Tomato fruit maturation specific expression promoter E8 and application thereof
By developing the tomato fruit ripening-specific expression promoter E8, the problem of varying tomato quality was solved, the precise expression of the target gene in tomato fruit and the improvement of protein purity were achieved, thereby improving tomato quality.
Patent Information
- Application Number
- CN202510875215.6
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-06-27
- Publication Date
- 2025-09-12
AI Technical Summary
In existing technologies, the quality of tomatoes varies and it is difficult to meet consumer demand. Constitutive promoters such as CaMV 35S cause exogenous genes to be expressed in non-target tissues, increasing the metabolic burden of plants and reducing the purity of the target protein.
The tomato fruit ripening-specific expression promoter E8 was developed. It is specifically activated during tomato fruit development and ripening in response to ethylene, driving the expression of downstream genes in the fruit and avoiding expression in other tissues.
The precise expression of the target gene in tomato fruit was achieved, the metabolic burden was reduced, the purity of the target protein was increased, and the quality of tomato fruit was improved.
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Abstract
Description
Technical Field
[0001] The present application relates to the technical field of plant genetic engineering, and in particular to a tomato fruit ripening-specific expression promoter E8 and its application. Background Art
[0002] Tomato (Solanum lycopersicum L.) is a widely cultivated commercial crop worldwide. Rich in nutrients such as vitamin C and vitamin A, and boasting antioxidant, lipid-lowering, and cardiovascular disease-preventing properties, it has become an essential food in people's daily diets. Widely used in cooking and food processing, it enjoys high market demand and economic value. However, the quality of tomatoes currently available on the market varies, and their taste is suboptimal, failing to meet consumers' daily needs. Consequently, improving tomato quality through plant genetic engineering has become a research hotspot in recent years. The use of specific promoters is crucial in plant genetic engineering, enabling precise induction of exogenous gene expression in specific tissues or physiological stages, thereby achieving targeted regulation of crop quality. Currently, constitutive promoters such as CaMV 35S are commonly used in tomato cultivar improvement. However, this can result in exogenous gene expression in non-target tissues, leading to a series of problems such as increased metabolic burden on the plant and reduced target protein purity. This, to a certain extent, limits the development of high-quality tomato varieties. Therefore, the development of tomato fruit-specific promoters for targeted and precise tomato breeding is imperative. Summary of the Invention
[0003] In light of the aforementioned shortcomings of the prior art, the present application aims to provide a tomato fruit ripening-specific expression promoter, E8, and its applications. The tomato fruit ripening-specific expression promoter, E8, can achieve sustained and stable expression of exogenous target genes in tomatoes, thereby achieving the goal of breeding high-quality tomato varieties. Furthermore, the E8 promoter can reduce the metabolic burden and other issues caused by heterologous expression in tomatoes, improve the purity of target proteins, and thus make tomato breeding more precise and efficient.
[0004] The technical solution of this application is as follows:
[0005] In a first aspect of the present application, a tomato fruit ripening-specific expression promoter E8 is provided, the gene sequence of the tomato fruit ripening-specific expression promoter E8 is shown in SEQ ID NO.1.
[0006] Preferably, the tomato fruit ripening-specific expression promoter E8 is ripening-specifically expressed in tomato fruit.
[0007] Preferably, the tomato fruit ripening-specific expression promoter E8 is used to regulate the specific expression of the target gene in tomato fruit.
[0008] Preferably, the use of the tomato fruit ripening-specific expression promoter E8 in plant genetic improvement is characterized in that the plant is tomato.
[0009] In a second aspect of the present application, an expression cassette containing the above-mentioned tomato fruit ripening-specific expression promoter E8 is provided.
[0010] The third aspect of the present application provides a recombinant expression vector containing the above-mentioned tomato fruit ripening-specific expression promoter E8.
[0011] The fourth aspect of the present application provides an engineered bacterium containing the above-mentioned tomato fruit ripening-specific expression promoter E8.
