Application of tomato SlWRKY2 gene in regulation and control of fruit coloring and size

Abstract

The invention discloses an application of a tomato SlWRKY2 gene in regulation and control of fruit coloring and size, and relates to the field of gene engineering and molecular biology. According to the application, accumulation of lycopene, beta-carotene and the like in tomato fruits can be promoted through overexpression of the tomato SlWRKY2 gene in the tomato, the tomato fruit coloring process is accelerated, and the fruit size is not changed; the SlWRKY2 gene is knocked out from the tomato by using a gene editing technology, so that the time from flowering to color breaking of the tomato can be delayed, the fruit coloring process is slowed down, and the fruit size is remarkably reduced. The invention discloses the dual regulation and control effect of the SlWRKY2 gene on the coloring and size of the fruits for the first time, provides a new gene resource for cultivating a new high-quality tomato variety, and has a relatively good potential application value.

Description

Technical Field

[0001] This application involves fields such as genetic engineering, molecular biology, and physiology, specifically the application of the SlWRKY2 gene in regulating tomato fruit coloring and size. Background Technology

[0002] tomato( Solanum lycopersicum As the world's leading vegetable crop with a total production of 170 million tons, the dynamic changes in color during the ripening process of tomatoes are one of the core characteristics of quality formation. Tomato fruit ripening goes through four stages: green ripening, color breaking, red ripening, and full ripening (Ye Bishun et al., 2019). During the green ripening stage, chlorophyll dominates, resulting in a green color. As ripening progresses, chlorophyll gradually degrades, and the content of carotenoids such as lycopene and β-carotene continuously accumulates, ultimately achieving the color change from green to red, which is an important marker of fruit ripening. Lycopene not only determines the degree of fruit coloring but also has extremely high nutritional value, playing a significant role in enhancing the body's antioxidant capacity, anti-inflammation, and reducing the risk of various tumors such as prostate cancer and cardiovascular diseases. Fruit size is a key indicator affecting the sensory quality and yield of tomatoes, and its regulation involves multiple factors such as the number of layers of pericarp cells, cell size, and the number of ventricles, but its genetic regulatory mechanism is not yet fully understood. Given that transcription factors play a central role in the regulation of tomato fruit coloring and size, screening and identifying novel transcription factors that regulate fruit coloring and size has become an important research direction for elucidating the mechanisms of tomato fruit quality formation and ripening.

[0003] WRKY transcription factors are DNA-binding proteins widely found in plants (Eulgem et al., 2000). Their most prominent feature is the highly conserved WRKY domain: composed of 60 amino acids, including one or two WRKYGQK amino acid sequences at the N-terminus and a zinc finger motif (C2-HC or C2H2) at the C-terminus. The W-box [(T)(T)TGAC(C / T)] in the gene promoter is the preferred binding region for WRKY transcription factors, with the core sequence being TGAC. Previous studies have confirmed that WRKY transcription factors participate in the regulation of tomato fruit ripening through multiple pathways, including chlorophyll degradation, lycopene synthesis, and ripening-related transcription factors such as ERF / RIN. For example, SlWRKY35 It can directly activate 1-deoxy-D-xyulose 5-phosphate synthase ( SlDXS1 The expression of this gene reprograms metabolism to the 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway, thereby enhancing carotenoid accumulation (Yuan et al., 2022). Twelve genes upregulated by ethylene... SlER-WRKYs Among them, 8 can be directly controlled. SlPAO , SlPPH , SlPSY1 and SlPDSColor-related genes (Wang et al., 2017). The above research results show that the WRKY family has a clear function in fruit ripening, especially in the coloring process, and is involved in a complex regulatory network. However, further exploration is needed for the WRKY family to participate in the regulation of tomato fruit coloring, and currently there is no research report on the involvement of WRKY in the regulation of tomato fruit size. As a member of the WRKY family, the function of SlWRKY2 in fruit coloring and size regulation has not been reported. The present application first clearly defines its dual regulation, filling the research gap. SUMMARY

[0004] The purpose of the embodiments of the present application is to provide a tomato SlWRKY2 gene in the regulation of tomato fruit coloring and size, so as to promote tomato fruit coloring and improve tomato nutritional and appearance fruit quality.

