Use of genes for promoting lycopene biosynthesis

Abstract

The present invention belongs to the field of plant genetics, and in particular relates to the use of a gene to promote lycopene biosynthesis in tomato fruits. The present invention discloses the use of a gene to promote lycopene biosynthesis in plants, the gene being Solyc05g004600 having the nucleotide sequence shown in SEQ ID NO: 1, and by knocking out the Solyc05g004600 gene in tomato, lycopene biosynthesis in plant fruits can be promoted.

Description

Technical Field

[0001] The present invention belongs to the field of plant genetics and particularly relates to the use of genes for promoting the biosynthesis of lycopene in tomato fruits.

Background Art

[0002] Lycopene is a natural pigment widely contained in mature red plant fruits such as tomatoes, carrots, watermelons, papayas, and pomegranates. Its content is the highest in tomato fruits and is also an important indicator for measuring the quality of tomato fruits. Lycopene is currently considered to be one of the most powerful antioxidant plant nutrients. It achieves an antioxidant effect by removing free radicals generated by human cells, can delay aging, and is also effective in preventing neurological diseases and suppressing tumors. In addition, it is effective in many aspects such as preventing cardiovascular diseases, improving immunity, and delaying osteoporosis, and is expected to have a wide range of applications in the development fields of health foods and nutritional foods.

[0003] Currently, the biosynthesis pathway of lycopene and its related enzymes has been relatively clearly studied. In plants, lycopene is preferentially biosynthesized through the 1-deoxy-D-xylulose-5-phosphate (DOXP) pathway. Its direct precursor is isopentenyl pyrophosphate (IPP), which is synthesized from DOXP by a series of enzymatic catalysis in plastids. IPP generates dimethylallyl pyrophosphate (DMAPP) under the action of isomerase. IPP and DMAPP form geranylgeranyl pyrophosphate (GGPP) under the action of geranylgeranyl pyrophosphate synthase (GGPS). Next, phytoene is synthesized by the catalysis of an important enzyme, phytoene synthase (PSY). Subsequently, phytoene undergoes dehydrogenation reactions catalyzed by phytoene dehydrogenase (PDS) and ζ-carotene dehydrogenase (ZDS) to form lycopene.

[0004] Research has shown that promoting the expression of important enzyme genes PSY, PDS, and ZDS in the lycopene synthesis pathway may significantly increase lycopene production. The cytokinin CKs synthesis gene SlIPT4 positively regulates lycopene biosynthesis by directly affecting the ζ-carotene isomerase gene ZISO and ZDS. The positive ripening regulator NOR-like1 of tomato fruits controls the expression of SlACS2 and promotes lycopene accumulation. Suppressing the CUL4 gene increases the number of chromoplasts and lycopene accumulates in tomato fruits. Suppressing the SlNAC1 gene controls the expression of the ACC synthase 2 gene ACS2, the lycopene β-cyclase gene LCYb, PSY1 and other related genes, and increases the lycopene content of tomato fruits. Therefore, discovering new genes that control lycopene biosynthesis and promoting lycopene production and developing breeding applications have important theoretical significance and production value.

[0005] In the Chinese patent "Use of Genes for Enhancing Resistance to Tomato Gray Mold" (Application No.: 202110058943.X), it was reported that the Solyc05g004600 gene has a resistance function against tomato gray mold.

Summary of the Invention

Problems to be Solved by the Invention

[0006] The technical problem to be solved by the present invention is how to effectively promote the biosynthesis of lycopene in plants.

Means for Solving the Problems

[0007] To solve the above technical problems, the present invention provides the use of a gene that promotes lycopene biosynthesis in plants (a gene that negatively regulates lycopene biosynthesis in plants). The gene is Solyc05g004600 having the nucleotide sequence shown in SEQ ID NO: 1. By knocking out the Solyc05g004600 gene of tomato, the lycopene biosynthesis amount in plant fruits is promoted (effectively promoted).

[0008] As an improvement in the use of the present invention, the plant is a tomato.

[0009] As a further improvement in the use of the present invention, the sgRNA sequence of the CRISPR / Cas9 editing target in the Solyc05g004600 gene: 5'-GTTGTTCAACATGAGCAGAG-3' is designed, primers are artificially synthesized based on the sgRNA sequence, and are constructed into the CRISPR / Cas9 vector.

