Application of RIN4 family gene in improving resistance of tomato to phytophthora infestans

Abstract

The invention belongs to the technical field of molecular biology breeding, and particularly relates to application of RIN4 family genes in improving resistance of tomatoes to phytophthora infestans. Two genes related to phytophthora infestans infection are found and verified in tomatoes, the genome nucleotide sequences of the two genes are shown as SEQ ID NO: 1 and SEQ ID NO: 2 respectively, and the two genes belong to RIN4 gene family members. The gene is knocked out or silenced through a gene editing technology, and it is found that the tomato strain with the gene knocked out or silenced has higher phytophthora infestans resistance compared with a wild type. Meanwhile, tomato strains edited by the genes are not obviously influenced in the aspects of growth and development. Therefore, the invention not only enriches the research of phytophthora infestans susceptibility related genes in tomatoes, but also develops a tomato strain with enhanced phytophthora infestans resistance by utilizing the characteristics of the genes, and provides a new technical strategy and gene resources for tomato breeding.

Description

Technical Field

[0001] The invention relates to the technical field of molecular biological breeding, and in particular to application of RIN4 family genes in improving resistance of tomatoes to Phytophthora infestans. Background Art

[0002] Phytophthora infestans de Bary is an oomycete that can cause late blight of 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 methods of late blight, cultivation techniques, breeding of resistant varieties and chemical pesticide spraying are relied on. Among them, breeding of resistant varieties is the main research direction of current scientific research. At present, many genes that can improve the resistance of plants to late blight have been identified, but due to the variation of physiological races, the resistance genes cannot play a role for a long time, and the loss of the function of the susceptible gene can enable the plant to obtain a broad spectrum of disease resistance. Therefore, the present invention starts with the relationship between the susceptible gene and late blight, analyzes the mechanism of tomato susceptibility at the molecular level, and thus improves the disease resistance of tomatoes, which becomes a new research idea. 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 RIN4 family genes in improving the resistance of tomatoes to Phytophthora infestans.

[0005] Preferably, the RIN4 family gene comprises a nucleotide sequence of any of the following groups:

[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] The third aspect of the present invention provides a recombinant vector, which comprises the above-mentioned sgRNA.

[0016] The fourth aspect of the present invention provides a transformant, which comprises the above-mentioned recombinant vector.

[0017] In some embodiments of the present invention, the transformant is transformed with the RIN4 family gene or the above-mentioned vector.

[0018] The 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 RIN4 family gene, and the RIN4 family gene is any one of SEQ ID NOs: 1-8.

[0019] 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 selected from at least one of the following:

[0020] 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;

[0021] ii) knocking out or silencing related genes in tomatoes or tomato cells using the above-mentioned sgRNA;

[0022] iii) a tomato or tomato cell comprising the above-mentioned recombinant vector;

[0023] iv) Tomato or a tomato cell comprising the above transformant.

[0024] 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 RIN4 family genes; the RIN4 family genes are shown in any one of SEQ ID NOs: 1-8.

[0025] In some embodiments of the present invention, gene editing technology is used to knock out or silence RIN4 family genes in tomatoes; or gene mutation technology is used to low-express or not express the RIN4 family genes in target tomatoes, so as to prepare tomatoes with resistance to Phytophthora infestans.

[0026] Preferably, the RIN4 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.

[0027] 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.

[0028] The beneficial effect of the present invention is that it provides the use of two RIN4 family genes in improving the resistance of tomatoes to Phytophthora infestans. Tobacco transiently expressing the target gene has a higher susceptibility to the disease than the control group; and the tomato experimental strain after the target gene is knocked out or silenced has a higher resistance to Phytophthora infestans than the wild type, and at the same time, the tomato strain with the target gene knocked out or silenced is not significantly affected in growth and development. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 Schematic diagram of gene editing vector.

[0030] 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 Solyc04G001683 is shown in Figure 2. Figure 2b This is the editing status of the target gene Solyc04G000477; the red part in Figure 2 represents the edited part.

