A method for promoting ripening and quality enhancement of tomatoes

Abstract

This invention discloses a method for promoting tomato ripening and improving its quality by silencing or knocking out the XERICO gene in tomatoes, wherein the XERICO gene is the XERICO1 gene and / or the XERICO3 gene. This method can promote tomato ripening and improve its quality, specifically by advancing tomato ripening, increasing fruit ethylene production, and improving tomato fruit quality.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to a method for promoting tomato ripening and improving its quality. Background Technology

[0002] The ubiquitin-proteasome pathway (UPS) is a highly selective protein degradation pathway in eukaryotes. This pathway involves: first, ubiquitin activator E1 activates ubiquitin under ATP-fueled conditions; then, ubiquitin is transferred to ubiquitin conjugator E2, where it is linked to a cysteine ​​residue at the active site of E2 via a thioester bond. E2 can then directly transfer ubiquitin to a lysine residue of the target protein. E3 ubiquitin ligases, which couple ubiquitin to homologous substrates, play a crucial role in the ubiquitin pathway. All E3 ligases possess the ability to link target proteins to specific E2 ligases. Protein-specific post-translational modifications often serve as markers for recognition by their corresponding ubiquitin ligase E3. E3 ubiquitin ligases can be classified into four types based on structure and function: HECT, RING, U-box, and RBR.

[0003] Tomato (Solanum lycopersicum L.) is an annual or perennial herbaceous plant belonging to the genus Solanum in the family Solanaceae. Also known as tomato or foreign persimmon, it is the most widely cultivated and consumed vegetable crop in the world. Due to its relatively short growth cycle, increasingly mature greenhouse cultivation technology, and its status as an important climacteric fruit, it has gradually become one of the important model plants for plant molecular research.

[0004] Previous research results indicate that E3 ubiquitin ligases may play different regulatory roles in different plants and at different stages of plant growth and development or under stress. For example, inhibiting AtATL78 in Arabidopsis can increase tolerance to cold stress and decrease tolerance to drought stress (Kim, Soo Jin and Kim, Woo Taek (2013), Suppression of Arabidopsis RING E3 ubiquitin ligase AtATL78 increases tolerance to cold stress and decreases tolerance to drought stress, FEBS Letters, 587, doi:10.1016 / j.febslet.2013.06.038); overexpression of CHYR1 can promote the regulation of stomatal movement and improve drought resistance (Shuangcheng Ding, Bin Zhang, Feng Qin, Arabidopsis RZFP34 / CHYR1, a Ubiquitin E3Ligase, Regulates Stomatal Movement and Drought Tolerance via SnRK2.6-Mediated Phosphorylation, The Plant Cell, Volume 27, Issue 11, November 2015, Pages 3228–3244, https: / / doi.org / 10.1105 / tpc.15.00321); Overexpression of XERICO can improve drought tolerance in Arabidopsis thaliana (Ko, J.-H., Yang, SHand Han, K.-H. (2006), Upregulation of an Arabidopsis RING-H2 gene, XERICO, confers drought tolerance through increased abscisic acid biosynthesis. The Plant Journal, 47:343-355. https: / / doi.org / 10.1111 / j.1365-313X.2006.02782.x); Overexpression of OsCTR1 in rice can improve drought tolerance (LIM, SD, LEE, C. and JANG, CS (2014), OsCTR1 in trafficking inhibition of interactors. Plant Cell Environ, 37:1097-1113.https: / / doi.org / 10.1111 / pce.12219; Inhibition of OsHTAS can suppress heat tolerance in rice (Jianping Liu, Cuicui Zhang, Chuchu Wei, Xin Liu, Mugui Wang, Feifei Yu, QiXie, Jumin Tu, The RING Finger Ubiquitin E3 Ligase OsHTAS Enhances Heat Tolerance by Promoting H2O2-Induced Stomatal Closure in Rice, Plant Physiology, Volume 170, Issue 1, January 2016, Pages 429–443, https: / / doi.org / 10.1104 / pp.15.00879).

[0005] However, the functions of the RING-type E3 ubiquitin ligase genes SlXERICO1 and SlXERICO3 in tomato leaves in fruit ripening and quality improvement have not yet been reported. Summary of the Invention

[0006] This invention provides a method for promoting tomato ripening and improving quality. It can inhibit the expression of the tomato XERICO1 gene and / or XERICO3 gene through CRISPR / Cas9 technology to produce XERICO1, XERICO3, and XERICO1 / 3 (which jointly inhibit the XERICO1 and XERICO3 genes) mutant plants, which can promote tomato ripening and improve quality. Specifically, it can promote earlier tomato ripening, increase fruit ethylene production, and improve tomato fruit quality.

[0007] A method for promoting tomato ripening and quality improvement involves silencing or knocking out the XERICO gene in tomatoes, wherein the XERICO gene is the XERICO1 gene and / or the XERICO3 gene.

[0008] The nucleotide sequence of the XERICO1 gene is shown in SEQ ID No. 1, and the nucleotide sequence of the XERICO3 gene is shown in SEQ ID No. 2.

