Application of EF Hand family genes in improving tomato resistance to Phytophthora infestans

Abstract

The application belongs to the technical field of molecular biology breeding, and particularly relates to application of EF Hand family genes in improving resistance of tomatoes to pathogenic Phytophthora. Two pathogenic Phytophthora disease-related genes are found and verified in tomatoes, the genomic nucleotide sequences of which are shown as SEQ ID NO: 1 and SEQ ID NO: 2 respectively, and the two genes both belong to members of the EF Hand gene family. By knocking out or silencing the above genes through gene editing technology, it is found that the tomato lines with the above genes knocked out or silenced have higher resistance to pathogenic Phytophthora compared with wild types. Meanwhile, the gene-edited tomato lines are not obviously affected in growth and development. Therefore, the application not only enriches the research on pathogenic Phytophthora disease-related genes in tomatoes, but also develops tomato lines with enhanced resistance to pathogenic Phytophthora by using the characteristics of the above genes, thereby providing new technical strategies and gene resources for tomato breeding.

Description

Technical Field

[0001] This invention relates to the field of molecular biology breeding technology, specifically to the application of EF Hand family genes in improving the resistance of tomatoes to pathogenic Phytophthora. Background Technology

[0002] *Phytophthora infestans de Bary* is an oomycete that causes late blight in tomatoes and potatoes. As one of the most destructive pathogens, it was a major contributor to the Irish famine of the 19th century. Control methods for late blight rely on cultivation techniques, the breeding of resistant varieties, and the application of chemical pesticides. Among these, the breeding of resistant varieties is currently a major research focus. Many genes that can enhance plant resistance to late blight have been identified, but variations in physiological races prevent these resistance genes from functioning long-term, while the loss of function in susceptibility genes can result in broad-spectrum resistance. Therefore, studying the response of susceptibility genes to late blight has become a new research approach. Compared to the study of resistance genes, the identification, pathogenic mechanisms, and functional studies of susceptibility genes require further in-depth exploration. Summary of the Invention

[0003] The purpose of this invention is to overcome the shortcomings of the prior art and provide target genes for improving the resistance of tomatoes to Phytophthora infestans and their applications.

[0004] The first aspect of the present invention provides the application of EF Hand family genes in improving the resistance of tomatoes to Phytophthora infestans.

[0005] Preferably, the EF Hand family gene comprises any of the following groups of nucleotide sequences:

[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 which is shown in any one of SEQ ID NO:9-48.

[0015] A third aspect of the present invention provides a recombinant vector comprising the above-described sgRNA.

[0016] A fourth aspect of the present invention provides a transformant comprising the recombinant vector described above.

[0017] In some embodiments of the present invention, the transformant is transformed using EF Hand family genes or the vectors described above.

[0018] The fifth aspect of the present invention provides the application of the above-mentioned sgRNA, the above-mentioned recombinant vector or the above-mentioned transformant in improving the resistance of tomatoes to Phytophthora infectica, wherein the sgRNA, vector or transformant specifically knocks out or silences EF Hand family genes, and the EF Hand family genes are any one of those shown in SEQ ID NO:1–8.

[0019] A sixth aspect of the present invention provides a tomato or tomato cell, said tomato or tomato cell being at least one of the following:

[0020] i) Knock out or silence any of the sequences shown in SEQ ID NO:1–8 in tomatoes or tomato cells;

[0021] ii) Tomato or tomato cells that use the above-mentioned sgRNA to knock out or silence related genes;

[0022] iii) Tomatoes or tomato cells containing the recombinant vector described above;

[0023] iv) Tomatoes or tomato cells containing the above-mentioned transformants.

[0024] The seventh aspect of the present invention provides a method for preparing tomatoes resistant to Phytophthora infectans, wherein the target tomato expresses low or no EF Hand family genes; the EF Hand family genes are shown in any one of SEQ ID NO:1–8.

[0025] In some embodiments of the present invention, gene editing technology is used to knock out or silence the EF Hand family gene in tomatoes; or gene mutation technology is used to make the target tomato express low or no expression of the EF Hand family gene, so as to prepare tomatoes with resistance to pathogenic Phytophthora.

[0026] Preferably, the EF Hand family gene is knocked out or silenced using CRISPR-Cas technology, T-DNA and transposon insertion, chemical mutagenesis, or physical mutagenesis. Chemical mutagenesis can be achieved using ethyl methanesulfonate, while physical mutagenesis can be achieved using high-energy radiation or plasma.

