JP2023513862A - Use of genes to improve tomato gray mold resistance - Google Patents
Use of genes to improve tomato gray mold resistance Download PDFInfo
- Publication number
- JP2023513862A JP2023513862A JP2022520494A JP2022520494A JP2023513862A JP 2023513862 A JP2023513862 A JP 2023513862A JP 2022520494 A JP2022520494 A JP 2022520494A JP 2022520494 A JP2022520494 A JP 2022520494A JP 2023513862 A JP2023513862 A JP 2023513862A
- Authority
- JP
- Japan
- Prior art keywords
- gene
- tomato
- plants
- botrytis
- resistance
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- 108090000623 proteins and genes Proteins 0.000 title claims abstract description 58
- 235000007688 Lycopersicon esculentum Nutrition 0.000 title claims abstract description 50
- 240000003768 Solanum lycopersicum Species 0.000 title claims description 57
- 241001465180 Botrytis Species 0.000 claims abstract description 16
- 238000000034 method Methods 0.000 claims abstract description 10
- 108091033409 CRISPR Proteins 0.000 claims description 28
- 238000010354 CRISPR gene editing Methods 0.000 claims description 13
- 239000013598 vector Substances 0.000 claims description 11
- 230000009261 transgenic effect Effects 0.000 claims description 10
- 239000002773 nucleotide Substances 0.000 claims description 9
- 125000003729 nucleotide group Chemical group 0.000 claims description 9
- 108091027544 Subgenomic mRNA Proteins 0.000 claims description 5
- 230000002194 synthesizing effect Effects 0.000 claims description 4
- 230000001131 transforming effect Effects 0.000 claims description 3
- FWMNVWWHGCHHJJ-SKKKGAJSSA-N 4-amino-1-[(2r)-6-amino-2-[[(2r)-2-[[(2r)-2-[[(2r)-2-amino-3-phenylpropanoyl]amino]-3-phenylpropanoyl]amino]-4-methylpentanoyl]amino]hexanoyl]piperidine-4-carboxylic acid Chemical compound C([C@H](C(=O)N[C@H](CC(C)C)C(=O)N[C@H](CCCCN)C(=O)N1CCC(N)(CC1)C(O)=O)NC(=O)[C@H](N)CC=1C=CC=CC=1)C1=CC=CC=C1 FWMNVWWHGCHHJJ-SKKKGAJSSA-N 0.000 claims description 2
- 208000035240 Disease Resistance Diseases 0.000 abstract description 9
- 238000009395 breeding Methods 0.000 abstract description 2
- 230000001488 breeding effect Effects 0.000 abstract description 2
- 241000227653 Lycopersicon Species 0.000 abstract 5
- 241000196324 Embryophyta Species 0.000 description 25
- 235000013399 edible fruits Nutrition 0.000 description 7
- 201000010099 disease Diseases 0.000 description 6
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 6
- 230000003902 lesion Effects 0.000 description 5
- 239000000243 solution Substances 0.000 description 5
- 238000012408 PCR amplification Methods 0.000 description 4
- 238000003209 gene knockout Methods 0.000 description 4
- 230000012010 growth Effects 0.000 description 4
- 230000035772 mutation Effects 0.000 description 4
- 108091026890 Coding region Proteins 0.000 description 3
- 108020004414 DNA Proteins 0.000 description 3
- 230000030279 gene silencing Effects 0.000 description 3
- 238000010362 genome editing Methods 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
- 239000006228 supernatant Substances 0.000 description 3
- 238000011144 upstream manufacturing Methods 0.000 description 3
- 229920001817 Agar Polymers 0.000 description 2
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- 241000123650 Botrytis cinerea Species 0.000 description 2
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 2
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 2
- VGGSQFUCUMXWEO-UHFFFAOYSA-N Ethene Chemical compound C=C VGGSQFUCUMXWEO-UHFFFAOYSA-N 0.000 description 2
- 239000005977 Ethylene Substances 0.000 description 2
- 101100278181 Homo sapiens DND1 gene Proteins 0.000 description 2
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 description 2
- 208000031888 Mycoses Diseases 0.000 description 2
- 238000012300 Sequence Analysis Methods 0.000 description 2
- 206010052428 Wound Diseases 0.000 description 2
- 208000027418 Wounds and injury Diseases 0.000 description 2
- 230000009471 action Effects 0.000 description 2
- 239000008272 agar Substances 0.000 description 2
- 230000032683 aging Effects 0.000 description 2
- 230000033228 biological regulation Effects 0.000 description 2
- 238000010276 construction Methods 0.000 description 2
- 230000007123 defense Effects 0.000 description 2
- 238000004925 denaturation Methods 0.000 description 2
- 230000036425 denaturation Effects 0.000 description 2
- 238000011081 inoculation Methods 0.000 description 2
- 230000002018 overexpression Effects 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 239000011550 stock solution Substances 0.000 description 2
- 239000000725 suspension Substances 0.000 description 2
- 235000013311 vegetables Nutrition 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- GEWDNTWNSAZUDX-WQMVXFAESA-N (-)-methyl jasmonate Chemical compound CC\C=C/C[C@@H]1[C@@H](CC(=O)OC)CCC1=O GEWDNTWNSAZUDX-WQMVXFAESA-N 0.000 description 1
- OWEGMIWEEQEYGQ-UHFFFAOYSA-N 100676-05-9 Natural products OC1C(O)C(O)C(CO)OC1OCC1C(O)C(O)C(O)C(OC2C(OC(O)C(O)C2O)CO)O1 OWEGMIWEEQEYGQ-UHFFFAOYSA-N 0.000 description 1
- 102100036945 Dead end protein homolog 1 Human genes 0.000 description 1
- 101000950194 Homo sapiens Dead end protein homolog 1 Proteins 0.000 description 1
- GUBGYTABKSRVRQ-PICCSMPSSA-N Maltose Natural products O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@@H](CO)OC(O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-PICCSMPSSA-N 0.000 description 1
- 239000001888 Peptone Substances 0.000 description 1
- 108010080698 Peptones Proteins 0.000 description 1
