SlMBP7 gene for regulating serotonin content in tomato fruits, method and application

Abstract

The invention provides an SlMBP7 gene for regulating and controlling serotonin content in tomato fruits, a method and application. The CDS sequence of the SlMBP7 gene is shown as SEQ ID NO. 1. A complete coding sequence of the SlMBP7 gene is obtained from a tomato genome, a specific sgRNA target is designed and constructed to a CRISPR / Cas9 gene editing vector, the recombinant vector is introduced into a tomato cotyledon explant by using an agrobacterium-mediated genetic transformation method, and finally a mutant plant with the SlMBP7 gene homozygous knockout is obtained. According to the invention, the negative regulation effect of the SlMBP7 gene in regulation of tomato fruit serotonin biosynthesis is disclosed for the first time, an efficient and stable genetic improvement method is established, and the serotonin content of tomato fruits is remarkably increased by accurately regulating the expression of the SlMBP7 gene, so that the fruit quality is improved, and the economic value of the tomato fruits is increased.

Description

Technical Field

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

[0002] According to the statistical data released by the Food and Agriculture Organization of the United Nations (FAO) in 2024, the total output of tomatoes in China reached 68 million tons in 2022, accounting for one-third of the total global tomato output, occupying an important position in agricultural production and international trade. From the perspective of nutritional value, tomato fruits are rich in various bioactive substances, among which the contents of carotenoid compounds (such as lycopene) and vitamin C and other nutrients are particularly prominent. These nutrients not only endow tomatoes with unique nutritional value, but also their unique sweet and sour flavor is deeply favored by global consumers. With the rapid development of the global tomato industry and the continuous improvement of residents' consumption levels, the market's requirements for tomato varieties have shifted from a single yield index to diversified demands, specifically manifested in: stricter requirements for fruit quality, more attention to flavor characteristics, continuous improvement of yield indexes, continuous enhancement of stress resistance, and especially increased attention to their nutritional value. Utilizing modern biological breeding techniques to cultivate high-quality tomato varieties that meet market demands is the key to promoting the sustainable development of the tomato industry.

[0003] Serotonin (5-hydroxytryptamine) was first discovered in the serum of blood. Its biosynthesis process is to catalyze the production of 5-hydroxytryptophan from tryptophan under the action of tryptophan hydroxylase (TPH), and then catalyze the production of 5-hydroxytryptamine under the action of L-amino acid decarboxylase (AADC). Serotonin can relieve anxiety and resist depression, and plays an important role in regulating nerve activities such as mood and behavior. In addition, serotonin also shows potential functions in inhibiting cardiovascular diseases and inhibiting tumor growth. By increasing the serotonin content in tomato fruits, serotonin can be ingested from daily fruits and vegetables to play a health care role. Therefore, developing a method for rapidly increasing the serotonin content in tomato fruits is a technical problem that needs to be solved urgently by those skilled in the art. Summary of the Invention

[0004] The present invention aims to provide the SlMBP7 gene, method and application for regulating the serotonin content in tomato fruits. By creating SlMBP7 gene mutant tomato materials through CRISPR / Cas9 gene editing technology, the serotonin content in tomato fruits is increased by 3 times, improving the quality of tomato fruits.

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

[0006] One object of the present invention is to provide the SlMBP7 gene for regulating the serotonin content in tomato fruits, and the CDS sequence of the SlMBP7 gene is shown in SEQ ID NO.1.

[0007] The second object of the present invention is to provide a protein for regulating the serotonin content in tomato fruits, and the amino acid sequence of the protein is shown in SEQ ID NO.2.

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

[0009] Step 1: Design sgRNA target-specific primers for the SlMBP7 gene, and construct a CRISPR / Cas9-SlMBP7 vector for knocking out the SlMBP7 gene. 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: Transfer the CRISPR / Cas9-SlMBP7 vector constructed in Step 1 into explants by Agrobacterium-mediated infection to obtain stable hereditary F2 generations of SlMBP7 gene mutants and their offspring seeds.

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

[0014] Preferably, in Step 1, the p1300-AtU6-35S-Cas9 vector is digested with BsaⅠ at 37°C for 4-6 h and then subjected to a ligation reaction.

[0015] Preferably, in Step 2, the Agrobacterium is Agrobacterium tumefaciens EHA105.

[0016] Preferably, in Step 2, the explant is the cotyledon of tomato germinated for 2 weeks.

