Application of TOE1 protein and its encoding gene in improving tomato tolerance to strong light stress or in strong light-tolerant breeding

Abstract

This invention discloses the application of the TOE1 protein and its encoding gene in improving the tolerance of tomatoes to strong light stress or in breeding for strong light tolerance, belonging to the field of biotechnology. This invention is the first to discover that the TOE1 protein and its encoding gene play a crucial role in regulating the strong light adaptation of tomatoes. By inactivating the TOE1 protein or knocking out its encoding gene, the anthocyanin accumulation response in tomatoes under strong light stress can be reduced, thus improving the tomato's tolerance to strong light stress. Using genome editing technologies such as CRISPR / Cas9, targeted knockout of the TOE1 protein was used to create TOE1 mutant materials. These materials can alleviate the growth burden that may be caused by the severe photosensitivity response induced by excessive light in summer or in high-light areas of greenhouse agriculture, helping plants maintain more stable photosynthetic growth under strong light conditions, and providing core germplasm resources for breeding new tomato varieties adapted to high-light environments.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to the application of the TOE1 protein and its encoding gene in improving the tolerance of tomatoes to strong light stress or in breeding for strong light tolerance. Background Technology

[0002] tomato( Solanum lycopersicum Tomato is an important vegetable crop in the Solanaceae family, possessing both nutritional and economic value, and plays a vital role in both greenhouse and open-field cultivation. Sufficient and suitable sunlight is crucial for ensuring normal photosynthesis in tomatoes. However, in actual production, especially during summer in open-field or greenhouse cultivation when ventilation and shading measures are inadequate, tomatoes often suffer from strong light stress. When the light intensity exceeds their suitable growth range, even reaching 500 μmol (photons) m... -2 s -1 The above conditions will have many adverse effects on the growth and development of tomatoes, becoming an important adverse factor that restricts the improvement of tomato yield and quality.

[0003] Strong light stress causes multi-dimensional and systemic damage to tomatoes. It not only directly causes morphological damage but also disrupts their physiological metabolic balance, ultimately leading to reduced yield and deterioration in quality. At the morphological level, direct sunlight can cause tomato leaves to turn pale and white, wither and turn yellow, and the leaf margins to dry out. The sun-facing side of the fruit is prone to sunburn, resulting in yellow-white spots, thinning of the skin, and shriveling, thus losing its commercial value. In addition, strong light stress can induce a large accumulation of reactive oxygen species (ROS), triggering oxidative stress and damaging cell structure.

[0004] To address the constraints of strong light stress on tomato production, breeding tomato varieties tolerant to strong light stress has become an important research direction in the field of tomato breeding. Accurate and rapid evaluation of the tolerance of tomato materials to strong light stress is the prerequisite and foundation for conducting strong light-tolerant breeding work. Anthocyanins, as an important class of water-soluble pigments in plants, not only give plant tissues diverse colors but also play a crucial protective role in the plant's response to abiotic stress. Under strong light stress conditions, anthocyanin synthesis in tomatoes is significantly induced, and its content changes systematically with increasing stress intensity and duration. This change is clearly correlated with the tomato's tolerance to strong light stress: tomato materials with strong light tolerance have stronger self-regulation capabilities and do not need to synthesize large amounts of anthocyanins to resist the damage caused by strong light stress, thus their anthocyanin content is lower; while tomato materials with weak light tolerance are unable to resist the damage caused by strong light stress and need to synthesize more anthocyanins to try to alleviate the stress damage, thus their anthocyanin content is relatively higher. Compared with traditional physiological and biochemical indicators and growth indicators, using anthocyanin content as a morphological marker of tomato's tolerance to strong light stress has the advantages of being simple, rapid, low-cost, and applicable on a large scale. By detecting the level of anthocyanin content, the strength of its resistance to strong light can be characterized—the lower the anthocyanin content, the stronger the tomato's resistance to strong light stress, and vice versa. This feature enables rapid screening of tomato materials resistant to strong light, significantly improving breeding efficiency.

[0005] TOE1 (Target of EAT1) is a known transcription factor in plants. The inventors discovered this in previous research (CN109423492A): SlTOE1 Genes are related to the flowering time and yield of tomatoes. SlTOE1 Gene silencing can promote early flowering and high yield in tomatoes. However, SlTOE1 The relationship between genes and tomato tolerance to strong light stress has not yet been reported. Summary of the Invention

[0006] In view of the above-mentioned prior art, the purpose of this invention is to provide the application of TOE1 protein and its encoding gene in improving the tolerance of tomatoes to strong light stress or in strong light-tolerant breeding.

[0007] To achieve the above objectives, the present invention adopts the following technical solution: In a first aspect, the present invention provides the application of TOE1 protein in improving the tolerance of tomatoes to strong light stress or in breeding for strong light tolerance; the amino acid sequence of said TOE1 protein is shown in SEQ ID NO.1.

