Tomato plant SlTOM2A allele and application thereof

Abstract

The invention discloses a tomato plant SlTOM2A allele and application thereof, and relates to the technical field of plant genetic engineering, the tomato plant SlTOM2A allele is obtained by editing a first target spot and a second target spot on a second exon of a tomato plant SlTOM2A gene; the nucleotide sequence of the first target spot is as shown in SEQ ID NO. 1, and the nucleotide sequence of the second target spot is as shown in SEQ ID NO. 2. Experiments prove that the tomato plant SlTOM2A allele provided by the invention has the effects of improving the tobacco mosaic virus resistance of the tomato plant and changing the growth form of the tomato plant.

Description

Technical Field

[0001] This application relates to the field of plant genetic engineering technology, and in particular to a tomato plant SlTOM2A allele and its application. Background Technology

[0002] Tobacco mosaic virus (TMV) is a pathogenic virus that can widely infect Solanaceae crops (including tomatoes). The mosaic disease it causes in tomatoes severely impacts yield and quality. For a long time, tomato breeding for TMV resistance has mainly relied on... Tm-1 , Tm-2 and Tm-2 -2 A few major resistance genes exist. However, with the continuous co-evolution of the TMV population and host resistance genes, new TMV lines capable of overcoming the aforementioned resistances have emerged in the field, posing a threat to existing resistant varieties. Therefore, it is urgent to discover gene resources with broad-spectrum or novel resistance to TMV to ensure the healthy and sustainable development of the tomato industry.

[0003] The stem, as a vital supporting organ of plants, directly influences a crop's lodging resistance, photosynthetic efficiency, and adaptability to mechanized harvesting. While most plant stems grow upright, some, such as climbing plants, exhibit a spiral growth pattern. Identifying genes in crops related to stem morphology is of great significance for crop architecture breeding. Summary of the Invention

[0004] The main purpose of this application is to propose an SlTOM2A allele for tomato plants and its application, aiming to provide a method for improving the resistance of tomato plants to tobacco mosaic virus.

[0005] To achieve the above objectives, the tomato plant proposed in this application SlTOM2A Alleles, through the influence of tomato plants SlTOM2A The gene was obtained by editing the first and second target sites on the second exon of the gene. The nucleotide sequence of the first target is shown in SEQ ID NO.1, and the nucleotide sequence of the second target is shown in SEQ ID NO.2.

[0006] Preferably, the SlTOM2A The amino acid sequence of the protein encoded by the allele is shown in SEQ ID NO.3.

[0007] Preferably, the SlTOM2A The nucleotide sequence of the allele is shown in SEQ ID NO.4.

[0008] This application also proposes a CRISPR / Cas9 vector system for editing tomato plants. SlTOM2A Genes, including first sgRNA and second sgRNA; The nucleotide sequence of the first sgRNA is shown in SEQ ID NO.5, and the nucleotide sequence of the second sgRNA is shown in SEQ ID NO.6.

[0009] This application also proposes a host cell for editing tomato plants. SlTOM2A Genes, including the CRISPR / Cas9 vector system proposed in this application.

[0010] This application also proposes a tomato plant as described in this application. SlTOM2A Application of alleles, the CRISPR / Cas9 vector system proposed in this application, or the host cell proposed in this application in improving the resistance of tomato plants to tobacco mosaic virus and / or altering the stem growth morphology of tomato plants.

[0011] This application also proposes a tobacco mosaic virus-resistant tomato plant or a portion thereof, wherein the genome of the tobacco mosaic virus-resistant tomato plant or the portion thereof includes the tomato plant proposed in this application. SlTOM2A Alleles.

[0012] This application also proposes a tomato plant or a portion thereof with a curved stem, wherein the genome of the tomato plant or the portion thereof includes the tomato plant proposed in this application. SlTOM2A Alleles.

[0013] This application also proposes a method for improving the resistance of tomato plants to tobacco mosaic virus, the method comprising using CRISPR / Cas9 technology to treat tomato plants. SlTOM2A Gene editing was performed to obtain the tomato plant proposed in this application. SlTOM2A Alleles.

