Slmbp7 gene for regulating serotonin content in tomato fruit, method and application

Abstract

The application provides a SlMBP7 gene for regulating the content of serotonin in tomato fruits, a method and application, and a CDS sequence of the SlMBP7 gene is shown as SEQ ID NO. 1. The application obtains a complete coding sequence of the SlMBP7 gene from a tomato genome, designs a specific sgRNA target point, and constructs the target point on a CRISPR / Cas9 gene editing vector. The recombinant vector is introduced into tomato cotyledon explants by using an agrobacterium-mediated genetic transformation method, and finally, mutant plant strains with homozygous knockout of the SlMBP7 gene are obtained. The application first discloses the negative regulation of the SlMBP7 gene in the regulation of serotonin biosynthesis in tomato fruits, and establishes an efficient and stable genetic improvement method. By accurately regulating the expression of the SlMBP7 gene, the content of serotonin in tomato fruits is significantly improved, so that the fruit quality is improved and the economic value is improved.

Description

Technical Field

[0001] This invention belongs to the field of plant genetic engineering technology, and relates to the SlMBP7 gene, method and application for regulating serotonin content in tomato fruits. Background Technology

[0002] According to statistics released by the Food and Agriculture Organization of the United Nations (FAO) in 2024, China's total tomato production reached 68 million tons in 2022, accounting for one-third of global tomato production, playing a vital role in agricultural production and international trade. Nutritionally, tomatoes are rich in various bioactive substances, with particularly high levels of carotenoids (such as lycopene) and vitamin C. These nutrients not only give tomatoes their unique nutritional value, but their distinctive sweet and sour flavor is also highly favored by consumers worldwide. With the rapid development of the global tomato industry and the continuous improvement of residents' consumption levels, market demands for tomato varieties have shifted from a single yield indicator to diversified needs. Specifically, this is reflected in: stricter requirements for fruit quality, greater emphasis on flavor characteristics, continuous improvement in yield indicators, and increasing resistance to adverse conditions, especially a greater focus on nutritional value. Utilizing modern bio-breeding technology to cultivate high-quality tomato varieties that meet market demands is key to promoting the sustainable development of the tomato industry.

[0003] Serotonin (5-hydroxytryptamine) was first discovered in blood serum. Its biosynthesis involves the catalytic production of 5-hydroxytryptamine from tryptophan by tryptophan hydroxylase (TPH), followed by the catalytic production of 5-hydroxytryptamine by L-amino acid decarboxylase (AADC). Serotonin can alleviate anxiety and combat depression, playing a crucial role in regulating mood, behavior, and other neurological activities. Furthermore, serotonin exhibits potential functions in inhibiting cardiovascular disease and tumor growth. Increasing the serotonin content in tomatoes allows for the intake of serotonin from everyday fruits and vegetables, thus providing health benefits. Therefore, developing a method to rapidly increase the serotonin content in tomatoes is a pressing technical problem that needs to be solved by those in this field. Summary of the Invention

[0004] This invention aims to provide the SlMBP7 gene, method, and application for regulating serotonin content in tomato fruits. By using CRISPR / Cas9 gene editing technology, SlMBP7 gene mutant tomato materials are created, which increases the serotonin content in tomato fruits by 3 times and improves the quality of tomato fruits.

[0005] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0006] One of the objectives of this invention is to provide the SlMBP7 gene, which regulates the serotonin content in tomato fruits, and the CDS sequence of the SlMBP7 gene is shown in SEQ ID NO.1.

[0007] A second objective of this invention is to provide a protein that regulates the serotonin content in tomato fruits, the amino acid sequence of which is shown in SEQ ID NO.2.

[0008] A third objective of this invention is to provide a method for regulating serotonin content in tomato fruits, comprising the following steps:

[0009] Step 1: Design sgRNA target-specific primers for the SlMBP7 gene and construct the SlMBP7 gene knockout vector CRISPR / Cas9-SlMBP7. The primer sequences are as follows:

[0010] SlMBP7-sgRNA1-F: 5′-TGATTGCATTATGCAGGAGAAGAGT-3′ (SEQ ID NO.3);

[0011] SlMBP7-sgRNA1-R: 5′-AAACACTCTTCTCCTGCATAATGCA-3′ (SEQ ID NO.4);

[0012] Step 2: The CRISPR / Cas9-SlMBP7 vector constructed in Step 1 was transferred into explants using Agrobacterium tumefaciens infection to obtain the F2 generation and offspring seeds of the stably inherited SlMBP7 gene mutation.

