Application of PP2C family gene in improving resistance of tomato to phytophthora infestans
By knocking out or silencing the PP2C family genes, the problem that the existing technology is difficult to effectively improve the resistance of tomatoes to pathogenic Phytophthora in the long term is solved, and the effect of significantly improving tomato resistance and not significantly affecting growth and development is achieved.
Patent Information
- Application Number
- CN202510032756.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-01-09
- Publication Date
- 2025-04-15
- Estimated Expiration
- Not applicable · inactive patent
AI Technical Summary
The prior art is difficult to effectively improve the resistance of tomatoes to Phytophthora pathogenic, and the research on the sensory genes has not yet been thorough.
By knocking out or silencing the PP2C family genes, and using CRISPR-Cas technology, T-DNA and transposon insertion and other methods, tomatoes with higher pathogenic Phytophthora resistance are prepared.
The resistance of tomatoes to pathogenic Phytophthora is significantly improved, the diameter and area of lesions are significantly reduced, and the relative biomass of Phytophthora is also significantly reduced, and it has little impact on the growth and development of tomatoes.
Smart Images
Figure CN119824003A_ABST
Abstract
Description
Technical Field
[0001] The invention relates to the technical field of molecular biological breeding, and in particular to the application of PP2C family genes in improving the resistance of tomatoes to Phytophthora infestans. Background Art
[0002] Phytophthora infestans de Bary is an oomycete that can cause late blight on tomatoes and potatoes. As one of the most destructive pathogens, it is the culprit of the Great Irish Famine in the 19th century. In the prevention and control of late blight, we rely on cultivation technology, breeding of resistant varieties and spraying of chemical pesticides. Among them, the breeding of resistant varieties is the main research direction of scientific research. At present, many genes that can improve the resistance of plants to late blight have been identified. However, due to the variation of physiological subspecies, the resistance genes cannot play a role for a long time, while the loss of susceptible gene function can enable plants to obtain broad-spectrum disease resistance. Therefore, studying the response of susceptible genes to late blight has become a new research idea. Compared with the study of resistance genes, the identification, pathogenic mechanism and function of susceptible genes need to be further explored. Summary of the invention
[0003] The purpose of the present invention is to overcome the deficiencies of the prior art and provide a target gene for improving the resistance of tomatoes to Phytophthora infestans and its application.
[0004] The first aspect of the present invention provides the use of PP2C family genes in improving the resistance of tomatoes to Phytophthora infestans.
[0005] Preferably, the above-mentioned PP2C family gene comprises a nucleotide sequence selected from the group consisting of:
[0006] 1) the genomic nucleotide sequence shown in SEQ ID NO: 1;
[0007] 2) the genomic nucleotide sequence shown in SEQ ID NO: 2;
[0008] 3) the coding region nucleotide sequence of SEQ ID NO: 1 shown in SEQ ID NO: 3;
[0009] 4) the coding region nucleotide sequence of SEQ ID NO: 2 shown in SEQ ID NO: 4;
[0010] 5) a nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 5;
[0011] 6) a nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 6;
[0012] 7) the promoter nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 3 shown in SEQ ID NO: 7;
[0013] 8) The promoter nucleotide sequence of SEQ ID NO:2 or SEQ ID NO:4 shown in SEQ ID NO:8.
[0014] A second aspect of the present invention provides an sgRNA, the nucleotide sequence of the sgRNA being shown in any one of SEQ ID NOs: 9-48.
[0015] Preferably, the sgRNA is designed based on the sequences shown in SEQ ID NO: 1 and SEQ ID NO: 2, and is used to specifically knock out or silence PP2C family genes.
[0016] The third aspect of the present invention provides a recombinant vector, which contains the above-mentioned sgRNA.
[0017] The fourth aspect of the present invention provides a transformant, which comprises the above-mentioned recombinant vector.
[0018] In some embodiments of the present invention, the transformant is transformed with the PP2C family gene or the above-mentioned vector.
[0019] A fifth aspect of the present invention provides the use of the above-mentioned sgRNA, the above-mentioned recombinant vector or the above-mentioned transformant in improving the resistance of tomatoes to Phytophthora infestans, wherein the sgRNA, vector or transformant specifically knocks out or silences a PP2C family gene, and the PP2C family gene is any one of SEQ ID NOs: 1-8.
