Csadap gene and application thereof in regulating fruit ripening process

Abstract

The application discloses a CsADAP gene and application thereof in regulating fruit ripening. The gene belongs to an AP2 / ERF family member, the CDS sequence of which is shown as SEQ ID NO. 1, the CDS sequence length is 1048 bp, and 348 amino acids are encoded, and the amino acid encoded by the gene is shown as SEQ ID NO. 2. A primer is designed to amplify the gene CsADAP on Fengjie navel orange, the gene is introduced into tomatoes and shanhuyan by using an agrobacterium-mediated genetic transformation method, and the obtained transgenic plants are verified by phenotype observation statistics and gene expression analysis, and it is shown that the CsADAP gene has the function of promoting fruit ripening, and provides a new gene resource for molecular design breeding of fruit ripening.

Description

Technical Field

[0001] This invention relates to the field of plant genetic engineering technology, and in particular to the gene CsADAP, which is related to the ripening of citrus fruits, and its application in regulating fruit ripening. Background Technology

[0002] China is a major citrus-growing country in the world, with the largest citrus planting area globally (Deng Xiuxin, 2022). The Chinese citrus market is primarily based on fresh fruit sales (Shen Zhaomin, 2020). However, the overall supply of fresh fruit suffers from uneven distribution of varieties, concentrated ripening periods, and a lack of high-quality early- and mid-maturing varieties. Therefore, breeding high-quality early- and mid-maturing citrus varieties and achieving year-round supply of early-maturing citrus is of great significance to the citrus industry.

[0003] Fruit ripening is a complex biological process involving sugar and acid metabolism, pigment accumulation, cell wall degradation, and hormone signal transduction. Related studies have shown that citrus fruit ripening is regulated by many factors, such as external conditions (light, temperature, and water), plant hormones, and transcriptional regulation (Ren, 2021). In citrus, abscisic acid (ABA) and ethylene are key hormones regulating fruit ripening. ABA plays a positive regulatory role in citrus fruit ripening, and its increased content is closely related to changes in fruit coloring and sugar-acid ratio. Multi-omics analysis of 'Fengjie 72-1' navel orange and its late-maturing mutant 'Fengjie Late Orange' revealed that ABA and ethylene biosynthesis and signal transduction pathways play crucial roles in the formation of ripening differences, with ERF family transcription factors being one of the most significantly differentiated transcription factor families (Wu, 2015).

[0004] The AP2 / ERF transcription factor family is a plant-specific transcriptional regulatory family whose members are widely involved in plant growth and development, hormone signal transduction, and stress responses. As terminal response genes in the ethylene signaling pathway, AP2 / ERF transcription factors can regulate the biosynthesis of ethylene, cytokinins, gibberellins, and abscisic acid through feedback regulation, while also participating in hormone signal responses such as auxin and jasmonic acid, constructing a cross-regulatory network model of plant hormone signals, thereby regulating fruit ripening (Gu, 2017). The AP2 / ERF family has been found to be closely related to fruit development, ripening, and quality regulation in many fruits, such as citrus (Xie, 2014), sand pear 'Huangguan' (XU, 2023), jujube (Zhang, 2018), and banana (Xie, 2024). Synthesizing research data on AP2 / ERF function in different fruits, it has been found that AP2 / ERF transcription factors play a crucial role in ethylene-mediated fruit ripening by regulating multiple quality attributes such as fruit color, texture, and flavor (Xie, 2016). In the regulation of citrus fruit ripening, AP2-type transcription factors influence ABA and ethylene synthesis by regulating the expression of downstream target genes, thereby regulating the fruit ripening process. Studies have shown that AP2 family transcription factors may affect citrus fruit development and ripening by participating in light and hormone signaling pathways (Zheng Saisai, 2017); the transcription factor FcrNAC22 can directly bind to the promoter of NCED5, a key gene in the ABA synthesis pathway, and activate its expression, promoting fruit ripening (Gong Jinli, 2021).

[0005] Traditional breeding methods are too time-consuming and inefficient to meet the urgent needs of the industry. Using molecular technology to improve the ripening period and analyze the ripening regulation mechanism of transcription factors may become an important research direction, which will have a decisive impact on achieving precise regulation of the ripening period of citrus fruits and improving the economic benefits of the industry. Summary of the Invention

[0006] One of the purposes of this application is to provide the CsADAP gene, whose encoding nucleic acid sequence is shown in SEQ ID NO.1.

[0007] Preferably, the CsADAP gene is subcellularly located on the cell nucleus and has transcriptional activation function.

[0008] The second objective of this application is to provide the protein encoded by the CsADAP gene, the amino acid sequence of which is shown in SEQ ID NO.2.

[0009] The third objective of this application is to provide an expression vector for the aforementioned CsADAP gene.

[0010] Preferably, this application uses the Gateway system to construct binary expression vectors, which include an introductory vector pDONR221 and an overexpression final vector pK7WG2D.

