WO2022151607A1 - Use of gene enhancement for tomato gray mold resistance - Google Patents
Use of gene enhancement for tomato gray mold resistance Download PDFInfo
- Publication number
- WO2022151607A1 WO2022151607A1 PCT/CN2021/087986 CN2021087986W WO2022151607A1 WO 2022151607 A1 WO2022151607 A1 WO 2022151607A1 CN 2021087986 W CN2021087986 W CN 2021087986W WO 2022151607 A1 WO2022151607 A1 WO 2022151607A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- gene
- tomato
- plants
- resistance
- seq
- Prior art date
Links
- 108090000623 proteins and genes Proteins 0.000 title claims abstract description 49
- 235000007688 Lycopersicon esculentum Nutrition 0.000 title claims abstract description 42
- 240000003768 Solanum lycopersicum Species 0.000 title claims description 47
- 238000000034 method Methods 0.000 claims abstract description 5
- 108091033409 CRISPR Proteins 0.000 claims description 25
- 241000123650 Botrytis cinerea Species 0.000 claims description 13
- 238000010354 CRISPR gene editing Methods 0.000 claims description 12
- 239000002773 nucleotide Substances 0.000 claims description 9
- 125000003729 nucleotide group Chemical group 0.000 claims description 9
- 230000009261 transgenic effect Effects 0.000 claims description 9
- 108091027544 Subgenomic mRNA Proteins 0.000 claims description 4
- 230000015572 biosynthetic process Effects 0.000 claims description 2
- 238000013461 design Methods 0.000 claims description 2
- 238000003786 synthesis reaction Methods 0.000 claims description 2
- 230000001131 transforming effect Effects 0.000 claims 1
- 208000035240 Disease Resistance Diseases 0.000 abstract description 5
- 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 18
- 238000012408 PCR amplification Methods 0.000 description 5
- 230000003902 lesion Effects 0.000 description 5
- 239000000203 mixture Substances 0.000 description 5
- 206010064571 Gene mutation Diseases 0.000 description 4
- 108091026890 Coding region Proteins 0.000 description 3
- 108020004414 DNA Proteins 0.000 description 3
- 235000013399 edible fruits Nutrition 0.000 description 3
- 238000003209 gene knockout Methods 0.000 description 3
- 239000000047 product Substances 0.000 description 3
- 238000012163 sequencing technique Methods 0.000 description 3
- 239000000243 solution Substances 0.000 description 3
- 239000006228 supernatant Substances 0.000 description 3
- 229910021642 ultra pure water Inorganic materials 0.000 description 3
- 239000012498 ultrapure water 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
- 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
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 239000008272 agar Substances 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 238000010276 construction Methods 0.000 description 2
- 201000010099 disease Diseases 0.000 description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 238000010362 genome editing Methods 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 230000035772 mutation Effects 0.000 description 2
- 239000000843 powder Substances 0.000 description 2
- 239000011550 stock solution Substances 0.000 description 2
- 239000000725 suspension Substances 0.000 description 2
- 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
- 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 description 1
- 241000894006 Bacteria Species 0.000 description 1
- VGGSQFUCUMXWEO-UHFFFAOYSA-N Ethene Chemical compound C=C VGGSQFUCUMXWEO-UHFFFAOYSA-N 0.000 description 1
- 239000005977 Ethylene Substances 0.000 description 1
- 101100278181 Homo sapiens DND1 gene 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
- 208000031888 Mycoses Diseases 0.000 description 1
- 238000000692 Student's t-test Methods 0.000 description 1
- 238000000137 annealing Methods 0.000 description 1
- 230000033228 biological regulation Effects 0.000 description 1
- 230000007123 defense Effects 0.000 description 1
- 238000004925 denaturation Methods 0.000 description 1
- 230000036425 denaturation Effects 0.000 description 1
- 239000000706 filtrate Substances 0.000 description 1
- 230000030279 gene silencing Effects 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 238000011081 inoculation Methods 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 230000002018 overexpression Effects 0.000 description 1
- 244000052769 pathogen Species 0.000 description 1
- 230000001717 pathogenic effect Effects 0.000 description 1
- 238000012257 pre-denaturation Methods 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 239000011780 sodium chloride Substances 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
- 235000013311 vegetables Nutrition 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 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
Definitions
- Figure 1 shows the CRISPR/Cas9 target site sequencing analysis of the tomato Solyc05g004600 gene mutant
Landscapes
- Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Organic Chemistry (AREA)
- Molecular Biology (AREA)
- Biotechnology (AREA)
- Biomedical Technology (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- Biophysics (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Plant Pathology (AREA)
- Physics & Mathematics (AREA)
- Cell Biology (AREA)
- Microbiology (AREA)
- Gastroenterology & Hepatology (AREA)
- Botany (AREA)
- Virology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Medicinal Chemistry (AREA)
- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
Abstract
The present invention relates to the field of crop disease resistance breeding, and provides a use of a gene for increasing tomato gray mold resistance. The use of a tomato gene Solyc05g004600 is provided, and the gene Solyc05g004600 is knocked out in the tomato, so that the resistance of the tomato leaf to the gray mold can be effectively increased. Also provided is a method for knocking out a mutant gene Solyc05g004600 in tomatoes.
