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

Abstract

The application 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 Phytophthora infestans 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 RIN4 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 Phytophthora infestans 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 Phytophthora infestans disease-related genes in tomatoes, but also develops tomato lines with enhanced resistance to Phytophthora infestans by using the characteristics of the above genes, thereby providing new technical strategies and gene resources for tomato breeding.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of molecular biology breeding technology, and particularly relates to application of RIN4 family genes in improving resistance of tomatoes to Phytophthora infestans. BACKGROUND

[0002] Phytophthora infestans de Bary is an oomycete that can cause tomato and potato late blight, and is one of the most destructive pathogens, and is the culprit of the Irish famine in the 19th century. In the prevention and treatment methods of late blight, measures such as cultivation technology, breeding of resistant varieties and chemical pesticide spraying are relied on. Breeding of resistant varieties is the main research direction of current scientific research, and 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 long-term role, and the loss of function of the susceptible gene can make the plant obtain broad-spectrum resistance. Therefore, starting from the relationship between the susceptible gene and the late blight, the mechanism of tomato susceptibility is analyzed from the molecular level, so that the improvement of tomato resistance becomes a new research idea. SUMMARY

[0003] The present application aims to overcome the shortcomings of the prior art, and provides a target gene for improving the resistance of tomatoes to Phytophthora infestans and application thereof.

[0004] The first aspect of the present application provides application of RIN4 family genes in improving the resistance of tomatoes to Phytophthora infestans.

[0005] Preferably, the RIN4 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) a promoter nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 3 as shown in SEQ ID NO: 7;

[0013] 8) a promoter nucleotide sequence of SEQ ID NO: 2 or SEQ ID NO: 4 as shown in SEQ ID NO: 8.

[0014] The second aspect of the present application provides a sgRNA, the nucleotide sequence of which is shown in any one of SEQ ID NO: 9 - 48.

[0015] The third aspect of the present application provides a recombinant vector, the vector comprising the sgRNA described above.

[0016] The fourth aspect of the present application provides a transformant, the transformant comprising the recombinant vector described above.

[0017] In some embodiments of the present application, the transformant is transformed with a RIN4 family gene or the vector described above.

[0018] The fifth aspect of the present application provides the use of the sgRNA described above, the recombinant vector described above or the transformant described above in improving the resistance of tomato to P. infestans, the sgRNA, the vector or the transformant specifically knocking out or silencing a RIN4 family gene, the RIN4 family gene being any one of SEQ ID NO: 1 - 8.

[0019] The sixth aspect of the present application provides a tomato or a tomato cell, the tomato or the tomato cell being a tomato or a tomato cell in at least one of:

[0020] i) a tomato or a tomato cell in which a sequence as shown in any one of SEQ ID NO: 1 - 8 is knocked out or silenced;

[0021] ii) a tomato or a tomato cell in which a related gene is knocked out or silenced by using the sgRNA described above;

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

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

[0024] The seventh aspect of the present application provides a method for preparing a tomato with resistance to P. infestans, the method comprising the step of making a target tomato to express a RIN4 family gene at a low level or not express the RIN4 family gene, the RIN4 family gene being any one of SEQ ID NO: 1 - 8.

[0025] In some embodiments of the present application, the RIN4 family genes in tomatoes are knocked out or silenced by gene editing technology; or the target tomato has low expression or no expression of the RIN4 family genes by gene mutation technology, so as to prepare tomatoes with resistance to P. infestans.

[0026] Preferably, the RIN4 family genes are knocked out or silenced by CRISPR-Cas technology, T-DNA and transposon insertion, chemical mutagenesis or physical mutagenesis. Here, 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 application, the sgRNA for specifically knocking out or silencing the nucleotide sequence as shown in SEQ ID NO: 1 or SEQ ID NO: 3 is any one of SEQ ID NO: 9-28; and the sgRNA for specifically knocking out or silencing the nucleotide sequence as shown in SEQ ID NO: 2 or SEQ ID NO: 4 is any one of SEQ ID NO: 29-48.

[0028] The present application has the beneficial effect of providing the application of two RIN4 family genes in improving the resistance of tomatoes to P. infestans. The tobacco transiently expressing the target gene has higher susceptibility than the control group; and the tomato experimental strain after knocking out or silencing the target gene has higher resistance to P. infestans than the wild type, and the tomato strain after knocking out or silencing the target gene is not significantly affected in growth and development. BRIEF DESCRIPTION OF DRAWINGS

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

[0030] Figure 2 is a schematic diagram of the nucleotide sequence of the editing site with a frameshift mutation; Figure 2a It is the editing of the target gene Solyc04G001683, Figure 2b It is the editing of the target gene Solyc04G000477; wherein the red part in Figure 2 represents the editing part.

[0031] Figure 3 It is to improve the susceptibility by transiently expressing the target gene; wherein Figure 3 a is the lesion diameter, Figure 3 b is UV irradiation. Single factor analysis of variance is used, and Duncan method is used to test the significance of difference (P<0.05).