[0011] The fourth objective of this application is to provide a host bacterium for the aforementioned CsADAP gene.

[0012] The fifth objective of this application is to provide primer pairs for amplifying the CsADAP gene described above, characterized in that their nucleic acid sequences are shown in SEQ ID NO. 3 and 4.

[0013] The sixth objective of this application is to provide the above-mentioned CsADAP gene, or the above-mentioned protein, or the above-mentioned expression vector, or the above-mentioned host bacterium, or the above-mentioned primer pair for any of the following (1)-(5):

[0014] (1) Prepare products that reduce the number of days it takes for fruit to lose its color;

[0015] (2) Prepare products that improve the rate of fruit color change;

[0016] (3) Prepare products that increase the carotenoid content in fruits;

[0017] (4) Prepare products that reduce the chlorophyll content in fruits;

[0018] (5) Prepare products that promote fruit ripening.

[0019] Preferably, the fruit is a fruit with a high carotenoid content after ripening, such as citrus or tomato.

[0020] The seventh objective of this application is to provide a method for promoting fruit ripening by overexpressing the aforementioned CsADAP gene in the plant.

[0021] The eighth objective of this application is to provide an expression vector for silencing the aforementioned CsADAP gene.

[0022] The ninth objective of this application is to provide a host bacterium that silences the aforementioned CsADAP gene.

[0023] The tenth objective of this application is to provide an expression vector that silences the CsADAP gene, or a host bacterium that silences the CsADAP gene, for any of the following applications (1)-(5):

[0024] (1) Prepare products that increase the number of days for fruit to break color;

[0025] (2) Prepare products that reduce the rate of fruit discoloration;

[0026] (3) Prepare products that reduce the carotenoid content in fruits;

[0027] (4) Prepare products that increase the chlorophyll content in fruits;

[0028] (5) Prepare products that delay fruit ripening.

[0029] Preferably, the fruit is a fruit with a high carotenoid content after ripening, such as citrus or tomato.

[0030] Beneficial effects:

[0031] This application utilizes gene cloning technology to isolate and clone the citrus fruit ripening gene CsADAP from 'Fengjie 72-1' navel orange. Based on this, a vector for CsADAP was constructed and genetically transformed into tomato and kumquat to verify the function of CsADAP in the fruit ripening process. The gene was transformed into tomato and kumquat using Agrobacterium-mediated transformation. Phenotypic observation and gene expression analysis of the resulting transgenic plants and fruits showed that the fruit of the CsADAP-overexpressing tomato lines broke color significantly earlier than that of the wild type. The average color-breaking time of the T2 generation overexpressing tomato lines was 32 days, about 5 days earlier than the wild type. Transient overexpression of the CsADAP gene in kumquats initiated color change about 5 days later, about 4 days earlier than the control group. The silent group initiated color change about 12 days later, about 4 days later than the control. The above results confirm that it has a significant function in promoting fruit ripening, providing excellent gene resources for molecular design breeding of citrus ripening period, and has important research significance and application value for elucidating the molecular mechanism of citrus fruit ripening regulation and the development of the citrus industry. Attached Figure Description

[0032] Figure 1 This is a schematic diagram illustrating the process of cloning, isolating, and functionally validating the CsADAP gene in this invention.

[0033] Figure 2 This is the subcellular localization result of the CsADAP gene in Example 2 of the present invention.

[0034] Figure 3 This is the result of the identification of the transcriptional activation activity of the CsADAP gene in Example 3 of this invention.

[0035] Figure 4 This is a semi-quantitative gel image of the tomato transgenic positive plants in Example 4 of the present invention.

[0036] Figure 5 This is a graph showing the quantitative results of the CsADAP gene in the tomato transgenic positive plants in Example 4 of this invention.

[0037] Figure 6a , Figure 6b The figures are statistical graphs showing the color changes and the number of days of fruit color damage in tomato fruits overexpressing the CsADAP gene in Example 4 of this invention.

[0038] Figure 7 The graph shows the changes in ethylene release, ABA content, carotenoid content, and chlorophyll content at different stages in tomato fruits overexpressing the CsADAP gene and control fruits in Example 4 of this invention.

[0039] Figure 8 The expression level of the CsADAP gene in the transiently transformed fruit of *Citrus aurantiacus* in Example 5 of this invention is given. Here, PK7 represents *Citrus aurantiacus* transformed with the pK7WG2D vector, PTRV represents *Citrus aurantiacus* transformed with the pTRV2 vector, #1 and #2 represent CsADAP gene overexpression lines, and PTRV-CsADAP represents CsADAP gene silencing lines.

[0040] Figure 9a , Figure 9b The above examples, in Example 5 of this invention, describe the phenotypic observation and color-changing day statistics of the CsADAP gene after transient transformation into Kumquat. CsADAP-OE represents the CsADAP gene overexpression line, and CsADAP-VIGS represents the CsADAP gene silencing line. Detailed Implementation

[0041] The present invention will be further explained below with reference to specific embodiments.