Description
本发明涉及一个增加番茄灰霉病抗性基因的用途，属于作物抗病育种领域。The invention relates to an application for increasing tomato gray mold resistance gene, belonging to the field of crop disease resistance breeding.
番茄因其较高的食用、药用和经济价值，成为世界上重要的蔬果作物。灰霉病是番茄中常见且危害较为严重的真菌性病害，植株的茎、叶、花、果都可发病。通常病菌从植株的伤口或衰老组织中侵入，并逐渐蔓延到其它健康部位，对番茄的产量和品质造成巨大的影响。利用抗病基因防治作物的病害是最经济有效的办法。近年来，越来越多与番茄抗病相关的基因被挖掘，例如Yu等(2018)发现乙烯应答因子SlERF2参与了番茄对灰霉病的防御调控，过表达SlERF2能增强番茄果实对灰霉病的抗性；Sun等(2017)通过在番茄中沉默DND1基因，发现沉默植株与对照植株相比，灰霉病斑的直径显著减小，叶片上的灰霉病菌分生孢子数显著下降，菌丝生长受到抑制。可见，利用基因编辑技术，发掘更多植物抗病相关基因，是提高作物抗性、降低经济损失的一种有效手段。Tomato has become an important vegetable and fruit crop in the world because of its high edible, medicinal and economic value. Botrytis cinerea is a common and serious fungal disease in tomatoes, which can affect the stems, leaves, flowers and fruits of plants. Usually the pathogen invades from the wound or senescent tissue of the plant, and gradually spreads to other healthy parts, which has a huge impact on the yield and quality of tomato. The use of disease-resistant genes to control crop diseases is the most economical and effective way. In recent years, more and more genes related to tomato disease resistance have been excavated. For example, Yu et al. (2018) found that the ethylene-responsive factor SlERF2 is involved in the regulation of tomato defense against gray mold, and overexpression of SlERF2 can enhance tomato fruit resistance to gray mold By silencing the DND1 gene in tomato, Sun et al. (2017) found that compared with the control plants, the diameter of Botrytis cinerea lesions was significantly reduced, the number of Botrytis cinerea conidia on leaves was significantly reduced, and the bacteria Filament growth is inhibited. It can be seen that using gene editing technology to discover more genes related to plant disease resistance is an effective means to improve crop resistance and reduce economic losses.
发明内容SUMMARY OF THE INVENTION
本发明要解决的技术问题是如何有效提高番茄对灰霉病的抗性。The technical problem to be solved by the present invention is how to effectively improve the resistance of tomato to Botrytis cinerea.
为了解决上述技术问题，本发明提供一个番茄的基因Solyc05g004600，该基因Solyc05g004600的核苷酸序列如SEQ ID NO:1所示。In order to solve the above-mentioned technical problems, the present invention provides a tomato gene Solyc05g004600, and the nucleotide sequence of the gene Solyc05g004600 is shown in SEQ ID NO: 1.
本发明还同时提供了上述基因的用途：在番茄中敲除Solyc05g004600基因，可有效增加番茄叶片对灰霉病的抗性。The present invention also provides the use of the above gene: knocking out the Solyc05g004600 gene in tomato can effectively increase the resistance of tomato leaves to Botrytis cinerea.