[0005] According to a first aspect of the embodiments of the present application, a tomato SlWRKY2 gene in the regulation of tomato fruit coloring and size, the SlWRKY2 nucleotide sequence of the gene is shown as SEQ ID NO: 1.

[0006] Optionally, the SlWRKY2 amino acid sequence of the protein encoded by the gene is shown as SEQ ID NO: 2.

[0007] Optionally, overexpression of the SlWRKY2 gene in tomato can promote tomato fruit coloring, and the fruit size remains unchanged; knockout of the SlWRKY2 gene in tomato can inhibit tomato fruit coloring and reduce fruit size.

[0008] Optionally, the gene overexpression technology comprises the following steps: extracting total RNA from tomato, reverse transcribing to obtain cDNA, using cDNA as a template, F and R as primers, amplifying SlWRKY2 gene, constructing the amplification product into a plant overexpression vector, the nucleotide sequences of the primers F and R are shown as SEQ ID NO: 3 and 4; introducing the plant overexpression vector into a host cell, then using it to infect the target plant, screening positive transgenic plants, and obtaining transgenic plants.

[0009] Optionally, the host cell is Agrobacterium.

[0010] Optionally, the Agrobacterium is GV3101.

[0011] Optionally, the plant expression vector is an expression vector with a 35S promoter.

[0012] Optionally, the plant overexpression vector is pFGC1008-HA.

[0013] According to a second aspect of the embodiments of the present application, a method for promoting coloration of tomato fruits is provided, the method comprising: transferring the overexpression SlWRKY2 gene vector into Agrobacterium; infesting tomato plants with the Agrobacterium, thereby promoting coloration of tomato fruits.

[0014] According to a third aspect of the embodiments of the present application, a method for inhibiting coloration of tomato fruits and reducing fruit size is provided, the method comprising steps of: amplifying a gene target fragment from the target primer SlWRKY2 inserting the to-be-transferred gene fragment into the linearized transformation vector, connecting, transferring into E. coli, extracting plasmid to obtain SlWRKY2 a gene knockout vector; infesting tomato plants with the Agrobacterium into which the gene knockout vector is transferred, to obtain tomato SlWRKY2 gene knockout plants. SlWRKY2 gene knockout plants.

[0015] The technical solutions provided by the embodiments of the present application can include the following beneficial effects: As can be seen from the above embodiments, the present application provides an overexpression vector of SlWRKY2 gene, which can be used to overexpress the SlWRKY2 gene, thereby promoting coloration of tomato fruits and improving quality of tomato fruits. Specifically, the content of lycopene, beta-carotene and lutein in tomato fruits increases, fruit coloration is accelerated, and fruit size remains unchanged. Knocking out the SlWRKY2 gene in tomatoes can delay the time of fruit coloration, slow down fruit coloration, and make the fruits smaller. The SlWRKY2 protein and its encoding gene provided by the present application provide gene resources for cultivating new varieties of tomatoes with higher nutritional value, have good potential application value, and can be used to adjust the nutritional and appearance quality of tomatoes, regulate the time of tomato fruits on the market, and lay a theoretical foundation and technical support for the molecular mechanism of regulating nutritional quality and ripening of tomatoes.

[0016] It should be understood that the foregoing general description and the following detailed description are only exemplary and explanatory, and cannot limit the present application. BRIEF DESCRIPTION OF DRAWINGS

[0017] The accompanying drawings, which are incorporated into and form part of the specification, illustrate embodiments consistent with the present application and, together with the specification, serve to explain the principles of the present application.

[0018] Figure 1 For the gene knockout vector in Example 3 of the present application SlWRKY2Western Blot detection results of plant proteins of the tomato lines with overexpression of the gene.