[0013] Preferably, in step one, the CDS sequence of the SlMBP7 gene is shown in SEQ ID NO.1.

[0014] Preferably, in step one, the p1300-AtU6-35S-Cas9 vector is digested with BsaⅠ enzyme at 37°C for 4-6 hours before the ligation reaction is performed.

[0015] Preferably, in step two, the Agrobacterium is EHA105 Agrobacterium tumefaciens.

[0016] Preferably, in step two, the explant is a tomato cotyledon that has sprouted for two weeks.

[0017] The fourth objective of this invention is to provide the application of the SlMBP7 gene in regulating serotonin content in tomato fruits.

[0018] Furthermore, the application includes increasing the serotonin content in tomato fruits by knocking out the SlMBP7 gene.

[0019] Preferably, the CDS sequence of the SlMBP7 gene is shown in SEQ ID NO.1.

[0020] The beneficial effects of this invention are as follows:

[0021] This invention obtained the complete coding sequence of the SlMBP7 gene from the tomato genome, designed a specific sgRNA target, and constructed it into a CRISPR / Cas9 gene editing vector. Using Agrobacterium-mediated genetic transformation, the recombinant vector was introduced into tomato cotyledon explants, ultimately obtaining homozygous knockout mutant plants of the SlMBP7 gene. Analysis of the fruits of the mutant plants showed that, compared with wild-type tomatoes, the serotonin content in the SlMBP7 gene knockout mutant fruits was significantly increased by 2.8–3.0 times. This invention reveals for the first time the negative regulatory role of the SlMBP7 gene in regulating serotonin biosynthesis in tomato fruits and establishes an efficient and stable genetic improvement method. By precisely regulating the expression of the SlMBP7 gene, the serotonin content of tomato fruits is significantly increased, thereby improving fruit quality and increasing its economic value. This invention provides important genetic resources and breeding strategies for cultivating high-quality tomato varieties with high serotonin content and has significant application prospects in the field of agricultural biotechnology. Attached Figure Description

[0022] Figure 1 Comparison of PCR sequencing results of leaves from wild tomato and SlMBP7 gene knockout tomato lines: AC is wild-type tomato, while mbp7-cr1 and mbp7-cr2 are two SlMBP7 gene knockout lines.

[0023] Figure 2 Wild tomato and SlMBP7 gene knockout tomato fruits. AC is a wild-type tomato, while mbp7-cr1 and mbp7-cr2 are two SlMBP7 gene knockout lines.

[0024] Figure 3 Results of serotonin content detection in wild tomato and SlMBP7 gene knockout tomato fruits. AC is a wild-type tomato, while mbp7-cr1 and mbp7-cr2 are two SlMBP7 gene knockout lines. Detailed Implementation

[0025] The present invention will now be described in detail with reference to specific embodiments. The following specific embodiments will help those skilled in the art to further understand the present invention, but do not limit the present invention in any way.

[0026] Example 1: Obtaining strain EHA105 containing CRISPR / Cas9-SlMBP7 plasmid

[0027] Step 1: Target sequence design for the SlMBP7 gene

[0028] The tomato SlMBP7 gene is 810 bp in length and consists of 8 exons. sgRNAs of the SlMBP7 gene were screened using the online CRISPR-P tool (http: / / cbi.hzau.edu.cn / cgi-bin / CRISPR). Preferred targets were those located in the first exon of the gene's CDS, with a GC content between 45% and 70%, and differing from other locations in the genome by at least three bases.

[0029] Step 2: Primer synthesis. The following primer sequences were synthesized by Shanghai Bioengineering Co., Ltd.:

[0030] SlMBP7-sgRNA1-F:5′- TGATTG CATTATGCAGGAGAAGAGT-3′ (SEQ ID NO. 3);

[0031] SlMBP7-sgRNA1-R: 5′- AAAC ACTCTTCTCCTGCATAATG CA -3′(SEQ ID NO.4);

[0032] The underlined part represents the connector sequence.

[0033] Step 3: Primer annealing

[0034] The forward and reverse primers from step 2 were treated at 95°C for 5 minutes in a PCR instrument, followed by annealing at room temperature.

