Application of SlCNR8 gene in regulation and control of drought resistance of tomatoes

Abstract

The invention relates to the technical field of biology, in particular to application of an SlCNR8 gene in regulation and control of drought resistance of tomatoes, and the amino acid sequence of protein coded by the SlCNR8 gene is shown as SEQ ID NO: 5. The gene is mutated through a CRISPR-Cas9 mediated gene editing technology, so that the function of the gene is lost, the drought stress resistance of a mutant plant is remarkably improved compared with that of a control plant, the stress resistance of a tomato plant can be improved by editing the gene, and cultivation of new varieties of stress-resistant crops is facilitated.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a... SlCNR8 Application of genes in regulating drought resistance in tomatoes. Background Technology

[0002] Tomatoes are one of the world's most important vegetable crops, occupying a vital position in the vegetable industry. With the continuous improvement of people's living standards, the demand for tomatoes is constantly increasing, which places higher demands on tomato breeding.

[0003] The growth, development, and productivity of tomatoes are severely affected by various abiotic stresses, such as salinity, drought, cold, and high temperatures. Drought is one of the most unfavorable abiotic stresses for plant growth and development, seriously threatening agricultural sustainability. Drought affects many aspects of plant physiology and causes abnormal changes in cellular processes. Therefore, improving abiotic resistance is becoming increasingly important for the sustainable production of tomatoes. Summary of the Invention

[0004] In view of this, the present invention provides a SlCNR8 The application of genes in regulating the drought resistance of tomatoes has improved the drought resistance of tomato plants.

[0005] To achieve the above objectives, the present invention adopts the following technical solution: In a first aspect, the present invention provides a SlCNR8 Gene-encoded proteins, SlCNR8 The amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO:5.

[0006] Secondly, the present invention provides an encoding of the aforementioned SlCNR8 Nucleotide molecules that encode proteins.

[0007] Preferably, the sequence of the nucleic acid molecule is selected from any one of the following (1)-(3): (1) The sequence of the nucleic acid molecule is as shown in SEQ ID NO:3 or its complementary sequence; (2) The sequence of the nucleic acid molecule is as shown in SEQ ID NO:4 or its complementary sequence; (3) A sequence containing the nucleotide sequence in (1) or (2).

[0008] Thirdly, the present invention provides an expression vector comprising the aforementioned nucleotide molecule.

[0009] Fourthly, the present invention provides a host cell containing the aforementioned nucleotide molecules, or containing the aforementioned expression vector.

[0010] Fifthly, the present invention provides the aforementioned SlCNR8 The application of proteins encoded by genes, or proteins encoded by the aforementioned nucleic acid molecules, or proteins encoded by the aforementioned expression vector containing the aforementioned nucleic acid molecules, or proteins encoded by the aforementioned host cell containing the aforementioned nucleic acid molecules, in regulating the drought resistance of tomatoes. This invention utilizes CRISPR-Cas9-mediated gene editing technology to regulate the drought resistance of tomatoes. SlCNR8 When a gene is mutated to the point of loss of function, tomato plants exhibit an increased drought resistance phenotype, thus confirming that the gene is related to the drought resistance of tomatoes.

[0011] Sixthly, the present invention provides SlCNR8 The application of the upstream sequence of the start codon or the downstream sequence of the stop codon in regulating the drought resistance of tomatoes, wherein the nucleotide sequence of the upstream sequence is shown in SEQ ID NO:1 and the nucleotide sequence of the downstream sequence is shown in SEQ ID NO:2.

[0012] In a seventh aspect, the present invention provides a method for improving the drought resistance of tomatoes by knocking out the nucleic acid molecules described in claim 2 or 3.