[0009] Preferably, the method may specifically include the following steps:

[0010] (1) Construct a gene silencing or knockout vector, wherein the gene silencing or knockout vector is a plant expression vector containing sequences for silencing or knocking out the XERICO1 gene with a base sequence as shown in SEQ ID No. 1 and / or the XERICO3 gene with a base sequence as shown in SEQ ID No. 2;

[0011] (2) Introduce the gene silencing or knockout vector from step (1) into tomato cells to silence or knock out the XERICO1 gene with the base sequence shown in SEQ ID No. 1 and / or the XERICO3 gene with the base sequence shown in SEQ ID No. 2.

[0012] In a preferred embodiment, the method for promoting tomato ripening and quality improvement involves introducing a CRISPR / Cas9 vector containing the tomato XERICO1 gene and / or XERICO3 gene into the target tomato to suppress its expression, thereby obtaining mutant plants with suppressed expression of the XERICO1 gene and / or XERICO3 gene.

[0013] Suppressing the expression of XERICO1, XERICO3, and XERICO1 / 3 genes in the target tomato can be achieved by any method that reduces the expression of these genes in the target tomato. In one specific embodiment of the invention, suppressing the expression of XERICO1, XERICO3, and XERICO1 / 3 genes in the target tomato is achieved by constructing a CRISPR / Cas9 vector containing these genes and then infecting them with Agrobacterium.

[0014] Further preferably, the method specifically includes the following steps:

[0015] 1) Construct an engineered Agrobacterium tumefaciens strain containing a CRISPR / Cas9 vector containing the tomato XERICO1 gene and / or XERICO3 gene;

[0016] 2) Transform the Agrobacterium tumefaciens engineered bacteria into the target tomato explants to prepare mutant plants with suppressed expression of the tomato XERICO1 gene and / or XERICO3 gene.

[0017] Preferably, in step 1), the engineered Agrobacterium tumefaciens strain is Agrobacterium GV3101.

[0018] Preferably, in step 2), the explant is a cotyledon that has been germinated for 6 to 8 days.

[0019] In the above specific method, preferably, the mutant plants with suppressed expression of the tomato XERICO1 gene and / or XERICO3 gene are subjected to normal growth management to obtain transgenic F2 generation and subsequent seeds with stable inheritance.

[0020] The specific manifestations of the method described in this invention in promoting tomato ripening include accelerating the ripening speed of tomato fruits and shortening the ripening time of tomato fruits. The specific manifestations of promoting tomato quality improvement include increasing the fructose and glucose content and decreasing the citric acid and malic acid content in tomato fruits.

[0021] In one experimental design, the specific steps are as follows:

[0022] 1. Constructing Agrobacterium tumefaciens engineered strains containing CRISPR / Cas9 vectors of tomato XERICO1, XERICO3 and XERICO1 / 3 genes;

[0023] 2. The *Agrobacterium tumefaciens* engineered bacteria were used to transform the target tomato explants to prepare XERICO1, XERICO3 and XERICO1 / 3 gene mutant plants;

[0024] 3. The XERICO1, XERICO3 and XERICO1 / 3 gene mutant plants were subjected to normal growth management to obtain transgenic F2 generation and subsequent seeds with stable inheritance.

[0025] 4. Experiments were conducted using transgenic F2 generation and later seeds to observe the fruit ripening stage of tomato plants.

[0026] The lower the expression levels of the XERICO1 and XERICO3 genes in tomatoes, the faster the tomato fruits mature and the better their quality.

[0027] The XERICO1, XERICO3, and XERICO1 / 3 genes can be introduced into target tomatoes using recombinant expression vectors containing these genes. Existing plant expression vectors such as pFGC1008, pFGC5941, pCAMBIA1300, and pBI121, or other derived plant expression vectors, can be used. When constructing recombinant vectors using plant expression vectors, constitutive, tissue-specific, or inducible promoters can be used.

[0028] The tomato XERICO1 and XERICO3 genes are specifically described below:

[0029] Mature sequences of tomato XERICO1 and XERICO3 were obtained from the Solanaceae Genomics Network (https: / / solgenomics.net / ) database. The base sequences are shown in SEQ ID NO: 1 and 2.

[0030] This invention provides the application of the tomato XERICO1 and XERICO3 genes in regulating tomato fruit ripening and quality. The nucleotide sequence of the XERICO1 gene is shown in SEQ ID No. 1, and the nucleotide sequence of the XERICO3 gene is shown in SEQ ID No. 2.

[0031] Compared with the prior art, the beneficial effects of this invention are as follows:

[0032] This invention uses CRISPR / Cas9 technology to suppress the expression of the tomato XERICO1 and XERICO3 genes, producing XERICO1, XERICO3, and XERICO1 / 3 mutant plants, which can promote tomato fruit ripening and improve fruit quality. Specifically, it promotes earlier ripening, increases fruit ethylene production, and improves tomato fruit quality. Attached Figure Description

[0033] Figure 1 This is a schematic diagram illustrating the construction of a CRISPR / Cas9 vector for large fragment deletion. Figure 1 A is a diagram showing the location and structure of the vector gRNAs; Figure 1 B. Figure 1 C Figure 1 D represents the location and sequence of two gRNAs in the tomato XERICO1, XERICO3, and XERICO1 / 3 genomes, with the gRNA-specific recognition sequence underlined.

