WO2017217508A1 - 植物体の耐塩性向上方法 - Google Patents
植物体の耐塩性向上方法 Download PDFInfo
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- A01H3/00—Processes for modifying phenotypes, e.g. symbiosis with bacteria
- A01H3/04—Processes for modifying phenotypes, e.g. symbiosis with bacteria by treatment with chemicals
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- A—HUMAN NECESSITIES
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- 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
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8216—Methods for controlling, regulating or enhancing expression of transgenes in plant cells
- C12N15/8218—Antisense, co-suppression, viral induced gene silencing [VIGS], post-transcriptional induced gene silencing [PTGS]
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- Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
- Y02A40/10—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in agriculture
- Y02A40/13—Abiotic stress
- Y02A40/135—Plants tolerant to salinity
Definitions
- the present invention relates to a method of improving the salt tolerance of a plant.
- the present application is applied to Japan in Japanese Patent Application No. 2016-121235 filed on June 17, 2016, Japanese Patent Application No. 2016-241469 filed on December 13, 2016, and April 25, 2017. Priority is claimed based on Japanese Patent Application No. 2017-086654 and Japanese Patent Application No. 2017-100286 filed on May 19, 2017, the contents of which are incorporated herein by reference.
- a method of enhancing salt tolerance of a plant there is a method of introducing a gene involved in a salt tolerance mechanism using genetic recombination technology. For example, there are halophytes that have acquired resistance to osmotic pressure by accumulating osmolytes (proline and betaine) in plant cells. It has been reported that a recombinant plant into which a gene for accumulating this osmolyte has been introduced has acquired salt tolerance.
- intracellular sodium ion concentration in plants is mainly a non-selective cation channel (NSCC) that controls intracellular uptake and cell membrane Na + that controls extracellular efflux.
- NSCC non-selective cation channel
- SOS1 Salt Overly Sensitive 1
- SOS1 SOS pathway, vacuolar Na + / H + antiporter controlling uptake into vacuoles, and high affinity allowing sodium ion to flow along with potassium ion from the conduit It is controlled by the active potassium transporter (High affinity K Transporter; HKT) (see Non-Patent Document 1).
- Patent Document 1 in the transformed plant overexpressing the SOS1 gene identified from a salt-tolerant plant, Thellungiella halophila, the excretion of sodium ions outside the cell is promoted and the salt tolerance is enhanced. Has been reported to improve.
- Patent Document 2 reports that salt tolerance has been improved even in a transformed plant in which the SOS2 gene, which is a protein kinase constituting the SOS pathway, is overexpressed.
- Patent Document 3 in the transformed plant in which the vacuolar Na + / H + antiporter (HvNHX1) gene of barley (Hordeum vulgare) is overexpressed, the uptake of sodium ion into vacuoles is promoted and the salt tolerance is enhanced. Has been reported to improve.
- Non-Patent Document 2 in transformed plants in which the HKT gene of Arabidopsis thaliana (Arabidopsis thaliana) is overexpressed, accumulation of sodium ion in the root is increased, and increase in shoot salt concentration is suppressed, and the whole plant body is It has been reported that salt tolerance has been improved.
- a method of enhancing the salt tolerance of a plant without using a genetic engineering technology a method of administering a drug or a microorganism having a salt tolerance imparting effect to a plant to a plant has been studied.
- a drug having a salt tolerance imparting effect for example, pyrroloquinoline quinone (see, for example, Patent Document 4), strigolactone of a plant hormone, and the like are known.
- a microorganism having a salt tolerance imparting effect for example, Paenibacillus fukuinensis (see, for example, Patent Document 5) is known.
- Non-Patent Document 1 the mechanism of controlling the sodium ion concentration in plants has been elucidated to some extent. Moreover, as described in Patent Document 1 etc., it is known that the salt tolerance of a plant can be improved by overexpressing a gene involved in the mechanism such as the SOS1 gene. However, most of transgenic plants into which a gene involved in the mechanism has been introduced have been confirmed to be resistant to sodium chloride of about 100 mM, and further improvement of salt tolerance is desired.
- An object of the present invention is to provide a method for improving the salt tolerance of a plant so that cultivation under a high salt concentration environment is possible.
- PERK13 Protein-rich extensin-like receptor kinase 13
- PERK13 Proline-rich extensin-like receptor kinase 13
- the present inventors have found that the salt resistance of a plant body is improved by the treatment, and the present invention has been completed.
- the method for improving the salt tolerance of a plant according to the present invention is the following [1] to [16].
- [1] A method for improving the salt tolerance of a plant, which suppresses or inhibits the function of PERK 13 (Proline-rich extensin-like receptor kinase 13) of the plant.
- [2] The method for improving salt tolerance of a plant of [1], wherein an antagonist of PERK13 is brought into contact with the root of the plant.
- the antagonist is one or more kinds of microorganisms, or a secretory substance thereof.
- [4] The method for improving salt tolerance of a plant of [2] or [3], comprising the step of immersing at least a part of the root of the plant in an aqueous solution containing the antagonist.
- [5] The method for improving salt tolerance of plants according to the above [1], wherein the function of PERK13 is suppressed by suppressing the expression of PERK13 gene, or the function of PERK13 is inhibited by suppressing the expression of PERK13 gene .
- [6] The method for improving salt tolerance of a plant of [1] or [5], wherein a PERK13 gene is deleted or a mutation that reduces the function of the PERK13 gene is introduced into the plant.
- the plant is selected from the group consisting of nonselective cation channel, cell membrane Na + / H + antiporter, vacuolar Na + / H + antiporter, and high affinity potassium transporter
- the plant is selected from the group consisting of non-selective cation channel, cell membrane Na + / H + antiporter, vacuolar Na + / H + antiporter, and high affinity potassium transporter
- the plant is a transformant into which a foreign gene has been introduced, The salt tolerance of any one of the above-mentioned [1] to [6], wherein the foreign gene is one or more selected from the group consisting of SOS1 gene, SOS2 gene, SOS3 gene, NHX1 gene, and HKT1 gene How to improve.
- the above plant body is a gramineous plant, a solanaceous plant, a cruciferous plant, a cucurbitaceous plant, a grapevine plant, a citrus family plant, a rosaceous plant, a leguminous plant, a lotus family [1] a plant selected from the group consisting of: plants of the Sesame family, plants of the family Sesaceous family, plants of the family Cinamiaceae, plants of the family Sacaceae; The salt tolerance improvement method of the plant body in any one of 1]-[9].
- the above-mentioned plant body is rice, corn, sorghum, wheat, barley, rye, hen, fox, tomato, eggplant, paprika, pepper, potato, tobacco, Arabidopsis thaliana, Brassica napus, Japanese radish, radish, cabbage, purple cabbage, Chinese cabbage (petit ver), Chinese cabbage, Chinese cabbage, kale, watercress, komatsuna, broccoli, cauliflower, cauliflower, turnip, wasabi, mustard, cucumber, bitter melon, pumpkin, melon, watermelon, grape, lemon, orange, navel orange, grapefruit, orange, lime , Sudachi, yuzu, shiikuwasha, tangan, apple, cherry, plum, peach, strawberry, loquat, apricot, plum (sumomo), prune, almond, pear, pear, pear, raspberry, blackberry, cassis, cranberry ⁇ Blueberries, soybeans, beans, peas, fava beans, fern beans, mung
- a method for cultivating a plant wherein the plant in which the microorganism is allowed to coexist is grown under an environment having a sodium chloride concentration of 1.5% by mass or more, and the survival rate of the plant is 10% or more.
- the salt tolerance of a plant having originally low salt tolerance can be improved by the method for improving salt tolerance of a plant according to the present invention. Therefore, plants having improved salt tolerance by the method can be grown even in an environment with a relatively high sodium chloride concentration.
- FIG. 1 is a fluorescence staining image by fluorescence sodium indicator of a wild strain of Arabidopsis thaliana after salt stress in Example 3.
- FIG. 2 is a fluorescence staining image by fluorescence sodium indicator of a functionally deficient mutant of PERK13 of Arabidopsis thaliana after salt stress in Example 3.
