3, (SEQ ID NO: 1) and NER; 5'-GCGAAGCTTACTCGAAG_3 '(SEQ ID NO: 2)), BamHI at the 5' end and Hindlll at the 3 'end Ndk-1 cDNA and ndk_l P72H having the above were amplified by RT-PCR from wild-type strain and ndk-l P72H RNA, respectively. These primers were designed to replace the first ATG codon of ndk- ⁇ with TGT (encoding cysteine). Ligate pBluescript II SK + to the amplified fragment and distribute it for fidelity The column was determined.
  • ndk-1 cDNA and ⁇ (3 ⁇ 4_1 ⁇ 72 ⁇ were cloned into the BamHI-Hindlll site of the Escherichia coli expression vector pQE32 (QI AGEN).
  • This expression vector contains 6 consecutive histidines at the amino terminus of the expressed protein. Residue (His-tag) is added! /, E.
  • histag NDK-1 protein was induced by adding 1 mM IPTG for 5 hours and the culture was harvested by centrifugation at 15,000 g for 15 minutes at 4 ° C. 20 mM sodium phosphate The pellet was resuspended in starting buffer containing (pH 7.4), 500 mM NaCl, and 10 mM imidazole and lysed by sonication. The lysate was centrifuged at 15,000 g for 15 minutes at 4 ° C. 200 ml The supernatant from the culture was used to purify His NDK-1 protein.
  • His-tag NDK-1 protein was purified by NTA chromatography. Purification was performed using HisTrap it (Amersham Pharmacia Biotech). An NTA Sepharose column (1 ml) was equilibrated with the starting buffer. Chelate the column with 0.5 ml of 0.1 M NiSO and add 5 column volumes
  • Histag protein is 20 mM NaPO (pH 7.4)
  • Histag NDK-1 and Histag NDK-1 P72H (Histag NDK-1 and Histag NDK-1 P72H bind to nickel chelate sepharose) using ( 32 P) NADH to these wild-type His tags NDK-1 Moreover, it was made to couple
  • ndk-l P72H and sod-1 mutants showed similar sensitivity, and the latter showed more sensitivity to riboflavin (singlet oxygen). This result suggests that the elimination of singlet oxygen is reduced by the reaction predicted by Process 1, Process 2, and Process 3, and the active oxygen is reduced. Even if other routes such as bypass exist, their activity is considered to be low.
  • the sod- ⁇ mutant of Akapan mold is clear! /,
  • the circadian rhythm of conidia formation I found it to show.
  • the traditionally used band (bd) strain was irradiated with light for 2 hours (20, 1 mol / m 2 / sec) after inoculation, and then at 30 ° C under constant temperature. Is 22.1 hours, but the sod-1 mutant is 20.3 hours, a little shorter. sod_l; The band does not show rhythm.
  • the band due to conidia formation of the sod-1 mutant almost disappeared when the antioxidant NAC (N-acetyl-L-cysteine) was added to the medium.
  • NAC N-acetyl-L-cysteine
  • frq (frequency) gene knockout mutant frq 1 is said to control circadian rhythms of akapan brute force. Under the same conditions as the above experiment, almost no band is formed (Fig. 49 A)
  • the frq gene is said to be homologous to the Drosophila period gene, and band; frq 1Q has also been reported to produce no rhythm under the same conditions. If Spoken to, sod-1; for band has no rhythm, sod-1 mutant can be said to be mutated circadian rhythms also sod-1 in 1-way;..
  • frq 1Q its band appears clearly that Rhythm cycle is a force with slight fluctuation S, 22.6 hours, not much different from 22.1 hours of the band strain, so in the mutation with the background of the band mutation, the frq gene mutation is a mutation of the rhythm cycle
  • the sod-1 mutation The sod-1; frq 1 Q mutation is not a clear circadian rhythm mutation, and sod-1 mutation as a reactive oxygen mutation is sod-1 -1; It can be judged that the force S to suppress the period and frequency variation, which has been the center of the circadian rhythm, can be determined so that the band appears clearly in frq 1Q .
  • Table 2 Wild strains, bd, sod-1 sod-1 can be adjusted to a 24-hour cycle by 12 hours light irradiation / 12 hours dark periodic light irradiation (3 O ⁇ mol / mVsec).
