SlECT1, a gene that regulates the synthesis of aromatic substances in tomato fruit, and its application.

Abstract

This invention provides a gene, SlECT1, that regulates the synthesis of aromatic compounds in tomato fruits and its applications. Using CRISPR / Cas9 technology, this invention edited the SlECT1 gene in tomatoes, obtaining the SlECT1 gene mutant lines slect1#1 and slect1#2. The aromatic compound content in the mature fruits of these two lines increased to 166.86% and 149.63% of the wild type, respectively, demonstrating that the SlECT1 gene has the function of regulating the synthesis of aromatic compounds in tomato fruits. The SlECT1 gene described in this invention can provide a candidate gene resource for improving the flavor of tomato fruits through genetic engineering breeding.

Description

Technical Field

[0001] This invention belongs to the fields of plant molecular biotechnology and genetic engineering technology, specifically relating to a gene SlECT1 that regulates the synthesis of aromatic substances in tomato fruits and its applications. Background Technology

[0002] Fruits are an important part of the human diet, and their quality mainly includes color, flavor, texture, and nutrition. Fruit quality is a crucial indicator affecting fruit consumption, with aroma quality being closely related to consumer preferences. With advancements in technology, research on fruit aroma quality has rapidly progressed, becoming a research hotspot in the field of fruit quality biology. Tomatoes, with their high-quality genome annotation, short growth cycle, and efficient genetic transformation system, are ideal materials for studying fruit quality regulation. Therefore, research on the synthesis and regulatory mechanisms of aromatic substances in tomato fruits has significant theoretical and practical value for improving fruit quality.

[0003] As the fruit ripens, aromatic substances gradually accumulate and are released. The aromatic compounds in tomato fruits are complex, with over 400 species identified to date, primarily including aldehydes, ketones, alcohols, and terpenes. Several genes involved in the synthesis of aromatic substances in tomato fruits have been successfully identified, including SlLIP1, SlLIP8, SlLOXC, SlHPL, SlADH2, and SlAAT1 in the fatty acid pathway; SlCCD1 in the carotenoid pathway; and SlBCAT1 and SlTNH1 in the branched-chain amino acid pathway. Currently, research on the mechanisms of aromatic quality formation in tomato fruits mainly focuses on structural gene screening, transcriptional regulation, and DNA methylation; whether other levels of synergistic regulation exist requires further exploration.

[0004] The central dogma states that genetic information is transcribed from DNA into RNA, and then translated from RNA into proteins with different functions; epigenetics also consists of these three parts. Each step of gene expression is finely regulated, and through epigenetic modifications, the same nucleotide sequence can transmit different heritable genetic information. Building on research into DNA methylation and histone modifications, reversible RNA methylation has gradually become a research hotspot in the field of epigenetics. 6 -Methyladenine (N 6 -methyladenosine,m 6 A) is the most common post-transcriptional RNA modification in eukaryotes, which can be dynamically regulated in time and space by methyltransferases and demethylases. 6 A-binding protein binds to m 6 The selective binding of A-modifications exerts biological functions, such as regulating mRNA cleavage, translation, nucleus export, and degradation. Previous studies have found that m... 6The A-binding protein SlECT2 plays a role in regulating the synthesis of aromatic substances in tomato fruit, but other m 6 The role of A-binding proteins in the formation of aroma quality in tomato fruits remains unclear. Summary of the Invention

[0005] The purpose of this invention is to provide and validate a gene, SlECT1, that regulates the synthesis of aromatic substances in tomato fruits.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0007] This invention provides a gene, SlECT1, that effectively regulates the synthesis of aromatic substances in tomato fruits. The nucleotide sequence of the SlECT1 gene is shown in SEQ ID NO.1; the amino acid sequence encoded by the SlECT1 gene is shown in SEQ ID NO.2.