- FIG. 3 is a diagram showing the measurement results of the fluorescence intensity of roots of a wild strain of Arabidopsis thaliana and a functionally deficient mutant of PERK13 after salt stress in Example 3.
- FIG. 1 is a fluorescence staining image by fluorescence sodium indicator of a wild strain of Arabidopsis thaliana after salt stress in Example 3.
- FIG. 2 is a fluorescence staining image by fluorescence sodium indicator of a functionally deficient mutant of PERK13 of Arabidopsis thaliana after salt stress in Example 3.
- FIG. 3 is a diagram showing the measurement results of the fluorescence intensity of roots of
- FIG. 4 shows the survival rate under each sodium chloride concentration when hydroponically cultivating a wild-type strain of Arabidopsis thaliana and a functionally deficient mutant of PERK13 in Example 4 under the symbiosis of the identified salt tolerance improving microorganism mixture in Example 4. It is the figure which showed the result of having investigated.
- FIG. 5 shows that in Example 5, salt stress (sodium concentration 2.5 mass) in the presence of the salt tolerance improving microorganism mixture obtained in Example 4 with respect to the wild strain of Arabidopsis thaliana and the functionally deficient mutant of PERK13. It is the figure which showed the measurement result of the fluorescence intensity of the root of each plant after applying%.
- FIG. 5 shows that in Example 5, salt stress (sodium concentration 2.5 mass) in the presence of the salt tolerance improving microorganism mixture obtained in Example 4 with respect to the wild strain of Arabidopsis thaliana and the functionally deficient mutant of PERK13. It is the figure which showed the measurement result of the fluorescence
- FIG. 6 shows that in Example 5, salt stress (sodium concentration 1.0 mass) in the presence of the salt tolerance-improved microorganism mixture obtained in Example 4 with respect to the wild strain of Arabidopsis thaliana and the functionally deficient mutant of PERK13. It is the figure which showed the measurement result of the fluorescence intensity of the root of each plant after applying%.
- FIG. 7 is a structural map of an RNAi vector targeting the tomato SlPERK gene (pBI-SlPERKs-sense, anti sense vector) constructed in Example 6.
- FIG. 8 is a photograph of the transformed tomato obtained in Example 6 at the time of hydroponic cultivation for 21 days in a 0.5 or 1.0% by mass sodium chloride environment.
- FIG. 7 is a structural map of an RNAi vector targeting the tomato SlPERK gene (pBI-SlPERKs-sense, anti sense vector) constructed in Example 6.
- FIG. 8 is a photograph of the transformed tomato obtained in Example 6 at the time of hydroponic cultivation for 21 days in
- FIG. 10 shows that the concentration of sodium chloride in the recombinantly redifferentiated plant (KO) into which the vector for knockout of non-recombinant redifferentiated rice (WT) and the PERK13 ortholog gene knockout vector (KO) in Example 7 was introduced was 1.5% by mass. It is a photograph at the time of making it grow for 2 weeks under environment.
- the method for improving salt tolerance of a plant according to the present invention is characterized by suppressing or inhibiting the function of PERK13 of a plant.
- PERK13 is a membrane protein specifically expressed in plant roots and has a kinase active site in cells.
- PERK13 is similar to PERK4 (Proline-rich extensins-like receptor kinase 4) (see, for example, Non-Patent Document 3), which is a receptor having an action to promote the calcium ion influx of NSCC due to the similarity in amino acid sequence. It has been estimated that it has an action effect, but the present inventors' research has revealed that it is involved in the control of the influx of sodium ions into the plant body.
- Non-Patent Document 1 the control mechanism of sodium ion concentration is widely common in plants.
- PERK13 which is an end of the mechanism, is also a protein conserved in a wide variety of plants, and has the function of controlling the influx of sodium ions into the plant body in many plants.
- PERK13 gene includes orthologs of PERK13 gene of Arabidopsis thaliana whose Gene ID of NCBI is At1g 70460 (Arabidopsis thaliana), and specifically, Gene ID of NCBI is 101266034 (Solanum lycopersicum), 107059185 (Solanum tuberosum) ), 107279382 (Oryza sativa Japonica Group), 4333279 (Oryza sativa Japonica Group), 102703815 (Oryza brachyantha), 103649394 (Zea mays), 106804357 (Setaria italica), 101775206 (Setaria italica), 106322706 (Brassica oleracea), Brassica napus), 106416704 (Brassica napus), 103852653 (Brassica rapa), 101215732 (Cucumis sativus), 100247217 (Vitis vinifera), 104882493 (Vitis vinifera), 10
- the PERK 13 whose function is suppressed or inhibited is not particularly limited as long as it retains the function as the PERK 13.
- PERK13 which suppresses or inhibits the function may be wild-type PERK13 present in a wild-type plant, and the PERK13 mutation produced by mutation. It may be a body, may be a PERK13 mutant into which a mutation has been introduced by various types of mutation treatments such as ultraviolet irradiation treatment, or may be a PERK13 variant which has been modified by gene modification technology or the like.
- the plant according to the present invention is a plant body which is a modified PERK13 in which the PERK13 possessed is a PERK13 modified product in which the wild type PERK13 is subjected to various modification treatments to enhance or attenuate the sodium ion influx promoting function by PERK13.
- Salt resistance can be improved by carrying out the method for improving the salt resistance of the body.
- the degree of suppression or inhibition of the function of PERK13 possessed by the plant to improve the salt tolerance of the plant includes, for example, hydroponicing the plant for 6 to 24 hours in a 1.0% by mass sodium chloride environment. 90% or less, preferably 100% or less, when the amount of sodium chloride in the root when cultivated is 100% when the plant before cultivation or function of PERK13 is cultivated under the same conditions 80% or less, more preferably 60% or less, further preferably 50% or less, still more preferably 40% or less, particularly preferably 30% or less.
- the relative amount of sodium chloride in the roots of plants can be measured, for example, using the fluorescence intensity when fluorescently staining the inside of the roots with a fluorescent substance that binds to sodium ions as an indicator.
- the method for suppressing or inhibiting the function of PERK13 originally possessed by the plant is not particularly limited, and may be a method for reducing the expression level of PERK13, and mutation to the PERK13 gene in genomic DNA may be used. May be a method of expressing a PERK13 mutant having a reduced function by introducing S. or a method of inhibiting intracellular signaling of PERK13.
- the expression level of PERK13 in the plant body after reduction is 100% of the expression level of PERK13 in the plant body before reduction 90% or less, preferably 80% or less, more preferably 60% or less, still more preferably 50% or less, still more preferably 30% or less, particularly preferably 0% (not fully expressed) It is preferable to make it become.
- the expression level of PERK13 in plants can be measured by various methods used when measuring the expression level of proteins in the art, such as RT-PCR.
- the function of PERK13 of the plant body after reduction is based on 100% function of PERK13 of the plant body before reduction. 90% or less, preferably 80% or less, more preferably 60% or less, still more preferably 50% or less, still more preferably 30% or less, particularly preferably 0% (completely lost function) It is preferable to do so.
- a method of modifying genomic DNA may be used, or a method which does not modify genomic DNA like RNA interference may be used.
- Methods of reducing the expression level of PERK13 by modifying genomic DNA include methods of deleting PERK13 gene, methods of introducing nonsense mutation into PERK13 gene, methods of modifying expression regulatory sequences such as promoter sequence of PERK13 gene, etc. It can be mentioned.
- mutations that reduce or delete the function of PERK13 include, for example, mutations in which the kinase activity is eliminated or reduced in the kinase domain in the intracellular domain of PERK13, and ligands in the ligand binding site in the extracellular domain of PERK13. And mutations that reduce the affinity to
- the PERK13 gene is deleted by replacing the region encoding the PERK13 gene with a DNA fragment encoding another gene, or replacing it with a DNA fragment encoding a mutated PERK13 mutant gene,
- the mutant PERK13 can be expressed instead of the type PERK13.
- the homology (sequence identity) of the nucleotide sequence required for homologous recombination is preferably 70% or more, more preferably 80% or more, still more preferably 90% or more, particularly preferably 95% or more.