- 244000061456 Solanum tuberosum Species 0.000 description 1
- 235000002595 Solanum tuberosum Nutrition 0.000 description 1
- 238000000692 Student's t-test Methods 0.000 description 1
- 238000007605 air drying Methods 0.000 description 1
- 238000000137 annealing Methods 0.000 description 1
- 230000004665 defense response Effects 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 239000000706 filtrate Substances 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 230000035784 germination Effects 0.000 description 1
- 230000028644 hyphal growth Effects 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 230000001404 mediated effect Effects 0.000 description 1
- GEWDNTWNSAZUDX-UHFFFAOYSA-N methyl 7-epi-jasmonate Natural products CCC=CCC1C(CC(=O)OC)CCC1=O GEWDNTWNSAZUDX-UHFFFAOYSA-N 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 235000019319 peptone Nutrition 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 238000012353 t test Methods 0.000 description 1
- 230000008685 targeting Effects 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
- 229910021642 ultra pure water Inorganic materials 0.000 description 1
- 239000012498 ultrapure water Substances 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
-
- 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
- C12N15/09—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]
-
- 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
- C12N15/09—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)
- C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
- C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
- C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
- C12N15/8279—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance
- C12N15/8282—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance for fungal resistance
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Genetics & Genomics (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biotechnology (AREA)
- General Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Biophysics (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Physics & Mathematics (AREA)
- Microbiology (AREA)
- Plant Pathology (AREA)
- Cell Biology (AREA)
- Botany (AREA)
- Gastroenterology & Hepatology (AREA)
- Medicinal Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Virology (AREA)
- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
Abstract
本発明は、トマト灰色かび病抵抗性の向上のための遺伝子の使用に関するものであり、作物の耐病性育種分野に属する。本発明では、トマトにおいて遺伝子Ｓｏｌｙｃ０５ｇ００４６００をノックアウトすることにより、トマトの葉の灰色かび病菌に対する抵抗性が効果的に向上されるトマト遺伝子Ｓｏｌｙｃ０５ｇ００４６００の使用が開示されている。さらに、本発明では、トマトにおいて遺伝子Ｓｏｌｙｃ０５ｇ００４６００をノックアウトする方法も開示されている。The present invention relates to the use of genes for improving tomato botrytis resistance, and belongs to the field of disease resistance breeding of crops. The present invention discloses the use of the tomato gene Solyc05g004600 to effectively improve the resistance of tomato leaves to botrytis by knocking out the gene Solyc05g004600 in tomato. Further, the present invention also discloses a method of knocking out the gene Solyc05g004600 in tomato.
Description
本発明は、トマト灰色かび病抵抗性の向上のための遺伝子の使用に関するものであり、作物の耐病性育種分野に属する。 The present invention relates to the use of genes for improving tomato botrytis resistance, and belongs to the field of disease resistance breeding of crops.
トマトは、食用、薬用、及び経済的価値が高いため、世界で重要な果菜類作物となっている。灰色かび病は、トマトにおいて一般的かつ被害が深刻な真菌病であり、植物の茎、葉、花、果実のいずれも発病する可能性がある。通常、病菌は植物の傷や老化組織から侵入し、徐々に他の健康な部分に広がり、トマトの収量や品質に多大なる影響を与える。作物の病害を防除するために耐病性遺伝子を使用することは、最も経済的で効果的な方法である。近年、トマトの耐病性に関連する遺伝子がますます多く発見されている。例えば、Ｙｕら（２０１８）は、エチレン応答因子ＳｌＥＲＦ２が灰色かび病に対するトマトの防御制御に関与し、ＳｌＥＲＦ２の過剰発現により、トマト果実の灰色かび病抵抗性が向上されることを発見した；Ｓｕｎら（２０１７）は、トマトにおけるＤＮＤ１遺伝子のサイレンシングにより、対照植物に比べて、サイレンシング植物は、灰色かび病の病斑の直径が顕著に減少し、葉における灰色かび病菌の分生子の数が顕著に低下し、菌糸成長が抑制されたことを発見した。これにより、植物の耐病性に関連するより多くの遺伝子を発見するための遺伝子編集技術の使用は、作物の抵抗性の向上や経済的損失の低減のための効果的な手段であることが分かる。 Tomatoes have become an important fruit vegetable crop in the world because of their high edible, medicinal and economic value. Botrytis is a common and devastating fungal disease in tomatoes, and can affect any of the plant's stems, leaves, flowers and fruits. The disease usually enters the plant through wounds and aging tissue and gradually spreads to other healthy parts, significantly affecting tomato yield and quality. The use of disease resistance genes to control crop diseases is the most economical and effective method. In recent years, more and more genes related to disease resistance in tomato have been discovered. For example, Yu et al. (2018) found that the ethylene responsive factor SlERF2 is involved in tomato defense regulation against gray mold, and overexpression of SlERF2 improves the resistance of tomato fruits to gray mold; (2017) showed that silencing the DND1 gene in tomato resulted in a marked reduction in the diameter of Botrytis blight lesions and the number of Botrytis conidia on leaves in silenced plants compared to control plants. was found to be significantly reduced and mycelial growth was suppressed. This indicates that the use of gene editing techniques to discover more genes associated with disease resistance in plants is an effective tool for improving crop resistance and reducing economic losses. .
本発明の解決しようとする課題は、如何にトマト灰色かび病抵抗性を効果的に向上させるかである。 The problem to be solved by the present invention is how to effectively improve tomato gray mold resistance.
上記課題を解決するために、本発明は、ヌクレオチド配列が配列番号１で示されるトマトの遺伝子Ｓｏｌｙｃ０５ｇ００４６００を提供する。 In order to solve the above problems, the present invention provides tomato gene Solyc05g004600 whose nucleotide sequence is shown in SEQ ID NO:1.