[0017] The fourth object of the present invention is to provide the application of the SlMBP7 gene in regulating the 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 the present invention are as follows:

[0021] The coding sequence of the complete SlMBP7 gene was obtained from the tomato genome, specific sgRNA target sites were designed and constructed onto the CRISPR / Cas9 gene editing vector. Using the Agrobacterium-mediated genetic transformation method, the recombinant vector was introduced into tomato cotyledon explants, and finally, a mutant plant with homozygous knockout of the SlMBP7 gene was obtained. Analysis of the fruits of the mutant plants showed that compared with wild-type tomatoes, the serotonin content in the fruits of the SlMBP7 gene knockout mutants was significantly increased by 2.8 - 3.0 times. The present invention first reveals 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 in tomato fruits was significantly increased, thereby improving the fruit quality and its economic value. The present invention provides important gene resources and breeding strategies for cultivating new high-quality tomato varieties with high serotonin content, and has important application prospects in the field of agricultural biotechnology. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 : Results of PCR sequencing alignment of tomato leaves between wild tomatoes and SlMBP7 gene knockout lines. AC is wild-type tomato, and mbp7-cr1 and mbp7-cr2 are two SlMBP7 gene knockout lines.

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

[0024] Figure 3 : Detection results of serotonin content in fruits of wild tomatoes and SlMBP7 gene knockout tomatoes. AC is wild-type tomato, and mbp7-cr1 and mbp7-cr2 are two SlMBP7 gene knockout lines. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0025] The present invention will be described in detail below in conjunction with the specific embodiments. The following specific examples are helpful for those skilled in the art to further understand the present invention, but do not limit the present invention in any form.

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

[0027] Step 1: Design of target sequence of SlMBP7 gene

[0028] The tomato SlMBP7 gene is 810 bp in length and consists of 8 exons. The sgRNA of the SlMBP7 gene was screened using the CRISPR-P online tool (http: / / cbi.hzau.edu.cn / cgi-bin / CRISPR). The target sites were preferably located on the first exon of the gene CDS, with a GC content in the range of 45%-70% and at least three base differences from other positions in the genome.

[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 is the adapter sequence.

[0033] Step 3: Primer annealing

[0034] The forward primer and reverse primer in Step 2 were treated at 95 °C for 5 min in a PCR instrument, and then cooled to room temperature to complete annealing.

[0035] Step 4: Vector digestion

[0036] The p1300-AtU6-35S-Cas9 vector was digested with BsaⅠ enzyme (NEB) at 37 °C for 4-6 h. The reaction system was as follows: 10 μL of p1300-AtU6-35S-Cas9, 2.5 μL of 10× BasⅠ CutSmart buffer, 1 μL of BsaⅠ enzyme (NEB), and ddH2O was added to make up to 25 μL.

[0037] Step 5: Ligation reaction

[0038] The product of Step 3 was ligated to the digested p1300-AtU6-35S-Cas9 vector using T4 ligase. The reaction system was as follows: 1 μL of the product of Step 3, 1 μL of the product of Step 4, 2.5 μL of T4 Ligase Buffer, and 0.5 μL of T4 Ligase, and reacted at 16 °C for 2 h.

[0039] Step 6: Escherichia coli transformation

[0040] Add 1 μL of the ligation product to 25 μL of DH5α competent cells. After incubation on ice for 25 min, 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 culture with shaking at 37 °C for 1 h. Centrifuge at 13000 rpm / s for 1 min, discard the supernatant, resuspend the remaining 50 μL of bacterial liquid by pipetting, and spread it evenly on an LB plate containing kanamycin. Culture at 37 °C for 16 h.

[0041] Step 7: Identification of positive monoclonal colonies

[0042] Pick monoclonal colonies for PCR verification. The upstream primer is the M13 universal primer. The downstream primer is SlMBP7-sgRNA-R: 5′-ACTCTTCTCCTGCATAATG-3′ (SEQ ID NO.5). After verification by agarose gel electrophoresis, extract the positive clone plasmid and send it for sequencing (sequenced by Shanghai Bioengineering Co., Ltd.) to obtain the CRISPR / Cas9-SlMBP7 recombinant plasmid.

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

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

[0045] Use the leaf disc method to transform tomato cotyledons with the CRISPR / Cas9-SlMBP7 vector. The specific steps are as follows:

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

[0047] 2) Differentiation culture: Transfer the explants to the differentiation medium and culture at 25 °C with a 16 h light / 8 h dark cycle. Replace the medium every 15 d.