[0032] Figure 4 It is to improve the resistance of tomatoes to P. infestans after editing the target gene; Figure 4 a is the lesion diameter, Figure 4 b is the lesion area,Figure 4 c is the relative biomass of Phytophthora, Figure 4 d is the UV irradiation photograph. Single factor variance analysis and Duncan test are used to determine the significance of difference (P<0.05).

[0033] Figure 5 The growth and development of the tomato strain after editing the target gene are not affected. Figure 5 a is the phenotype of the tomato after the target gene Solyc04G001683 edited strain grows for 4 weeks, Figure 5 b is the phenotype of the tomato after the target gene Solyc04G000477 edited strain grows for 4 weeks. DETAILED DESCRIPTION

[0034] The application provides two target genes Solyc04G001683 and Solyc04G000477 for improving the resistance of tomatoes to Phytophthora infestans, and both of the two genes are members of the RIN4 family. 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 application also includes amino acid sequences with equivalent functions formed by replacing, deleting, or adding one or several amino acids of the amino acid sequences shown in SEQ ID NO:5 or SEQ ID NO:6 by artificial engineering or natural nucleic acid polymorphism, and nucleotide sequences encoding these amino acid sequences.

[0036] In some embodiments of the present application, after knocking out the target gene in tomato (the sequence of which is shown in SEQ ID NO: 1 or SEQ ID NO: 2), the resistance of tomato to P. infestans is significantly increased. The knockout and editing of the target gene can be achieved by CRISPR-Cas technology (and its individual derivative technologies, such as prime editing, etc.), T-DNA and transposon insertion, treatment with chemical mutagens such as ethyl methanesulfonate, high-energy radiation or plasma physical mutagenesis, etc. The gene mutations 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 amplification products are subjected to nucleotide sequencing for verification. Based on the sequencing results obtained, it can be predicted whether the alleles after mutagenesis can still encode functional proteins corresponding to the amino acid sequence shown in SEQ ID NO: 3 or SEQ ID NO: 4.

[0037] The technical solutions of the present application will be described clearly and completely in connection with the embodiments. Obviously, the described embodiments are only some of the embodiments of the present application, but not all. The embodiments are carried out according to conventional experimental conditions or according to the conditions suggested by the manufacturer's instructions. Unless otherwise defined, all professional terms used in the present application have the same meaning as generally understood by those skilled in the art. In addition, any methods and materials similar or identical to those described can be used in the present application, and all other embodiments obtained by those skilled in the art without creative labor based on the embodiments in the present application are within the scope of protection of the present application.

[0038] In the following examples, the experimental methods were carried out according to the conventional methods or the manufacturer's recommendations unless otherwise specified. The materials, reagents and consumables used, unless otherwise specified, can be obtained commercially.

[0039] In this embodiment, the tomato variety used is microtom, and other varieties of tomato can also 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, a suitable gene editing target is selected and designed, and a corresponding sgRNA (such as the sequence shown in SEQ ID NO: 9-48 in the sequence listing) is synthesized, which is then cloned into a cloning vector containing a kanamycin (Kan) resistance gene, and then transfected into an E. coli strain. The E. coli strain used in this embodiment is DH5a, and other suitable E. coli strains can also be used in other embodiments. The transfected E. coli is incubated on LB solid medium containing kanamycin (concentration 50 μg / mL), and positive clones are selected and transferred to liquid LB medium for culture and expansion. Plasmid DNA is extracted using a plasmid extraction kit (TIANGEN: #DP103), and the target fragment containing the sgRNA is recombined 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 in Figure 1 . The vector is transfected into a suitable Agrobacterium strain, and the Agrobacterium strain used in this embodiment is GV3101, and EHA105 or other suitable Agrobacterium strains can also be used in other embodiments. Then, the successfully transfected positive strains are cultured and expanded using LB medium containing hygromycin (hyg) and kanamycin (Kan) (working concentration 50 μg / mL), and used for subsequent Agrobacterium infiltration 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 is used for co-culture with tomato leaves that have been surface sterilized and slightly rubbed to cause injury under sterile conditions on Murashige-Skoog medium. The differentiated bud tissues after co-culture are transferred to new MS medium containing hygromycin (hyg) for resistance screening. The resistant positive differentiated buds obtained by screening are transferred to a rooting-promoting medium, and after confirming root development and colonization, they are removed from the bottle and transplanted into nutrient soil for further identification and harvesting of T0 generation seeds.

[0044] Corresponding upstream and downstream amplification nucleotide primers are designed and synthesized for different sgRNA target sequences used in Example 1, and the oligonucleotide amplification fragments containing the editing target sites are obtained by PCR amplification. Sequencing of the obtained fragments can verify whether a frameshift mutation (i.e., addition or deletion of nucleotides that are not multiples of 3) has occurred at the editing target. Figure 2 shows an example of a nucleotide sequence of an editing site that has undergone a coding mutation. The gene editing strain in which a frameshift mutation has occurred at the editing target is determined as the desired positive strain, and homozygous gene editing plants are continuously selected for subsequent physiological and pathological index determination.