[0027] In some embodiments of the present invention, sgRNAs for specifically knocking out or silencing nucleotide sequences such as those shown in SEQ ID NO:1 or SEQ ID NO:3 are any one of those shown in SEQ ID NO:9-28; sgRNAs for specifically knocking out or silencing nucleotide sequences such as those shown in SEQ ID NO:2 or SEQ ID NO:4 are any one of those shown in SEQ ID NO:29-48.

[0028] The beneficial effect of this invention lies in providing the application of two EF Hand family genes in improving the resistance of tomatoes to Phytophthora infestans. Tobacco varieties transiently expressing the target gene showed higher susceptibility to the disease compared to the control group; while tomato experimental lines with the target gene knocked out or silenced showed higher resistance to Phytophthora infestans compared to the wild type. At the same time, the growth and development of tomato lines with the target gene knocked out or silenced were not significantly affected. Attached Figure Description

[0029] Figure 1 This is a schematic diagram of a gene editing vector.

[0030] Figure 2 is a schematic diagram of the nucleotide sequence of the edit site where the frameshift mutation occurred; Figure 2a The editing status of the target gene Solyc02G002917. Figure 2b The editing status of the target gene Solyc03G000005 is shown in Figure 2; the red part in Figure 2 represents the edited part.

[0031] Figure 3 To enhance susceptibility through transient expression of target genes; among which Figure 3 a is the diameter of the lesion. Figure 3 b represents UV irradiation. One-way ANOVA was used, and Duncan's test was used to test for significance (P < 0.05).

[0032] Figure 4 Editing the target gene improved the resistance of tomatoes to Phytophthora infestans. Figure 4 a is the diameter of the lesion. Figure 4b represents the area of ​​the lesion. Figure 4 c represents the relative biomass of Phytophthora. Figure 4 Image d is taken under UV irradiation. One-way ANOVA was used, and Duncan's test was used to test for significance (P < 0.05).

[0033] Figure 5 The growth and development of tomato lines after the target gene was edited were not affected. Figure 5 'a' represents the tomato phenotype of the target gene Solyc02G002917 edited line after 4 weeks of growth. Figure 5 b represents the tomato phenotype of the target gene Solyc03G000005 edited line after 4 weeks of growth. Detailed Implementation

[0034] This invention provides two target genes, Solyc02G002917 and Solyc03G000005, to enhance resistance to Phytophthora in tomatoes, both of which are members of the EF Hand family. The genomic nucleotide sequence of target gene Solyc02G002917 is shown in SEQ ID NO:1, the coding region nucleotide sequence in SEQ ID NO:3, the protein amino acid sequence in SEQ ID NO:5, and the promoter nucleotide sequence in SEQ ID NO:7. The genomic nucleotide sequence of target gene Solyc03G000005 is shown in SEQ ID NO:2, the coding region nucleotide sequence in SEQ ID NO:4, the protein amino acid sequence in SEQ ID NO:6, and the promoter nucleotide sequence in SEQ ID NO:8.

[0035] The present invention also includes amino acid sequences with equivalent functions formed by substitution, deletion, or addition of one or more amino acids resulting from artificial engineering modification or natural nucleic acid polymorphism of the amino acid sequences shown in sequence listing SEQ ID NO:5 or SEQ ID NO:6, as well as nucleotide sequences encoding these amino acid sequences.

[0036] In some embodiments of the present invention, knocking out the target gene in tomato (whose sequence is shown in SEQ ID NO:1 or SEQ ID NO:2) significantly increases the tomato's resistance to Phytophthora infestans. Knockout and editing of the target gene can be achieved through CRISPR-Cas technology (and its derivative technologies, such as prime editing), T-DNA and transposon insertion, treatment with chemical mutagens such as ethyl methanesulfonate, or physical mutagenesis using high-energy radiation or plasma. The gene mutation generated by the above methods can be amplified by PCR using oligonucleotide primers designed based on the sequence of SEQ ID NO:1 or SEQ ID NO:2, 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] The technical solution of the present invention will be clearly and completely described below with reference to embodiments. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. All embodiments were carried out under conventional experimental conditions or conditions recommended in the manufacturer's instructions. Unless otherwise defined, all technical terms used in this invention have the same meaning as commonly understood by those skilled in the art. Furthermore, any methods and materials similar to or identical to those described can be used in the methods of the present invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort are within the scope of protection of the present invention.