[0042]

[0043] The protein encoded by the CsADAP gene has an amino acid sequence such as SEQ ID MAKTSRQSQKNTTTNPNNNNTVTTKTKRTRKSVPRDSPPQRSSIYRGVTRHRWTGRYEAHLWDKNCWNESQNKKGRQVYLGAYDNEEAAARAYDLAALKYWGHDTILNFPLSNYEEELVEMEGQSKEEYIGSLRRKSSGFSRGVSKYRGVARHHHNGRWEARIGRVFGNKY shown in NO.2 LYLGTYATQEEAAQAYDRAAIEYRGLNAVTNFDLSKYIKWLRPNNNQNNPKPSNPQQNPNSDDTSPIPKLNQETSSGSETSAPPPRSGATAGGSGSASSALGLLLQSSKYKEMVEKTSTDCLSTSEPGSSHRIFPDDIQTMFFDCQDSSSYTEGDDVLLGDLNPYIFPTFYSELDN.

[0044] Example 1: Cloning of the full-length cDNA of the citrus fruit development and maturation gene CsADAP

[0045] The applicant obtained the CDS sequence of the sweet orange from the Citrus genome database (http: / / citrus.hzau.edu.cn / orange / ) and designed primers in the 5' untranslated region and 3' untranslated region of the sequence.

[0046] Forward primer CsADAP-F1: AAAAAGCAGGCTCCATGGCGAAAACCTCAAGGCA

[0047] Reverse primer CsADAP-R1:AGAAAGCTGGGTTCTAATTATCAAGTTCACTAT

[0048] Then, using wild-type Fengjie navel orange cDNA as a template, a high-fidelity enzyme was used for amplification reaction. The kit used was Phanta Max Super-Fidelity DNA Polymerase.

[0049] Leaves of wild-type Fengjie navel oranges stored at -80℃ were used to extract RNA using the Novozymes RNA reagent (FastPure PlantTotal RNA Isolation kit). The specific RNA extraction method is as follows:

[0050] 1) Take an appropriate amount of plant tissue that has been ground with liquid nitrogen and immediately add 600 μL Buffer EL or 600 μL Buffer PSL. Vortex vigorously for 30 seconds to ensure that the sample and lysis buffer are thoroughly mixed. Centrifuge at 12000 rpm (13400 × g) for 5 min and proceed with subsequent operations immediately.

[0051] 2) Take about 500 μL of supernatant into FastPure gDNA-Filter Columns III (FastPure gDNA-Filter Columns III has been placed in the collection tube), centrifuge at 12000 rpm (13400×g) for 30 seconds, discard FastPure gDNA-Filter Columns III, and collect the filtrate;

[0052] 3) Add 0.5 times the volume of the filtrate to the collection tube, and shake to mix for 15 seconds.

[0053] 4) Transfer the above mixture to FastPure RNA Columns V (FastPure RNA Columns V has been placed in the collection tube), centrifuge at 12000 rpm (13400×g) for 30 seconds, and discard the filtrate;

[0054] 5) Add 700 μL of Buffer RWA to FastPure RNA Columns V, centrifuge at 12000 rpm (13400×g) for 30 seconds, and discard the filtrate;

[0055] 6) Add 500 μL of Buffer RWB to FastPure RNA Columns V (please check that 48 mL of anhydrous ethanol has been added before use), centrifuge at 12000 rpm (13400 × g) for 30 seconds, and discard the filtrate;

[0056] 7) Repeat step 6).

[0057] 8) Place the FastPure RNA Columns V back into the collection tube and centrifuge at 12000 rpm (13400×g) for 2 min;

[0058] 9) Transfer FastPure RNA Columns V to a new RNase-free Collection Tubes 1.5mL centrifuge tube, add 30-100μL of RNase-free ddH2O to the center of the adsorption column membrane, and centrifuge at 12000rpm (13400×g) for 1min;

[0059] 10) The extracted RNA can be used directly in downstream experiments or stored at -20℃.

[0060] cDNA was synthesized by reverse transcription using RNA from Fengjie navel oranges as a template.

[0061] cDNA synthesis was performed using the Hiscript III RT SuperMix for qPCR reverse transcription kit. Specific procedures and synthesis methods were followed according to the manufacturer's instructions. The resulting cDNA was used for PCR amplification of the CsADAP gene. PCR amplification used the designed CsADAP-F1 and CsADAP-R1 primers as primers.

[0062] The detailed steps for PCR amplification are as follows: 95℃ pre-denaturation for 3 min; 95℃ denaturation for 15 s, 58℃ annealing for 15 s, 72℃ extension for 1 min, 35 cycles, followed by 72℃ extension for 5 min after each cycle.