[0013] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention is designed SlCNR8 The target of the gene was identified, and a U6pro-Target-gRNA expression cassette was constructed. This cassette was then ligated into a pTX binary vector using homologous recombination and transformed into Agrobacterium tumefaciens. This transformed the cassette into tomato cotyledon explants, which further differentiated into shoots and induced roots, resulting in transgenic plants. Statistical analysis of the target editing and the phenotype of the mutant plants revealed that, compared to the control plants, the mutant plants exhibited significantly enhanced drought resistance. This indicates that the gene plays a crucial role in regulating the drought resistance pathway in tomatoes, and can, to some extent, improve crop stress resistance, providing genetic resources for the cultivation of high-quality tomato varieties. Attached Figure Description

[0014] Figure 1 This is a schematic diagram of mutation types provided in Embodiment 2 of the present invention; Figure 2 The AC and provided in Embodiment 2 of the present invention SlCNR8 Phenotypic diagram of mutant plants after drought treatment; Figure 3 The AC and provided in Embodiment 2 of the present invention SlCNR8 Image of NBT staining results of mutant plants after drought treatment; Figure 4 The AC and provided in Embodiment 2 of the present invention SlCNR8 Image of DAB staining results of mutant plants after drought treatment; Figure 5 The AC and provided in Embodiment 2 of the present invention SlCNR8 Statistical graph of relative electrical conductivity of mutant plants before and after drought treatment; Figure 6 The AC and provided in Embodiment 2 of the present invention SlCNR8 Statistical graph of chlorophyll content in mutant plants before and after drought treatment; Figure 7 The AC and provided in Embodiment 2 of the present invention SlCNR8 Statistical chart of malondialdehyde (MDA) content in mutant plants before and after drought treatment. Detailed Implementation

[0015] The present invention will now be described in further detail with reference to specific embodiments, so that those skilled in the art can more clearly understand the present invention.

[0016] The following embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. All other embodiments obtained by those skilled in the art based on the specific embodiments of the invention without inventive effort are within the protection scope of the invention.

[0017] Example 1 This embodiment provides a method related to the drought resistance of tomatoes. SlCNR8 The gene, the sequence of which is 5000 bp upstream of the start codon is shown in SEQ ID NO:1, and the sequence of which is 1000 bp downstream of the stop codon is shown in SEQ ID NO:2. SlCNR8 The genome sequence of the gene is shown in SEQ ID NO:3, the coding sequence is shown in SEQ ID NO:4, and the encoded protein sequence is shown in SEQ ID NO:5.

[0018] SEQ ID NO:4:ATGGCAAATACCGATGAATCGAACCCACTTTTACCCAATCAACAATCTGAGGTGAAAGATGAGAAGAATCCCAATAAACCCATTTCTTCAACAACCCCAGTTCCACCGTTTCCGGCGGATCCGGTGAAGCCTTTATCAGCCGTTGTTCCGATGGGATGGACAGTTGAAGGGGTCCCCATGGGTCATGGAGTTGTCGTTGATCCGATTATGAATAGGGCTCAGTGGGATTCTGGACTTTGTGCTTGTTTTGGAAGGACTGATGAGTTCTGCAGCAGTGATATTGAAGTTTGTTTACTAGGTAGCATGGCCCCATGCGTGCTCTACGGGAGCAATGCTGAGAGACTTGGTTCTGCTCCAGGGACTTTTGCCAATCACTGCTTGCCTTACACTGGCCTCTTCCTAATTGGACAATCCTTCTTTGGCTCGAACTGTGTGGCACCTTGCTTTACATATCCCAGTCGTACAGCTATCCGCCGGAAGTTCAATCTTGAGGGGAGCTGTGAGGCCTTCAACAGGTCTAGTGGGTGCTGCGGGAGCTTTATTGAGGACGAGGTGCAACGCGAGCAATGTGAGTCAGTTTGTGACTTTGCAACTCATTTCTTCTGCCACCCTTGTGCTCTTTGCCAAGAAGGTCGTGAGCTTCGTCGCAGGCTTCCTCATCCTGGCTTTAAGGCCCAACAAGTGCTCGTTATGATCCCCCCTAACGAGCAAACCATGGGCCGCTAA; SEQ ID NO:5:MANTDESNPLLPNQQSEVKDEKNPNKPISSTTPVPPFPADPVKPLSAVVPMGWTVEGVPMGHGVVVDPIMNRAQWDSGLCACFGRTDEFCSSDIEVCLLGSMAPCVLYGSNAERLGSA PGTFANHCLPYTGLFLIGQSFFGSNCVAPCFTYPSRTAIRRKFNLEGSCEAFNRSSSGCCGSFIEDEVQREQCESVCDFATHFFCHPCALCQEGRELRRRLPHPGFKAQQVLVMIPPPNEQTMGR.

