PpMYB60 gene for regulating and controlling synthesis of wax on epidermis of peach fruit and application of PpMYB60 gene

Abstract

The invention discloses a PpMYB60 gene for regulating and controlling peach fruit epidermis wax synthesis and application thereof, and is characterized in that the nucleotide sequence of the PpMYB60 gene is as shown in SEQ ID NO.1, and the amino acid sequence of protein coded by the PpMYB60 gene is as shown in SEQ ID NO.2. The invention further provides application of the PpMYB60 gene in regulating and controlling peach fruit epidermis wax synthesis. The PpMYB60 gene in the peach fruit is over-expressed, so that the content of wax synthesized on the epidermis of the peach fruit is increased, and the expression method of the PpMYB60 gene comprises the following steps: constructing an over-expression vector of the PpMYB60 gene of the peach fruit, transforming the over-expression vector into agrobacterium, and then infecting the peach peel; the method has the advantages that the contents of epidermis waxy alkane and triterpenes in the fruits are increased, the expression of wax synthesis key genes is up-regulated, the water loss of the fruits is obviously delayed, and the permeability is reduced.

Description

Technical Field

[0001] This invention belongs to the fields of plant molecular biotechnology and genetic engineering, and specifically relates to a method for regulating the synthesis of waxy substances in peach fruit epidermis. PpMYB60 Genes and their applications. Background Technology

[0002] Peach( Prunus persica Prunus (L.) is a plant belonging to the genus Prunus in the family Rosaceae. The waxy layer on the epidermis of the peach fruit serves as its natural protective barrier, playing a crucial role in reducing fruit water loss, resisting pests and diseases, and enhancing the fruit's resilience. The waxy layer adheres to the outer surface of the plant's epidermal cells, hindering the diffusion of water from the fruit's interior to the environment, effectively reducing water loss, maintaining fruit firmness and freshness, and extending post-harvest storage. Furthermore, the hydrophobic properties of the cuticle create a dry environment unfavorable to pathogen growth. Additionally, certain cuticle components secreted by the epidermal cells, such as unsaturated fatty acids and terpenes, have antibacterial effects.

[0003] Studies have shown that wax synthesis is regulated by transcription factors, with the MYB transcription factor family being the most studied regulatory genes for wax synthesis. For example, in Arabidopsis thaliana... AtMYB49 , AtMYB96 ,apple MdMYB30 and grapes VcMYB30 All of these factors are related to the regulation of wax biosynthesis. However, due to the abundance of trichomes on the peach fruit epidermis, the presence of a cuticle and its role as a barrier have been overlooked by many experts and scholars. Currently, research on the role of the peach fruit PpMYB transcription factor in regulating wax synthesis remains relatively scarce. Summary of the Invention

[0004] The technical problem to be solved by this invention is to provide a method for regulating the synthesis of wax on the peach fruit peel that can significantly delay fruit water loss and reduce permeability. PpMYB60 Genes and their applications can positively regulate the synthesis of waxy substances in peach skin.

[0005] The technical solution adopted by this invention to solve the above-mentioned technical problems is: a method for regulating the synthesis of wax on the skin of peach fruits. PpMYB60 Genes, as described PpMYB60 The nucleotide sequence of the gene is shown in SEQ ID NO.1.

[0006] Furthermore, the amino acid sequence of the protein encoded by the PpMYB60 is shown in SEQ ID NO.2.

[0007] Furthermore, the aforementioned PpMYB60The nucleotide sequence of the upstream amplification primer is shown in SEQ ID NO.3: tttggagaggacacgctcgagATGGGAAGGCCACCTTGC. PpMYB60 The nucleotide sequence of the downstream amplification primer is shown in SEQ ID NO.4: gcccttgctcaccatctcgagAAACAAGACAGATGTGTTCTCTAATGAC.

[0008] Another aspect of the present invention also provides the above-mentioned PpMYB60 The application of genes in regulating the synthesis of waxy substances in peach fruit skin, as described above PpMYB60 The nucleotide sequence of the gene is shown in SEQ ID NO.1. This gene was expressed by overexpression in peach fruit. PpMYB60 The gene causes it to synthesize an increased amount of wax on the surface of peach fruit.