[0019] Figure 2 For the overexpression of the gene in Example 3 of the present application SlWRKY2 Sequencing results of the target site of the gene knockout tomato plant.

[0020] Figure 3 For the overexpression of the gene in Example 4 of the present application SlWRKY2 , SlWRKY2 Fruit coloring of the gene mutation and wild type.

[0021] Figure 4 For the overexpression of the gene in Example 4 of the present application SlWRKY2 , SlWRKY2 Fruit flowering to color breaking time results of the gene mutation and wild type.

[0022] Figure 5 For the overexpression of the gene in Example 5 of the present application SlWRKY2 , SlWRKY2 Carotenoid content change results of the gene mutation and wild type fruit.

[0023] Figure 6 For the overexpression of the gene in Example 6 of the present application SlWRKY2 , SlWRKY2 Fruit size and weight results of the gene mutation and wild type. DETAILED DESCRIPTION

[0024] The exemplary embodiments will be described in detail herein with reference to the attached drawings. The following description is made with reference to the accompanying drawings in which like reference numerals refer to like elements in the several figures. The following exemplary embodiments described therein represent implementations consistent with the present application. Alternate embodiments consistent with the application will be apparent to those skilled in the art from review of the disclosure herein. The following exemplary embodiments described herein are intended to be illustrative only and are not intended to limit the scope of the present application. Accordingly, the exemplary embodiments are merely examples and should not be construed as limiting the scope of the application.

[0025] The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the present application. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise. It will be further understood that the terms "comprises" and / or "comprising," when used in this specification, specify the presence of stated features, integers, steps, operations, elements, and / or components, but do not preclude the presence or addition of one or more other features, integers, steps, operations, elements, components, and / or groups thereof.

[0026] Example 1: SlWRKY2 Construction of the gene overexpression vector To regulate the fruit coloring process and the time to market of tomato, the gene was cloned from the tomato genome. SlWRKY2 According to the coding region sequence analysis, specific primersSlWRKY2 -F and SlWRKY2 -R, and add restriction enzyme sites to the primers respectively ( Asc I and Kpn I The sequences are shown in SEQ ID NO:3 and 4. Amplification was performed using PrimerSTAR high-fidelity enzyme PCR. SlWRKY2 The fragments were then digested with enzymes, and the PCR amplification fragments and vector were further digested. SlWRKY2 The fragment was ligated into pFGC1008-HA to obtain the overexpression vector pFGC1008:: SlWRKY2 -HA. The above recombinant plasmid was sent to Shangya Company for sequencing confirmation, and the obtained gene... SlWRKY2 The nucleotide sequence is shown in SEQ ID NO:1; the amino acid sequence of the protein encoded by this gene is shown in SEQ ID NO:2. The results show that the cloned sequence is consistent with the sequence published in Solgenomics (Solyc07g066220).

[0027] Example 2: SlWRKY2 Construction of gene mutation vectors Design using the website (http: / / crispor.tefor.net / ) SlWRKY2 The target gene sequence is GCCCTTAGACCTGCTGATCTG. After annealing, the synthesized target sequence is ligated to the Bbs I site of the AtU6-sgRNA-AtUBQ-Cas9 vector. Then, the newly obtained AtU6-sgRNA-AtUBQ-Cas9 fragment is ligated to the Hind III / Kpn I site of the pCAMBIA1301 vector to construct the tomato gene. SlWRKY2 Gene CRISPR expression vector. The recombinant plasmid was sent to Shangya Company for sequencing confirmation.