作为本发明的番茄基因Solyc05g004600的用途的改进：As an improvement of the use of the tomato gene Solyc05g004600 of the present invention:
mu-1的Solyc05g004600基因的核苷酸序列如SEQ ID NO:2所示，The nucleotide sequence of the Solyc05g004600 gene of mu-1 is shown in SEQ ID NO:2,
mu-2的Solyc05g004600基因的核苷酸序列如SEQ ID NO:3所示。The nucleotide sequence of the Solyc05g004600 gene of mu-2 is shown in SEQ ID NO:3.
本发明还同时提供了番茄中敲除突变基因Solyc05g004600的方法，包括以下步骤：The present invention also provides a method for knocking out the mutant gene Solyc05g004600 in tomato, comprising the following steps:
1)、设计CRISPR/Cas9编辑的靶点序列sgRNA：1) Design the target sequence sgRNA for CRISPR/Cas9 editing:
5'-GTTGTTCAACATGAGCAGAG-3’；5'-GTTGTTCAACATGAGCAGAG-3';
2)、利用步骤1)所得的序列合成引物，并构建到CRISPR/Cas9载体中；2), utilize the sequence synthesis primer of step 1) gained, and build in CRISPR/Cas9 carrier;
3)、将步骤2)所得的载体转化番茄(野生型番茄品种MicroTom)，从而获得相应的转基因番茄植株(从所述转基因番茄植株中鉴定出Solyc05g004600基因突变的植株)：mu-1和 mu-2；所述转基因番茄植株为Solyc05g004600基因被敲除的番茄植株。3), transform the vector obtained in step 2) into tomato (wild-type tomato variety MicroTom), so as to obtain corresponding transgenic tomato plants (plants with Solyc05g004600 gene mutation were identified from the transgenic tomato plants): mu-1 and mu- 2. The transgenic tomato plant is a tomato plant in which the Solyc05g004600 gene has been knocked out.
基因Solyc05g004600序列虽然已登录在数据库中，但目前还没有该基因相关功能和作用的研究报道，因此本发明首次发现了该基因具有抗番茄灰霉菌的功能。Although the sequence of the gene Solyc05g004600 has been registered in the database, there is no research report on the related function and function of the gene. Therefore, the present invention discovers for the first time that the gene has the function of resisting tomato Botrytis cinerea.
本发明的技术方案具体如下：The technical scheme of the present invention is as follows:
采用CRISPR/Cas9基因编辑技术，根据Solyc05g004600基因的核苷酸序列(SEQ ID NO:1)，合成特异性靶向Solyc05g004600基因的sgRNA序列，构建相应的CRISPR/Cas9载体，并遗传转化到野生型番茄品种MicroTom中，使其对基因组中的Solyc05g004600基因进行定向编辑，获得转基因植株，对些转基因植株中Solyc05g004600基因进行PCR扩增与测序，鉴定获得了Solyc05g004600基因的2种不同类型的突变株系mu-1与mu-2(图1)。mu-1植株中的Solyc05g004600基因突变序列为SEQ ID NO:2；mu-2植株中的Solyc05g004600基因突变序列为SEQ ID NO:3。接种番茄的灰霉病菌后，野生型对照品种MicroTom发生感病，而Solyc05g004600基因突变的两个株系均提高了抗病性(图2)，其叶片的病斑面积显著性低于对照品种MicroTom(图3)，表明在番茄中突变Solyc05g004600基因能有效提高番茄对灰霉病的抗性。Using CRISPR/Cas9 gene editing technology, according to the nucleotide sequence of Solyc05g004600 gene (SEQ ID NO: 1), the sgRNA sequence specifically targeting Solyc05g004600 gene was synthesized, the corresponding CRISPR/Cas9 vector was constructed, and genetically transformed into wild-type tomato In the variety MicroTom, the Solyc05g004600 gene in the genome was directionally edited to obtain transgenic plants. PCR amplification and sequencing of the Solyc05g004600 gene in some transgenic plants were performed, and two different types of mutant lines mu- 1 and mu-2 (Fig. 1). The Solyc05g004600 gene mutation sequence in mu-1 plants is SEQ ID NO:2; the Solyc05g004600 gene mutation sequence in mu-2 plants is SEQ ID NO:3. After inoculation with Botrytis cinerea of tomato, the wild-type control variety MicroTom became susceptible, while the two lines with Solyc05g004600 gene mutation both improved the disease resistance (Fig. 2), and the lesion area of the leaves was significantly lower than that of the control variety MicroTom ( FIG. 3 ), indicating that the mutation of the Solyc05g004600 gene in tomato can effectively improve the resistance of tomato to Botrytis cinerea.