[0009] Furthermore, the aforementioned PpMYB60 The nucleotide sequence of the upstream amplification primer for gene overexpression is shown in SEQ ID NO. 5: tactattctagtcgagaattcATGGGAAGGCCACCTTGC; PpMYB60 The nucleotide sequence of the downstream amplification primer for gene overexpression is shown in SEQ ID NO.6: caggtcgactctagaggatccTCAAAACAAGACAGATGTGTTCTCTAAT.

[0010] Furthermore, the aforementioned expression was overexpressed in the peach fruit peel. PpMYB60 Methods for gene expression, including constructing the peach fruit PpMYB60 The steps include: overexpressing the gene using a vector, transforming the overexpression vector into Agrobacterium, and then infecting the peach peel.

[0011] Compared with the prior art, the advantages of the present invention are as follows: The present invention discloses a transcription factor PpMYB60 that participates in the synthesis of waxy substances in peach fruit epidermis. PpMYB60 The base sequence is shown in SEQ ID No:1, and the amino acid sequence of the protein encoded by PpMYB60 is shown in SEQ ID No:2. This protein was obtained by overexpression in peach fruit. PpMYB60 The gene increases the content of waxy alkanes and triterpenes in the fruit peel, and upregulates the expression of key genes for wax synthesis. The advantage is that it can significantly delay fruit water loss and reduce permeability. Attached Figure Description

[0012] Figure 1 peach fruit PpMYB60 Gene cloning and pART-CAM PpMYB60 Electrophoretic gel image of the construction of the -GFP recombinant vector, where A is...PpMYB60 Gene amplification; B is the linearized electrophoresis diagram of the pART-CAM-GFP vector, 1 is the circular plasmid, 2 is the linearized plasmid; C is the Escherichia coli colony PCR identification results, 1-4 are single Escherichia coli colonies, - is the negative control; D is the Agrobacterium colony PCR identification results, 1-3 are single Agrobacterium colonies, - is the negative control; Figure 2 Subcellular localization results of PpMYB60 in tobacco epidermal cells; pART-CAM-GFP: Subcellular localization results of the control group; pART-CAM- PpMYB60 -GFP: Subcellular localization results of the experimental group; Figure 3 For overexpression PpMYB60 The effects on the composition and content of waxy substances in peach fruit peel, where A represents waxes; B represents alkanes; and C represents triterpenes. Figure 4 For overexpression PpMYB60 Effects on the microstructure of waxy crystals on peach fruit skin; Figure 5 peach fruit PpMYB60 Gene cloning and BG Plant express- PpMYB60 Construction of the recombinant vector, where A is PpMYB60 Gene amplification; B represents the PCR identification results of Escherichia coli colonies, 1-5 are single Escherichia coli colonies, and - is the negative control; C represents the PCR identification results of Agrobacterium colonies, 1-5 are single Agrobacterium colonies, + is the positive control, and - is the negative control; Figure 6 For semi-quantitative PCR identification PpMYB60 Expression levels in transgenic Arabidopsis thaliana; Figure 7 For overexpression PpMYB60 Effects on the water permeability of Arabidopsis thaliana epidermis; Figure 8 For overexpression PpMYB60 The effect on water loss from detached Arabidopsis leaves. Detailed Implementation

[0013] The present invention will be further described in detail below with reference to the accompanying drawings and embodiments.

[0014] Specific Implementation Example 1: In peach fruit PpMYB60 Gene cloning and sequence analysis.

[0015] Step 1: Extract RNA from peach peel Total RNA was extracted from peaches using the Eastep Super Total RNA Extraction Kit (LS1040, Promega). The procedure was followed according to the kit's instructions, and the RNA concentration and purity were determined using a micro spectrophotometer.

[0016] Step 2: RNA is reverse transcribed into cDNA The samples were reverse transcribed using the HiScript II Q RT SuperMix for qPCR (+gDNA wiper) kit (R223-01, Vazyme), and the products can be stored at -20°C.