  • the power sod-1 is 30 times more sensitive to the ability to form conidial bands.
  • Standard errors were calculated from three independent experiments.
  • mutants with early flower bud formation were isolated by Mv treatment.
  • the second self-bred was seeded on December 6th as an autumn sowing and investigated.
  • the R80-4-5 -2 strain was confirmed on the 26th of May. Wild strains headed on May 10 and are about two weeks earlier.
  • the number of leaves until the green heading was deeper than that of the wild strain was 6 to 7 compared to 9 wild strains.
  • the number of wild heads was 6 compared to an average of 10 wild strains.
  • the yield is often reduced.
  • the ability to harvest before the rainy season is an important factor in maintaining the quality of the wheat.
  • Alaska Endu has a late-blooming force. Its harvest is approximately doubled and can be harvested in May. It can be fully used for agriculture.
  • Example 2 Isolation of a strain resistant to active oxygen containing singlet oxygen of sweet potato
  • Satsumaimo is a tropical plant. In Japan, when it is frosty from autumn to early winter, it leaves and becomes the limit of field cropping. In addition, it is necessary to keep warm about 10 ° C for long-term storage. In Japan, it is almost impossible to place flowers outside the Kanto region. Satsumaimo uses root shoots as seedlings rather than buds as rhizomes. The seedlings are cut out, planted in the field from May to June, and harvested in October. Mutant strains were isolated using seedlings of cultivated sweet potato, Benjazma (purchased from Watanabe Seed).
  • seedlings are used in this example, the theoretical background thereof will be considered.
  • mutations were induced in cells corresponding to embryonic stem cells existing around the growth point (shoot tip split tissue) using an active oxygen generator such as Mv.
  • Mv active oxygen generator
  • seedlings were used in this example, it can be considered that the growth point of each seedling is not so different from the shoot apical meristem of germinated seeds.
  • the force Mv that generates reduced ferredoxin from photosystem I as a result of photosynthesis receives electrons instead and generates reduced Mv. Reduced Mv passes electrons to triplet oxygen and produces superoxide.
  • Superoxide is converted to hydrogen peroxide by superoxide dismutase (SOD).
  • SOD superoxide dismutase
  • Ascorbic acid is not regenerated and ascorbate peroxidase (Apx) does not work. Therefore, a large amount of hydrogen peroxide is generated, and the strain that does not mutate resistance is killed.
  • plants produce large amounts of singlet oxygen under strong light. Furthermore, when transplanted to a field, it will receive strong light from the sun exceeding 2 000 H mole m— 2 sec— 1, and the energy of singlet oxygen will be dissipated as sufficient heat, and only strains that can be detoxified can grow. It becomes.
  • MSi night body medium (Sigma; Murashige and Skoog basal salt mixture (MS)) was each prepared in a plastic dish at 1 000 ml, Mv concentration was 0, 4, 8, 40, 80 ⁇ M, and 5 dishes were prepared. Five dishes were prepared with hydrogen peroxide concentrations of 0, 0.1, 1, 10, and 100 mM. Each dish was filled with plastic mushrooms, and 50 seedlings were stacked and adjusted so that they could grow. From early to mid-June, grown for 12 days under continuous light irradiation (SO ⁇ mole m— 2 sec— at 23 ° C. Then transplanted to the field.45 days after transplanting (early August), vine growth Its head was measured.
  • continuous light irradiation SO ⁇ mole m— 2 sec— at 23 ° C.
  • FIG. 4 shows the correlation between the initial vine length and the weight of the cocoon of the R0 strain (number of survivors, 44).
  • the R4 mutant has 39 survivors ( Figure 5)
  • the R8 mutant has 39 survivors ( Figure 6)
  • the R40 mutant has 19 survivors ( Figure 7)
  • the R80 mutant The number of survivors was 7 (Fig. 8).
  • Each figure shows the R0 strain as an internal standard.
