SlTAGL1 gene for regulating and controlling lutein content of tomato fruits, method and application

Abstract

The invention provides an SlTAGL1 gene for regulating and controlling the lutein content of tomato fruits, a method and application, a coding sequence of the SlTAGL1 gene is obtained from a tomato genome, a target spot is connected to CRISPR / Cas9 by designing the target spot, the tomato is successfully infected by using agrobacterium competent cells, a CRISPR / Cas9 gene editing plant of the tomato is obtained, the gene editing plant is analyzed, and the content of lutein in the tomato fruits is regulated and controlled. The result shows that compared with the wild tomato, the lutein content in the tomato fruits after the SlTAGL1 is knocked out is obviously increased by 1.6-2.2 times. According to the invention, an efficient and stable genetic method is adopted to improve the content of xanthophyll in tomato fruits, so that the quality of the tomato fruits is improved, and gene resources and strategies are provided for culturing high-xanthophyll tomato varieties.

Description

Technical Field

[0001] The invention belongs to the technical field of plant genetic engineering and relates to a SlTAGL1 gene, method and application for regulating the lutein content of tomato fruit. Background Art

[0002] Tomato is one of the three largest traded vegetables in the world. According to the statistics of the Food and Agriculture Organization of the United Nations in 2023, the total global tomato production in 2022 was 186 million tons, and China's tomato production accounted for one-third of the global production. Tomato fruits are rich in carotenoid compounds and vitamin C and other nutrients, and their sweet and sour flavor is popular among consumers. In recent years, with the development of the tomato industry and the improvement of people's living standards, the demand for the quality, flavor, yield, resistance and nutrients of tomato varieties has gradually increased.

[0003] Lutein is a tetraterpenoid carotenoid and the main pigment in the macular region of the human retina. It can filter blue light and protect the retina from oxidative damage, and plays an important role in maintaining visual health. Lutein also plays an important role in blocking the occurrence and development of tumors and cardiovascular diseases, inhibiting the proliferation of human gastric cancer and breast cancer cells, and enhancing the body's immunity. It can also be used as a natural pigment. In recent years, its role has been increasingly valued in the health food, cosmetics and pharmaceutical industries. With the popularization of electronic devices such as mobile phones and computers, the prevalence of visual fatigue among people of all ages in my country has greatly increased. Lutein cannot be synthesized in the human body and can only be supplemented by ingestion.

[0004] In plants, lutein is synthesized through the carotenoid biosynthesis pathway. Tomato fruits are rich in carotenoids. Increasing the lutein content in tomato fruits and taking lutein from daily fruits and vegetables can relieve visual fatigue and protect the retina. Therefore, developing a method for quickly increasing the lutein content in tomato fruits is a technical problem that people in this field need to solve urgently. Summary of the invention

[0005] The present invention provides a SlTAGL1 gene, method and application for regulating the lutein content of tomato fruit. The endogenous tomato gene SlTAGL1 is knocked out by using CRISPR / cas9 gene editing technology, so as to obtain tomato fruit with high lutein content, thereby significantly improving the quality and economic value of the tomato fruit.

[0006] To achieve the above object, the technical solution adopted by the present invention is:

[0007] One of the purposes of the present invention is to provide a SlTAGL1 gene for regulating the lutein content in tomato fruit, wherein the nucleotide sequence of the SlTAGL1 gene is shown in SEQ ID NO.1.

[0008] The second object of the present invention is to provide a protein for regulating the lutein content in tomato fruit, wherein the amino acid sequence of the protein is shown in SEQ ID NO.2.