- the gene manipulation method by the homologous recombination method has already been established in many plants.
- a transformation vector containing a DNA fragment to be substituted by homologous recombination is introduced into the callus of a plant for the purpose of improving salt tolerance to prepare a transformant, and the resulting transformant is differentiated.
- Undifferentiated calli can be prepared by a conventional method.
- the transformation vector may be linear DNA or may be a plasmid.
- Transfer of vectors into plant cells such as callus can be carried out by those skilled in the art such as the Agrobacterium method, particle gun method, polyethylene glycol method, electroporation method, liposome method, calcium phosphate precipitation method, lipofection method, microinjection method and the like. Various methods known to those skilled in the art can be used.
- RNA interference is a small interfering RNA (siRNA) that has a double-stranded structure consisting of a sense strand and an antisense strand of a partial region (RNAi (RNA interference) target region) of a PERK13 gene cDNA (shutter) in plants. It can be carried out by introducing RNA) or miRNA (micro RNA). An RNAi-inducing vector capable of producing siRNA or the like may be introduced into a target plant cell. Preparation of siRNA, shRNA, miRNA, and RNAi induction vector can be designed and manufactured by a conventional method from the base sequence information of the cDNA of PERK13 gene to be targeted.
- the RNAi-inducing vector can also be prepared by inserting the base sequence of the RNAi target region into the base sequences of various commercially available RNAi vectors.
- the introduction of the RNAi-inducing vector can be carried out in the same manner as the introduction of the above-mentioned transformation vector.
- the intracellular signal transduction of PERK13 may be inhibited by contacting an antagonist of PERK13 with the surface of the root of the plant, and an inhibitor that inhibits the kinase activity of PERK13 may be intracellular in the root of the plant. May be introduced into the
- the antagonist of PERK13 means a substance that inhibits binding of PERK13 ligand to PERK13 by binding to the extracellular domain of PERK13.
- a method for improving the salt tolerance of a plant according to the present invention a method in which an antagonist of PERK13 is brought into contact with the surface of the root of a plant is particularly preferable, from the viewpoint that there is no need to modify genomic DNA and processing is more simple.
- the antagonist of PERK13 used in the present invention may be a compound, may be one or more types of microorganisms, or may be a secreted substance of one or more types of microorganisms.
- the antagonist is a substance secreted by a specific microorganism
- the culture supernatant of the microorganism may be used as it is or a crude product thereof may be brought into contact with the surface of the root of the plant.
- an antagonist of PERK13 As a method of bringing an antagonist of PERK13 into contact with the surface of the root of a plant, in the case of hydroponics where cultivation is carried out in a state where at least a part of the root of the plant is immersed in a solution for cultivation, By immersing at least a part of the root of the plant in a treatment solution containing an antagonist of PERK13 for a certain period of time, the antagonist is bound to PERK13 on the surface of the root of the plant, and the function of PERK13 is inhibited. It suppresses the influx of sodium ions from the roots and improves the salt tolerance of the plant.
- the composition of the treatment solution in particular the composition of the salt, may be the same as or different from the cultivation solution.
- the cultivation solution may be mixed with an antagonist of PERK13 directly.
- the soil on which the plant is planted may be wetted with an aqueous solution containing the antagonist, and granules containing the antagonist may be placed near the roots in the soil.
- the plant that improves salt tolerance is not particularly limited as long as it is a plant originally having the PERK13 gene or its homolog gene in genomic DNA, and may be angiosperm, and It may be a plant or may be a fern or moss. Also, it may be a monocotyledonous plant or a dicotyledonous plant.
- gramineous plants such as rice, corn, sorghum, wheat, barley, rye, barn and so on; solanaceous plants such as tomato, eggplant, paprika, peppers, potatoes and tobacco; Arabidopsis thaliana, Brassica napus, Plants of the Brassicaceae family such as Chinese cabbage, Japanese radish, cabbage, purple cabbage, cabbage (Japanese cabbage), Chinese cabbage, Chinese cabbage, kale, watercress, komatsuna, broccoli, cauliflower, turnip, wasabi, mustard, etc .; cucumber, bitter melon, pumpkin, melon, watermelon Plants of the family Uraceae, such as grapes, etc .; Plants of the family Vitis, such as grapes; Plants of the family Cimicidae, such as lemon, orange, navel orange, grapefruit, oranges, limes, sudachi, jujubes, shikwashas, tankans; apples, cherry blossoms, plums , Strawberry, loquat, a
- palm such as date palm, oil palm, coconut palm, acai etc Plants of the family Family Family; plants of the family Family Family Family, such as bananas, plants and manila hemp; plants of family Family Family Family, such as cotton and okra; plants of the Family Family Family Group, such as Eucalyptus;
- the treatment for reducing the sodium ion concentration in the root of a plant include non-selective cation channel of a plant, cell membrane Na + / H + antiporter, vacuolar Na + / H + antiporter And a treatment that enhances the function of one or more proteins selected from the group consisting of high affinity potassium transporters.
- these proteins include SOS1, SOS2, SOS3, NHX1, and HKT1 (Non-patent Document 1). The function of these proteins can be enhanced by increasing the expression level of the protein.
- the expression level of the protein in the plant can be increased by introducing and transforming a foreign gene encoding the protein.
- the foreign gene may be a gene derived from an organism of the same species as the plant to be introduced, or may be a gene derived from a different organism.
- the method is selected from the group consisting of SOS1 gene, SOS2 gene, SOS3 gene, NHX1 gene, and HKT1 gene which are the same or different from the target plant to improve salt tolerance. It is preferable to suppress or inhibit the function of PERK13 in a transformant into which one or more types of foreign genes have been introduced.
- the introduction of the foreign gene into the plant can be carried out using the transformation vector into which the DNA fragment encoding the foreign gene has been incorporated, in the same manner as the introduction of the transformation vector.
- the method for improving the salt tolerance of a plant according to the present invention it is possible to obtain a plant having improved salt tolerance over a plant before suppressing or inhibiting the function of PERK13.
- the salt tolerance be improved to such an extent that it can be grown even in an environment of a sodium concentration that can grow no more than 10%.
- the plant obtained by the method for improving salt tolerance of a plant according to the present invention is grown by hydroponic culture using a culture solution having a high sodium ion concentration or by soil culture using a soil having a high sodium ion concentration.
- the sodium ion concentration is 0.2 mass% or more, preferably 0.5 mass% or more, more preferably 1 mass% or more, further preferably 1.5 mass
- plants that can be grown can be obtained even by water culture using a culture solution of% or more, more preferably 2.0% by mass or more, particularly preferably 2.5% by mass or more.
- magnesium chloride as a cultivation solution used for cultivation of the plant which improved salt tolerance, and it is more preferable to contain 0.5 mass% or less of magnesium chloride, and 0. More preferably, it contains 1 to 0.5% by mass of magnesium chloride.
- the said solution for cultivation contains various nutrient components required for growth of a plant body besides sodium chloride and magnesium chloride.
- the said nutrient component can be suitably adjusted according to the kind of plant body to grow.
- elements such as aluminum and silicon may be contained as salts.
- the composition of the culture solution may be changed according to the growth stage of the plant.
- the culture solution for example, a solution obtained by adding a deficient salt such as sodium chloride to a commercially available liquid fertilizer, or a solution obtained by diluting a commercially available concentrated liquid fertilizer with water and diluting it with seawater is used. It can be used. In addition, a solution obtained by appropriately adding insufficient salts such as phosphorus to seawater can also be used.
- a deficient salt such as sodium chloride
- a solution obtained by diluting a commercially available concentrated liquid fertilizer with water and diluting it with seawater can be used. It can be used.
- a solution obtained by appropriately adding insufficient salts such as phosphorus to seawater can also be used.
- Hydroponic cultivation of the plant body which improved salt tolerance can be performed by a general hydroponic cultivation method.
- a relatively large amount of culture solution may be stored in a culture tank using a solution-type hydroponic method, or a thin-film hydroponic method in which the culture solution is allowed to flow down little by little on a gently sloping plane. Good.