[0038] Unless otherwise specified, the experimental methods used in the following examples shall be performed according to conventional methods or the manufacturer's recommendations. Unless otherwise specified, all materials, reagents, and consumables used are commercially available.

[0039] In this embodiment, the tomato variety used is Microtom, but other tomato varieties may be used in other embodiments.

[0040] Example 1: Construction of gene editing vector targeting the target gene

[0041] Based on the target gene sequence shown in SEQ ID NO:1 or SEQ ID NO:2, an appropriate gene editing target site was selected and the corresponding sgRNA was synthesized (as shown in SEQ ID NO:9–48 in the sequence listing). This sgRNA was then cloned into a cloning vector containing the kanamycin (Kan) resistance gene, and then transfected into an *E. coli* strain. In this example, the *E. coli* strain used was DH5α; other suitable *E. coli* strains can be used in other examples. The transfected *E. coli* were cultured on LB solid medium containing kanamycin (50 μg / mL), and positive clones were selected and transferred to liquid LB medium for propagation. Plasmid DNA was extracted using a plasmid miniprep kit (TIANGEN:#DP103), and the target fragment containing the sgRNA was recombined into a plant binary expression vector containing the hygromycin resistance gene (hyg) and the Cas9 protein expression cassette. A schematic diagram of this gene editing vector is shown below. Figure 1 As shown. The vector was transfected into a suitable Agrobacterium strain. In this example, Agrobacterium strain GV3101 was used. In other examples, EHA105 or other suitable Agrobacterium strains may also be used. Then, the successfully transfected positive strains were screened and cultured in LB medium containing hygromycin (hyg) and kanamycin (Kan) (both at a working concentration of 50 μg / mL) and propagated for subsequent Agrobacterium infection of explants.

[0042] Example 2: Transfection and Screening of Tomato Lines with Target Gene Editing

[0043] The transgenic Agrobacterium strain containing a plant binary expression vector constructed in Example 1 was co-cultured with surface-sterilized and slightly abraded tomato leaves under aseptic conditions on Murashige-Skoog medium. The differentiated shoot tissues after co-culture were transferred to fresh MS medium containing hygromycin (hyg) for resistance screening. The selected resistant positive differentiated shoots were transferred to root-promoting medium, and after confirming root development and establishment, they were transplanted into nutrient soil for further identification and harvesting of T0 generation seeds.

[0044] For the different sgRNA target sequences used in Example 1, corresponding upstream and downstream amplification nucleotide primers were designed and synthesized, and oligonucleotide amplification fragments containing the editing target site were obtained by PCR amplification. Sequencing of the obtained fragments can verify whether a frameshift mutation (i.e., the addition or deletion of nucleotides not being a multiple of 3) has occurred at the editing target. Figure 2 shows an example of the nucleotide sequence of the editing site with the coding mutation. Gene-edited lines with frameshift mutations at the editing target site were identified as the desired positive lines, and homozygous gene-edited plants were further screened for subsequent physiological and pathological index determination.

[0045] Example 3: Transient expression of tobacco

[0046] The transgenic Agrobacterium containing the plant binary expression vector constructed in Example 1 was resuspended in a tobacco resuspension and placed in the dark for about 3 hours to allow for full adsorption. The treated Agrobacterium was then injected into tobacco leaves. Lesions were statistically analyzed on detached tobacco leaves treated with a suspension of pathogenic Phytophthora spores (converted 5-7 days after inoculation), and images of the lesions were simultaneously taken using a UV lamp.

[0047] Test results as follows Figure 3 As shown, compared to the control group, tobacco leaves transiently expressing the target gene Solyc02G002917 and Solyc03G000005 both exhibited significantly increased lesion diameter. Images taken under UV irradiation also showed obvious lesions in the tobacco plants transiently expressing the target gene. These results indicate that, under controlled environmental conditions, tobacco plants transiently expressing the target gene have a higher susceptibility to disease compared to the control group.

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

[0049] Lesions were counted on detached tomato leaves containing a suspension of Phytophthora spores that had been inoculated with the fungus for 5-7 days, and images of the lesions were taken by irradiation with a UV lamp.

