A gene affecting the release of aromatic substances in tomato fruits and its application

Abstract

The present invention provides a gene that affects the release of aromatic substances in tomato fruits and its applications. By using gene editing technology to knockout a gene in tomatoes, a homozygous mutant is obtained, which shows an increase in the release of aromatic substances in mature fruits. Compared with wild-type fruits, the release amounts of aromatic substances in mutant fruits are significantly increased. For example, the release amounts of hexanal, 2-isobutylthiazole, 6-methyl-5-hepten-2-ol, and phenylacetaldehyde are increased by approximately 1.9, 2.3, 2.4, and 3.7 times, respectively. The gene described in the present invention can effectively affect the release of aromatic substances in tomato fruits, has guiding significance for improving the aromatic quality of tomato fruits through agricultural breeding, and can provide new candidate gene resources for cultivating new high-quality tomato varieties with rich flavors.

Description

Technical Field

[0001] The present invention belongs to the technical fields of plant molecular biotechnology and genetic engineering, and particularly relates to a gene for regulating the release of aromatic substances in tomato fruits and its application. Background Art

[0002] Fruits are important foods for humans, and their quality mainly includes color, aroma, taste, texture, and nutrition. Since the aromatic quality of fruits is closely related to consumer preferences, the formation and regulation of fruit aromatic substances have attracted much attention. Tomato is a model material for studying the formation of fruit aromatic substances, with a clear genetic background, a mature genetic transformation system, and an analytical method for aromatic substances. Therefore, in-depth research on the synthesis, storage, and transport of tomato fruit aromatic substances has important theoretical value and practical significance for improving and maintaining fruit aromatic quality.

[0003] As tomato fruits mature, the content of aromatic substances increases. Currently, genes involved in the synthesis of important aromatic substances in tomatoes have been identified, including SlLOXC, SlHPL, SlADH2, and SlAAT1 in the fatty acid pathway, SlCCD1 in the carotenoid pathway, and SlBCAT1 in the branched-chain amino acid pathway, etc. Free aromatic substances will be glycosylated and stored in vacuoles through the UGT (UDP-glucuronosyltransferase, UGT) family. Currently, the research on tomato fruit aromatic substances mostly focuses on the synthesis of aromatic substances, and further exploration is needed on how aromatic substances are released from fruits to the outside.

[0004] ABC transporters (ATP-binding cassette transporter, ABC) are one of the largest and oldest protein families discovered so far. There are a large number of ABC transporters in plants, with complex structures and diverse functions, which are involved in almost all processes of life activities. Currently, it is known that this protein is involved in the transport of hormones, lipids, metal ions, secondary metabolites, and exogenous substances in the body. Currently, it has only been proven in flower organs that ABC transporters can actively transport aromatic substances, or affect the distribution of aromatic substances by changing the cuticle thickness. The function of ABC transporters in fruits is still unclear, and their role in the release of fruit aromatic substances is also not clear. Summary of the Invention

[0005] The object of the present invention is to provide a gene for regulating the release of aromatic substances in tomato fruits, and the gene is SlABCG35, which can affect the release of aromatic substances in tomato fruits.

[0006] To achieve the above object of the invention, the present invention provides the following technical solutions:

[0007] The present invention provides a gene SlABCG35 that effectively regulates the release of aromatic substances in tomato fruits. The nucleotide sequence of the SlABCG35 gene is shown in SEQ ID NO.1; the amino acid sequence of the protein encoded by the SlABCG35 gene is shown in SEQ ID NO.2.

[0008] The present invention also provides a CRISPR / Cas9 gene editing vector for mutating the SlABCG35 gene, and the vector is pYLCRISPR / Cas9-SlABCG35. The vector can express an sgRNA targeting the nucleic acid molecule of the SEQ ID NO.1 sequence. The sequence of the sgRNA can be T1, T2, T3. The T1 sequence is the 1830-1849th nucleotides of SEQ ID NO.1, the T2 sequence is the 2393-2412th nucleotides of SEQ ID NO.1, and the T3 sequence is the 5160-5179th nucleotides of SEQ ID NO.1. The pYLCRISPR / Cas9-SlABCG35 gene editing vector is a recombinant vector obtained by sequentially inserting AtU3d-T1-gRNA, AtU3b-T2-gRNA, and AtU6-1-T3-gRNA between the multiple cloning sites of pYLCRISPR / Cas9-P ubi -H.