[0008] In the above applications, the tolerance of tomatoes to strong light stress is improved by inactivating the TOE1 protein.

[0009] In the above applications, the light intensity of the intense light stress is greater than or equal to 500 μmol m. -2 s -1 .

[0010] As a feasible approach, the synthesis of TOE1 protein can be inhibited at its source by gene editing or gene silencing technology, thereby inactivating the TOE1 protein. Alternatively, a specific agent can be sprayed exogenously to directly act on the TOE1 protein in plant cells, destroying its structure or inhibiting its activity, thereby inactivating the TOE1 protein.

[0011] In a second aspect, the invention provides the application of the gene encoding the TOE1 protein in improving the tolerance of tomatoes to strong light stress or in breeding for strong light tolerance. The gene encoding the TOE1 protein is a nucleic acid molecule as shown in (i) or (ii) below: (i) Nucleic acid molecules with nucleotide sequences as shown in SEQ ID NO.2; (ii) Nucleic acid molecules other than (i) that encode the amino acid sequence shown in SEQ ID NO.1.

[0012] In the above applications, the tolerance of tomatoes to strong light stress was improved by targeting and knocking out the gene encoding the TOE1 protein.

[0013] Preferably, the gene encoding the TOE1 protein is knocked out using the following substances: e1) Nucleic acid molecules that target and knock out the gene encoding the TOE1 protein; e2) An expression cassette containing the nucleic acid molecule described in e1); e3) A recombinant vector containing the nucleic acid molecule described in e1), or a recombinant vector containing the expression cassette described in e2); e4) Recombinant microorganisms containing the nucleic acid molecules described in e1), or recombinant microorganisms containing the expression cassette described in e2), or recombinant microorganisms containing the recombinant vector described in e3).

[0014] In a preferred embodiment of the present invention, a CRISPR / Cas9 editing vector carrying a specific target is used to target and knock out the gene encoding the TOE1 protein; The nucleotide sequence of the specific target is shown in SEQ ID NO.3.

[0015] In the above applications, the light intensity of the intense light stress is greater than or equal to 500 μmol m. -2 s -1 .

[0016] The beneficial effects of this invention are: (1) This invention is the first to discover that the TOE1 protein and its encoding gene play a key role in regulating the strong light adaptation of tomatoes. By inactivating the TOE1 protein or knocking out the encoding gene of the TOE1 protein, the anthocyanin accumulation response of tomatoes under strong light stress can be reduced, thereby improving the tolerance of tomatoes to strong light stress.

[0017] (2) Using the gene encoding the TOE1 protein as a target, the gene was knocked out using CRISPR / Cas9 and other genome editing technologies to create a toe1 mutant material. This can reduce the growth burden that may be caused by the intense photoprotective response induced by excessive light in summer or high-light areas of facility agriculture (such as the use of a large amount of resources for anthocyanin synthesis), and help the plant maintain more stable photosynthetic growth under strong light conditions, providing core germplasm resources for the cultivation of new tomato varieties adapted to high-light environments. Attached Figure Description

[0018] Figure 1 CRISPR / Cas9 vector targeted editing TOE1 A schematic diagram of a gene.

[0019] Figure 2 : TOE1 Genotyping of the gene-edited mutant (toe1) shows a deletion mutation at the target site; in the figure, A is the sequencing peak of toe1-2; B is the sequencing peak of toe1-25.

[0020] Figure 3 Wild-type tomatoes (WT) and TOE1 Anthocyanin content in hypocotyls of gene-edited mutant seedlings (toe1-2, toe1-25) under normal light and strong light treatment.

[0021] Figure 4 Wild-type tomatoes (WT) and TOE1 ROS fluorescence imaging of gene-edited mutant seedlings (toe1-2, toe1-25) under normal light and strong light treatment.

[0022] Figure 5 Wild-type tomatoes (WT) and TOE1 Bar chart showing the quantitative determination of ROS fluorescence intensity of gene-edited mutant seedlings (toe1-2, toe1-25) under normal light and strong light treatment. Detailed Implementation

[0023] It should be noted that the following detailed descriptions are illustrative and intended to provide further explanation of this application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.