  • Satsumaimo (benjazuma) seedlings (purchased from Watanabe Seed) are divided into 5 groups of 40 each, grown in a greenhouse (10-23 ° C) for 2 days, and then in a clean bench for 4 minutes 15W X 2 UV irradiator at a distance of about 30 cm, irradiation (7 mole 'm- 2' sec- was. 1 ⁇ 4 rotated by a minute, after irradiated. irradiation in uniform, riboflavin final concentration 0, 20, 40
  • MS was added to MS medium at 80 M. Control was performed without UV irradiation, and the cells were grown in a greenhouse for 4 days (3 days of clear weather) and then transplanted in a single treatment group per 1 cm.
  • Figure 9 shows the riboflavin addition and the growth condition under sunlight of sweet potato seedlings after UV irradiation.
  • Table 4 summarizes the total stem length, cocoon weight, and cocoon weight ratio (mutant / wild type) of wild and mutant strains.
  • Figures 10 to 15 show how the weight of the dug up is measured.
  • R0-1-7 39 36 81 16 R0-1-8 35 25 88 1 1.
  • mutant strains F80-l-l_l, F80-l-l_2, and F80-1-1-3 grow on one cocoon and have high productivity despite competition for growth. It was shown to be the strain shown.
  • F80-1-1-1 55 47 48 94 F80-1-1-2 53 45 46 ⁇ 8 F80-1-1-3 69 50 60 97 F80-2 (0 shares / 3 shares)
  • the symbols are F0_0, F0_4, F20_4, F40_4, F80_4, and F80-0.
  • One ear of the wild strain was designated as 0 group and designated as 0F0-0.
  • the results are shown in Table 7.
  • the seeds from the head ⁇ ⁇ 3 had 1.4 times the weight of the ears after growth, the head No.ll was 2.1 times the wild strains, and the head ⁇ ⁇ .13 was 1.3 times the wild strains. This means that, as expected, the frequency of the mutant cells is different in the ear cells.
  • ear No. 11 shows that it contains abnormal cells with high frequency. From Pan No.3, only 0 mutant strains were isolated, and from Pan No. 11, mutant strain 11F40-4-2-2 was isolated. From Shishiki and Shiho No. 13, four mutant strains, 13F0-4-3_3, 13F20_4-2-2, 13F40_4-3_1, and 13F80-4-3-1 were isolated. In particular, 13 F80-4-3- Zhu has a weight of 3.8 times that of the wild strain. Ear No. 3 and Ear No. ll were sufficiently resistant to active oxygen, indicating that mutations could not be selected successfully. In this sense, ear No. 13 is considered suitable for inducing mutation and selection.
  • Example 1 In mid-July (Example 1) or mid-August (Example 2), 50 seeds were placed in 20 Petri dishes and sterilized with 1/4 diluted sodium hypochlorite. After washing with sterilized water 5 times, the sterilized seeds were kept under 4 ° C black for 1 day. 25 ml of MS medium was placed in each of 4 dishes. Four plates of 0, 1, 2, 3, 4 M Methyl viologen (Mv; paraquat) were placed in the MS medium. The germinated seeds were grown for 7 days under conditions of 8 ° C for 8 hours of light irradiation (150 ⁇ mol / mVsec) /? C for 16 hours in the dark. In late August, 50 seeds per seed were sown in a plastic dish containing soil.
  • Mv Methyl viologen
  • a biochemical analysis was performed to determine the biochemical characteristics of these high-yield mutants.
  • the leaves of the second-breeding plant were cut out, wrapped in oil, frozen with liquid nitrogen, and stored at -80 ° C. All work was performed at 0-4 ° C under dark or dark green safety light.
  • the crude extract was centrifuged at 1,000 rpm (l, 000xg) for 10 minutes, and the precipitate was treated as a chloroplast fraction (Chi).
  • the supernatant was further centrifuged at 20,000 rpm (2,000 ⁇ g) for 20 minutes, and the supernatant was used as a soluble fraction.
  • the precipitate was suspended in Re-suspension buffer, and the mixture was centrifuged at 1,00 Orpm (l, 000xg) for 10 minutes to remove chloroplast contamination.
  • the supernatant was centrifuged at 20,000 ⁇ g for 10 minutes to obtain a mitochondrial and peroxisome fraction as a precipitate.
