Application of MdZFP3-like gene in regulating fruit ripening and softening

Abstract

This invention discloses the application of the MdZFP3-like gene in regulating fruit ripening and softening, belonging to the field of molecular biology. This invention is the first to discover a close linkage between the MdZFP3-like gene and climacteric fruit ripening and softening. The CDS sequence of the MdZFP3-like gene is shown in SEQ ID NO.1, and the amino acid sequence is shown in SEQ ID NO.2. Overexpression of the MdZFP3-like gene inhibits fruit ripening and softening, while reduced expression promotes fruit ripening and softening. Transient transformation of apples can achieve the effect of regulating fruit ripening and softening. Based on this experiment, recombinant vectors that overexpress or silence the MdZFP3-like gene can be used to prepare storage formulations and shelf-life preservatives for climacteric fruits, with higher safety levels. The provided MdZFP3-like gene can be used for the breeding of climacteric fruit varieties, providing a new approach for creating fruit varieties with fast or slow ripening rates, and is of great significance for shortening breeding years, simplifying the breeding process, and reducing breeding workload.

Description

Technical Field

[0001] This invention relates to the field of molecular biology, and in particular to the application of the MdZFP3-like gene in regulating fruit ripening and softening. Background Technology

[0002] Apples (Malus domestica Borkh.) are an important global fruit, widely cultivated and loved by consumers, used for both fresh consumption and processing. As a major global apple producer, my country accounts for over 50% of the world's apple cultivation and production. The apple industry is a pillar industry in its advantageous production areas, playing a significant role in regional economies.

[0003] The ripening and softening of apples are key physiological processes in fruit quality formation and storage. In-depth analysis of this mechanism can not only optimize production management and post-harvest preservation techniques, but also provide theoretical support for improving the quality and efficiency of the industry. Fruit ripening is a complex process synergistically regulated by hormones, enzymes, and signaling molecules, manifested as reddening, increased sugar content, and softening. Among these processes, cell wall degrading enzymes (such as cellulase and pectinase) cause fruit softening by breaking down cell wall components, while ethylene, as a core hormone, dominates the ripening process by regulating the expression of related genes.

[0004] In recent years, molecular biology techniques have propelled research progress in this field, leading to the identification of several key genes and proteins and revealing the molecular regulatory network of fruit ripening and softening. However, many unknown mechanisms remain to be explored, leaving ample room for future research. Identifying and discovering functional genes involved in fruit ripening and softening is of great significance for the sustainable protection and high-quality utilization of apple germplasm resources, fruit quality regulation (such as texture improvement and ripening delay), molecular breeding, revealing the regulatory mechanisms of fruit firmness and ethylene metabolism, identifying downstream target genes and signaling networks, and contributing to improved economic benefits and sustainable development of the industry. Summary of the Invention

[0005] The purpose of this invention is to provide the application of the MdZFP3-like gene in regulating fruit ripening and softening, so as to provide a functional gene linked to fruit ripening and softening, which can be used to prepare safer fruit preservation agents and preservatives.

[0006] To achieve the above objectives, this invention provides the application of the MdZFP3-like gene in regulating fruit ripening and softening. The CDS sequence of the MdZFP3-like gene is shown in SEQ ID NO.1, and the amino acid sequence is shown in SEQ ID NO.2.

[0007] Preferably, overexpression of the MdZFP3-like gene inhibits fruit ripening and softening, while reducing MdZFP3-like gene expression promotes fruit ripening and softening.

[0008] Preferably, the fruit is a climacteric fruit.

[0009] Application of recombinant vectors containing MdZFP3-like genes in regulating fruit ripening and softening, wherein the recombinant vectors are vectors that overexpress, knock out, or silence the aforementioned MdZFP3-like genes.

[0010] The application of engineered bacteria containing recombinant vectors containing the MdZFP3-like gene in regulating fruit ripening and softening; engineered bacteria include Escherichia coli and Agrobacterium.

[0011] Application of the MdZFP3-like gene in the preparation of fruit storage preparations, wherein the fruit storage preparations contain the recombinant vector containing the MdZFP3-like gene.

[0012] Preferably, the formulation containing a recombinant vector overexpressing the MdZFP3-like gene is introduced into the fruit to inhibit fruit ripening and softening; the recombinant vector containing the inhibition of MdZFP3-like gene expression is introduced into the fruit to promote fruit ripening and softening.

[0013] Application of the MdZFP3-like gene in the preparation of fruit preservatives, wherein the fruit preservatives contain recombinant vectors that overexpress the MdZFP3-like gene.

[0014] As mentioned above, the MdZFP3-like gene is used in variety breeding, which refers to the selection of fruit varieties that improve the ripening rate and fruit varieties that delay the ripening rate.

[0015] The preferred and selected varieties are climacteric fruit varieties.

[0016] Therefore, the specific technical effects of the MdZFP3-like gene provided by this invention in regulating fruit ripening and softening are as follows:

[0017] (1) This invention first discovered that the MdZFP3-like gene is closely linked to the fruit ripening and softening of the respiratory climacteric type. The CDS sequence of the MdZFP3-like gene is shown in SEQ ID NO.1, and the amino acid sequence is shown in SEQ ID NO.2. Overexpression of the MdZFP3-like gene inhibits fruit ripening and softening, while reducing the expression of the MdZFP3-like gene promotes fruit ripening and softening.