[0010] As a further improvement in the use of the present invention, the nucleotide sequence of the homozygous mutant obtained after knocking out the Solyc05g004600 gene is shown in SEQ ID NO: 2.

[0011] As a further improvement in the use of the present invention, compared with the wild-type tomato variety Micro-Tom, the lycopene content of tomato fruits after knocking out the Solyc05g004600 gene is significantly increased, and the expression level of PSY, a gene encoding an important enzyme in the lycopene biosynthesis pathway, is also significantly increased.

[0012] The present invention provides the Solyc05g004600 gene of tomato, and the nucleotide sequence of the protein encoded by the gene is shown in SEQ ID NO: 1.

[0013] The present invention also provides a method for knocking out the Solyc05g004600 gene of tomato, which includes the following steps. 1) Design the sequence of the CRISPR / Cas9 editing target: 5'-GTTGTTCAACATGAGCAGAG-3'; 2) Using the sequence obtained in step 1), construct a CRISPR / Cas9 gene editing vector for the Solyc05g004600 gene. 3) The vector obtained in step 2) was used to genetically transform wild-type tomatoes, specifically editing the Solyc05g004600 gene in their genomes to obtain mutant plants in which the Solyc05g004600 gene was knocked out. The amount of lycopene biosynthesis in the fruits of these mutant plants was significantly higher than that in the wild-type control variety.

[0014] The details of the technical solution of the present invention are as follows. First, using the CRISPR / Cas9 gene editing technology, an sgRNA sequence (SEQ ID NO: 1) specifically targeting the gene encoding Solyc05g004600 was designed, a CRISPR / Cas9 vector was constructed, and the knockout vector was genetically transformed into the wild-type tomato variety Micro-Tom to obtain genetically modified plants.

[0015] Next, the Solyc05g004600 gene of the genetically modified plants was amplified and sequenced using PCR technology to identify mutant strains KO-#1 and KO-#2 in which the gene was knocked out. The sequencing results of the mutation sites are shown in Figure 1. These two mutant strains were derived from different transformed calli, and the sequence of the Solyc05g004600 gene in them is SEQ ID NO: 2. After the tomatoes ripened, the lycopene contents of the fruits of wild-type Micro-Tom and mutant strains KO-#1 and KO-#2 were detected, and it was found that the lycopene contents of the mutant strains were significantly higher than that of wild-type Micro-Tom (Figure 2).

[0016] To prove that the Solyc05g004600 gene regulates the biosynthesis of lycopene in tomatoes, the gene Solyc05g004600 sequence (SEQ ID NO: 1) was obtained using PCR amplification, an overexpression vector of the Solyc05g004600 gene was constructed, and then the overexpression vector was genetically transformed into the wild-type tomato variety Micro-Tom to obtain the corresponding genetically modified plants. Next, the qRT-PCR technique was used to detect the expression level of the Solyc05g004600 gene in the genetically modified plants, and the overexpression lines OE-#1 and OE-#2 of this gene were identified and shown in Figure 3. After the tomato fruits matured, by detecting the lycopene content of the plant fruits of wild-type Micro-Tom and overexpression lines OE-#1 and OE-#2, it was found that the lycopene content of overexpression lines OE-#1 and OE-#2 was significantly lower than that of wild-type Micro-Tom (Figure 4).

[0017] At the same time, the gene encoding PSY, an important enzyme in the lycopene biosynthesis pathway (phytoene synthase), was selected, and the qRT-PCR technique was used to analyze the expression level of the PSY gene in the fruits of wild-type Micro-Tom, mutant strains KO-#1 and KO-#2, and overexpression lines OE-#1 and OE-#2. It was shown that the expression of the PSY gene in the mutant plants was significantly higher than that of wild-type Micro-Tom, while the expression of the PSY gene in the overexpression plants was significantly lower than that of wild-type Micro-Tom (Figure 5).

Advantages of the Invention

[0018] These results further indicate that the Solyc05g004600 gene negatively regulates the biosynthesis of lycopene in tomatoes. Therefore, knocking out the Solyc05g004600 gene in tomatoes may promote the biosynthesis of lycopene and increase the lycopene content in fruits. The Solyc05g004600 gene and its use have important application values.

[0019] It should be emphasized that the Chinese patent "Use of Genes for Enhancing Resistance to Tomato Gray Mold" (Application No.: 202110058943.X) only reported that the Solyc05g004600 gene has a resistance function against tomato gray mold, which has nothing to do with the use for promoting lycopene biosynthesis included in the present invention.