下面结合附图对本发明的具体实施方式作进一步详细说明。The specific embodiments of the present invention will be further described in detail below with reference to the accompanying drawings.
图1为番茄Solyc05g004600基因突变体的CRISPR/Cas9靶位点测序分析；Figure 1 shows the CRISPR/Cas9 target site sequencing analysis of the tomato Solyc05g004600 gene mutant;
MicroTom：野生型对照品种；mu-1与mu-2：Solyc05g004600基因2种不同类型的突变株系。MicroTom: wild-type control variety; mu-1 and mu-2: 2 different types of mutant lines of Solyc05g004600 gene.
图2为番茄Solyc05g004600基因突变植株与野生型对照MicroTom叶片接灰霉菌表型对比图。Figure 2 is a graph showing the comparison of the phenotype of Botrytis cinerea in the leaves of the tomato Solyc05g004600 gene mutant plant and the wild-type control MicroTom.
图3为番茄Solyc05g004600基因突变植株与野生型对照MicroTom叶片病斑统计；Figure 3 shows the statistics of leaf lesions of the tomato Solyc05g004600 gene mutant plants and the wild-type control MicroTom;
图3中的数值为平均值±标准差，**表示在番茄Solyc05g004600基因突变植株与野生型对照MicroTom比较中，t检验存在极显著性(P<0.01)差异。The values in Figure 3 are the mean ± standard deviation, ** indicates that there is a very significant (P<0.01) difference in the t-test between the tomato Solyc05g004600 gene mutant plants and the wild-type control MicroTom.
下面结合具体实施例对本发明进行进一步描述，但本发明的保护范围并不仅限于此：The present invention is further described below in conjunction with specific embodiment, but the protection scope of the present invention is not limited to this:
实施例1：Example 1:
步骤1、Solyc05g004600基因敲除载体的构建 Step 1. Construction of Solyc05g004600 gene knockout vector
根据Solyc05g004600基因的编码序列(SEQ ID NO:1)，利用CRISPR/Cas9靶点分析软件(http://crispr.mit.edu/)，在Solyc05g004600基因中设计CRISPR/Cas9编辑的sgRNA序列： 5'-GTTGTTCAACATGAGCAGAG-3'；并根据该序列合成相应的引物：According to the coding sequence of the Solyc05g004600 gene (SEQ ID NO: 1), the CRISPR/Cas9 edited sgRNA sequence was designed in the Solyc05g004600 gene using CRISPR/Cas9 target analysis software (http://crispr.mit.edu/): 5' - GTTGTTCAACATGAGCAGAG-3'; and synthesize the corresponding primers according to this sequence:
上游5'-TGATTGTTGTTCAACATGAGCAGAG-3'，upstream 5'-TGATTGTTGTTCAACATGAGCAGAG-3',
下游5'-AAACCTCTGCTCATGTTGAACAACA-3'。Downstream 5'-AAACCTCTGCTCATGTTGAACAACA-3'.
然后采用CRISPR/Cas9试剂盒(Biogle，China)构建相应的CRISPR/Cas9载体，构建方法按照产品说明进行操作。Then, the corresponding CRISPR/Cas9 vector was constructed using the CRISPR/Cas9 kit (Biogle, China), and the construction method was operated according to the product instructions.
步骤2、Solyc05g004600基因敲除载体的番茄遗传转化 Step 2. Tomato genetic transformation of Solyc05g004600 gene knockout vector
将步骤1构建的CRISPR/Cas9载体遗传转化到番茄品种MicroTom，转基因方法根据采用Kimura等方法(Kimura S et al,CHS Protoc,2008)，获得一系列相应的未鉴定转基因番茄植株。The CRISPR/Cas9 vector constructed in step 1 was genetically transformed into the tomato variety MicroTom, and a series of corresponding unidentified transgenic tomato plants were obtained according to the method of Kimura et al. (Kimura S et al, CHS Protoc, 2008).