[0020] A sixth aspect of the present invention provides a tomato or a tomato cell, wherein the tomato or the tomato cell is a tomato or a tomato cell of at least one of the following:
[0021] i) a tomato or tomato cell in which any one of the sequences shown in SEQ ID NO: 1-8 is knocked out or silenced;
[0022] ii) knocking out or silencing related genes in tomatoes or tomato cells using the above-mentioned sgRNA;
[0023] iii) a tomato or tomato cell comprising the above-mentioned recombinant vector;
[0024] iv) Tomato or a tomato cell comprising the above transformant.
[0025] A seventh aspect of the present invention provides a method for preparing tomatoes resistant to Phytophthora infestans, such that the target tomatoes low-express or do not express PP2C family genes; the PP2C family genes are shown in any one of SEQ ID NOs: 1-8.
[0026] In some embodiments of the present invention, gene editing technology is used to knock out or silence PP2C family genes in tomatoes; or gene mutation technology is used to low-express or not express the PP2C family genes in target tomatoes, so as to prepare tomatoes with resistance to Phytophthora infestans.
[0027] Preferably, the PP2C family gene is knocked out or silenced by using CRISPR-Cas technology, T-DNA and transposon insertion, chemical mutagenesis or physical mutagenesis. Chemical mutagenesis can be achieved by using ethyl methanesulfonate, and physical mutagenesis can be achieved by using high-energy rays or plasma.
[0028] In some embodiments of the present invention, the sgRNA for specifically knocking out or silencing the nucleotide sequence shown in SEQ ID NO: 1 or SEQ ID NO: 3 is any one of SEQ ID NOs: 9-28; the sgRNA for specifically knocking out or silencing the nucleotide sequence shown in SEQ ID NO: 2 or SEQ ID NO: 4 is any one of SEQ ID NOs: 29-48.
[0029] The beneficial effect of the present invention is to provide the application of PP2C family genes in improving the resistance of tomatoes to Phytophthora infestans. By knocking out or silencing the target gene, the edited or regulated tomato experimental strain has higher resistance to Phytophthora infestans than the wild type. At the same time, the growth and development of the tomato strain with the target gene knocked out or silenced is not significantly affected. BRIEF DESCRIPTION OF THE DRAWINGS
[0030] Figure 1 Schematic diagram of gene editing vector.
[0031] FIG2 is a schematic diagram of the nucleotide sequence of the editing site where a frameshift mutation occurs; Figure 2a The editing status of the target gene Solyc01G003420 is shown in Figure 2. Figure 2b This is the editing status of the target gene Solyc03G003340; the red part in Figure 2 represents the edited part.
[0032] Figure 3 Editing the target gene improved the resistance of tomatoes to Phytophthora infestans. Figure 3 a is the diameter of the lesion, Figure 3 b is the lesion area, Figure 3 c is the relative biomass of Phytophthora, Figure 3d is the image taken under UV irradiation; where, Figure 3 The data were analyzed by one-way analysis of variance and Duncan's method was used to test the significance of the difference (P<0.05).
[0033] Figure 4 The growth and development of tomato lines after editing the target gene were not affected; Figure 4 a is the tomato phenotype of the target gene Solyc01G003420 edited strain after 4 weeks of growth. Figure 4 b: Tomato phenotype of the target gene Solyc03G003340 edited line after 4 weeks of growth. DETAILED DESCRIPTION
[0034] The present invention provides two target genes Solyc01G003420 and Solyc03G003340 for improving the resistance of tomatoes to Phytophthora infestans, and both genes are members of the PP2C family gene. The genomic nucleotide sequence of the target gene Solyc01G003420 is shown in SEQ ID NO: 1, the coding region nucleotide sequence is shown in SEQ ID NO: 3, the protein amino acid sequence is shown in SEQ ID NO: 5, and the promoter nucleotide sequence is shown in SEQ ID NO: 7. The genomic nucleotide sequence of the target gene Solyc03G003340 is shown in SEQ ID NO: 2, the coding region nucleotide sequence is shown in SEQ ID NO: 4, the protein amino acid sequence is shown in SEQ ID NO: 6, and the promoter nucleotide sequence is shown in SEQ ID NO: 8.
[0035] The present invention also includes amino acid sequences with equivalent functions formed by replacing, deleting, or adding one or more amino acids in the amino acid sequence shown in the sequence listing SEQ ID NO: 5 or SEQ ID NO: 6 through artificial engineering or natural nucleic acid polymorphism, as well as nucleotide sequences encoding these amino acid sequences.
