Application of the SlCSLA2 gene in improving the storage and transportability of tomato fruits

Abstract

This invention belongs to the field of biological breeding technology, specifically involving SlCSLA2 Application of genes in improving the storage and transportability of tomato fruit. This invention addresses the industry problem of severe storage and transport losses in tomatoes due to excessive softening after harvest. For the first time, it utilizes CRISPR / Cas9 gene editing technology to knock out the cell wall hemicellulose (mannan) synthase gene. SlCSLA2 This invention has led to the creation of a tomato mutant with significantly enhanced fruit firmness. The firmness of the entire fruit during the red-ripe stage is 35%–42% higher than that of the wild type, and the firmness can be maintained for about a week after harvest, effectively delaying the softening process and significantly improving storage and transportability. This invention provides new gene targets and technical approaches for breeding tomato varieties with improved storage and transportability.

Description

Technical Field

[0001] This invention belongs to the field of biological breeding technology, specifically involving SlCSLA2 Application of genes in improving the storage and transportability of tomato fruits. Background Technology

[0002] tomato( Solanum lycopersicum As a widely grown and consumed fruit and vegetable crop globally, the loss caused by excessive softening after harvest has always been a huge economic, resource, and environmental challenge in the industry's development. Therefore, cultivating storage-resistant varieties with softening-delaying properties is one of the important means to improve the firmness of tomato fruits and extend their storage and transportation period.

[0003] Softening is one of the most prominent characteristics of fleshy fruit ripening. It is a biological process of cell wall remodeling, with cell wall materials mainly including pectin, hemicellulose, cellulose, and some cell wall proteins. The fruit softening process involves pectin dissolution, hemicellulose depolymerization, and reduced cell adhesion. Previous research on softening has focused on primary cell wall degrading enzymes and their corresponding encoding genes, such as pectin methyl esterase (PME), polygalacturonase (PG), pectin lyase (PL), and β-galactosidase (β-GAL) involved in pectin degradation, and xylo-glucan endosyltransferase XET / hydrolase XTH and glucanase (EG) involved in cellulose and hemicellulose depolymerization. Extensin (EXP) can also participate in cell wall relaxation and promote fruit softening. In tomatoes, the transgenic functions of genes encoding related degradation enzymes, such as SlPME2, SlPG2a, SlPL, SlTBG4, SlXTH5, SlEXP1, and SlCEL2, have been verified. However, the relationship between genes encoding cell wall substance synthases and fruit softening remains largely unknown.

[0004] CSL belongs to the cellulose synthase gene superfamily and encodes a type II glycosyltransferase, primarily involved in the synthesis of the β-1,4-linked glycan backbone of hemicellulose polysaccharides. CSL contains several subfamilies, among which the CSLA subfamily is mainly involved in mannan synthesis; for example, it is knocked out in Arabidopsis thaliana. AtCSLA2 By reducing galactomannan, the structure of seed mucilage is affected, and this is related to the structure of tomato mucilage. CSLA There have been no reports on the functional studies of subfamilies. Summary of the Invention

[0005] The purpose of this invention is to provide SlCSLA2 Application of genes in improving the storage and transportability of tomato fruits.

[0006] To achieve the above objectives, the present invention provides the following technical solution: This invention provides a method for improving the storage and transportability of tomato fruits by using gene editing techniques. SlCSLA2The gene function is lost, resulting in tomato plants with increased fruit firmness.

[0007] Preferably, the gene editing is performed using the CRISPR / Cas9 system.

[0008] Preferably, it includes the following steps: (1) According to SlCSLA2 The gene sequence should include at least one sgRNA; (2) Construct sgRNA into a CRISPR / Cas9 expression vector; (3) The recombinant vector was introduced into tomatoes through Agrobacterium-mediated genetic transformation; (4) Screening SlCSLA2 Mutant plants with edited genes.

[0009] Preferably, the SlCSLA2 The nucleotide sequence of the gene is shown in SEQ ID NO. 1, and the amino acid sequence it encodes is shown in SEQ ID NO. 2.