[0028] Example 3: Tomato SlWRKY2 Construction and detection of genetically modified materials The overexpression vector pFGC1008:: SlWRKY2 -HA and gene editing vector pCAMBIA1301::AtU6-sgRNA ( SlWRKY2 )-AtUBQ-Cas9. Agrobacterium GV3101 was transformed and tomato cotyledons were infected. Tissue culture seedlings were obtained through callus induction, resistance-induced differentiation, and rooting culture. T1 generation mutant seeds and overexpression seeds were tested for kanamycin and chloramphenicol resistance, respectively. Lines with 3 / 4 resistant genes and the remaining 1 / 4 non-resistant genes were selected, indicating that the overexpression vector carrying the target gene was inserted as a single copy in these lines. These plants were removed, and individual seed harvesting was performed. Western blotting was used for verification. SlWRKY2Overexpression positive transgenic plants, the results show that wild type has no protein band, and overexpression lines have SlWRKY2 band of HA Figure 1 , PCR and sequencing techniques to verify positive SlWRKY2 mutant transgenic plants, found wrky2 #1 deletion of 1 base Figure 2 .

[0029] Example 4: SlWRKY2 Tomato fruit coloration time statistics of gene transgenic materials Tomato materials after flowering, the full bloom flowers were marked, and the date of the day when the tomato fruit broke color was marked. Photographs were taken on the day when the color broke (Br) and the first, third, fifth, and seventh days after the color broke (Br+1, Br+3, Br+5, Br+7) to record the color of tomato fruit wild type WT, gene overexpression material WRKY2 -OE #4, gene knockout mutant wrky2 #1 color change Figure 3 ), the number of days from flowering to color breaking of different tomato materials Figure 4 ) was counted. SlWRKY2 Gene overexpression tomato fruit WRKY2 -OE #4 coloration was significantly accelerated compared with wild type fruit WT, SlWRKY2 Gene mutant tomato fruit wrky2 #1 coloration was significantly delayed compared with wild type fruit WT, which indicated that SlWRKY2 Gene positively promotes tomato fruit coloration.

[0030] Example 5: SlWRKY2 Determination of the pigment content of tomato fruit of gene transgenic materials The carotenoid content of fruits from different materials was determined on days 5 and 7 after color breaking. Peeled tomato pulp was placed in liquid nitrogen, freeze-dried, and then thoroughly ground. 0.1 g of the pulp was weighed and transferred to a 2 mL centrifuge tube. 700 μL of chloroform, 350 μL of ddH₂O, and 350 μL of methanol were added, vortexed thoroughly, and centrifuged at 10,000 g for 10 min at 4 ℃. The chloroform phase was collected. Another 700 μL of chloroform was added to the remaining residue tube until the chloroform phase became colorless. The chloroform phases were combined and dried under nitrogen. Then, 350 μL of a methanol solution containing 6% KOH (w / v) was added to dissolve the precipitate, and derivatized at 60 ℃ in the dark for approximately 30 min. 350 μL of ddH₂O and 700 μL of chloroform were added, vortexed thoroughly, and centrifuged at 10,000 g for 5 min at 4 ℃. The chloroform phase was collected in a 10 mL centrifuge tube. Extract the chloroform phase again with 700 μL ddH2O, repeating the process multiple times. Dry the collected chloroform phase under nitrogen, then dissolve it in 100 μL of chromatographic-grade ethyl acetate. Centrifuge at 14000 g for 20 min at 4 °C, and then use 150 μL of the supernatant for high-performance liquid chromatography (HPLC) analysis. At least five biological replicates should be performed for this procedure.

[0031] The results show ( Figure 5 Overexpression SlWRKY2 In tomato fruits, the accumulation of lycopene and other carotenoids is greater in the 7 days following color breaking (Br+7) than in wild-type fruits. SlWRKY2 Mutant tomatoes accumulate less lycopene and other carotenoids than wild-type tomatoes.

[0032] Example 6: SlWRKY2 Determination of tomato fruit size and weight of genetically modified materials Harvest ripe tomato fruits of different varieties, measure the transverse and longitudinal diameters of the tomato fruits using vernier calipers, and determine the weight of each individual tomato fruit using an electronic balance. At least 10 biological replicates should be set up for the above operations.