[0008] This invention provides a CRISPR / Cas9 gene editing vector for SlECT1 gene mutation, the vector being pYLCRISPR / Cas9-SlECT1. The vector can express sgRNA targeting the sequence SEQ ID NO.1. The sgRNA sequence is T1, T2, T3, and T4, where T1 is nucleotides 207-226 of SEQ ID NO.1, T2 is nucleotides 776-795 of SEQ ID NO.1, T3 is nucleotides 942-961 of SEQ ID NO.1, and T4 is nucleotides 1554-1573 of SEQ ID NO.1.

[0009] The pYLCRISPR / Cas9-SlECT1 gene editing vector utilizes the BsaI restriction endonuclease in pYLCRISPR / Cas9-P ubi The recombinant vectors were obtained by sequentially inserting AtU3d-T1-gRNA, AtU3b-T2-gRNA, AtU6-1-T3-gRNA, and AtU6-29-T4-gRNA into the -H multiple cloning site.

[0010] The present invention provides a microorganism containing the pYLCRISPR / Cas9-SlECT1 recombinant vector, wherein the microorganism is Agrobacterium GV3101.

[0011] The purpose of this invention is to provide the application of the SlECT1 gene in regulating the synthesis of aromatic substances in tomato fruit. The SlECT1 gene in mutant tomatoes can significantly increase the content of aromatic substances in tomato fruit.

[0012] Another objective of this invention is to provide the application of the gene SlECT1 as an important candidate gene for tomato genetic engineering breeding in improving the aroma quality of tomato fruits.

[0013] The beneficial effects of this invention are as follows: This invention provides the application of the SlECT1 gene in regulating the synthesis of aromatic substances in tomato fruits. Knocking out the SlECT1 gene can significantly increase the content of aromatic substances in tomato fruits, and can provide candidate gene resources for genetic engineering to cultivate tomato germplasm with excellent aromatic quality. Attached Figure Description

[0014] Figure 1 The four target sites of SlECT1 (T1, T2, T3, T4) and their locations on the SlECT1 gene are shown.

[0015] Figure 2 The gene editing methods used in the SlECT1 gene mutant lines slect1#1 and slect1#2.

[0016] Figure 3 The results show the protein mutations in the SlECT1 gene mutant lines slect1#1 and slect1#2.

[0017] Figure 4 The results of electronic nose analysis on mature fruits of wild-type and SlECT1 gene mutant lines.

[0018] Figure 5 The statistical results show the content of aromatic substances in mature fruits of wild-type and SlECT1 gene mutant lines. Detailed Implementation

[0019] The present invention will be further described in detail below with reference to specific embodiments. These embodiments are merely illustrative and not intended to limit the scope of the invention. Unless otherwise specified, the experimental methods in the following embodiments are conventional methods. Unless otherwise specified, the materials, reagents, instruments, etc., used are all commercially available.

[0020] The wild-type tomato variety Solanum lycopersicum cv Ailsa Craig, abbreviated as AC (Marian Bemer et al., The Tomato FRUITFULL Homologs TDR4 / FUL1 and MBP7 / FUL2 Regulate Ethylene-Independent Aspects of Fruit Ripening[J].The Plant Cell,2012,24(11):4437-4451), is available to the public from the applicant. This biological material is only used to repeat the relevant experiments of this invention and may not be used for other purposes.

[0021] Example: SlECT1 gene mutation leads to increased aromatic substance content in tomato fruit

[0022] This embodiment uses CRISPR / Cas9 technology to edit the SlECT1 gene in tomatoes. After mutation, the content of aromatic substances in mature tomato fruits is significantly increased compared with wild type. The DNA sequence of the SlECT1 gene is SEQ ID NO.1 in the sequence listing, and the amino acid sequence is SEQ ID NO.2 in the sequence listing. The specific steps are as follows:

[0023] (I) Construction of pYLCRISPR / Cas9-SlECT1 gene editing vector

[0024] Select target sequences: target site T1 is nucleotides 207-226 of SEQ ID NO.1, target site T2 is nucleotides 776-795 of SEQ ID NO.1, target site T3 is nucleotides 942-961 of SEQ ID NO.1, and target site T4 is nucleotides 1554-1573 of SEQ ID NO.1.