また、本発明は、トマトにおいてＳｏｌｙｃ０５ｇ００４６００遺伝子をノックアウトすることにより、トマトの葉の灰色かび病抵抗性が効果的に向上される前記遺伝子の使用、及び
ｍｕ－１のＳｏｌｙｃ０５ｇ００４６００遺伝子のヌクレオチド配列が配列番号２で示され、ｍｕ－２のＳｏｌｙｃ０５ｇ００４６００遺伝子のヌクレオチド配列が配列番号３で示される、本発明のトマト遺伝子Ｓｏｌｙｃ０５ｇ００４６００の使用の改良を提供する。
In addition, the present invention also provides the use of the Solyc05g004600 gene, which effectively improves the botrytis resistance of tomato leaves, by knocking out the Solyc05g004600 gene in tomato, and the nucleotide sequence of the Solyc05g004600 gene of mu-1 is SEQ ID NO: 2 and the nucleotide sequence of the mu-2 Solyc05g004600 gene is shown in SEQ ID NO:3.
さらに、本発明は、以下のステップを含む、トマトにおいて変異遺伝子Ｓｏｌｙｃ０５ｇ００４６００をノックアウトする方法を提供する。
１）、ＣＲＩＳＰＲ／Ｃａｓ９編集における標的配列ｓｇＲＮＡ：５’－ＧＴＴＧＴＴＣＡＡＣＡＴＧＡＧＣＡＧＡＧ－３’を設計するステップ、
２）、ステップ１）で得られた配列でプライマーを合成し、ＣＲＩＳＰＲ／Ｃａｓ９ベクターを構築するステップ、
３）、ステップ２）で得られたベクターでトマト（野生型トマト品種ＭｉｃｒｏＴｏｍ）を形質転換して、相応のＳｏｌｙｃ０５ｇ００４６００遺伝子がノックアウトされたトマト植物であるトランスジェニックトマト植物ｍｕ－１とｍｕ－２を得る（前記トランスジェニックトマト植物からＳｏｌｙｃ０５ｇ００４６００遺伝子の変異が生じた植物が同定される）ステップ。
Further, the present invention provides a method of knocking out the mutant gene Solyc05g004600 in tomato, comprising the following steps.
1), designing the target sequence sgRNA in CRISPR/Cas9 editing: 5′-GTTGTTCAACATGAGCAGAG-3′;
2), synthesizing a primer with the sequence obtained in step 1) and constructing a CRISPR/Cas9 vector;
3), transforming tomatoes (wild-type tomato cultivar MicroTom) with the vectors obtained in step 2) to produce transgenic tomato plants mu-1 and mu-2, which are tomato plants in which the corresponding Solyc05g004600 gene has been knocked out; obtaining (identifying a plant having a mutation of the Solyc05g004600 gene from said transgenic tomato plant).
遺伝子Ｓｏｌｙｃ０５ｇ００４６００の配列が既にデータベースに登録されているが、現在、当該遺伝子に関する機能や作用について研究も報告もされていないため、当該遺伝子がトマトの灰色かび病菌に対して抵抗性があることは、本発明が初めて発見したことである。 Although the sequence of the gene Solyc05g004600 has already been registered in the database, there is currently no research or report on the function or action of this gene. This is the first discovery made by the present invention.
本発明の技術案は、以下のとおりである。
ＣＲＩＳＰＲ／Ｃａｓ９遺伝子編集技術により、Ｓｏｌｙｃ０５ｇ００４６００遺伝子のヌクレオチド配列（配列番号１）に基づき、Ｓｏｌｙｃ０５ｇ００４６００遺伝子を特異的に標的とするｓｇＲＮＡ配列を合成し、相応のＣＲＩＳＰＲ／Ｃａｓ９ベクターを構築し、野生型トマト品種ＭｉｃｒｏＴｏｍを遺伝形質転換し、ゲノム内のＳｏｌｙｃ０５ｇ００４６００遺伝子を指向性編集し、トランスジェニック植物を得て、これらのトランスジェニック植物内のＳｏｌｙｃ０５ｇ００４６００遺伝子に対してＰＣＲ増幅と配列決定を行い、同定した結果、Ｓｏｌｙｃ０５ｇ００４６００遺伝子の２種のタイプの異なる変異株ｍｕ－１とｍｕ－２（図１）が得られた。ｍｕ－１植物内のＳｏｌｙｃ０５ｇ００４６００遺伝子の変異配列は配列番号２であり、ｍｕ－２植物内のＳｏｌｙｃ０５ｇ００４６００遺伝子の変異配列は配列番号３である。トマトの灰色かび病菌を接種した後、野生型対照品種ＭｉｃｒｏＴｏｍが発病したのに対し、Ｓｏｌｙｃ０５ｇ００４６００遺伝子の変異が生じた２つの株は、いずれも耐病性が向上し（図２）、葉における病斑の面積が対照品種ＭｉｃｒｏＴｏｍに比べて顕著に小さい（図３）。トマトにおけるＳｏｌｙｃ０５ｇ００４６００遺伝子の変異は、トマトの灰色かび病抵抗性を効果的に向上させることができることが明らかとなった。
The technical solution of the present invention is as follows.
Using CRISPR/Cas9 gene editing technology, based on the nucleotide sequence of Solyc05g004600 gene (SEQ ID NO: 1), synthesizing an sgRNA sequence specifically targeting the Solyc05g004600 gene, constructing a corresponding CRISPR/Cas9 vector, wild-type tomato varieties MicroTom was genetically transformed, the Solyc05g004600 gene in the genome was directed edited to obtain transgenic plants, and the Solyc05g004600 gene in these transgenic plants was PCR amplified and sequenced to identify Solyc05g004600. Mutants mu-1 and mu-2 (Fig. 1) with two different types of genes were obtained. The mutated sequence of the Solyc05g004600 gene in mu-1 plants is SEQ ID NO:2 and the mutated sequence of the Solyc05g004600 gene in mu-2 plants is SEQ ID NO:3. After inoculation with botrytis of tomato, the wild-type control cultivar MicroTom developed the disease, whereas the two strains in which the Solyc05g004600 gene was mutated showed improved disease resistance (Fig. area is significantly smaller than that of the control cultivar MicroTom (Fig. 3). It was found that mutation of the Solyc05g004600 gene in tomato can effectively improve the resistance to botrytis in tomato.