[0034] Figure 2 Identification of mutant plants. Figure 2 A represents two mutant lines obtained from SlXERICO1, namely slxerico1#3 and slxerico1#5. Figure 2 B represents two mutant lines obtained from SlXERICO3, namely slxerico3#2 and slxerico3#4. Figure 2 C represents two mutant lines obtained from SlXERICO1 and SlXERICO3, namely slxerico1 / 3#1 and slxerico1 / 3#2.

[0035] Figure 3 The ripening status of wild-type tomatoes and SlXERICO1, SlXERICO3, and SlXERICO1 / 3 mutants. Figure 3In the series, Slxerico1-1 corresponds to the mutant line slxerico1#3, Slxerico1-2 corresponds to the mutant line slxerico1#5; Slxerico3-1 corresponds to the mutant line slxerico3#2, Slxerico3-2 corresponds to the mutant line slxerico3#4; and Slxerico1 / 3 corresponds to the mutant line slxerico1 / 3#1. Figure 3 A represents the fruit ripening time of SlXERICO1, SlXERICO3, and SlXERICO1 / 3 mutants, dpa represents the number of days after flowering, and the data shown in the figure is the average of four replicates. The standard error is shown by the vertical line. Figure 3 B represents the fruit phenotypes of SlXERICO1, SlXERICO3, and SlXERICO1 / 3 mutants, with a scale bar of 1 cm.

[0036] Figure 4 Ethylene yield in fruits of wild-type and tomato mutants XERICO1, XERICO3, and XERICO1 / 3. dpa represents days after flowering. The data shown in the figure are the average of four replicates, with standard error indicated by vertical lines. The Turkey test was used; an asterisk indicates a 5% significance level difference between different lines and the wild type.

[0037] Figure 5 Fruit firmness was measured in wild-type and tomato mutants XERICO1, XERICO3, and XERICO1 / 3. dpa represents days after flowering. The data shown in the figure are the average of four replicates, with the standard error indicated by the vertical line. The Turkey test was used, with an asterisk indicating a 5% significance level difference between different lines and the wild type.

[0038] Figure 6 The pigment content was measured in wild-type and tomato XERICO1, XERICO3, and XERICO1 / 3 mutant plants. Figure 6 A represents the carotenoid content. Figure 6 B represents lycopene content. dpa represents the number of days after flowering. The data shown in the figure is the average of three replicates, and the standard error is indicated by the vertical line. The Turkey test was used; an asterisk indicates a 5% significance level difference between different strains and the wild type.

[0039] Figure 7 The sugar and acid content of wild-type and tomato XERICO1, XERICO3, and XERICO1 / 3 mutant plants. Figure 7 A represents the soluble sugar content. Figure 7 B represents the organic acid content. Figure 7 C represents the sugar-acid ratio. dpa represents the number of days after flowering. The data shown in the figure is the average of three replicates, and the standard error is indicated by the vertical line. The Turkey test was used; an asterisk indicates a 5% significance level difference between different strains and the wild type. Detailed Implementation

[0040] The present invention will be further described below with reference to the accompanying drawings and specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Operating methods not specifically specified in the following embodiments are generally performed under conventional conditions or as recommended by the manufacturer.

[0041] Unless otherwise specified, all experimental materials and reagents used in the following examples are commercially available. The experimental material used was the tomato cultivar Alisa Craig (Solanum lycopersicum L.cv).

[0042] Example 1

[0043] Cloning and vector construction of the tomato SlXERICO1 / 3 gene.

[0044] 1. Construction of CRISPR / Cas9 vectors

[0045] The tomato SlXERICO1 and SlXERICO3 genes, with full lengths of 459 bp and 456 bp respectively, are both composed of a single exon. Using the CRISPR-P website (http: / / crispr.hzau.edu.cn / CRISPR2 / ), the U6 promoter was selected to screen for sgRNAs in the SlXERICO1 and SlXERICO3 genes. For the SlXERICO1 and SlXERICO3 genes, two sgRNAs from their respective exons were selected for the CRISPR / Cas9 vectors. Each sgRNA had one forward primer and one reverse primer for PCR to obtain fragments containing the sgRNA. The forward primer for the first sgRNA used in each vector was a universal forward primer. The PCR primer sequences are shown in Table 1. Figure 1 This is a schematic diagram illustrating the construction of a CRISPR / Cas9 vector for large fragment deletion. Figure 1 A is a diagram showing the location and structure of the vector gRNAs; Figure 1 B. Figure 1 C Figure 1D represents the location and sequence of the two gRNAs in the tomato XERICO1, XERICO3, and XERICO1 / 3 genomes, with the gRNA-specific recognition sequence underlined. The specific construction process was as follows: using the tRNA vector as a template, PCR was performed on both target sites using KOD high-fidelity enzyme, and the PCR products were purified to a fragment size of approximately 200 bp. Multi-fragment recombination was performed using the Golden Gate Assembly Kit (BsaI-HFv2) (NEB, E1601). The recombination system included Golden Gate Assembly Mix, PHEE401 vector, target PCR purified products, and T4 DNA ligase buffer, brought to a final volume of 20 μL with ddH2O. PCR program settings were performed according to the kit instructions. The recombinant product was heat-transferred to *E. coli* competent cells (TransGen, CD201). Following the instructions, the cells were activated at 37°C and shaken at 200 rpm for 1 hour. The supernatant was discarded by centrifugation, leaving 100–200 μL of liquid. The precipitate was resuspended and evenly spread onto solid LB medium containing 50 mg / L kanamycin, and incubated overnight at 37°C. Once single colonies appeared, colonies were picked and gently shaken. Positive clones were identified by colony PCR using universal M13-F and M13-R primers and sequenced. The positive plasmids were named pHEE401-SlXERICO1, pHEE401-SlXERICO3, and pHEE401-SlXERICO1 / 3, respectively.