[0025] Promoter selection: AtU3d, an endogenous Arabidopsis thaliana, was selected as the promoter for target site T1; AtU3b was selected as the promoter for target site T2; AtU6-1 was selected as the promoter for target site T3; and AtU6-29 was selected as the promoter for target site T4.

[0026] Preparation of sgRNA expression cassettes containing target sites: After two rounds of PCR, four DNA fragments were obtained: AtU3d-T1-gRNA, AtU3b-T2-gRNA, AtU6-1-T3-gRNA, and AtU6-29-T4-gRNA. AtU3d-T1-gRNA is the sgRNA expression cassette for AtU3d initiating target site T1, AtU3b-T2-gRNA is the sgRNA expression cassette for AtU3b initiating target site T2, AtU6-1-T3-gRNA is the sgRNA expression cassette for AtU6-1 initiating target site T3, and AtU6-29-T4-gRNA is the sgRNA expression cassette for AtU6-29 initiating target site T4. The preparation method is as follows: The first round of PCR reaction is a 25 μl system: KOD enzyme (TOYOBO) 0.5 μL, 10×KODPlus Buffer (TOYOBO) 2.5 μL, dNTP 2.5 μL, MgSO4 1.5 μL, pYLgRNA-LacZ-AtU3d / AtU3b / AtU6-1 / AtU6-29 plasmid 1 μL, UF primer (5 μmol / L) 1 μL, gR-R primer (5 μmol / L) 1.0 μL, gR-SlECT1-T1 / T2 / T3 / T4 primer 2.5 μL, AtU-SlECT1-T1 / T2 / T3 / T4 primer 2.5 μL, ddH2O 10 μL. The pYLgRNA-LacZ-AtU3d, pYLgRNA-AtU3b, pYLgRNA-AtU6-1, and pYLgRNA-AtU6-29 plasmids are all described in the literature (Maet al., A Robust CRISPR / Cas9 System for Convenient, High-Efficiency Multiplex Genome Editing in Monocot and Dicot Plants[J]. Molecular Plant, 2015, 8(8):1274-1284). The first round of PCR reaction program was: 94℃ for 60s; 94℃ for 10s, 58℃ for 15s, 68℃ for 20s, 28 cycles; 68℃ for 7min. After the first round of PCR reaction, four PCR products were obtained, and then the second round of PCR reaction was performed.The second round of PCR consisted of a 20 μl system: 0.5 μL KOD enzyme (TOYOBO), 2.0 μL 10×KOD Plus Buffer (TOYOBO), 2.0 μL dNTPs, 1.2 μL MgSO4, 1.0 μL of the first round PCR product (diluted 10-fold), 2.0 μL of specific primer pairs (Pps-GGL, Pgs-GG2 or Pps-GG2, Pgs-GG3 or Pps-GG3, Pgs-GG4 or Pps-GG4, Pgs-GGR, 1.5 μmol / L), and 11.3 μL ddH2O. The second round PCR program was: 94℃ for 60 s; 94℃ for 10 s, 58℃ for 15 s, 68℃ for 20 s, 25 cycles; 68℃ for 7 min. After the second round of PCR, the four PCR products were mixed in equal volumes and purified. The primer sequences used in the two rounds of PCR reactions are shown in Table 1.