[0063] After amplification, a single-band PCR product was generated. Following 1% agarose gel electrophoresis, the amplified gel product was purified and recovered using an Omega gel extraction kit. The purified product was then ligated into the entry vector pDOR221 in a total reaction volume of 10 μL. After incubation at room temperature for 5 min, the mixture was transformed into *E. coli* competent cells DH5α.

[0064] Then, the bacterial culture was identified by PCR using primers for the target gene sequence (i.e., CsADAP-F1 and CsADAP-R1) and sent to the company for sequencing. Finally, based on the sequencing results, single-clone bacterial cultures with the correct sequence were selected, and plasmids were extracted using Kangwei Century CWO500M. Then, the plasmids were ligated with the final vector pK7WG2D, and the ligation product was transformed into Agrobacterium GV3101.

[0065] The plasmid contains the CDS sequence of the gene CsADAP, the nucleotide sequence of which is shown in SEQ ID NO.1, and the amino acid sequence of the protein it encodes is shown in SEQ ID NO.2.

[0066] The CDS sequence of the CsADAP gene is 1048 bp in length, containing a coding reading frame that encodes 348 amino acids, with an isoelectric point of 8.73. MEGAX analysis (https: / / www.megasoftware.net) revealed its evolutionary relationship with the AP2 / ERF family in Arabidopsis thaliana, identifying it as belonging to the AP2 subfamily. Based on its homologous gene AtADAP in Arabidopsis thaliana, it was named CsADAP.

[0067] Example 2: Subcellular localization of the CsADAP gene

[0068] A localization vector for the CsADAP gene was constructed using the PRI101-GFP vector. Primers were designed based on the gene sequence, and SaII / KPnI restriction sites were added to the forward and reverse primers according to the multiple cloning site of the PRI101-GFP vector. The CDS with the stop codon removed was fused into the PRI101-GFP vector to construct the fusion protein CsADAP:GFP. The recombinant plasmid was identified by PCR in bacterial culture. The recombinant plasmid and the empty plasmid were transformed into Agrobacterium strain GV3101, and then into the lower epidermal cells of Nicotiana benthamiana for transient expression.

[0069] Agrobacterium infection of tobacco epidermis was carried out using the following method:

[0070] 1) Cell activation: Agrobacterium GV3101 bacterial culture, which was stored at -80℃ and transformed with recombinant plasmid and empty vector PRI101-GFP plasmid, was streaked on LB solid medium (containing 25 mg / L Kana) and cultured at 28℃ for 2 days to activate the cells;

[0071] 2) Small-scale shaking of bacterial culture: Pick a single colony and place it in 5 mL of LB liquid medium, shake at 28℃ and 220 r / min for 24 h;

[0072] 3) Large-scale shaking of bacterial culture: On the day of the experiment, take 300 μL of the bacterial culture after small-scale shaking and add it to an Erlenmeyer flask containing 30 mL of fresh liquid LB. Shake at 220 r / min for about 10 hours at 28℃ until the bacterial culture reaches OD. 600 The value is 0.7;

[0073] 4) Collection of bacterial cells: After shaking, the bacterial solution is placed into a 50mL sterile centrifuge tube, centrifuged at 4000r / min for 5min, and the liquid is removed to ensure complete drainage;

[0074] 5) Cell washing: Add 10 mL of washing solution (10 mM MES, 10 mM MgCl2, freshly prepared) to the cells, suspend them thoroughly, centrifuge at 4000 r / min for 5 min, discard the liquid, add 5 mL of washing solution for a second washing, and then dissolve in 3 mL of washing solution.

[0075] 6) OD of suspended bacterial solution 600 Value determination: Dilute the bacterial suspension 20 times (e.g., 150 μL suspension + 2850 μL washing solution) to achieve the final OD value of P19 in the bacterial suspension. 600 The ratio was 0.7:0.5. The solution was added to 8 mL with washing buffer, and 8 μL of acetylsalicylic acid (50 mg / mL) was added and mixed well. The mixture was then incubated at 30 °C for 3 h.

[0076] 7) Injecting tobacco leaves: Select 3 tobacco plants with uniform growth and no disease for injection. Inject 2 leaves from each plant. When injecting, select the back of the leaf (the back of the leaf has more stomata, making it easier to inject the bacterial solution).

[0077] 8) Culture and observation: Fluorescence observation was performed using laser confocal microscopy after 24 hours.

[0078] The results are as follows Figure 2 (Fluorescence detection results) show that the fluorescence of the CsADAP:GFP recombinant fusion protein only appeared in the cell nucleus, while the fluorescence of the empty vector was distributed throughout the cell tissue. These results indicate that the CsADAP gene is located in the cell nucleus and is a nuclear localized protein.

[0079] Example 3: Analysis of CsADAP gene transcriptional activation

[0080] Transcriptional activation activity is a fundamental characteristic of transcription factors. The applicant used the pGBKT7 vector for recombination to verify whether CsADAP has transcriptional activation activity.