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

[0049] Specific medium preparation method:

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

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

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

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

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

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

[0056] Example 3: Molecular identification of transgenic plants

[0057] 1) DNA extraction and PCR analysis: Extract the DNA of T0 generation plants by CTAB method. Design specific primers upstream and downstream near the sequence position of the sgRNA 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, continue sowing the T0 generation seeds with heterozygous PCR sequencing results, analyze the editing situation of T1 generation plants, and the PCR sequencing alignment results show that mbp7-cr1 has a 1 bp deletion in the 4th exon, and mbp7-cr2 has a 197 bp deletion in the 4th exon, see Figure 1 .

[0061] 2) Serotonin content determination: Collect the fruit samples of wild-type AC (Ailsa Craig) and tomato mbp7 mutants (cr1, cr2) at 17 days, 25 days after flowering and at the red-ripe stage for serotonin content determination.

[0062] The specific operations are as follows: Weigh 50 mg of the 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 evenly, and then place it in a refrigerator at 20 °C for 30 min; subsequently, centrifuge at 4 °C and 12,000 r / min for 10 min, take 250 μL of the supernatant, and repeat centrifugation and then take 150 μL of the supernatant. The data acquisition instrument system mainly includes ultra-high performance liquid chromatography (UPLC) and tandem mass spectrometry (MS / MS). The liquid phase conditions include: Waters ACQUITY UPLC HSS T3 C18 column (1.8 μm, 100 mm × 2.1 mm i.d.); 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: at 0 min, A / B is 90:10 (V / V), at 1 min, A / B is 90:10 (V / V), at 6 min, it is 5:95 (V / V), at 7 min, A / B is 5:95 (V / V), at 7.1 min, it is 90:10 (V / V), and at 10 min, it is 90:10 (V / V). The determination of the serotonin content result is as Figure 3 shown.

[0063] Figure 2 In which AC is wild-type tomato, and mbp7-cr1 and mbp7-cr2 are two SlMBP7 gene knockout lines. According to Figure 3 the detection results, after knocking out the tomato SlMBP7 gene, the serotonin content in the tomato fruit development process and the mature stage is significantly increased.

[0064] Obviously, the above embodiments of the present invention are merely examples for more clearly explaining the present invention, rather than limiting the implementation manners of the present invention. For those of ordinary skill in the art, based on the above description, other different forms of changes or modifications can be made. It is impossible to list all the implementation methods here. Any obvious changes or modifications derived from the technical solutions of the present invention are still within the protection scope of the present invention.

Claims

1. The SlMBP7 gene for regulating the serotonin content in tomato fruits, wherein the CDS sequence of the SlMBP7 gene is as shown in SEQ ID NO.

1.

2. The protein for regulating the serotonin content in tomato fruits, wherein the amino acid sequence of the protein is as shown in SEQ ID NO.

2.

3. A method for regulating the serotonin content in tomato fruits, comprising the following steps: Step 1: Design sgRNA target-specific primers for the SlMBP7 gene, and construct the SlMBP7 gene knockout vector CRISPR / Cas9-SlMBP7 vector. The primer sequences are: SlMBP7-sgRNA1-F: 5′-TGATTGCATTATGCAGGAGAAGAGT-3′ (SEQ ID NO.3); SlMBP7-sgRNA1-R: 5′-AAACACTCTTCTCCTGCATAATGCA-3′ (SEQ ID NO.4); Step 2: Transfer the CRISPR / Cas9-SlMBP7 vector constructed in Step 1 into explants by Agrobacterium-mediated infection to obtain the stably inherited F2 generation and progeny seeds of the SlMBP7 gene mutation.

4. The method according to claim 3, wherein In Step 1, the CDS sequence of the SlMBP7 gene is as shown in SEQ ID NO.

1.

5. The method according to claim 3 or 4, characterized in that, In Step 1, use BsaⅠ enzyme to digest the p1300-AtU6-35S-Cas9 vector at 37°C for 4-6 h and then perform a ligation reaction.

6. The method according to claim 3, characterized in that, In Step 2, the Agrobacterium is Agrobacterium tumefaciens EHA105.

7. The method according to claim 3 or 6, characterized in that, In Step 2, the explant is the cotyledon of a tomato germinated for 2 weeks.

8. The application of the SlMBP7 gene in regulating the serotonin content in tomato fruits.

9. The application according to claim 8, wherein, The application includes: increasing the serotonin content in tomato fruits by knocking out the SlMBP7 gene.

10. The application according to claim 8 or 9, characterized in that, The CDS sequence of the SlMBP7 gene is as shown in SEQ ID NO.1.

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