Gene SlERF5 for regulating tomato carotenoid as well as encoding protein and application thereof

Abstract

The invention discloses a gene SlERF5 for regulating and controlling tomato carotenoid as well as an encoding protein and application of the gene SlERF5. A novel gene SlERF5 for regulating tomato carotenoid is identified, an SlERF5 tomato genome editing material is constructed for the first time, and it is found that the gene plays a regulating role in carotenoid biosynthesis, and the content of carotenoid in tomato fruits can be effectively increased. Compared with a wild type, the knockout of the SlERF5 gene leads to up-regulation of expression of carotenoid metabolism related genes, so that accumulation of carotenoid in tomato fruits is promoted. The invention provides an important scientific basis and a technical path for increasing the carotenoid content of tomatoes and improving the nutritional quality of fruits through genetic means.

Description

Technical Field

[0001] The invention belongs to the field of molecular biology, and in particular relates to a tomato carotenoid regulating gene SlERF5 and a coded protein and application thereof. Background Art

[0002] Tomato (Solanum lycopersicum) is one of the important horticultural crops, and its fruit is rich in carotenoid components such as lycopene, β-carotene, and lutein. The above carotenoid components not only give tomato fruits red, orange or yellow colors, but also have important value in nutrition and health. Among them, lycopene is a powerful antioxidant that is closely related to reducing the risk of cardiovascular disease and certain cancers; β-carotene is a synthetic precursor of vitamin A, which plays a key role in maintaining vision, promoting immune function, and anti-oxidation. The biosynthesis of carotenoids involves a complex regulatory network, and its mechanism is affected by multiple genes and signaling pathways.

[0003] Current methods for regulating tomato and carrot varieties include gene editing technology and plant hormone regulation. Plant hormone regulation can affect abnormal plant growth, and in severe cases can lead to a decline in the quality of agricultural products. However, gene editing regulation may also result in unclear regulatory effects.

[0004] In recent years, genome editing technologies such as CRISPR / Cas9 have provided powerful tools for functional gene research and crop genetic improvement. By knocking out genes through genome editing technology, its function can be studied and its effect on the synthesis of various biological factors can be verified. Summary of the invention

[0005] Purpose of the invention: In order to solve the above technical problems, the present invention aims to provide a tomato carotenoid regulating gene SlERF5. The present invention identifies a new tomato carotenoid regulating gene SlERF5. Knocking out the gene using CRISPR / Cas9 gene editing technology will increase the carotenoid content of tomatoes. Therefore, it has good application prospects in breeding high tomato carotenoid varieties.

[0006] The present invention also provides a protein for regulating the coding of tomato carotenoid gene SlERF5 and its application.

[0007] Technical solution: In order to achieve the above-mentioned purpose, the present invention provides a tomato carotenoid gene SlERF5 for regulation, wherein the sequence of the gene SlERF5 is shown in SEQ ID NO.1.

[0008] Further, the primer pair used to amplify the gene S1ERF5 is:

[0009] ERF5-F:5'-ATGGAAGGAGAGAAGAGAAAAC-3',

[0010] ERF5-R:5'-TCAGTTATCTTCGTCGCAACTTTC-3'.

[0011] The amino acid sequence of the tomato carotenoid regulating gene SlERF5 of the present invention is shown in SEQ ID NO.2.

[0012] The knockout vector pToCas9-ERF5 for regulating the tomato carotenoid gene SlERF5 of the present invention.

[0013] Furthermore, the gene knockout vector construction method is to design an sgRNA that recognizes the target site, connect it to the pToCas9 vector after enzyme cutting, and finally obtain the gene knockout vector pToCas9-ERF5 through transformation.

[0014] Furthermore, the sgRNA of the target site includes target 1 and target 2, the target 1 sequence is 5'-AACGCTCTCGAATTTGGCT-3', and the target 2 sequence is 5'-AATTTCCCTGAATGTATAG-3'.

[0015] The invention discloses an application of the gene SlERF5 or the encoded protein or the knockout vector in regulating carotenoids in tomato fruits.

[0016] Furthermore, the tomato SlERF5 gene was edited by CRISPR / Cas9 to increase the carotenoid content in tomato fruits.