[0009] The third object of the present invention is to provide a method for regulating the lutein content of tomato fruit, comprising the following steps:

[0010] Step 1: Design specific primers for the sgRNA target according to the SlTAGL1 gene, connect the target to CRISPR / Cas9, and construct a CRISPR / Cas9-SlTAGL1 vector. The sequence of the specific primer is:

[0011] SlTAGL1-sgRNA1-F: 5′- TGATTG TATTCATAGAGACGGCCG-3′ (SEQ ID NO. 3);

[0012] SlTAGL1-sgRNA1-R: 5′- AAAC CGGCCGTCTCTATGAATA CA -3′ (SEQ ID NO. 4);

[0013] Step 2: Introduce the CRISPR / Cas9-SlTAGL1 vector constructed in step 1 into tomato cells by Agrobacterium infection to obtain tomato mutant plants with SlTAGL1 gene mutation.

[0014] Preferably, step one comprises: annealing the forward primer and reverse primer of the target in a PCR instrument at 95° C. for 5 min, and then ligating the cleaved p1300-AtU6-35S-Cas9 vector with T4 ligase.

[0015] Preferably, step two comprises: transforming the CRISPR / Cas9-SlTAGL1 vector into EHA105 Agrobacterium competent cells, and then infecting tomato explants with the EHA105 strain containing the CRISPR / Cas9-SlTAGL1 plasmid to prepare a tomato SlTAGL1 mutant material.

[0016] More preferably, the explants used are tomato cotyledons from seeds germinated for 2 weeks.

[0017] Preferably, the above method further comprises: subjecting the tomato mutant plants obtained in step 2 to normal growth management to obtain stably inherited SlTAGL1 gene mutant materials.

[0018] More preferably, the specific steps of the above method are as follows:

[0019] 1) Construction of Agrobacterium tumefaciens expressing tomato SlTAGL1 gene CRISPR / Cas9 vector, using EHA105 strain of Agrobacterium;

[0020] 2) using the Agrobacterium infection method to transform the EHA105 strain containing the CRISPR / Cas9-SlTAGL1 plasmid obtained in step 1) into the target tomato explants to prepare SlTAGL1 gene mutant plants;

[0021] 3) subjecting the SlTAGL1 gene mutant plants obtained in step 2) to normal growth management to obtain stably inherited SlTAGL1 gene mutant materials and offspring homozygous seeds.

[0022] A fourth object of the present invention is to provide an application of the SlTAGL1 gene in regulating the lutein content in tomato fruit.

[0023] The beneficial effects of the present invention are:

[0024] The present invention obtains the coding sequence of the SlTAGL1 gene from the tomato genome, connects the target to CRISPR / Cas9 by designing the target, and successfully infects tomatoes with Agrobacterium competent cells to obtain CRISPR / Cas9 gene-edited plants of tomatoes. The gene-edited plants are analyzed, and the results show that compared with wild-type tomatoes, the lutein content in tomato fruits after SlTAGL1 knockout is significantly increased by 1.6 to 2.2 times. The present invention adopts an efficient and stable genetic method to increase the lutein content in tomato fruits, thereby improving the quality of tomato fruits, and providing gene resources and strategies for cultivating high-lutein tomato varieties. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 : PCR sequencing comparison results of tomato fruits of wild tomato and SlTAGL1 gene knockout lines. WT is wild-type tomato, and tagl1-cr1 and tagl1-cr2 are two SlTAGL1 gene knockout lines.

[0026] Figure 2 : Tomato fruits at maturity of wild tomato and SlTAGL1 gene knockout lines. AC are wild-type tomatoes, and tagl1-cr1 and tagl1-cr2 are two SlTAGL1 gene knockout lines.

[0027] Figure 3 : Detection results of lutein content in wild tomato and SlTAGL1 gene knockout tomato fruits, AC is wild-type tomato, tagl1-cr1 and tagl1-cr2 are two SlTAGL1 gene knockout lines. DETAILED DESCRIPTION

[0028] In order to explain the present invention more clearly, the present invention is further described in detail below in conjunction with embodiments and with reference to the accompanying drawings. It should be understood by those skilled in the art that the content described below is illustrative rather than restrictive, and should not be used to limit the scope of protection of the present invention.