  • the precipitate was suspended in Re-suspension buffer, aliquoted, and stored at -80 ° C. 100 ag of the protein of each of these fractions was fractionated by 12.5% SDS-PAGE, and the gel was stained with CBB to give a protein band.
  • the results are shown in FIG. Based on the molecular weight markers, the two protein bands around 15 kDa are considered to be NDK isomers, and both R4-2-4 and R4-5-8 strains are clearly darker than the corresponding wild-type protein bands. Show. The high amount of NDK protein can be detected in the protein band. Furthermore, there is a protein band around 68 kDa, which is considered to be catalase from its molecular weight.
  • catalase activity was performed using native dye electrophoresis after native gel electrophoresis for soluble fraction, chloroplast fraction, and mitochondrial fraction. .
  • the catalase activity in each fraction was clearly higher in the mutant strain than in the wild strain.
  • the activity of catalase in native gel electrophoresis reflects the high mobility of catalase bound to singlet oxygen.
  • the crude extract was diluted and compared with the wild type and the mutant strain (R4-5-8) so that the catalase activity could be quantified as a whole.
  • FIG. 34 catalase activity with high mobility is observed in the mutant strain, and the ratio of the activity reaches 2 to 5 times in the mutant strain compared to the wild strain. Therefore, the 68 kDa protein band was identified as catalase.
  • NDK-isomer3 is a chloroplast fraction in Alaska Endu, but no activity is observed in wild-type and mutant strains, and may be an NDK in chloroplasts.
  • the pods are used as vegetables, and the beans are used as food as cereals.
  • Seeds were purchased from Watanabe Seed. In a conventional manner, 25 seeds were dispensed into 20 dishes in a Petri dish, and the seeds were sterilized with hypochlorous acid. Five stainless steel bags with paper towels were sterilized with foam. Thereto, 100 ml of MS medium was added liquid medium containing 0, 4, 8, 40, and 80 ⁇ ⁇ , respectively.
  • the dry weight of the pods was 1.5 and 1.8 times that of the wild type.
  • these two mutant strains were found to have had large increases that had never been reported before. These large increases in production include fermentations related to the elimination of active oxygen.
  • the element may be highly expressed. It is thought that the high expression of the active oxygen scavenging enzyme led to a rapid increase in the efficiency of photosynthesis in order to quickly remove the active oxygen generated in the leaves by sunlight, resulting in many productions.
  • the plant height is 78% and 76% of the wild strains, and it is rather suitable for low production!
  • Alaska Endu seeds (purchased from Watanabe Seed) were placed in 25 grains / petri dish, and 20 dishes and 500 grains were sterilized by a conventional method. Put a thick paper towel on 5 stainless steel bats, And sterilized. 100 ml of MS medium containing 0, 4, 8, 40, 80 HM Mv was added thereto. 100 sterilized seeds were spread on each vat, and after 30 spectral irradiations on a clean bench, they were placed under black at 4 ° for 3 days. Then, at 23 ° C, under the light irradiation of 80, 1 mole / mVsec, 1 part of the file was opened and allowed to grow for 7 days. Approximately 250 germinating seeds that had grown were transplanted into planters. The experiment was conducted twice and was cultivated in the open field in early March and late April. Among them, about 100 pods (seed) with good harvest were harvested for further analysis.
  • the node of flower bud initiation is section 8 in the wild strain and sections 15 and 13 in the two mutants, respectively.
  • the results shown in (E) are different from the wild type. Different strains have differences in the number of nodes, length between nodes, and plant height.
  • the wild strains have an average plant height of 126 cm and 19 nodes.
  • the two mutants have average plant heights of 99 cm and 97 cm, respectively, and the number of nodes is 25 and 23, respectively. This suggests that the mutant strains have short internodes and that the number of nodes with flower buds (usually two flower buds are attached to one node) increases.
  • (G) it was further found that the number of stems and branches in the mutant strain was 2 to 2.2 times that in the wild strain.
  • Figure 39 summarizes the harvesting of seeds as an energy accumulation site.
  • A As can be seen from the number of pods (fruit yield), the two mutants have 2.2 and 2.7 times the pods of the wild type.
  • B add 2 and 2.5 times the number of seeds.
