A gene for increasing the number of tomato fruits and a method for regulating the same

Abstract

A gene for increasing the number of tomato fruits and a method thereof, wherein the nucleotide sequence of the gene is shown in Fig. Seq No. 1. The present application is obtained by obtaining the complete coding sequence of the gene from tomato SlGRF The gene is amplified by PCR technology, then the target fragment is connected to the pBG-GFP vector, the plant is transformed by using the agrobacterium infection method, the high expression plant is obtained, and the high expression plant is analyzed, and the results show that the high expression of the gene can increase the fruit setting number of tomato.

Description

Technical Field

[0001] This invention belongs to the field of bioengineering technology, and relates to a method for regulating tomato fruit setting, particularly a GRF family transcription factor. SlGRF The application of this gene in increasing tomato fruit yield. Background Technology

[0002] Tomato fruit is a fleshy berry, making it a classic model material for studying fruit development. Tomato fruit development can be divided into four stages: (1) fruit set, the stage of mature flower formation; (2) a period of intense cell division, lasting two weeks after fertilization; (3) a period of rapid cell expansion, where cell volume can increase 20-fold; and (4) the ripening stage, involving the formation of fruit qualities such as aroma, color, and texture. It is evident that the key event in the second stage of fruit development is intense cell division, driven by the cell cycle, in which cyclin D (CycD3.1 / 3.2 / 3.3) and cyclin-dependent protein kinase CDKB (B-type cyclin-dependent kinase) play a role in rapid cell division. The key event in the third stage of fruit development is nuclear replication, but without mitosis, resulting in an increase in chromosome number and cell volume. Therefore, cell division in the second stage and cell expansion in the third stage of fruit development jointly promote the formation of tomato fruit.

[0003] The number of tomatoes is a key factor determining tomato yield and an important trait of interest during tomato domestication. Currently, several genes have been identified that control fruit size, but research on genes controlling fruit number is scarce. During tomato domestication, a small gene family—growth-regulating factors (GRFs)—has gradually gained attention for their function in regulating plant growth and development. GRFs are plant-specific transcription factors that play a wide range of roles in stem and leaf development, floral organ formation, seed development, and the synergistic regulation of stress and growth. GRFs are typically expressed at higher levels in rapidly growing tissues and at lower levels in mature tissues, suggesting that this gene family may be involved in the early stages of growth and development in different plant tissues. For example, high expression of AtGRF1-3 in Arabidopsis thaliana promotes leaf development by regulating cell proliferation, while triple mutations in AtGRF1-3 result in smaller organs; in rice, OsGRF6 and OsGRF10 also regulate growth and development, and mutations in these genes lead to dwarf plants. However, how GRF regulates tomato fruit development remains unclear. Searching for new functional genes involved in regulating fruit number could help improve crop yield. Summary of the Invention

[0004] The purpose of this invention is to provide a gene that increases the number of tomato fruits and a method for regulating it.

[0005] To achieve the above and other related objectives, the technical solution provided by the present invention is: a gene for increasing the number of tomato fruits, the nucleotide sequence of which is shown in Seq No. 1.

[0006] To achieve the above and other related objectives, the technical solution provided by this invention is: a method for regulating the increase of tomato fruit quantity, comprising the following steps:

[0007] Step 1: Dilute the forward primer-F and reverse primer-R with sterile ddH2O; PCR amplification 50 μL system: 32 μL ddH2O, 10 μL SF buffer, 2 μL forward primer-F, 2 μL reverse primer-R, 1 μL dNTP, 1 μL SF DNA Polymerase, 2 μL cDNA, then place in a PCR instrument: 95℃, 3 min; 95℃, 10 s; 55℃, 10 s; 72℃, 45 s; 35 cycles; 72℃, 7 min, 4℃, ∞;

[0008] Forward primer-F: 5'-tactattctagtcgagaattcATGGACTTTGGGGGTTTGGA-3';

[0009] Reverse primer-R: 5'-accatcccgggtaccgagctcCATGGCAGGTAGGGTTGGATT-3';

[0010] Step 2: Purify the PCR amplification product obtained in Step 1 using a DNA product purification kit to obtain purified DNA. SlGRF Target fragment; take 2 μL of purified fragment. SlGRF The target fragment was subjected to agarose gel electrophoresis to check the purification effect of the product. The concentration was not less than 5 ng / μL and the next step could be carried out.