[0050] Inoculation of *Phytophthora infestans*: Take *Phytophthora infestans* grown on oat medium plates (grown for 2-3 weeks), activate the spores with distilled water for about 3 hours (4℃), then scrape off the water with a spreader and collect it. Centrifuge at 3000 rpm for 10 minutes to collect the spores, discard the supernatant and keep an appropriate spore suspension. Observe the number of spores under a microscope. Under 40x magnification, about 50 spores are sufficient. Inoculate 10 μL of the inoculum on each leaf and treat in the dark at 28℃ for 5-7 days.

[0051] Test results as follows Figure 4 As shown, compared to the wild type, the tomato leaves of the Solyc02G002917 and Solyc03G000005 edited lines exhibited significantly reduced lesion diameter and area; the relative biomass of Phytophthora in the leaves was also significantly reduced. Images taken under UV irradiation further validated these conclusions. In summary, the tomato lines with the target gene knocked out exhibited higher resistance to Phytophthora than the wild-type tomato lines.

[0052] Example 5: Measurement of Tomato Growth and Development Indicators

[0053] To observe whether there are differences in growth phenotype between the target gene-edited lines and the wild type. The target gene-edited lines Solyc02G002917 and Solyc03G000005 were cultured under suitable conditions for 4 weeks, with the wild type used as a control. Figure 4 As shown, there were no significant phenotypic differences between wild-type and target gene-edited lines. This means that the growth and development of tomato lines edited with the target gene were not affected.

[0054] The above description is merely a preferred embodiment of the present invention. It should be understood that the present invention is not limited to the forms disclosed herein and should not be construed as excluding other embodiments. It can be used in various other combinations, modifications, and environments, and can be altered within the scope of the concept described herein through the above teachings or related technologies or knowledge. Modifications and variations made by those skilled in the art that do not depart from the spirit and scope of the present invention should be within the protection scope of the appended claims.

Claims

1. The application of EF Hand family genes in improving tomato resistance to Phytophthora infestans, characterized by: The nucleotide sequence of the EF Hand family gene is any of the following: 1) The genomic nucleotide sequence shown in SEQ ID NO:1; 2) The genomic nucleotide sequence shown in SEQ ID NO:2; 3) The nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO:5; 4) The nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO:6; The application involves knocking out or silencing EF Hand family genes to improve the resistance of tomatoes to Phytophthora infestans.

2. The application according to claim 1, characterized in that, The nucleotide sequences of the EF Hand family genes are shown in SEQ ID NO:3 or SEQ ID NO:

4.

3. An sgRNA, characterized in that, The nucleotide sequence of the sgRNA is shown in any one of SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11 or SEQ ID NO:

33.

4. An sgRNA combination, characterized in that, The sgRNA combination consists of the sgRNA described in SEQ ID NO:33, the sgRNA described in SEQ ID NO:30, and the sgRNA described in SEQ ID NO:

31.

5. A recombinant vector, characterized in that, The vector comprises the sgRNA as described in claim 3 or the combination of sgRNAs as described in claim 4.

6. A transformant, characterized in that, The transformant comprises the recombinant vector as described in claim 5.

7. The application of the sgRNA of claim 3, the sgRNA combination of claim 4, the recombinant vector of claim 5, or the transformant of claim 6 in improving the resistance of tomato to Phytophthora infestans, characterized in that, The sgRNA, sgRNA combination, recombinant vector, or transformant specifically knocks out or silences EF Hand family genes, wherein the nucleotide sequence of the EF Hand family genes is selected from any of the following: (a1) The nucleotide sequence shown in SEQ ID NO:1 or SEQ ID NO:2; (a2) A nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO:5 or SEQ ID NO:

6.

8. The application according to claim 7, characterized in that, The nucleotide sequences of the EF Hand family genes are shown in any one of SEQ ID NO:3-4.

9. A method for preparing tomatoes resistant to Phytophthora infestans, characterized in that, The target tomato does not express EF Hand family genes, wherein the nucleotide sequence of the EF Hand family genes is selected from any of the following: (b1) The nucleotide sequence shown in SEQ ID NO:1 or SEQ ID NO:2; (b2) A nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO:5 or SEQ ID NO:

6.

10. The method according to claim 9, characterized in that, The nucleotide sequences of the EF Hand family genes are shown in any one of SEQ ID NO:3-4.

11. The method according to claim 9, characterized in that, Gene editing technology can be used to knock out or silence the EF Hand family gene in tomatoes; or gene mutation technology can be used to prevent the target tomato from expressing the EF Hand family gene.

12. The method according to claim 11, characterized in that, The gene editing technology is at least one of CRISPR-Cas technology, T-DNA, and transposon insertion.

Images