[0024] As mentioned earlier, TOE1 (Target of EAT1) is a known transcription factor in plants. The amino acid sequence of the TOE1 protein in tomato is shown in SEQ ID NO.1; its encoding gene... TOE1 The nucleotide sequence is shown in SEQ ID NO.2; specifically as follows: TOE1 protein: MLDLNVSVIYNNDLPQVSLLDESATSNSSLRNAEATTSAGDEDSCAGELFAFNFGILKVEGAETSRSSNNDDEEGYGKNQRVTHSQFVTRQLFPVDDGELNRKQTDRVILSSARSGTS IGFGDVRIIQQQQTEQPKQQVKKSRRGPRSRSSQYRGVTFYRRRTGRWESHIWDCGKQVYLGGFDTAHTAARAYDRAAIKFRGVDADINFSLSDYEEDMQQMKNLGKEEFVHLLRRHSTG FSRGSSKFRGVTLHKCGRWEARMGQFLGKKYIYLGLFDSEVEAARAYDKAAIKTSGREAVTNFEPSSYEGETMSLPQSEGSQHDLDLNLGISTTSSKENDRLGGSRYHPYDMQDATKP KMDKPGSVIVGSSHLKGLPMSSQQAQLWTGIYSNFSSSYEGRAYDKRKDTGSSQGPPNWALQMPSQVDTNSPLTMFCTAASSGFFIPSTTTSITSSTSALATSTNASQCFYQINPRLPLP .

[0025] TOE1 Gene:

[0026] The inventors discovered in their previous research that RNAi technology can partially silence the red blood cells in tomatoes. TOE1 Genes can promote earlier flowering in tomatoes and increase tomato yield.

[0027] To further develop TOE1 This invention addresses the problem of light stress caused by excessive sunlight in greenhouse agriculture during summer or in high-light areas by utilizing CRISPR / Cas9 gene editing technology to modify the gene in tomatoes. TOE1 Gene knockout, an unexpected discovery: Knockout TOE1 The tomato mutant material after gene modification showed significantly enhanced tolerance to strong light stress.

[0028] To enable those skilled in the art to better understand the technical solution of this application, the technical solution of this application will be described in detail below with reference to specific embodiments.

[0029] The test materials used in the embodiments of this invention are all conventional test materials in the art and can be purchased through commercial channels. Experimental methods without specified detailed conditions are performed according to conventional test methods or the supplier's recommended operating instructions. Wherein: The wild-type tomato uses the conventional tomato variety "M82", which is recorded in patent CN109423492A.

[0030] Differentiation medium: MS powder 4.43 g / L, sucrose 30 g / L, kanamycin 50 mg / L, auxin 1 mg / L, cytokinin 1.75 mg / L.

[0031] Rooting medium: MS powder 4.43 g / L, sucrose 30 g / L, kanamycin 50 mg / L.

[0032] Example 1: Construction of a CRISPR / Cas9 editing vector targeting the TOE1 gene According to publicly available information about tomatoes TOE1 The gene sequence (SEQ ID NO.2) was used to select a highly specific target sequence from its first exon region (target sequences: GCGGAAGCTACAACCAGTGC; ATCGGGTCATTCTCTCTCTC; SEQ ID NO.3). The corresponding oligonucleotide sequence was synthesized, annealed, and ligated into the BsaI-digested pTX041 vector to construct the recombinant editing vector pTX041-TOE1-sgRNA. Sequencing verified the correct vector construction.

[0033] Example 2: Genetic transformation and obtaining toe1 mutant plants Genetic transformation: The correctly constructed pTX041-TOE1-sgRNA vector was electroporated into Agrobacterium tumefaciens GV3101 competent cells. Using the Agrobacterium-mediated tomato cotyledon transformation method (refer to Deng L, Wang H, Sun C, Li Q, Jiang H, Du M, Li CB, Li C. Efficient generation of pink-fruited tomatoes using CRISPR / Cas9 system. J Genet Genomics. 2018;45(1):51-54. doi: 10.1016 / j.jgg.2017.10.002.), the above-mentioned Agrobacterium was used to infect cotyledonary explants of cultivated tomato 'M82'. After co-culture, the explants were transferred to differentiation medium and cultured until callus tissue differentiated into seedlings. The seedlings were then transferred to rooting medium to induce rooting, obtaining resistant regenerated plants (T0 generation).

[0034] Mutant screening and identification: Genomic DNA was extracted from leaves of T0 generation plants and used to screen for mutants. TOE1 PCR amplification was performed using primers designed to flank the gene editing target. The primer sequences are shown below: F: 5'-GCGGAAGCTACAACCAGTGC-3'; (SEQ ID NO.4) R: 5'-ATCGGGTCATTCTCTCCTC-3'. (SEQ ID NO.5) PCR products were sequenced and compared with wild-type sequences to screen for plants with frameshift mutations (such as base deletions or insertions) at the target site. Figure 1 , Figure 2 Through self-pollination, homozygous toe1 mutant lines (toe1-2 and toe1-25) without the CRISPR / Cas9 transgenic backbone were obtained for subsequent phenotypic analysis.

