Application of Tomato Transcription Factor SlMYB1 in Regulating Tomato Fruit Quality, Botrytis cinerea Resistance and Fruit Shape

Abstract

The present invention discloses the application of tomato transcription factor SlMYB1 in regulating tomato fruit quality, Botrytis cinerea resistance and changing fruit shape, belonging to the field of biotechnology. The research of the present invention finds that overexpressing the transcription factor SlMYB1 gene in the fruits of wild-type tomato MicroTom, the fruit color of the tomato overexpression lines is more red and bright at the red-ripe stage, and the top of the fruit is smoother. By measuring the fruit quality, it is found that the contents of secondary metabolites such as vitamin C, carotenoids and flavonoids increase. At the same time, by identifying the Botrytis cinerea resistance of the fruits at the red-ripe stage, it is found that the resistance of the SlMYB1 overexpression lines is enhanced. In summary, the tomato transcription factor SlMYB1 gene positively regulates the quality of tomato fruits and the resistance to Botrytis cinerea, as well as changes the fruit shape, providing a basis for further in-depth study of the function of MYB transcription factors.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and specifically relates to the application of the tomato transcription factor SlMYB1 in regulating tomato fruit quality, gray mold resistance and fruit shape. Background Art

[0002] Tomato (Solanum lycopersicum L.) is one of the most important vegetables in the world. It contains rich nutrients and beneficial secondary metabolites, such as lycopene, flavonoids and vitamin C, etc. (Li et al., 2015, Wang et al., 2018).

[0003] Global climate change has an increasingly adverse impact on tomato production, including biotic (such as pathogens and insects) and abiotic stresses (such as drought) (Fedoroff et al., 2010, Xia et al., 2019, Zhu et al., 2018). Tomato gray mold is a necrotrophic disease caused by the fungal pathogen Botrytis cinerea, which seriously affects tomato fruits during maturation and postharvest storage (Soulie et al., 2020). In addition, since tomato is a fleshy and soft fruit, it will quickly lose its firm texture during the ripening process, thus becoming sensitive to diseases during storage and transportation. Therefore, identifying genes related to stress resistance and excellent fruit quality is of great significance for cultivating high-quality and disease-resistant tomato varieties (Chen et al., 2019, Kourelis et al., 2016).

[0004] MYB transcription factors are widely present in organisms such as animals, plants and fungi. They are one of the largest gene families and play different roles in plant growth and development, metabolism and stress resistance (Ramsay and Gonda, 2008). At present, the functions of some MYB transcription factors have been identified in different species. For example, SlMYB72 regulates the metabolism of chlorophyll, carotenoids and flavonoids in tomato fruits (Wu et al., 2020), SlMYB102 can improve the salt tolerance of tomatoes (Zhang et al., 2020), OsMYB1 regulates phosphate homeostasis to regulate root development, and StMYB122 can be induced by benzothiadiazole (BTH) and may regulate the plant's response to pathogens (Li et al., 2019). However, there is no report on MYB transcription factors that can simultaneously regulate tomato fruit quality, improve gray mold resistance and change fruit shape. Summary of the Invention

[0005] In view of the above-mentioned prior art, the object of the present invention is to provide the application of tomato transcription factor SlMYB1 in regulating tomato fruit quality, resistance to Botrytis cinerea, and changing fruit shape. The present invention has found through research that the expression level of SlMYB1 in fruits continuously increases with fruit ripening, positively regulates the accumulation of secondary metabolites such as flavonoids and carotenoids, improves the resistance to Botrytis cinerea, and at the same time changes the fruit shape, making the top smoother. The mode of action of SlMYB1 in regulating fruit quality and resistance provides a basis for further in-depth study of the functions of MYB transcription factors.