[0017] Step 3 PpMYB60 Cloning of the full gene Using the cDNA from step 2, "RNA reverse transcription to cDNA," as a template and XhoI as the restriction enzyme site, a design was developed. PpMYB60 Upstream and downstream primers for gene amplification, PpMYB60 upstream amplification primers for the gene (pART-CAM-) PpMYB60 The sequence of -F) is shown in SEQ ID NO.3: tttggagaggacacgctcgagATGGGAAGGCCACCTTGC; PpMYB60 Downstream amplification primers for the gene (pART-CAM-) PpMYB60 The sequence of -R) is shown in SEQ ID NO.4: gcccttgctcaccatctcgagAAACAAGACAGATGTGTTCTCTAATGAC. Amplification was performed using PhantaMax Super-Fidelity DNA Polymerase (P505, Vazyme) high-fidelity enzyme. Analysis of the amplification product by agarose gel electrophoresis yielded a band approximately 1131 bp in size, as shown below. Figure 1 As shown in (A), the target band and brightness meet the requirements.

[0018] PpMYB60

[0019] PpMYB60 The amino acid sequence of the encoded protein such as SEQ ID Shown in NO.2: MGRPPCCDKVGVKKGPWTPEEDIILVSYIQEHPGGNWRSVPTNTGLLRCSKSCRLRWTNYLRPGIKRGNFTDHEEKMIIHLQALLGNRWAAIASYLPQRTDNDIKNYWNTHLKKKLRKLQTGLDGHDHRNSQDGFSGNSHDQPISKGQWERRLQTDIHMAKQALCEALSLDKPPTHDL HLQDLKPSINLGYNNNDQPNTNTCSRPHQASTYASNTENIAKLLESWMKNSPKGPSSAQNHNPTNSETNLHQINSFKNKKNTAAGCSMSTSSEGAQSATTTPEQAFDSLFSFNSSTSDVSQSMSVDENNANFTAETSCLFQDESKPNLEGQVPLTLLEKWLFDDAAPHAHEDLIDMSLENTSVLF.

[0020] Specific Example 2: pART-CAM in peach fruit PpMYB60- Construction of GFP recombinant vector.

[0021] Step 1: Linearization of pART-CAM-GFP vector The empty vector pART-CAM-GFP was digested with the restriction endonuclease XhoI. The digestion system was as follows: 1 μL XhoI, 2 μL FD buffer, X μL pART-CAM-GFP plasmid (calculated based on 1 μg plasmid), and RNase-free ddH2O to a final volume of 20 μL. The digestion was performed at 37℃ for 20 min and then at 80℃ for 5 min. The PCR products were then subjected to agarose gel electrophoresis. The vector linearization results are shown below. Figure 1 As shown in (B).

[0022] Step 2: Purification of cloning and enzyme digestion products The gel was recovered and purified using the EZNAGel Extraction Kit (D2500, Omega). The specific operating procedures were performed according to the instruction manual.

[0023] Step 3: Connect the target fragment to the linearized vector The pART-CAM-GFP linearized vector and the target gene PpMYB60 fragment obtained in Example 1 were ligated using recombinase. Recombination ligation was performed according to the kit (C116, Vazyme) instructions, with the vector (0.03 pmol) and the insert fragment (0.06 pmol) to obtain the recombinant product.

[0024] Step 4: Transform the recombinant product into E. coli. 100 μL of competent *E. coli* cells were thawed on ice. The recombinant product obtained in step 3 was added to the competent *E. coli* cells. After centrifugation, ice bath, and heat shock, the recombinant product was transferred into *E. coli* DH5α competent cells. Subsequently, the bacterial culture was plated onto LB agar medium supplemented with 25 mg / L streptomycin and incubated upside down at 37°C for 14–16 h. Single colonies of *E. coli* with round shape and bright white color were selected for colony PCR identification. The results are as follows: Figure 1 As shown in (C), the band position is correct. After selecting positive clones and verifying their accuracy through sequencing, the correct recombinant plasmid pART-CAM- was obtained. PpMYB60 -GFP.