  • the seed number (Fecundity) in the cicada is around 4, and there is no difference between wild and mutant strains! /,
  • C The weight (g) of the seeds with dried seeds was 1.5 and 1.8 times that of the wild type.
  • productivity improvement by normal breeding the maximum force S is 30%.
  • the improvement in productivity by this method can be increased by about 2 times as a whole. This means that this method will become an indispensable technology for improving biomass productivity.
  • the wild-type strain was 1.3 cm when treated with Mv at 8 ⁇ M, whereas the mutant strain was 2.6 cm and 2.4 cm, respectively.
  • the root elongation is 2.4 cm in the wild-type strain in the control experiment, while the mutant strain is 2.3 cm in both cases.
  • the stems were 1.0 cm in the wild type and 3.1 cm and 2.4 cm in the mutants, respectively. It showed such strong resistance.
  • Mv treatment is a resistance test against superoxide. Furthermore, as shown in the lower part of FIG. 40, seeds of wild type and two mutants are grown on a medium containing riboflavin. Riboflavin generates singlet oxygen by irradiation with strong light (lSO ⁇ mole / mVsec). By this method, singlet oxygen tolerance can be investigated. As shown in the bottom row (A), (B), and (C) of Fig. 40, 10 sterilized seeds were placed in a beaker containing 100 ml MS medium (with a paper towel underneath).
  • Riboflavin was changed to 0, 200, 400, 800, 1,600, 3,200 M in the MS medium, and the effects on seed (A) germination, (B) stem elongation, and (C) root elongation were observed. .
  • riboflavin was 1,600 M, and both wild and mutant strains showed maximum.
  • Riboflavin 800, 1,600, and 3,200 were more resistant to mutants than the wild type.
  • the mutant strains were more resistant to stem growth than the wild strains at 200, 400, 800, 1,600, 3,200. From these results, it was found that the mutant strain was resistant to singlet oxygen generated by intense light.
  • the detoxification process of singlet oxygen is considered to be a reduction process.
  • the second and third leaves from the top buds were harvested from the wild and two mutant strains, quickly wrapped in foam, and placed in liquid nitrogen. It was stored at -80 ° C.
  • a crude extract was prepared by the following method and further divided into mitochondria, peroxisome fraction, chloroplast fraction, and soluble fraction. The various treatments were carried out at 0-4 ° C under dark green safety light.
  • An extraction buffer solution (30 mM Mops (pH 7.3), 3 mM EDTA, 25 mM Cystein, 0.3 M Manitol) 5 times the weight of the leaves was placed in a chilled beak and ground with a pestle 30 times. It was squeezed with a double nylon cloth. Chloroplasts were precipitated at 4000 rpm (4,000xg) for 5 minutes by high-speed centrifugation.
  • the supernatant was further precipitated at 10,000 rpm (10,000 ⁇ g) for 10 minutes, and used as a fraction of mitochondria and peroxisome.
  • the supernatant was used as a soluble fraction. In this experimental example, only this soluble fraction was used. These are subdivided and wrapped in oil, -80. Saved at the same time. As shown in Figure 41, this is used to activate the active oxygen scavenging enzyme, (A) superoxide dismutase activity, (B) ascorbate peroxidase activity, (C) force tarase activity, and (D ) NDK activity was investigated.
  • Isomer 2 had higher phosphorylation activity in the mutant strain.
  • fP NADH was then mixed with the soluble fraction, and 3 minutes later, UV irradiation was performed for another 3 minutes. The samples were separated by SDS-PAGE and photoradiographed. From the run, it moved to the location corresponding to isomer 2. It is considered that isomer 2 has strong ( 32 P) NADH! / Binding ability.
  • (C) ( 32 P) NADH was mixed with the soluble fraction and electrophoresed on a native gel after 3 minutes. After electrophoresis, autoradiography was taken, and the portion surrounded by the red box was cut out. We also observed catalase activity in duplicated gels.
  • the corresponding part was cut out from the corresponding green box.
  • a control gel was cut from the purple box. Proteins from these gels were extracted and NDK activity was measured. From the catalase active part and the fP) NADH binding active part, it was possible to see / develop NDK activity. No activity was seen from the control gel!