[0019] Example 2 SlCNR8 Verification of gene function This embodiment provides a SlCNR8 Methods for verifying gene function. The background material used for tomato genetic transformation was Ailsa Craig, and the germplasm was derived from TGRC. Specific methods mainly included constructing... SlCNR8 Gene editing vectors, tomato genetic transformation SlCNR8 The detailed steps for detecting gene-edited plants and observing and statistically analyzing their phenotypic characteristics are as follows: (1) Construction SlCNR8 Gene editing vector Designed using the CRISPR direct website (http: / / crispr.dbcls.jp / ). SlCNR8 The gene has two target sites, both located in the first exon. The target fragment length is 20 bp, and the interval between the two target sites does not exceed 800 bp. Homologous recombination sequences are added to the 5' end of the target primers, and intermediate vector primers are added to the 3' end. The sequences of the target primers SlCNR8-DT1 and SlCNR8-DT2 are as follows: SlCNR8-DT1: GAATCTAACAGTGTAGTTTGCCCAGTTCCACCGTTTCCGGGTTTTAGAGCTAGAAATAG (SEQ ID NO. 6); SlCNR8-DT2: GCTATTTCTAGCTCTAAAACATGGGGACCCCTTCAACTGTCAAACTACACTGTTAGATT (SEQ ID NO. 7).

[0020] Using the intermediate vector pTX043 plasmid (Deng L, Wang H, Sun C, Li Q, Jiang H, Du M, LiC-B, Li C (2018) Efficient generation of pink-fruited tomatoes using CRISPR / Cas9 system. Journal of Genetics and Genomics 45 (1):51-54. doi:10.1016 / j.jgg.2017.10.002) as the amplification template, and SlCNR8-DT1 and SlCNR8-DT2 as amplification primers, the amplification included... SlCNR8 The gRNA sequence targeting two gene sites was obtained. The amplification system was as follows: PCR system (50 μL): 1 μL 100-200 ng / μL DNA template, 25 μL 2×Phanta Max buffer, 1 μL 10 mM dNTP Mix, 2 μL each of 10 μM primers, 1 μL Phanta Max Super-Fidelity DNA Polymerase, and 18 μL ddH2O. The amplification program was: 95℃ pre-denaturation for 3 min; 95℃ denaturation for 15 s, 56℃ annealing for 15 s, 72℃ extension for 30 s, for 35 cycles; 72℃ extension for 5 min, 12℃ for 5 min. The amplified sequence is shown in SEQ ID NO:8.

[0021] SEQ ID NO:8:

[0022] After amplification, the target fragment was recovered by gel digestion. The binary expression vector pTX041 (Deng L, Wang H, Sun C, Li Q, Jiang H, Du M, Li CB, Li C (2018) Efficientgeneration of pink-fruited tomatoes using CRISPR / Cas9 system. Journal of Genetics and Genomics 45 (1):51-54. doi:10.1016 / j.jgg.2017.10.002) was digested with Bsa1 restriction endonuclease at 37℃ for at least 3 hours. The linearized vector was recovered by gel digestion, and homologous recombination was performed on the recovered target fragment and the linearized vector. The two gRNA sequences were then ligated into the digested binary expression vector pTX041.

[0023] The recombinant expression vector was heat-transformed into Escherichia coli Trans-T1 (purchased from Beijing TransGen Biotech Co., Ltd.). After verifying the correct single clone, the bacteria were shaken and the plasmid was extracted. The recombinant plasmid was then transformed into Agrobacterium GV3101 (purchased from Shanghai Weidi Biotechnology Co., Ltd.) by electroporation or liquid nitrogen method. After verification, it can be used for subsequent tomato genetic transformation.