[0035] Example 3: Tolerance of the toe1 mutant to strong light stress Material treatment: Homozygous toe1 mutants (toe1-2 and toe1-25) obtained in Example 2 and their corresponding wild-type (WT) tomato seeds were simultaneously disinfected, germinated, and sown. After the seedlings grew under normal white light for 5 days, seedlings with uniform growth were selected and placed under normal light (control, light intensity 100 μmol / m²). -2 s -1 ) and strong light (light intensity 500 μmol m -2 s -1 Continue culturing for 3 days under the following conditions.

[0036] Phenotypic observation: Under strong light conditions, the leaves of the toe1 mutant are dark green and uniform, with thick, flat leaves, compact plant type, thick stems and short internodes; while the leaves of the wild-type tomato ("M82") are light green, with some curling and wilting, taller and more elongated plant height, and thin and long internodes.

[0037] Anthocyanin quantitative determination: Hypocotyls of the two groups of materials were collected separately, and anthocyanins were extracted with 1 mL of extraction solution (propanol: hydrochloric acid: water = 18:1:81 = V:V:V) in the dark. The absorbance values ​​at wavelengths of 535 nm and 650 nm were measured, and the anthocyanin content (A535–A650) / g material was calculated.

[0038] Anthocyanin content serves as a morphological marker of tomato tolerance to strong light stress. The anthocyanin content determination results for different materials are as follows: Figure 3 As shown, the results indicated that compared to wild-type tomato (“M82”), the toe1 mutant exhibited significantly lower anthocyanin content under strong light conditions, with a statistically significant difference (P<0.01). Therefore, knockout... TOE1 Genes can improve the tolerance of tomato seedlings to strong light stress.

[0039] ROS quantification: The total reactive oxygen species level of the hypocotyl was detected using 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA). The procedure is as follows: Hypocotyls were stained in 10 mmol / L DCFH-DA solution for 30 minutes in the dark, followed by rinsing at least twice with 10 mmol / L phosphate buffer (pH 7.4). The stained hypocotyls were observed and photographed using a Nikon Eclipse Ni fluorescence microscope, and the green fluorescence intensity of the hypocotyls was analyzed using ImageJ software.

[0040] ROS fluorescence imaging, such as Figure 4 As shown in the bar chart, the quantitative determination of fluorescence intensity is as follows: Figure 5 As shown in the figure. The results showed that under strong light conditions, the ROS fluorescence intensity of the toe1 mutant was significantly lower than that of the wild-type control, and the statistical difference was extremely significant (P<0.01).

[0041] In summary, strong light stress leads to a large accumulation of anthocyanins and ROS in tomato seedlings. Targeted knockout of these substances can help address this issue. TOE1 The gene can significantly slow down the rise of anthocyanins and ROS, thereby improving the tolerance of tomato seedlings to strong light stress.

[0042] Example 4: Application of the toe1 mutant in strong light tolerance breeding The homozygous toe1 mutant lines (toe1-2 and toe1-25) obtained in Example 2 were used as high-light-tolerant germplasm resources and hybridized with high-yielding, high-quality commercial tomato cultivars. In the subsequent segregating populations, new tomato lines or varieties possessing both high-light tolerance and excellent agronomic traits can be rapidly bred for use in open-field or greenhouse production in high-light-intensity areas.

[0043] The above description is merely a preferred embodiment of this application and is not intended to limit this application. Various modifications and variations can be made to this application by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this application should be included within the protection scope of this application.

Claims

1. The application of TOE1 protein inactivation in improving tomato tolerance to strong light stress or in strong light tolerance breeding, characterized by, The amino acid sequence of the TOE1 protein is shown in SEQ ID NO.1; The intensity of the intense light stress is greater than or equal to 500 μmol / m³. -2 s -1 .

2. The application of targeted knockout of the gene encoding the TOE1 protein in improving the tolerance of tomatoes to strong light stress or in strong light tolerance breeding, characterized in that... The gene encoding the TOE1 protein is a nucleic acid molecule as shown in (i) or (ii) below: (i) Nucleic acid molecules with nucleotide sequences as shown in SEQ ID NO.2; (ii) Nucleic acid molecules other than (i) that encode the amino acid sequence shown in SEQ ID NO.1; The intensity of the intense light stress is greater than or equal to 500 μmol / m³. -2 s -1 .

3. The application according to claim 2, characterized in that, The gene encoding the TOE1 protein can be knocked out using the following substances: e1) Nucleic acid molecules that target and knock out the gene encoding the TOE1 protein; e2) An expression cassette containing the nucleic acid molecule described in e1); e3) A recombinant vector containing the nucleic acid molecule described in e1), or a recombinant vector containing the expression cassette described in e2); e4) Recombinant microorganisms containing the nucleic acid molecules described in e1), or recombinant microorganisms containing the expression cassette described in e2), or recombinant microorganisms containing the recombinant vector described in e3).

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