[0014] This application also proposes a method for altering the stem growth shape of tomato plants, the method comprising using CRISPR / Cas9 technology to modify the tomato plant... SlTOM2A Gene editing was performed to obtain the tomato plant proposed in this application. SlTOM2A Alleles.

[0015] The tomato plants proposed in this application SlTOM2AThe allele, experimentally verified, has been shown to enhance the resistance of tomato plants to tobacco mosaic virus (TMV) and alter the growth morphology of tomato plants. It is a novel gene with broad-spectrum or new resistance to TMV, solving the problem of poor disease resistance in tomato plants caused by existing resistance genes to TMV. Simultaneously, the tomato plants... SlTOM2A The effect of alleles on altering the growth morphology of tomato plants is also of great significance for crop plant type breeding. Attached Figure Description

[0016] To more clearly illustrate the technical solutions in the embodiments of this application or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on the structures shown in these drawings without creative effort.

[0017] Figure 1 The tomato in Example 1 of this invention SlTOM2A A schematic diagram of a CRISPR / Cas9 gene editing vector; Figure 2 The tomato in Example 1 of this invention SlTOM2A Schematic diagram of the mutation site sequence in gene knockout strains; Figure 3 The tomato in Example 2 of this invention SlTOM2A Image showing TMV resistance identification in gene knockout plants and wild-type plants; Figure 4 The tomato in Example 2 of this invention SlTOM2A Expression levels of the gene encoding the TMV coat protein in gene knockout plants and wild-type plants: A is the inoculated leaf; B is the system leaf; Figure 5 The tomato in Example 3 of this invention SlTOM2A Phenotypic diagrams of stems from gene knockout plants and wild-type plants; Figure 6 The tomato in Example 3 of this invention SlTOM2A Figures showing the auxin and gibberellin content in the stems of gene knockout plants and wild-type plants: A represents the auxin content (IAA-Asp); B represents the gibberellin content (GA3).

[0018] The realization of the purpose, functional features and advantages of this application will be further explained in conjunction with the embodiments and with reference to the accompanying drawings. Detailed Implementation

[0019] The technical solutions of the embodiments of this application will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only a part of the embodiments of this application, and not all of the embodiments. Based on the embodiments of this application, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of this application.

[0020] It should be noted that if the embodiments of this application involve directional indicators (such as up, down, left, right, front, back, etc.), the directional indicators are only used to explain the relative positional relationship and movement of the components in a specific posture. If the specific posture changes, the directional indicators will also change accordingly.

[0021] Furthermore, if the embodiments of this application involve descriptions such as "first" or "second," these descriptions are for descriptive purposes only and should not be construed as indicating or implying their relative importance or implicitly specifying the number of technical features indicated. Therefore, a feature defined with "first" or "second" may explicitly or implicitly include at least one of those features. Additionally, the use of "and / or" or "and / or" throughout the text includes three parallel solutions. For example, "A and / or B" includes solution A, solution B, or a solution that simultaneously satisfies A and B. Furthermore, the technical solutions of the various embodiments can be combined with each other, but this must be based on the ability of those skilled in the art to implement them. When the combination of technical solutions is contradictory or impossible to implement, it should be considered that such a combination of technical solutions does not exist and is not within the scope of protection claimed in this application.

[0022] tomato( Solanum lycopersicum L.) is an annual herbaceous plant belonging to the genus Solanum in the family Solanaceae. It is one of the important economic and vegetable crops in my country and was introduced to my country from South America. At present, the research on the regulation mechanism of stem morphology mainly focuses on the following two aspects: (1) genes related to cell wall synthesis and modification (such as the CesA family of cellulose synthases, PAL and CAD, key enzymes in lignin synthesis, etc.), whose expression level can change the mechanical strength of stem; (2) plant hormone signaling network (such as auxin polar transport carrier PIN protein, gibberellin biosynthesis gene GA20ox, etc.), which affect the length of internodes by regulating cell elongation. However, the above studies mostly focus on cell wall components or longitudinal growth regulation, and there is still a gap in the understanding of the molecular mechanism of stem three-dimensional morphology construction. Traditional stem improvement technology relies on natural variation screening or quantitative trait locus (QTL) mining, which has the disadvantages of long breeding cycle (usually 6-8 generations) and limited trait improvement.

