Regulation of tomato carotenoid gene slerf5, protein encoded by the gene and application thereof

Abstract

The application discloses a kind of regulation tomato carotene gene SlERF5 and its coding protein and application.This application identifies a new regulation tomato carotene gene SlERF5, and first constructs SlERF5 tomato genome editing material, finds that the gene plays a regulatory role in carotene biosynthesis, can effectively improve the carotene content in tomato fruit.Compared with wild type, knockout SlERF5 gene causes the expression of carotene metabolism related gene to be up-regulated, to promote the accumulation of carotene in tomato fruit.This provides important scientific basis and technical path for improving the carotene content of tomato by genetic means, improving fruit nutritional quality.

Description

Technical Field

[0001] This invention belongs to the field of molecular biology, and in particular relates to a gene SlERF5 that regulates carotenoids in tomatoes, its encoded protein, and its applications. Background Technology

[0002] Tomato (Solanum lycopersicum) is an important horticultural crop, its fruit rich in carotenoids such as lycopene, beta-carotene, and lutein. These carotenoids not only give tomatoes their red, orange, or yellow color but also have significant nutritional and health benefits. Lycopene is a potent antioxidant closely associated with reducing the risk of cardiovascular disease and certain cancers; beta-carotene is a precursor to vitamin A synthesis and plays a crucial role in maintaining vision, promoting immune function, and antioxidation. The biosynthesis of carotenoids involves a complex regulatory network, and its mechanisms are influenced by multiple genes and signaling pathways.

[0003] Current methods for regulating tomato and carotenoids include gene editing technology and plant hormone regulation. Plant hormone regulation can cause abnormal plant growth, which can lead to a decline in the quality of agricultural products. However, gene editing regulation may not have a significant regulatory effect.

[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, their functions can be studied and their effects 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-mentioned technical problems, the present invention aims to provide a gene SlERF5 that regulates tomato carotenoids. The present invention has identified a new gene SlERF5 that regulates tomato carotenoids. Knocking out this gene using CRISPR / Cas9 gene editing technology will increase the carotenoid content of tomatoes, and therefore has good application prospects in breeding high-carotenoid tomato varieties.

[0006] This invention also provides a protein that regulates the coding of the tomato carotenoid gene SlERF5 and its application.

[0007] Technical solution: In order to achieve the above objectives, the present invention provides a gene SlERF5 that regulates tomato carotenoids, the sequence of which is shown in SEQ ID NO.1.

[0008] Furthermore, the primer pair used to amplify the SlERF5 gene is:

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

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

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

[0012] The present invention describes the knockout vector pToCas9-ERF5, which regulates the tomato carotenoid gene SlERF5.

[0013] Furthermore, the gene knockout vector construction method involves designing an sgRNA that recognizes the target site, digesting it with enzymes and ligating it into the pToCas9 vector, and then transforming it to finally obtain the gene knockout vector pToCas9-ERF5.

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

[0015] The application of the gene SlERF5 or its encoded protein or knockout vector described in this invention in regulating carotenoids in tomato fruit.

[0016] Furthermore, the carotenoid content in tomato fruits was increased by editing the SlERF5 gene in tomato using CRISPR / Cas9.

[0017] The present invention relates to the application of the gene SlERF5, which regulates tomato carotenoids, or its encoded protein or knockout vector, in the cultivation of tomato varieties with high carotenoid content in the fruit.

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

[0019] This invention is the first to construct the SlERF5 tomato genome-edited material and conduct functional studies, discovering that this gene plays a regulatory role in carotenoid biosynthesis and can effectively increase the carotenoid content in tomato fruits. This provides important scientific evidence and technical pathways for improving tomato carotenoid content and enhancing fruit nutritional quality through genetic means.

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

[0021] This invention utilizes CRISPR / Cas9 gene editing technology to construct a mutant of the tomato SlERF5 gene and conducts in-depth research on its function. Experimental verification revealed that knocking out the SlERF5 gene significantly increased the carotenoid content in tomato fruits, indicating that the SlERF5 gene plays a negative regulatory role in the regulation of carotenoid biosynthesis. The discovery of this novel 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, demonstrating broad application prospects. Attached Figure Description

[0022] Figure 1 This is a diagram of the pToCas9 carrier.

[0023] Figure 2 A schematic diagram of the mutant DNA sequence obtained by knocking out the target sequence of the SlERF5 gene in CRISPR / Cas9.