[0010] Preferably, the sgRNA used is selected from: sgRNA1: Its sequence is shown in SEQ ID NO. 3; sgRNA2: Its sequence is shown in SEQ ID NO. 4.

[0011] Preferably, the CRISPR / Cas9 expression vector is the CDC45-1300 vector.

[0012] This invention also provides a CRISPR / Cas9 gene editing vector for improving the storage and transportability of tomato fruits, the vector containing [a specific gene editing vector]. SlCSLA2 The sgRNA expression cassette of the gene, wherein the sgRNA is selected from at least one sequence shown in SEQ ID NO. 3-4.

[0013] The present invention also provides SlCSLA2 Gene-edited tomato mutant plants, in their genome SlCSLA2 The gene coding sequence was mutated, resulting in loss of function, and its fruit firmness was higher than that of the wild-type control; the plant was obtained by any of the methods described above.

[0014] This invention also provides SlCSLA2 Application of genes in screening tomato varieties that are resistant to storage and transportation.

[0015] This invention also provides SlCSLA2 The application of gene editing in regulating tomato fruit firmness, using CRISPR / Cas9 technology to knock out... SlCSLA2Genes that improve the firmness of tomato fruits at all developmental stages and post-harvest firmness.

[0016] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention involves knocking out the cell wall synthase gene. SlCSLA2 The following significant and beneficial effects were achieved: This invention breaks away from the traditional approach of slowing down cell wall softening by inhibiting cell wall degrading enzymes. For the first time, it modifies the cell wall chassis material at its source by editing the synthase gene. slcsla2 The mutant fruit firmness was higher than that of the wild type at all developmental stages, especially at the red ripe stage where its commercial value was highest, with the whole fruit pressing firmness significantly increased by 35%–42%. This effect was verified in two independent homozygous mutant lines, indicating that it has genetic stability and reproducibility.

[0017] Postharvest placement experiments have shown that slcsla2 The mutant fruit retains its firmness for about a week after harvesting, softening significantly slower than the wild type. This suggests that the fruit may be better able to withstand the pressure and jolting during long-distance transportation, facilitating post-harvest storage, reducing post-harvest losses, and improving economic benefits.

[0018] The above results indicate that knocking out cell wall synthase genes has a positive effect on improving fruit firmness, providing a new entry point for exploring cell wall remodeling and offering a new solution for improving postharvest storage and transportation of fruits. Attached Figure Description

[0019] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0020] Figure 1 for SlCSLA2 A diagram illustrating the spatiotemporal expression pattern of genes in tomato fruits.

[0021] Figure 2 for slcsla2 The target site editing status on the mutant nucleotide sequence, (A) indicates the insertion of 1 base A, and - indicates the deletion of 2 bases.

[0022] Figure 3 for slcsla2 The truncation of the amino acid sequence in the mutant.

[0023] Figure 4 for slcsla2Analysis of the firmness of mutant tomato fruits during their developmental stages; the left figure shows the results of the whole fruit compression experiment; the right figure shows the results of the peel puncture experiment.

[0024] Figure 5 for slcsla2 Analysis of the firmness of mutant tomato fruits after harvest; the left figure shows the results of the whole fruit compression experiment; the right figure shows the results of the peel puncture experiment. Detailed Implementation

[0025] To further illustrate the present invention, the technical solutions provided by the present invention will be described in detail below with reference to the accompanying drawings and embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0026] Unless otherwise specified, the production processes, experimental methods, or testing methods involved in the embodiments of this invention are all conventional methods in the prior art, and their names and / or abbreviations are all conventional names in the field, which are very clear and distinct in the relevant application areas. Those skilled in the art can understand the conventional process steps based on the names and apply the corresponding equipment, and implement them according to conventional conditions or the conditions recommended by the manufacturer.

[0027] The various instruments, equipment, raw materials or reagents used in the embodiments of this invention are not subject to any special restrictions on their source. They are all conventional products that can be purchased through regular commercial channels and can be prepared according to conventional methods known to those skilled in the art.