[0006] To achieve the above object, the present invention adopts the following technical solutions:

[0007] In the first aspect of the present invention, there is provided the application of the tomato transcription factor SlMYB1 gene as a positive regulatory gene in at least one of the following (1)-(4):

[0008] (1) Improving tomato fruit quality;

[0009] (2) Improving the resistance of tomato fruits to Botrytis cinerea;

[0010] (3) Changing fruit shape;

[0011] (4) Cultivating high-quality and highly resistant tomato varieties;

[0012] The tomato transcription factor SlMYB1 gene is a nucleic acid molecule as shown in the following i), ii), or iii):

[0013] i) A nucleic acid molecule whose nucleotide sequence is the nucleic acid molecule shown in SEQ ID NO.1;

[0014] ii) A nucleic acid molecule whose nucleotide sequence is the nucleic acid molecule shown in SEQ ID NO.2;

[0015] iii) A nucleic acid molecule encoding the amino acid sequence shown in SEQ ID NO.3 other than i) or ii).

[0016] In the second aspect of the present invention, there is provided the application of tomato transcription factor SlMYB1 as a positive regulatory factor in at least one of the following (1)-(4):

[0017] (1) Improving tomato fruit quality;

[0018] (2) Improving the resistance of tomato fruits to Botrytis cinerea;

[0019] (3) Changing fruit shape;

[0020] (4) Cultivating high-quality and highly resistant tomato varieties;

[0021] The amino acid sequence of the tomato transcription factor SlMYB1 is as shown in SEQ ID NO.3.

[0022] In the above application, the tomato transcription factor SlMYB1 gene or the tomato transcription factor SlMYB1 is used as a target. By overexpressing the tomato transcription factor SlMYB1 gene, or increasing the expression level or activity of the tomato transcription factor SlMYB1, the accumulation of secondary metabolites such as soluble sugars, soluble proteins, flavonoids, and carotenoids in tomato fruits can be positively regulated, thereby improving the quality of tomato fruits. At the same time, overexpressing the tomato transcription factor SlMYB1 gene or increasing the expression level or activity of the tomato transcription factor SlMYB1 can also improve the resistance of tomato fruits to Botrytis cinerea. In addition, overexpressing the tomato transcription factor SlMYB1 gene makes the top of its fruit smoother.

[0023] In the third aspect of the present invention, there is provided the use of a recombinant expression vector, transgenic cell line or engineered bacterium containing the tomato transcription factor SlMYB1 gene in any one of the following (1)-(4):

[0024] (1) Improving the quality of tomato fruits;

[0025] (2) Improving the resistance of tomato fruits to Botrytis cinerea;

[0026] (3) Changing the fruit shape;

[0027] (4) Cultivating high-quality and highly resistant tomato varieties.

[0028] In the fourth aspect of the present invention, there is provided a method for simultaneously improving the quality of tomato fruits and the resistance to Botrytis cinerea, including: the step of overexpressing the tomato transcription factor SlMYB1 gene in tomatoes.

[0029] In the above method, overexpressing the tomato transcription factor SlMYB1 gene in tomatoes can be achieved by the method of exogenous transfer of the tomato transcription factor SlMYB1 gene; or upregulating the expression of the tomato transcription factor SlMYB1 gene in the tomato genome.

[0030] In the fifth aspect of the present invention, there is provided a method for cultivating high-quality and highly resistant tomato varieties, including the following steps:

[0031] Transfer the tomato transcription factor SlMYB1 gene into wild tomato plants to overexpress the tomato transcription factor SlMYB1 gene and obtain transgenic tomato plants.

[0032] In the above method, compared with wild tomato plants, the transgenic tomato plants have at least one of the following characteristics 1)-3):

[0033] 1) The contents of vitamin C, carotenoids, flavonoids, and carotenoids in the fruits of the transgenic tomato plants are higher than those in the fruits of the wild tomato plants;

[0034] 2) The incidence of Botrytis cinerea infection in the fruits of the transgenic tomato plants is lower than that in the wild tomato plants.

[0035] 3) The fruits of the transgenic tomato plants are smoother than those of the wild tomato plants.