Brief Description of the Drawings

[0020] In the following figures, ** in the figures indicates a highly significant difference from the wild-type control variety.

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Modes for Carrying Out the Invention

[0021] (Step 1. Construction of the CRISPR / Cas9 Vector for Solyc05g004600 Gene Knockout) (Refer to Chinese Patent 202110058943.X "Use of Genes for Enhancing Resistance to Tomato Gray Mold")

[0022] Based on the coding sequence of the Solyc05g004600 gene (SEQ ID NO: 1), the sgRNA sequence: 5’-GTTGTTCAACATGAGCAGAG-3’ is designed using the Guide Design Resources online software (http: / / crispr.mit.edu / ). Corresponding primers are synthesized based on this sequence. Upstream 5’-TGATTGTTGTTCAACATGAGCAGAG-3’, Downstream 5’-AAACCTCTGCTCATGTTGAACAACA-3’. For the construction of the corresponding CRISPR / Cas9 vector, a CRISPR / Cas9 kit (Biogle) is used, and the construction method is carried out according to the product manual.

[0023] (Step 2. Genetic transformation of tomatoes using the Solyc05g004600 gene CRISPR / Cas9 vector) The CRISPR / Cas9 vector constructed in Step 1 is genetically transformed into the tomato variety Micro-Tom to enable targeted editing of the Solyc05g004600 gene in the genome. The gene recombination method adopted is the method of Kimura et al (Kimura S et al, CHS Protoc, 2008) to obtain the corresponding genetically modified tomato plants.

[0024] (Step 3. Identification of the Solyc05g004600 gene editing site in genetically modified tomatoes) Using the SDS method, genomic DNA of transgenic tomato plants and their wild-type Micro-Tom was extracted. 0.1 g of tomato leaves were collected, ground in liquid nitrogen, and 600 μl of extraction buffer (15.76 g of Tris-cl, 29.22 g of Nacl, 15.0 g of SDS powder, added with ultrapure water to a final volume of 1 L and adjusted to pH 8.0) was added, followed by incubation at 65 °C for 60 minutes. 200 μl of KAC (5 mol / L) was added, mixed, and carried out in an ice bath for 10 minutes. Further, 500 μl of chloroform was added, mixed, centrifuged at 10,000 rpm for 5 minutes, the supernatant was collected, 500 μl of isopropanol was added, mixed well, centrifuged at 12,000 rpm for 3 minutes, and the supernatant was discarded. The precipitate was washed with 75% ethanol, centrifuged at 12,000 rpm for 3 minutes, and the supernatant was discarded. After drying the DNA for 15 minutes, 30 μl of pure water was added to dissolve the DNA.

[0025] Using the extracted tomato genomic DNA as a template, PCR amplification was performed using PrimeSTAR® HS DNA Polymerase (TaKaRa). The PCR primers used were F1: 5’-TAGAGTTGGAACCTTTGTAAT-3’ and R1: 5’-TTGTTCCTCCAAAGTCAATAT-3’. The PCR amplification system was as follows: 25 μl of PrimeSTAR HS (Premix), 1 μl each of F1 and R1 primers (10 μM), 2 μl of template DNA (<200 ng), and 21 μl of sterilized water. The PCR amplification process was as follows: pre-denaturation at 95 °C for 5 minutes, denaturation at 98 °C for 10 seconds, annealing at 58 °C for 15 seconds, extension at 72 °C for 60 seconds, 30 cycles, and extension at 72 °C for 5 minutes.

[0026] The PCR products were sent to a biotechnology company for sequencing analysis. The sequencing primer was F1. Two homozygous mutant strains KO-#1 and KO-#2 of the Solyc05g004600 gene were identified.

[0027] These two mutant strains are derived from different transformed calli, are the gene recombination products of different transformation events, and are two independent gene recombination plants. A base A is inserted into the coding region of the Solyc05g004600 gene, and its nucleotide sequence is as described in SEQ ID NO: 2, causing a frameshift mutation in the gene. As a result, the gene is knocked out in both strains.