[0009] The present invention provides a microorganism containing the pYLCRISPR / Cas9-SlABCG35 recombinant vector, and the microorganism is Agrobacterium tumefaciens GV3101.

[0010] The application of the gene SlABCG35 provided by the present invention in effectively affecting the release of aromatic substances in tomato fruits includes that mutating the SlABCG35 gene in tomatoes can significantly increase the content of aromatic substances released by tomato fruits.

[0011] The application of the gene SlABCG35 provided by the present invention as an important candidate gene for genetic engineering of tomato fruits in tomato breeding and improving the aromatic quality of tomato fruits.

[0012] The beneficial effects of the present invention: The present invention provides the application of the SlABCG35 gene in affecting the release of aromatic substances in tomato fruits. In the mutant tomatoes of the SlABCG35 gene, the content of aromatic substances released by tomato fruits is significantly increased, indicating that SlABCG35 can effectively regulate the release of aromatic substances in tomato fruits, and can provide new candidate gene resources for cultivating high-quality tomato new varieties with improved aromatic quality. Description of the Drawings

[0013] Figure 1Three target sequences (T1, T2, T3) of SlABCG35 and their specific positions on the SlABCG35 gene.

[0014] Figure 2 Gene editing methods of the homozygous mutants slabcg35#2 and slabcg35#3 of the SlABCG35 gene.

[0015] Figure 3 Protein mutation results of the homozygous mutants slabcg35#2 and slabcg35#3 of the SlABCG35 gene.

[0016] Figure 4 Results of electronic nose analysis of mature fruits of wild type and homozygous mutants of the SlABCG35 gene.

[0017] Figure 5 Results of analysis of the contents of aromatic substances released by mature fruits of wild type and homozygous mutants of the SlABCG35 gene. Detailed implementation mode

[0018] The present invention will be further described in detail below in conjunction with the specific implementation mode. The embodiments are only for clarifying the present invention and not for limiting the scope of the present invention. The experimental methods in the following embodiments are all conventional methods unless otherwise specified. The materials, reagents, instruments, etc. used are all commercially available unless otherwise specified. In the following embodiments, quantitative tests are all set with three biological replicates, and the results are the average of the three.

[0019] The wild-type tomato variety Solanum lycopersicum cv Ailsa Craig, hereinafter referred to 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) can be obtained by the public from the applicant. This biological material is only used for repeating the relevant experiments of the present invention and cannot be used for other purposes.

[0020] Example 1: Obtaining of SlABCG35 gene mutant tomatoes

[0021] In this example, the SlABCG35 gene in tomatoes was mutated by the CRISPR / Cas9 technology, and it was found that the content of aromatic substances released from mature tomato fruits increased after the gene mutation. The DNA sequence of the SlABCG35 gene is SEQ ID NO.1 in the sequence listing, and it encodes the protein shown in SEQ ID NO.2 in the sequence listing. The specific steps are as follows:

[0022] (1) Construction of the pYLCRISPR / Cas9-SlABCG35 gene editing vector

[0023] Selection of target sequences: The selected target site sequences are T1 (nucleotides 1830-1849 of SEQ ID NO.1), T2 (nucleotides 2393-2412 of SEQ ID NO.1), and T3 (nucleotides 5160-5179 of SEQ ID NO.1). The positions of T1, T2, and T3 in SEQ ID NO.1 are as Figure 1 shown.

[0024] Selection of promoters: The AtU3d from Arabidopsis thaliana was used to initiate the T1 target site, AtU3b to initiate the T2 target site, and AtU6-1 to initiate the T3 target site.