以下、図面を参照しながら、本発明の具体的な実施形態を更に詳細に説明する。
以下、具体的な実施例を参照しながら、本発明を更に説明するが、本発明の保護範囲はそれらに限定されない。 The present invention is further described below with reference to specific examples, but the scope of protection of the present invention is not limited thereto.
実施例１：
ステップ１、Ｓｏｌｙｃ０５ｇ００４６００遺伝子ノックアウトベクターの構築
Ｓｏｌｙｃ０５ｇ００４６００遺伝子のコード配列（配列番号１）に基づき、ＣＲＩＳＰＲ／Ｃａｓ９標的部位解析ソフトウェア（ｈｔｔｐ：／／ｃｒｉｓｐｒ．ｍｉｔ．ｅｄｕ／）により、Ｓｏｌｙｃ０５ｇ００４６００遺伝子に対してＣＲＩＳＰＲ／Ｃａｓ９編集におけるｓｇＲＮＡ配列：５’－ＧＴＴＧＴＴＣＡＡＣＡＴＧＡＧＣＡＧＡＧ－３’を設計し、当該配列に基づいて相応のプライマーを合成した。
Example 1:
上流５’－ＴＧＡＴＴＧＴＴＧＴＴＣＡＡＣＡＴＧＡＧＣＡＧＡＧ－３’、
下流５’－ＡＡＡＣＣＴＣＴＧＣＴＣＡＴＧＴＴＧＡＡＣＡＡＣＡ－３’。
upstream 5′-TGATTGTTGTTCAACATGAGCAGAG-3′,
Downstream 5′-AAACCTCTGCTCATGTTGAACAACA-3′.
次に、ＣＲＩＳＰＲ／Ｃａｓ９キット（Ｂｉｏｇｌｅ，Ｃｈｉｎａ）で相応のＣＲＩＳＰＲ／Ｃａｓ９ベクターを構築した。構築方法は、製品の説明書に準じて行った。 Then, the corresponding CRISPR/Cas9 vector was constructed with CRISPR/Cas9 kit (Biogle, China). The construction method was performed according to the instruction manual of the product.
ステップ２、Ｓｏｌｙｃ０５ｇ００４６００遺伝子ノックアウトベクターのトマトへの遺伝形質転換
ステップ１で構築したＣＲＩＳＰＲ／Ｃａｓ９ベクターでトマト品種ＭｉｃｒｏＴｏｍを遺伝形質転換した。トランスジェニック方法については、Ｋｉｍｕｒａらの方法（Ｋｉｍｕｒａ Ｓ ｅｔ ａｌ， ＣＨＳ Ｐｒｏｔｏｃ， ２００８）により、相応の一連の未同定のトランスジェニックトマト植物が得られた。
ステップ３、Ｓｏｌｙｃ０５ｇ００４６００遺伝子ノックアウト植物の同定
トマトの葉０．１ｇを取り、液体窒素で粉砕した後、抽出液（Ｔｒｉｓ－ｃｌ １５．７６ｇ、Ｎａｃｌ ２９．２２ｇ、ＳＤＳ粉末１５．０ｇを超純水に添加して１Ｌに定容し、ｐＨ＝８．０となるように調節したもの）６００μｌを加え、６５℃で６０分インキュベートした。５Ｍ ＫＡＣ ２００μｌを加え、均一に混合した後、氷浴で１０分冷却した。更に、クロロホルム５００μｌを加え、均一に混合し、１００００ ｒｐｍで５分遠心した。上澄みを取り、イソプロパノール５００μｌを加え、均一に混合し、１２０００ ｒｐｍで３分遠心し、上澄みを捨てた。７５％エタノールで沈殿を洗浄し、１２０００ ｒｐｍで３分遠心し、上澄みを捨てた。室温で上下を反転させて置き、ＤＮＡを風乾した後、純水３０μｌを添加してＤＮＡを溶解した。
Ｓｏｌｙｃ０５ｇ００４６００遺伝子のＰＣＲ増幅のプライマー（上流５’－ＧＣＣＴＣＴＡＴＴＡＡＴＧＡＣＡＴＣ－３’、下流５’－ＡＴＡＡＧＣＴＡＡＡＡＴＣＧＡＣＴＡ－３’）を合成し、トランスジェニックトマト植物およびその対照品種ＭｉｃｒｏＴｏｍのゲノムＤＮＡをテンプレートとし、ＧｏＴａｑ（商標登録） Ｍａｓｔｅｒ Ｍｉｘ（Ｐｒｏｍｅｇａ， ＵＳＡ）によりＰＣＲ増幅を行った。ＰＣＲ増幅系は、製品の説明書に準じて操作した。 Primers for PCR amplification of the Solyc05g004600 gene (upstream 5'-GCCTCTATTAATGACATC-3', downstream 5'-ATAAGCTAAATCGACTA-3') were synthesized, and genomic DNAs of transgenic tomato plants and their control variety MicroTom were used as templates, and GoTaq (registered trademark) was used. ) PCR amplification was performed by Master Mix (Promega, USA). The PCR amplification system was operated according to the manufacturer's instructions.
ＰＣＲ増幅プログラムにおいて、９４℃で５分初期変性を行い；９４℃での３０秒の変性、５５℃での３０秒のアニーリング、７２℃での３０秒の伸長を３５サイクル行い；７２℃で１０分伸長させる。 In the PCR amplification program, initial denaturation at 94°C for 5 minutes; 35 cycles of denaturation at 94°C for 30 seconds, annealing at 55°C for 30 seconds, extension at 72°C for 30 seconds; minutes.