[0018] (2) The experiment of this invention confirms that by instantaneously transforming apples, the effect of regulating fruit ripening and softening can be achieved. Based on this experiment, the recombinant vector that overexpresses or silences the MdZFP3-like gene can be used to prepare storage preparations and shelf-extending preservatives for respiratory climacteric fruits. The prepared storage preparations and preservatives are safer.

[0019] (3) The MdZFP3-like gene provided by this invention can be used for the breeding of climacteric fruit varieties, providing a new idea for creating fruit varieties with fast or slow maturation rates. It is of great significance for shortening the breeding period, simplifying the breeding process, and reducing the breeding workload.

[0020] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. Attached Figure Description

[0021] To more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings used in the description of the embodiments of the present invention will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0022] Figure 1 The results of genetic transformation of apple callus tissue in Example 2 of this invention are shown below; where A is the PCR verification result; B is the immunoblotting result; C is the expression level measurement result; *** indicates P<0.001; **** indicates P<0.0001;

[0023] Figure 2 The results of the identification of transgenic 'Micro-Tom' tomatoes in Example 3 of this invention are shown below; where A is the PCR identification result; B is a photograph of tomatoes at different stages; C is the ethylene release rate detection result; D is the fruit firmness measurement result; MG is the green ripening stage; BR is the color breaking stage; B5 is the slightly red stage; B10 is the fully red stage; ** is P<0.01; *** is P<0.001; **** is P<0.0001; ns is no difference.

[0024] Figure 3 The results of the instantaneous conversion of apples in Example 5 of this invention are shown below; where A represents apple photographs taken at different times after conversion; B represents the ethylene release rate detection results; C represents the fruit firmness measurement results; D represents the expression level measurement results; * represents P<0.01; *** represents P<0.001; **** represents P<0.0001; ns represents no difference.

[0025] Figure 4 The results of the MdZFP3-like binding to the MdPG2a promoter experiment in Example 6 of this invention are shown below; where A is yeast monohybrid (Y1H); B is the electrophoretic mobility assay (EMSA) result; C is a tobacco photograph of the luciferase complementation experiment; D is the statistical result of the relative luciferase activity of the luciferase complementation experiment; a, b, c, and d in part D indicate that the differences between treatments are significant, P < 0.05. Detailed Implementation

[0026] The technical solution of the present invention will be further described below with reference to the accompanying drawings and embodiments.

[0027] To make the objectives, technical solutions, and advantages of this application clearer, more thorough, and more complete, the technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings and embodiments. The following detailed descriptions are all illustrations of embodiments, intended to provide further detailed explanation of the present invention. Unless otherwise specified, all technical terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.

[0028] The instruments, equipment, reagents and materials used in the embodiments were all obtained through commercial means; the methods and steps not described in detail in the embodiments are all conventional techniques in the art.

[0029] The culture medium components used in the examples are as follows:

[0030] Co-culture medium: solid MS medium containing 1 mg / L 2,4-D and 0.5 mg / L 6-BA;

[0031] Screening medium: solid MS medium containing 50 mg / L kanamycin, 250 mg / L carbenicillin, 1 mg / L 2,4-D and 0.5 mg / L 6-BA;

[0032] MS basal medium: 4.48 g / L MS, 30 g / L sucrose, 7 g / L agar, 150 mg / L kanamycin, 300 mg / L carbenicillin, 0.5 mg / L auxin, 2 mg / L zeatin;

[0033] Pre-medium: MS + 100 μM acetylsalicylic acid;

[0034] Proliferation medium: MS basal medium containing 0.5 mg / L 6-BA and 2 mg / L zeatin;

[0035] Rooting medium: 4.48 g / L MS + 30 g / L sucrose + 6 g / L agar + 0.2 mg / ml IAA + 50 mg / L kanamycin + 200 mg / L carbenicillin.

[0036] Example 1

[0037] The MdZFP3-like gene overexpression vector was constructed as follows:

[0038] (1) The MdZFP3-like gene is the encoding gene of the transcription factor MdZFP3-like. The MdZFP3-like gene has the accession number XP_017183172.3 on NCBI.

[0039] The CDS sequence of the MdZFP3-like gene is shown in SEQ ID NO.1, and the amino acid sequence is shown in SEQ ID NO.2.

[0040] SEQ ID NO.1:

[0041] ATGGCTAACATATCAACCCTCTTCAACAAGTTCCTCTCTCTCCTCATCCTCAT

[0042] CCTCCACCTCGGCTGTTTCTCTTCCACCGCCACTGCCAACCACCCAAAAAA

[0043] GCCTCATCACCGCCGTAAACTCTCCACTCACCTCTCCAAACCCAGCACCCT

[0044] AAAACCCCAAAAAGCCCTCTCCACCTCCTGGTCCTATCTCAAACGCATTTT

[0045] CACCTCCAAATCCTACAAAATCACCTGCACCAACATCATCCAATCCCACCTC

[0046] TCCACGCCACCTCGATCCTCCCACCACTCCATCGTCTCCCTCGTCCTACCTG

[0047] ACTCCGACCCCAAATACCTACCTGGGTCGCTCCCCGAATCAGATATCTCGGC

[0048] CGATTCCCACCAATTGTTCCCTCTCCGAAACGATATCTTCCCCTGCACCGCC

[0049] TGCGGGGAAATCTTCCCGAAACCCGAGACTCTCGATCACCACCAGGCGAT

[0050] CCACCACGCCGTTTCGGAACTTCATGACGGAGACTCGGGCAAAAACATTG

[0051] TCCGAATCATATTCAAAACAGGTTGGACCGATACGCGAAAAGCCCCCGAAA

[0052] TTCACCGGATCCTGAAGATCCACAACAGCGGAAAAATCCTGTCGAGGTTC

[0053] GAGGAGTACAGAGAGCTGGTCAAGTCCAAGGCGGCGCGAAACGGCACCG

[0054] TTCGGAGGAGGGATGAGCGGTGCATCGCCGACGGCAACGAGCTTCTCAGA

[0055] TTTCACTGCTCAACTTTCGTTTGCGATTTGGGACTCAACGGGAATTCTGGG

[0056] ATTTGTAATCACGAGTACTGCAGTGTTTGTGGAATTATTAAATCTGGATTCTC

[0057] ACCCAAGTTGGACGGAATTTCCACGCTGTCGAGTAGCTCCAGAGCACACG

[0058] TGGCAATTCCAGAGGATATCGAGGAGGAGTTTCAGTTCATGAACGTGAAGC

[0059] GGGCTATGCTGGTCTGCCGGGTCGTGGCGGGTCGGGTCGGGTGTGACACT

[0060] GAGGAGGATATTGACGACGTGGACAAAGAGGGCGGCGGATTCGACTCGGT

[0061] TGTCGGCAGAGAAGGCAGCGGGGTCCACACGAGGGTGGACGAGGAGGAG

[0062] CTTTTGGTGTTTAATCCAAGAGCCGTTCTTCCTTGCTTTGTGATCGTATATAC

[0063] CGTGTAA

[0064] SEQ ID NO.2:

[0065] MANISTLFNKFLSLLILILHLGCFSSTATANHPKKPHHRRKLSTHLSKPSTLKPQ

[0066] KALSTSWSYLKRIFTSKSYKITCTNIIQSHLSTPPRSSHHSIVSLVLPDSDPKYLP

[0067] GSLPESDISADSHQLFPLRNDIFPCTACGEIFPKPETLDHHQAIHHAVSELHDGD

[0068] SGKNIVRIIFKTGWTDTRKAPEIHRILKIHNSGKILSRFEEYRELVKSKAARNGT

[0069] VRRRDERCIADGNELLRFHCSTFVCDLGLNGNSGICNHEYCSVCGIIKSGFSPK

[0070] LDGISTLSSSSRAHVAIPEDIEEEFQFMNVKRAMLVCRVVAGRVGCDTEEDIDD

[0071] VDKEGGGFDSVVGREGSGVHTRVDEEELLVFNPRAVLPCFVIVYTV

[0072] (2) Send the sequence information shown in SEQ ID NO.1 to the company to synthesize the CDS sequence of the MdZFP3-like gene. Dissolve the received DNA sequence in sterile ddH2O according to the attached instructions to prepare a 100mM stock solution. Then take a small amount of the stock solution and dilute it to a 10mM working solution.

[0073] Using the CDS working solution of the MdZFP3-like gene in (1) as a template, PCR amplification was performed using primer pairs consisting of MdZFP3-like-F (sequence shown in SEQ ID NO.3) and MdZFP3-like-R (sequence shown in SEQ ID NO.4). The amplification system was prepared according to the instructions accompanying the high-fidelity enzyme. The amplification program was as follows: 95℃ pre-denaturation for 30 seconds; 95℃ denaturation for 5 seconds, 60℃ annealing for 1 min, 72℃ extension for 1 min, 38 cycles; 72℃ final extension for 10 min; storage at 4℃. The PCR amplification products were subjected to electrophoresis, and the target fragment was then recovered using a kit gel.

[0074] SEQ ID NO.3:ttgatacatatgcccgtcgacATGGCTAACATATCAACCCTCTTCA

[0075] SEQ ID NO.4:tcagaattcggtacccccgggCACGGTATATACGATCACAAAGCA

[0076] (3) Take the PRI101 vector and perform double digestion with restriction endonucleases SalI and SmaI (the digestion system and digestion conditions are in accordance with the instructions attached to the enzymes). The vector backbone is recovered by gel extraction using the kit.

[0077] (4) The vector backbone recovered from the gel in step (3) and the PCR amplification product recovered from the gel in step (2) are ligated by homologous recombination. The homologous recombination ligation product is then transfected with Escherichia coli DH5α- for culture PCR (using the 35S-F (sequence shown in SEQ ID NO.5) and the amplification program in (2), in a Taq enzyme system). The culture with PCR amplification bands that meet the expected size is sent to the company for sequencing. The plasmid extracted from the correctly sequenced culture is the MdZFP3-like overexpression vector.

[0078] SEQ ID NO.5:GACGCACAATCCCACTATCC

[0079] (4) Transform the MdZFP3-like overexpression vector into Agrobacterium (LBA4404), add 50% glycerol and store at -80℃.

[0080] Example 2

[0081] The MdZFP3-like gene overexpression vector obtained in Example 1 was genetically transformed into apple callus, as detailed below:

[0082] (1) Preparation of wild-type callus.

[0083] Callus tissue from 'Wanglin' ​​apples (preparation method is described in Zhang Shuhui's 2023 doctoral dissertation at Shandong Agricultural University) was inoculated into solid MS medium containing 1 mg / L 2,4-D and 0.5 mg / L 6-BA for subculture.