[0011] Step 3: Double digest the pBG-GFP plasmid, and then purify it using a DNA product purification kit to obtain the digested pBG-GFP plasmid; take the digested pBG-GFP plasmid for agarose gel electrophoresis to check the purification effect. If the concentration is not less than 5 ng / μL, proceed to the next step.

[0012] Step 4: Purify SlGRF The target fragment is ligated with the enzyme-digested pBG-GFP plasmid to obtain the ligation product;

[0013] Step 5: Take the ligation product obtained in Step 4, transform it into E. coli DH5α by chemical heat shock, extract the plasmid, and sequence it.

[0014] Step 6: Add pBG-GFP- SlGRF Plasmid was transformed into EHA105 Agrobacterium competent cells;

[0015] Step 7: Add pBG-GFP- SlGRF Agrobacterium tumefaciens plasmid EHA105 infected tomato cotyledons and obtained transgenic positive seedlings. SlGRF .

[0016] The preferred technical solution is as follows: In step 3, the enzyme digestion reaction solution is: 5 μL 10×cutsmart buffer, 20 ngpBG-GFP plasmid, 2 μL EcoRI, 2 μL SacI, and ddH2O to make up to 50 μL; the process parameters are: 37℃, 30 min.

[0017] The preferred technical solution is as follows: In step 5, 1 μL of the ligation product obtained in step 4 is added to 100 µL of *E. coli* DH5α competent cells, incubated on ice for 25 min, heat-shocked at 42℃ for 45 s, and then incubated on ice for 2 min; 700 µL of antibiotic-free LB liquid medium is added to a centrifuge tube, and the tube is incubated at 37℃ and 200 rpm for 60 min; the cultured bacterial solution is centrifuged at 5000 rpm for 1 min, and about 100 μL of supernatant is collected, resuspended by pipetting, and evenly spread on LB solid medium containing spectinomycin hydrochloride, and incubated upside down at 37℃ for 16 h; single colonies are picked, mixed by pipetting in 10 µL of sterile water, and 2 µL of bacterial solution is used for colony identification. The colony PCR system is 25 μL: 2 μL bacterial solution, 1 μL each of forward primer-F and forward primer-R, 12.5 μL of 2×Rapid Taq Master Mix, and 8.5 μL of ddH2O. μL, mixed well and placed in a PCR instrument; PCR conditions were: pre-denaturation 95℃ 3 min; denaturation 95℃ 15 s, annealing 55℃ 1 min, extension 72℃ 45 s, 35 cycles; final extension 72℃ 5 min; 4℃ ∞; after the reaction was completed, the size of the PCR product bands was checked on an agarose gel to see if it met the theoretical value. The remaining bacterial culture with the correct band size was transferred to 3 mL of LB liquid medium containing spectinomycin hydrochloride and cultured in a shaker at 37℃ and 200 rpm for 16 h. The plasmid was extracted using a plasmid miniprep kit and the plasmid was run on an agarose gel to verify the success of the plasmid extraction. The extracted plasmid was sent to a biotechnology company for further sequencing and identification.

[0018] The preferred technical solution is as follows: In step 6, take 100 μL of Agrobacterium competent cells EHA105, thaw them in an ice bath, and add 1 μL of correctly sequenced pBG-GFP- SlGRF To collect the plasmid, gently tap the bottom of the centrifuge tube to mix it. Then, incubate the tube sequentially on ice for 5 min, in liquid nitrogen for 5 min, in a 37°C water bath for 5 min, and in an ice bath for 5 min. Add 700 μL of antibiotic-free LB liquid medium and incubate at 28°C with shaking for 2-3 h. Centrifuge at 6000 rpm for 1 min to collect the bacteria. Collect approximately 100 μL of the supernatant, mix it thoroughly by pipetting, resuspend the bacteria, and spread it evenly on LB-sp+ / Rif solid medium. Invert the tube and incubate at 28°C for 2-3 days.