- Example 1 Mutants with improved salt tolerance were screened against a library of mutants in which random mutations were introduced into Arabidopsis thaliana, and genes contributing to improvement in salt tolerance were examined.
- Paenibacillus a microorganism of the genus Paenibacillus (Paenibacillus) was used. Paenibacillus bacteria promote the influx of sodium chloride into cells of Arabidopsis thaliana. Therefore, Arabidopsis thaliana in the presence of Paenibacillus spp. Also dies in a 0.5% by mass sodium chloride environment which does not usually die. Using this property, we screened for genes that could improve salt tolerance even in the presence of Paenibacillus spp.
- Example 2 In Example 1, two strains out of the four strains confirmed to be a functionally deficient mutant of PERK13 were grown in a 1.5% by mass sodium chloride environment, and salt tolerance was examined.
- the seeds are sterilized with hypochlorous acid and then seeded on a gel plate medium of MS medium, and this gel plate medium is subjected to hydroponic culture with at least the bottom portion being in contact with the liquid medium for hydroponic culture.
- a liquid medium a 1/2 MS medium was used.
- 24 seeds were inoculated for sodium chloride treatment and 24 seeds for control treatment, respectively.
- sodium chloride was added to a plant for sodium chloride treatment in an amount such that the final concentration would be 1.5% by mass, and water culture was carried out for one week. None was added to the control treatment plants, and hydroponic culture was performed for one more week.
- hydroponic cultivation was similarly performed on wild strains.
- control-treated plants ie, plants grown in 1/2 MS medium without addition of sodium chloride, did not wither all of the 24 wild-type and PERK13 functionally deficient mutants. It grew normally.
- Example 3 Among the functionally deleted mutants of PERK13, the amount of sodium in the roots was examined for one of the two strains examined for salt tolerance in Example 2.
- the seeds are sown on a gel plate medium of MS medium containing 1% sucrose, and at least the bottom of the gel plate medium is for hydroponic culture.
- Hydroponic cultivation was carried out in an artificial weather apparatus in a state of being in contact with a liquid medium (1/2 MS medium). The weather conditions were 25 ° C., 5000 lux, 16 hours light, 8 hours dark. 10 to 14 days after germination, the liquid medium in contact with the bottom of the gel plate medium is replaced with a 1/2 mass medium containing sodium chloride at a final concentration of 1.0% by mass, and water is further continued for 6 to 24 hours Cultivation and salt stress was applied. The culture was carried out for the same time without adding anything to the liquid medium of the control treatment plant.
- CoroNa-Green AM is a sodium indicator whose green fluorescence intensity is increased by binding to sodium ions.
- PERK13 is involved in the influx of sodium ions in the root, and the improvement of salt stress tolerance in the functionally deleted mutant of PERK13 is due to the influx of sodium ions into the plant under high salt concentration environment It turned out that it was.
- Example 4 Using a wild-type Arabidopsis thaliana, plant symbionts having a symbiotic effect to enhance salt tolerance were selected from microorganisms extracted from soil.
- Sucrose-containing MS agar medium (MS medium containing 0.5% (w / v) sucrose and 0.9% (w / v) agar) was injected into a cylindrical pot with an open top and bottom. By setting and hardening, the pot for growing a plant body was produced. A plurality of the pots were placed in each of eight containers containing sucrose-containing MS medium (liquid medium obtained by adding 0.5% (w / v) sucrose to MS medium).
- hypochlorous acid treatment of seeds Arabidopsis thaliana seed was purchased from LEHLE (Round Rock, TX, USA). The seeds were sterilized by immersion for 1 minute while immersed in 1% hypochlorous acid, and then the hypochlorous acid was removed by centrifugation. The hypochlorous acid-treated seeds were washed three times with sterile water, seeded at the top of the pot, and stored at 4 ° C. for 24 hours in the dark.
- a sucrose-containing MS medium a liquid medium obtained by adding 0.5% (w / v) sucrose to MS medium.
- Each pot was placed so that the bottom surface was immersed in sucrose-containing MS medium but the top surface was not.
- seeds of wild type after hypochlorous acid treatment and washing three times with sterile water are sown, and in an incubator at 25 ° C, 16 hours of light and 8 days of dark. Nurtured for 14 days.
- the same procedure was carried out except that 100 ⁇ L of the first microorganism recovery solution was added to a sucrose-containing MS medium to which sodium chloride was added so that the final concentration of sodium chloride would be 1.5% by mass as a solution for cultivation. Hydroponic culture, and after 14 days of culture under salt stress, cut the roots and above-ground parts (leaves and stems) of the growing plants, collect the roots, homogenize, and carry out the second microorganism It was a recovery solution.
- the symbiosis of the microorganism mixture contained in the third microorganism recovery solution with the roots of the plant made it possible to grow the plant under salt stress. That is, the microorganism mixture or the secretory substance contained in the third microorganism recovery solution has an effect of improving the salt tolerance of the plant, that is, the microorganism mixture is a plant under salt stress. It was found that it is a plant symbionts that enables the growth of
- Microorganisms that constitute plant symbionts capable of growing plants under selected salt stress were identified.
- cells are recovered from the third microorganism recovery solution, and genomic DNA is obtained from a portion of the recovered cells using GenElute Bacterial Genomic DNA kit (Sigma-Aldrich, St. Louis, MO, USA).
- GenElute Bacterial Genomic DNA kit Sigma-Aldrich, St. Louis, MO, USA.
- a 16S rDNA is amplified by PCR using the recovered genomic DNA as a template and the forward primer (5'-AGAGTTTGATCATGGCTCAG-3 ', SEQ ID NO: 1) and the reverse primer (5'-TACGGTTACCTTGTTACGACTT-3', SEQ ID NO: 2) did.
- the temperature condition of PCR is a cycle consisting of a heating step of 95 ° C. for 3 minutes, a denaturation step of 95 ° C. for 30 seconds, an annealing step of 50 ° C. for 30 seconds, and an elongation step of 72 ° C. for 1 minute 30 seconds. After cycling, the reaction was carried out under the conditions that an extension reaction at 72 ° C. for 5 minutes was finally added.
- the resulting PCR products were confirmed by 1.2% agarose gel electrophoresis and extracted from the gel using QIAquick gel extraction kit (Quiagen, Germantown, MD, USA).
- the extracted PCR product was inserted into a plasmid using TOPO-TA cloning kit (Life Technologies, Carlsbad, CA, USA) and transformed into E. coli.
- 30 E. coli colonies grown overnight on LB medium containing ampicillin were randomly picked, transferred to LB liquid medium containing ampicillin, and cultured.
- the plasmid was purified from E. coli cultured using QIAprep spin miniprep kit (Quiagen).
- the purified plasmid was subjected to Thermalcycle reaction using BigDye terminator v3.1 Cycle sequence kit (Life Technologies), and the nucleotide sequence of 16S rDNA incorporated into the plasmid was determined with a DNA sequencer (ABI 3130 x L) . As a result, two types of 16S rDNA (strains YROK-1 and YROK-2) were identified.
- the YROK-1 strain (SEQ ID NO: 3) was identical in sequence identity to Paenarthrobacter nitroguajacolicus (accession number: AJ512504)
- the YROK-2 strain (SEQ ID NO: 4) had a sequence identity of 97.14% with Arthrobacter psychrochitiniphilus (Accession number: AJ810896). From these results, it was found that the YROK-1 strain is a new strain of Paenarthrobacter nitroguajacolicus, and the YROK-2 strain is a new strain of Arthrobacter psychrochitiniphilus.
- Paenarthrobacter nitroguajacolicus strain YROK-1 was 98.0%
- Arthrobacter psychrochitiniphilus was 2.0%.
- the final concentration of sodium chloride is adjusted to 0, 0.5, 1.0, 1.5, 2.0, 2.5, or 3.0 mass% in the sucrose-containing MS medium in which the bottom of the pot is immersed
- a sterile 5 M sodium chloride aqueous solution was added thereto, and the above-mentioned microorganism mixture for salt tolerance improvement was further added to carry out hydroponic cultivation, and the survival rate after culture for 14 days was examined.