[0084] (2) Preparation of apple callus overexpressing 'Wang Lin'.

[0085] 1) The MdZFP3-like gene overexpression vector obtained in Example 1 was introduced into Agrobacterium LBA4404 to obtain recombinant Agrobacterium. The recombinant Agrobacterium cells were washed with ddH2O and then resuspended in 30 mL of liquid MS medium to obtain OD. 600nm A bacterial suspension with a pH of 0.8 is prepared by adding 30 μL of 100 mM acetylsuccinone solution (in DMSO) to form the infection solution.

[0086] 2) Take the callus tissue from the subcultured 'Wanglin' ​​apple in step (1) and inoculate it onto solid MS medium plates. Incubate in the dark at 25°C for 15 days. After the incubation period, immerse the callus tissue in the infection solution obtained in step (1) and shake at 120 rpm at room temperature for 30 min. After drying the callus surface with sterile filter paper, place it on a co-culture medium plate and incubate in the dark at 25°C for 36 hours. Then place the callus tissue on a selection medium plate and incubate in the dark at 25°C for 20-30 days.

[0087] 3) Take resistant callus tissue that can grow on the selection medium, extract genomic DNA using a kit, and perform PCR amplification and identification using 35S-F (sequence shown in SEQ ID NO.5) and ZFP3-R (sequence shown in SEQ ID NO.6) as primers. The amplification system was prepared according to the instructions accompanying the Taq enzyme. The amplification program was as follows: 95℃ pre-denaturation for 30 sec; 95℃ denaturation for 25 sec, 60℃ annealing for 30 sec, 72℃ extension for 1 min, 27 cycles; 72℃ final extension for 5 min; and storage at 4℃.

[0088] The PCR amplification products were subjected to agarose gel electrophoresis. The callus tissue corresponding to the 1128bp electrophoretic band was the MdZFP3-like gene transgenic callus tissue.

[0089] SEQ ID NO.6:CACGGTATATACGATCACAAAGCA

[0090] 4) The MdZFP3-like gene-transgenic callus obtained in step 3) was inoculated onto selection medium plates and subcultured in the dark at 25°C. Callus obtained from the same callus is called a line.

[0091] (3) RNA was extracted from the callus tissue transfected with the MdZFP3-like gene obtained in (2) using a kit. The RNA meeting quality requirements was reverse transcribed into cDNA. Using MdActin-F (sequence shown in SEQ ID NO.7) and MdActin-R (sequence shown in SEQ ID NO.8) as references, and MdZFP3-like quantitative primer-F (sequence shown in SEQ ID NO.9) and MdZFP3-like quantitative primer-R (sequence shown in SEQ ID NO.10) as references, qRT-PCR was performed. The expression levels of the three callus tissues with the highest expression levels (OE1-OE3) were as follows: Figure 1 As shown in C, the electrophoresis results of the PCR products of OE1-OE3 transgenic callus are as follows: Figure 1As shown in Figure A. Genomic DNA was extracted and verified by PCR amplification using primers specifically targeting the overexpression vector. The results showed that all transgenic lines amplified specific bands of the expected size, which fully confirmed that the MdZFP3-like gene was stably expressed at the transcriptional level.

[0092] SEQ ID NO.7:TGACCGAATGAGCAAGGAAATTACT

[0093] SEQ ID NO.8:TACTCAGCTTTTGGCAATCCACATC

[0094] SEQ ID NO.9:GACACTGAGGAGGATATTGA

[0095] SEQ ID NO.10:AGAACGGCTCTTGGATTA

[0096] (4) Proteins were extracted from the transgenic callus using a kit, and immunoblotting analysis was performed using the following method:

[0097] ① Protein electrophoretic separation: Following the standard SDS-PAGE procedure, proteins were separated according to their molecular weight differences using sodium dodecyl sulfate-polyacrylamide gel electrophoresis. After denaturation with Laemmli buffer, protein samples were electrophoretically migrated at a constant voltage (80V for stacking gel / 120V for separating gel). Electrophoresis was terminated when the bromophenol blue indicator reached the bottom of the gel.

[0098] ② Transfer System Assembly: Construct a transfer sandwich structure using the wet transfer method. Activate the PVDF membrane with methanol for 2 min to enhance its hydrophobic binding capacity, then equilibrate it in transfer buffer (25 mM Tris-192 mM glycine-20% methanol, pH 8.3) for 10 min. Stack the soaked fiber pad, filter paper, separating gel, PVDF membrane, and the upper filter paper / fiber pad in sequence, using rollers to remove interfacial air bubbles layer by layer before fixing it in the transfer tank.

[0099] ③ Low-temperature electroblotting: The transfer device is placed in a pre-cooled electrophoresis tank, and electroblotting is performed at a constant current of 120mA for 3 hours at 4℃. Under these conditions, the negatively charged SDS-protein complex migrates directionally from the gel to the PVDF membrane surface under the drive of the electric field. For high molecular weight proteins (>100kDa), the transfer time needs to be extended to 90-120 minutes to ensure sufficient transfer.

[0100] ④ Blocking non-specific binding: The transfer membrane was immersed in TBST blocking solution containing 5% skim milk powder (10mM Tris-HCl, 150mM NaCl, 0.1% Tween-20, pH 7.6) and blocked on a horizontal shaker at 4°C for 3 hours, which effectively blocked the protein binding sites remaining on the membrane surface.