[0025] Step 5: Transformation of Agrobacterium and colony PCR identification The extracted recombinant plasmid pART-CAM- PpMYB60 -GFP and the empty vector pART-CAM-GFP were mixed with 50 μL of Agrobacterium tumefaciens competent cells (pSoup), and the mixture was gently stirred at the bottom of the tube. The mixture was then incubated sequentially on ice for 5 min, in liquid nitrogen for 5 min, in a 37°C water bath for 5 min, and then cooled on ice for 5 min. Antibiotic-free LB broth was then added, and the cells were cultured in a shaker at 28°C for 2 h to activate them. 100 µL of the bacterial culture was spread onto LB solid medium (containing 100 mg / L spectinomycin, 20 mg / L rifampin, and 40 mg / L gentamicin), and incubated upside down at 28°C for 3 days. Positive colonies were picked for colony PCR identification. The results are as follows: Figure 1 As shown in (D), the bands are correctly positioned. Further culture in LB liquid medium (containing 100 mg / L spectinomycin, 20 mg / L rifampin, and 40 mg / L gentamicin) yielded the corresponding bands with pART-CAM- PpMYB60 Agrobacterium-1, GFP and pART-CAM-GFP recombinant plasmids.

[0026] Specific Example 3: Subcellular localization of the PpMYB60 gene in peach fruit.

[0027] Step 1: Preparation of Agrobacterium infection solution Will carry pART-CAM-GFP and pART-CAM- PpMYB60Agrobacterium-mediated transformation of the GFP recombinant plasmid was followed by amplification culture to OD200. 600 Value = 0.8-1.0. Centrifuge the bacterial culture at 5000×g for 10 min, discard the supernatant, resuspend the bacterial cells in freshly prepared infection buffer (containing 10 mM 2-morpholinoethanesulfonic acid (MES), 10 mM MgCl2, and 200 μM acetylsuccinone (AS)), and adjust OD. 600 Approximately 0.75, after standing in the dark for 1 h, it was used. Agrobacterium carrying the empty subcellular localization plasmid pART-CAM-GFP was used as the control group. The recombinant plasmid pART-CAM-GFP carrying the target gene was used... PpMYB60 Agrobacterium with GFP was used as the experimental group.

[0028] Step 2: Tobacco injection and observation using laser confocal microscopy Using a 1 ml sterile syringe, aspirate the Agrobacterium tumefaciens permeate solution prepared in step 1 and inject it into leaves of 1-month-old Nicotiana benthamiana plants. Inject 3-4 leaves per plant (the 3rd-6th leaves in the order of growth). Incubate in darkness for 1 day, then transfer to a normal light environment (24℃ / 16 h light, 18℃ / 8 h darkness). Two days later, using a 1 cm diameter punch, collect tobacco leaf tissue from the injection area. After removing the air from the leaves with a syringe, place the tobacco leaves on a glass slide and observe them using a laser confocal microscope. The excitation wavelength for green fluorescent protein (GFP) is 488 nm, and the excitation wavelength for chlorophyll is 633 nm.

[0029] The subcellular localization results of PpMYB60 in tobacco leaves are as follows: Figure 2 As shown, GFP fluorescence in the control group (pART-CAM-GFP) was observed in the cell nucleus, cell membrane, and cytoplasm, while in the experimental group (pART-CAM-... PpMYB60 Strong green fluorescence signals were observed only in the nucleus of leaf cells containing PpMYB60 (-GFP), indicating that PpMYB60 is mainly located in the nucleus.

[0030] Specific Implementation Example 4: Peach Fruit PpMYB60 Genes are instantaneously transformed into peach fruits.