[0025] Preparation of sgRNA expression cassette containing target sites: Through two rounds of PCR, three DNA fragments, namely AtU3d-T1-gRNA, AtU3b-T2-gRNA, and AtU6-1-T3-gRNA, were obtained respectively. AtU3d-T1-gRNA is an expression cassette containing sgRNA targeting the T1 site, and its expression is driven by the AtU3d promoter; AtU3b-T2-gRNA is an expression cassette containing sgRNA targeting the T2 site, and its expression is driven by the AtU3b promoter, and AtU6-1-T3-gRNA is an expression cassette containing sgRNA targeting the T3 site, and its expression is driven by the AtU6-1 promoter. The preparation method is as follows: The reaction system for the first round of PCR is 25 μL, including 0.5 μL of KOD enzyme (TOYOBO), 2.5 μL of 10×KOD Plus Buffer (TOYOBO), 2.5 μL of dNTP, 1.5 μL of MgSO4, 1 μL of pYLgRNA-LacZ-AtU3d / AtU3b / AtU6-1 plasmid, 1 μL of U-F primer (5 μmol / L), 1.0 μL of gR-R primer (5 μmol / L), 2.5 μL of gR-SlABCG35-T1 / T2 / T3 primer, 2.5 μL of AtU-SlABCG35-T1 / T2 / T3 primer, and 10 μL of ddH2O. The pYLgRNA-LacZ-AtU3d plasmid, PYLgRNA-AtU3b plasmid, and PYLgRNA-AtU6-1 plasmid are all described in the literature (Ma et 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 reaction program for the first round of PCR is: 94°C for 60 s; 94°C for 10 s, 58°C for 15 s, 68°C for 20 s, for 28 cycles; 68°C for 7 min. After the PCR reaction, three kinds of PCR products were obtained. The reaction system for the second round of PCR is 20 μL, including 0.5 μL of KOD enzyme (TOYOBO), 2.0 μL of 10×KOD Plus Buffer (TOYOBO), 2.0 μL of dNTP, 1.2 μL of MgSO4, 1.0 μL of the first-round PCR reaction product (diluted 10 times), 2.0 μL of specific primer pairs (Pps-GGL, Pgs-GG2 or Pps-GG2, Pgs-GG3 or Pps-GG3, Pgs-GGR, 1.5 μmol / L), and 11.3 μL of ddH2O.The procedure of the second-round PCR reaction was as follows: 94°C for 60 s; 94°C for 10 s, 58°C for 15 s, 68°C for 20 s, for 25 cycles; 68°C for 7 min. After the second-round PCR reaction, the three kinds of PCR products were mixed in equal amounts and then purified. The primer sequences used in the two-round PCR reactions are shown in Table 1.

[0026] Table 1. Primer sequences for vector construction

[0027]

[0028]

[0029] Preparation of the pYLCRISPR / Cas9-SlABCG35 recombinant vector: The enzymatic digestion and ligation reaction of the target sgRNA expression cassette and the pYLCRISPR / Cas9-P ubi -H vector was completed 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). The reaction system was: 1.5 μL of 10× CutSmart Buffer (NEB), 2.0 μL of pYLCRISPR / Cas9-P ubi -H vector, 1.0 μL of the mixture of the three purified DNA fragments of AtU3d-T1-gRNA, AtU3b-T2-gRNA, and AtU6-1-T3-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), 7.5 μL of ddH2O. The reaction conditions were: 37°C for 10 min, 10°C for 5 min, 20°C for 5 min, for 3 cycles; 37°C for 3 min, 10°C for 5 min, 20°C for 5 min, for 10 cycles; 37°C for 5 min. The ligation product was transformed into Escherichia coli DH5α competent cells, positive colonies were picked to extract plasmids, and after correct sequencing, it was transformed into Agrobacterium tumefaciens GV3101 competent cells.

[0030] (II) Genetic transformation of tomatoes

[0031] The pYLCRISPR / Cas9-SlABCG35 gene editing vector was transformed into wild-type AC tomatoes using the Agrobacterium-mediated leaf disc transformation method. All operations were carried out in a sterile workbench, and the specific operation steps are as follows:

[0032] (1) Sowing: Take an appropriate amount of AC tomato seeds in a sterile Petri dish, disinfect them with 75% ethanol and 4% sodium hypochlorite solution for 5 min and 8 min respectively, wash them several times with sterile water, and then sow the seeds on the seed germination medium 1 / 2MS.

[0033] Seed germination: Place the seeds in a dark room for 3 days until germination, then transfer them to the light for cultivation. Wait until the cotyledons are fully unfolded before using them for the next step.

[0034] Pre-culture: Cut the tomato cotyledons into 5×5 mm squares, place them with the back side up on the KCMS medium covered with a layer of filter paper.

[0035] Prepare the infection solution: Pick a single colony of Agrobacterium tumefaciens transformed with the pYLCRISPR / Cas9-SlABCG35 gene editing vector into 3 mL of LB medium containing the corresponding antibiotics, and culture it overnight with shaking at 28°C. The next day, pipette 300 μL of the bacterial solution into 20 mL of LB medium containing the corresponding antibiotics, and culture it with shaking at 28°C until the OD 600 reaches 0.5 - 0.6. Centrifuge at 5000 rpm for 10 min to collect the bacteria, and dilute the bacterial solution with 30 mL of liquid KCMS solution.