[0081] Specific primers were designed according to the gene sequence, and EcoRI / SmaI restriction sites were added to the forward and reverse primers of the pGBKT7 vector according to the pGBKT7 vector sequence information to amplify the full-length CDS region of the gene. The amplified region was then ligated into the pGBKT7 vector to construct a recombinant plasmid, resulting in the fusion expression vector pGBKT7-CsADAP.

[0082] After sequencing confirmed the sequences were correct, the fusion expression vector and the empty vector (pGBKT7) were transformed into the same yeast strain AH109 (purchased from Kangwei Century). Finally, the positive bacterial cultures were evenly spread on three solid media: SD / -Trp, SD / -Trp-His, and SD / -Trp-His-Ade, and cultured on different deletion media to detect the survival of the transformants.

[0083] The results are as follows Figure 3 The results showed that yeast cells transformed with the empty vector could only grow on the SD / -Trp medium lacking the vector, while yeast cells transformed with the recombinant plasmid pGBKT7-CsADAP grew normally.

[0084] This result indicates that the transcription factor CsADAP can bind to GAL4-BD, activate the transcription of downstream reporter genes, and enable yeast to grow normally on incomplete culture media, that is, CsADAP has a transcriptional activation function.

[0085] Example 4: Overexpression of gene CsADAP in tomato

[0086] 1. Construction of plant transformation overexpression vectors

[0087] A binary expression vector was constructed using the Gateway system, with wild-type Fengjie navel orange cDNA as a template. The primers were designed as follows:

[0088] F1:AAAAAGCAGGCTCCATGGCGAAAACCTCAAGGCA

[0089] R1:AGAAAGCTGGGTCTAATTATCAAGTTCACTAT

[0090] The cDNA obtained by reverse transcription was used for PCR amplification of the CsADAP gene. After amplification, a single-band PCR product was generated. After electrophoresis on a 1% agarose gel, the amplified gel product was purified and recovered using an Omega gel recovery kit. The product was then subjected to a BP reaction with the entry vector pDOR221 and transformed into DH5α Escherichia coli.

[0091] The following day, single clones were selected for positive identification. Positive single clones were then cultured in LB broth containing kanamycin (Kan) resistance medium, and positive clones were detected and sequenced. Once the sequencing results were correct, the ligation product was transformed into *E. coli* competent cells DH5α. Plasmids were extracted from positive strains using Kangwei Century CWO500M, thus completing the construction of the overexpression vector pK7WG2D-CsADAP.

[0092] 2. Genetic transformation in tomatoes

[0093] Tomatoes are considered a good material for gene function verification in scientific research due to their short growth cycle and ease of transformation. 'Mic-Tom' is a dwarf tomato plant that is shorter than ordinary tomatoes, making it easier to plant and manage at high density. The steps of Agrobacterium-mediated tomato genetic transformation are as follows:

[0094] 2.1 Vaccination

[0095] 1) Select plump seeds and soak them in tap water for 1-2 hours;

[0096] 2) Disinfect seeds with 75% ethanol for 1 minute (stirring constantly);

[0097] 3) Shake the solution with 50% 84 disinfectant for 15 minutes;

[0098] 4) Wash the seeds three times with sterile water, blot dry the surface moisture with sterile filter paper, and sow them evenly on S1 medium.

[0099] 5) Culture in a culture room with 16 hours of light and 8 hours of darkness for about one week.

[0100] 2.2 Cut cotyledons and Agrobacterium infection

[0101] 1) Activation and propagation of bacterial strains: Agrobacterium tumefaciens containing the overexpression vector pK7WG2D-CsADAP was streaked onto LB (SPE resistant) solid medium and incubated at 28°C for 48 h; after activation, the bacterial cells were re-stripped onto new LB (SPE resistant) solid medium and propagated at 28°C for 24 h.

[0102] 2) About 6-10 days after sowing, when the tomatoes have grown two cotyledons, use a sterile scalpel to cut off the leaf tip and the junction of the leaf blade and petiole. Divide the middle section into two halves (about 0.4cm long). Place the middle section of the leaf with the back facing up in S2 co-culture medium and incubate in the dark at 25±2℃ for 24h.

[0103] 3) Using a sterile blade, scrape the propagated Agrobacterium and resuspend it in 50 mL of Agrobacterium suspension. Incubate at 28°C with shaking for about 30 minutes until OD reaches 50%. 600 The concentration is 0.2–0.6, and 50 mg / L AS (acetyleugenol) is added for later use;

[0104] 4) Place the cotyledons cultured in the pre-culture medium into a sterile Erlenmeyer flask, pour in the prepared Agrobacterium suspension, and shake to inoculate for 5 minutes;

[0105] 5) Discard the suspension, blot the surface moisture of the cotyledons with sterile filter paper, and place them back in the S2 co-culture medium (with the back of the leaves facing up) and co-culture in the dark at 25±2℃ for 2 days.