[0036] In the above method, the method of transferring the tomato transcription factor SlMYB1 gene into wild tomato plants includes: polyethylene glycol method, Agrobacterium-mediated method or gene gun bombardment method.

[0037] Advantages of the present invention:

[0038] The present invention for the first time studies and discovers that overexpressing the transcription factor SlMYB1 gene in the fruits of wild-type tomato MicroTom, the fruit color of the tomato overexpression lines is more red and bright at the red-ripe stage. The fruit quality is measured, and it is found that the contents of secondary metabolites such as vitamin C, carotenoids, and flavonoids increase. At the same time, the resistance to Botrytis cinerea of the fruits at the red-ripe stage is identified, and it is found that the resistance of the SlMYB1 overexpression lines is enhanced. In summary, the tomato transcription factor SlMYB1 gene positively regulates the quality of tomato fruits and the resistance to Botrytis cinerea, providing a basis for further in-depth study of the function of MYB transcription factors. Brief Description of the Drawings

[0039] Figure 1 : Identification of the transcriptional activation function and subcellular localization of the SlMYB1 transcription factor. Figure A shows the identification of the transcriptional activation function. Figure B shows the subcellular localization results, GFP is the green fluorescence signal, DAPI is the nuclear staining signal, BF is the cell structure under bright field, and Merge is the mixed field.

[0040] Figure 2 : Creation and phenotypic identification of SlMYB1 fruit-specific overexpression lines. Figure A shows the quantification of the SlMYB1 expression level, WT is wild-type MicroTom, and OE-1 to OE-8 are SlMYB1 overexpression lines. Figure B shows the phenotypic identification at different fruit stages, DPA is the number of days after flowering.

[0041] Figure 3 : Quality determination of SlMYB1 fruit-specific overexpression lines. The contents of soluble sugar, soluble protein, titratable acid, vitamin C, carotenoids, and flavonoids in wild-type and SlMYB1 overexpression lines (OE-1, OE-2, and OE-3) are measured.

[0042] Figure 4 : Disease resistance determination of SlMYB1 fruit-specific overexpression lines. Figure A shows the inoculation of spore suspension of Botrytis cinerea on the fruits at the red-ripe stage of wild-type and SlMYB1 overexpression lines. Figure B shows the statistical analysis of the lesion diameter. Figure C shows the quantitative detection of the biomass of Botrytis cinerea. Detailed Embodiments

[0043] It should be noted that the following detailed description is illustrative and aims to provide further explanation of the present application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the technical field to which the present application belongs.

[0044] As mentioned above, the quality of tomato fruits and their resistance to Botrytis cinerea are crucial for tomato cultivation. MYB transcription factors are widely present in organisms such as animals, plants, and fungi. They are one of the largest gene families and play different roles in plant growth and development, metabolism, and stress resistance. Therefore, it is of great significance to develop MYB transcription factors that can regulate the quality of tomato fruits and their resistance to Botrytis cinerea.

[0045] Based on this, the present invention has conducted in-depth research on tomato MYB transcription factors and found that overexpression of SlMYB1 can positively regulate the accumulation of substances such as soluble sugars, soluble proteins, flavonoids, and carotenoids in tomato fruits, improving the quality of tomato fruits; at the same time, it can also enhance the resistance of tomatoes to Botrytis cinerea.

[0046] The full-length sequence of the tomato transcription factor SlMYB1 gene is shown in SEQ ID NO.1; the nucleotide sequence of the coding region is shown in SEQ ID NO.2; the amino acid sequence of the encoded SlMYB1 transcription factor is shown in SEQ ID NO.3.

[0047] In order to enable those skilled in the art to more clearly understand the technical solution of the present application, the technical solution of the present application will be described in detail below with reference to specific embodiments. The test materials used in the embodiments of the present invention are all conventional test materials in the art and can be obtained through commercial channels.