[0019] The preferred technical solution is as follows: In step 7, a single colony of Agrobacterium tumefaciens containing the pBG-GFP vector constructed in step 6 is picked and placed in 3 mL of LB liquid medium containing sp+ and Rif. After incubation at 200 rpm and 28℃ for 12-16 h, 500 µL is added to 30 mL of LB liquid medium containing sp+ and Rif. The culture is then incubated with shaking at 200 rpm and 28℃ for 12-16 h. The OD of the bacterial culture is detected using a spectrophotometer. 600 Centrifuge at 5000 rpm for 5 min at room temperature to collect bacterial cells, and dilute the bacterial cells with sterile water to an OD value of 0.6-0.8. 600 =0.1-0.15, freshly prepared and used immediately. Tomato cotyledons and stem segments were pre-cultured in the dark for 2 days, then immersed in diluted Agrobacterium infection solution, shaken, and inoculated for 5 minutes. The infection solution was then discarded, and excess solution was aspirated with a pipette tip. The cotyledons and stem segments were then subjected to germination, shoot elongation, and rooting processes on culture media containing different plant hormones. Rooted explants were transferred to nutrient soil for subsequent sequencing and identification, thus obtaining transformed tomato plants. Using tag detection, GFP tags were detected by PCR. Transformed tomato plants were obtained through plant tissue culture. 0.1-0.2 g of tomato leaves were ground in liquid nitrogen, and plant DNA was extracted using a plant genomic DNA extraction kit. DNA amplification was performed, and agarose gel electrophoresis was used to analyze the presence of GFP tags in the transgenic plants. Subsequently, RNA from the transgenic tomato plants was extracted using quantitative upstream and downstream primers, amplified by qPCR, and sequenced for identification and analysis. SlGRF Whether the gene is overexpressed.

[0020] The preferred technical solution is as follows: In step 4, the ligation system is: 1 μL CE ligase, 2 μL 5×CE buffer, vector and fragment are added according to the ratio, and ddH2O is added to make up to 10 μL; the process parameters are: 37℃, 30 min.

[0021] Due to the application of the above technical solution, the advantages of this invention compared with the prior art are:

[0022] This invention obtains from tomatoes SlGRF The complete coding sequence of the gene was obtained, primers were designed, and the target fragment was ligated into the vector pBG-GFP. Plants were transformed using Agrobacterium tumefaciens infection to obtain transgenic plants. The transgenic plants were analyzed, and the results showed that overexpression of this gene could accelerate the ripening of tomato fruits. Attached Figure Description

[0023] Figure 1 for SlGRF Sequence alignment diagram of the overexpression vector.

[0024] Figure 2 for SlGRF Results of qRT-PCR and tag detection of overexpressing plants.

[0025] Figure 3 For WT and SlGRF Overall image of a tomato plant that has overexpressed the plant's characteristics.

[0026] Figure 4 For WT and SlERFD2 The results showed that overexpression affected the number of tomato fruits in the plant. Detailed Implementation

[0027] The following specific embodiments illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in these embodiments.

[0028] Please see Figures 1-4 It should be noted that the structures, proportions, sizes, etc., illustrated in the accompanying drawings are merely for illustrative purposes and to aid those skilled in the art in understanding and reading the invention. They are not intended to limit the scope of the invention and therefore have no substantial technical significance. Any modifications to the structure, changes in proportions, or adjustments to the size are not permitted. The following embodiments are provided to better understand the invention, but are not intended to limit it. Unless otherwise specified, the experimental materials used in the following embodiments were purchased from conventional biochemical reagent stores.

[0029] Example 1: A gene that promotes tomato fruit ripening and its regulation method

[0030] A gene that promotes the ripening of tomato fruit, the nucleotide sequence of which is shown in Seq No. 1.

[0031] Seq No. 1:

[0032]

[0033] Step 1: Dilute the forward primer-F and reverse primer-R with sterile ddH2O to a concentration of 10 μM. Perform PCR amplification in a 50 μL system: 32 μL ddH2O, 10 μL SF buffer, 2 μL F primer, 2 μL R primer, 1 μL dNTP, 1 μL SFDNA Polymerase, and 2 μL cDNA. Then place the system in a PCR instrument: 95℃ for 3 min; 95℃ for 10 s, 55℃ for 10 s, 72℃ for 45 s, for 35 cycles; 72℃ for 7 min, 4℃ at ∞.