[0036] Through experiments, it was found that after knocking out the target gene in tomatoes (its sequence is shown in SEQ ID NO:1 or SEQ ID NO:2), the resistance of tomatoes to Phytophthora infestans increased significantly. The knockout and editing of the target gene can be achieved by CRISPR-Cas technology (and its derivative technologies, such as prime editing, etc.), T-DNA and transposon insertion, chemical mutagen treatment such as ethyl methanesulfonate, high-energy ray or plasma physical mutagenesis, etc. The gene mutation produced by the above method can be amplified by PCR method using oligonucleotide primers designed based on SEQ ID NO:1 or SEQ ID NO:2 sequence, and the amplified product can be verified by nucleotide sequencing. Based on the obtained sequencing results, it can be predicted whether the mutagenized allele can still encode the functional protein corresponding to the amino acid sequence shown in SEQ ID NO:3 or SEQ ID NO:4.
[0037] Below in conjunction with embodiment, technical scheme of the present invention is clearly and completely described, obviously, described embodiment is only a part of embodiment of the present invention, rather than all embodiments. Embodiment is carried out according to conventional experimental conditions or according to the condition suggested by manufacturer's specification. Unless otherwise defined, all professional terms used in the present invention have the same meaning as those skilled in the art are generally understood. In addition, any method and material similar or identical to the described content can be used in the inventive method, based on the embodiment in the present invention, all other embodiments obtained by those skilled in the art under the premise of not paying creative work, all belong to the scope of protection of the present invention.
[0038] The experimental methods used in the following examples were carried out according to conventional methods or those recommended by the manufacturer unless otherwise specified. The materials, reagents and consumables used were all commercially available unless otherwise specified.
[0039] In this embodiment, the tomato variety used is microtom, and other varieties of tomatoes may also be used in other embodiments.
[0040] Example 1. Construction of gene editing vector targeting target gene
[0041] Based on the target gene sequence shown in SEQ ID NO:1 or SEQ ID NO:2, select and design the appropriate gene editing target and synthesize the corresponding sgRNA (such as the sequence shown in SEQ ID NO:9-48 in the sequence list), and then clone it into a cloning vector containing the kanamycin (Kan) resistance gene, and then transfect it into the Escherichia coli strain. The Escherichia coli strain used in this embodiment is DH5α, and other suitable Escherichia coli strains can also be used in other embodiments. The transfected Escherichia coli is cultivated on LB solid medium containing kanamycin (concentration 50μg / mL), and the positive clones are selected and transferred to liquid LB medium for culture and expansion. Use a plasmid mini-extraction kit (TIANGEN:#DP103) to extract plasmid DNA, and recombinant the target fragment containing sgRNA into a plant binary expression vector containing a hygromycin resistance gene (hyg) and a Cas9 protein expression cassette. The schematic diagram of the gene editing vector is shown as follows Figure 1 As shown. The vector is transfected into a suitable Agrobacterium strain. The Agrobacterium strain used in this embodiment is GV3101. In other embodiments, EHA105 or other suitable Agrobacterium strains may also be used. Then, LB medium containing hygromycin (hyg) and kanamycin (Kan) (working concentrations are both 50 μg / mL) is used to screen and culture the positive strains that have been successfully transfected and expand them for subsequent Agrobacterium infection of explants.
[0042] Example 2: Transfection and screening of target gene-edited tomato lines
[0043] The transgenic Agrobacterium strain containing the plant binary expression vector constructed in Example 1 was co-cultured with tomato leaves that had been surface-sterilized and slightly rubbed to cause wounding under sterile conditions on Murashige-Skoog medium. The bud tissue differentiated after co-culture was transferred to a new MS medium containing hygromycin (hyg) for resistance screening. The resistant positive differentiated buds obtained by screening were transferred to a root-promoting medium, and after confirming that the root system had developed and colonized, they were taken out of the bottle and transplanted into nutrient soil for further identification and harvesting of T0 generation seeds.
[0044] The corresponding upstream and downstream amplification nucleotide primers were designed and synthesized for the different sgRNA target sequences used in Example 1, and the oligonucleotide amplification fragments containing the editing target site were obtained by PCR amplification. Sequencing the obtained fragments can verify whether the editing target has undergone a frameshift mutation (i.e., the addition or deletion of nucleotides that is not an integer multiple of 3). Figure 2 is an example of an editing site nucleotide sequence with a coding mutation. The gene-edited strains that have undergone frameshift mutations at the editing target site are determined as the desired positive strains, and the homozygous gene-edited plants are further screened for subsequent physiological and pathological index determination.
[0045] Example 3: Functional identification of tomato resistance to Phytophthora infestans
[0046] The lesions on the detached tomato leaves inoculated with the spore suspension of Phytophthora infestans for 5-7 days were counted and the lesion images were taken by irradiation with UV light.