[0101] ⑤ Primary antibody specific recognition: Mouse polyclonal antibody (1:5000 diluted in blocking buffer) was incubated at 4°C with shaking for 12-16 hours to form antigen-antibody complexes. The primary antibody working solution was stored at -20°C for reuse (≤3 times).

[0102] ⑥ Membrane regeneration and washing: After recovering the primary antibody, perform 5 rounds of rinsing with TBST buffer (5 min / rinse) to thoroughly remove unbound antibody and impurities. It is recommended to briefly immerse the membrane in high-salt buffer (TBST containing 500 mM NaCl) after the first wash to dissociate weakly bound antibody.

[0103] ⑦ Enzyme-labeled secondary antibody binding: Incubate with HRP-labeled goat anti-mouse IgG secondary antibody (1:10000 dilution) at room temperature for 2 hours to construct an antigen-primary antibody-enzyme-labeled secondary antibody complex.

[0104] ⑧ Strict elution procedure: After recovering the secondary antibody, perform 5 rounds of TBST rinsing (5 min / time). After the last wash, rinse twice with TBS buffer (without Tween-20) to eliminate the quenching effect of detergent residue on chemiluminescence.

[0105] ⑨ Chemiluminescence imaging: The ECL substrate working solution (solution A:solution B = 1:1) is uniformly applied to the membrane surface. After reacting for 30 seconds, images are captured using a chemiluminescence imaging system (Tanon 5200). The exposure time (30-300 seconds) is adjusted according to the signal intensity. For strong signal samples, a stepped exposure mode (10 seconds / 30 seconds / 60 seconds) is recommended to avoid overexposure.

[0106] The results are as follows Figure 1 As shown in Figure B, the MdZFP3-like protein fused with the GFP tag was detected by immunoblotting. The results showed that the overexpression line showed a specific signal at the corresponding molecular weight position, while no related signal was detected in the wild-type control (WL).

[0107] Example 3

[0108] The MdZFP3-like gene overexpression vector obtained in Example 1 was genetically transformed into 'Micro-Tom' tomatoes, as follows:

[0109] (1) Seed pretreatment and sterilization: Select plump tomato seeds without mechanical damage, place the seeds in a constant temperature water bath shaker at 40±0.5℃ and soak for 30 min to activate metabolic activity, then surface sterilize with 70% ethanol solution for 30±2s, rinse 4 times with sterile deionized water, and then deep sterilize with 8% sodium hypochlorite solution for 10±1 min. After sterilization, rinse 7 times with sterile water, and finally use sterile filter paper to absorb the surface moisture.

[0110] (2) Aseptic germination and explant preparation: The seeds sterilized in step (1) were inoculated into MS basal medium and cultured in the dark at 24±1℃ for 3-4 days until the radicle broke through the seed coat; then cultured under a photoperiod of 16h light / 8h dark and a light intensity of 2000±200lx until the cotyledons were fully expanded. The cotyledon tissue was aseptically removed in a clean bench, the petiole and apical meristem were removed, and the wound side was laid flat on the pre-medium and cultured in the dark at 25±1℃ for 48h to enhance cell competence.

[0111] (3) Propagation of Agrobacterium engineered strain and preparation of infection solution: The bacterial solution stored at -80℃ in Example 1, after being thawed on ice and mixed, was placed in YEP liquid medium containing 50 mg / L kanamycin + 50 mg / L rifampin and cultured at 28±0.5℃ with shaking at 200 rpm until OD. 600 =0.6±0.05; transferred to fresh medium at a 1:100 volume ratio for amplification, OD 600 When the concentration of bacteria reached 0.6, the cells were collected by centrifugation at 4°C and 5000×g for 10 min; the cells were then resuspended in MS liquid medium containing 100 μM acetylsyleugenol to OD0.6. 600 =0.8±0.05, to obtain the working solution for contamination.

[0112] (4) Genetic transformation and plant regeneration: On a clean bench, explants were immersed in the infection solution for 10±1 min. After removing the surface bacterial film with sterile filter paper, they were transferred to co-culture medium and cultured upside down in the dark for 48±2 h. Then, they were transferred to selection medium, with the wound side facing up, and the medium was replaced with fresh medium every 14 days. When the callus differentiated and formed 1-2 mm green buds, they were transferred to proliferation medium and cultured at 25±1℃ under dark / 8h light conditions with a photoperiod of 16 h.

[0113] (5) Identification of transgenic plants is the same as step (2) of Example 2, part 3). A total of 2 transgenic plants were obtained, and the electrophoretic identification results are as follows: Figure 2 As shown in Figure A, WT represents the electrophoresis results of the PCR products of 'Micro-Tom' tomatoes grown from non-transgenic callus tissue.

[0114] (6) When the tender shoots of the transgenic plants obtained in step (5) grow to 2-4 cm, transplant them to a rooting medium until the seedlings root. Then transplant them into a substrate, using 'Micro-Tom' tomatoes grown from non-transgenic callus tissue as the rootstock (WT). Photos of tomatoes at the green-ripe stage (MG), color-changing stage (BR), 5 days after color-changing stage (B5), and 10 days after color-changing stage (10B) are shown below. Figure 2 As shown in B.

[0115] Gas chromatography was used to determine the ethylene content in tomatoes at the MG, BR, B5, and B10 stages, respectively. Three tomatoes were tested for each stage. The results are as follows: Figure 2 As shown in Figure C, the ethylene release rate of tomato fruit exhibits a trend of first increasing and then decreasing during the ripening process, reaching a peak at stage B5. However, we found that tomato fruits overexpressing MdZFP3-like showed a lower ethylene release rate at the same developmental stage compared to the wild type.