[0034] Forward primer-F: 5'-tactattctagtcgagaattcATGGACTTTGGGGGTTTGGA-3';

[0035] Reverse primer-R: 5'-accatcccgggtaccgagctcCATGGCAGGTAGGGTTGGATT-3';

[0036] Step 2: After purifying the PCR product obtained in Step 1 using a DNA product purification kit, take 2 μL of the purified product for agarose gel electrophoresis to check the purification effect. If the concentration is not less than 5 ng / μL, proceed to the next step.

[0037] Step 3: Double digest the pBG-GFP plasmid, then purify it using a DNA product purification kit. Take 2 μL of the purified product and perform agarose gel electrophoresis to check the purification effect. If the concentration is not less than 5 ng / μL, proceed to the next step.

[0038] Step 4: Purify SlGRF The target fragment is ligated to the enzyme-digested pBG-GFP plasmid;

[0039] The ligation system consisted of 1 μL CE ligase, 2 μL 5×CE buffer, vector and fragment added according to the ratio, and ddH2O added to a final volume of 10 μL. The process parameters were: 37℃, 30 min.

[0040] Optimal cloning vector usage (number of cloning vector base pairs × 0.01) / vector concentration.

[0041] Optimal amount of insert fragment used (number of base pairs in insert fragment × 0.02) / fragment concentration.

[0042] Step 5: After ligation, the ligation product from Step 4 is used to transform E. coli DH5α using the chemical heat shock method, plasmid is extracted, and sequencing is performed.

[0043] Step 6: Add pBG-GFP-SlGRF Plasmid was transformed into EHA105 Agrobacterium competent cells;

[0044] Step 7: Add pBG-GFP- SlGRF Agrobacterium tumefaciens plasmid EHA105 infected tomato cotyledons and obtained transgenic positive seedlings. SlGRF .

[0045] The preferred technical solution is as follows: In step 1, the PCR amplification reaction is as follows: 50 μL system: 32 μL ddH2O, 10 μL SF buffer, 2 μL F primer, 2 μL R primer, 1 μL dNTP, 1 μL SF DNA Polymerase, 2 μL cDNA, and then placed in a PCR instrument: 95℃ for 3 min; 95℃ for 10 s, 55℃ for 10 s, 72℃ for 45 s, for 35 cycles; 72℃ for 7 min, 4℃ ∞.

[0046] The preferred technical solution is as follows: In step 3, the enzyme digestion and ligation reaction solution is: 5 μL 10×cutsmart buffer, 20 ng pBG-GFP plasmid, 2 μL EcoRI, 2 μL SacI, and ddH2O to a final volume of 50 μL; the PCR amplification parameters are: 37℃, 30 min.

[0047] The preferred technical solution is as follows: In step 5, 1 μL of the ligation product obtained in step 4 is added to 100 µL of *E. coli* DH5α competent cells, incubated on ice for 25 min, heat-shocked at 42℃ for 45 s, and then incubated on ice for 2 min; 700 µL of antibiotic-free LB liquid medium is added to a centrifuge tube and cultured at 37℃ and 200 rpm for 60 min; the cultured bacterial solution is centrifuged at 5000 rpm for 1 min, and about 100 μL of supernatant is collected, resuspended by pipetting, and evenly spread on LB solid medium containing spectinomycin hydrochloride (500 µL of 10 mg / mL spectinomycin hydrochloride per 100 mL of LB solid medium), and incubated upside down at 37℃ for 16 h; single colonies are picked, mixed by pipetting in 10 µL of sterile water, and 2 µL of bacterial solution is taken for colony identification. The colony PCR system is 25 μL: 2 μL bacterial solution, 1 μL each of F / R primers, and 2×Rapid Taq Master Mix. 12.5 μL of ddH2O and 8.5 μL of ddH2O were mixed and placed in a PCR instrument. PCR conditions were as follows: pre-denaturation at 95℃ for 3 min; denaturation at 95℃ for 15 s, annealing at 55℃ for 1 min, extension at 72℃ for 45 s, 35 cycles; final extension at 72℃ for 5 min; 4℃ ∞. After the reaction, the PCR product band size was checked on an agarose gel to see if it met the theoretical value. The remaining bacterial culture with the correct band size was transferred to 3 mL of LB liquid medium containing spectinomycin hydrochloride and cultured at 37℃ and 200 rpm for 16 h. Plasmids were extracted using a plasmid miniprep kit and run on an agarose gel to verify successful extraction. The extracted plasmids were sent to a biotechnology company for further sequencing and identification.