[0028] Example 1 1.1 Plant materials and spatiotemporal gene expression patterns In this embodiment, Ailsa Craig tomatoes were used as the material and cultivated in the plant factory of Zhejiang University. The cultivation conditions were 16 hours of light (temperature 26-29℃) and 8 hours of darkness (temperature 17-22℃).

[0029] Gene expression analysis: SlCSLA2 The results of gene expression analysis at different developmental stages and in different tissues of tomato fruit are as follows: Figure 1 As shown in the figure. This gene is highly expressed in the early pericarp, and the transcriptome data were obtained from the SGN-TEA database.

[0030] 1.2 Construction of recombinant vectors according to SlCSLA2 The genomic nucleotide sequence: SlCSLA2 amino acid sequence MSTVQATASDIAGQIGMMWEVLKAPLLVPLLKSAVYICIVMELMLFVERLYMGIVIVLVKIFMKKPDKRYKWIPMADDDLEIGSADFPKVLVQIPMFNEKEVYKISIGAACNLSWPSDRLVIQVLDDSTDP IVKDMVETECLRWASKGLNITYQIRETRGGYKAGALKEGLKHNYVKDCEYVVIFDADFRPEPDFLRRSIPFLIHNPEIALVQGRWRFVNSNECLLTRMQEMSLDYHFTVEQEVGSSTHAFFGFNGTGGIWR IAAIDEAGGWKDRTTVEDMDLAVRASLKGWKFVYLGDLQVKSELPSTFKAFRFQQHRWSCGPANLFRKMVMEIIRNKRVNFWKKFYVIYSFFFVRKIIAHMVTFFFFCVVLPLTLLVPEVEVPIWAAIYIP CIITTLNSVGTPRSIHLLFYWILFENVMAYHRTKATFIGLLEAKRANEWVVTEKLGDALKNKDKSKPVKKARGPLFGDRILPQELGFAAFLFFCGLYDVLYGKRQYFVYVFLQVITFTIAGFGYIGTIVPS (SEQ ID NO. 2); Using online software tools, two sgRNAs with high scores and high specificity were selected from the exons, and these sequences were annealed and fused with their corresponding inverse complementary sequences. sgRNA1: CAACTGCCTCGGACATAGCG (SEQ ID NO.3) sgRNA2: CGGACATAGCGGGGCAAATT (SEQ ID NO.4) The fusion products of sgRNA1 and sgRNA2 were ligated into the intermediate vectors AtU6-18T and AtU3-18T, respectively, and transformed into DH5α competent E. coli cells by heat shock. Single colonies were selected by plate screening and sequencing. After sequencing verification, the successfully ligated plasmids were used as templates for amplification using primers U6-T FP / U6-T RP and U3-T FP / U3-T RP, respectively. The primer sequences are as follows: U6-T FP: ACGACGGCCAGTGCCAAGCTTCATTCGGAGTTTTTG (SEQ ID NO.5) U6-T RP: CATCACAGGCTCGAGCTCGAGCCATTTGTCTGCAG (SEQ ID NO.6) U3-T FP:GACAAATGGCTCGAGCTCGAGCCTGTGATGGATAAC (SEQ ID NO.7) U3-T RP: CTTTATCATCAGGAGCCCGGGAGCTCCATTTGTC (SEQ ID NO.8) After gel extraction and recovery, the amplified products were ligated at a 1:1 ratio. The ligation products were used as templates for PCR amplification using U6-T FP / U3-T RP. The products were ligated overnight with the CDC45-1300 vector and then transformed into DH5α Escherichia coli competent cells. Single colonies were screened by plate selection and sequencing. The successfully ligated plasmids were heat-transformed into GV3101 Agrobacterium competent cells for subsequent tomato genetic transformation.