[0023] On the other hand, Tobacco mosaic virus (TMV) is a pathogenic virus that can widely infect Solanaceae crops (including tomatoes), and the mosaic disease it causes in tomatoes severely affects their yield and quality. For a long time, tomato TMV resistance breeding has mainly relied on... Tm-1 , Tm-2 and Tm-2 -2 A few major resistance genes, etc. With the continuous co-evolution of the TMV population and host resistance genes, new TMV lines capable of overcoming the above resistances have emerged in the field, posing a threat to existing resistant varieties.

[0024] Based on this, this application proposes tomato plants SlTOM2A Alleles are genes obtained by genetically modifying tomato plants. SlTOM2A The gene (NCBI Gene ID: LOC101262238) was obtained by editing the first and second target sites on the second exon. The nucleotide sequence of the first target site is shown in SEQ ID NO.1, and the nucleotide sequence of the second target site is shown in SEQ ID NO.2.

[0025] In some implementations... SlTOM2A Alleles (named) SlTOM2A_a The amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO.3.

[0026] In some implementations... SlTOM2A_a The nucleotide sequence of the gene is shown in SEQ ID NO.4.

[0027] The tomato plants proposed in this application SlTOM2A The allele, experimentally verified, has been shown to enhance the resistance of tomato plants to tobacco mosaic virus (TMV) and alter the growth morphology of tomato plants. It is a novel gene with broad-spectrum or new resistance to TMV, solving the problem of poor disease resistance in tomato plants caused by existing resistance genes to TMV. Simultaneously, the tomato plants... SlTOM2A The effect of alleles on altering the growth morphology of tomato plants is also of great significance for crop plant type breeding.

[0028] This application also proposes a method for editing tomato plants. SlTOM2A The CRISPR / Cas9 vector system for the gene includes a first sgRNA and a second sgRNA. The nucleotide sequence of the first sgRNA is shown in SEQ ID NO.5, and the nucleotide sequence of the second sgRNA is shown in SEQ ID NO.6.

[0029] The term "CRISPR / Cas9 vector system" refers to one or more nucleic acid constructs designed for the expression of CRISPR-Cas9 gene editing components within host cells. The system comprises at least one vector containing a Cas9 coding sequence and a guide RNA expression cassette. Optionally, the system includes a donor DNA template. The term "vector system" includes, but is not limited to, plasmid vectors, viral vectors (such as lentiviral vectors and adeno-associated virus vectors), and linear DNA fragments.

[0030] The term "Cas9 coding sequence" refers to any nucleotide sequence that encodes a Cas9 protein or a functionally active fragment thereof that has DNA endonuclease activity. This includes, but is not limited to, sequences derived from Streptococcus pyogenes (Streptococcus pyogenes). Streptococcus pyogenes The coding sequence of wild-type Cas9 (SpCas9), its orthologs, paralogs, variants (such as variant xCas9 with different PAM specificity, SpCas9-VQR), mutants (e.g., nickase variants D10A or H840A, or the completely inactivated variant dCas9), and sequences having at least 80%, 85%, 90%, 95%, or 99% sequence identity with the stated sequence and retaining DNA binding and / or cleavage function. The coding sequence is typically codon-optimized for expression in a specific host (e.g., human cells) and may contain coding sequences for one or more nuclear localization signals (NLS).

[0031] The term "guide RNA expression cassette" refers to a DNA unit contained within a vector that is used to transcribe guide RNA (gRNA) into the host cell. It contains at least: Guide RNA (gRNA): A chimeric RNA molecule comprising: (i) a target region, which is a sequence of about 15 to 30 nucleotides in length (e.g., 17, 18, 19, 20, 21, 22, 23 nucleotides) having at least 80%, 90%, 95%, or 100% complementarity to a target genomic DNA site; and (ii) a scaffold region, which is a conserved RNA backbone sequence capable of binding to the Cas9 protein and forming a functional ribonucleoprotein complex. The gRNA may be a single RNA molecule (single-stranded guide RNA, sgRNA) or a bimolecular form of crRNA and tracrRNA.