[0048] Example 1: Cloning and transcriptional activation function identification of the tomato transcription factor SlMYB1 gene

[0049] RNA of tomato 'MicroTom' leaves was extracted using the Plant RNA Kit R6827 (OMEGA, USA) and reverse transcribed into cDNA using the HiFiScript cDNA Synthesis Kit (ComWin Biotech, China). Using the 'MicroTom' cDNA as a template, amplification was carried out using the KOD-Plus-Mutagenesis Kit (TOYOBO, Japan) according to specific primers (forward: 5'-ATGGAAAACGATACACCGGAAG-3', reverse 5'-TTAGAATGGTCTGCTTGGCAAA-3'). The PCR reaction conditions were: pre-denaturation at 95°C for 3 min; denaturation at 95°C for 30 s, annealing at 55°C for 30 s, extension at 68°C for 1 min, for a total of 33 cycles; final extension at 68°C for 10 min. The target band was recovered by agarose gel electrophoresis with a mass-volume fraction of 0.9%, ligated to the pMD18-T vector (Takara Bio Inc., China), transformed into Escherichia coli DH5α, and positive clones were picked after plasmid extraction and PCR detection and sent to Qingdao Tsingke Biotechnology Co., Ltd. for sequencing.

[0050] According to specific primers (forward: 5’-tcagaggaggacctgcatatgATGGAAAACGATACACCGGAAG-3’, reverse 5’-tcgacggatccccgggaattcTTAGAATGGTCTGCTTGGCAAA-3’), a fragment shown in SEQ ID NO.2 was amplified. The amplified product was recovered using the KOD-Plus-Mutagenesis Kit (TOYOBO, Japan) and ligated into the pGBKT7-BD vector digested with NdeI and EcoRI using the High Efficiency Seamless Cloning Kit (Vazyme, China) to construct the pGBKT7-SlMYB1 recombinant vector. Escherichia coli DH5α was transformed, and after correct sequencing, the plasmid was extracted using the Plasmid Mini Kit (Vazyme, China) and transformed into competent yeast AH109. Cultivation was carried out on yeast SD medium lacking Trp and Trp, His. It was found that yeast transfected with the pGBKT7-SlMYB1 recombinant vector could grow on the medium lacking Trp, His ( Figure 1 A), indicating that it has transcriptional activation activity and the function of a transcription factor.

[0051] The fragment containing SEQ ID NO.2 was amplified using specific primers (forward: 5’-acgatagccggtacccccgggATGGAAAACGATACACCGGAAG-3’, reverse: 5’-gcccttgctcaccatcccgggTTAGAATGGTCTGCTTGGCAAA-3’), and ligated into the SmaI-digested pBIN-GFP plant green fluorescence overexpression vector using the above protocol to create the SlMYB1-pBIN-GFP recombinant vector. After transformation of Agrobacterium, tobacco was injected, and after 3 days, it was found by laser confocal microscopy that SlMYB1 had nuclear localization ( Figure 1 B).

[0052] In summary, SlMYB1 is a classical transcription factor with nuclear localization and activation function.

[0053] Example 2: Creation and phenotypic identification of SlMYB1 fruit-specific overexpression lines

[0054] The product containing the SEQ ID NO.2 sequence was amplified using specific primers (forward: 5’-GGACTAGTATGGAAAACGATACACCGG-3’, reverse: 5’-GGACTAGTTTAGAATGGTCTGCTTGGC-3’), and the product was ligated to the pX6 vector, transformed into Escherichia coli DH5α. After correct sequencing, the plasmid was extracted and transformed into Agrobacterium competent GV3101. The SlMYB1 fruit-specific overexpression lines (OE1 - OE8) were created by infecting the callus of tomato 'MicroTom' using the Agrobacterium-mediated genetic transformation system. At the seedling stage, specific primers (forward: 5’-ATTCTTTTACAACCTCCATGCC-3’, reverse: 5’-GCGTTTAACAGCAGGGTCAAGC-3’) were used for positive identification, and specific primers (forward: 5’-TCACCTGAAGAAGACGCTATAC-3’, reverse: 5’-GGTAAAGGGTTTGCGTTTAACA-3’) were used for quantification of the SlMYB1 expression level.