[0027] Table 1. Primer sequences for vector construction

[0028]

[0029] Preparation of pYLCRISPR / Cas9-SlECT1 recombinant vector: The sgRNA expression cassette and pYLCRISPR / Cas9-SlECT1 recombinant vector were prepared using the Golden Gate cloning method (Ma et al., Robust CRISPR / Cas9 System for Convenient, High-Efficiency Multiplex Genome Editing in Monocot and Dicot Plants[J]. Molecular Plant, 2015, 8(8):1274-1284). ubi Enzymatic digestion and ligation reaction of the -H vector. The reaction system was: 1.5 μL of 10×CutSmartBuffer (NEB) and pYLCRISPR / Cas9-P. ubiThe following ingredients were added: 2.0 μL of -H vector, 1.0 μL of a mixture of four DNA fragments (AtU3d-T1-gRNA, AtU3b-T2-gRNA, AtU6-1-T3-gRNA, and AtU6-29-T4-gRNA), 0.5 μL of Bsai-HF (NEB), 1.0 μL of T4 DNA ligase (Promaga), 1.5 μL of 10×T4 DNA ligase buffer (Promaga), and 7.5 μL of ddH2O. The reaction conditions were: 37℃ for 10 min, 10℃ for 5 min, 20℃ for 5 min, 3 cycles; 37℃ for 3 min, 10℃ for 5 min, 20℃ for 5 min, 10 cycles; 37℃ for 5 min. The ligation product was transformed into *E. coli* DH5α competent cells and plated onto cells containing the corresponding antibiotic (Kan...). + Incubate overnight at 37°C on LB medium. Pick single colonies, extract plasmids and sequence them. Transform Agrobacterium GV3101 competent cells with correctly sequenced recombinant plasmids.

[0030] (II) Genetic transformation of tomatoes

[0031] Genetic transformation of tomatoes was carried out using Agrobacterium-mediated leaf disc transformation. All operations were performed in a sterile laminar flow hood, and the specific operational steps are as follows:

[0032] Sowing: Take an appropriate amount of AC tomato seeds into a sterile petri dish, disinfect with 75% ethanol for 30 seconds, then disinfect with 4% sodium hypochlorite solution for 8 minutes, rinse several times with sterile water, and then evenly place the seeds on 1 / 2 MS medium.

[0033] Seed germination: Place the seeds in the dark at 25℃ until germination (approximately 3 days). Then, place them in the light for cultivation, with seedling growth conditions of 25℃, 16 hours of light / 8 hours of darkness. After 3-5 days, the cotyledons of the seedlings will be fully expanded, ready for the next step.

[0034] Pre-culture: Cut tomato cotyledons into 5×5mm squares with a scalpel, place them evenly on a KCMS medium lined with a layer of filter paper with the back of the leaves facing up, and incubate in the dark overnight.

[0035] Preparation of infection solution: Pick a single colony of Agrobacterium that has been transformed into the pYLCRISPR / Cas9-SlECT1 gene editing vector and inoculate it into 3 mL of solution containing the corresponding antibiotic resistance (Kan). + Get + Incubate the culture in LB medium at 28°C with shaking overnight. The next day, transfer 300-500 μL of the bacterial culture to 20 mL of medium containing the appropriate antibiotic resistance (Kan). + Get + In LB medium, cultured at 28°C with shaking until OD 600=0.6-0.8. Centrifuge at 5000 rpm for 10 min to collect bacterial cells, and dilute the bacterial solution with sterile water to OD0.6. 600 =0.1-0.2.

[0036] Infection: Scrape tomato cotyledons from the dark cultured medium onto a sterile petri dish, pour in the infection solution, and infect for 5 minutes, gently shaking the petri dish during this time. After infection, discard the infection solution and aspirate any remaining infection solution from the cotyledons. Place the cotyledons back onto KCMS medium and incubate in the dark for 2 days.

[0037] Selection culture and regeneration: After 2 days of dark culture, the explants were carefully transferred to 2Z medium and cultured at 25°C under 16h light / 8h dark conditions for 2 weeks. Then, they were transferred to 0.2Z medium for subculture, and subcultured every 2 weeks thereafter. During the selection culture process, contaminated material was cleaned up in a timely manner until the explants grew callus tissue and regenerated shoots.