[0032] gRNA promoter: A promoter operatively linked upstream of the coding sequence of the gRNA, configured to drive transcription of the gRNA in eukaryotic cells. RNA polymerase III promoters, such as the U6 or H1 promoter, are typically used.

[0033] This application also proposes a method for editing tomato plants. SlTOM2AThe host cell for the gene, including the CRISPR / Cas9 vector system proposed in this application.

[0034] The term "host cell" refers to any cell that has been introduced into or can be introduced into the CRISPR-Cas9 vector system proposed in this application. The host cell may be a prokaryotic cell (such as Escherichia coli used for vector amplification) or a eukaryotic cell, including but not limited to: mammalian cells (such as HEK293, HeLa, mouse embryonic stem cells), plant cells, fungal cells or in vivo animal cells.

[0035] This application also proposes a tomato plant as described in this application. SlTOM2A Application of alleles, the CRISPR / Cas9 vector system proposed in this application, or the host cell proposed in this application in improving the resistance of tomato plants to tobacco mosaic virus and / or altering the stem growth morphology of tomato plants.

[0036] In conjunction with the foregoing, the tomato plant proposed in this application SlTOM2A The allele, experimentally verified, has been shown to enhance the resistance of tomato plants to tobacco mosaic virus and alter the growth morphology of tomato plants. Therefore, including tomato plants... SlTOM2A Alleles, used to edit tomato plants SlTOM2A CRISPR / Cas9 vector system for gene editing in tomato plants SlTOM2A The host cells of the gene can be used to improve the resistance of tomato plants to tobacco mosaic virus and / or change the stem growth morphology of tomato plants.

[0037] This application also proposes a tobacco mosaic virus-resistant tomato plant or a portion thereof, wherein the genome of the tobacco mosaic virus-resistant tomato plant or the portion thereof includes the tomato plant proposed in this application. SlTOM2A Alleles.

[0038] This application also proposes a tomato plant or a portion thereof with a curved stem, wherein the genome of the tomato plant or the portion thereof includes the tomato plant proposed in this application. SlTOM2A Alleles.

[0039] This application also proposes a method for improving the resistance of tomato plants to tobacco mosaic virus, the method comprising using CRISPR / Cas9 technology to treat tomato plants. SlTOM2A Gene editing was performed to obtain the tomato plant proposed in this application. SlTOM2A Alleles.

[0040] In conjunction with the foregoing, the tomato plant proposed in this application SlTOM2A Alleles were experimentally verified to enhance the resistance of tomato plants to tobacco mosaic virus. Therefore, CRISPR / Cas9 technology was used to enhance the resistance of tomato plants to tobacco mosaic virus.SlTOM2A Gene editing was performed to obtain the tomato plant proposed in this application. SlTOM2A Alleles can enhance the resistance of tomato plants to tobacco mosaic virus.

[0041] This application also proposes a method for altering the stem growth shape of tomato plants, the method comprising using CRISPR / Cas9 technology to modify the tomato plant... SlTOM2A Gene editing was performed to obtain the tomato plant proposed in this application. SlTOM2A Alleles.

[0042] In conjunction with the foregoing, the tomato plant proposed in this application SlTOM2A Alleles were experimentally verified to alter the stem growth shape of tomato plants. Therefore, CRISPR / Cas9 technology was used to modify the stem shape of tomato plants. SlTOM2A Gene editing was performed to obtain the tomato plant proposed in this application. SlTOM2A Alleles can alter the shape of tomato plant stems.

[0043] The following specific examples provide further details.

[0044] It should be noted that, unless otherwise specified, the experimental methods and reagents used in the following embodiments are conventional experimental methods and reagents used by those skilled in the art.