[0045] Example 3, Tobacco Transient Expression

[0046] The transgenic Agrobacterium containing the constructed expression vector of the target gene was resuspended with tobacco resuspension solution, and was placed in the dark for about 3 hours to allow sufficient adsorption. Then the treated Agrobacterium was injected onto tobacco leaves. The in vitro tobacco leaves inoculated with 5-7 day-old P. infestans spore suspension were counted for lesion, and the lesion images were taken by UV irradiation.

[0047] The test results are shown in Table 1. Figure 3 As shown in Table 1, compared with the control group, the tobacco leaves transiently expressing the target gene Solyc04G001683 and the tobacco leaves transiently expressing the target gene Solyc04G000477 both showed a significant increase in lesion diameter; the images taken by UV irradiation also showed that the tobacco transiently expressing the target gene all showed obvious lesions. The above results show that under controlled environmental conditions, the tobacco transiently expressing the target gene has higher susceptibility than the control group.

[0048] Example 4, Functional identification of tomato resistance to P. infestans

[0049] The in vitro tomato leaves inoculated with 5-7 day-old P. infestans spore suspension were counted for lesion, and the lesion images were taken by UV irradiation.

[0050] phytophthora infestans inoculation: Take phytophthora infestans grown on oat medium plate (grown for 2-3 weeks) and activate spores in distilled water for about 3 hours (4°C), then scrape the water with a coating rod and collect it, centrifuge at 3000 rpm for 10 minutes to collect spores, discard the supernatant and leave appropriate spore suspension, observe the number of spores under a microscope, about 50 spores under a 40x microscope, inoculate 10 μL on each leaf, and treat at 28°C in the dark for 5-7 days.

[0051] The test results are shown in Table 2. Figure 4 As shown in Table 2, compared with the wild type, the tomato leaves of the target gene Solyc04G001683 edited strain and the target gene Solyc04G000477 edited strain both showed a significant decrease in lesion diameter and lesion area; the relative biomass of P. infestans contained in the leaves was significantly reduced. The above conclusion was further verified by taking images by UV irradiation. In summary, the tomato strain after knocking out the target gene has higher resistance to P. infestans than the wild type tomato strain.

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

[0053] To observe whether there is a difference in growth phenotype between the target gene edited line and the wild type, the target gene Solyc04G001683 edited line and the target gene Solyc04G000477 edited line were cultured for 4 weeks under suitable conditions, with the wild type as a control. As shown in FIG. 12, there was no significant difference in phenotype between the wild type and the target gene edited line. That is, the tomato line after editing the target gene was not affected in growth and development. Figure 4

[0054] The above description is only preferred embodiments of the present application, and it should be understood that the present application is not limited to the forms disclosed herein, and should not be considered as excluding other embodiments, but can be used in various other combinations, modifications and environments, and can be modified within the scope of the concepts described herein, by the above teachings or related art or knowledge. Any modification and change made by those skilled in the art without departing from the spirit and scope of the present application shall be within the scope of protection of the appended claims of the present application.​

Claims

1. Use of a RIN4 family gene to increase resistance of tomato to P. infestans, characterized in that, The nucleotide sequence of the RIN4 family gene is any one of the following 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 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 is to improve the resistance of tomato to Phytophthora infestans by knocking out or silencing the RINA gene.

2. Use according to claim 1, characterized in that, The nucleotide sequence of the RIN4 family gene is shown in SEQ ID NO: 3 or SEQ ID NO:

4.

3. An sgRNA, characterized in that, The nucleotide sequence of the sgRNA is any one of SEQ ID NO: 20, SEQ ID NO: 33, or SEQ ID NO:

38.

4. A combination of sgRNAs, characterized in that, The sgRNA combination consists of the sgRNA shown in SEQ ID NO: 12 and the sgRNA shown in SEQ ID NO:

17.

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

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

7. Use of the sgRNA according to claim 3, of the combination of sgRNAs according to claim 4, of the recombinant vector according to claim 5 or of the transformant according to claim 6 for increasing the resistance of tomato to the pathogenic P. infestans, characterized in that, The sgRNA, sgRNA combination, recombinant vector, or transformant specifically knocks out or silences the RIN4 family gene, and the nucleotide sequence of the RIN4 family gene is selected from any one of the following: (a1) the nucleotide sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2; (a2) the nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 5 or SEQ ID NO:

6.

8. Use according to claim 7, characterized in that, The nucleotide sequence of the RIN4 family gene is any one of SEQ ID NO: 3-4.

9. A method of making a tomato plant having resistance to Phytophthora infestans, characterized in that, The target tomato does not express the RIN4 family gene, and the nucleotide sequence of the RIN4 family gene is selected from any one of the following: (b1) the nucleotide sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2; (b2) the nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 5 or SEQ ID NO:

6.

10. The method of claim 9, wherein, The nucleotide sequence of the RIN4 family gene is any one of SEQ ID NO: 3-4.

11. The method of claim 9, wherein, The RIN4 family gene in the tomato is knocked out or silenced using gene editing technology; or the target tomato does not express the RIN4 family gene using gene mutation technology.

12. The method of claim 11, wherein, The gene editing technology is at least one of CRISPR-Cas technology, T-DNA, and transposon insertion.

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