[0035] Step 4: Vector digestion

[0036] The p1300-AtU6-35S-Cas9 vector was digested with BsaⅠ enzyme (NEB) at 37℃ for 4-6 hours. The reaction system was as follows: 10 μL p1300-AtU6-35S-Cas9, 2.5 μL 10×BasⅠCutSmart buffer, 1 μL BsaⅠ enzyme (NEB), and ddH2O to a final volume of 25 μL.

[0037] Step 5: Ligation Reaction

[0038] The product from step 3 was ligated into the digested p1300-AtU6-35S-Cas9 vector using T4 ligase. The reaction mixture consisted of 1 μL of the product from step 3, 1 μL of the product from step 4, 2.5 μL of T4 Ligase Buffer, and 0.5 μL of T4 Ligase. The mixture was reacted at 16 °C for 2 h.

[0039] Step 6: E. coli transformation

[0040] Add 1 μL of the ligation product to 25 μL of DH5α competent cells, incubate on ice for 25 min, then heat shock at 42°C for 45 s, and immediately incubate on ice for 2 min. Add 900 μL of antibiotic-free LB medium and incubate at 37°C with shaking for 1 h. Centrifuge at 13000 rpm / s for 1 min, discard the supernatant, and aspirate the remaining 50 μL of bacterial culture, spread it evenly on LB agar plates containing kanamycin-resistant culture, and incubate at 37°C for 16 h.

[0041] Step 7: Identification of positive monoclonal colonies

[0042] Single colonies were selected for PCR verification. The upstream primer was the universal M13 primer. The downstream primer was SlMBP7-sgRNA-R: 5′-ACTCTTCTCCTGCATAATG-3′ (SEQ ID NO.5). After verification by agarose gel electrophoresis, positive clone plasmids were extracted and sent for sequencing (Shanghai Bioengineering Co., Ltd.), yielding the CRISPR / Cas9-SlMBP7 recombinant plasmid.

[0043] Step 8: Transform the CRISPR / Cas9-SlMBP7 recombinant plasmid into EHA105 Agrobacterium competent cells to obtain EHA105 strain containing the CRISPR / Cas9-SlMBP7 plasmid.

[0044] Example 2: Construction of SlMBP7 gene mutant plants

[0045] The CRISPR / Cas9-SlMBP7 vector was transformed into tomato cotyledons using the leaf disc method. The specific steps are as follows:

[0046] 1) Agrobacterium infection: Soak pre-cultured tomato cotyledons in 15 mL of infection solution for 15 min, blot dry with sterile filter paper, and place in new pre-culture medium. Co-culture at 25°C in the dark for 48 h.

[0047] 2) Differentiation culture: Transfer the explants to the differentiation medium and culture them at 25°C with 16 hours of light and 8 hours of darkness. Change the medium every 15 days.

[0048] 3) Rooting culture: Cut off differentiated buds and insert them into rooting culture medium. After rooting, transplant them into nutrient pots.

[0049] Specific methods for preparing culture media:

[0050] Seed germination medium: MS powder 2.15 g / L, sucrose 30 g / L, agar 7.4 g / L, pH 5.5.

[0051] Pre-medium: MS powder 2.15 g / L, sucrose 10 g / L, agar 8 g / L, 2,4-D 0.2 mg / L, pH 5.8.

[0052] Differentiation medium: MS powder 4.3 g / L, sucrose 20 g / L, agar 7.4 g / L, Zeatin 2 mg / L, Timentin 300 mg / L, hygromycin 6 mg / L, pH 6.

[0053] Rooting medium: MS powder 2.15 g / L, sucrose 30 g / L, agar 7.4 g / L, Timentin 300 mg / L, hygromycin 6 mg / L, pH 6.

[0054] MS infection solution: MS(519) powder 4.3 g / L, sucrose 20 g / L, pH 6.

[0055] LB liquid: NaCl 10g / L, Trypyone 10g / L, Yeast Extract 5g / L, pH 7.

[0056] Example 3: Molecular identification of transgenic plants

[0057] 1) DNA extraction and PCR analysis: DNA was extracted from T0 generation plants using the CTAB method. Specific primers were designed upstream and downstream of the sgRNA sequence location of the SlMBP7 gene.