[0024] (2) Genetic transformation of tomatoes a. Seed preparation: Select clean, mold-free, plump seeds with a high and consistent germination rate. After separating the seeds, soak them in distilled water for about 1 hour. b. Seed disinfection and inoculation: Discard the distilled water, disinfect the seeds with 75% anhydrous ethanol for about 45 seconds, transfer the disinfected seeds to a sterile Erlenmeyer flask, and then disinfect them with a solution of sodium hypochlorite and distilled water in equal proportions by shaking for 15 minutes. After that, wash the seeds with sterile water 3-4 times and inoculate them into 1 / 2 MS medium. c. Explant preparation: 6-7 days after inoculation, when the cotyledons are fully expanded and the true leaves have not yet emerged, cut the cotyledons into small segments, place them flat on KCMS medium (purchased from Beijing Cooler Biotechnology Co., Ltd.) with the back of the leaves facing up, and incubate in the dark for about 12 hours. d. Agrobacterium infection: Centrifuge the activated Agrobacterium to retain the bacterial cells, resuspend the bacterial cells in a suspension, put the explants into the prepared infection suspension, add the Agrobacterium resuspended in the suspension, gently shake to infect for 4 minutes, discard the infection solution, blot dry the bacterial solution on the surface of the explants with sterile filter paper, and then put the explants back on KCMS medium and incubate in the dark for 2 days; e. Screening culture: Place the explants in the screening medium (purchased from Beijing Coolbo Biotechnology Co., Ltd.) and culture them for 15-20 days in a constant temperature of 25℃, 16h light and 8h dark environment. Select the explant materials that sprout green from the callus and continue to subculture until new shoots appear. f. Rooting: Once the new shoots have grown to an appropriate size, they can be transferred to a rooting medium (purchased from Beijing Coolbot Biotechnology Co., Ltd.) to induce rooting and obtain transgenic seedlings; g. Transplanting: Once the transgenic seedlings have grown to a suitable size, they can be transplanted into pots containing equal proportions of nutrient soil and vermiculite.

[0025] (3) Detection of gene-edited plants DNA was extracted from leaf samples of transgenic plants using the CTAB method. The detailed procedure was as follows: 50-100 mg of young tomato tissue was placed in a 2 mL centrifuge tube containing a steel ball, and 750 μL of extraction buffer was added. The tube was then ground at 60 Hz for 90 seconds. The ground sample was placed in a 65°C water bath for 45-90 minutes, inverting the tube approximately every 20 minutes to mix. 750 μL of a 24:1 mixture of chloroform and isoamyl alcohol was added to the centrifuge tube, and the tube was inverted approximately 100 times. After thorough mixing, the tube was centrifuged at 1000 rpm for 10 minutes. Approximately 500 μL of the supernatant was transferred to a new 1.5 mL centrifuge tube, and an equal volume of isopropanol was added. The mixture was mixed and placed at -20°C for 10 minutes. The tube was then centrifuged at 12000 rpm for 10 minutes. The supernatant was poured off, and the milky white gel-like precipitate was the DNA sample. Wash the precipitate with 500-700 μL of 75% anhydrous ethanol. After thorough washing, briefly centrifuge and discard the supernatant. Centrifuge again briefly and aspirate the supernatant with a pipette. Place the centrifuge tube open in a fume hood and air dry the milky white gel-like DNA sample until it becomes transparent. Then, add approximately 200 μL of ddH2O to dissolve the DNA. After thorough dissolution, store at -20°C for later use.

[0026] Using DNA samples as templates, vector insertion was detected using pTX-F and pTX-R primers (SEQ ID NO: 9~10). After confirming vector insertion, in... SlCNR8 Primers SlCNR8-det-F and SlCNR8-det-R (SEQ ID NO: 11~12) were designed before and after gene target editing to detect gene target editing. pTX-F: AGCGGATAACAATTTCACACAGGA (SEQ ID NO:9); pTX-R: GCAGGCATGCAAGCTTATTGG (SEQ ID NO: 10); SlCNR8-det-F: GACAAAGGACCAATTCCACACG (SEQ ID NO: 11); SlCNR8-det-R:GGTTAACAAAGCAAAGATCATCCC (SEQ ID NO: 12).

[0027] (4) Observation and statistics SlCNR8 Phenotype of gene-edited plants.