- hydroponic cultivation was carried out similarly with the cultivation solution to which the microorganism mixture for salt tolerance improvement was not added, and the survival rate after cultivation for 14 days was examined.
- non-microbe WT is the result of a wild strain grown in the state where the salt mixture for improving salt tolerance is not coexisting
- microorganism-containing WT is cultivated in the state where the mixture for salt tolerance improvement is in symbiosis
- Microbial MT indicates the results of functionally deficient mutants of PERK13 grown in the state where the salt mixture for improving salt tolerance coexists.
- the sodium chloride concentration is 1% by mass and the survival rate is 10% or less
- the survival rate at a sodium chloride concentration of 1% by mass is as high as 90% or more
- the survival rate at a sodium chloride concentration of 3% by mass is 30% or more as in the functionally deficient mutant of PERK13 It was high.
- the functionally deficient mutant of PERK13 was slightly higher than the survival rate of the wild strain. It was speculated that this may be because the salt mixture for improving salt tolerance has some effect not only on the pathway via the functional loss of PERK13 but also on other routes for improving salt tolerance.
- the salt tolerance improving effect of the mixture for salt tolerance for the wild strain on the wild strain Is considered to be the salt tolerance improving effect brought about by the functional defect of PERK13. That is, the salt mixture for improving salt tolerance or the secretion thereof is an antagonist of PERK13, and the function of PERK13 is suppressed or inhibited by symbiosis with the root of the plant with the salt mixture for improving salt tolerance. It has been suggested that bringing the antagonist of PERK13 into contact with the surface of the root of the plant also suggests that the same salt tolerance improvement effect as the gene deficient form of PERK13 can be obtained.
- Example 5 With respect to a wild strain of Arabidopsis thaliana and a functionally deficient mutant of PERK13, the effect of the mixture of the microorganism for improving salt tolerance obtained in Example 4 on sodium influx to the roots of plants under salt stress was examined.
- the liquid medium in contact with the bottom of the gel plate medium on which the plant body is placed is sodium chloride at a final concentration of 2.5% by mass or 1.0% by mass sodium chloride
- the medium was changed to a contained 1/2 MS medium, and the above-mentioned microorganism mixture for salt tolerance improvement was further added. Thereafter, it was subjected to hydroponic cultivation for 6 hours to apply salt stress in the presence of the above-mentioned microorganism for improving salt tolerance.
- the roots of plants after salt stress are stained with a fluorescent sodium indicator (CoroNa-Green AM) in the same manner as in Example 3.
- a fluorescent sodium indicator CoroNa-Green AM
- roots after salt stress are stained.
- the fluorescence intensity per cross-sectional area was measured.
- the results of plants hydroponically grown at a sodium chloride concentration of 2.5% by mass are shown in FIG. 5, and the results of plants hydroponically grown at a sodium chloride concentration of 1.0% by mass are shown in FIG.
- Example 6 A PERK13 functionally deleted mutant of tomato (Solanum lycopersicum) was prepared and its salt tolerance was examined.
- the target sequence of S1PERK10 gene (SEQ ID NO: 5), the target sequence of S1PERK9a gene (SEQ ID NO: 6), and the target sequence of S1PERK9b gene (SEQ ID NO: 6)
- the chimeric gene consisting of the base sequence which connected 7) was produced.
- RNAi vector pBI-SlPERKs-sense, anti sense vector targeting the SlPERK gene was constructed. The structural map of the vector is shown in FIG.
- the RNA of the chimeric gene transcribed under the control of the CaMV 35S promoter formed a double-stranded RNA consisting of a sense RNA and an antisense RNA through intron cleavage.
- RNAi vector was introduced into Agrobacterium (Agrobacterium tumefaciens) GV3101 strain by a conventional method to obtain a recombinant Agrobacterium.
- Agrobacterium Agrobacterium tumefaciens
- GV3101 strain Agrobacterium tumefaciens
- the cotyledon pieces from tomato variety Microtom were infected with the obtained recombinant Agrobacterium, and callus formation was induced in callus formation medium. Thereafter, drug resistant calli were selected and allowed to redifferentiate.
- DNA was extracted from the leaf of a tomato individual obtained by redifferentiation, and PCR was performed to select a transformed tomato into which the chimeric gene had been introduced. DNA extraction from leaves and PCR were performed as follows.
- extraction buffer 100 mM Tris, 50 mM EDTA, 500 mM NaCl (p
- PCR reaction solution so that the final concentration of the forward primer (5'-GTTCTTCTACACCATTTGCAGC, SEQ ID NO: 8) and the reverse primer (5'-ATTGTGGTAGTGTTGGTAAGGC, SEQ ID NO: 9) becomes 0.2 ⁇ M using GoTaq polymerase (Promega)
- GoTaq polymerase GoTaq polymerase (Promega)
- the PCR is maintained at 95 ° C. for 3 minutes, repeated 35 cycles of 95 ° C. for 30 seconds, then 55 ° C. for 30 seconds, then 72 ° C. for 30 seconds, and finally held at 72 ° C. for 3 minutes It did, on condition of doing.
- the resulting transformed tomato is a tomato in which the function of PERK13 (SlPERK) of tomato has been deleted by the introduced chimeric gene.
- the PERK13 deficient tomato was hydroponically cultured in a 1/2 MS medium containing sodium chloride to a final concentration of 0.5, 1.0, 1.5 or 2.0 mass%. Hydroponic cultivation was carried out in an artificial weather apparatus (25 ° C., 16 hours light, 8 hours dark). In the case of wild type tomato, when hydroponic culture is performed with 1/2 MS medium containing sodium chloride so that the final concentration is 0.5% by mass, on the 21st day of cultivation, sodium chloride Unable to survive, leaves turn white, roots turn brown and wither (not shown).
- Example 7 A PERK13 functional deletion mutant of rice (Oryza sativa) was prepared and its salt tolerance was examined.
- target gene is OsPERK13 (Os03g056880, NCBI GeneID 4333279), a gene with 70% or more amino acid sequence identity to PERK13 in Arabidopsis thaliana (PERK13 orthologous gene in rice)
- a knockout vector was constructed.
- a polynucleotide consisting of the target sequence of OsPERK13 gene was produced by gene artificial synthesis.
- the knockout vector pOsPERK-KO1 targeting rice PERK13 orthologous gene is a modified pRIT1 vector (Terada et al., Nature Biotechnology, 2002, vol. 20, p. It was introduced and constructed in 1030-1034.
- the transformation of rice was performed according to the method of Toki et al. (Plant Journal, Vol. 47, pp. 69-76, 2006).
- the knockout vector was introduced into Agrobacterium strain EHA101 strain or LBA 4404 strain by a conventional method to obtain a recombinant Agrobacterium.
- the obtained recombinant Agrobacterium was infected with scutella-derived calli of the rice variety "Nipponbare".
- the infected rice calli were cultured in a medium containing 0.25 ⁇ M bispyribac salt and bispyribac salt resistant calli were selected.
- genomic DNA is extracted using a DNA extraction kit "Maxwell 16 LEV Plant DNA kit (manufactured by Promega)", and PCR is performed to introduce the knockout vector. Callus was selected. PCR was carried out at 94 ° C. for 1 minute using a DNA polymerase (Tks Gflex, manufactured by Takara Bio Inc.), a forward primer (5′-AAGCTCAAGCTCCAATACGCAAACCCCTC, SEQ ID NO: 11) and a reverse primer (5 ′ The cycle is repeated at 35 ° C for 10 seconds at 98 ° C, then 15 seconds at 60 ° C, then 1 minute at 68 ° C, and finally held for 7 minutes at 68 ° C. The bispyribac-resistant calli from which a PCR product of the size was obtained was selected as a transformed calli into which the knockout vector was introduced.
- a DNA polymerase Tks Gflex, manufactured by Takara Bio Inc.