[0024] Figure 3 The content of each carotenoid component in the fruit of the obtained SlERF5 mutant strain. Detailed Implementation

[0025] The technical solution of the present invention will be further described below with reference to the accompanying drawings.

[0026] Unless otherwise specified, all materials and reagents used in the following examples are commercially available. Experimental methods not specifically described in the examples are generally performed under standard conditions or as recommended by the manufacturer.

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

[0028] In this invention, the various carriers pCE2 TA / Blunt-Zero and pCBC-DT1T2 are all commercially available or publicly disclosed carriers.

[0029] Example 1

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

[0031] Using cDNA from the tissue of tomato variety 'Ailsa Craig' (AC) as a template, primers ERF5-F and ERF5-R were designed and cloned using the high-fidelity enzyme PhantaMax Super-Fidelity DNA Polymerase (Vazyme).

[0032] The primer sequences are:

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

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

[0035] The reaction mixture consisted of 18 μL ddH₂O, 25 μL buffer, 1 μL dNTP, 2 μL each of forward and reverse primers, 1 μL cDNA, and 1 μL enzyme. The reaction program was as follows: 95°C pre-denaturation for 3 minutes; 95°C denaturation for 15 seconds, 55°C annealing for 15 seconds, 72°C extension for 30 seconds, for a total of 35 cycles; and a final extension at 72°C for 5 minutes. The PCR product was purified using the FastPure Gel DNA Extraction Mini Kit (Vazyme) and ligated into pCE2 TA / Blunt-Zero (Vazyme). The plasmid was sequenced, and the nucleotide sequence of the SlERF5 gene is shown in SEQ ID No. 1, while the amino acid sequence encoded by this sequence is shown in SEQ ID No. 2.

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

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

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

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

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

[0041] The Golden Gate ligation system consisted of: 2 μL T4 DNA ligase buffer, 1 μL T4 DNA ligase (Promega), 1 μL Bsa I-HF-V (NEB), 1 μL PCR-purified fragment, 1 μL pCBC-DT1T2, and 14 μL ddH2O. The ligation product was transformed into *E. coli* Trans5α competent cells and incubated overnight at 37°C in Kans resistant medium. Positive clones were screened by PCR using the following primers: pToCas9-F: 5'-GCAGGCATGCAAGCTTATTGG-3' and pToCas9-Seq-R: 5'-CAGCTGGCGAAAGGGGGAT-3'. Positive clones were subjected to Sanger sequencing using pToCas9-Seq-R primers. Correctly sequenced positive clones were extracted, and recombinant plasmids were extracted using a plasmid miniprep kit (Takara). The plasmids were named pToCas9-ERF5, and the corresponding E. coli strains were flash-frozen in liquid nitrogen at -80°C after being treated with glycerol.

[0042] III. Construction and Detection of Genome Editing Materials:

[0043] The constructed pToCas9-ERF5 plasmid was transformed into Agrobacterium strain LBA4404 (Weidi Biotechnology). Positive clones were screened by PCR for infection of cotyledons of tomato variety 'AC'. Before infection, the cotyledons needed to be pre-cultured. The pre-culture process was as follows: tomato cotyledons were cut one week after sowing and soaked in pre-culture solution (MS liquid medium + 0.2 mg / L 2,4-D + 0.1 mg / L Kinetin) for 1 hour. After drying, they were placed on pre-culture medium (MS solid medium + 1 mg / L IAA + 1.75 mg / L zeatin). The infection process was as follows: the pre-cultured cotyledons were soaked in Agrobacterium bacterial solution for 15 minutes (OD value of bacterial solution 2.0), excess bacterial solution was blotted with filter paper, and they were returned to the original medium for co-culture for 2 days. After two days of co-culturing, the cotyledons were transferred to resistance medium (MS solid medium + 1.0 mg / L IAA + 1.75 mg / L zeatin + 75 mg / L kanamycin + 200 mg / L termetidine). Once callus formation occurred, the cotyledons were transferred to shoot-forming medium (MS solid medium + 1.0 mg / L zeatin + 50 mg / L kanamycin + 200 mg / L termetidine). After shoot emergence, they were excised and transferred to rooting medium (MS solid medium + 200 mg / L termetidine). PCR detection of the Cas9 and npt genes in the obtained tissue culture seedlings was performed. The PCR detection primers were zCas9-F: 5'-CCACATGATTAAGTTCAGGGGCCAT-3' and zCas9-R: 5'-GAGCCTTCGTAATCTCGGTGTTC-3', as well as npt-F: 5'-GGCCGCTTGGG TGGAGAG-3' and npt-R: 5'-GGTAGCCGGATCAAGCGTATG-3'. Plants positive for both Cas9 and npt sites were then amplified using the SlERF5 editing site detection primers CR-SlERF5-genotyping-F: 5'-ATGGA AGGAGAGAAGAGAAAACAG-3' and CR-SlERF5-genotyping-R: 5'-GTTATCTTCGTCGC AACTTTCC-3'. The PCR products were Sanger sequenced, and the mutant plants were successfully constructed. Five gene-editing positive plants were obtained, and the seeds of the T1 generation were sown and self-pollinated for seed production. PCR was performed again using primers zCas9-F and zCas9-R. Three homozygous T2 mutant lines without the Cas9 gene sequence were selected and named CR-erf5-1, CR-erf5-2, and CR-erf5-3, respectively. Their gene editing site sequences are as follows: Figure 2As shown in the figure, CR-erf5-1 lost one base at the first target site, CR-erf5-2 lost five bases at the second target site, and CR-erf5-3 lost one base at each of the first and second target sites. In all three plants, the loss of bases caused changes in the reading frame, resulting in premature termination of translation.