步骤3、Solyc05g004600基因敲除植株的鉴定 Step 3. Identification of Solyc05g004600 knockout plants
取番茄叶片0.1g，用液氮研磨后，加600μl提取液(15.76gTris-cl，29.22gNacl，15.0gSDS粉末加超纯水定容至1L，调节pH＝8.0)，65℃温育60min；加200μl 5M KAC，混匀后，冰浴10min；再加500μl氯仿，混匀，10000rpm离心5min；取上清，加入500μl异丙醇，混匀，12000rpm离心3min，弃去上清；用75％乙醇洗涤沉淀，12000rpm离心3min，弃去上清；倒置室温风干DNA后，加30μl纯水溶解DNA。Take 0.1 g of tomato leaves, grind with liquid nitrogen, add 600 μl of extract (15.76 g Tris-cl, 29.22 g NaCl, 15.0 g SDS powder, add ultrapure water to make the volume to 1 L, adjust pH=8.0), incubate at 65°C for 60 min; add 200μl 5M KAC, after mixing, ice bath for 10min; add 500μl chloroform, mix well, centrifuge at 10000rpm for 5min; take the supernatant, add 500μl isopropanol, mix well, centrifuge at 12000rpm for 3min, discard the supernatant; use 75% ethanol The precipitate was washed, centrifuged at 12,000 rpm for 3 min, and the supernatant was discarded; after the DNA was air-dried at room temperature by inversion, 30 μl of pure water was added to dissolve the DNA.
合成Solyc05g004600基因PCR扩增的引物：上游5'-GCCTCTATTAATGACATC-3'，下游5'-ATAAGCTAAAATCGACTA-3'，以转基因番茄植株及其对照品种MicroTom的基因组DNA为模板，用
Master Mix(Promega,USA)进行PCR扩增，PCR扩增体系按照产品的说明书操作；
The primers for the PCR amplification of the Solyc05g004600 gene were synthesized: upstream 5'-GCCTCTATTAATGACATC-3', downstream 5'-ATAAGCTAAAATCGACTA-3', using the genomic DNA of the transgenic tomato plant and its control variety MicroTom as the template, using Master Mix (Promega, USA) was used for PCR amplification, and the PCR amplification system was operated according to the product instructions;
PCR扩增程序为：94℃预变性5min；94℃变性30sec，55℃退火30sec，72℃延伸30sec，35个循环；72℃延伸10min。The PCR amplification program was as follows: pre-denaturation at 94°C for 5 min; denaturation at 94°C for 30 sec, annealing at 55°C for 30 sec, extension at 72°C for 30 sec, 35 cycles; extension at 72°C for 10 min.
PCR扩增体系为：2×Mix10μl，上游引物0.5μl，下游引物0.5μl，模板2μl，ddH
2O 7μl。
The PCR amplification system is: 2×Mix 10 μl, upstream primer 0.5 μl, downstream primer 0.5 μl, template 2 μl, ddH 2 O 7 μl.
PCR产物经测序分析后，成功鉴定了Solyc05g004600基因的2种突变类型：mu-1与mu-2。在mu-1植株中，Solyc05g004600基因编码区插入1个碱基(图1)，其核苷酸序列为SEQ ID NO:2；在mu-2植株中，Solyc05g004600基因编码区缺失了3个碱基(图1)，其核苷酸序列为SEQ ID NO:3。After sequencing and analysis of PCR products, two mutation types of Solyc05g004600 gene were successfully identified: mu-1 and mu-2. In the mu-1 plant, 1 base was inserted into the coding region of the Solyc05g004600 gene (Figure 1), and its nucleotide sequence was SEQ ID NO: 2; in the mu-2 plant, the coding region of the Solyc05g004600 gene was deleted by 3 bases (Fig. 1), its nucleotide sequence is SEQ ID NO:3.