[0012] In a fifth aspect of the present application, a transgenic plant containing the above-mentioned tomato fruit ripening-specific expression promoter E8 is provided.
[0013] The sixth aspect of the present application provides a method for cultivating transgenic plants, comprising the steps of transferring the tomato fruit ripening-specific expression promoter E8, the expression cassette, the recombinant expression vector or the engineered bacteria into a plant; the plant is a tomato.
[0014] The seventh aspect of the present application provides an application containing the expression cassette, the recombinant expression vector, and the engineered bacteria for regulating the specific expression of the target gene during tomato fruit ripening.
[0015] Beneficial effects of this application:
[0016] The E8 promoter is a 1.1kb DNA sequence derived from the tomato genome. Its gene sequence contains several important functional regions, such as the TATA box, CAAT box, GC-box, and AT-rich region. It also contains cis-acting elements that respond to ethylene and regulate fruit development and ripening. Therefore, the E8 promoter exhibits highly ripening-specific expression in tomato fruit. It is activated at specific stages of fruit development, driving the preferential expression of downstream genes in the fruit. Compared to other tissues such as roots, stems, and leaves, the E8 promoter exhibits significantly higher activity in tomato fruit, making it suitable for precise regulation of specific genes in the fruit. By linking a target gene to the E8 promoter, expression of the target gene can be restricted to the tomato fruit, avoiding unwanted effects in other tissues. This approach is of great significance for improving the quality and characteristics of tomato fruit, and offers broad application prospects and economic benefits. BRIEF DESCRIPTION OF THE DRAWINGS
[0017] In order to more clearly illustrate the technical solutions in the embodiments of the present application, the following briefly introduces the drawings required for describing the embodiments.
[0018] Figure 1 A map of the pCAMBIA1301 plasmid vector containing the E8 promoter provided in the examples of this application;
[0019] Figure 2 Western blot analysis of transgenic tomato fruit provided in the examples of this application:
[0020] Figure 3 Western blot analysis of transgenic tomato leaves provided in the examples of this application:
[0021] Figure 4 Western blot analysis of transgenic tomato flowers provided in the examples of this application:
[0022] Figure 5 The actual diagram of the apparent metabolism of transgenic tomato plants provided in the examples of this application:
[0023] Figure 6 Statistical graph of epigenetic metabolic analysis of transgenic tomato plants provided in the examples of this application:
[0024] (A) Statistical analysis of fruit weight of transgenic tomato plants; (B) Statistical analysis of leaf length of transgenic tomato plants; (C) Statistical analysis of height of transgenic tomato plants;
[0025] Figure 7 Graph showing glucose content detection and analysis of transgenic tomato plants provided in the examples of this application: DETAILED DESCRIPTION
[0026] To make the purpose, technical solutions and advantages of this application clearer, the technical solutions in the embodiments of this application will be clearly and completely described below in conjunction with the drawings and embodiments of this application. Obviously, the described embodiments are only part of the embodiments of this application, not all of the embodiments. Based on the embodiments of this application, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of this application. In the absence of conflict, the features in the following embodiments and embodiments can be combined with each other.
[0027] It should be noted that if there are descriptions involving "first", "second", etc. in the implementation of this application, the descriptions such as "first", "second", etc. are only used for descriptive purposes and cannot be understood as indicating or suggesting their relative importance and implicitly indicating the number of the indicated technical features. Therefore, the features defined as "first" and "second" may explicitly or implicitly include at least one of such features. In addition, the technical solutions between the various embodiments can be combined with each other, but they must be based on the fact that ordinary technicians in this field can implement them. When the combination of technical solutions is contradictory or cannot be implemented, it should be deemed that such a combination of technical solutions does not exist and is not within the scope of protection required by this application.
[0028] The following is further described with reference to specific examples.