[0029] Example

[0030] 1. Obtaining EHA105 strain containing CRISPR / Cas9-SlTAGL1 plasmid

[0031] Step 1: Target sequence design of SlTAGL1 gene

[0032] The tomato SlTAGL1 gene is 810 bp in total and consists of 9 exons. The sgRNA of the SlTAGL1 gene was screened using the CRISPR-P (http: / / cbi.hzau.edu.cn / cgi-bin / CRISPR) website. Try to select a target site that is located in the first exon of the gene CDS, has a GC content in the range of 45%-70%, and has at least three base mismatches with other locations in the genome.

[0033] Step 2: The primers were synthesized by Shanghai Bioengineering Co., Ltd. The synthetic sequences are as follows:

[0034] SlTAGL1-sgRNA1-F: 5′- TGATTG TATTCATAGAGACGGCCG-3′ (SEQ ID NO. 3);

[0035] SlTAGL1-sgRNA1-R: 5′- AAAC CGGCCGTCTCTATGAATA CA -3′ (SEQ ID NO. 4);

[0036] The underlined part is the linker sequence.

[0037] Step 3: Place the forward primer and reverse primer of the target in step 2 in a PCR instrument at 95°C for 5 minutes, then cool to room temperature to complete annealing.

[0038] Step 4: Use the U6 promoter and BsaⅠ enzyme (NEB) to cut the p1300-AtU6-35S-Cas9 vector at 37°C for 4 to 6 hours. The specific system is: 10μL p1300-AtU6-35S-Cas9, 2.5μL 10×BasⅠCutSmartbuffer, 1μL BsaⅠ enzyme (NEB), and use ddH2O to make up to 25μL.

[0039] Step 5: Use T4 ligase to connect the product of step 3 to the cut p1300-AtU6-35S-Cas9 vector. The ligation system is: take 1 μL of the products of step 3 and step 4, 2.5 μL T4 Ligase Buffer, and 0.5 μL T4 Ligase and place them at 16°C for 2 hours.

[0040] Step 6: Transformation clones by chemical heat shock method: Add 1 μL of CRISPR / Cas9-SlTAGL1 plasmid connected in step 5 to 25 μL of E. coli DH5a competent cells placed on ice, let stand for 25 minutes, then heat shock in a 42°C metal bath for 45 seconds, then quickly return to ice and let stand for 2 minutes. Add 900 μL of antibiotic-free LB solution in a sterile environment, shake and culture at 37°C shaker at 220 rpm / min for 1 hour, then centrifuge at 13000 rpm / s for 1 minute to collect the bacterial solution, discard the supernatant, gently pipette and mix with the remaining 50 μL of bacterial solution, and evenly spread it on the LB medium containing anti-kanamycin. Incubate inverted overnight at 37°C incubator for 16 hours.

[0041] Step 7: Identification of monoclonal colonies: The positive monoclonal colonies obtained in step 6 were identified by PCR. The upstream primer was the universal M13 primer and the downstream primer was SlTAGL1-sgRNA-R: 5′-CGGCCGTCTCTATGAATA-3′.

[0042] The obtained PCR products were tested by agarose gel electrophoresis to check whether the band size of the PCR products was consistent with the theoretical value. The monoclonal colonies with the correct band size were subjected to plasmid extraction and sent for testing (Shanghai Bioengineering Co., Ltd. for sequencing). The plasmid was extracted using the Nanjing Bio-Novozyme plasmid extraction kit. The CRISPR / Cas9-SlTAGL1 plasmid was obtained.

[0043] Step 8: The recombinant CRISPR / Cas9-SlTAGL1 plasmid is transformed into EHA105 Agrobacterium competent cells to obtain the EHA105 strain containing the CRISPR / Cas9-SlTAGL1 plasmid.

[0044] 2. Construction of SlTAGL1 gene mutant plants

[0045] The CRISPR / Cas9-SlTAGL1 vector was transformed into tomato cotyledons using the leaf disc method, screened using hygromycin, and mutant plants were screened using PCR amplification and first-generation sequencing.