[0046] Table 1. PCR primer sequences for constructing CRISPR / Cas9 vectors

[0047]

[0048] 2. Total RNA extraction from tomatoes

[0049] Total RNA was extracted from tomatoes using a plant total RNA extraction kit (Tiangen, DP419). The steps were as follows:

[0050] (1) Take 0.1g of leaf and grind it in liquid nitrogen, add 1mL of lysis buffer RZ, and vortex mix well;

[0051] (2) Let it stand at room temperature for 5 minutes to allow the nucleic acid-protein complex to completely separate;

[0052] (3) Centrifuge at 4℃ and 12000rpm for 5min, remove the supernatant and transfer to a new RNase-free centrifuge tube;

[0053] (4) Add 200 μL of chloroform, cap the tube, shake vigorously for 15 seconds, and let stand at room temperature for 3 minutes.

[0054] (5) Centrifuge at 4℃ and 12000rpm for 10min. The sample will separate into three layers: a yellow organic phase, a middle layer, and a colorless aqueous phase. RNA is mainly in the upper aqueous phase, and the volume of the aqueous phase is approximately 50% of the RZ lysis buffer used. Transfer the aqueous phase to a new tube for the next step.

[0055] (6) Slowly add 0.5 times the volume of anhydrous ethanol and mix well (precipitation may occur at this time). Transfer the resulting solution and precipitate together into the adsorption column CR3, centrifuge at 12000 rpm for 30 s at 4℃, and discard the waste liquid in the collection tube;

[0056] (7) Add 500 μL of protein removal solution RD to the adsorption column CR3, centrifuge at 12000 rpm for 30 s at 4℃, and discard the waste liquid.

[0057] (8) Add 600 μL of washing solution RW to the adsorption column CR3, let it stand at room temperature for 2 min, centrifuge at 12000 rpm for 30 s at 4℃, and discard the waste liquid.

[0058] (9) Repeat step (8);

[0059] (10) Place the adsorption column into a 2mL collection tube, centrifuge at 4℃ and 12000rpm for 2min to remove residual waste liquid;

[0060] (11) After the adsorption column was dried in the clean bench for 5 min, it was transferred to a new RNase-free centrifuge tube, 50 μL of RNase-Free ddH2O was added, and the tube was placed at room temperature for 2 min. Then, it was centrifuged at 12000 rpm for 2 min at 4℃.

[0061] (12) OD was measured using an ultraviolet spectrophotometer. 260 / OD 280 The RNA sample was tested for content and purity at a concentration of 781 ng / μL, OD 260 / OD 280 =2.10.

[0062] 3. Gene cloning and construction of engineered Agrobacterium tumefaciens bacteria

[0063] A reverse transcription kit (Vazyme, R223) was used to remove genomic DNA from total RNA and reverse it to cDNA. The obtained CRISPR / Cas9 vectors pHEE401-SlXERICO1, pHEE401-SlXERICO3, and pHEE401-SlXERICO1 / 3 were transformed into Agrobacterium tumefaciens GV3101, resulting in Agrobacterium tumefaciens engineered strain A containing the tomato SlXERICO1 gene CRISPR / Cas9 vector, Agrobacterium tumefaciens engineered strain B containing the tomato SlXERICO3 gene CRISPR / Cas9 vector, and Agrobacterium tumefaciens engineered strain C containing the tomato SlXERICO1 / 3 gene CRISPR / Cas9 vector, respectively.

[0064] Example 2

[0065] Tomato SlXERICO1 / 3 gene mutant plants were constructed.

[0066] Using the leaf disc method, tomato cotyledons were infected with Agrobacterium-mediated transformation. The target vectors CRISPR / Cas9 vectors pHEE401-SlXERICO1, pHEE401-SlXERICO3, and pHEE401-SlXERICO1 / 3 were transformed into tomato cotyledons, respectively. Hygromycin was used for screening, and PCR amplification and first-generation sequencing were used to screen mutant plants.

[0067] The specific steps are as follows:

[0068] 1) Preparation of culture medium

[0069] Sowing medium: 2.15 g / L MS powder + 100 mg / L inositol + 10 g / L sucrose + 8 g / L agar. pH 5.8.

[0070] Nursing medium: 4.44 g / L MS powder + 30 g / L sucrose + 100 mg / L inositol + 1.3 g / L thiamine hydrochloride + 0.2 mg / L 2,4-D + 200 mg / L KH2PO4 + 0.1 mg / L KT + 7.5 g / L agar. pH 5.8.

[0071] 2Z regeneration medium: 4.44 g / L MS powder + 30 g / L sucrose + 100 mg / L inositol + 2 mg / L ZR + 300 mg / L timentin + 6 mg / L hygromycin. pH 5.8.