[0028] (Step 4. Measurement of lycopene content in fruits of Solyc05g004600 gene knockout plants) The two mutant strains KO-#1 and KO-#2 of the Solyc05g004600 gene identified above and their wild-type control variety Micro-Tom were planted in a greenhouse at 25°C, with 16 hours of light and 8 hours of darkness. When the tomato fruits matured, three plants were randomly selected from each variety, and three mature fruits were harvested from each plant. Weighed 0.2 g of fruit tissue, ground it in liquid nitrogen, added 8 mL of a hexane:ethanol:acetone (2:1:1, V:V:V) mixture, and placed it on a shaker (100 rpm) overnight at room temperature. Added 1 mL of H2O, vortexed, used a spectrophotometer, used hexane as a control, took out the solvent layer (upper liquid), measured the absorbance at 503 nm, and repeated the measurement three times. The lycopene content in tomato fruits is determined by the following calculation formula. Lycopene component (μg / g) = (x / y) × A503 × 3.12, where x is the amount of hexane (ml), y is the weight of the fruit tissue (g), A503 is the absorbance at 503 nm, and the extinction coefficient is 3.12. The measurement results are analyzed using a t-test for significant differences between the mutants and the wild-type control.

[0029] The results obtained were that the lycopene contents of the mutant strains KO-#1 and KO-#2 of the Solyc05g004600 gene were significantly higher than the lycopene content of the control variety Micro-Tom (Figure 2).

[0030] (Step 5. Construction of tomato plants overexpressing the Solyc05g004600 gene) Extract total RNA from the leaves of the wild-type tomato variety Micro-Tom using the RNeasy Plant Mini Kit (QIAGEN) according to the product instructions. Then, reverse transcribe it into cDNA using the PrimeScript TM 1st Strand cDNA Synthesis Kit (TaKaRa). The operating method follows the product instructions. Using the cDNA obtained above, amplify the Solyc05g004600 gene by PCR with PrimeSTAR (registered trademark) HS DNA Polymerase (manufactured by TaKaRa). The composition of the reaction system is carried out according to the product instructions. The sequences of the PCR primers are F2: 5’- cggggtacc AAGTATACACTATGGGTGATTC-3’, R2: 5’- aaaactgcag GCTAATTAATTAGTGCTATGG-3’ (the underlined letters are restriction endonuclease recognition sequences). The PCR amplification program is pre-denaturation at 95°C for 5 minutes, denaturation at 98°C for 10 seconds, annealing at 58°C for 15 seconds, extension at 72°C for 30 seconds, 30 cycles, and extension at 72°C for 5 minutes.

[0031] The pCAMBIA1300-2×35S vector is digested with the restriction endonucleases Kpn I and Pst I (TaKaRa). The reaction system is as follows: add 1 μl each of Kpn I and Pst I, 4 μl of Buffer (attached to the product), 15 μl of pCAMBIA1300-2×35S vector plasmid, and ddH2O to make 40 μl, and digest at 37°C for 4 hours. The digestion products are purified using the AxyPrep PCR Cleaning Kit (Axygen) according to the product manual. The amplification products of the Solyc05g004600 gene are also purified in the same way. The digested and purified vector and PCR product above are ligated using the T4 ligase kit (Promega). The ligation system is as follows: add 1 μl of digested vector plasmid, 2 μl of target gene fragment, 0.5 μl of T4 ligase, 1 μl of buffer, and ddH2O to make 10 μl, and incubate at 4°C overnight (12 hours). The reaction products are transformed into JM109 competent cells by the heat shock method. After obtaining positive clones, sequence analysis is requested from a biotechnology company to confirm the inserted fragment (SEQ ID NO: 1) in the vector and obtain the expression vector of the Solyc05g004600 gene. According to the method in Step 2, the vector is genetically transformed into the wild-type tomato variety Micro-Tom to obtain the corresponding transgenic tomato plants.