[0058] SlMBP7-CHECK-F: 5′-GCTGAAAGGCAGCTTAATGC-3′ (SEQ ID NO. 6);

[0059] SlMBP7-CHECK-R: 5'-TGCCTTGTCCTGTGTCATGT-3' (SEQ ID NO. 7).

[0060] Mutant screening was conducted. Seeds from the T0 generation, which were heterozygous according to PCR sequencing results, were sown again to analyze the editing status of the T1 generation plants. PCR sequencing alignment results showed that mbp7-cr1 had a 1bp deletion in exon 4, and mbp7-cr2 had a 197bp deletion in exon 4. (See attached image) Figure 1 .

[0061] 2) Serotonin content determination: Serotonin content was determined in fruit samples collected from wild-type AC (Ailsa Craig) and tomato mbp7 mutants (cr1,cr2) at 17 days, 25 days after flowering and at the red ripening stage.

[0062] The specific procedure is as follows: Weigh 50 mg of fruit, add 0.5 mL of methanol solution containing 20 μL of internal standard working solution (250 ng / mL), vortex for 3 min to mix, and then place in a 20℃ refrigerator for 30 min; subsequently, centrifuge at 4℃, 12000 r / min for 10 min, and collect 250 μL of supernatant. Repeat the centrifugation process and collect 150 μL of supernatant. The data acquisition instrument system mainly includes ultra-high performance liquid chromatography (UPLC) and tandem mass spectrometry (MS / MS). The liquid chromatography conditions included: Waters ACQUITY UPLC HSS T3 C18 column (1.8 μm, 100 mm × 2.1 mm id); mobile phase: Phase A, ultrapure water (containing 0.1% formic acid); Phase B, acetonitrile (containing 0.1% formic acid); flow rate 0.35 mL / min; column temperature 40 °C; injection volume 2 μL; mobile phase gradient: 0 min A / B 90:10 (V / V), 1 min A / B 90:10 (V / V), 6 min 5:95 (V / V), 7 min A / B 5:95 (V / V), 7.1 min 90:10 (V / V), 10 min 90:10 (V / V). Serotonin levels were determined as follows: Figure 3 As shown.

[0063] Figure 2 AC is a wild-type tomato, while mbp7-cr1 and mbp7-cr2 are two SlMBP7 gene knockout lines. According to... Figure 3 The test results showed that after knocking out the SlMBP7 gene in tomatoes, the serotonin content in tomatoes during fruit development and ripening was significantly increased.

[0064] Obviously, the above embodiments of the present invention are merely examples to illustrate the present invention more clearly, and are not intended to limit the implementation of the present invention. For those skilled in the art, other variations or modifications can be made based on the above description. It is impossible to exhaustively list all implementation methods here. Any obvious variations or modifications derived from the technical solutions of the present invention are still within the protection scope of the present invention.

Claims

1. A method to increase the serotonin content in tomato fruits includes the following steps: Step 1: Design SlMBP7 Constructing sgRNA target-specific primers for genes SlMBP7 The gene knockout vector is CRISPR / Cas9-SlMBP7, and the primer sequences are as follows: SlMBP7 -sgRNA1-F:5′-TGATTGCATTATGCAGGAGAAGAGT-3′(SEQ ID NO.3); SlMBP7 -sgRNA1-R:5′-AAACACTCTTCTCCTGCATAATGCA-3′(SEQ ID NO.4)? Step 2: The CRISPR / Cas9-SlMBP7 vector constructed in Step 1 was transferred into explants using Agrobacterium infection to obtain stably inherited vectors. SlMBP7 Seeds of the F2 generation and offspring of the gene mutation.

2. The method according to claim 1, characterized in that, In step one, the SlMBP7 The CDS sequence of the gene is shown in SEQ ID NO.

1.

3. The method according to claim 1 or 2, characterized in that, In step one, the p1300-AtU6-35S-Cas9 vector was digested with BsaⅠ enzyme at 37℃ for 4-6 h before the ligation reaction was carried out.

4. The method according to claim 1, characterized in that, In step two, the Agrobacterium is EHA105 Agrobacterium tumefaciens.

5. The method according to claim 1 or 4, characterized in that, In step two, the explant is a tomato cotyledon that has been germinated for two weeks.

6. Knockout SlMBP7 Application of genes in increasing serotonin content in tomato fruits.

7. The application according to claim 6, characterized in that, The SlMBP7 The CDS sequence of the gene is shown in SEQ ID NO.1.

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