[0031] Will carry pART-CAM-GFP and pART-CAM- PpMYB60Agrobacterium-mediated transformation of the GFP recombinant plasmid was streaked onto LB solid medium (containing 100 mg / L spectinomycin, 20 mg / L rifampin, and 40 mg / L gentamicin) and incubated at 28°C for 3 days for the first activation. The bacteria were then scraped onto LB liquid medium (containing the corresponding antibiotics) and cultured overnight with shaking for the second activation. A suitable amount of fresh bacterial culture was added to fresh LB liquid medium (containing 100 mg / L spectinomycin, 20 mg / L rifampin, and 40 mg / L gentamicin) and cultured at 28°C with shaking at 220 rpm until OD (out of control) was reached. 600 Value = 0.8-1.0. Centrifuge the bacterial suspension at 5000×g for 10 min, discard the supernatant, resuspend Agrobacterium in freshly prepared infection buffer (containing 10 mM MES, 10 mM MgCl2, and 200 μM AS), and adjust OD value. 600 =3.0, and used after standing at room temperature in the dark for 1 h. Agrobacterium carrying the empty vector plasmid pART-CAM-GFP was used as the control group. The bacteria carrying the target gene recombinant plasmid pART-CAM-... PpMYB60 Agrobacterium with GFP was used as the experimental group.

[0032] Using green-ripe peaches as experimental material, 1 mL of prepared Agrobacterium tumefaciens permeation solution was drawn into the fruit using a sterile syringe. With the needle tip angled inwards towards the inside of the peel, the needle was inserted into the fruit at approximately a 20° angle and injected slowly until water droplets appeared on the surface. After drying, the fruit was placed in the dark at 25°C and 85±5% humidity. After 24 h, the fruit was placed under light to promote transformation, resulting in the transient transformation experimental group (pART-CAM-). PpMYB60 Peach fruits of the control group (pART-CAM-GFP) and the control group (pART-CAM-GFP).

[0033] Specific Example 5: Determination of wax components and content in instantaneously converted peach fruit.

[0034] Step 1: Extraction and determination of fruit wax Five biological replicates were set up, and each was drilled using a 2cm diameter circular punch to enlarge the transient overexpression isolate prepared in Specific Example 4. PpMYB60 The surface of the peaches in both the control and control groups was perforated, with four round perforations constituting one replicate. The surface area was calculated using the formula: S = πr². 2The wafer discs were immersed in chloroform for 1 min to extract the cuticle wax. 10 µL of n-tetracosane (1 mg / mL) was added as an internal standard. The mixture was concentrated to 1 mL under nitrogen, filtered through a 0.45 µm nylon filter, and dried under nitrogen. 100 µL of pyridine and 100 µL of N,O-bis(trimethylsilyl)trifluoroacetamide (containing 1 wt% trimethylchlorosilane) were added to the sample, and the mixture was derivatized in an 80℃ drying oven for 1 h. The residual derivatizing reagents were dried under nitrogen at room temperature. 1 mL of chloroform was added to dissolve the sample. The wax composition and content of peach peel were determined using GC-MS. The column model was HP-5MS, with dimensions of 30 m × 0.25 mm × 0.25 μm. An autosampler was used with an injection volume of 4 µL, an injection port temperature of 280℃, a detector temperature of 300℃, and a transfer line temperature of 320℃. Temperature program: Initial temperature 50℃, hold for 2 min, increase to 200℃ at a rate of 20℃ / min and hold for 2 min; then increase to 320℃ at a rate of 3℃ / min and hold for 15 min. Wax components were identified using the NIST 17 database, and wax content was quantified using peak area. Wax content was calculated using the following formula: Wax content (μg / cm) 2 = (Area of ​​peak of analyte / Area of ​​internal standard peak) × Amount of internal standard added.

[0035] Step 2: Analyze the wax components and content by GC-MS. The results are as follows Figure 3 China A Figure 3 China B and Figure 3 As shown in Figure C, overexpression was found. PpMYB60 It promoted the accumulation of wax in the epidermal cuticle, with a significant increase in the content of alkanes and triterpenes. Alkanes were predominantly C23 and C25, while α-amyrin was the dominant triterpenoid. These results indicate that the PpMYB60 transcription factor activates the biosynthesis of wax in peach fruit epidermis.