步骤4、Solyc05g004600基因敲除植株的灰霉菌抗性鉴定Step 4. Identification of Botrytis cinerea resistance of Solyc05g004600 gene knockout plants
将以上鉴定出的Solyc05g004600基因突变株系mu-1、mu-2以及对照品种MicroTom种植在温室，25℃，16h光照，8h黑暗。待生长45天左右，mu-1、mu-2以及对照品种随机选取3株，每株取4片叶子固定在0.8％的琼脂(8g琼脂粉加超纯水定容至1L)培养皿上。挑取大 约2ml体积的灰霉菌，放入5ml1％的SMB(10g霉菌蛋白胨，40g麦芽糖加超纯水定容至1L)溶液中震荡混匀，过滤，所得滤液即为孢子原液。使用25×16规格的血细胞计数板计算孢子原液浓度，用1％的SMB溶液稀释，直到25个中格中，每个中格大约有16个孢子，接种时再次稀释4倍，稀释后所得即为孢子悬浮液。以无孢子的1％SMB溶液为对照，每张叶片滴加5μl孢子悬浮液，加盖置于适于番茄生长的环境中。2天后，摘取接菌叶片，使用Image J软件计算病斑面积。所得结果为Solyc05g004600基因突变株系mu-1、mu-2的叶片感病程度要低于对照品种(图2)，叶片病斑面积显著小于对照品种(图3)。The Solyc05g004600 gene mutant lines identified above, mu-1, mu-2, and the control variety MicroTom were grown in a greenhouse at 25°C, 16 hours of light, and 8 hours of darkness. After about 45 days of growth, 3 plants of mu-1, mu-2 and control varieties were randomly selected, and 4 leaves of each plant were fixed on 0.8% agar (8g agar powder and ultrapure water to 1L) petri dish. Pick about 2ml of Botrytis cinerea, put into 5ml of 1% SMB (10g of mycopeptone, 40g of maltose and ultrapure water to dilute to 1L) solution, shake and mix well, filter, and the obtained filtrate is the spore stock solution. Use a 25 × 16 hemocytometer to calculate the concentration of the spore stock solution, and dilute it with 1% SMB solution until there are about 16 spores in 25 medium cells. When inoculating, dilute 4 times again. for spore suspension. Using 1% SMB solution without spores as a control, 5 μl of spore suspension was added dropwise to each leaf, and the leaves were covered and placed in an environment suitable for tomato growth. After 2 days, the inoculated leaves were picked, and the lesion area was calculated using Image J software. The results obtained showed that the leaf susceptibility of Solyc05g004600 gene mutant lines mu-1 and mu-2 was lower than that of the control variety (Fig. 2), and the leaf lesion area was significantly smaller than that of the control variety (Fig. 3).
根据上述内容能证明：Solyc05g004600基因被敲除的番茄植株mu-1、mu-2的叶片对有效增加对灰霉菌的抗性。According to the above content, it can be proved that the leaves of the tomato plants mu-1 and mu-2 in which the Solyc05g004600 gene has been knocked out can effectively increase the resistance to Botrytis cinerea.
最后，还需要注意的是，以上列举的仅是本发明的若干个具体实施例。显然，本发明不限于以上实施例，还可以有许多变形。本领域的普通技术人员能从本发明公开的内容直接导出或联想到的所有变形，均应认为是本发明的保护范围。Finally, it should also be noted that the above enumeration is only a few specific embodiments of the present invention. Obviously, the present invention is not limited to the above embodiments, and many modifications are possible. All modifications that those of ordinary skill in the art can directly derive or associate from the disclosure of the present invention shall be considered as the protection scope of the present invention.
Claims (4)
- 番茄基因Solyc05g004600，其特征是：该基因的核苷酸序列如SEQ ID NO：1所示。The tomato gene Solyc05g004600 is characterized in that: the nucleotide sequence of the gene is shown in SEQ ID NO: 1.
- 如权利要求1所述的番茄基因Solyc05g004600的用途，其特征是：在番茄中敲除基因Solyc05g004600，能有效增加番茄叶片对灰霉菌的抗性。The use of the tomato gene Solyc05g004600 according to claim 1, characterized in that: knocking out the gene Solyc05g004600 in tomato can effectively increase the resistance of tomato leaves to Botrytis cinerea.