[0045] Example 1 This embodiment uses wild-type tomatoes, namely Ailsa Craig ( Solanum lycopersicum L.cv. AilsaCraig) was used to construct the SlTOM2A knockout line using CRISPR. The specific construction method is as follows: Using the online target site identification software CRIPSRMultiTargeter (http: / / www.multicrispr.net / index.html), target site sequences were designed: sgRNA1: CACTCATAGGTGGTGCAAC (SEQ ID NO.1), and primer A2F: 5'-ATATATGGTCTCGATTGCACTCATAGGTGGTGCAACGTTTTAGAGCTAGAAATAGC-3' (SEQ ID NO. 7); sgRNA2: ATGCTGATGGCTGTATCGT (SEQ ID NO. 2), and primer A2R: 5'-ATTATTTGGTCTCGAAAC ACGATACAGCCATCAGCAT CAATCTCTTAGTCGACTCTAC-3' (SEQ ID NO.8).

[0046] Knockout vector construction: Single-stranded OligoDNA was annealed to form double-stranded DNA. The synthesized two single-stranded primers were diluted to 50 μM and then annealed.

[0047] The annealing reaction system was 50 µL, including 10 µL Annealing Buffer for DNA Oligos (5×), 10 µL each of upstream and downstream primers, and 20 µL ddH2O.

[0048] The annealing reaction was carried out on a Genesy 96T instrument under the following conditions: pre-denaturation at 95℃ for 2 min. Cool to 25℃ in 0.1℃ for 8 seconds, then store at 4℃.

[0049] Competent cells were transformed, and positive clones were detected and sequenced for verification. Agrobacterium EHA105 was then transformed. Genetic transformation was performed using the leaf disc method. Stable homozygous lines were obtained through hygromycin resistance screening combined with PCR (e.g., forward primer 5'-ATGGCGTGCAAAGGGTTTTGG-3' as shown in SEQ ID NO.9, and reverse primer 5'-CCTTTTTAACTGTGACATTCCTCC-3' as shown in SEQ ID NO.10), and after sequencing verification. Two independent edited lines (Mut #5 and Mut #13) were selected for subsequent experiments. The identification diagram of the edited tomato lines is shown below. Figure 2 As shown.

[0050] Example 2 When the tomato seedlings have grown to the stage of six leaves and one bud, the friction inoculation method was used to inoculate the seedlings in Example 1. SlTOM2A Gene-edited lines (Mut #5 and Mut #13) and the wild-type tomato line Ailsa Craig were inoculated with tobacco mosaic virus (TMW), and resistance was assessed 10 days later. The results are as follows: Figure 3 As shown.

[0051] from Figure 3 As can be seen, compared with the wild-type tomato strain Ailsa Craig (WT), SlTOM2A The gene-edited strain Mut #5 exhibited obvious TMV symptoms, while SlTOM2A The gene-edited line Mut #13 showed no obvious TMV symptoms, and its leaves were free of mosaic patterns and malformations. SlTOM2A In gene-edited strain Mut #13 SlTOM2A Gene mutations can significantly increase the resistance of tomatoes to TMV, reaching an immune level.

[0052] Example 3 For quantitative purposes SlTOM2AThe resistance level of gene-edited lines to TMV, compared to the TMV-inoculated lines in Example 2. SlTOM2A The expression levels of the TMV coat protein gene in the inoculated leaves and systemic leaves of gene-edited lines (Mut #5 and Mut #13) and the wild-type tomato line Ailsa Craig inoculated with TMV were analyzed by quantitative real-time analysis. The results are as follows: Figure 4 As shown.

[0053] from Figure 4 It can be seen from this that, compared with wild-type strains and SlTOM2A Compared to the gene-edited strain Mut #5 mutant (which lacks only 19 amino acids), SlTOM2A The systemically infected leaves of the gene-edited strain Mut #13 showed extremely low viral expression levels, only 0.078% of the wild type. Further experimental results indicate that... SlTOM2A Loss of gene function significantly enhances tomato resistance to TMV, indicating that... SlTOM2A_a Genes play an important role in tomato resistance to TMV infection.

[0054] Example 4 The wild-type tomato strain Ailsa Craig (WT), in Example 1 SlTOM2A Gene-edited lines Mut #5 and Mut #13 were planted in pots, and stem growth phenotypes were collected during the flowering and fruiting stages. The results are as follows: Figure 5 As shown.