[0036] Specific Implementation Example Six: Overexpression PpMYB60 Effects on the microstructure of waxy crystals on peach fruit skin The transient overexpression prepared in Specific Example 4 PpMYB60 The peels of peaches from both the control and control groups were perforated using a sampler and then soaked in 3% (v / v) glutaraldehyde for storage. The treated samples were then frozen at -80°C for at least 1 hour, followed by freeze-drying for 48 hours to completely remove moisture. Afterward, the samples were fixed and sputtered with gold, and finally observed using a scanning electron microscope (SEM). The results are as follows: Figure 4 As shown in the SEM image, overexpression can be observed. PpMYB60This resulted in a denser distribution of cuticle crystals, exhibiting more layered wax crystals, and simultaneously promoted wax deposition on the peach fruit epidermis; in contrast, the CK group showed larger and sparser epidermal wax pores. These results confirm that... PpMYB60 It strengthens the physical barrier function of the cuticle layer of peach fruit epidermis.

[0037] Specific Implementation Example 7 PpMYB60 Permeability test of Arabidopsis thaliana overexpression.

[0038] Step 1, BG Plant express- PpMYB60 Vector construction and transformation of competent cells Using the cDNA from "Specific Example 1, Step 2, RNA reverse transcription into cDNA" as a template, and EcoRI and BamHI as restriction enzyme sites, the DNA was amplified using PhantaMax Super-Fidelity DNA Polymerase (P505, Vazyme) high-fidelity enzyme. PpMYB60 The upstream and downstream amplification primers for gene overexpression are as follows: BG Plant express- ​ -F: tactattctagtcgagaattcATGGGAAGGCCACCTTGC (SEQ ID NO.5); BG Plant express- ​ -R: caggtcgactctagaggatccTCAAAACAAGACAGATGTGTTCTCTAAT (SEQ ID NO. 6). Refer to "Specific Embodiment 1, Step 3", ​ The gene cloning method of "cloning the full length of the gene" was obtained. ​ Gene overexpression fragment. Agarose gel electrophoresis was performed on the amplification product, yielding a band approximately 1134 bp in size, as shown below. ​As shown in Figure A, the target band and brightness meet the requirements. The empty vector BG Plant express MCS was then digested with restriction endonucleases EcoRI and BamHI, following the same digestion system and PCR procedure as in "Specific Example 2, Step 1: Linearization of the pART-CAM-GFP Vector". After digestion, the PCR product was subjected to agarose gel electrophoresis and purified using the EZNAGel Extraction Kit (Omega), following the manufacturer's instructions. The target fragment was ligated to the linearized vector using the recombinant system described in "Specific Example 2, Step 3: Ligation of the Target Fragment and the Linearized Vector", yielding the recombinant product. Following the method described in "Specific Example 2, Step 4: Transformation of the Recombinant Product into E. coli", the obtained recombinant product was transformed into E. coli DH5, and the bacterial culture was then plated onto LB agar containing 100 mg / L spectinomycin. The results of E. coli colony PCR identification are shown. ​ As shown in Figure B, the band position is correct. The positive clone was sequenced for verification. BG Plant express extraction was performed. ​ The recombinant plasmid was used to transform Agrobacterium tumefaciens according to the method described in "Specific Example 2, Step 5: Transformation and Colony PCR Identification of Agrobacterium". The activated cells were then spread onto LB solid medium (containing 100 mg / L spectinomycin, 20 mg / L rifampin, and 40 mg / L gentamicin) and incubated upside down at 28°C for 2-3 days. Single colonies were picked for Agrobacterium tumefaciens colony PCR identification. The results are as follows: ​ As shown in Figure C, the band position is correct. Further culture in LB liquid medium (containing 100 mg / L spectinomycin, 20 mg / L rifampin, and 40 mg / L gentamicin) yielded the BG Plant express- ​ Agrobacterium recombinant plasmid.