[0106] 2.3 Screening and Culture

[0107] Transfer the leaves to S3 selection medium. After 48 hours of pre-culture, inoculate the cotyledons face up onto the S3 selection medium, ensuring that the cut surfaces are in full contact with the medium. Culture at 25±2℃ under light.

[0108] 2.4 Rooting Culture

[0109] After about 7 days of cultivation, white callus tissue will grow on the edge of the tomato leaves. After 12 days, it will be transferred to S3 subculture medium. After 10-14 days, the callus tissue will differentiate into buds. After about 30 days, the buds will grow into growth points and then be transferred to S4 rooting medium (at which point positive results can be identified). Roots will grow in about 7 days.

[0110] Table 1. Culture medium used for tomato seedling transformation

[0111] culture medium formula S1 Macroelements (20×) 25 mL / L, microelements (200×) 2.5 mL / L, organic components (200×) 2.5 mL / L, iron salts (200×) 2.5 mL / L, sucrose 15 g / L, agar 7.4 g / L S2 Macroelements (20×) 50 mL / L, microelements (200×) 5 mL / L, iron salts (200×) 5 mL / L, inositol 100 mg / L, thiamine hydrochloride 1.3 mg / L, 2,4-D 0.2 mg / L, KH₂PO₄ 2 mg / L, KT 0.1 mg / L, sucrose 30 g / L, agar 7.4 g / L S3 Macroelements (30×) 50mL / L, Microelements (200×) 5mL / L, Organic Components (200×) 5mL / L, Iron Salts (200×) 5mL / L, Organic Components (200×) 5mL / L, Sucrose 30g / L, Agar 7.4g / L, LIAA 0.1mg / L, ZR 2mg / L, Kana 100mg / L, Termetidine 360mg / L S4 MS 4.4 g / L, sucrose 30 g / L, vitamin 2.5 mL / L, gel 3 g, kanamycin 100 mg / L, IAA 0.1 mg / L, termethin 360 mg / L Agrobacterium suspension Macroelements (20×) 50 mL / L, microelements (200×) 5 mL / L, iron salts (200×) 5 mL / L, organic components (200×) 5 mL / L, malt extract 0.5 g / L, glutamine 1.5 g / L, sucrose 40 g / L, agar 7.4 g / L

[0112] 3. Screening and identification of transgenic tomato seedlings with positive results

[0113] Transgenic tomato plants with the CsADAP gene were obtained using the above method. Genomic DNA was extracted from the plant leaves. Forward and reverse primers were designed based on the 35S sequence on the vector and the gene sequence to amplify the DNA and verify whether the exogenous target gene had been inserted into the genome of the transformed material.

[0114] 3.1 DNA extraction from tomato leaves

[0115] 1) Preparation of DNA buffer (1L): 100mL of 1M Tris-HCl (pH 8.0), 100mL of 0.5M EDTA (pH 8.0), 300mL of 5M NaCl, and 500mL of H2O.

[0116] For 1M Tris-HCl (pH 8.0): Weigh 121g of Tris-Base, add 800mL of water and stir on a magnetic stirrer until fully dissolved. Then add concentrated hydrochloric acid to adjust the pH to 8.0, add water to bring the volume to 1000mL, sterilize, and store at room temperature.

[0117] 0.5M EDTA (pH 8.0): Weigh 187g of EDTA-2Na salt and add it to about 800mL of water. While stirring on a magnetic stirrer, add solid NaOH. When both EDTA and NaOH are completely dissolved and the solution becomes clear, the pH will be around 8.0. Adjust the pH slightly with pH paper. After sterilization, store at room temperature.

[0118] 5M NaCl: Weigh 300g of NaCl, add about 800mL of distilled water and stir thoroughly on a magnetic stirrer. After about 5 minutes, stop stirring, let stand for 2-3 minutes, pour off the supernatant, add another 100mL of distilled water and repeat the previous steps until the NaCl is fully dissolved. Then, bring the volume to 1000mL, sterilize, and store at room temperature.

[0119] Preparation of phenol:chloroform:isoamyl alcohol (25:24:1): Mix water-saturated phenol, chloroform and isoamyl alcohol in a volume ratio of 25:24:1 and store in a brown bottle.

[0120] Preparation of 70% anhydrous ethanol: Mix anhydrous ethanol and water in a volume ratio of 7:3 and set aside.

[0121] The specific extraction steps are as follows:

[0122] 1) Weigh (0.64N×1%) g PVP, (0.64N×2%) g CTAB, and 0.64×N mL DNA buffer to prepare a 0.64×N mL CTAB buffer solution, and transfer it to a 10 mL centrifuge tube. Dissolve the solution in a 65°C water bath (N is the number of samples).

[0123] 2) Weigh approximately 0.1g of the sample into a 1.5mL centrifuge tube and grind it with liquid nitrogen;

[0124] 3) Add 100µL of β-mercaptoethanol (1%-4%) to the CTAB buffer solution;

[0125] 4) Add 640µL of the above CTAB buffer mixture to each sample and shake up and down (or place on a vortex mixer to mix).