[0046] The specific steps are as follows:

[0047] Use Agrobacterium EHA105 strain containing CRISPR / Cas9-SlTAGL1 plasmid to infect tomato cotyledons: soak the pre-prepared tomato cotyledons in a sterile culture dish containing 15mL MS infection solution for 15 minutes, then transfer the explants to sterile filter paper, absorb the remaining infection solution and transfer the leaves to new pre-culture medium, place them with the reverse side facing up at 25℃ in the dark for 48 hours.

[0048] After co-cultivation, the explants were transferred to differentiation medium and cultured at 25°C with 16h / 8h light / dark conditions. The differentiation medium was replaced every 15 days.

[0049] After differentiation and budding, cut off the bud with independent main stem and insert it into rooting medium. After rooting, transplant it into nutrient pot.

[0050] Specific culture medium preparation:

[0051] Seed germination medium: 2.15g / L MS powder + 30g / L sucrose + 7.4g / L agar. pH value is 5.5.

[0052] Pre-culture medium: 2.15g / L MS powder + 10g / L sucrose + 8g / L agar + 0.2mg / L 2,4-D mother solution (1mg / mL). pH value is 5.8.

[0053] Differentiation medium: 4.3g / L MS powder + 20g / L sucrose + 7.4g / L agar + 2mg / L Zeatin stock + 300mg / L Timentin + 6mg / L hygromycin. pH value is 6.

[0054] Rooting medium: 2.15g / L MS powder + 30g / L sucrose + 7.4g / L agar + 300mg / L Timentin + 6mg / L hygromycin. pH value is 6.

[0055] MS infection solution: 4.3g / L MS (519) powder + 20g / L sucrose. pH value is 6.

[0056] LB solution: 10g / L Nacl + 10g / L Trypyone + 5g / L Yeast Extract. pH value is 7.

[0057] 3. Identification of transgenic positive plants at the DNA level using PCR

[0058] Using T0 generation seedlings as templates, DNA was extracted using the CTAB method, and specific primers were designed upstream and downstream of the sgRNA sequence position of the SlTAGL1 gene to detect changes in the target gene sequence. The primers (synthesized by Shanghai Bioengineering Co., Ltd.) are as follows:

[0059] SlTAGL1-CHECK-F: 5′-GGAGGAGGTAGAGGGAAGAT-3′ (SEQ ID NO. 5);

[0060] SlTAGL1-CHECK-R: 5'-ACACCATAGTCACCCTCTTT-3' (SEQ ID NO. 6).

[0061] The T0 generation seeds that were heterozygous according to PCR sequencing results were sown continuously, and the editing status of T1 generation plants was analyzed. The PCR sequencing comparison results showed that tagl1-cr1 had a 2bp deletion in the first exon, and tagl1-cr2 had a 1bp deletion in the first exon, see Figure 1 . Thus, a stably inherited SlTAGL1 mutant material was obtained.

[0062] 4. Determination of lutein content in fruits of wild-type and SlTAGL1 mutant plants

[0063] After obtaining homozygous mutants, red-ripe fruit samples of wild-type AC (Ailsa Craig) and tomato SlTAGL1 mutant (tagl1-cr1, tagl1-cr2) plants were collected. Figure 2 As shown, the lutein content was determined.

[0064] The specific operation is as follows: weigh 50 mg of the powder after freezing and grinding, add 0.5 mL of a mixed solution of n-hexane / acetone / ethanol (1:1:1, v / v / v) containing 0.01% BHT (g / mL), vortex at room temperature for 20 minutes, centrifuge at 12000r / min for 5 minutes at 4°C, take the supernatant, repeat the extraction once, centrifuge and combine the supernatants; then add 100 μL of dichloromethane to dissolve, filter through a 0.22 μm filter membrane, and store in a brown injection bottle for LC-MS / MS analysis. The data acquisition instrument system mainly includes ultra-high performance liquid chromatography (UPLC) and tandem mass spectrometry (MS / MS). The liquid phase conditions were as follows: chromatographic column: YMC C30 (3 μm, 100 mm × 2.0 mm id); mobile phase: phase A, methanol / acetonitrile (1:3, v / v) with 0.01% BHT and 0.1% formic acid added; phase B, methyl tert-butyl ether with 0.01% BHT added; gradient elution program: 0 min A / B is 100:0 (V / V), 3 min is 100:0 (V / V), 5 min is 30:70 (V / V), 9 min is 5:95 (V / V), 10 min is 100:0 (V / V), 11 min is 100:0 (V / V); flow rate is 0.8 mL / min; column temperature is 28 °C; injection volume is 2 μL. The results of lutein content determination are as follows Figure 3shown.