  • NDK has an activity of binding NADH and further has an activity of forming a complex with catalase. NDK binds and transports NADH and donates two electrons to two molecules of singlet oxygen bound to force talase. Two molecules of singlet oxygen become two molecules of superoxide, which is the first step in detoxifying singlet oxygen.
  • the leaf extract of the wild strain, R3-KHIY1), R3-2 (HIY2) mutant was prepared by the method described in the above experimental example, and soluble fraction (Cs), chloroplast fraction (Cl), Mitochondrial fraction (including peroxisome fraction) (MP) was prepared.
  • Table 11 shows the protein content per raw leaf weight. In the two mutants, the same results were obtained in the soluble fraction (Cs), chloroplast fraction (C1), and mitochondrial fraction (including peroxisome fraction) (MP), which contained a large amount of protein per fresh leaf weight. I got it. The results are shown in Fig.
  • Table 11 Protein content in each cell fraction of wild-type and high-yield mutants Protein content of soluble fraction (Cs), chloroplast fraction (C1), and mitochondrial-peroxisome fraction (MP) Indicated in g leaf fresh weight.
  • Table 12 shows the activities of (A) superoxide dismutase (SOD), (B) ascorbate peroxidase (APX), (C) catalase (CAT), and (D) nucleoside diphosphate kinase (NDK) per mg protein. It was. This is shown in Figure 45 as a histogram. Mutants contain protein Due to the high amount, the difference between the wild strain and the mutant strain does not appear very clearly on this display. The method for comparatively displaying enzyme activity is usually expressed per g of fresh leaf weight. The results are shown in Table 13 and shown in histogram in FIG. In the soluble fraction, SOD and APX activities did not differ much between wild-type and mutant strains, but CAT and NDK showed clear differences as described above.
  • SOD superoxide dismutase
  • APX ascorbate peroxidase
  • CAT catalase
  • NDK nucleoside diphosphate kinase
  • Table 13 Activity of active oxygen metabolism enzyme / g leaf fresh weight.
  • the protein content of soluble fraction (Cs), chloroplast fraction (C1), and mitochondrial-peroxisome fraction (MP) is shown in mg / g leaf weight.
  • the soluble fraction (Cs), chloroplast fraction (Cl), and mitochondrial fraction (including peroxisome fraction) (MP) were prepared and phosphorylated by the method described above. It was. The results are shown in FIG. In NDK, the mobility of isomer 1 was increased in the mutant strains, from 18 kDa to 17 kDa. In all fractions, phosphorylation levels were increased in the mutant strains with similar results. Isomer 2 is 16 kDa, and the mobility does not change, but the mutants showed higher phosphorylation than the wild type in all fractions. In all fractions, Isomer 2 was shown to bind ( 32 P) NA DH.
  • Isomer 3 was 15 kDa, and the soluble fraction (Cs) of the mutant showed rather decreased phosphorylation. It was not present in chloroplasts, and only wild strains showed phosphorylation activity in the mitochondrial fraction. In addition, there are histidine kinase-like phosphorylated proteins that are specifically phosphorylated in mutant strains at the molecular weights of the soluble fraction (Cs), 47 kDa, 68 kDa, and 70 kDa. In the chloroplast fraction, phosphorylation of the 68 kDa protein was particularly promoted in the R3-2 (HIY2) mutant.
  • NDK is a typical histidine kinase, and it is suggested that it functions together with 47 kDa, 68 kDa, and 70 kDa histidine kinase to function to eliminate singlet oxygen.
  • Akaban mold when catalase was knocked out, not only NDK-1 but also 70 kDa histidine kinase having a large molecular weight was found to increase phosphorylation activity. It is suggested that 70 kDa histidine kinase also forms a complex with catalase, controls phosphorylation, and functions in scavenging singlet oxygen.
  • the seeds of this wheat were distributed by Kihara Biology Institute and Prof. Yasunari Sugawara.
  • sodium hypochlorite diluted 1/4 was used for seed sterilization as usual.
  • 50 wheat seeds were added and 20 Petri dishes were prepared. After sterilizing the wheat seeds in each Petri dish, the sterilized seeds were placed in the dark at 4 ° C for 3 days. Five 100 ml MS media were prepared. Methyl viologen (Mv) was added so that the concentrations would be 0, 4, 8, 40, and 80 M, respectively. Dispense 25 ml into 4 Petri dishes.