[0031] Example 2: Genetic transformation using tomato cotyledon infection method This embodiment uses the tomato cotyledon infection method for genetic transformation. The specific steps are as follows: Seed disinfection and germination: Wild-type tomato seeds were soaked in tap water and shaken for 6 hours. After disinfection and cleaning with sodium hypochlorite, they were inoculated onto 1 / 2 MS solid medium (containing 2.2 g / L MS, 10 g / L sucrose, 8 g / L agar powder, and 0.1 g / L inositol). The seeds were first cultured in the dark for about 3 days until germination, and then transferred to light (25℃, 16 h light / 8 h dark) for 3-4 days until the cotyledons were fully expanded and the true leaves were just emerging.

[0032] Explant preparation and pre-culture: Cotyledons were cut off with a scalpel and placed upside down on KCMS solid medium (4.44 g / L MS, 30 g / L sucrose, 8 g / L agar powder, 0.1 g / L inositol, 1 mL / L acetylsyl syringone) lined with filter paper and cultured in the dark for 1 day.

[0033] Agrobacterium infection: Transfer cotyledons to a culture dish containing KCMS liquid medium (same composition as above, without agar), and add activated to OD... 600 Prepare a 1.0 μL Agrobacterium bacterial suspension by gently shaking the petri dish to immerse the cotyledons for 2–3 minutes. After discarding the bacterial suspension, place the infected cotyledons on sterile filter paper to blot away any remaining bacterial suspension.

[0034] Co-culture: Inoculate the cotyledons with the underside facing up onto KCMS solid medium and co-culture in the dark for 2 days.

[0035] Induction and selection of resistant shoots: After co-culture, the cotyledons were transferred face up to 2Z selection medium (4.44 g / L MS, 20 g / L sucrose, 7.4 g / L agar powder, 0.1 g / L inositol, 1 mL / L zeatin, 1.2 mL / L termethin, 60 μL / L hygromycin). After 2 weeks of culture, the samples were transferred to 0.2Z selection medium (zeatin concentration reduced to 0.1 mL / L, other components the same as 2Z). Subculture was then performed every 2 weeks until the regenerated shoots reached a length of approximately 1 cm.

[0036] Rooting and hardening off: Regenerated shoots were cut and inoculated onto rooting medium (4.44 g / L MS, 30 g / L sucrose, 8 g / L agar powder, 1.2 mL / L termethin, 60 μL / L hygromycin). After the root system was well developed and the plants were growing vigorously, they were transplanted into the soil and used as T0 generation transformation lines for subsequent identification.

[0037] Example 3: Identification and Acquisition of Mutant Materials GMO testing: In SlCSLA2 Transgenic detection primers were designed 200 bp upstream and downstream of the sgRNA. DNA was extracted from T0 generation transgenic tomato leaves using the CTAB method and used as a template for PCR amplification. The amplified product was ligated into the pGEM-T easy vector overnight at 4°C. The ligation product was then heat-shocked into DH5α competent E. coli cells, and single colonies were selected by plate screening and sequencing. The sequencing results were compared with... SlCSLA2 Nucleotide sequence alignment was used to determine the editing method of the T0 generation heterozygous transgenic material.

[0038] Homozygous screening: After the T0 generation is harvested, T1 generation transgenic materials that are homozygous and Cas9 negative are sown. DNA is extracted from the leaves of T1 generation plants and used as a template for PCR amplification. Sequencing is performed to obtain the editing mode of homozygous mutants. The DNA of the homozygous mutant is then diluted to 20 ng / μL and quantitatively analyzed by RT-qPCR using Cas9 primers. The DNA of wild-type plants at the same dilution is used as a control to screen for Cas9 negative homozygous mutants.

[0039] Genetically modified organism (GMO) detection primers: slcsla2 -FP:ACTAAACACTACCCCCTCTCT (SEQ ID NO.9) slcsla2 -RP:GGGTCTGAACAGAACAGAACGG (SEQ ID NO.10) Cas9 detection primers: Cas9-FP: CAAGGGCTACAAAGAAGTG (SEQ ID NO.11) Cas9-RP: AGTTCACATATTTGGAGGG (SEQ ID NO.12) Through screening, this invention obtained two homozygous and Cas9-negative mutant lines, which were named respectively. slcsla2 -3、 slcsla2 -14, the target editing status of its nucleotide sequence and the truncation status of its amino acid sequence are as follows: Figure 2 , Figure 3 As shown.