[0072] 0.2Z selective regeneration medium: 4.44 g / L MS powder + 30 g / L sucrose + 100 mg / L inositol + 0.2 mg / L ZR + 300 mg / L timentin + 6 mg / L hygromycin. pH 5.8.

[0073] Rooting medium: 4.44 g / L MS powder + 30 g / L sucrose + 100 mg / L inositol + 300 mg / L timentin + 6 mg / L hygromycin. pH 5.8.

[0074] Liquid MS 0.2 medium: 4.44 g / L MS powder + 20 g / L sucrose + 100 mg / L inositol + 0.2 mg / L thiamine hydrochloride. pH 5.8. Used for suspension infection of Agrobacterium.

[0075] YEB medium: 5g beef extract, 5g peptone, 1g yeast extract, 5g sucrose, 0.5g MgSO4·7H2O, bring to a final volume of 1L with distilled water, adjust pH to 7.0, and autoclave at 121℃ for 20 minutes. For YEB solid medium, add 15g agar powder per liter, and other components are the same as for liquid medium.

[0076] 2) Cultivation of sterile seedlings

[0077] Tomato seeds were soaked in tap water (or shaken at 28℃ and 200 rpm) for 6–8 hours, then disinfected with 75% alcohol for 30 seconds, followed by disinfection in 10% NaClO for 15 minutes (shaking at 28℃ and 200 rpm). The seeds were rinsed three times with sterile distilled water and transferred to sterile containers, inoculated onto half a volume of MS medium. After culturing in the dark at 25℃ until seedlings showed signs of sprouting, they were transferred to a light-controlled culture chamber. Seedling growth conditions were 25℃, 16 hours of light / 8 hours of darkness, and a light intensity of 1800 lx.

[0078] 3) Prepare explants and culture Agrobacterium.

[0079] About a week after seed germination, before the cotyledons have unfolded but the true leaves have emerged, cut the cotyledons of the sterile seedlings into two sections with a new scalpel, leaving a small section of petiole attached. Lay these sections flat on a nurturing medium and pre-culture for 24 hours (avoid light, overnight is sufficient; prolonged nurturing can lead to over-infection). Pick a single colony of Agrobacterium from an LB agar plate containing antibiotics and inoculate it into 30 mL of LB agar (or a 150 mL Erlenmeyer flask) containing antibiotics. Incubate overnight at 28°C and 200 rpm until mid-log phase (OD600≈1.0, approximately 16–24 hours). Shake the culture first, then cut the cotyledons (inoculate between 12 and 20 hours).

[0080] 4) Conversion and Regeneration

[0081] The engineered Agrobacterium tumefaciens strains A-C containing the target vector plasmid were removed from a -80℃ freezer and activated on YEB plates containing the corresponding antibiotics. Single colonies of Agrobacterium were picked and inoculated into 2 mL of YEB containing the antibiotics. The culture was incubated overnight at 28℃ with shaking at 200 rpm. Then, the culture was expanded at a 1:100 ratio to 30 mL and incubated overnight at 28℃ with shaking at 200 rpm until OD reached [value missing]. 600 =0.8~1.0. Centrifuge the cultured Agrobacterium at 4000 rpm for 10 min at 4℃; discard the supernatant, and add 15 mL of MS 0.2 suspension medium to resuspend the bacteria for later use. Transfer the pre-cultured cotyledon explants to a sterile petri dish containing 15 mL of MS 0.2, pour in the resuspended bacterial solution, and incubate in the dark for 2–3 min, gently agitating the dish. Gently lift the explants with tweezers, transfer them to sterile filter paper, blot off any remaining bacterial solution, and then place them back into the original nucleating medium, reverse side up. Co-culture at 22℃ in the dark for 48 h.

[0082] After co-culturing, the explants were transferred face up onto 2Z medium and cultured at 25°C under a 16-hour light / 8-hour dark light cycle. The 2Z medium was replaced with fresh medium every two weeks. Once shoots differentiated, the browned explants were removed, and the differentiated shoots were transferred to 0.2Z medium for selective culture. The medium was replaced with fresh medium every three weeks.

[0083] 5) Rooting culture and transplanting

[0084] When the regenerated shoots grow to about 1cm, cut them off (or leave them uncut to avoid damaging the rooting area) and place them in a rooting medium to root. Two weeks later, harden off the well-rooted seedlings that have grown to about 5cm and transplant them into nutrient pots with a peat moss:vermiculite = 3:1 substrate to obtain tomato SlXERICO1 / 3 gene mutant plants.

[0085] Example 3

[0086] Molecular detection of transgenic plants: PCR was used to detect the SlXERICO1 / 3 mutant tomato plants at the DNA level.

[0087] DNA was rapidly extracted from transgenic tomato plants in small quantities using the CTAB method, as follows:

[0088] 1. Take 50-100 mg of tomato leaves into a 1.5 mL centrifuge tube, add steel balls, quickly freeze in liquid nitrogen, grind the sample into powder, and add 500 μL of CTAB buffer.