[0036] Co-culture: Place the pre-cultured explants in the infection solution for 2 - 3 min, then pour out the infection solution, and put the explants back on the pre-culture medium KCMS, and co-culture them in the dark room for 2 days.

[0037] Callus induction culture: Take the explants out of the dark room, place them with the back side down on the callus induction medium 2Z, and culture them under the conditions of 25°C, 16 h light / 8 h dark for 2 - 3 weeks. Transfer them to fresh 2Z medium every 7 - 10 days until callus tissue grows on the explants.

[0038] Bud induction culture: After the explants grow callus, transfer them to the bud induction medium 0.2Z until small buds with a stem length of 1 - 2 cm grow.

[0039] Rooting culture: After the small buds elongate, cut off the callus tissue, and transfer them to the rooting medium R until the roots are well-developed.

[0040] Soil culture: Remove the small seedlings from the R medium, wash the medium, and transfer the small seedlings to a moist soil pot for soil culture, paying attention to keeping moisture.

[0041] The medium formula is as follows (in units of 1 L):

[0042] 1 / 2MS: 2.2 g of MS salts, 10 g of sucrose, 100 mg of inositol, adjust the pH to 5.8, 8 g of agar;

[0043] Solid KCMS: 4.44 g of MS salts, 30 g of sucrose, 100 mg of inositol, adjust the pH to 5.8, 8 g of agar, add AS to a concentration of 100 mg / L after sterilization;

[0044] Liquid KCMS: 4.44 g of MS salts, 30 g of sucrose, 100 mg of inositol, adjust the pH to 5.8, add AS to a concentration of 100 mg / L after sterilization;

[0045] 2Z: 4.44 g of MS salts, 20 g of sucrose, 100 mg of inositol, adjust the pH to 5.8, 7.4 g of agar, add hygromycin to a concentration of 10 mg / L, ticarcillin to a concentration of 200 mg / L, and zeatin to a concentration of 2 mg / L after sterilization;

[0046] 0.2Z: 4.44 g of MS salts, 20 g of sucrose, 100 mg of inositol, adjust the pH to 5.8, 7.4 g of agar, add hygromycin to a concentration of 10 mg / L, ticarcillin to a concentration of 200 mg / L, and zeatin to a concentration of 0.2 mg / L after sterilization;

[0047] R: 4.44 g of MS salts, 20 g of sucrose, adjust the pH to 5.8, 8 g of agar, add hygromycin to a concentration of 5 mg / L, ticarcillin to a concentration of 150 mg / L, and IAA to a concentration of 1 mg / L after sterilization.

[0048] (III) Screening of homozygous SlABCG35 gene mutants

[0049] Using the genomic DNA of the SlABCG35 gene-edited plants obtained in step (II) as a template, PCR primers designed around approximately 300 bp upstream and downstream of each target sequence were used to amplify a DNA sequence of approximately 600 bp near the target sequence, and sequencing was performed to detect the SlABCG35 gene editing method. The target PCR primers are shown in Table 2.

[0050] Table 2. Primer sequences for target detection

[0051]

[0052]

[0053] After successful sequencing, analyze through the CRISPR target editing method analysis website DSDecode (http: / / dsdecode.scgene.com / ) and combine with the manual peak reading map to align with the gene standard sequence, and analyze the editing methods of each target sequence and its upstream and downstream sequences.

[0054] The gene editing methods of the SlABCG35 gene homozygous mutants slabcg35#2 and slabcg35#3 screened by the above method are as Figure 2 shown. In the slabcg35#2 mutant, 1 nucleotide was inserted at the T1 site, that is, a base T was inserted after the 1833rd nucleotide of SEQ ID NO.1. In the slabcg35#3 mutant, 1 nucleotide was inserted at the T1 site, that is, a base A was inserted after the 1833rd nucleotide of SEQ ID NO.1. The above nucleotide deletions both caused frameshift mutations in the SlABCG35 gene, premature termination of protein translation, and domain deletion. slabcg35#2 obtained a truncated protein with the remaining 164 amino acids; slabcg35#3 obtained a truncated protein with the remaining 165 amino acids, as Figure 3 shown.