[0017] The invention discloses an application of the tomato carotenoid regulating gene SlERF5 or the encoding protein or the knockout vector in cultivating high carotenoid varieties in tomato fruits.

[0018] Furthermore, the carotenoids include any one of lycopene, β-carotene or total carotenoids.

[0019] The present invention first constructed the SlERF5 tomato genome editing material and conducted functional studies, finding that the gene plays a regulatory role in carotenoid biosynthesis and can effectively increase the carotenoid content in tomato fruits. This provides an important scientific basis and technical path for increasing the carotenoid content of tomatoes and improving the nutritional quality of fruits through genetic means.

[0020] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages:

[0021] The present invention uses CRISPR / Cas9 gene editing technology to construct a tomato SlERF5 gene mutant and conducts in-depth research on its function. Experimental verification shows that knocking out the SlERF5 gene significantly increases the content of carotenoids in tomato fruits, indicating that the SlERF5 gene plays a negative regulatory role in the regulation of carotenoid biosynthesis. The discovery of the new function of the SlERF5 gene provides a scientific basis for improving the nutritional quality of tomato fruits and creating high-quality tomato germplasm materials with high carotenoid content, showing broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is a map of the pToCas9 vector;

[0023] Figure 2 Schematic diagram of the mutant DNA sequence obtained for CRISPR / Cas9 knockout of the SlERF5 gene target sequence;

[0024] Figure 3 The contents of various carotenoid components in the fruits of the obtained SlERF5 mutant strain. DETAILED DESCRIPTION

[0025] The technical solution of the present invention is further described below in conjunction with the accompanying drawings.

[0026] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources. Experimental methods without specific conditions specified in the examples are usually carried out under conventional conditions or under conditions recommended by the manufacturer.

[0027] The tomato variety 'Ailsa Craig' (AC) (Sun et al. A Transscriptional Network Promotes Anthocyanin Biosynthesis in Tomato Flesh. Mol Plant, 2020, 13(1): 42-58.) in the present invention was provided by Yangzhou University.

[0028] The vectors pCE2 TA / Blunt-Zero and pCBC-DT1T2 in the present invention are commercially available or disclosed vectors.

[0029] Example 1

[0030] 1. Obtain the full-length sequence of SlERF5:

[0031] The cDNA of tomato variety 'Ailsa Craig' (AC) tissue was used as a template to design primers ERF5-F and ERF5-R, and the high-fidelity enzyme PhantaMax Super-Fidelity DNA Polymerase (Vazyme) was used for cloning.

[0032] The primer sequences are:

[0033] ERF5-F:5'-ATGGAAGGAGAGAAGAGAAAAC-3',

[0034] ERF5-R:5'-TCAGTTATCTTCGTCGCAACTTTC-3';

[0035] The reaction system was: ddH2O 18μL, Buffer 25μL, dNTP 1μL, upstream and downstream primers 2μL each, cDNA 1μL, enzyme 1μL. The reaction procedure was: 95℃ pre-denaturation for 3 minutes; 95℃ denaturation for 15 seconds, 55℃ annealing for 15 seconds, 72℃ extension for 30 seconds, a total of 35 cycles; 72℃ thorough extension for 5 minutes. The PCR reaction product was purified with FastPure Gel DNAExtraction Mini Kit (Vazyme), and the product was connected to pCE2 TA / Blunt-Zero (Vazyme). The plasmid was sequenced, and the nucleotide sequence of the SlERF5 gene was shown in SEQ ID No.1, and the amino acid sequence encoded by the sequence was shown in SEQ ID No.2.

[0036] 2. Construction of CRISPR / Cas9 genome editing vector for SlERF5 gene

[0037] Two 19 bp target gene sites were selected in the coding sequence of the SlERF5 gene and primers were designed. The target site 1 sequence was: 5'-AACGCTCTCGAATTTGGCT-3', and the target site 2 sequence was: 5'-AATTTCCCTGAATGTATAG-3'. A pair of primers containing the target site sequences were designed for the construction of the genome editing vector. The primer sequences were:

[0038] SlERF5-CR-F: 5'-ATATATGGTCTCGTTTGAACGCTCTCGAATTTGGCTGTTTTAGAGCTAGAAATAGC-3',

[0039] SlERF5-CR-R: 5'-ATTATTGGTCTCGAAACCTATACATTCAGGGAAATTCCAAACTACACTGTTAGATTC-3'.