[0029] The specific steps are as follows:
[0030] Example 1 Construction of gene expression plasmid
[0031] (1) The gene sequences of the tomato fruit ripening-specific E8 promoter and the thaumatin gene were selected and used to design a gene recombinant expression vector.
[0032] (2) The gene sequence obtained in (1) was connected to the plasmid vector pCAMBIA1301 by homologous recombination technology to obtain a gene recombination expression plasmid vector. Figure 1 shown.
[0033] Example 2 Construction and Detection of Agrobacterium
[0034] (1) Preparation of Agrobacterium: 1 μL of the plasmid vector constructed in Example 1 was added to 50 μL of GV3101 Agrobacterium competent cells, mixed thoroughly, and then pipetted into an electroporation cuvette. After electroporation, 1 mL of LB liquid medium was added, mixed thoroughly, and then pipetted into a 1.5 mL centrifuge tube. The cells were shaken and cultured on a shaker at 30°C and 180 rpm for 30 min. 50 μL of the activated Agrobacterium culture solution was inoculated onto LB solid culture medium and cultured in the dark at 30°C for 48 h.
[0035] (2) Agrobacterium detection: Synthesize forward primers and negative primers, prepare PCR amplification system, mix thoroughly after preparation, and use PCR instrument for amplification. The amplification program is set accordingly according to the primer information, etc.
[0036] The PCR reaction system is as follows:
[0037]
[0038] Amplification parameters were as follows: 98°C for 3 min, 98°C for 20 s (34 cycles), 56°C for 20 s (34 cycles), 72°C for 40 s (34 cycles), and 72°C for 5 min.
[0039] (3) Gel electrophoresis detection: Prepare 1% agarose gel, weigh 0.5g agarose powder and dissolve it in 50mL 1×TAE buffer, heat in a microwave oven until boiling, the solution becomes transparent, cool slightly and add 5μL SuperRed (10000x). Pour the gel into the gel membrane tool inserted with the comb and wait for it to solidify, and add the mixed sample and marker to the gel well. The sample loading volume is determined by the specific experiment. The marker loading volume is based on the following standards: 11-well gel corresponds to 2μL, 8-well gel corresponds to 5μL, and 6-well gel corresponds to 10μL. Perform electrophoresis at 150V until the color band of the loading buffer is electrophoresed to the middle or two-thirds of the gel, and stop electrophoresis;
[0040] (4) Check the PCR amplification results. If a positive result is obtained, proceed to the next step.
[0041] Example 3 Cultivation of transgenic tomatoes
[0042] (1) Seed disinfection: Tomato seeds were disinfected in the following order: washing with sterile water for 2 minutes, disinfecting with 75% alcohol for 40 seconds, washing with 84 disinfectant for 7 minutes, washing with sterile water for 3 times, and soaking in sterile water for 1 hour.
[0043] (2) Sowing: Sow the sterilized tomato seeds on the germination medium, culture them in the dark for 3-4 days, and then place them in a lighted tissue culture box for 4-5 days after the seeds turn white and germinate.
[0044] (3) Preparation and pre-culture of explants: After the cotyledons of the germinated tomato seedlings are fully expanded, the petioles and tips of the cotyledons are removed with a scalpel, and the middle part is cut into 2 to 3 sections and inoculated into the pre-culture medium. The explants are pre-cultured at 23 ± 2 °C for 2 to 3 days.
[0045] (4) Agrobacterium infection and co-cultivation: The Agrobacterium in Example 3 was picked up and placed in the infection solution to prepare an Agrobacterium resuspension with an OD (600) of 0.1; the infection was carried out for 10 to 15 minutes, and the dried explants were inoculated into the co-cultivation medium and cultured in the dark at 23 ± 2°C for 2 days.
[0046] (5) Screening and Rooting: The recovered calli were inoculated on screening medium and cultured at 23°C with 16h / 8h light / darkness for 15-30 days. The selected calli were inoculated on differentiation medium and cultured at 23°C with 16h / 8h light / darkness for 30-40 days. When the seedlings to be differentiated grew to about 2-3 cm, they were excised from the calli and inoculated on rooting medium and cultured at 23°C with 16h / 8h light / darkness for 10-15 days.