[0065] according to Figure 3 The test results showed that after knocking out the tomato SlTAGL1 gene, the lutein content in tomato fruits increased significantly.

[0066] Obviously, the above embodiments of the present invention are merely examples to more clearly illustrate the present invention, and are not limitations on the implementation methods of the present invention. For ordinary technicians in the relevant field, other different forms of changes or modifications can be made on the basis of the above description. It is impossible to list all the implementation methods here. All obvious changes or modifications derived from the technical solution of the present invention are still within the protection scope of the present invention.

Claims

1. A SlTAGL1 gene for regulating the lutein content in tomato fruit, wherein the nucleotide sequence of the SlTAGL1 gene is shown in SEQ ID NO.

1.

2. A protein for regulating the lutein content in tomato fruit, the amino acid sequence of the protein being shown in SEQ ID NO.

2.

3. A method for regulating the lutein content of tomato fruit, comprising the following steps: Step 1: Design specific primers for the sgRNA target according to the SlTAGL1 gene, connect the target to CRISPR / Cas9, and construct a CRISPR / Cas9-SlTAGL1 vector. The sequence of the specific primer is: SlTAGL1-sgRNA1-F:5′- TGATTG TATTCATAGAGACGGCCG-3′(SEQ ID NO.3); SlTAGL1-sgRNA1-R:5′- AAAC CGGCCGTCTCTATGAATA CA -3′ (SEQ ID NO.4) Step 2: Introduce the CRISPR / Cas9-SlTAGL1 vector constructed in step 1 into tomato cells by Agrobacterium infection to obtain tomato mutant plants with SlTAGL1 gene mutation.

4. The method according to claim 3, characterized in that Step 1 includes: annealing the forward primer and reverse primer of the target in a PCR instrument at 95°C for 5 minutes, and then connecting them to the cut p1300-AtU6-35S-Cas9 vector using T4 ligase.

5. The method according to claim 3, characterized in that: Step 2 includes: transforming the CRISPR / Cas9-SlTAGL1 vector into EHA105 Agrobacterium competent cells, and then infecting tomato explants with the EHA105 strain containing the CRISPR / Cas9-SlTAGL1 plasmid to prepare a tomato SlTAGL1 mutant material.

6. The method according to claim 5, characterized in that The tomato explants used were cotyledons of tomato seeds germinated for 2 weeks.

7. The method according to claim 3, characterized in that Also includes: The tomato mutant plants obtained in step 2 are subjected to normal growth management to obtain stably inherited SlTAGL1 gene mutant materials.

8. The method according to claim 3, characterized in that The specific steps of the method are as follows: 1) Construction of Agrobacterium tumefaciens expressing tomato SlTAGL1 gene CRISPR / Cas9 vector, using EHA105 strain of Agrobacterium; 2) using the Agrobacterium infection method to transform the EHA105 strain containing the CRISPR / Cas9-SlTAGL1 plasmid obtained in step 1) into the target tomato explants to prepare SlTAGL1 gene mutant plants; 3) subjecting the SlTAGL1 gene mutant plants obtained in step 2) to normal growth management to obtain stably inherited SlTAGL1 gene mutant materials and offspring homozygous seeds.

9. Application of SlTAGL1 gene in regulating lutein content in tomato fruit.

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