[0044] IV. Analysis of Carotenoid Content:

[0045] Fruits from CR-erf5-1, CR-erf5-2, CR-erf5-3, and the control 'AC' cultivated to red-ripe stage were used for carotenoid component and content analysis. Under liquid nitrogen pre-cooling conditions, the red-ripe fruit samples were pulverized into powder. 0.5 g of the powder was used for carotenoid extraction. 30 mL of extraction buffer (a 1:1:1 mixture of hexane, acetone, and ethanol, v / v) was added, and the mixture was shaken at 150 rpm for 30 minutes to mix thoroughly. 15 mL of ddH2O was added, and the mixture was centrifuged at 1500 g for 10 minutes. The supernatant was collected, filtered, and concentrated using a nitrogen blower. Residues were dissolved three times using 0.5 mL of dissolving buffer (tetrahydrofuran:acetonitrile:methanol = 15:30:55, v / v). The solutions were then mixed in sample vials for high-performance liquid chromatography (HPLC) analysis. Carotenoids were determined using a Shimadzu HPLC instrument equipped with an SPD-M20A diode array detector. A 20 μL 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, with 0.05% triethylamine added. The absorption wavelength was set to 475 nm. The carotenoid content was calculated using a carotenoid standard (Sigma) as a reference. 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, with the total carotenoid content increasing by 24.2%, 22.4% and 24.0%, respectively. This indicates that knocking out the SlERF5 gene significantly increased the carotenoid content in tomato fruit, suggesting that the SlERF5 gene plays a negative regulatory role in the regulation of carotenoid biosynthesis.

Claims

1. SlERF5 The application of gene knockout vectors in increasing the carotenoid content in tomato fruits, wherein the carotenoids are lycopene or β-carotene; SlERF5 The gene sequence is shown in SEQ ID NO.1; the gene knockout vector is... pToCas9-ERF5 .

2. Application of the SlERF5 gene knockout vector in increasing the carotenoid content in tomato fruit, wherein the carotenoids are total carotenoids; SlERF5 The gene sequence is shown in SEQ ID NO.1; the gene knockout vector is... pToCas9-ERF5 .

3. The application according to claim 1 or 2, characterized in that, The gene knockout vector construction method involves designing an sgRNA that recognizes the target site, digesting it with enzymes and ligating it into a pToCas9 vector, and then transforming it to obtain the gene knockout vector. pToCas9-ERF5 .

4. The application according to claim 3, characterized in that, The sgRNA at the target site includes target site 1 and target site 2, wherein the sequence of target site 1 is 5'-AACGCTCTCGAATTTGGCT-3' and the sequence of target site 2 is 5'-AATTTCCCTGAATGTATAG-3'.

5. SlERF5 The application of gene knockout vectors in cultivating tomato varieties with high carotenoid content in the fruit, wherein the carotenoids are lycopene or β-carotene; SlERF5 The gene sequence is shown in SEQ ID NO.1; the gene knockout vector is... pToCas9-ERF5 .

6. Application of the SlERF5 gene knockout vector in cultivating tomato varieties with high carotenoid content in the fruit, wherein the carotenoids are total carotenoids; SlERF5 The gene sequence is shown in SEQ ID NO.1; the gene knockout vector is... pToCas9-ERF5 .

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