[0040] The plasmid pCBC-DT1T2 (Addgene, Plasmid #50590) was used as a template and cloned using the high-fidelity enzyme Phanta Max Super-Fidelity DNA Polymerase (Vazyme). The PCR-purified fragment was connected to the pToCas9 vector using the Golden Gate system (pToCas9 vector is as follows Figure 1 The sequence is shown in SEQ ID No. 3, synthesized by a biological company).

[0041] The Golden Gate system was as follows: T4 DNA ligase buffer 2 μL, T4 DNA ligase 1 μL (Promega), Bsa I-HF-V2 1 μL (NEB), PCR-purified fragment 1 μL, pCBC-DT1T2 1 μL, ddH2O 14 μL. The ligation product was transformed into E. coli Trans5α competent cells, Kan resistance medium was incubated at 37°C overnight, and positive clones were screened by PCR. The colony PCR primers were: pToCas9-F: 5'-GCAGGCATGCAAGCTTATTGG-3' and pToCas9-Seq-R: 5'-CAGCTGGCGAAAGGGGGAT-3'. The positive clones were subjected to Sanger sequencing, and the sequencing primer was pToCas9-Seq-R. The positive clones with correct sequencing were extracted, and the recombinant plasmid was extracted using a plasmid extraction kit (Takara). The plasmid was named pToCas9-ERF5, and the corresponding Escherichia coli strain was added with glycerol and quickly frozen with liquid nitrogen and stored at -80°C.

[0042] 3. Construction and testing of genome editing materials:

[0043] The constructed pToCas9-ERF5 plasmid was transferred into the Agrobacterium strain LBA4404 (Weidi Biotech), and the positive clones were screened by PCR to infect the cotyledons of the tomato variety 'AC'. Before infection, the cotyledons need to be pre-cultured. The pre-culture process is as follows: cut the tomato cotyledons one week after sowing, soak them in the pre-culture solution (MS liquid medium + 0.2mg / L 2,4-D + 0.1mg / L Kinetin) for 1h, and then place them on the pre-culture medium (MS solid medium + 1mg / L IA A + 1.75mg / L Zeatin) after drying. The infection process is as follows: soak the pre-cultured cotyledons in the Agrobacterium bacterial solution for 15 minutes (the OD value of the bacterial solution is 2.0), use filter paper to dry the excess bacterial solution, and return them to the original culture medium for co-culture for 2 days. After 2 days of co-cultivation, the cotyledons were transferred to a resistance medium (MS solid medium + 1.0 mg / L IAA + 1.75 mg / L zeatin + 75 mg / L kanamycin + 200 mg / L timentin). After callus formation, the cotyledons were transferred to a budding medium (MS solid medium + 1.0 mg / L zeatin + 50 mg / L kanamycin + 200 mg / L timentin). After the buds grew out, they were cut onto a rooting medium (MS solid medium + 200 mg / L timentin). The Cas9 gene and npt gene of the obtained tissue culture seedlings were tested by PCR. The PCR detection primers were zCas9-F: 5'-CCACATGATTAAGTTCAGGGGCCAT-3' and zCas9-R: 5'-GAGCCTTCGTAATCTCGGTGTTC-3' and npt-F: 5'-GGCCGCTTGGG TGGAGAG-3' and npt-R: 5'-GGTAGCCGGATCAAGCGTATG-3'. Plants that were positive for both the Cas9 site and the npt site were amplified using the detection primers CR-SlERF5-genotyping-F: 5'-ATGGA AGGAGAGAAGAGAAAACAG-3' and CR-SlERF5-genotyping-R: 5'-GTTATCTTCGTCGC AACTTTCC-3' of the SlERF5 editing site. The PCR products were sequenced by Sanger sequencing, and mutant plants were successfully constructed. Finally, 5 gene-edited positive plants were obtained. The above 5 plants were kept as seeds to obtain T1 generation seeds for sowing and self-pollination. PCR detection was performed again using primers zCas9-F and zCas9-R, and three homozygous T2 mutant strains without Cas9 gene sequences were selected and named CR-erf5-1, CR-erf5-2, and CR-erf5-3, respectively. The gene editing site sequences were as follows Figure 2Among them, CR-erf5-1 lacked 1 base at the first target site, CR-erf5-2 lacked 5 bases at the second target site, and CR-erf5-3 lacked 1 base at the first and second target sites respectively. The deletion of bases in all three plants caused the reading frame to change, resulting in premature termination of translation.