  • Mv Methyl viologen
  • the final concentrations were 0, 2, 4, 6, and 8 M Methyl viologen (Mv) liquid media, respectively. 25 ml each was dispensed into 4 Petri dishes containing sterile wheat seeds. It was germinated for 8 days in the dark at 8 ° C for 8 hours (163 ⁇ 01016 01 — 2 sec—, 7 ° C, 16 hours in 4 petri dishes at each concentration. Then transplanted to the planter, The plants were grown under a greenhouse bench (in the shade) for 10 days at 10-23 ° C and then exposed to the open ground (December 27) . The growth status at that time is shown in Table 15. The germination numbers for each drug Concentrations were R0: 57/200, R2: 100/200, R4: 73/200, R6; 42/197, R8: 30/200, which were then transplanted into the field (January 29).
  • Fig. 18 shows the results of the wild-type strain and the grown mutant strain (June 26). Of the 32 wild-type strains, the top 11 strains with the largest number of divisions were used, and the average and standard error of the wild-type strains were used. Eight strains have been isolated, which are apparently likely to have increased numbers.
  • Figure 19 shows the total weight of the ear.
  • Four mutant strains (R2-1-1, R2-1-3, R2-3-9, R8-1-1) were isolated that showed 2 to 3.5 times the total ear weight of the wild strain. The photographs are shown in Figs. Similar experiment using cv Nambu wheat I went. The result is shown in FIG. There are 5 strains (R4-1-8, R4-2-8, R4-4-13, R6-2-6, R6-4-2) that show a 2-2.5-fold increase in ear weight even with Nambu wheat. ) Obtained.
  • useful mutations can be induced in fungi and plants with high efficiency.
  • this research is a technology that includes the possibility of improving the situation where the earth is placed.
  • Toyota Motor Corporation for example, is developing plant plastics, and Brazil produces corn, sugarcane, and other plant alcohol, and has a national system that does not depend on oil.
  • Such research is necessary in Japan, which is highly dependent on fossil fuels, which have few resources.
  • This study will contribute to agriculture (cereals, vegetables). In addition, it provides a large amount of raw materials for fuel ethanol and plant plastics as an alternative energy source for health food, vegetable oil industry, and fossil fuel resources. It will also improve the growth of pasture and provide large amounts of livestock feed.
  • SEQ ID NOs: 1 and 2 show the sequences of a set of oligonucleotide primers (NEF and NER).

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Abstract

L'invention concerne un procédé de production d'une souche mutante utile d'une bactérie ou d'une plante. De manière spécifique, l'invention concerne : une bactérie mutante ou une plante mutante résistant à l'oxygène singulet ou d'un descendant de celui-ci ; et un procédé de production d'une souche mutante utile d'une bactérie ou d'une plante, qui comprend les étapes consistant à provoquer une mutagénèse dans la bactérie ou la plante, cultiver la bactérie ou la plante dans des conditions permettant de générer l'oxygène singulet, et sélectionner une souche en pleine croissance de la bactérie ou de la plante.
PCT/JP2007/070253 2006-10-18 2007-10-17 Procédé de production d'une nouvelle souche mutante utile de bactérie et/ou de plante WO2008047834A1 (fr)

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WO2009096443A1 (fr) * 2008-01-31 2009-08-06 Public University Corporation Yokohama City University Nouveau procédé d'isolement très efficace de mutants fongiques et/ou végétaux par un procédé de neutralisation par clarification d'un oxygène singulet et d'amélioration des molécules participant au procédé
KR101408975B1 (ko) * 2013-08-13 2014-07-02 주식회사 케이엠티알 활성 산소를 이용한 미생물 배양방법

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2010018598A1 (fr) * 2008-08-11 2010-02-18 Ashwani Pareek Gène de l'histidine kinase de type hybride isolé à partir du riz indica ir64 et clones produits par celui-ci
US9234189B2 (en) 2008-08-11 2016-01-12 Ashwani Pareek Hybrid type histidine kinase gene isolated from indica rice IR64

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