[0032] (Step 6. Identification of Solyc05g004600 gene overexpression plants) Extract the total RNA of the transformed tomato plants obtained in Step 5 according to the method described in Step 5 and reverse transcribe it into cDNA. Next, perform qPCR using the TB GreenTM Premix Ex TaqTM kit (TaKaRa). The sequences of the PCR primers for the Solyc05g004600 gene are F3: 5’-ATGGGTGATTCTTCGGCTCA-3’ and R3: 5’-TCCAACTCTTTCCACACTGTG-3’. Use the tomato housekeeping gene actin as an internal reference, and the sequences of its PCR primers are F4: 5’-CAGCAGATGTGGATCTCAAA-3’ and R4: 5’-CTGTGGACAATGGAAGGAC-3’. The PCR reaction system is as follows. 2 μL of cDNA, 10 μL of Green Premix Ex Taq, 1 μL each of F3 and R3 primers (or F4 and R4, both at a concentration of 10 μM), 5.6 μL of ddH2O, and 0.4 μL of ROX Reference Dye. PCR is performed on a StepOne Plus TM real-time PCR system (Applied Biosystems). The execution program is as follows. Pre-denaturation at 95°C for 30 seconds; 95°C for 5 seconds, 60°C for 30 seconds, for 40 cycles, and 2 -ΔΔCt methods are used to analyze the expression level of the Solyc05g004600 gene. The measurement results are analyzed using a t-test for significant differences between the mutant and wild-type controls.

[0033] The results are shown in Figure 3. The expression levels of the Solyc05g004600 gene in the overexpression lines OE-#1 and OE-#2 plants are much higher than those of the wild-type variety Micro-Tom.

[0034] Note: As common sense, the role of the internal reference is as follows. When detecting the expression of a target gene, a specific internal reference gene is used to eliminate differences in RNA yield, quality, and reverse transcription efficiency between different samples and obtain the true differences in the specific expression of the target gene. Usually, it is selected for calibration and normalization.

[0035] (Step 7. Determination of Lycopene Content in Solyc05g004600 Gene Overexpression Plants) According to the method described in Step 4, the results of measuring the lycopene in Solyc05g004600 gene overexpression lines OE-#1 and OE-#2 plants and their wild-type control variety Micro-Tom fruits are shown in Figure 4. The lycopene contents of overexpression lines OE-#1 and OE-#2 are significantly lower than that of wild-type Micro-Tom.

[0036] The expression level of the Solyc05g004600 gene is inversely proportional to the lycopene content and the expression levels of its synthetic genes.

[0037] (Step 8. Expression Analysis of the Key Enzyme Gene PSY for Lycopene Synthesis) According to the method described in Step 6, the expression levels of the PSY gene in the fruits of Solyc05g004600 gene mutant lines KO-#1 and KO-#2, overexpression lines OE-#1 and OE-#2, and their wild-type control variety Micro-Tom are detected. The measurement results are analyzed using a t-test for significant differences between mutants and wild-type controls. The sequences of the PCR primers for the PSY gene are F5: 5’-ACGAAACAGAAATACTTGGC-3’, R5: 5’-CTTCCGACAACTTCTTTTGG-3’. The results are shown in Figure 5. The expression levels of the PSY gene in the fruits of mutant strains KO-#1 and KO-#2 were significantly higher than that of wild-type Micro-Tom, while the expression levels of the PSY gene in the fruits of overexpression strains OE-#1 and OE-#2 plants were significantly lower than the expression level of wild-type Micro-Tom, indicating that the Solyc05g004600 gene is involved in the expression regulation of the key enzyme gene PSY for lycopene synthesis, thereby affecting the biosynthesis of lycopene.

[0038] Finally, it should also be noted that the above enumeration only represents some specific embodiments of the present invention. It goes without saying that the present invention is not limited to the above embodiments and various modifications are possible. All changes that can be directly derived or associated by those skilled in the art from the disclosure of the present invention should be considered to be within the protection scope of the present invention.

Claims

A method for using a gene for promoting lycopene biosynthesis in tomatoes, comprising: the gene being Solyc05g004600 having the nucleotide sequence shown in SEQ ID NO: 1, and promoting the biosynthesis of lycopene in tomatoes by knocking out the gene; designing an sgRNA sequence for use as a CRISPR / Cas9 editing target for the gene, the sgRNA sequence being 5'-GTTGTTCAACATGAGCAGAG-3', synthesizing primers based on the sgRNA sequence, constructing them into a CRISPR / Cas9 vector to obtain a knockout vector, genetically transforming the knockout vector into the wild-type tomato variety Micro-Tom, and obtaining a genetically modified plant in which the gene is knocked out; the nucleotide sequence of the homozygous mutant, which is a genetically modified plant obtained by knocking out the gene, is shown in SEQ ID NO: 2; a method for using a gene, characterized in that, compared with the wild-type tomato variety Micro-Tom, the lycopene content of tomato fruits after knocking out the gene is increased, and the expression level of the gene PSY encoding an important enzyme in the lycopene biosynthesis pathway is also increased.

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