- 根据权利要求2所述的番茄基因Solyc05g004600的用途，其特征是：The purposes of tomato gene Solyc05g004600 according to claim 2, is characterized in that:mu-1的Solyc05g004600基因的核苷酸序列如SEQ ID NO:2所示，The nucleotide sequence of the Solyc05g004600 gene of mu-1 is shown in SEQ ID NO:2,mu-2的Solyc05g004600基因的核苷酸序列如SEQ ID NO:3所示。The nucleotide sequence of the Solyc05g004600 gene of mu-2 is shown in SEQ ID NO:3.
- 番茄中敲除突变基因Solyc05g004600的方法，其特征是包括以下步骤：The method for knocking out mutant gene Solyc05g004600 in tomato is characterized by comprising the following steps:1)、设计CRISPR/Cas9编辑的靶点序列sgRNA：1) Design the target sequence sgRNA for CRISPR/Cas9 editing:5'-GTTGTTCAACATGAGCAGAG-3’；5'-GTTGTTCAACATGAGCAGAG-3';2)、利用步骤1)所得的序列合成引物，并构建到CRISPR/Cas9载体中；2), utilize the sequence synthesis primer of step 1) gained, and build in CRISPR/Cas9 carrier;3)、将步骤2)所得的载体转化番茄，从而获得相应的转基因番茄植株：mu-1和mu-2；所述转基因番茄植株为Solyc05g004600基因被敲除的番茄植株。3), transforming the vector obtained in step 2) into tomato to obtain corresponding transgenic tomato plants: mu-1 and mu-2; the transgenic tomato plants are tomato plants with the Solyc05g004600 gene knocked out.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2022520494A JP7383136B2 (en) | 2021-01-17 | 2021-04-19 | Use of genes to improve tomato Botrytis blight resistance |
Applications Claiming Priority (2)
| 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 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| WO2022151607A1 true WO2022151607A1 (en) | 2022-07-21 |
Family
ID=75368393
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| PCT/CN2021/087986 WO2022151607A1 (en) | 2021-01-17 | 2021-04-19 | Use of gene enhancement for tomato gray mold 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 (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103667265A (en) * | 2012-09-06 | 2014-03-26 | 中国科学院遗传与发育生物学研究所 | Application of Lox (lipoxygenase) D gene of tomato in regulating and controlling plant resistance |
| 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 |
| CN110734481A (en) * | 2019-11-12 | 2020-01-31 | 中国农业大学 | Application of tomato SlMIP protein and coding gene thereof in regulation and control of plant gray mold resistance |
| CN111118039A (en) * | 2020-02-07 | 2020-05-08 | 浙江大学 | Pathogenicity-related botrytis cinerea genes Bcmet3 and Bcmet16 and application |
| CN111635908A (en) * | 2020-05-11 | 2020-09-08 | 青岛农业大学 | Application of KDA2 gene in improving resistance of plants to botrytis cinerea infection |
| CN112646819A (en) * | 2021-01-17 | 2021-04-13 | 浙江师范大学 | Use of gene to enhance resistance to tomato gray mold |
Family Cites Families (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| 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 |
-
2021
- 2021-01-17 CN CN202110058943.XA patent/CN112646819B/en active Active
- 2021-04-19 JP JP2022520494A patent/JP7383136B2/en active Active
- 2021-04-19 WO PCT/CN2021/087986 patent/WO2022151607A1/en active Application Filing
Patent Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103667265A (en) * | 2012-09-06 | 2014-03-26 | 中国科学院遗传与发育生物学研究所 | Application of Lox (lipoxygenase) D gene of tomato in regulating and controlling plant resistance |
| 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 |
| CN110734481A (en) * | 2019-11-12 | 2020-01-31 | 中国农业大学 | Application of tomato SlMIP protein and coding gene thereof in regulation and control of plant gray mold resistance |
| CN111118039A (en) * | 2020-02-07 | 2020-05-08 | 浙江大学 | Pathogenicity-related botrytis cinerea genes Bcmet3 and Bcmet16 and application |
| CN111635908A (en) * | 2020-05-11 | 2020-09-08 | 青岛农业大学 | Application of KDA2 gene in improving resistance of plants to botrytis cinerea infection |
| CN112646819A (en) * | 2021-01-17 | 2021-04-13 | 浙江师范大学 | Use of gene to enhance resistance to tomato gray mold |
Non-Patent Citations (2)
| Title |
|---|