[0039] Step 2: Preparation of transgenic Arabidopsis thaliana First, in a sterile laminar flow hood, Arabidopsis seeds were disinfected by soaking in a 75% (v / v) ethanol solution for 5 minutes, then rinsed three times with sterile water. They were then evenly sown on 1 / 2 MS solid medium (containing 50 mg / L kanamycin, pH 5.7-5.9) and vernalized at 4℃ for 2 days before being transferred to a light incubator (24℃ / 16 h light, 18℃ / 8 h darkness). After true leaves emerged in 9-12 days, the seeds were transplanted into nutrient soil. After about 3 weeks of cultivation, initial inflorescences appeared, and infection was carried out during the peak flowering period. BG Plantexpress- ​ Agrobacterium tumefaciens was activated by shaking culture to OD 600The OD value is approximately 0.6-0.8. Centrifuge at 5000×g for 10 min, collect the bacterial cells, resuspend the cells in freshly prepared infection solution (containing 5% sucrose and 0.025% (v / v) Silwet-77), and adjust the OD value of the bacterial solution. 600 The concentration is approximately 0.8-1.0. After standing in the dark for 1 hour, use the solution. Then, cut off the pods and fully open flowers from the Arabidopsis plants and water them thoroughly before infiltration to ensure the stomata are fully open. Completely immerse the flower buds in the bacterial solution and gently shake for 1 minute. After briefly drying, seal and maintain a high humidity environment. Culture in the dark for 1 day, then transfer to a normal culture environment (24℃ / 16 h light, 18℃ / 8 h dark). Seal for 2-3 days, then remove the sealed bag. Repeat the infection 2-3 times depending on the growth of the Arabidopsis. Harvest the seeds after the pods mature; this is the T0 generation.

[0040] Step 3 ​ Screening and identification of homozygous Arabidopsis thaliana plants overexpressing Arabidopsis thaliana The harvested Arabidopsis seeds were planted in 1 / 2 MS solid medium (containing 50 mg / L kanamycin, pH=5.7-5.9) to screen for positive plants up to the T3 generation for subsequent experiments.

[0041] Semi-quantitative PCR detection was used. ​ Expression in wild-type (WT) and transgenic Arabidopsis thaliana. Four-week-old Arabidopsis thaliana were freeze-ground into powder under liquid nitrogen. Total RNA was extracted from Arabidopsis leaves according to the method described in step 1 of Example 1, and cDNA was synthesized. Using Arabidopsis leaf cDNA as a template, ​ As a reference gene, the PCR products were electrophoresed on a 1% (w / v) agarose gel, and the band brightness was observed. ​ Quantitative upstream primers for genes (q ​ The sequence of -F) is shown in SEQ ID NO.7: GACCACCGTAACAGCCAAGA; ​ Quantitative downstream primers for genes (q) ​ The sequence of -R) is shown in SEQ ID NO.8: ACAGAGCCTCACAAAGAGCC; ​ Quantitative upstream primers for genes (q ​ The sequence of -F) is shown in SEQ ID NO.9: CATCAGGAAGGACTTGTACGG, ​ Quantitative downstream primers for genes (q) ​ The sequence of -R) is shown in SEQ ID NO.10: GATGGACCTGACTCGTCATAC. The agarose gel electrophoresis results are as follows: ​ As shown, wild-type Arabidopsis thaliana and ​ -OE-L4、​ -OE-L6 internal reference gene ​ The electrophoretic bands have similar brightness. For ​ Genes were almost undetectable in WT, while ​ -OE-L4 and ​ The two strains, -OE-L6 and -OE-L6, show distinct bands, indicating that... ​ -OE-L4 and ​ -OE-L6 material ​ Successful expression.

[0042] Step 4 ​ Permeability analysis of Arabidopsis thaliana overexpression Wild type and overexpression ​ Five biological replicates were set up for each Arabidopsis thaliana strain. Four-week-old Arabidopsis thaliana rosette leaves were immersed in a 1% (w / v) toluidine blue (TB) solution, ensuring that the fruit was completely submerged, for 15-20 minutes. After immersion, the surface residual liquid was rinsed off with sterile water, and the water permeability of the leaf epidermal cuticle was observed.