[0116] Tomato firmness was measured using a hardness tester at MG, BR, B5, and B10 stages. Three tomatoes were tested for each stage. The results are as follows: Figure 2 As shown in D, the firmness of the fruit typically decreases gradually as it ripens. This indicates that overexpression of MdZFP3-like compounds in tomatoes can effectively increase the firmness of tomato fruits.

[0117] Example 4

[0118] The MdZFP3-like gene silencing vector was constructed as follows:

[0119] The 300bp conserved target sequence of the single-stranded DNA molecule in the CDS sequence of the MdZFP3-like gene shown in SEQ ID NO.1 was obtained from the SGN-VIGS website. The 300bp sequence was reverse-complemented using DNAMAN software and then inserted between the EcoRI and KpnI restriction sites of the pTRV2 vector. Primers were synthesized by the sequencing company as in Example 1. After homologous recombination and transformation into DH5α, the sequence was sent for sequencing (using universal primers for the pTRV2 vector from the sequencing company). After comparing the sequence results, the recombinant plasmid was obtained.

[0120] Example 5

[0121] The MdZFP3-like gene silencing vector obtained in Example 4 was transiently transformed into apples. The specific steps are as follows:

[0122] (1) The MdZFP3-like gene silencing vector obtained in Example 4 was transformed into Agrobacterium GV3101-p19 strain to obtain GV3101-p19-MdZFP3-like engineered strain.

[0123] (2) Preparation of inoculation materials: Select 'Golden Delicious' apples with consistent physiological state as inoculation materials, with the following requirements:

[0124] Maturity: Select Golden Delicious apples about 120 days after flowering, with a maturity of about 80% and a green skin; Quality characteristics: Fresh, free from pests and diseases, and uniform in size.

[0125] (3) Preparation of infection solution: The GV3101-p19-MdZFP3-like engineered strain was washed with ddH2O and then suspended in 30 mL of liquid MS medium to obtain OD. 600nm A bacterial suspension with a pH of 0.8 is prepared by adding 30 μL of 100 mM acetylsuccinone solution (in DMSO) to form the infection solution.

[0126] (4) Vacuum permeation treatment: After surface disinfection of apple fruits with 75% ethanol for 30 seconds, rinse with sterile water, and then completely immerse them in a sealed container containing the infiltration solution. Start the vacuum pump to gradually reduce the pressure inside the container to -0.08 MPa to -0.1 MPa, and maintain this vacuum level for 3 ± 0.5 minutes. Then slowly open the gas valve to restore the pressure to normal pressure. Repeat the above vacuum-recompression process once, and record it as MdZFP3-like-pTRV2. Use the no-load switch as a control, and record it as pTRV2.

[0127] (5) Post-infection treatment: Remove the apples from the infection solution, blot the surface bacterial solution with sterile filter paper, and store them in the dark at room temperature (25±1℃) for 9 days. Photos of storage on day 0, day 3, day 6, and day 9 are shown below. Figure 3 As shown in A, the fruit gradually matures and softens as the storage time increases, with MdZFP3-like-pTRV2 maturing faster.

[0128] The ethylene content of apples in the MdZFP3-like-pTRV2 group and the pTRV2 group was determined by gas chromatography at storage days 0, 3, 6, and 9. The results are as follows: Figure 3 As shown in B, the ethylene (ETH) release rate indicates that the silent MdZFP3-like fruit first reaches the peak ethylene release at 3.8 (μL / h / kg FW).

[0129] The firmness of apples from the MdZFP3-like-pTRV2 group and the pTRV2 group was measured using a texture analyzer at storage days 0, 3, 6, and 9. The results are as follows: Figure 3 As shown in C, compared with the control fruit (pTRV2), the fruit firmness of the MdZFP3-like silent group (pTRV2-MdZFP3-like) was lower than that of the control, indicating that the fruit softening rate was higher than that of the control.

[0130] RNA was extracted from apples in the MdZFP3-like-pTRV2 and pTRV2 groups at storage days 0, 3, 6, and 9, respectively. The RNA meeting quality requirements was reverse transcribed into cDNA using a kit. Using the obtained cDNA as a template, and with MdZFP3-like-F (sequence shown in SEQ ID NO. 9) and MdZFP3-like-R (sequence shown in SEQ ID NO. 10) as primers, the relative expression level of the MdZFP3-like gene was detected by qRT-PCR. The MdActin gene was used as an internal reference gene. -ΔΔCT The method was used for analysis. The primers for the MdActin gene consisted of MdActin-F (sequence shown in SEQ ID NO.7) and MdActin-R (sequence shown in SEQ ID NO.8).

[0131] The relative expression levels of MdZFP3-like genes are as follows: Figure 3 As shown in D, this indicates successful infection.

[0132] Example 6

[0133] The interaction between MdZFP3-like genes and apple fruit ripening and softening genes was verified by yeast one-hybrid assay, as follows:

[0134] (1) Construct a reorganization vehicle.