[0048] The preferred technical solution is as follows: In step 6, take 100 μL of Agrobacterium competent cells EHA105, thaw them in an ice bath, and add 1 μL of correctly sequenced pBG-GFP. -SlGRF To collect the plasmid, gently tap the bottom of the centrifuge tube to mix it. Incubate the tube sequentially on ice for 5 min, in liquid nitrogen for 5 min, in a 37°C water bath for 5 min, and in an ice bath for 5 min. Add 700 μL of antibiotic-free LB liquid medium and incubate at 28°C with shaking for 2-3 h. Centrifuge at 6000 rpm for 1 min to collect the bacteria. Collect approximately 100 μL of supernatant, mix it thoroughly by pipetting, resuspend the bacteria, spread it on LB-sp+ / Rif solid medium, and incubate upside down in a 28°C incubator for 2-3 days.

[0049] The preferred technical solution is as follows: In step 7, a single colony of Agrobacterium tumefaciens containing the pBG-GFP vector constructed in step 6 is picked and placed in 3 mL of LB liquid medium containing sp+ and Rif. After incubation at 200 rpm and 28℃ for 12-16 h, 500 µL is added to 30 mL of LB liquid medium containing sp+ and Rif. The culture is then incubated with shaking at 200 rpm and 28℃ for 12-16 h. The OD of the bacterial culture is detected using a spectrophotometer. 600 Centrifuge at 5000 rpm for 5 min at room temperature to a final OD value of 0.6-0.8. Collect bacterial cells and dilute with sterile water to a final OD value of 0.6-0.8. 600 =0.1-0.15, freshly prepared and used immediately. Tomato cotyledons and stem segments were pre-cultured in the dark for 2 days, then immersed in diluted Agrobacterium infection solution, shaken, and inoculated for 5 minutes. The infection solution was then discarded, and excess solution was aspirated with a pipette tip. The cotyledons and stem segments were then subjected to germination, shoot elongation, and rooting processes on culture media containing different plant hormones. Rooted explants were transferred to nutrient soil for subsequent sequencing and identification, resulting in transformed tomato plants. Subsequently, quantitative upstream and downstream primers were designed to extract RNA from the transgenic tomato plants, which was then amplified by qRT-PCR and sequenced for identification and analysis. SlGRF Whether the gene is overexpressed.

[0050] Using tag detection, PCR detection of GFP tags was performed. Transformed tomato plants were obtained through plant tissue culture. 0.1-0.2 g of tomato leaves were ground in liquid nitrogen, and plant DNA was extracted using a plant genomic DNA extraction kit. The DNA was amplified and detected using agarose gel electrophoresis to analyze whether there were GFP tags in the transgenic plants.

[0051] one, SlGRF Identification of overexpression tomato plants

[0052] pBG-GFP- was constructed using homologous recombination. SlGRF Vectors are constructed using transgenic and tissue culture techniques. SlGRF Overexpressing tomato plants ( Figure 1 ), will grow to generation T0 SlGRF RNA was extracted from 0.1 g leaves of overexpressing tomato plants, reverse transcribed into cDNA, and then analyzed by qRT-PCR. SlGRF The amount of expression. Compared to WT, SlGRF Overexpression in plants SlGRF The expression level was 15 times higher than that of WT. Figure 2 A). Simultaneously, using immunoblotting to detect the GFP tag, it was found that... SlGRF Overexpressing plants have GFP tags ( Figure 2 (B), indicating that this plant is SlGRF Overexpressing plants.