[0055] from Figure 5 It can be seen from this that SlTOM2A The stems of the gene-edited lines Mut #5 and Mut #13 both exhibited a curved growth pattern, while the stems of the wild-type tomato line Ailsa Craig (WT) showed an upright growth pattern, indicating that... SlTOM2A The gene itself plays an important role in tomato stem development, and the mutation in Mut #5 happens to occur at a site related to stem growth and development.

[0056] Example 5 To clarify the reasons for the differences in stem morphology between the gene-edited strains and the wild-type strains, the stems planted in flowerpots in Example 4 were analyzed. SlTOM2A The content and distribution of auxin and gibberellin in the stems of the gene-edited strain Mut #13 and the wild-type tomato strain Ailsa Craig (WT) grown in pots were examined and tested. The results are as follows: Figure 6 As shown.

[0057] from Figure 6As can be seen, compared with the wild-type tomato line (WT), the Mut #13 line contains more gibberellin GA3 in its shoot tip tissue and more auxin IAA-Asp on the inner side of the curved main stem. The difference in the content and distribution of these two hormones may be the reason for the curved stem growth.

[0058] In summary, the tomato plant proposed in this application... SlTOM2A The alleles, as verified by experiments, have been shown to enhance the resistance of tomato plants to tobacco mosaic virus and alter the growth morphology of tomato plants.

[0059] The above description is merely an exemplary embodiment of this application and does not limit the patent scope of this application. Any equivalent structural transformations made based on the technical concept of this application and the contents of the specification and drawings of this application, or direct / indirect applications in other related technical fields, are included within the patent protection scope of this application.

Claims

1. A tomato plant SlTOM2A Alleles, characterized by, The SlTOM2A Alleles influence tomato plants SlTOM2A The gene was obtained by editing the first and second target sites on the second exon of the gene. The nucleotide sequence of the first target is shown in SEQ ID NO.1, and the nucleotide sequence of the second target is shown in SEQ ID NO.

2.

2. The tomato plant as described in claim 1 SlTOM2A Alleles, characterized by, The SlTOM2A The amino acid sequence of the protein encoded by the allele is shown in SEQ ID NO.

3.

3. The tomato plant as described in claim 2 SlTOM2A Alleles, characterized by, The SlTOM2A The nucleotide sequence of the allele is shown in SEQ ID NO.

4.

4. A CRISPR / Cas9 vector system, characterized in that, The CRISPR / Cas9 vector system is used to edit tomato plants. SlTOM2A Genes, including first sgRNA and second sgRNA; The nucleotide sequence of the first sgRNA is shown in SEQ ID NO.5, and the nucleotide sequence of the second sgRNA is shown in SEQ ID NO.

6.

5. A host cell, characterized in that, The host cell is used to edit the tomato plant. SlTOM2A Genes, including the CRISPR / Cas9 vector system as described in claim 4.

6. The tomato plant according to any one of claims 1 to 3 SlTOM2A The application of alleles, the CRISPR / Cas9 vector system of claim 4, or the host cell of claim 5 in improving the resistance of tomato plants to tobacco mosaic virus and / or altering the stem growth morphology of tomato plants.

7. A tobacco mosaic virus-resistant tomato plant or a part thereof, characterized in that, The genome of the tobacco mosaic virus-resistant tomato plant or a portion thereof includes the tomato plant described in any one of claims 1 to 3. SlTOM2A Alleles.

8. A tomato plant or a portion thereof with a curved stem, characterized in that, The genome of the tomato plant with curved stem growth or a portion thereof includes the tomato plant described in any one of claims 1 to 3. SlTOM2A Alleles.

9. A method for improving the resistance of tomato plants to tobacco mosaic virus, characterized in that, The method includes using CRISPR / Cas9 technology to control tomato plants. SlTOM2A Genes are edited to obtain tomato plants as described in any one of claims 1 to 3. SlTOM2A Alleles.

10. A method for altering the growth shape of tomato plant stems, characterized in that, The method includes using CRISPR / Cas9 technology to control tomato plants. SlTOM2A Genes are edited to obtain tomato plants as described in any one of claims 1 to 3. SlTOM2A Alleles.

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