- a forward primer 5′-AAGCTCAAGCTCCAATACGCAAACCCC
- PCR was performed using 3g 05688 No1-F primer (5'-AGTCAAGCTTCGCCGGCGCCAATGCCGATGTGAGCCGCGC, SEQ ID NO: 13) and 3g 05688 No1-R primer (5'-TGACGAATTCGCTCGGCACGAGACGAGGGTTCTCCTCGCG, SEQ ID NO: 14).
- the resulting PCR amplified product was purified using a nucleic acid purification kit "DNA Cleaner (manufactured by Wako Pure Chemical Industries, Ltd.)", treated with a restriction enzyme (TspRI), and the state of cleavage of the DNA fragment was confirmed by agarose electrophoresis.
- FIG. 9 shows the results of agarose electrophoresis of a digested product obtained by TspRI-treated PCR-amplified fragment derived from the OsPERK13 gene.
- FIG. 10 (A) and 10 (C) are photographs of the above-ground part of the individual plant, and FIG. 10 (B) is a photograph of the underground part of the individual plant shown in FIG. 10 (A). ) Is a photograph of the underground part of the individual plant shown in FIG. 10 (C). As shown in FIGS.
Abstract
Description
本願は、日本国に、2016年6月17日に出願された特願2016-121235号、2016年12月13日に出願された特願2016-241469号、2017年4月25日に出願された特願2017-086654号、及び2017年5月19日に出願された特願2017-100286号に基づき優先権を主張し、その内容をここに援用する。
[1] 植物体のPERK13(Proline-rich extensin-like receptor kinase 13)の機能を抑制又は阻害する、植物体の耐塩性向上方法。
[2] PERK13のアンタゴニストを前記植物体の根に接触させる、前記[1]の植物体の耐塩性向上方法。
[3] 前記アンタゴニストが、1種若しくは2種以上の微生物、又はこれらの分泌物質である、前記[2]の植物体の耐塩性向上方法。
[4] 前記アンタゴニストを含む水溶液に、前記植物体の根の少なくとも一部を浸漬させる工程を含む、前記[2]又は[3]の植物体の耐塩性向上方法。
[5] PERK13の機能の抑制をPERK13遺伝子の発現を抑制することによって行う、又はPERK13の機能の阻害をPERK13遺伝子の発現を阻害することによって行う、前記[1]の植物体の耐塩性向上方法。
[6] 前記植物体に対して、PERK13遺伝子を欠損させる、又はPERK13遺伝子にその機能を低下させる変異を導入する、前記[1]又は[5]の植物体の耐塩性向上方法。
[7] 前記植物体が、非選択性陽イオンチャネル、細胞膜型Na+/H+アンチポーター、液胞型Na+/H+アンチポーター、及び高親和性カリウムトランスポーターからなる群より選択される1種以上の蛋白質の機能が亢進している、前記[1]~[6]のいずれかの植物体の耐塩性向上方法。
[8] 前記植物体が、非選択性陽イオンチャネル、細胞膜型Na+/H+アンチポーター、液胞型Na+/H+アンチポーター、及び高親和性カリウムトランスポーターからなる群より選択される1種以上の蛋白質が過剰発現している、前記[1]~[6]のいずれかの植物体の耐塩性向上方法。
[9] 前記植物体が、外来遺伝子が導入された形質転換体であり、
前記外来遺伝子が、SOS1遺伝子、SOS2遺伝子、SOS3遺伝子、NHX1遺伝子、及びHKT1遺伝子からなる群より選択される1種以上である、前記[1]~[6]のいずれかの植物体の耐塩性向上方法。
[10] 前記植物体が、双子葉植物である、前記[1]~[9]のいずれかの植物体の耐塩性向上方法。
[11] 前記植物体が、単子葉植物である、前記[1]~[9]のいずれかの植物体の耐塩性向上方法。
[12] 前記植物体が、イネ科の植物、ナス科の植物、アブラナ科の植物、ウリ科の植物、ブドウ科の植物、ミカン科の植物、バラ科の植物、マメ科の植物、ハス科の植物、ゴマ科の植物、アカザ科の植物、ヤシ科の植物、バショウ科の植物、アオイ科の植物、フトモモ科の植物、及びフウチョウソウ科の植物より選ばれる1種の植物である、前記[1]~[9]のいずれかの植物体の耐塩性向上方法。
[13] 前記植物体が、イネ、トウモロコシ、モロコシ、コムギ、オオムギ、ライムギ、ヒエ、アワ、トマト、ナス、パプリカ、ピーマン、ジャガイモ、タバコ、シロイヌナズナ、セイヨウアブラナ、ナズナ、ダイコン、キャベツ、紫キャベツ、メキャベツ(プチヴェール)、ハクサイ、チンゲンサイ、ケール、クレソン、小松菜、ブロッコリー、カリフラワー、カブ、ワサビ、マスタード、キュウリ、ニガウリ、カボチャ、メロン、スイカ、ブドウ、レモン、オレンジ、ネーブルオレンジ、グレープフルーツ、ミカン、ライム、スダチ、ユズ、シイクワシャー、タンカン、リンゴ、サクラ、ウメ、モモ、イチゴ、ビワ、アンズ、プラム(スモモ)、プルーン、アーモンド、ナシ、洋ナシ、ラズベリー、ブラックベリー、カシス、クランベリー、ブルーベリー、ダイズ、インゲンマメ、エンドウマメ、ソラマメ、エダマメ、リョクトウ、ヒヨコマメ、ハス(レンコン)、ゴマ、ホウレンソウ、ビート、テンサイ、キヌア、ヒユ、アマランサス、ケイトウ、ナツメヤシ、アブラヤシ、ココヤシ、アサイー、バナナ、バショウ、マニラアサ、ワタ、オクラ、ユーカリ、フウチョウソウ 、及びセイヨウフウチョウソウより選ばれる1種の植物である、前記[1]~[9]のいずれかの植物体の耐塩性向上方法。
[14] 微生物を共生させた植物体を、塩化ナトリウム濃度が1.5質量%以上の環境下で栽培し、当該植物体の生存率が10%以上である、植物体の栽培方法。
[15] PERK13の機能が抑制又は阻害された、植物体。
[16] 植物体のPERK13の機能を抑制又は阻害する、耐塩性植物体の製造方法。
シロイヌナズナにランダム変異を導入した変異体のライブラリーに対して、耐塩性が向上している変異体をスクリーニングし、耐塩性向上に寄与している遺伝子を調べた。このスクリーニングには、パエニバシラス(Paenibacillus)属の微生物を用いた。パエニバシラス属菌は、シロイヌナズナの細胞内への塩化ナトリウムの流入を促進する。このため、パエニバシラス属菌共存下のシロイヌナズナは、通常は枯死することはない0.5質量%の塩化ナトリウム環境下でも枯死する。この性質を利用し、パエニバシラス属菌共存下でも耐塩性を向上させることができる遺伝子をスクリーニングした。
実施例1において、PERK13の機能欠失変異体であることを確認した4株のうちの2株について、1.5質量%の塩化ナトリウム環境下で栽培し、耐塩性を調べた。
PERK13の機能欠失変異体のうち、実施例2において耐塩性を調べた2株のうちの1株について、根におけるナトリウムの量を調べた。
野生型シロイヌナズナを用い、土壌から抽出された微生物から、耐塩性を高める共生効果を有する植物共生菌群を選抜した。
沖縄県にて採取された土壌1gを、緩衝液で懸濁し、十分に撹拌し、微生物懸濁液として用いた。
天面と底面が開口した円柱状のポットに、スクロース含有MS寒天培地(MS培地に0.5%(w/v)スクロースと0.9%(w/v)アガーを加えた培地)を注入して固めることにより、植物体を育成するためのポットを作製した。当該ポットを、スクロース含有MS培地(MS培地に0.5%(w/v)スクロースを加えた液体培地)を入れた8つの容器にそれぞれ複数個ずつ設置した。