[0038] Rooting culture: When the regenerated shoots elongate to about 1 cm, cut them off and transfer them to R rooting medium for culture until roots are formed.

[0039] Soil cultivation: Remove the seedlings with good roots from the R medium, wash off the medium, and transplant them into flower pots for soil cultivation, paying attention to keeping them moist.

[0040] The culture medium formula is as follows:

[0041] 1 / 2MS: MS salt 2.22 g / L, sucrose 10 g / L, inositol 100 mg / L, agar 8 g / L, adjust pH to 5.8 ± 0.03;

[0042] KCMS: MS salt 4.44 g / L, sucrose 30 g / L, inositol 100 mg / L, agar 8 g / L, adjust pH to 5.8 ± 0.03, and add AS to a concentration of 0.1 mg / L after sterilization;

[0043] 2Z: MS salt 4.44 g / L, sucrose 20 g / L, inositol 100 mg / L, agar 7.4 g / L, adjust pH to 6.0 ± 0.03, after sterilization add hygromycin to a concentration of 6 mg / L, termethin to a concentration of 300 mg / L, and zeatin to a concentration of 2 mg / L;

[0044] 0.2Z: MS salt 4.44 g / L, sucrose 20 g / L, inositol 100 mg / L, agar 7.4 g / L, adjust pH to 6.0 ± 0.03, after sterilization add hygromycin to a concentration of 6 mg / L, termethin to a concentration of 300 mg / L, and zeatin to a concentration of 0.2 mg / L;

[0045] R: MS salt 4.44 g / L, sucrose 30 g / L, agar 8 g / L, adjust pH to 6.0 ± 0.03, after sterilization add hygromycin to a concentration of 6 mg / L, termethin to a concentration of 300 mg / L, and NV to a concentration of 2 mg / L.

[0046] (III) Screening for SlECT1 gene mutants

[0047] Genomic DNA was extracted from the SlECT1 gene-edited plants obtained in step (II). PCR primers were designed approximately 200 bp upstream and downstream of each target site to amplify the DNA sequence near the target site and then sequenced to detect the editing method of the SlECT1 gene-edited plants. Target PCR primers and sequencing primers are shown in Table 2.

[0048] Table 2. Primer sequences for target detection

[0049]

[0050] After successful sequencing, the CRISPR target editing method was analyzed using the website DSDecode.

[0051] (http: / / dsdecode.scgene.com / ) and compared with standard sequences using manual peak reading methods to analyze the editing methods of each target site.

[0052] The gene editing methods for the SlECT1 gene knockout mutants slect1#1 and slect1#2, selected through the above methods, are as follows: Figure 2 As shown. In the slect1#1 mutant, a 5-nucleotide deletion occurs at the T3 site, specifically nucleotides 954-958 of SEQ ID NO. 1. In the slect1#2 mutant, deletions of 3 and 65 nucleotides occur near the T4 site, specifically nucleotides 1666-1668 and 1534-1598 of SEQ ID NO. 1. slect1#1 yields a truncated protein with 48 remaining amino acids, while slect1#2 yields truncated proteins with 705 and 144 remaining amino acids, respectively. Figure 3 As shown.

[0053] (iv) Sensory evaluation analysis of tomato fruit based on electronic nose

[0054] Mature fruits (7 days after color breakage) from wild-type, select1#1, and select1#2 mutants were selected, with two biological replicates per group and six fruits randomly selected per biological replicate. Fruits were flash-frozen in liquid nitrogen and ground into powder. 2g of powder was weighed into a 10mL centrifuge tube, and 5mL of saturated NaCl solution was added, followed by thorough vortexing. 2mL of the fruit juice mixture was transferred to a 10mL vial (two technical replicates per biological replicate) and placed on ice. The vials were sequentially heated at 40℃ for 30min, and 2mL of headspace gas was extracted. Aromatic differences between samples were detected using an electronic nose (αFOX4000, Alpha-MOS, France), with air (Hangzhou Jingong Special Gases Co., Ltd.) as the carrier gas. Acquisition time: 120s, acquisition period: 1s, delay time: 240s, (carrier gas) flow rate: 150mL / min, injection volume: 500μL, injection rate: 500μL / min. Cluster analysis was performed using the principal component analysis (PCA) method built into the electronic nose system.