[0047] Example 4: Verification of E8 promoter-induced exogenous gene expression
[0048] (1) Prepare a gel for protein blotting, and then. Take the ripe tomato fruit, tomato leaves and tomato flowers in Example 3 to extract protein to obtain protein samples. Take 40 μL of each sample and add 10 μL of 5x SDS-PAGE loading buffer. Boil the protein sample at 95-100°C for 5-10 minutes using a PCR machine. Immediately place the sample on ice to cool for 5 minutes. Add 20 μL of each prepared sample to the gel well. In addition, add a pre-stained molecular weight marker to one of the wells. Perform electrophoresis at 150 volts for 60 minutes.
[0049] (2) Transfer the gel obtained after electrophoresis to a clean tray containing EB solution for 3 minutes. Prepare filter paper and cut the PVDF membrane into the required size. Place the PVDF membrane in methanol for 1 minute to activate it. After activation, transfer the membrane to a clean tray containing EB solution for 3 minutes. Soak the filter paper in EB solution. On a semi-dry transfer instrument, place the components from the negative electrode to the positive electrode in the following order: filter paper, gel, membrane, filter paper. When placing the components, make sure to eliminate any bubbles. Cover the electrode cover of the electrotransfer instrument and transfer at 15V for 42 minutes.
[0050] (3) After electroporation, remove the PVDF membrane and block the membrane with 5% BSA solution at room temperature for 2 hours. Dilute the primary antibody (1:5000) in blocking solution. Once the blocking solution is removed, immediately add the diluted primary antibody. Incubate the membrane at room temperature or on a shaker at 4°C for 15 hours. It is best to incubate at a lower temperature. If incubating the primary antibody overnight in blocking solution, make sure to do it at 4°C to prevent contamination and protein degradation. After the primary antibody incubation is completed, carefully remove the primary antibody solution. Place the membrane in a square container (such as a plastic container) and add TBST (tris-buffered saline and Tween) to completely cover the PVDF membrane. Shake on a low-speed shaker for 10 minutes. Repeat this washing step three times, changing the TBST solution each time. Dilute the secondary antibody (1:5000) in blocking solution. Immerse the PVDF membrane containing the transferred protein in the square container containing the diluted secondary antibody. Gently shake and incubate the membrane for 2 hours. After the incubation is complete, carefully remove the secondary antibody solution. Place the membrane in a square container and add TBST to completely cover the PVDF membrane. Shake on a low-speed shaker for 10 minutes. Repeat this wash step three times, replacing the TBST solution each time.
[0051] (4) Then, equal volumes of DAB solutions A and B were mixed to prepare DAB (3,3′-diaminophenfetamine) working solution (Note: Handle in the dark). Using a pipette, carefully apply an appropriate amount of DAB working solution to the PVDF membrane. Allow the reaction to proceed for an appropriate duration depending on the staining requirements. After the desired staining intensity is achieved, remove the excess DAB solution and rinse the membrane with deionized water. The membrane was placed in an ECL (enhanced chemiluminescence) imaging system for imaging analysis to obtain the test results. The test results are shown in Figure 4. Figure 2 As shown, from Figure 2 As can be seen from the results, there is a clear band around 26-30kD, which is consistent with the expected experimental results; this shows that the E8 promoter successfully achieves fruit ripening-specific expression in tomatoes, and Figure 3 as well as Figure 4 In the experimental results, no obvious band appeared near 26-30kD, indicating that the E8 promoter would not induce the specific expression of the target gene in non-fruit parts of tomatoes, further demonstrating that E8 can reduce the metabolic burden brought by heterologous expression to other tissues of tomatoes.