[0089] Incubate in a 2.55℃ water bath for 15 minutes, inverting and mixing several times during the process;

[0090] 3. Add 500 μL of chloroform:isoamyl alcohol (24:1), vortex to mix, and centrifuge at 12000 rpm for 5 min;

[0091] 4. Transfer the supernatant to a new centrifuge tube, add 1 / 10 volume of sodium acetate (3M) and 2 volumes of ice-cold anhydrous ethanol, vortex to mix, and precipitate at -20°C for 1 hour.

[0092] 5. Centrifuge at 12000 rpm for 3 minutes. A white precipitate will be visible at the bottom, which is DNA. Discard the supernatant.

[0093] 6. Add 1 mL of pre-cooled 70% ethanol, shake and wash, centrifuge at 12000 rpm for 1 min, and discard the supernatant;

[0094] 7. Repeat the washing once to remove as much residual liquid as possible, dry on a clean bench, dissolve in 50 μL ddH2O, and store at -20℃.

[0095] Specific primers were designed near the sgRNA sequence positions of the tomato SlXERICO1 / 3 gene to detect changes in the target gene sequence. The primers are shown below, with SlXERICO1 / 3 detection fragment lengths of 731 bp and 453 bp, respectively. Homozygous mutants of SlXERICO1 / 3 were screened based on sequencing results of the PCR products. PCR results were submitted for analysis, and the results are shown below. Figure 2 A, 2B, and 2C. Figure 2 Figure A shows two lines of SlXERICO1 mutant plants, slxerico1#3 and slxerico1#5. slxerico1#3 has one base inserted at the first exon, while slxerico1#5 has 16 bases deleted at the first exon, causing the SlXERICO1 protein to terminate prematurely at amino acids 93 and 52, respectively. Figure 2 Figure B shows two lines of the SlXERICO3 mutant plant, slxerico3#2 and slxerico3#4. slxerico3#2 has a deletion of 1 base in the first exon, while slxerico3#4 has a deletion of 2 bases in the first exon, causing the SlXERICO3 protein to terminate prematurely at 106 and 86 amino acids, respectively. Figure 2Figure C shows two lines of the SlXERICO1 / 3 mutant plant, slxerico1 / 3#1 and slxerico1 / 3#2. slxerico1 / 3#1 has a deletion of one base in the first exon of SlXERICO1 and a deletion of one base in the first exon of SlXERICO3, causing premature termination of the SlXERICO1 and SlXERICO3 proteins at amino acids 98 and 106, respectively. slxerico1 / 3#2 has an insertion of one base in the first exon of SlXERICO1 and a deletion of two bases in the first exon of SlXERICO3, causing premature termination of the SlXERICO1 and SlXERICO3 proteins at amino acids 59 and 86, respectively.

[0096] The specific primer sequences are as follows:

[0097] CRISPR-SlXERICO1-F: 5′-TACTTGAAGTCCTATGTTTCTTCTCTA-3′ (SEQ ID No. 13);

[0098] CRISPR-SlXERICO1-R: 5′-CATTGGACATGTATCGTCCTCTTC-3′ (SEQ ID No. 14);

[0099] CRISPR-SlXERICO3-F: 5′-ATGGGACTCTCACCATATACGACTC-3′ (SEQ ID No. 15);

[0100] CRISPR-SlXERICO3-R: 5′-CATTGGACAAGTATCTTCCTCACCT-3′ (SEQ ID No. 16).

[0101] Example 4

[0102] The fruit ripening stage of the obtained tomato XERICO1, XERICO3, and XERICO1 / 3 gene mutant plants was observed. The experimental materials were: wild-type WT, and mutant plants XERICO1-1, XERICO1-2, XERICO3-1, XERICO3-2, and XERICO1 / 3. For all transgenic plants, F2 generation seeds with stable inheritance were used for subsequent experimental observation.

[0103] Phenotypic observation and physiological index measurement of the plants during the fruiting period were conducted. Seedlings were managed routinely in the growth chamber. Once the wild-type WT plants reached six leaves and one bud, all lines were transplanted to an artificial greenhouse. The culture conditions were: 200 μmol / L... -2 s-1 Light intensity (PPFD), 12-hour photocycle, temperature 25 / 20℃ (day / night), water with Hoagland nutrient solution every 2-3 days.

[0104] (1) Statistics on fruit ripening

[0105] The number of days from sowing to the opening of the first inflorescence and the number of days for fruit color development were recorded. The number of days from flowering to fruit color development was also recorded for different strains. Starting with the fully enlarged, green-ripe fruit of the wild type, the fruit phenotypes of different strains in this invention were photographed and recorded. The results are as follows: Figure 3 As shown.

[0106] Note: Figure 3 The data shown in A is the average of four replicates. A Turkey test was used, and the standard error is shown by a vertical line.

[0107] Phenotypic records were made by photographing the fruit from its green-ripe to red-ripe stage, such as... Figure 3 As shown, the fruit ripening process of the XERICO1, XERICO3, and XERICO1 / 3 mutant lines was faster than that of the wild type.