[0126] 5) Water bath at 65℃ for 60-90 minutes;

[0127] 6) Add 700µL of phenol:chloroform:isoamyl alcohol (25:24:1), shake up and down for about 5 minutes, then centrifuge at 20℃ and 13000rpm for 8 minutes;

[0128] 7) Take 500µL of the supernatant (using a yellow pipette tip), add 60µL of 5M NaCl and 1mL of pre-cooled anhydrous ethanol, mix by inverting, and incubate in an ice-water bath at -20℃ for 30min.

[0129] 8) Centrifuge at 4℃ and 13000 rpm for 6 minutes;

[0130] 9) Discard the supernatant, add 1 mL of 70% anhydrous ethanol, and let stand at -20℃ for 2 hours;

[0131] 10) Centrifuge at 4℃ and 10000rpm for 5 minutes;

[0132] 11) Discard the supernatant and dry it in the clean bench (not too dry);

[0133] 12) Add 100µL TE and 1.5µL 10µM RNase enzyme to each centrifuge tube (clean bench operation).

[0134] 13) Bath in a 37°C water bath overnight.

[0135] 3.2 DNA Positive Identification

[0136] Positive plants were identified using specific primer 35S and a reverse primer. Among the selected transgenic lines, those that amplified the expected size fragment were considered positive transgenic lines. Ultimately, eight positive plants were verified (e.g., ...). Figure 5 (As shown).

[0137] 35S: 5'-GACGCACAATCCCACTAT-3';

[0138] CsADAP-R1: 5'-AGAAAGCTGGGTCTAATTATCAAGTTCACTAT-3'.

[0139] 4. Overexpression analysis of transgenic tomato seedlings

[0140] RNA was extracted from the transplanted and viable transgenic positive seedlings (referred to as #1, #2, ... #8) and reverse transcribed to synthesize cDNA (RNA extraction method is the same as in Example 1). The cDNA obtained by reverse transcription was diluted 5 times with ddH2O as a template. Quantitative primers were designed using the online website Primer Premier 5.

[0141] The CsADAP quantitative primers are:

[0142] CsADAP-qPCR-F: 5'-AGCCAAAAGAACACTACTACAAACC-3';

[0143] CsADAP-qPCR-R: 5'-AAGCACCTAAATATACTTGTCTCCC-3'.

[0144] The primers for the tomato Actin gene, used as an internal reference gene, are:

[0145] SlActin-F: 5'-GTCCTCTCCAGCCATCCAT-3';

[0146] SlActin-R: 5'-ACCACTGAGCACAATGTTACCG-3'.

[0147] The expression level of the CsADAP gene was identified by qRT-PCR, which showed that the expression level of the CsADAP gene was relatively high in positive transgenic tomatoes (e.g., Figure 5 (As shown).

[0148] 5. Observation of fruit phenotype and determination of physiological indicators of transgenic tomato seedlings

[0149] The color change of the fruit peel signifies the beginning of ripening and is often used to determine the onset time of fruit ripening. Phenotypic observations were conducted on wild-type (WT) and T2 generation CsADAP-overexpressing tomatoes, and the time required from full bloom to the onset of fruit coloring was statistically analyzed, i.e., the number of days until color breakage.

[0150] As shown in Figure 6, the fruits of the CsADAP overexpressing lines began to color earlier than those of the wild type (WT). Wild-type fruits began to color around 37 DAF (Days after flowering), while fruits of the CsADAP overexpressing lines began to color around 32 DAF. Figure 6 indicates that the tomato fruits overexpressing the CsADAP gene began to color about 5 days earlier than those of the wild type (WT).

[0151] Tomato fruits overexpressing the CsADAP gene had significantly higher carotenoid content than those overwhelmed (WT) fruits, while chlorophyll content was the opposite. The peak ABA content occurred earlier than that of WT fruits, and the peak ABA content occurred before ethylene.

[0152] Example 5: Overexpression and silencing of the CsADAP gene in Citrus aurantiacus

[0153] 1. Construction of VIGS silencing vector

[0154] The vectors used were pTRV1 and pTRV2. Based on the characteristics of the pTRV2 vector, EcoRI and SmaI enzymes were selected as the forward and reverse restriction sites. Primers were designed using the first 300 bp sequence of the CsADAP gene CDS as a template and wild-type Fengjie navel orange cDNA as a template. The primers are as follows:

[0155] F2: 5'-GTGAGTAAGGTTACCGAATTCATGGCGAAAACCTCAAGGCAAAGCC-3';

[0156] R2: 5'-TGCTCGACGACAAGACCCGGGGACCCTCCATTTCTACGAGC-3'.