ＰＣＲ増幅系を、２×Ｍｉｘ １０μｌ、上流プライマー０．５μｌ、下流プライマー０．５μｌ、テンプレート２μｌ、ｄｄＨ２Ｏ ７μｌのものとする。
The PCR amplification system is 10
ＰＣＲ産物に対してシーケンス解析を行ったところ、Ｓｏｌｙｃ０５ｇ００４６００遺伝子の、ｍｕ－１とｍｕ－２という２種の変異タイプの同定に成功した。ｍｕ－１植物では、Ｓｏｌｙｃ０５ｇ００４６００遺伝子のコード領域に１つの塩基が挿入され（図１）、そのヌクレオチド配列は配列番号２である。ｍｕ－２植物では、Ｓｏｌｙｃ０５ｇ００４６００遺伝子のコード領域の３つの塩基が欠失し（図１）、そのヌクレオチド配列は配列番号３である。 Sequence analysis of the PCR product successfully identified two mutation types, mu-1 and mu-2, in the Solyc05g004600 gene. In mu-1 plants, a single base was inserted into the coding region of the Solyc05g004600 gene (FIG. 1), the nucleotide sequence of which is SEQ ID NO:2. In mu-2 plants, 3 bases in the coding region of the Solyc05g004600 gene are deleted (Fig. 1), the nucleotide sequence of which is SEQ ID NO:3.
ステップ４、Ｓｏｌｙｃ０５ｇ００４６００遺伝子ノックアウト植物の灰色かび病菌に対する抵抗性の同定
上記で同定されたＳｏｌｙｃ０５ｇ００４６００遺伝子変異株ｍｕ－１、ｍｕ－２および対照品種ＭｉｃｒｏＴｏｍを温室で植え、２５℃、１６時間光照射、８時間暗黒条件で培養した。約４５日間成長したのち、ｍｕ－１、ｍｕ－２および対照品種から３株を無作為に選択し、株あたり４枚の葉を取り、それらを０．８％の寒天（８ｇ寒天粉を超純水に加えて１Ｌに定容したもの）シャーレに固定させた。体積が約２ｍｌの灰色かび病菌を選択し、５ｍｌの１％ＳＭＢ（１０ｇカビ用ペプトン、４０ｇマルトースを超純水に加えて１Ｌに定容したもの）溶液に入れ、均一に振とうし、濾過して、濾液として胞子原液が得られた。２５×１６規格の血球計算盤で胞子原液の濃度を計算し、２５個の中ブロックのうちの各中ブロックに約１６個の胞子があるように１％ＳＭＢ溶液で希釈し、接種時にさらに４倍希釈した後、胞子懸濁液が得られた。胞子無しの１％ＳＭＢ溶液を対照とし、各一枚の葉に５μｌの胞子懸濁液を滴下し、蓋をし、トマトの成長に適した環境に置いた。２日後、菌が接種された葉を摘み取り、Ｉｍａｇｅ Ｊソフトウェアで病斑の面積を計算した。その結果、Ｓｏｌｙｃ０５ｇ００４６００遺伝子変異株ｍｕ－１、ｍｕ－２は、対照品種に比べて、葉の発病程度が低く（図２）、葉における病斑の面積が顕著に小さい（図３）ことが示された。
Step 4, Identification of resistance of Solyc05g004600 gene knockout plants to botrytis fungus Solyc05g004600 gene mutant strains mu-1, mu-2 and control variety MicroTom identified above were planted in a greenhouse, exposed to light at 25°C for 16 hours, 8 Cultured in the dark for hours. After about 45 days of growth, 3 plants were randomly selected from mu-1, mu-2 and control cultivars, 4 leaves were taken per plant and they were sprinkled with 0.8% agar (more than 8 g agar powder). It was added to pure water and fixed to 1 L) and fixed in a Petri dish. Select a botrytis with a volume of about 2 ml, put it in 5 ml of 1% SMB (10 g mold peptone, 40 g maltose added to ultrapure water to make 1 L) solution, shake evenly, and filter. A spore stock solution was obtained as a filtrate. Calculate the concentration of the spore stock solution in a 25×16 standard hemocytometer, dilute with a 1% SMB solution so that each of the 25 medium blocks has approximately 16 spores, and add an additional 4 at the time of inoculation. After 2-fold dilution a spore suspension was obtained. A 1% SMB solution without spores served as a control, and each single leaf was dripped with 5 μl of the spore suspension, covered and placed in an environment suitable for tomato growth. Two days later, the fungus-inoculated leaves were picked and the lesion area was calculated with Image J software. As a result, the Solyc05g004600 gene mutant strains mu-1 and mu-2 showed a lower degree of disease on the leaves (Fig. 2) and a significantly smaller area of lesions on the leaves (Fig. 3) than the control cultivar. was done.
上記のことにより、Ｓｏｌｙｃ０５ｇ００４６００遺伝子がノックアウトされたトマト植物ｍｕ－１、ｍｕ－２は、葉の灰色かび病菌に対する抵抗性が効果的に向上したことが証明された。 From the above, it was proved that tomato plants mu-1 and mu-2 in which the Solyc05g004600 gene was knocked out had effectively improved resistance to botrytis in the leaves.
なお、上記で挙げたものは、本発明のいくつかの具体的な実施例にすぎない。本発明が上記の実施例に限定されず、多くの変形が可能であることは明らかである。当業者が本発明の開示内容に基づいて直接に導き出す、または想到する変形は、いずれも本発明の保護範囲に属する。 It should be noted that the above are only some specific examples of the present invention. It is clear that the invention is not limited to the examples described above, but that many variants are possible. Any variation directly derived or conceived by a person skilled in the art based on the disclosure content of the present invention shall fall within the protection scope of the present invention.