[0135] 1) Design specific primers for the MdZFP3-like gene (sequences shown in SEQ ID NO.11 and SEQ ID NO.12). Double digestion of the CDS of the MdZFP3-like gene and the pGADT7 vector with NdeI and BamHI, respectively, was performed. The recovered target fragment was then subjected to homologous recombination, inserting the full-length CDS of the MdZFP3-like gene into the pGADT7 vector to obtain the MdZFP3-like-pGADT7 recombinant vector. SEQ ID ON.11:gtaccagattacgctcatatgATGGCTAACATATCAACCCTCTTCA SEQ ID ON.12:cagctcgagctcgatggatccCACGGTATATACGATCACAAAGCA

[0136] 2) Design specific primers (sequences shown in SEQ ID NO.13 and SEQ ID NO.14) to clone the promoter sequence of MdPG2a, and construct the target promoter sequence into the pHIS2 (EcoRI / SacI) vector to obtain the proMdPG2a-pHIS2 recombinant vector.

[0137] SEQ ID ON.13:gactcactatagggcgaattcTCATAATAATATATTTTAGAACCCCCCASEQ IDON.14:gattcgcgaacgcgtgagctcGGTACTAAAAAAATGGTGGTTATTCAA

[0138] (2) Yeast Y187Gold strain was transformed using Yeast Transformation Kit (Coolaber, China).

[0139] 1) Preparation of Y187 yeast competent cells.

[0140] Strawberry strain Y187 was streaked onto YPDA solid medium and incubated upside down in a 30°C incubator for 2-3 days until single colonies appeared. A single colony was picked and inoculated into 3 mL of YPDA liquid medium and incubated overnight at 30°C and 220 rpm. 20 μL of the bacterial culture was then transferred to 50 mL of YPDA liquid medium and incubated at 28°C and 220 rpm until OD500 was reached. 600nm Once the OD value reaches 0.2-0.4, transfer the entire culture system to a 100mL centrifuge tube, centrifuge at 5000rpm for 5min, collect the bacterial cells, add 100mL of YPDA liquid medium, transfer to a 250mL Erlenmeyer flask, and incubate at 220rpm for 4-5h until OD reaches 0.2-0.4. 600nm The value reached 0.4-0.6; then, the culture system was aliquoted into two 100mL centrifuge tubes, centrifuged at 5000rpm for 5min, the supernatant was discarded, 30mL of sterile ddH2O was added to the bacterial pellet to resuspend the bacterial cells, centrifuged at 5000rpm for 5min, the supernatant was discarded, 2mL of TE / LiAC solution was added to the bacterial pellet to resuspend the bacterial cells, the resuspended bacterial cells were transferred to four 1.5mL centrifuge tubes, centrifuged at 5000rpm for 5min, the supernatant was discarded, 250μL of TE / LiAC solution was added to each tube to dissolve the bacteria, and the tubes were placed on ice for transformation.

[0141] 3) Transformation of Y187Gold yeast competent cells and functional verification of MdZFP3-like cells.

[0142] The recombinant plasmid obtained in (1) was transformed into the Y187Gold yeast cells prepared in step 2) using the conventional PEG / LiAc transformation method. The positive control: ProMdPG2a-pHIS2+pGADT7-MdZFP3-like and the negative control: ProMdPG2a-pHIS2+pGADT7 were transformed into the Y187Gold yeast cells prepared in step 2).

[0143] Transformed yeast cells were plated onto yeast SD / -Trp-Leu-His medium and SD / -Trp-Leu-His medium containing different concentrations of 3-AT, respectively. The cells were incubated upside down at 30°C, and colony growth was observed. Results are shown below. Figure 4 As shown in Figure A, all combinations grew normally on SD / -Trp-Leu-His medium, while only the negative control combination grew normally on SD / -Trp-Leu-His medium containing the lowest corresponding concentration of 3-AT inhibitor. The growth of positive control yeast cells was inhibited, indicating that there is an interaction effect between the promoters of MdZFP3-like and MdPG2a.

[0144] (3) Electrophoretic mobility experiments were conducted (for specific methods, please refer to Zhang Shuhui's 2023 doctoral dissertation from Shandong Agricultural University). The results are as follows: Figure 4 As shown in B, MdZFP3-like can bind to the MdPG2a promoter.

[0145] (4) Verification by dual-luciferase experiment.

[0146] 1) Promoter linking to LUC reporter gene.

[0147] Design specific primers for the MdZFP3-like gene (sequences shown in SEQ ID NO.15 and SEQ ID NO.16), construct the MdZFP3-like gene into the pGreenII 62-SK vector (BamHI / HindIII), and obtain the MdZFP3-like-62-SK recombinant vector;

[0148] SEQ ID ON.15:cgctctagaactagtggatccATGGCTAACATATCAACCCTCTTCA SEQ ID ON.16:gtcgacggtatcgataagcttCACGGTATATACGATCACAAAGCA

[0149] Design specific primers (sequences shown in SEQ ID NO.17 and SEQ ID NO.18) to clone the promoter of MdPG2a. Insert the cloned MdPG2a promoter into the pGreenII 0800-LUC vector (SalI / BamHI) to obtain the MdPG2a-LUC promoter recombinant vector.

[0150] SEQ ID ON.17:

[0151] gggccccccctcgaggtcgacTCATAATAATATATTTTAGAACCCCCCA SEQ ID ON.18:cgctctagaactagtggatccGGTACTAAAAAAATGGTGGTTATTCAA

[0152] The recombinant vectors MdZFP3-like-62-SK and MdPG2a-LUC were transformed into Agrobacterium GV3101 (pSoup-p19) competent cells, respectively, to obtain Agrobacterium MdZFP3-like-62-SK and ProMdPG2a-LUC.