[0053] two,SlGRF Development process of tomato fruit

[0054] By recording WT and SlGRF The development process of tomato fruit at different stages after flowering, to determine SlGRF The function of genes in tomato fruit development. The growth of the entire tomato plant was recorded at 30 and 60 days after flowering. A top-down view of the plant was also taken at 60 days after flowering. The images show that, compared to WT, SlGRF More tomatoes on the tree ( Figure 3 To more accurately reflect the difference between the two, the number of fruits per plant was recorded. SlGRF The number of tomato fruits was more than double that of WT, indicating that SlGRF Gene overexpression increases the number of tomatoes that bear fruit. Figure 4 ).

[0055] The above description is merely a preferred embodiment for explaining the present invention and is not intended to limit the present invention in any way. Therefore, any modifications or changes made to the present invention under the same inventive spirit should still be included within the scope of protection intended by the present invention.

Claims

1. A method for regulating the number of tomato fruits, characterized in that: Includes the following steps: Step 1: Dilute the forward primer-F and reverse primer-R with sterile ddH2O; PCR amplification 50 μL system: 32 μL ddH2O, 10 μL SF buffer, 2 μL forward primer-F, 2 μL reverse primer-R, 1 μL dNTP, 1 μL SF DNA Polymerase, 2 μL cDNA, then place in a PCR instrument: 95℃, 3 min; 95℃, 10 s; 55℃, 10 s; 72℃, 45 s; 35 cycles; 72℃, 7 min, 4℃, ∞; Forward primer-F: 5'-tactattctagtcgagaattcATGGACTTTGGGGGTTTGGA-3'; Reverse primer-R: 5'-accatcccgggtaccgagctcCATGGCAGGTAGGGTTGGATT-3'; Step 2: Purify the PCR amplification product obtained in Step 1 using a DNA product purification kit to obtain purified DNA. SlGRF Target fragment; take 2 μL of purified fragment. SlGRF The target fragment was subjected to agarose gel electrophoresis to check the purification effect of the product. If the concentration was not lower than 5 ng / μL, the next step could be carried out. Step 3: Double digest the pBG-GFP plasmid, and then purify it using a DNA product purification kit to obtain the digested pBG-GFP plasmid; take the digested pBG-GFP plasmid for agarose gel electrophoresis to check the purification effect of the product. If the concentration is not less than 5 ng / μL, proceed to the next step. Step 4: Purify SlGRF The target fragment is ligated with the enzyme-digested pBG-GFP plasmid to obtain the ligation product; Step 5: Take the ligation product obtained in Step 4, transform it into E. coli DH5α by chemical heat shock, extract the plasmid, and sequence it. Step 6: Add pBG-GFP- SlGRF Plasmid was transformed into EHA105 Agrobacterium competent cells; Step 7: Add pBG-GFP- SlGRF Agrobacterium tumefaciens plasmid EHA105 infected tomato cotyledons and obtained transgenic positive seedlings. SlGRF .

2. The method for controlling the increase of tomato fruit quantity according to claim 1, characterized in that: In step 3, the enzyme digestion reaction solution was: 5 μL 10×cutsmart buffer, 20 ng pBG-GFP plasmid, 2 μL EcoRI, 2 μL SacI, and ddH2O to a final volume of 50 μL; the process parameters were: 37℃, 30 min.