[0055] like Figure 4 The PCA analysis results show that, in the PC1 dimension, the select1#1 and select1#2 mutants cluster together and can be clearly distinguished from the wild type (WT), proving that there are significant differences in the olfactory characteristics of the select1 mutant and wild type fruits.

[0056] (V) Analysis of the content of volatile aromatic substances in tomato fruit

[0057] Mature fruits (7 days after color breakdown) of wild-type, select1#1, and select1#2 mutants were selected, with three biological replicates for each group, and each biological replicate consisting of six randomly selected fruits. The fruits were flash-frozen in liquid nitrogen and ground into powder. 5 g of powder was weighed into a 20 mL sample vial, and 5 mL of saturated NaCl solution and 20 μL of internal standard 2-octanol (0.8 mg / mL) were added, followed by thorough vortexing. The composition and content of volatile aromatic compounds in the fruits were determined using headspace solid-phase microextraction (HS-SPME) combined with gas chromatography-mass spectrometry (GC-MS). After equilibration at 40 °C for 30 min, solid-phase microextraction was performed for 30 min using a 50 / 30 μm DVB / CAR / PDMS extraction head. After desorption for 15 min at the GC-MS (Agilent 7890-5975) inlet, the extractor was separated using a DB-WAX capillary column (0.25 mm, 30 m, 0.25 μm, J&W Scientific). The temperature program was as follows: from 40 °C to 230 °C at a rate of 4 °C / min, then to 260 °C at a rate of 100 °C / min, and held for 11.7 min. Helium (Hangzhou Jingong Special Gases Co., Ltd.) was used as the carrier gas, and the MS ion source temperature was 230 °C. Electron impact ionization was employed with an electron energy of 70 eV. Aromatic substances were qualitatively identified by comparison with the NIST-8 (NIST / EPA / NIH, USA) standard library; the content of substances was quantitatively calculated by referring to the peak area of ​​the internal standard 2-octanol.

[0058] The aromatic substance content of mature fruits of wild type and slect1 mutant are shown in the table. Figure 5 The aromatic compounds in the mature fruits of the slect1#1 and slect1#2 mutants increased to 166.86% and 149.63% of the wild type, respectively. This demonstrates that the slect1 gene is involved in regulating the synthesis of aromatic compounds in tomato fruits.

Claims

1. A kind SlECT1 The application of gene mutants in enhancing the synthesis of aromatic substances in tomato fruits is characterized by, By editing tomatoes SlECT1 Gene gain mutant slect1 #1 and slect1 #2, by deleting nucleotides 954-958 or 1666-1668 and 1534-1598, increases the content of aromatic substances in tomato fruit. SlECT1 The nucleotide sequence of the gene is shown in SEQ ID NO.1; SlECT1 The encoded amino acid sequence is shown in SEQ ID NO.

2.

2. A method for SlECT1 CRISPR / Cas9 gene editing vectors with gene mutations are characterized by, The vector is pYLCRISPR / Cas9- SlECT1 The vector expresses sgRNA targeting the sequence of SEQ ID NO.1, wherein the sequence of the sgRNA is T1, T2, T3, and T4, wherein the T1 sequence is nucleotides 207-226 of SEQ ID NO.1, the T2 sequence is nucleotides 776-795 of SEQ ID NO.1, the T3 sequence is nucleotides 942-961 of SEQ ID NO.1, and the T4 sequence is nucleotides 1554-1573 of SEQ ID NO.

1.

3. The gene according to claim 1 SlECT1 mutant slect1 #1 and slect1 #2 Application in improving the aroma quality of tomato fruit.

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