[0055] The results showed that: compared with the wild-type "Micro-Tom" tomato (WT), the expression level of SlMYB1 was significantly up-regulated in the fruits of the SlMYB1 fruit-specific overexpression lines ( Figure 2 A).

[0056] The SlMYB1 fruit-specific overexpression lines OE1 - OE3 with a significant increase in SlMYB1 expression level were selected as the research objects. During the fruiting period, trait observation and statistics were carried out, and it was found that the fruits of the SlMYB1 overexpression lines were more red and bright. Figure 2B) and the top of the fruit is smoother. Figure 2 B).

[0057] Example 3: Quality determination of SlMYB1 fruit-specific overexpression lines

[0058] The tomato SlMYB1 fruit-specific expression lines (OE1 - OE3) and the wild-type "Micro-Tom" tomatoes were obtained and planted in plastic pots with a diameter of 7 cm. The potting substrate was healthy soil and vermiculite (2:1, volume ratio). The plants were grown under standard greenhouse conditions with a day / night cycle of 16 hours / 8 hours and a temperature regime of 25°C / 20°C. At the red-ripe stage, the fruits were taken and entrusted to Shandong Yihui Biotechnology Company to detect the fruit quality, including soluble sugar, soluble protein, titratable acid, flavonoid, carotenoids, vitamin C and other substances. It was found that the content of related beneficial secondary metabolites in the SlMYB1 overexpression lines was more than that of the wild type ( Figure 3 ), indicating that SlMYB1 can improve the fruit quality and increase the content of substances such as flavonoids and carotenoids.

[0059] Example 4: Disease resistance determination of SlMYB1 fruit-specific overexpression lines

[0060] The Botrytis cinerea used in this invention is recorded in the literature "Study on Multiple Drug Resistance of Botrytis cinerea in Tomatoes" (Journal of Shandong Agricultural University (Natural Science Edition), 2001). The public can obtain the Botrytis cinerea from the applicant within 20 years from the application date for repeating this experiment. The Botrytis cinerea was cultured on PDA medium for 7 days at 25°C with a 14-hour / 10-hour light / dark cycle. The spores produced on the surface of the medium were gently scraped with a brush and suspended in sterile distilled water, and then a spore suspension (2×10 5 conidia mL -1 ) was prepared. Using the wild-type "Micro-Tom" tomatoes and the SlMYB1 overexpression tomato lines (OE1 - OE3) as materials, the spore suspension was injected into the four sides of the red-ripe fruits, and each inoculation site received 10 μL of the spore suspension ( Figure 4 A). After inoculation, the plants were placed in a climate chamber at 25 ± 2°C with a relative humidity of 95% for 24 hours. By counting the diameter of the disease spots, it was found that the diameter of the disease spots in the SlMYB1 overexpression lines was smaller ( Figure 4B); In addition, specific primers (forward: 5’-ACAGAGTTCATGCCCGAAAG-3’, reverse 5’-TTGGTATTCTCTGGCGAGCA-3’) were used to count the biomass of Botrytis cinerea, and it was found that the biomass of the pathogen in the SlMYB1 overexpression lines was less than that in the wild type after inoculation ( Figure 4 C). In summary, SlMYB1 positively regulates the resistance of tomatoes to Botrytis cinerea.

[0061] The above are only the preferred embodiments of the present application and are not intended to limit the present application. For those skilled in the art, various changes and modifications can be made to the present application. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. Application of tomato transcription factor SlMYB1 gene as a positive regulatory gene in at least one of the following (1)-(3): (1) Improve the quality of tomato fruits; (2) Improve the resistance of tomato fruits to Botrytis cinerea; (3) Breed tomato varieties resistant to Botrytis cinerea; The tomato transcription factor SlMYB1 gene is a nucleic acid molecule as shown in the following i), ii), or iii): i) The nucleotide sequence is the nucleic acid molecule shown in SEQ ID NO.1; ii) The nucleotide sequence is the nucleic acid molecule shown in SEQ ID NO.2; iii) A nucleic acid molecule encoding the amino acid sequence shown in SEQ ID NO.3 other than i) or ii).