[0055] Example 2: Discrimination of SlABCG35 mutants and wild-type tomato fruits by electronic nose

[0056] Sensory evaluation analysis of tomato fruits based on electronic nose

[0057] Wild-type, slabcg35#2 and slabcg35#3 mutant mature fruits (7 days after breaker) were selected, with 3 biological replicates for each sample. Each biological replicate was a mixture of 6 randomly selected fruits, ground into powder with liquid nitrogen, 1 g of powder was weighed into a 10 mL centrifuge tube, and 5 mL of saturated NaCl solution was added, followed by thorough vortex mixing. 2 mL was aspirated into a sample vial (2 technical replicates were set for each biological replicate) and placed on ice for later use. After heating each sample vial at 40 °C for 30 min, 2 mL of headspace gas was extracted and the fragrance differences between samples were detected using an electronic nose (αFOX4000, Alpha-MOS, France). The carrier gas was air (Hangzhou Jingong Special Gas Co., Ltd.). Sampling time: 120 s, sampling period: 1 s, delay time: 240 s, (carrier gas) flow rate: 150 mL / min, injection volume: 500 μL, injection speed: 500 μL / min. The Discriminant Function Analysis (DFA) method built into the system was used for data analysis.

[0058] As Figure 4 seen from the DFA analysis results of, in the DF1 dimension, the slabcg35#2 and slabcg35#3 mutants clustered together and could be clearly distinguished from the wild type (WT), indicating that there were significant differences in aroma between the slabcg35#2 and slabcg35#3 mutants and wild-type mature fruits.

[0059] Example 3: The mutation of SlABCG35 gene leads to an increase in the content of aromatic substances released from tomato fruits

[0060] Analysis of the content of endogenous and released volatile aromatic substances in tomato fruits

[0061] Select mature fruits (7 days after breaker) of wild type, slabcg35#2 and slabcg35#3 mutants. There are 5 biological replicates for each sample, and each biological replicate consists of 3 or 4 fruits. After weighing the fruit weight, put them into a sealed glass jar. Drop 10 μL of internal standard 2-octanol (1 mg / ml) on a filter paper of 1 cm × 1 cm, seal and let stand for 30 min, then insert an extraction head of 50 / 30 μm DVB / CAR / PDMS for 30 min of headspace solid-phase microextraction. Use headspace solid-phase microextraction (HS-SPME) combined with gas chromatography-mass spectrometry (GC-MS) technology to determine the composition and content of aromatic substances released from the fruits. The extraction head is desorbed in the injection port of GC-MS (Agilent 7890-5975) for 5 min and then separated by a DB-WAX capillary column (0.25 mm, 30 m, 0.25 μm, J&W Scientific). The temperature programming is from 40 °C at a rate of 4 °C·min -1 rate to 230 °C, and then at a rate of 100 °C·min -1 rate to 260 °C and hold for 11.7 min. Use helium (Hangzhou Jingong Special Gases Co., Ltd.) as the carrier gas at a flow rate of 1.0 mL·min -1 The temperature of the MS ion source is 230 °C. The electron impact ionization mode is adopted, and the electron energy is 70 eV. The qualitative determination of aromatic substances is determined by comparing with the NIST-8 (NIST / EPA / NIH, USA) standard spectral library, and the quantitative calculation uses the peak area of the internal standard as a reference.

[0062] The content of aromatic substances released from mature fruits of wild type and slabcg35 mutants is shown in Figure 5 , and the content of aromatic substances released from mature fruits of slabcg35#2 and slabcg35#3 mutants is significantly higher than that of the wild type. Specifically, it increases by about 36% compared with the wild type. Among them, the release amounts of hexanal, 2-isobutylthiazole, 6-methyl-5-hepten-2-ol, and phenylacetaldehyde increase by about 1.9, 2.3, 2.4, and 3.7 times respectively. It shows that the mutation of SlABCG35 gene can significantly increase the content of aromatic substances released from tomato fruits, and the SlABCG35 gene may affect the release of aromatic substances in tomato fruits.

Claims

1. Use of a gene in increasing the release content of aromatic substances in tomato fruits, characterized in that, The gene is SlABCG35 , the nucleotide sequence of the gene is shown as SEQ ID NO.1, the amino acid sequence of the protein encoded by the gene is shown as SEQ ID NO.2, and the application is to insert a base T or A after the 1833rd nucleotide of SEQ ID NO.

1.

2. A vector for gene mutation, characterized in that, The vector simultaneously expresses three sgRNAs, namely T1, T2, and T3, targeting the nucleic acid molecule of SEQ ID NO.

1. The T1 sequence is nucleotides 1830-1849 of SEQ ID NO.1, the T2 sequence is nucleotides 2393-2412 of SEQ ID NO.1, and the T3 sequence is nucleotides 5160-5179 of SEQ ID NO.1.

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