[0153] 2) Set up different treatment groups, with 3 parallel experiments in each group. Inject the reagents from group 1 to group 4 into different locations on the same leaf of the same tobacco plant. The group information is as follows:

[0154] Group 1: Empty vector (pGreenII 62-SK empty Agrobacterium) + LUC (pGreenII 0800-LUC empty Agrobacterium);

[0155] Group 2: ProMdPG2a-LUC+Empty vector;

[0156] Group 3: MdZFP3-like-62-SK+LUC;

[0157] Group 4: ProMdPG2a-LUC+MdZFP3-like-62-SK.

[0158] Tobacco leaves injected with different treatment groups were cultured in the dark at 24℃ for 36-48 hours. The leaves were then observed and photographed using a live imaging system. Simultaneously, the fluorescence intensity under different treatments was measured. Figure 4 As shown in C. The statistical results are as follows. Figure 4 As shown in Figure D, the addition of MdZFP3-like promoters promoted the activation activity of the MdPG2a promoter. This indicates that the transcriptional activation activity of the MdPG2a promoter significantly increased after the addition of MdZFP3-like promoters. These results further demonstrate that MdZFP3-like promoters are positive regulators of the MdPG2a promoter, promoting its transcriptional activity.

[0159] Therefore, this invention is the first to discover that the MdZFP3-like gene is closely linked to the ripening and softening of climacteric fruits. The CDS sequence of the MdZFP3-like gene is shown in SEQ ID NO.1, and the amino acid sequence is shown in SEQ ID NO.2. Overexpression of the MdZFP3-like gene inhibits fruit ripening and softening, while reducing MdZFP3-like gene expression promotes fruit ripening and softening. By transiently transforming apples, the effect of regulating fruit ripening and softening can be achieved. Based on this experiment, recombinant vectors that overexpress or silence the MdZFP3-like gene can be used to prepare storage preparations and shelf-life preservatives for climacteric fruits, and the prepared storage preparations and preservatives have higher safety. The provided MdZFP3-like gene can be used for the breeding of climacteric fruit varieties, providing a new approach for creating fruit varieties with fast or slow ripening rates, which is of great significance for shortening the breeding cycle, simplifying the breeding process, and reducing the breeding workload.

[0160] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the technical solutions of the present invention, and these modifications or equivalent substitutions cannot cause the modified technical solutions to deviate from the spirit and scope of the technical solutions of the present invention.

Claims

1. MdZFP3-like The application of genes in regulating apple ripening and softening is characterized by: MdZFP3-like The CDS sequence of the gene is shown as SEQ ID NO. 1, and the amino acid sequence is shown as SEQ ID NO. 2; overexpression MdZFP3-like The gene inhibits apple ripening softening, reduces MdZFP3-like The gene expression promotes apple ripening softening.

2. Overexpression, knockout, or silencing MdZFP3-like The application of gene recombinant vectors in regulating apple ripening and softening is characterized by: MdZFP3-like The CDS sequence of the gene is shown as SEQ ID NO. 1, and the amino acid sequence is shown as SEQ ID NO. 2; the recombinant vector containing overexpression MdZFP3-like The recombinant vector containing the gene expression is introduced into the apple to inhibit the apple ripening softening, and the recombinant vector containing the gene expression is introduced into the apple to reduce MdZFP3-like The recombinant vector containing the gene expression is introduced into the apple to promote the apple ripening softening.

3. Overexpression, knockout, or silence MdZFP3-like The application of engineered bacteria using recombinant gene vectors in regulating apple ripening and softening is characterized by: Engineered bacteria include Escherichia coli and Agrobacterium; MdZFP3-like The CDS sequence of the gene is shown in SEQ ID NO.1, and the amino acid sequence is shown in SEQ ID NO.2; a gene containing overexpression was used. MdZFP3-like Genetic transformation of apples using recombinant gene vectors inhibits apple ripening and softening, employing bacteria containing [a substance that reduces] [the ripening and softening process]. MdZFP3-like Genetic transformation of engineered bacteria using recombinant vectors for gene expression promotes apple ripening and softening.

4. MdZFP3-like The application of genes in the preparation of apple storage preparations is characterized by: MdZFP3-like The CDS sequence of the gene is shown in SEQ ID NO.1, and the amino acid sequence is shown in SEQ ID NO.2; [The gene containing overexpression...] MdZFP3-like The preparation of the gene recombinant vector was introduced into apples to inhibit the ripening and softening of apples.

5. MdZFP3-like The application of genes in the preparation of apple preservatives is characterized by: MdZFP3-like The CDS sequence of the gene is shown in SEQ ID NO.1, and the amino acid sequence is shown in SEQ ID NO.2; [The gene containing overexpression...] MdZFP3-like The recombinant vector of the gene is introduced into apples to inhibit the ripening and softening of the apples.

6. MdZFP3-like The application of genes in apple variety breeding is characterized by: MdZFP3-like The CDS sequence of the gene is shown in SEQ ID NO.1, and the amino acid sequence is shown in SEQ ID NO.2; Variety breeding refers to the selection and breeding of apple varieties that improve the ripening rate and apple varieties that delay the ripening rate; overexpression MdZFP3-like Genes inhibit apple ripening and softening, reducing MdZFP3-like Gene expression promotes apple ripening and softening.

Images