[0108] (2) Determination of ethylene release from fruit

[0109] The ethylene release rate of tomato fruits at different ripening stages was determined. At least two fruits from each stage were weighed and placed in a 550 mL sealed container at room temperature, protected from light, for 1 hour. Using a syringe marked with 1 mL graduations, more than 1 mL of headspace gas was drawn from the rubber tube at the top of each container, four times per container, constituting four biological replicates. The needles were inserted into prepared corks according to their corresponding labels. After the samples from each stage were collected, they were injected sequentially into a gas chromatograph (Philips, UNICAM pro.GC) for analysis. A 1500 × 4 mm alumina glass column was used. The injector, detector, and column temperature were 130℃, 130℃, and 200℃, respectively. For each measurement, 1 mL of gas was injected quantitatively for detection. Based on the reference peak position of standard ethylene gas (10 μL / L), the corresponding peak time and peak area were obtained using the analysis software provided with the gas chromatograph. The ethylene concentration in each sample was calculated, and the ethylene release per kilogram of sample per hour was then calculated. The results are as follows: Figure 4 As shown in the figure. The data displayed is the average of four repetitions, and the standard error is shown by the vertical line.

[0110] Depend on Figure 4 It can be seen that at 41 and 45 days after flowering, the ethylene production of XERICO1, XERICO3, and XERICO1 / 3 mutant lines was higher than that of the wild type, indicating that the XERICO1 and XERICO3 genes affect the release of ethylene from the fruit to some extent.

[0111] (3) Quality index determination

[0112] ① Fruit firmness test

[0113] The ethylene release rate of tomato fruits at different ripening stages was measured. At least three fruits at different stages were used. About 1 cm of peel was removed from the top of the fruit and the stem end. Using a GY-1 type fruit hardness tester, the probe of the hardness tester was held perpendicular to the fruit surface and pressed down with steady force so that the probe penetrated 2 mm into the diaphragm. The reading shown by the hardness tester at this time is the hardness of the flesh of the fruit. The test was performed on both sides.

[0114] Figure 5 Fruit firmness was measured in wild-type and tomato mutants XERICO1, XERICO3, and XERICO1 / 3.

[0115] Note: dpa refers to days after flowering. The data shown in the figure is the average of four replicates, and the standard error is indicated by the vertical line. The Turkey test was used; an asterisk indicates a 5% significance level difference between different strains and the wild type.

[0116] Depend on Figure 5 It can be seen that, 41 and 43 days after flowering, the firmness of XERICO1, XERICO3 and XERICO1 / 3 mutant lines decreased faster than that of wild type, indicating that the XERICO1 and XERICO3 genes affect the firmness and quality of fruit to some extent.

[0117] ② Extraction and content detection of carotenoids from fruits

[0118] Weigh 0.5g of tomato pulp and grind it thoroughly with liquid nitrogen, then transfer it to a 2mL centrifuge tube. Add 350μL of methanol, 700μL of chloroform, and 350μL of ddH2O in sequence, then vortex to mix. Centrifuge at 10000g for 10 min at 4℃, and collect the chloroform phase. Add another 700μL of chloroform to the remaining residue tube, repeating this extraction process several times until colorless. Combine the collected chloroform phases and dry the collection tube with nitrogen. Then add 350μL of methanol solution (w / v) containing 6% KOH to dissolve the precipitate, and derivatize at 60℃ in the dark for 30 min. Add 700μL of chloroform and 350μL of water, vortex to mix, and centrifuge at 10000g for 5 min at 4℃, collecting the chloroform phase. Add 700μL of water to the chloroform phase and extract again, repeating this process several times until the aqueous layer is neutral. The collected chloroform phase was dried under nitrogen, dissolved in 100 μL of chromatographic grade ethyl acetate, and centrifuged at 14000 g for 20 min at 4 °C to ensure complete settling of the precipitate. 150 μL of the supernatant was collected for high-performance liquid chromatography (HPLC) analysis. At least five biological replicates should be performed for the above procedure.

[0119] The injection procedure for carotenoids is described below. An Alliance 2695 system (Waters Corporation, USA) was used, containing the 2695 separation module and the 2996 PDA detector, equipped with a 5 μm C30 reversed-phase column (250 mm × 4.6 mm) and a 20 mm × 4.5 mm C30 pre-column (Waters Corp.). The column temperature was set to 25 °C, the flow rate to 1 mL / min, the injection volume to 20 μL, and the detection wavelength range to 220–600 nm. The mobile phase used a gradient elution with solvents A (methanol), B (80% methanol), and C (MTBE): 95% A + 5% B for 0–6 min, 80% A + 5% B + 15% C for 7–11 min, 30% A + 5% B + 65% C for 12–32 min, and 95% A + 5% B for 48–50 min, remaining unchanged until the end of the procedure, which lasted approximately 60 min. Data analysis was then performed using Waters Empower software. Based on the retention time and absorption spectrum curves of the standards, the various carotenoid components were systematically analyzed and identified. The results are as follows: Figure 6 As shown.

[0120] Figure 6 The pigment content was measured in wild-type and tomato XERICO1, XERICO3, and XERICO1 / 3 mutant plants. Figure 6 A represents the carotenoid content. Figure 6 B represents the lycopene content.

[0121] Note: dpa represents the number of days after flowering. The data shown in the figure is the average of three replicates, and the standard error is indicated by the vertical line. The Turkey test was used; an asterisk indicates that the mutant plants differed from the wild type at a 5% significance level.