3. The method for controlling the increase of tomato fruit quantity according to claim 1, characterized in that: In step 5, 1 μL of the ligation product obtained in step 4 was added to 100 µL of *E. coli* DH5α competent cells, incubated on ice for 25 min, heat-shocked at 42℃ for 45 s, and then incubated on ice for 2 min. 700 µL of antibiotic-free LB broth was added to a centrifuge tube, and the tube was incubated at 37℃ and 200 rpm for 60 min. The resulting bacterial culture was centrifuged at 5000 rpm for 1 min, and approximately 100 μL of supernatant was collected. The cells were resuspended by pipetting and spreading evenly on LB solid medium containing spectinomycin hydrochloride, and incubated upside down at 37℃ for 16 h. Single colonies were picked, mixed with 10 µL of sterile water, and 2 µL of the bacterial culture was used for colony identification. The colony PCR system was 25 μL: 2 μL bacterial culture, 1 μL each of forward primer-F and forward primer-R, 12.5 μL of 2×Rapid Taq Master Mix, and 8.5 μL of ddH2O. μL, mixed well and placed in a PCR instrument; PCR conditions were: pre-denaturation 95℃ 3 min; denaturation 95℃ 15 s, annealing 55℃ 1 min, extension 72℃ 45 s, 35 cycles; final extension 72℃ 5 min; 4℃ ∞; after the reaction was completed, the size of the PCR product bands was checked on an agarose gel to see if it met the theoretical value. The remaining bacterial culture with the correct band size was transferred to 3 mL of LB liquid medium containing spectinomycin hydrochloride and cultured in a shaker at 37℃ and 200 rpm for 16 h. The plasmid was extracted using a plasmid mini-prep kit and the plasmid was run on an agarose gel to verify the success of the plasmid extraction. The extracted plasmid was sent to a biotechnology company for further sequencing and identification.

4. The method for controlling the increase of tomato fruit quantity according to claim 1, characterized in that: In step 6, take 100 μL of Agrobacterium competent cells EHA105, thaw them in an ice bath, and add 1 μL of correctly sequenced pBG-GFP- SlGRF To collect the plasmid, gently tap the bottom of the centrifuge tube to mix it. Then, incubate the tube sequentially on ice for 5 min, in liquid nitrogen for 5 min, in a 37°C water bath for 5 min, and in an ice bath for 5 min. Add 700 μL of antibiotic-free LB liquid medium and incubate at 28°C with shaking for 2-3 h. Centrifuge at 6000 rpm for 1 min to collect the bacteria. Collect approximately 100 μL of the supernatant, mix it thoroughly by pipetting, resuspend the bacteria, and spread it evenly on LB-sp+ / Rif solid medium. Invert the tube and incubate at 28°C for 2-3 days.

5. The method for increasing the number of tomato fruits according to claim 1, characterized in that: In step 7, a single colony of Agrobacterium with the pBG-GFP vector constructed in step 6 was picked and placed in 3 mL of LB liquid medium containing sp+ and Rif. The culture was incubated at 28°C for 12-16 h at 200 rpm. Then, 500 µL of the culture was transferred to 30 mL of LB liquid medium containing sp+ and Rif and incubated with shaking at 28°C for 12-16 h at 200 rpm. The OD of the bacterial culture was then measured using a spectrophotometer. 600 Centrifuge at 5000 rpm for 5 min at room temperature to collect bacterial cells, and dilute the bacterial cells with sterile water to an OD value of 0.6-0.

8. 600 =0.1-0.15, freshly prepared and used. Tomato cotyledons and stem segments were pre-cultured in the dark for 2 days, then immersed in diluted Agrobacterium infection solution, shaken, and inoculated for 5 minutes. The infection solution was then discarded, and excess solution was aspirated with a pipette tip. The cotyledons and stem segments were then subjected to germination, shoot elongation, and rooting processes on culture media containing different plant hormones. Rooted explants were transferred to nutrient soil for subsequent sequencing and identification, resulting in transformed tomato plants. Using tag detection, PCR detection of the GFP tag was performed. Transformed tomato plants were obtained through plant tissue culture. 0.1-0.2 g of tomato leaves were ground in liquid nitrogen, and plant DNA was extracted using a plant genomic DNA extraction kit. DNA amplification was performed, and agarose gel electrophoresis was used to analyze the presence of the GFP tag in the transgenic plants. Subsequently, RNA from the transgenic tomato plants was extracted using designed quantitative upstream and downstream primers, amplified by qPCR, and sequenced for identification and analysis. SlGRF Whether the gene is overexpressed.

6. The method for controlling the increase of tomato fruit quantity according to claim 1, characterized in that: In step 4, the ligation system is: 1 μL CE ligase, 2 μL 5×CE buffer, vector and fragment added according to the ratio, and ddH2O added to make up to 10 μL; the process parameters are: 37℃, 30 min.

Images