| DATABASE NUCLEOTIDE 8 August 2018 (2018-08-08), ANONYMOUS : "PREDICTED: Solanum lycopersicum MATH and LRR domain-containing protein PFE0570w (LOC101259009), mRNA", XP055950987, retrieved from NCBI Database accession no. XM_004249477 * |
| SHU PAN, LI ZIYE, MIN DEDONG, ZHANG XINHUA, AI WEN, LI JIAOZHUO, ZHOU JINGXIANG, LI ZILONG, LI FUJUN, LI XIAOAN: "CRISPR/Cas9-Mediated SlMYC2 Mutagenesis Adverse to Tomato Plant Growth and MeJA-Induced Fruit Resistance to Botrytis cinerea", JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY, AMERICAN CHEMICAL SOCIETY, US, vol. 68, no. 20, 20 May 2020 (2020-05-20), US , pages 5529 - 5538, XP055950985, ISSN: 0021-8561, DOI: 10.1021/acs.jafc.9b08069 * |
Also Published As
| Publication number | Publication date |
|---|---|
| JP7383136B2 (en) | 2023-11-17 |
| CN112646819A (en) | 2021-04-13 |
| CN112646819B (en) | 2023-04-18 |
| JP2023513862A (en) | 2023-04-04 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| WO2022151607A1 (en) | Use of gene enhancement for tomato gray mold resistance | |
| US20240158787A1 (en) | USE OF miRNA408 IN REGULATION OF CADMIUM ACCUMULATION IN CROP | |
| CN114457106B (en) | Application of tomato gene SlCIPK7 in regulation and control of drought resistance of plants | |
| CN116479038B (en) | Method for obtaining non-transgenic quinoa with increased fragrance | |
| CN105420273A (en) | Method for cultivating transgenic plants with blooming ahead of time | |
| CN109161552B (en) | Gene for increasing seed yield of arabidopsis thaliana and use thereof | |
| CN107698672B (en) | Rice male sterility related protein and coding gene and application thereof | |
| CN110468138B (en) | Gene TSG2 for controlling cold resistance of rice and application thereof | |
| CN104946652B (en) | Plant gynoecium specific expression promoter OsPis1 and its derivative | |
| CN115942868A (en) | Cannabis plant with increased yield | |
| CN108668884A (en) | The method for formulating ornamental type rice germplasm using two kinds of transcription factor genes | |
| CN116041466B (en) | Barley grain black character HvBlp gene and related molecular marker and application thereof | |
| CN114478732B (en) | Application of rice MST6 gene and homologous gene MST3 thereof in regulation and control of plant branching or tillering | |
| CN103665129A (en) | Plant heading period associated protein TaMYB72 and application thereof | |
| CN112126633B (en) | Tomato cyclin dependent kinase SlCDK8 gene and application thereof | |
| CN116554292B (en) | Negative regulation plant broad-spectrum disease-resistant protein, and coding gene and application thereof | |
| CN104540943A (en) | Parthenocarpy regulation gene and use thereof | |
| CN117821503B (en) | Application of OsWD gene 40-115 gene in regulation and control of rice quality | |
| CN114181951B (en) | Corn sheath blight disease resistance related gene Zmbzip45 and application thereof | |
| CN117304288B (en) | Rice tillering angle related protein OsITAND, coding gene and application thereof | |
| CN114164228B (en) | Method for improving disease resistance of rice through gene editing and application thereof | |
| CN116064567B (en) | Corn small kernel mutant and application thereof | |
| CN115094072B (en) | Populus tomentosa PtYABBY7 gene and application thereof | |
| WO2021193865A1 (en) | Method for producing temperature-sensitive male sterile plant | |
| US20230235348A1 (en) | Control of meiotic crossover in maize |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| ENP | Entry into the national phase |
Ref document number: 2022520494 Country of ref document: JP Kind code of ref document: A |
|
| 121 | Ep: the epo has been informed by wipo that ep was designated in this application |
Ref document number: 21918806 Country of ref document: EP Kind code of ref document: A1 |
|
| NENP | Non-entry into the national phase |
Ref country code: DE |
|
| 122 | Ep: pct application non-entry in european phase |
Ref document number: 21918806 Country of ref document: EP Kind code of ref document: A1 |