[0028] right SlCNR8Phenotypic observations were conducted on the mutant plants, with AC materials serving as a control. After seed germination, seeds with uniform germination were selected and sown in seedling trays. When the plants grew to 2-3 true leaves, they were transplanted and managed using a standardized cultivation method. When the plants grew to 5-7 true leaves, they were thoroughly irrigated the day before drought stress to ensure the soil reached saturation. The excess water was then drained the following day, and drought treatment was initiated.

[0029] See results Figure 1 , Figure 1 Targeting CRISPR-Cas9 SlCNR8 Gene targets and SlCNR8 A schematic diagram of target mutation types in mutants. SlCNR8 The target editing status of the two mutant lines (CNR8-CR-1 and CNR8-CR-2) was detected. Compared with the control WT (AC), the mutant line CNR8-CR-1 had 1 base insertion and 1 base deletion, while the mutant line CNR8-CR-2 had 267 base deletions. Figure 2 For AC and SlCNR8 Phenotypic characteristics of mutant plants after drought treatment. After 5 days of drought, AC plants began to show leaf wilting phenotypes, while SlCNR8 The mutant plants are generally healthy. Figure 3 For AC and SlCNR8 NBT staining results of the mutant plants after drought treatment showed that the superoxide anion content of AC plants was significantly increased. Figure 4 For AC and SlCNR8 DAB staining results of mutant plants after drought treatment showed that the hydrogen peroxide content of AC plants was significantly increased. Figure 5 For AC and SlCNR8 The relative electrical conductivity of mutant plants before and after drought treatment was significantly higher after drought treatment than that of AC plants. SlCNR8 The mutant plants indicate that the AC plants suffered greater cell damage. Figure 6 For AC and SlCNR8 Chlorophyll content of mutant plants before and after drought treatment; after drought treatment, the chlorophyll content of AC plants was lower than that of other mutant plants. SlCNR8 Mutant plant. Figure 7 For AC and SlCNR8 The malondialdehyde (MDA) content of mutant plants before and after drought treatment was significantly higher in AC plants after drought treatment than in AC plants. SlCNR8 The mutant plants indicate that the AC plants suffered greater cellular damage. According to... Figure 1-7 The results in Tables 1-3 show that SlCNR8 The mutant plants exhibit significantly enhanced drought resistance compared to the AC plants. All materials not specifically described in this invention are existing substances that can be purchased directly from the market.

[0030] Table 1. Statistical results of relative electrical conductivity content in tomato plant leaves

[0031] Table 2 Statistical results of chlorophyll content in tomato plant leaves

[0032] Table 3. Statistical results of malondialdehyde content in tomato plant leaves

[0033] The above are merely preferred embodiments of the present invention and are not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A kind SlCNR8 Gene-encoded proteins are characterized by, SlCNR8 The amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO:

5.

2. A coding scheme as described in claim 1 SlCNR8 Nucleotide molecules that encode proteins.

3. The nucleotide molecule according to claim 2, characterized in that, The sequence of the nucleic acid molecule is selected from any one of the following (1)-(3): (1) The sequence of the nucleic acid molecule is as shown in SEQ ID NO:3 or its complementary sequence; (2) The sequence of the nucleic acid molecule is as shown in SEQ ID NO:4 or its complementary sequence; (3) A sequence containing the nucleotide sequence in (1) or (2).

4. An expression vector comprising the nucleotide molecule of claim 2 or 3.

5. A host cell, characterized in that, It contains the nucleotide molecule as described in claim 2 or 3, or the expression vector as described in claim 4.

6. The method according to claim 1 SlCNR8 The application of a gene-encoded protein, or a protein encoded by a nucleic acid molecule as described in claim 2 or 3, or a protein encoded by an expression vector containing the nucleic acid molecule as described in claim 4, or a protein encoded by a host cell containing the nucleic acid molecule as described in claim 5, in regulating the drought resistance of tomatoes.

7. A kind SlCNR8 The application of the upstream sequence of the start codon or the downstream sequence of the stop codon in regulating the drought resistance of tomatoes, wherein the nucleotide sequence of the upstream sequence is shown in SEQ ID NO:1 and the nucleotide sequence of the downstream sequence is shown in SEQ ID NO:

2.

8. A method for improving the drought resistance of tomatoes, characterized in that, Knock out the nucleic acid molecule described in claim 2 or 3.

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