[0031] Figure 3 To transiently express the target gene to increase susceptibility to disease; Figure 3 a is the diameter of the lesion, Figure 3 b UV irradiation. One-way ANOVA and Duncan's test were used to test the significant difference (P<0.05).

[0032] Figure 4 The target gene was edited to improve the resistance of tomatoes to Phytophthora infestans. Figure 4 a is the diameter of the lesion, Figure 4 b is the lesion area, Figure 4 c is the relative biomass of Phytophthora, Figure 4 d is the image taken by UV irradiation. One-way analysis of variance and Duncan's method were used to test the significant difference (P<0.05).

[0033] Figure 5 The growth and development of tomato lines after editing the target gene were not affected. Figure 5 a is the tomato phenotype of the target gene Solyc04G001683 edited strain after 4 weeks of growth. Figure 5 b: Tomato phenotype of the target gene Solyc04G000477 edited line after 4 weeks of growth. DETAILED DESCRIPTION

[0034] The present invention provides two target genes Solyc04G001683 and Solyc04G000477 for improving the resistance of tomatoes to Phytophthora infestans, and both genes are members of the RIN4 family gene. Among them, the genomic nucleotide sequence of the target gene Solyc04G001683 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 nucleotide sequence of the promoter is shown in SEQ ID NO: 7. The genomic nucleotide sequence of the target gene Solyc04G000477 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 nucleotide sequence of the promoter 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] In some embodiments of the present invention, after knocking out the target gene in tomatoes (whose sequence is shown in SEQ ID NO: 1 or SEQ ID NO: 2), the resistance of tomatoes to Phytophthora infestans increases 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 using oligonucleotide primers designed based on SEQ ID NO: 1 or SEQ ID NO: 2 sequence, and the amplified product is 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 an 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 cultured 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, Tobacco transient expression

[0046] The transgenic Agrobacterium containing the plant binary expression vector constructed in Example 1 was resuspended in tobacco resuspension and placed in the dark for about 3 hours to allow it to fully adsorb. The treated Agrobacterium was then injected into tobacco leaves. The lesions of the isolated tobacco leaves that were inoculated with the spore suspension of Phytophthora infestans for 5-7 days were counted, and the lesion images were taken using UV light.

[0047] Test results such as Figure 3 As shown in the figure, compared with the control group, tobacco leaves transiently expressing the target gene Solyc04G001683 and tobacco leaves transiently expressing the target gene Solyc04G000477 showed a significant increase in lesion diameter; UV irradiation images also showed that tobacco transiently expressing the target gene showed obvious lesions. The above results show that under controlled environmental conditions, tobacco transiently expressing the target gene has a higher susceptibility to disease than the control group.

[0048] Example 4: Functional identification of tomato resistance to Phytophthora infestans

[0049] 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.

[0050] 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.

[0051] Test results such as Figure 4 As shown in the figure, compared with the wild type, the tomato leaves of the target gene Solyc04G001683 edited strain and the target gene Solyc04G000477 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.

[0052] Example 5: Determination of tomato growth and development indicators

[0053] Observe whether there is any difference in the growth phenotype between the target gene edited strain and the wild type. The target gene Solyc04G001683 edited strain and the target gene Solyc04G000477 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.

[0054] 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 shall not deviate from the spirit and scope of the present invention, and shall be within the scope of protection of the claims attached to the present invention.

Claims

1. Application of RIN4 family genes in improving tomato resistance to Phytophthora infestans.

2. The use according to claim 1, characterized in that: The RIN4 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 RIN4 family gene, and the RIN4 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 RIN4 family gene, wherein the RIN4 family gene is shown in any one of SEQ ID NOs: 1-8.

9. The method according to claim 8, characterized in that Gene editing technology is used to knock out or silence the RIN4 family genes in tomatoes; or gene mutation technology is used to make the RIN4 family genes low-expressed or not expressed in the target tomatoes.

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.

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