トマトは、食用、薬用、及び経済的価値が高いため、世界で重要な果菜類作物となっている。灰色かび病は、トマトにおいて一般的かつ被害が深刻な真菌病であり、植物の茎、葉、花、果実のいずれも発病する可能性がある。通常、病菌は植物の傷や老化組織から侵入し、徐々に他の健康な部分に広がり、トマトの収量や品質に多大なる影響を与える。作物の病害を防除するために耐病性遺伝子を使用することは、最も経済的で効果的な方法である。近年、トマトの耐病性に関連する遺伝子がますます多く発見されている。例えば、Ｙｕら（２０１８）（Ｙｕ ＷＱ， Ｚｈａｏ ＲＲ， Ｓｈｅｎｇ ＪＰ， ｅｔ ａｌ． ＳｌＥＲＦ２ ｉｓ ａｓｓｏｃｉａｔｅｄ ｗｉｔｈ Ｍｅｔｈｙｌ Ｊａｓｍｏｎａｔｅ－ｍｅｄｉａｔｅｄ ｄｅｆｅｎｓｅ ｒｅｓｐｏｎｓｅ ａｇａｉｎｓｔ Ｂｏｔｒｙｔｉｓ ｃｉｎｅｒｅａ ｉｎ ｔｏｍａｔｏ ｆｒｕｉｔ． Ｊ Ａｇｒｉｃ Ｆｏｏｄ Ｃｈｅｍ， ２０１８， ６６（３８）： ９９２３－９９３２．）は、エチレン応答因子ＳｌＥＲＦ２が灰色かび病に対するトマトの防御制御に関与し、ＳｌＥＲＦ２の過剰発現により、トマト果実の灰色かび病抵抗性が向上されることを発見した；Ｓｕｎら（２０１７）（Ｓｕｎ Ｋ， Ｔｕｉｎｅｎ Ａ， ｖａｎ Ｋａｎ ＪＡ， ｅｔ ａｌ． Ｓｉｌｅｎｃｉｎｇ ｏｆ ＤＮＤ１ ｉｎ ｐｏｔａｔｏ ａｎｄ ｔｏｍａｔｏ ｉｍｐｅｄｅｓ ｃｏｎｉｄｉａｌ ｇｅｒｍｉｎａｔｉｏｎ， ａｔｔａｃｈｍｅｎｔ ａｎｄ ｈｙｐｈａｌ ｇｒｏｗｔｈ ｏｆ Ｂｏｔｒｙｔｉｓ ｃｉｎｅｒｅａ． ＢＭＣ Ｐｌａｎｔ Ｂｉｏｌ， ２０１７， １７（１）： ２３５．）は、トマトにおけるＤＮＤ１遺伝子のサイレンシングにより、対照植物に比べて、サイレンシング植物は、灰色かび病の病斑の直径が顕著に減少し、葉における灰色かび病菌の分生子の数が顕著に低下し、菌糸成長が抑制されたことを発見した。これにより、植物の耐病性に関連するより多くの遺伝子を発見するための遺伝子編集技術の使用は、作物の抵抗性の向上や経済的損失の低減のための効果的な手段であることが分かる。 Tomatoes have become an important fruit vegetable crop in the world because of their high edible, medicinal and economic value. Botrytis is a common and devastating fungal disease in tomatoes, and can affect any of the plant's stems, leaves, flowers and fruits. The disease usually enters the plant through wounds and aging tissue and gradually spreads to other healthy parts, significantly affecting tomato yield and quality. The use of disease resistance genes to control crop diseases is the most economical and effective method. In recent years, more and more genes related to disease resistance in tomato have been discovered.例えば、Ｙｕら（２０１８） （Ｙｕ ＷＱ， Ｚｈａｏ ＲＲ， Ｓｈｅｎｇ ＪＰ， ｅｔ ａｌ． ＳｌＥＲＦ２ ｉｓ ａｓｓｏｃｉａｔｅｄ ｗｉｔｈ Ｍｅｔｈｙｌ Ｊａｓｍｏｎａｔｅ－ｍｅｄｉａｔｅｄ ｄｅｆｅｎｓｅ ｒｅｓｐｏｎｓｅ ａｇａｉｎｓｔ Ｂｏｔｒｙｔｉｓ ｃｉｎｅｒｅａ ｉｎ ｔｏｍａｔｏ ｆｒｕｉｔ． Ｊ Ａｇｒｉｃ Ｆｏｏｄ Ｃｈｅｍ， ２０１８， ６６（３８）： ９９２３ -9932.) found that the ethylene responsive factor SlERF2 is involved in tomato defense regulation against gray mold, and overexpression of SlERF2 enhances the resistance of tomato fruits to gray mold; Sun et al. (2017). ） （Ｓｕｎ Ｋ， Ｔｕｉｎｅｎ Ａ， ｖａｎ Ｋａｎ ＪＡ， ｅｔ ａｌ． Ｓｉｌｅｎｃｉｎｇ ｏｆ ＤＮＤ１ ｉｎ ｐｏｔａｔｏ ａｎｄ ｔｏｍａｔｏ ｉｍｐｅｄｅｓ ｃｏｎｉｄｉａｌ ｇｅｒｍｉｎａｔｉｏｎ， ａｔｔａｃｈｍｅｎｔ ａｎｄ ｈｙｐｈａｌ ｇｒｏｗｔｈ ｏｆ Ｂｏｔｒｙｔｉｓ ｃｉｎｅｒｅａ． ＢＭＣ Ｐｌａｎｔ Ｂｉｏｌ， ２０１７， １７（１）： ２３５．）は、 Silencing of the DND1 gene in tomato resulted in significantly reduced Botrytis lesion diameters and a significantly reduced number of Botrytis conidia on leaves in silenced plants compared to control plants; It was found that mycelial growth was suppressed. This indicates that the use of gene editing techniques to discover more genes associated with disease resistance in plants is an effective tool for improving crop resistance and reducing economic losses. .