[0157] After PCR amplification, the amplified gel product was purified and recovered using an Omega gel extraction kit. Then, the linearized pTRV2 vector, which was digested with double enzymes, was homologously recombinated with the gel product. After homologous recombination, it was transformed into E. coli DH5α and sent to the company for sequencing. The plasmid of the correctly sequenced bacterial culture was extracted, and the successfully constructed silencing vector was named pTRV2-CsADAP. Finally, it was transformed into Agrobacterium GV3101. The bacterial culture with the correct positive verification was placed in glycerol and stored at -80°C.

[0158] 2. Instant transformation of kumquat

[0159] Agrobacterium culture containing the overexpression vector pK7WG2D-CsADAP and the empty vector pK7WG2D was activated on LB (SPE) solid medium for 2 days, while Agrobacterium culture containing the silencing vector pTRV2-CsADAP and the empty vector pTRV2 was activated on LB (Kan) fixation medium for 2 days, followed by a second activation for 1 day, for subsequent genetic transformation.

[0160] The detailed steps of Agrobacterium-mediated transient transformation of kumquat are as follows:

[0161] 1) For fruit injection, after washing once with suspension buffer, mix the target bacterial solution with the P19 helper plasmid bacterial solution (P19 is a gene silencing repressor that can prevent post-transcriptional gene silencing in transgenic citrus fruits and promote high-level expression of target proteins), and adjust the OD of the mixed bacterial solution. 600 = Around 0.5.

[0162] 2) Inject 0.1–0.15 mL of infection solution into each fruit. The injection process should be slow and even, and the extent to which the infection solution reaches the subcutaneous layer of the fruit should be visible to the naked eye. Aspirate any excess Agrobacterium and take photographs. Photograph the fruit's condition every two days. Detect the CsADAP gene expression level 14 days after infection.

[0163] Buffer formulation (10 mL): glucose (0.05 g), MES (500 mM, 1 mL), Na3PO4 (20 mM, 1 mL), acetylsylgenone (1 M, 1 μL) and H2O (to 10 mL).

[0164] 1M Acetyleugenol (AS): Dissolve 0.0392g AS in 0.2mL DMSO, aliquot into 10μL portions and store at -20℃.

[0165] 20mM Na3PO4: Dissolve 0.17g of anhydrous Na3PO4 in 50mL of water and store at 4℃.

[0166] 500mM MES: Dissolve 4.88g of MES in 50mL of water and store at 4℃.

[0167] 3. The expression level of the gene CsADAP in the transformed fruit of *Citrus aurantiacus* was identified using qRT-PCR. For example... Figure 8 As shown, the expression level in the hyperphenotype of Kumquat fruit was significantly higher than that in the PK7 control, and the expression level in the silent line fruit was significantly lower than that in the TRV empty vector. This indicates that the CsADAP gene was successfully transiently transformed into the fruit.

[0168] 4. Phenotypic observation of transgenic kumquat fruit

[0169] The fruit color changes of the empty control group, CsADAP gene overexpressing lines, and silent lines were statistically analyzed on the day of injection and every two days. Results are as follows: Figure 9a The results showed that the overexpression strains exhibited faster color decay in their fruits than the wild type, while the silent strains showed slower color decay in their fruits than the wild type.

[0170] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. The CsADAP gene, characterized by, The encoding nucleic acid sequence is shown in SEQ ID NO.

1.

2. The protein encoded by the CsADAP gene as described in claim 1, characterized in that, Its amino acid sequence is shown in SEQ ID NO.

2.

3. An expression vector containing the CsADAP gene as described in claim 1.

4. A host bacterium containing the CsADAP gene as described in claim 1.

5. A primer pair for amplifying the CsADAP gene as described in claim 1, characterized in that, Its nucleic acid sequences are shown in SEQ ID NO. 3 and 4.

6. The use of the CsADAP gene as claimed in claim 1, or the protein as claimed in claim 2, or the expression vector as claimed in claim 3, or the host bacterium as claimed in claim 4, or the primer pair as claimed in claim 5, in any of the following (1)-(5): (1) Prepare products that reduce the number of days it takes for fruit to lose its color; (2) To prepare products that improve the rate of fruit color change; (3) Prepare products that increase the carotenoid content in fruits; (4) Prepare products that reduce the chlorophyll content in fruits; (5) Prepare products that promote fruit ripening.

7. A method for promoting fruit ripening, characterized in that, Overexpression of the CsADAP gene as described in claim 1 in plants.

8. Silencing the expression vector of the CsADAP gene as described in claim 1.

9. Silencing the host bacteria of the CsADAP gene as described in claim 1.

10. Or the expression vector as described in claim 8, or the host bacterium as described in claim 9, in any of the following (1)-(5): (1) Prepare products that increase the number of days for fruit to break color; (2) Prepare products that reduce the rate of fruit discoloration; (3) Prepare products that reduce the carotenoid content in fruits; (4) Prepare products that increase the chlorophyll content in fruits; (5) Prepare products that delay fruit ripening.

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