[0033] The results show ( Figure 6 ), SlWRKY2 Mutant tomato fruit wrky2 #1 Horizontal Diameter ( Figure 6 (a) and longitudinal diameter (a) are given in the middle. Figure 6 (b) and single fruit weight (given in the middle) Figure 6 The WT of the medium-sized fruit (c) was significantly smaller than that of the wild-type fruit. SlWRKY2 Overexpression of tomato fruit WRKY2 -OE#4 transverse diameter ( Figure 6 (a) and longitudinal diameter (a) are given in the middle. Figure 6 (b) and single fruit weight (given in the middle) Figure 6 The results from the middle-type (c) were not significantly different from those from the wild-type fruit (WT).

[0034] This invention is the first to construct a tomato SlWRKY2 Transgenic plants with gene overexpression and gene knockout were studied for their functional characteristics. By marking flowering and color-breaking times, [the study] revealed... SlWRKY2 The gene can promote the accumulation of carotenoids in tomato fruit and promote the coloring and ripening of tomato fruit. Therefore, the present invention provides... SlWRKY2 This gene is a key gene regulating tomato fruit ripening and carotenoid accumulation, and can be constructed using transgenic technology. SlWRKY2 Gene overexpression materials can promote tomato fruit coloring and the accumulation of nutrients such as carotenoids, thus realizing molecular design breeding and having good application value.

[0035] Other embodiments of this application will readily conceive of by those skilled in the art upon consideration of the specification and practical disclosure. This application is intended to cover any variations, uses, or adaptations of this application that follow the general principles of this application and include common knowledge or customary techniques in the art not disclosed herein. The specification and embodiments are to be considered exemplary only, and the true scope and spirit of this application are indicated by the foregoing claims.

[0036] It should be understood that this application is not limited to the precise structure described above and shown in the accompanying drawings, and various modifications and changes can be made without departing from its scope. The scope of this application is limited only by the appended claims.

Claims

1. Solanum lycopersicum SlWRKY2 application of the gene in regulating fruit color and size of tomato, and the SlWRKY2 The nucleotide sequence of the gene is shown as SEQ ID NO:

1.

2. Use according to claim 1, characterized in that, The SlWRKY2 The amino acid sequence of the protein encoded by the gene is set forth in SEQ ID NO:

2.

3. Use according to claim 1 or 2, characterized in that, By overexpressing the gene in tomato SlWRKY2 the fruit size is not affected; by knocking out the gene in tomato SlWRKY2 the fruit color is inhibited and the fruit size is reduced.

4. Use according to claim 3, characterized in that, The gene overexpression technology comprises the following steps: Total RNA of tomato was extracted, cDNA was obtained by reverse transcription, and cDNA was taken as a template, F and R were taken as primers, and a gene was amplified SlWRKY2 The nucleotide sequences of the primers F and R are shown as SEQ ID NO: 3 and 4. The plant overexpression vector is introduced into host cells, and the host cells are used to infect target plants, and positive transgenic plants are screened to obtain transgenic plants.

5. Use according to claim 4, characterized in that, The host cells are Agrobacterium.

6. Use according to claim 5, characterized in that, The Agrobacterium is GV3101.

7. Use according to claim 4, characterized in that, The plant expression vector is an expression vector with a 35S promoter.

8. Use according to claim 7, characterized in that, The plant overexpression vector is pFGC1008-HA.

9. A method for promoting the coloration of tomato fruits, characterized in that, The method comprises the following steps: The overexpression SlWRKY2 gene vector is introduced into Agrobacterium; The Agrobacterium is used to infect tomato plants, thereby promoting the coloring of tomato fruits.

10. A method of inhibiting the colouring of tomato fruits and reducing the size of the fruits, characterized in that, The method comprises the following steps: The target point primer is used to amplify the gene target point fragment SlWRKY2 The gene fragment to be transformed is inserted into the linearized transformation vector, and after connection, it is transformed into E. coli. After extracting the plasmid, the SlWRKY2 Gene knockout vector is obtained. Tomato plants were transformed with the gene knockout construct and the SlWRKY2 Agrobacterium infected tomato plants with the gene knockout construct and the SlWRKY2 gene knockout plants.

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