シロイヌナズナの種子(Col-0)は、LEHLE社(Round Rock, TX, USA)より購入した。種子は、1%次亜塩素酸に浸漬させた状態で1分間撹拌をすることによって表面を滅菌した後、遠心分離処理により次亜塩素酸を除いた。次亜塩素酸処理後の種子は、滅菌水にて3回水洗した後、前記ポットの上部に播種して、4℃で24時間暗所にて保存した。
前記ポットを複数個用意し、スクロース含有MS培地(MS培地に0.5%(w/v)スクロースを加えた液体培地)を入れた1つの容器に全て設置した。各ポットは、底面はスクロース含有MS培地に浸っているが天面は浸っていない状態となるように設置した。これらのポットの上部に、次亜塩素酸処理後に滅菌水にて3回水洗した後の野生型の種子を播種し、25℃、明期16時間と暗期8時間の長日条件のインキュベーター内で14日間育成した。
14日間水耕栽培後に、当該ポットの底面を浸したスクロース含有MS培地に、塩化ナトリウムの最終濃度が1質量%となるように滅菌済の5M 塩化ナトリウム水溶液を添加し、さらに100μLの微生物懸濁液を添加した。その後、当該ポットを14日間培養した。
塩ストレス下での14日間の培養後、生育している植物体の根と地上部(葉と茎)を切断し、根を回収し、ホモジナイズして第1の微生物回収溶液とした。
選抜された塩ストレス下での植物体の生育を可能とする植物共生菌群(耐塩性向上用微生物混合物)を構成する微生物を同定した。
まず、前記第3の微生物回収溶液から菌体を回収し、回収した菌体の一部からゲノムDNAを、GenElute Bacterial Genomic DNA kit(Sigma-Aldrich、St. Louis, MO, USA)を用いて得た。
シロイヌナズナの野生株とPERK13の機能欠失変異体について、同定した耐塩性向上用微生物混合物の耐塩性向上効果を調べた。
まず、前記<植物体の水耕栽培>と同様にして、ポットにて14日間水耕栽培した植物体を用意した。当該ポットの底面を浸したスクロース含有MS培地に、塩化ナトリウムの最終濃度が0、0.5、1.0、1.5、2.0、2.5、又は3.0質量%となるように滅菌済の5M 塩化ナトリウム水溶液を添加し、さらに前記耐塩性向上用微生物混合物を添加して水耕栽培を行い、14日間培養後の生存率を調べた。対照として、耐塩性向上用微生物混合物を添加していない栽培用溶液で同様に水耕栽培し、14日間栽培後の生存率を調べた。
シロイヌナズナの野生株とPERK13の機能欠失変異体について、実施例4で取得した耐塩性向上用微生物混合物の、塩ストレス下における植物体の根へのナトリウム流入に対する影響を調べた。
トマト(Solanum lycopersicum)のPERK13機能欠失変異体を作製し、その耐塩性を調べた。
トマトのゲノムDNAに含まれている遺伝子のうち、シロイヌナズナのPERK13に60%以上の配列同一性のある3種の遺伝子(トマトのPERK13オーソログ遺伝子)、SlPERK9b(Solyc05g010140.2.1)、SlPERK10(Solyc01g010030.2.1)、SlPERK9a(Solyc04g006930.2.1)を標的遺伝子とするRNAiのベクターを構築した。これらは、互いにアミノ酸配列の配列同一性が98%以上であり、トマトのPERK13オーソログ遺伝子(NCBIのGene IDが101266034)のパラログと考えられる。標的配列は各遺伝子の遺伝子翻訳領域の5’末端側の200塩基とした。また、各遺伝子を同時にRNAiの標的配列とするため、遺伝子人工合成によって、SlPERK10遺伝子の標的配列(配列番号5)、SlPERK9a遺伝子の標的配列(配列番号6)、及びSlPERK9b遺伝子の標的配列(配列番号7)を連結した塩基配列からなるキメラ遺伝子を作製した。
人工合成した前記キメラ遺伝子を、相同組換えによって、pBI-sense, anti sense-GWベクター(Clontech社製)の改変ベクターに導入した。具体的には、当該キメラ遺伝子を、当該改変ベクターのカリフラワーモザイクウイルス35S(CaMV 35S)プロモーターとノパリン合成酵素遺伝子ターミネーター配列(NOS)の発現カセットの間に、センス方向とアンチセンス方向にそれぞれクローニングし、SlPERK遺伝子を標的とするRNAi用ベクター(pBI-SlPERKs-sense, anti senseベクター)を構築した。当該ベクターの構造マップを図7に示す。CaMV 35Sプロモーター制御下で転写された前記キメラ遺伝子のRNAは、イントロンの切断を介して、センスRNAとアンチセンスRNAからなる2本鎖RNAを形成した。
作製したRNAi用ベクターをアグロバクテリウム(Agrobacterium tumefaciens)GV3101系統に常法により導入し、組換えアグロバクテリウムを得た。トマト品種マイクロトム由来の子葉片に、得られた組換えアグロバクテリウムを感染させ、カルス形成培地にてカルス形成を誘導した。その後、薬剤耐性カルスを選抜し、再分化させた。
植物サンプル100mgを、液体窒素で凍結させて粉末状にした。この粉末に、300μLの抽出緩衝液(100mM Tris、50mM EDTA、500mM NaCl (pH8.0))と15μLの20% SDSを加えて混合してサンプル溶液を調製し、このサンプル溶液を65℃、10分間インキュベートした。インキュベート後のサンプル溶液は、90μLの5M 酢酸カリウムを添加した後、14,000rpmで10分間遠心分離を行なった。上清を新しいチューブに移し、400μLのイソプロパノールを添加して、室温で2分間静置した後、14,000rpmで2分間の遠心分離を行なった。得られたペレットを、500μLの70%エタノールで洗い、乾燥後に100μLの水で溶解したものを、DNAサンプルとした。
GoTaqポリメラーゼ(Promega社製)を用いて、フォワードプライマー(5’-GTTCTTCTACACCATTTGCAGC、配列番号8)とリバースプライマー(5’-ATTGTGGTAGTGTTGGTAAGGC、配列番号9)の終濃度が0.2μMになるようにPCR反応液を調製して、PCRを行なった。PCRは、95℃で3分間保持した後、95℃で30秒間、次いで55℃で30秒間、次いで72℃で30秒間を1サイクルとするサイクルを35サイクル繰り返し、最後に72℃で3分間保持する、という条件で行なった。
得られた形質転換トマトは、導入された前記キメラ遺伝子により、トマトのPERK13(SlPERK)の機能が欠失したトマトである。このPERK13機能欠失トマトを、塩化ナトリウムを終濃度が0.5、1.0、1.5、又は2.0質量%となるように含有させた1/2MS培地にて水耕栽培した。水耕栽培は、人工気象器内(25℃、16時間明期、8時間暗期)で行なった。野生型のトマトの場合、塩化ナトリウムを終濃度が0.5質量%となるように含有させた1/2MS培地で水耕栽培を行うと、栽培21日目には、高濃度の塩化ナトリウムに耐え切れず、葉が白化し、根が褐変して枯れてしまう(図示せず。)。これに対して、PERK13機能欠失トマトでは、栽培21日目において、0.5及び1.0質量%の塩化ナトリウム含有1/2MS培地では、いずれも葉が白化せずに生育している個体が確認された(図8)。また、1.5及び2.0質量%の塩化ナトリウム含有1/2MS培地では、葉の白化が観察されたが、根の褐変は観察されなかった。これらの結果から、トマトにおいても、PERK13の機能を抑制又は阻害することにより、植物体の耐塩性を向上させられることがわかった。
イネ(Oryza sativa)のPERK13機能欠失変異体を作製し、その耐塩性を調べた。
イネのゲノムDNAに含まれている遺伝子のうち、シロイヌナズナのPERK13に70%以上のアミノ酸配列の配列同一性のある遺伝子(イネのPERK13オーソログ遺伝子)OsPERK13(Os03g056880、NCBI GeneID 4333279)を標的遺伝子とするノックアウト用ベクターを構築した。当該遺伝子をノックアウトの標的配列とするため、遺伝子人工合成によってOsPERK13遺伝子の標的配列(配列番号10)からなるポリヌクレオチドを作製した。イネPERK13オーソログ遺伝子を標的とするノックアウト用ベクターpOsPERK-KO1は、人工合成した前記ポリヌクレオチドを、相同組換えによって、改変型pRIT1ベクター(Terada et al.,Nature Biotechnology,2002,vol.20,p.1030-1034)に導入して構築した。