[0122] ③ Determination of soluble sugar and organic acid content in fruit

[0123] Grind 0.2g of fruit pulp into powder using liquid nitrogen, add 1mL of chromatographic methanol, vortex to mix, heat in a rotating metal bath at 70℃ for 15min at 950rpm, centrifuge at 10000g for 10min at room temperature, transfer the supernatant to a 1.5mL centrifuge tube, take 100μL of the supernatant into a new 1.5mL centrifuge tube, add 20μL of ribitol (0.2mg / mL) as an internal standard, vacuum dry at room temperature for several hours, then add 400μL of freshly prepared methoxyamine hydrochloride solution (20mg / mL, pyridine soluble), heat in a rotating metal bath at 37℃ for 1.5h at 950rpm, then add 600μL of bis(trimethylsilyl)trifluoroacetamide (1% trimethylchlorosilane), and continue heating at 37℃ for 30min at 950rpm. After extraction, the analytical instrument used was a Shimadzu GC-MS-QP2010 plus, with a VF-5MS column (30m × 0.25mm × 0.25μm). The carrier gas was helium (He), and the column flow rate was 1 mL / min. -1 Weighing was performed using a Metteller balance with a weight of 0.01%.

[0124] Figure 7 The sugar and acid content of wild-type and tomato XERICO1, XERICO3, and XERICO1 / 3 mutant plants. Figure 7 A represents the soluble sugar content. Figure 7 B represents the organic acid content. Figure 7 C represents the sugar-acid ratio. Compared with the wild type, the XERICO1, XERICO3, and XERICO1 / 3 mutant lines had higher fructose and glucose contents, no significant difference in sucrose, and lower citric acid and malic acid contents.

[0125] Note: dpa represents the number of days after flowering. The data shown in the figure is the average of three replicates, and the standard error is indicated by the vertical line. The Turkey test was used; an asterisk indicates that the mutant plants differed from the wild type at a 5% significance level.

[0126] Depend on Figure 6 and Figure 7 It can be seen that, 41 and 45 days after flowering, compared with the wild type, the XERICO1, XERICO3 and XERICO1 / 3 mutant lines had higher contents of carotenoids and lycopene, higher contents of soluble sugars, lower contents of organic acids, and a larger sugar-acid ratio, indicating that the XERICO1 and XERICO3 genes have affected the color and flavor quality of the fruit to a certain extent.

[0127] The results of this invention show that the lower the expression levels of the XERICO1 and XERICO3 genes in tomatoes, the faster the tomato fruits mature and the better the fruit quality.

[0128] Furthermore, it should be understood that after reading the above description of the present invention, those skilled in the art can make various alterations or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. A method of promoting ripening of tomatoes, characterized in that, Tomato in XERICO gene silencing or knock-out, said XERICO gene is XERICO1 genes and / or XERICO3 genes.

2. The method of claim 1, wherein, The XERICO1 The nucleotide sequence of the gene is shown in SEQ ID No.

1. XERICO3 The nucleotide sequence of the gene is shown in SEQ ID No.

2.

3. The method of claim 1, wherein, The method includes the following steps: (1) Construct a gene silencing or knockout vector, wherein the gene silencing or knockout vector is a vector containing a base sequence as shown in SEQ ID No.

1. XERICO1 Gene and / or base sequence as shown in SEQ ID No. 2 XERICO3 Plant expression vectors containing sequences for silencing or knocking out genes; (2) Introduce the gene silencing or knockout vector from step (1) into tomato cells, and make the base sequence as shown in SEQ ID No.

1. XERICO1 Gene and / or base sequence as shown in SEQ ID No. 2 XERICO3 Gene silencing or knockout.

4. The method of claim 1, wherein, Import tomatoes into the target tomatoes XERICO1 Genes and / or XERICO3 The gene was repressed using a CRISPR / Cas9 vector to obtain XERICO1 Genes and / or XERICO3 Mutant plants with suppressed gene expression.

5. The method of claim 4, wherein, The method includes the following steps: 1) construction of Agrobacterium tumefaciens engineering bacteria containing CRISPR / Cas9 vector of tomato SlMYB12 gene and / or SlMYB58 gene XERICO1 XERICO3 gene and / or​ 2) The engineered Agrobacterium tumefaciens was transformed into the target tomato explants to prepare tomatoes. XERICO1 Genes and / or XERICO3 Mutant plants with suppressed gene expression.

6. The method of claim 5, wherein, In step 1), the engineered Agrobacterium tumefaciens strain is Agrobacterium GV3101.

7. The method according to claim 5, characterized in that, In step 2), the explant is the cotyledon of the seed 6-8 days after germination.

8. The method of claim 5, wherein, The tomatoes XERICO1 Genes and / or XERICO3 Seeds of the F2 generation and beyond, which are transgenic plants with suppressed gene expression, are obtained by normal growth management.

9. The method according to claim 1, characterized in that, The specific ways in which the method promotes tomato ripening include accelerating the ripening speed of tomato fruits and shortening the ripening time of tomato fruits.

10. Silencing or knocking out tomato XERICO1 genes, XERICO3 genes in promoting ripening of tomato fruits, characterized in that, The XERICO1 The nucleotide sequence of the gene is shown in SEQ ID No.

1. XERICO3 The nucleotide sequence of the gene is shown in SEQ ID No. 2.

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