2. The application according to claim 1, wherein The improvement of the quality of tomato fruits is to increase the contents of soluble sugar, soluble protein, vitamin C, flavonoids and carotenoids in tomato fruits.

3. The application of tomato transcription factor SlMYB1 as a positive regulator in at least one of the following (1)-(3): (1) Improve the quality of tomato fruits; (2) Improve the resistance of tomato fruits to Botrytis cinerea; (3) Breed tomato varieties resistant to Botrytis cinerea; The amino acid sequence of the tomato transcription factor SlMYB1 is shown in SEQ ID NO.

3.

4. The application according to claim 3, characterized in that, The improvement of the quality of tomato fruits is to increase the contents of soluble sugar, soluble protein, vitamin C, flavonoids and carotenoids in tomato fruits.

5. A recombinant expression vector, transgenic cell line or engineered bacterium containing a tomato transcription factor SlMYB1 Use in any one of the following (1)-(3): (1) Improve the quality of tomato fruits; (2) Improve the resistance of tomato fruits to Botrytis cinerea; (3) Breed tomato varieties resistant to Botrytis cinerea; The tomato transcription factor SlMYB1 The gene is a nucleic acid molecule as shown in i) or ii) or iii) below: i) The nucleotide sequence is the nucleic acid molecule shown in SEQ ID NO.1; ii) The nucleotide sequence is the nucleic acid molecule shown in SEQ ID NO.2; iii) A nucleic acid molecule encoding the amino acid sequence shown in SEQ ID NO.3 other than i) or ii).

6. A method for simultaneously improving the quality of tomato fruits and the resistance to Botrytis cinerea, comprising: Steps for overexpressing the tomato transcription factor SlMYB1 gene in tomatoes; The tomato transcription factor SlMYB1 The gene is a nucleic acid molecule shown in any of the following i), ii), or iii): i) The nucleotide sequence is the nucleic acid molecule shown in SEQ ID NO.1; ii) The nucleotide sequence is the nucleic acid molecule shown in SEQ ID NO.2; iii) A nucleic acid molecule encoding the amino acid sequence shown in SEQ ID NO.3 other than i) or ii).

7. The method according to claim 6, wherein Methods for overexpressing the tomato transcription factor SlMYB1 gene in tomatoes include: exogenously transferring the tomato transcription factor SlMYB1 gene; or upregulating the expression of the tomato transcription factor SlMYB1 gene in the tomato genome.

8. A method for cultivating a tomato variety resistant to Botrytis cinerea, characterized in that, It includes the following steps: Transfer the tomato transcription factor SlMYB1 gene into wild tomato plants to overexpress the tomato transcription factor SlMYB1 gene and obtain transgenic tomato plants; The tomato transcription factor SlMYB1 The gene is a nucleic acid molecule as shown in any of the following i), ii), or iii): i) The nucleotide sequence is the nucleic acid molecule shown in SEQ ID NO.1; ii) The nucleotide sequence is the nucleic acid molecule shown in SEQ ID NO.2; iii) A nucleic acid molecule encoding the amino acid sequence shown in SEQ ID NO.3 other than i) or ii).

9. The method according to claim 8, wherein Compared with the wild tomato plants, the transgenic tomato plants have at least one of the following characteristics 1)-2): 1) The contents of vitamin C, flavonoids and carotenoids in the fruits of the transgenic tomato plants are higher than those in the fruits of the wild tomato plants; 2) The incidence of Botrytis cinerea in the fruits of the transgenic tomato plants is lower than that in the wild tomato plants after infection.

10. The method according to claim 8, characterized in that, The method of transferring the tomato transcription factor SlMYB1 gene into wild tomato plants includes: polyethylene glycol method, Agrobacterium-mediated method or gene gun bombardment method.

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