[0047] Inoculation of phytophthora infestans: Take phytophthora infestans grown on oatmeal medium plates (grown for 2-3 weeks) and activate the spores with distilled water for about 3 hours (4°C). Then scrape the water off with a spreading stick and collect the spores. Centrifuge at 3000 rpm for 10 minutes to collect the spores. Discard the supernatant and keep an appropriate amount of spore suspension. Observe the number of spores under a microscope. About 50 spores are sufficient under a 40x microscope. Inoculate 10 μL of the bacteria on each leaf and treat in the dark at 28°C for 5-7 days.
[0048] The results of the test are as follows Figure 3 As shown in the figure, compared with the wild type, the leaves of the target gene Solyc01G003420 edited strain and the target gene Solyc03G003340 edited strain showed a significant decrease in lesion diameter and lesion area; the relative biomass of Phytophthora in the leaves was significantly reduced. The above conclusion was further verified by taking images through UV irradiation. In summary, the tomato strains after the target gene was knocked out have higher disease resistance to Phytophthora infestans than the wild type tomato strains.
[0049] Example 4: Determination of tomato growth and development indicators
[0050] Observe whether there is any difference in the growth phenotype between the target gene edited strain and the wild type. The target gene Solyc01G003420 edited strain and the target gene Solyc03G003340 edited strain were cultured under appropriate conditions for 4 weeks, and the wild type was used as a control. Figure 4 As shown in the figure, there is no significant difference in phenotype between the wild type and the target gene edited strains. In other words, the growth and development of the tomato strains after the target gene was edited were not affected.
[0051] The above is only a preferred embodiment of the present invention. It should be understood that the present invention is not limited to the form disclosed herein, and should not be regarded as excluding other embodiments, but can be used in various other combinations, modifications and environments, and can be modified within the scope of the concept described herein through the above teachings or the technology or knowledge of the relevant field. The changes and modifications made by those skilled in the art do not deviate from the spirit and scope of the present invention, and should be within the scope of protection of the claims attached to the present invention.
Claims
1. Application of PP2C family genes in improving tomato resistance to Phytophthora infestans.
2. The use according to claim 1, characterized in that: The PP2C family gene comprises any of the following groups of nucleotide sequences: 1) the genomic nucleotide sequence shown in SEQ ID NO: 1; 2) the genomic nucleotide sequence shown in SEQ ID NO: 2; 3) the coding region nucleotide sequence of SEQ ID NO: 1 shown in SEQ ID NO: 3; 4) the coding region nucleotide sequence of SEQ ID NO: 2 shown in SEQ ID NO: 4; 5) a nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 5; 6) a nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 6; 7) the promoter nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 3 shown in SEQ ID NO: 7; 8) The promoter nucleotide sequence of SEQ ID NO:2 or SEQ ID NO:4 shown in SEQ ID NO:
8.
3. A sgRNA, characterized in that The nucleotide sequence of the sgRNA is shown in any one of SEQ ID NOs: 9-48.
4. A recombinant vector, characterized in that: The vector comprises the sgRNA according to claim 3.
5. A transformant, characterized in that: The transformant comprises the recombinant vector according to claim 4.
6. Use of the sgRNA according to claim 3, the recombinant vector according to claim 4 or the transformant according to claim 5 in improving the resistance of tomatoes to Phytophthora infestans, characterized in that: The sgRNA, vector or transformant specifically knocks out or silences a PP2C family gene, wherein the PP2C family gene is any one of SEQ ID NOs: 1-8.
7. A tomato or tomato cell, characterized in that: The tomato or tomato cell is a tomato or tomato cell selected from at least one of the following: i) a tomato or tomato cell in which any one of the sequences shown in SEQ ID NO: 1-8 is knocked out or silenced; ii) Tomato or tomato cell for knocking out or silencing related genes using the sgRNA according to claim 3; iii) a tomato or a tomato cell comprising the recombinant vector according to claim 4; iv) a tomato or a tomato cell comprising the transformant according to claim 5.
8. A method for preparing tomatoes resistant to Phytophthora infestans, characterized in that: The target tomato is made to have low expression or no expression of a PP2C family gene, wherein the PP2C family gene is shown in any one of SEQ ID NOs: 1-8.
9. The method according to claim 8, characterized in that The PP2C family genes in tomatoes are knocked out or silenced using gene editing technology; or the PP2C family genes are low-expressed or not expressed in the target tomatoes using gene mutation technology.
10. The method according to claim 9, characterized in that The gene editing technology is at least one of CRISPR-Cas technology, T-DNA and transposon insertion, chemical mutagenesis, and physical mutagenesis.