[0052] Example 5 Tomato Metabolism Detection:
[0053] (1) Tomato apparent metabolic detection: The transgenic tomato plants in Example 3 were taken and observed. The results are as follows Figure 5 As shown in Figure 2, the E8 promoter has no adverse effects on tomato epigenetic metabolism. Statistical analysis was then performed on the fruit weight, leaf length, and plant height of the transgenic tomato plants. Figure 6 As shown, from Figure 6 It can be seen that there are no significant inter-group differences in the three epigenetic traits (fruit weight, leaf length and plant height) of tomato plants in different groups, indicating that the use of E8 promoter to induce the expression of exogenous genes has no significant negative impact on the metabolism of tomato plants.
[0054] (2) Glucose content test: Purchase Glucose (Glu) colorimetric test kit, prepare enzyme working solution and control working solution according to the instructions of the kit. Take the transgenic tomato fruit in Example 2 and perform conventional homogenization treatment (the homogenization medium is physiological saline (0.9% NaCl)). After homogenization, centrifuge at 4°C, 10000xg for 10 minutes, take the supernatant and place it on ice for testing. Reserve part of the supernatant for protein concentration determination. Take 3μL of 8 standards of different concentrations and add them to the corresponding standard wells respectively; measurement well: take 3μL of the sample to be tested, add it to the sample well to the bottom; control well: take 3μL of the sample to be tested, add it to the sample well to the bottom. Add 300μL of enzyme working solution to the standard well and measurement well. Add 300μL of control working solution to the control well. Cover with a film and incubate at 37°С for 15 minutes. Measure the OD value of each well with an enzyme reader at 505nm. The experimental results are as follows Figure 7 As shown, from Figure 7 It can be seen that there is no significant difference in the glucose content of tomato fruits in each group, and the glucose content of tomatoes in each group is within the range of glucose content found in the literature, indicating that using the E8 promoter to induce the expression of exogenous genes has no significant negative impact on tomato glucose metabolism.
[0055] In summary, the tomato fruit ripening-specific promoter E8 described in this application achieves specific expression in tomato fruit by responding to ethylene. Linking a target gene to the E8 promoter allows for expression only in tomato fruit, avoiding unwanted effects in other tissues and improving the purity of the target protein. This is of great significance for improving the quality and characteristics of tomato fruit, and has broad application prospects and economic benefits.
[0056] It should be understood that the application of this application is not limited to the above examples. For ordinary technicians in this field, they can make improvements or changes based on the above description. All these improvements and changes should fall within the scope of protection of the claims attached to this application.
Claims
1. A tomato fruit ripening-specific expression promoter E8, characterized in that: The gene sequence of the tomato fruit ripening-specific expression promoter E8 is shown in SEQ ID NO.
1.
2. The tomato fruit ripening-specific expression promoter E8 according to claim 1, characterized in that: The tomato fruit ripening-specific expression promoter E8 is specifically expressed in tomato fruits at maturity.
3. Use of the tomato fruit ripening-specific expression promoter E8 according to claim 1 in regulating the specific expression of a target gene in tomato fruit.
4. The use of the tomato fruit ripening specific expression promoter E8 in plant genetic improvement according to claim 1, characterized in that: The plant is tomato.
5. An expression cassette containing the tomato fruit ripening-specific expression promoter E8 according to claim 1.
6. A recombinant expression vector containing the tomato fruit ripening-specific expression promoter E8 according to claim 1.
7. An engineered bacterium containing the tomato fruit ripening-specific expression promoter E8 according to claim 1.
8. A method for cultivating transgenic tomatoes, characterized in that: The method comprises the steps of transferring the tomato fruit ripening-specific expression promoter E8 according to claim 1, the expression cassette according to claim 5, the recombinant expression vector according to claim 6 or the engineered bacteria according to claim 7 into a plant; the plant is a tomato.
9. Use of the expression cassette according to claim 5, the recombinant expression vector according to claim 6, and the engineered bacteria according to claim 7 in regulating the specific expression of a target gene during tomato fruit ripening.