[0040] Example 4: Fruit firmness analysis of wild-type and mutant materials Ten days after flowering, when the fruit diameter is about 1cm, the fruit is tagged and marked. Fruit firmness analysis at different development stages: Fruits from wild-type and mutant plants at the immature green stage (about 13 days after the fruit is 1cm in diameter), green-ripe stage (about 3-5 days before the fruit breaks color), color-changing stage (the bottom of the fruit begins to change color), and red-ripe stage (seven days after the fruit breaks color) were picked and their firmness was measured.

[0041] Postharvest fruit firmness analysis: Red-ripe fruits from wild-type and mutant plants were harvested and placed on shelves. Firmness was measured on the day of harvest, 7 days post-harvest, and 14 days post-harvest. The TA-XT plus texture analyzer was used with the following parameters: pre-test speed 10 mm / s, testing speed 1 mm / s, post-test speed 10 mm / s, and measurement depth 1.5 mm. Each fruit underwent a whole-fruit compression test (P100 plate), and two puncture tests were performed at symmetrical points on the equatorial plane of the fruit using a P2 probe. The average value was recorded. At least eight individual fruits were replicated for each stage of the test. Firmness was expressed in Newtons (N).

[0042] Fruit firmness analysis results are as follows Figure 4 , Figure 5 As shown, compared with wild-type fruit, the firmness of mutant fruit was improved in the immature green fruit stage, green ripe stage, color change stage and red ripe stage. The significant difference was concentrated in the late ripening stage. For example, the whole fruit compression firmness of red ripe fruit was significantly increased (p≤0.001), with an increase of 35% to 42%. Red ripe fruit can also maintain firmness for nearly a week after harvest.

[0043] Therefore, the present invention provides knockout SlCSLA2 It is an application that delays the softening of tomato fruits and improves their storage and transportability.

[0044] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A method for improving the storage and transportability of tomato fruits, characterized in that, Tomatoes were genetically edited SlCSLA2 The gene function is lost, resulting in tomato plants with increased fruit firmness.

2. The method according to claim 1, characterized in that, The gene editing was performed using the CRISPR / Cas9 system.

3. The method according to claim 2, characterized in that, Includes the following steps: (1) According to SlCSLA2 The gene sequence should include at least one sgRNA; (2) Construct sgRNA into a CRISPR / Cas9 expression vector; (3) The recombinant vector was introduced into tomatoes through Agrobacterium-mediated genetic transformation; (4) Screening SlCSLA2 Mutant plants with edited genes.

4. The method according to claim 3, characterized in that, The SlCSLA2 The nucleotide sequence of the gene is shown in SEQ ID NO. 1, and the amino acid sequence it encodes is shown in SEQ ID NO.

2.

5. The method according to claim 3, characterized in that, The sgRNA used was selected from: sgRNA1: Its sequence is shown in SEQ ID NO. 3; sgRNA2: Its sequence is shown in SEQ ID NO.

4.

6. The method according to claim 3, characterized in that, The CRISPR / Cas9 expression vector is the CDC45-1300 vector.

7. A CRISPR / Cas9 gene editing vector for improving the storage and transportability of tomato fruits, characterized in that, The carrier contains a target SlCSLA2 The sgRNA expression cassette of the gene, wherein the sgRNA is selected from at least one sequence shown in SEQ ID NO. 3-4.

8. A kind SlCSLA2 Gene-edited tomato mutant plants are characterized by, In its genome SlCSLA2 The gene coding sequence is mutated, resulting in loss of function, and its fruit firmness is higher than that of the wild-type control; the plant is obtained by the method described in any one of claims 1-6.

9. SlCSLA2 Application of genes in screening tomato varieties that are resistant to storage and transportation.

10. SlCSLA2 The application of gene editing in regulating tomato fruit firmness is characterized by, Knockout using CRISPR / Cas9 technology SlCSLA2 Genes that improve the firmness of tomato fruits at all developmental stages and post-harvest firmness.

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