さらに、本発明は、以下のステップを含む、トマトにおいて遺伝子Ｓｏｌｙｃ０５ｇ００４６００をノックアウトする方法を提供する。
１）、ＣＲＩＳＰＲ／Ｃａｓ９編集における標的配列ｓｇＲＮＡ：５’－ＧＴＴＧＴＴＣＡＡＣＡＴＧＡＧＣＡＧＡＧ－３’を設計するステップ、
２）、ステップ１）で得られた配列でプライマーを合成し、ＣＲＩＳＰＲ／Ｃａｓ９ベクターを構築するステップ、
３）、ステップ２）で得られたベクターでトマト（野生型トマト品種ＭｉｃｒｏＴｏｍ）を形質転換して、相応のＳｏｌｙｃ０５ｇ００４６００遺伝子がノックアウトされたトマト植物であるトランスジェニックトマト植物ｍｕ－１とｍｕ－２を得る（前記トランスジェニックトマト植物からＳｏｌｙｃ０５ｇ００４６００遺伝子の変異が生じた植物が同定される）ステップ。
Further, the present invention provides a method of knocking out gene Solyc05g004600 in tomato, comprising the following steps.
1), designing the target sequence sgRNA in CRISPR/Cas9 editing: 5′-GTTGTTCAACATGAGCAGAG-3′;
2), synthesizing a primer with the sequence obtained in step 1) and constructing a CRISPR/Cas9 vector;
3), transforming tomatoes (wild-type tomato cultivar MicroTom) with the vectors obtained in step 2) to produce transgenic tomato plants mu-1 and mu-2, which are tomato plants in which the corresponding Solyc05g004600 gene has been knocked out; obtaining (identifying a plant having a mutation of the Solyc05g004600 gene from said transgenic tomato plant).
遺伝子Ｓｏｌｙｃ０５ｇ００４６００の配列が既にデータベースに登録されているが、現在、当該遺伝子に関する機能や作用について研究も報告もされていないため、当該遺伝子がトマトの灰色かび病菌に対する抵抗性に関係することは、本発明が初めて発見したことである。 Although the sequence of the gene Solyc05g004600 has already been registered in a database, there is currently no research or report on the function or action of this gene. This is the first discovery made by the present invention.
Claims (4)
ｍｕ－２のＳｏｌｙｃ０５ｇ００４６００遺伝子のヌクレオチド配列が配列番号３で示されることを特徴とする、請求項２に記載のトマトの遺伝子Ｓｏｌｙｃ０５ｇ００４６００の使用。 The nucleotide sequence of the mu-1 Solyc05g004600 gene is shown in SEQ ID NO: 2,
Use of tomato gene Solyc05g004600 according to claim 2, characterized in that the nucleotide sequence of the Solyc05g004600 gene of mu-2 is given in SEQ ID NO:3.
１）、ＣＲＩＳＰＲ／Ｃａｓ９編集における標的配列ｓｇＲＮＡ：５’－ＧＴＴＧＴＴＣＡＡＣＡＴＧＡＧＣＡＧＡＧ－３’を設計するステップ、
２）、ステップ１）で得られた配列でプライマーを合成し、ＣＲＩＳＰＲ／Ｃａｓ９ベクターを構築するステップ、
３）、ステップ２）で得られたベクターでトマトを形質転換して、相応のＳｏｌｙｃ０５ｇ００４６００遺伝子がノックアウトされたトマト植物であるトランスジェニックトマト植物ｍｕ－１とｍｕ－２を得るステップ。 A method of knocking out the mutant gene Solyc05g004600 in tomato, characterized by comprising the following steps.
1), designing the target sequence sgRNA in CRISPR/Cas9 editing: 5′-GTTGTTCAACATGAGCAGAG-3′;
2), synthesizing a primer with the sequence obtained in step 1) and constructing a CRISPR/Cas9 vector;
3), transforming tomatoes with the vector obtained in step 2) to obtain transgenic tomato plants mu-1 and mu-2, which are tomato plants in which the corresponding Solyc05g004600 gene is knocked out.
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110058943.XA CN112646819B (en) | 2021-01-17 | 2021-01-17 | Use of gene to enhance resistance to tomato gray mold |
| CN202110058943.X | 2021-01-17 | ||
| PCT/CN2021/087986 WO2022151607A1 (en) | 2021-01-17 | 2021-04-19 | Use of gene enhancement for tomato gray mold resistance |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JP2023513862A true JP2023513862A (en) | 2023-04-04 |
| JP7383136B2 JP7383136B2 (en) | 2023-11-17 |
Family
ID=75368393
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP2022520494A Active JP7383136B2 (en) | 2021-01-17 | 2021-04-19 | Use of genes to improve tomato Botrytis blight resistance |
Country Status (3)
| Country | Link |
|---|---|
| JP (1) | JP7383136B2 (en) |
| CN (1) | CN112646819B (en) |
| WO (1) | WO2022151607A1 (en) |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112646819B (en) * | 2021-01-17 | 2023-04-18 | 浙江师范大学 | Use of gene to enhance resistance to tomato gray mold |
| CN116286942A (en) * | 2022-07-15 | 2023-06-23 | 浙江师范大学 | Gene use for promoting lycopene biosynthesis |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2017212315A1 (en) * | 2016-06-08 | 2017-12-14 | Lotan Agrosciences Llc | Rnai for control of fungi by cytb gene inhibition |
| WO2019041034A1 (en) * | 2017-08-30 | 2019-03-07 | The Governing Council Of The University Of Toronto | Methods of increasing disease resistance in a plant |
Family Cites Families (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103667265B (en) * | 2012-09-06 | 2015-10-28 | 中国科学院遗传与发育生物学研究所 | The application of tomato LoxD gene in regulating plant resistance |
| CN107164403A (en) * | 2017-06-16 | 2017-09-15 | 浙江理工大学 | Applications of the miR319 in botrytis resistant plant is cultivated |
| CN107699579B (en) * | 2017-11-03 | 2020-12-15 | 南京农业大学 | Gene for improving disease resistance of plants and application thereof |
| CN110734481B (en) * | 2019-11-12 | 2021-02-19 | 中国农业大学 | Application of tomato SlMIP protein and coding gene thereof in regulation and control of plant gray mold resistance |
| CN112646821B (en) * | 2020-02-07 | 2022-04-01 | 浙江大学 | Pathogenicity-related botrytis cinerea gene Bcmet16 and application thereof |
| CN111635908B (en) * | 2020-05-11 | 2021-03-23 | 青岛农业大学 | A kind ofKDA2Application of gene in improving plant resistance to botrytis cinerea infection |
| CN112048510B (en) * | 2020-09-11 | 2022-12-20 | 浙江师范大学 | Application of one gene in enlarging tomato fruit |
| CN112195186B (en) * | 2020-10-06 | 2022-05-27 | 华中农业大学 | Application of SlBBX20 gene in regulation and control of tomato gray mold resistance |
| CN112646819B (en) * | 2021-01-17 | 2023-04-18 | 浙江师范大学 | Use of gene to enhance resistance to tomato gray mold |