[0044] 4. Analysis of carotenoid content:

[0045] The fruits of CR-erf5-1, CR-erf5-2, CR-erf5-3 and control 'AC' grown to the red-ripe stage were used for carotenoid composition and content analysis. Under liquid nitrogen precooling conditions, the red-ripe fruit samples were crushed into powder, and 0.5g of powder was taken for carotenoid extraction. 30mL of extracting solution (the extracting solution is a mixed solution of hexane: acetone: ethanol with a volume ratio of 1:1:1) was added, and the mixture was shaken at 150r / min for 30 minutes to mix thoroughly. 15mL of ddH2O was added, and the mixture was centrifuged at 1500g for 10 minutes. The supernatant was aspirated, filtered, and concentrated by nitrogen blowing. 0.5mL of dissolving solution (tetrahydrofuran: acetonitrile: methanol = 15:30:55, volume ratio) was used to dissolve the residue each time, for a total of three times. The above solutions were mixed into sample bottles for HPLC analysis. Carotenoids were determined using a Shimadzu HPLC instrument (Shimadzu) with a SPD-M20A diode array detector. 20 μL of sample was injected into a C18 column (4.6 mm × 250 mm) at a flow rate of 1.2 mL / min. The mobile phase was methanol:acetonitrile = 90:10, and 0.05% triethylamine was added. The absorption wavelength was set to 475 nm. The carotenoid content was calculated using a carotenoid standard (Sigma). The results showed that Figure 3 As shown, compared with 'AC', the contents of lycopene, β-carotene and total carotenoids in CR-erf5-1, CR-erf5-2 and CR-erf5-3 were significantly increased, and the increase rate of total carotenoids was 24.2%, 22.4% and 24.0%, respectively, indicating that knocking out the SlERF5 gene significantly increased the content of carotenoids in tomato fruit, indicating that the SlERF5 gene plays a negative regulatory role in the regulation of carotenoid biosynthesis.

Claims

1. A method for regulating tomato carotenoid gene SlERF5, characterized in that: The sequence of the gene SlERF5 is shown as SEQ ID NO.

1.

2. The method for regulating the tomato carotenoid gene S1ERF5 according to claim 1, characterized in that: The primer pair used to amplify the gene SlERF5 is: ERF5-F:5'-ATGGAAGGAGAGAAGAGAAAAC-3', ERF5-R:5'-TCAGTTATCTTCGTCGCAACTTTC-3'.

3. A tomato carotenoid regulating gene S1ERF5 according to claim 1, characterized in that: The amino acid sequence is shown in SEQ ID NO.

2.

4. A gene knockout vector pToCas9-ERF5 for regulating tomato carotenoid gene SlERF5 according to claim 1.

5. The gene knockout vector pToCas9-ERF5 according to claim 4, characterized in that The gene knockout vector construction method comprises designing an sgRNA that recognizes the target site, enzymatically connecting it to a pToCas9 vector, and then transforming the vector to finally obtain the gene knockout vector pToCas9-ERF5.

6. The gene knockout vector pToCas9-ERF5 according to claim 5, characterized in that The sgRNA of the target site includes target 1 and target 2, the target 1 sequence is 5'-AACGCTCTCGAATTTGGCT-3', and the target 2 sequence is 5'-AATTTCCCTGAATGTATAG-3'.

7. Use of the gene S1ERF5 according to claim 1, or the encoded protein according to claim 3, or the knockout vector according to claim 4 in regulating carotenoids in tomato fruit.

8. The use according to claim 7, characterized in that: Editing the tomato SlERF5 gene through CRISPR / Cas9 can increase the carotenoid content in tomato fruits.

9. Use of the tomato carotenoid regulating gene S1ERF5 according to claim 1, the encoded protein according to claim 3, or the knockout vector according to claim 4 in cultivating high carotenoid varieties in tomato fruits.

10. The use according to claim 9, characterized in that: The carotenoids include any one of lycopene, β-carotene or total carotenoids.

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