イネの形質転換は、Toki らの方法(Plant Journal、2006年、第47巻、第69~76ページ)の手法に沿って行った。まず、前記ノックアウト用ベクターをアグロバクテリウム菌EHA101系統又はLBA4404系統に常法により導入し、組換えアグロバクテリウムを得た。得られた組換えアグロバクテリウムを、イネ品種「日本晴」の胚盤由来カルスに感染させた。感染させたイネカルスを0.25μM ビスピリバック塩を含む培地で培養し、ビスピリバック塩耐性カルスを選抜した。
前記ノックアウト用ベクターの導入が確認できたイネカルスを再分化培地へ継代し、25℃の明所で約3週間培養した。その結果、PERK13オーソログ遺伝子ノックアウト用ベクターを導入した組換え再分化植物体と、非組換え再分化植物体とを得た。同様に、非組換えイネカルスからも再分化植物体を誘導した。
組換え再分化植物体におけるOsPERK13遺伝子のノックアウト状況は、CAPS(Cleaved Amplified Polymorphic Sequences)法により解析した。まず、各植物体からDNA抽出キット「Maxwell 16 LEV Plant DNA kit(Promega社製)」を用いてゲノムDNAを抽出した。続いて、3g05688特異的プライマーである3g05688 No1-Fプライマー(5’-AGTCAAGCTTCGCCGGCGCCAATGCCGATGTGAGCCCGGC、配列番号13)と3g05688 No1-Rプライマー(5’-TGACGAATTCGCTCCGGCACGACGAGGGTTCTCCTGCGCG、配列番号14)を用いたPCRを行った。得られたPCR増幅産物は核酸精製キット「DNA Cleaner(和光純薬社製)」を用いて精製した後、制限酵素(TspRI)処理し、アガロース電気泳動によりDNA断片の切断状況を確認した。
PERK13オーソログ遺伝子ノックアウト用ベクターを導入した組換え再分化植物体、及び非組換え再分化植物体は、個体ごとに1.5質量%の塩化ナトリウムを含む発根培地(固形1/2MS)に継代した。その後、人工気象器(25℃、常時明期)にて約2週間育成し、植物体の表現型を観察した。
Claims (16)
- 植物体のPERK13(Proline-rich extensin-like receptor kinase 13)の機能を抑制又は阻害する、植物体の耐塩性向上方法。
- PERK13のアンタゴニストを前記植物体の根に接触させる、請求項1に記載の植物体の耐塩性向上方法。
- 前記アンタゴニストが、1種若しくは2種以上の微生物、又はこれらの分泌物質である、請求項2に記載の植物体の耐塩性向上方法。
- 前記アンタゴニストを含む水溶液に、前記植物体の根の少なくとも一部を浸漬させる工程を含む、請求項2又は3に記載の植物体の耐塩性向上方法。
- PERK13の機能の抑制をPERK13遺伝子の発現を抑制することによって行う、又はPERK13の機能の阻害をPERK13遺伝子の発現を阻害することによって行う、請求項1に記載の植物体の耐塩性向上方法。
- 前記植物体に対して、PERK13遺伝子を欠損させる、又はPERK13遺伝子にその機能を低下させる変異を導入する、請求項1又は5に記載の植物体の耐塩性向上方法。
- 前記植物体が、非選択性陽イオンチャネル、細胞膜型Na+/H+アンチポーター、液胞型Na+/H+アンチポーター、及び高親和性カリウムトランスポーターからなる群より選択される1種以上の蛋白質の機能が亢進している、請求項1~6のいずれか一項に記載の植物体の耐塩性向上方法。
- 前記植物体が、非選択性陽イオンチャネル、細胞膜型Na+/H+アンチポーター、液胞型Na+/H+アンチポーター、及び高親和性カリウムトランスポーターからなる群より選択される1種以上の蛋白質が過剰発現している、請求項1~6のいずれか一項に記載の植物体の耐塩性向上方法。
- 前記植物体が、外来遺伝子が導入された形質転換体であり、
前記外来遺伝子が、SOS1遺伝子、SOS2遺伝子、SOS3遺伝子、NHX1遺伝子、及びHKT1遺伝子からなる群より選択される1種以上である、請求項1~6のいずれか一項に記載の植物体の耐塩性向上方法。 - 前記植物体が、双子葉植物である、請求項1~9のいずれか一項に記載の植物体の耐塩性向上方法。
- 前記植物体が、単子葉植物である、請求項1~9のいずれか一項に記載の植物体の耐塩性向上方法。
- 前記植物体が、イネ科の植物、ナス科の植物、アブラナ科の植物、ウリ科の植物、ブドウ科の植物、ミカン科の植物、バラ科の植物、マメ科の植物、ハス科の植物、ゴマ科の植物、アカザ科の植物、ヤシ科の植物、バショウ科の植物、アオイ科の植物、フトモモ科の植物、及びフウチョウソウ科の植物より選ばれる1種の植物である、請求項1~9のいずれか一項に記載の植物体の耐塩性向上方法。
- 前記植物体が、イネ、トウモロコシ、モロコシ、コムギ、オオムギ、ライムギ、ヒエ、アワ、トマト、ナス、パプリカ、ピーマン、ジャガイモ、タバコ、シロイヌナズナ、セイヨウアブラナ、ナズナ、ダイコン、キャベツ、紫キャベツ、メキャベツ、ハクサイ、チンゲンサイ、ケール、クレソン、小松菜、ブロッコリー、カリフラワー、カブ、ワサビ、マスタード、キュウリ、ニガウリ、カボチャ、メロン、スイカ、ブドウ、レモン、オレンジ、ネーブルオレンジ、グレープフルーツ、ミカン、ライム、スダチ、ユズ、シイクワシャー、タンカン、リンゴ、サクラ、ウメ、モモ、イチゴ、ビワ、アンズ、プラム、プルーン、アーモンド、ナシ、洋ナシ、ラズベリー、ブラックベリー、カシス、クランベリー、ブルーベリー、ダイズ、インゲンマメ、エンドウマメ、ソラマメ、エダマメ、リョクトウ、ヒヨコマメ、ハス、ゴマ、ホウレンソウ、ビート、テンサイ、キヌア、ヒユ、アマランサス、ケイトウ、ナツメヤシ、アブラヤシ、ココヤシ、アサイー、バナナ、バショウ、マニラアサ、ワタ、オクラ、ユーカリ、フウチョウソウ 、及びセイヨウフウチョウソウより選ばれる1種の植物である、請求項1~9のいずれか一項に記載の植物体の耐塩性向上方法。
- 微生物を共生させた植物体を、塩化ナトリウム濃度が1.5質量%以上の環境下で栽培し、当該植物体の生存率が10%以上である、植物体の栽培方法。
- PERK13の機能が抑制又は阻害された、植物体。
- 植物体のPERK13の機能を抑制又は阻害する、耐塩性植物体の製造方法。
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CN114885835A (zh) * | 2022-05-27 | 2022-08-12 | 广西壮族自治区林业科学研究院 | 一种利用甲基磺酸乙酯探究桃金娘种子诱变效应的方法 |
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- 2017-06-15 CN CN201780028506.5A patent/CN109640631A/zh active Pending
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JP2018186834A (ja) | 2018-11-29 |
EP3409105A4 (en) | 2019-09-25 |
JP6435060B2 (ja) | 2018-12-05 |
AU2017285758A1 (en) | 2018-11-22 |
US20220411812A1 (en) | 2022-12-29 |
JP2018186835A (ja) | 2018-11-29 |
CN109640631A (zh) | 2019-04-16 |
SG11201809744YA (en) | 2019-01-30 |
US20190345508A1 (en) | 2019-11-14 |
US20190169631A1 (en) | 2019-06-06 |
JPWO2017217508A1 (ja) | 2018-06-28 |
EP3409105A1 (en) | 2018-12-05 |
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