-
2021
- 2021-01-17 CN CN202110058943.XA patent/CN112646819B/en active Active
- 2021-04-19 WO PCT/CN2021/087986 patent/WO2022151607A1/en active Application Filing
- 2021-04-19 JP JP2022520494A patent/JP7383136B2/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2017212315A1 (en) * | 2016-06-08 | 2017-12-14 | Lotan Agrosciences Llc | Rnai for control of fungi by cytb gene inhibition |
| WO2019041034A1 (en) * | 2017-08-30 | 2019-03-07 | The Governing Council Of The University Of Toronto | Methods of increasing disease resistance in a plant |
Non-Patent Citations (1)
| Title |
|---|
| DATABASE GENBANK [ONLINE], ACCESSION NO.XM_004249477 08-AUG-2018, [検索日 2023.4.11], JPN6023015346, ISSN: 0005046053 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN112646819A (en) | 2021-04-13 |
| WO2022151607A1 (en) | 2022-07-21 |
| JP7383136B2 (en) | 2023-11-17 |
| CN112646819B (en) | 2023-04-18 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| JP2023513862A (en) | Use of genes to improve tomato gray mold resistance | |
| CN107475210B (en) | Rice bacterial leaf blight resistance related gene OsABA2 and application thereof | |
| CN107034223B (en) | The anti-plant hopper gene BGIOSGA015651 of adjusting and controlling rice and its application | |
| CN111303259B (en) | Application of rice transcription factor gene OsBEAR1 in cultivation of rice variety with increased coleoptile or suitable for direct seeding in field | |
| CN107245495A (en) | The method for creating of the common line with genic sterile of paddy rice and application | |
| CN111909936B (en) | Application of GT1 gene in regulation and control of maize male inflorescence sex determination and/or multi-spike development | |
| CN109609527A (en) | CDPK18L gene is improving the application in tomato bacterial leaf spot resistance and high temperature resistance as negative regulatory factor | |
| Xu et al. | Different effects of DEP1 on vascular bundle-and panicle-related traits under indica and japonica genetic backgrounds | |
| CN110577964A (en) | Application of UB2/UB3 gene in regulation and control of multiple-ear development of corn | |
| JP2011120597A (en) | Method for selecting genomic dna fragment | |
| CN113373166A (en) | Application of tobacco NtAAP3 gene in tobacco | |
| CN115125219B (en) | Application of OsJMJ718 gene and encoding protein thereof in regulation and control of rice grain types | |
| CN110592255B (en) | Indel molecular marker closely linked with pepper cluster inflorescence gene, primer and application | |
| CN115942868A (en) | Cannabis plant with increased yield | |
| CN108668884A (en) | The method for formulating ornamental type rice germplasm using two kinds of transcription factor genes | |
| CN117304288B (en) | Rice tillering angle related protein OsITAND, coding gene and application thereof | |
| CN116064567B (en) | Corn small kernel mutant and application thereof | |
| CN113322266B (en) | Application of rice OsRHD1-1 gene in rice male sterile line cultivation | |
| CN114164228B (en) | Method for improving disease resistance of rice through gene editing and application thereof | |
| CN114317597B (en) | Application of gene OsBEAR1 in cultivation of early-flowering and early-maturing crop variety | |
| CN114292859B (en) | Cucumber MYB111 MULE-InDel Gene and application thereof | |
| Cui et al. | Dissecting the subtropical adaptation traits and cuticle synthesis pathways via the genome of the subtropical blueberry Vaccinium darrowii | |
| CN116042637A (en) | Novel glufosinate-ammonium-resistant herbicide genetic locus for rice and application thereof | |
| CN116042638A (en) | Novel glufosinate-ammonium-resistant herbicide genetic locus for rice and application thereof | |
| CN115896084A (en) | Application of rice OsRHD1-3 gene in regulation and control of rice pollen fertility |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| A521 | Request for written amendment filed |
Free format text: JAPANESE INTERMEDIATE CODE: A523 Effective date: 20220401 |
|
| A621 | Written request for application examination |
Free format text: JAPANESE INTERMEDIATE CODE: A621 Effective date: 20220401 |
|
| A131 | Notification of reasons for refusal |
Free format text: JAPANESE INTERMEDIATE CODE: A131 Effective date: 20230425 |
|
| A521 | Request for written amendment filed |
Free format text: JAPANESE INTERMEDIATE CODE: A523 Effective date: 20230712 |
|
| A131 | Notification of reasons for refusal |
Free format text: JAPANESE INTERMEDIATE CODE: A131 Effective date: 20230808 |
|
| A521 | Request for written amendment filed |
Free format text: JAPANESE INTERMEDIATE CODE: A523 Effective date: 20231013 |
|
| TRDD | Decision of grant or rejection written | ||
| A01 | Written decision to grant a patent or to grant a registration (utility model) |
Free format text: JAPANESE INTERMEDIATE CODE: A01 Effective date: 20231024 |
|
| A61 | First payment of annual fees (during grant procedure) |
Free format text: JAPANESE INTERMEDIATE CODE: A61 Effective date: 20231107 |
|
| R150 | Certificate of patent or registration of utility model |
Ref document number: 7383136 Country of ref document: JP Free format text: JAPANESE INTERMEDIATE CODE: R150 |