[0043] Experimental results are as follows ​ The results showed that the blue spots on the leaves of wild-type Arabidopsis thaliana soaked with TB covered a larger area and were significantly more numerous than those on wild-type Arabidopsis thaliana. ​ -OE-L4 and ​ -OE-L6 leaves, which indicates ​ Overexpression in Arabidopsis thaliana reduced the water permeability of the leaf epidermal cuticle.

[0044] Specific Implementation Example 8 ​ Dehydration test of Arabidopsis thaliana overexpression.

[0045] Four-week-old Arabidopsis thaliana plants were dehydrated for two days prior to the water loss experiment and placed in darkness for 24 hours to induce complete stomata closure. Rosette leaves of similar size were selected and cut from the petiole, with at least six leaves per plant line. The petioles were immediately sealed with paraffin to prevent water loss through the wound. The weight of freshly picked leaves was measured, followed by leaf weight measurements at 60, 120, 180, and 240 minutes in darkness at 25°C and 50% humidity. Leaf surface area was measured using ImageJ software after scanning the leaves flat, and the water loss per unit area was calculated. The calculation formula is as follows: Leaf water loss rate (mg / cm²). 2 = (M1-M2) / S, where M1: initial weight of the leaf; M2: weight of the leaf during storage; S: surface area of ​​the leaf.

[0046] Experimental results are as follows ​ As shown, ​ -OE-L4 and ​The detached leaves of -OE-L6 showed a significantly lower water loss rate than those of wild-type Arabidopsis thaliana, indicating that overexpression of -OE-L6... ​ It slowed down the water loss from Arabidopsis leaves.

[0047] The foregoing description is not intended to limit the invention, nor is the invention limited to the examples given. Any changes, modifications, additions, or substitutions made by those skilled in the art within the scope of the invention should also be considered within the protection scope of the invention.

Claims

1. A method for regulating the synthesis of wax on the skin of peach fruit. PpMYB60 Genes are characterized by: The aforementioned PpMYB60 The nucleotide sequence of the gene is shown in SEQ ID NO.

1.

2. The method for regulating the synthesis of wax on the skin of peach fruit according to claim 1 PpMYB60 Genes are characterized by: The amino acid sequence of the protein encoded by PpMYB60 is shown in SEQ ID NO.

2.

3. The method for regulating the synthesis of wax on the skin of peach fruit according to claim 1. PpMYB60 Genes are characterized by: The aforementioned PpMYB60 The nucleotide sequence of the upstream amplification primer is shown in SEQ ID NO.3: tttggagaggacacgctcgagATGGGAAGGCCACCTTGC. PpMYB60 The nucleotide sequence of the downstream amplification primer is shown in SEQ ID NO.4: gcccttgctcaccatctcgagAAACAAGACAGATGTGTTCTCTAATGAC.

4. A method according to claim 1 PpMYB60 The application of genes in regulating the synthesis of waxy substances in peach fruit epidermis is characterized by: The aforementioned PpMYB60 The nucleotide sequence of the gene is shown in SEQ ID NO.

1. This gene was expressed by overexpression in peach fruit. PpMYB60 The gene causes it to synthesize an increased amount of wax on the surface of peach fruit.

5. The application according to claim 4, characterized in that: The aforementioned PpMYB60 The nucleotide sequence of the upstream amplification primer for gene overexpression is shown in SEQ ID NO. 5: tactattctagtcgagaattcATGGGAAGGCCACCTTGC; PpMYB60 The nucleotide sequence of the downstream amplification primer for gene overexpression is shown in SEQ ID NO.6: caggtcgactctagaggatccTCAAAACAAGACAGATGTGTTCTCTAAT.

6. The application according to claim 4, characterized in that: Overexpression of the above in peach fruit epidermis PpMYB60 Methods for gene expression, including constructing the peach fruit PpMYB60 The steps include: overexpressing the gene using a vector, transforming the overexpression vector into Agrobacterium, and then infecting the peach peel.

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