Peach ERF transcription factor PpERF2 gene and application thereof

Abstract

The invention belongs to the field of plant molecular biology, and discloses a peach ERF transcription factor PpERF2 gene of peach pulp monosaccharide transporter protein PpERDL16 and application of the peach ERF transcription factor PpERF2 gene. The amino acid sequence of the protein coded by the gene is as shown in SEQ ID NO: 2. The invention proves that the gene can be combined with a monosaccharide transporter PpERDL16 promoter, and the accumulation of fructose is influenced by regulating the activity of the monosaccharide transporter PpERDL16 promoter. Further, an overexpression system of the gene is successfully constructed in peach pulp, and accumulation of fructose in the pulp can be effectively increased, so that the sweetness of the pulp is increased. Therefore, the sweetness of peach pulp can be improved through over-expression of the gene, and theoretical support is provided for cultivation of high-quality fruits.

Description

Technical Field

[0001] This invention belongs to the field of plant molecular biology, and in particular relates to the peach ERF transcription factor PpERF2 gene, which is a peach pulp monosaccharide transporter PpERDL16, and its application. Background Technology

[0002] The peach (Prumus persica (L.) Batsch.), originating in my country, belongs to the Rosaceae family, Prunus genus, and is a diploid fruit tree. The sweetness of a ripe peach mainly depends on the content of soluble sugars in its vacuoles. The ratio of soluble sugars and sugar alcohols varies significantly at different growth and development stages of the peach fruit. At maturity, the main soluble sugars in peaches are sucrose, glucose, fructose, and sorbitol. Fructose has the highest sweetness, being 1.73 times sweeter than sucrose and 2.34 times sweeter than glucose, making it a crucial factor influencing peach fruit quality. Monosaccharide transport proteins (ERDLs) located on the vacuolar membrane play a role in regulating the sugar content of the pulp. The PpERDL16 gene shows low expression levels in peaches with high fructose content; overexpression of the PpERDL16 gene significantly reduces the fructose content in the peach pulp, highlighting its important role in fruit quality formation.

[0003] The yeast one-hybrid (Y1H) system is a classic in vitro identification technique that translates DNA-protein interaction events into survival signals for yeast cells. A target DNA fragment is constructed upstream of a reporter gene as "bait," and a potential transcription factor is fused with its activation domain (AD). Once the transcription factor binds to the target DNA fragment, it activates the reporter gene, and the yeast cells grow positive plaques on a selection plate lacking a specific amino acid. The yeast one-hybrid system can effectively screen for proteins that regulate the expression level of the PpERDL16 gene.

[0004] Transient overexpression is a technique that induces temporary high expression of a target gene by introducing a specific vector, reflecting the gene's function. We artificially construct regulatory elements and add them upstream of the target gene, allowing for large-scale transcription and translation under controlled conditions to achieve overexpression of the gene product. Currently, Agrobacterium-mediated transient gene expression is simple, rapid, and widely used. However, there has been no successful application of transient overexpression of PpERF2 in peach pulp. Therefore, transient overexpression of the target gene PpERF2 in peach pulp using Agrobacterium transformation is particularly important for gene function analysis and application.

[0005] PpERF2 belongs to the AP2 / ERF transcription factor family. Members of this family are generally located in the cell nucleus and can specifically recognize and bind to the GCC-box (or DRE / CRT cis-acting element) of the promoter region of target genes, thereby activating or inhibiting the expression of a series of downstream functional genes. It plays a key "regulatory switch" role in the growth and development of peaches, especially in the process of fruit ripening and quality formation. Summary of the Invention

[0006] The technical problem to be solved by the present invention is to provide a peach ERF transcription factor PpERF2 gene, which is a monosaccharide transporter of peach pulp PpERDL16, and its application. The PpERF2 gene negatively regulates the monosaccharide transporter of peach pulp PpERDL16 and participates in sugar accumulation. The present invention discloses the PpERF2 gene, its cloning method, and its application.

[0007] The technical solution adopted by the present invention to solve the above-mentioned technical problems is as follows: This invention provides a peach ERF transcription factor PpERF2 gene, whose nucleotide sequence encodes the amino acid sequence shown in SEQ ID NO: 2, or has more than 99% identity with its nucleotide sequence, and still has the same function and is derived from a homologous gene of peach.

[0008] The present invention relates to a peach ERF transcription factor, which is a protein encoded by the peach ERF transcription factor PpERF2 gene.

[0009] Preferably, its amino acid sequence is as shown in SEQ ID NO: 2.

[0010] This invention provides an expression vector containing the PpERF2 gene, a type of ERF transcription factor from peach fruit, and a recombinant host cell.

[0011] This invention also provides a method for cloning the PpERF2 gene, an ERF transcription factor from peach fruit, comprising the following steps: (1) Total RNA was extracted from peach fruits and reverse transcribed into cDNA as a template; (2) Primers were designed based on the PpERF2 gene sequence: upstream primer sequence: 5`-ATGGACTCTAGTATTCTATGTCC-3`, downstream primer sequence: 5`-GAGGACAACAAGGGGGTCTG-3`; (3) PCR amplification: The amplified product of the PpERF2 gene of peach fruit ERF transcription factor was obtained by PCR amplification.

[0012] The present invention also provides the application of the peach fruit ERF transcription factor PpERF2 gene in the preparation of an activity promoter of peach pulp monosaccharide transporter PpERDL16.

[0013] The present invention further provides the application of the aforementioned peach fruit ERF transcription factor PpERF2 gene in the cultivation of peach varieties with PpERF2 gene overexpression.

[0014] The present invention further provides the application of the PpERF2 gene, a type of ERF transcription factor in peach fruit, in the breeding of peach varieties with improved flesh quality, wherein the improvement of peach flesh quality is to increase the sweetness of peach flesh.

[0015] This invention provides the application of the PpERF2 gene and / or its encoded protein in regulating the synthesis of the monosaccharide transporter PpERDL16.

[0016] The present invention also provides a method for regulating the synthesis of monosaccharide transporter PpERDL16 in plants, the method comprising overexpressing the PpERF2 gene in plants to reduce the synthesis of monosaccharide transporter PpERDL16, or knocking out the PpERF2 gene or reducing the expression of the PpERF2 gene in plants to increase the synthesis of monosaccharide transporter PpERDL16.

[0017] The present invention also provides the application of the PpERF2 gene and / or the protein encoded by it in improving the quality of plant fruit pulp, preferably, the improvement of plant fruit pulp quality is to change the sweetness of plant fruit pulp.

[0018] The present invention also provides the application of the PpERF2 gene and / or the protein encoded therein in the breeding of plant varieties in which the flesh quality is improved compared to wild-type plants, preferably wherein the improvement in flesh quality is an alteration of the sweetness of the plant flesh.

[0019] The present invention also provides a method for regulating the quality of plant pulp or cultivating plant varieties whose pulp quality can be altered. The method includes changing the expression of the PpERF2 gene. Preferably, the PpERF2 gene is overexpressed in the plant to increase the sweetness of the plant pulp, or the PpERF2 gene is knocked out or its expression is weakened in the plant to reduce the sweetness of the plant pulp, thereby providing plant fruits that are more suitable for sugar control to meet the needs of different groups of people.

[0020] In the application or method described above, the plant is a peach or other plant with the PpERF2 gene.

[0021] In the applications or methods described above, the amino acid sequence of the protein encoded by the PpERF2 gene is as shown in SEQ ID No. 2, or has 85% or 90% or more of the same amino acid sequence as shown in SEQ ID No. 2 and has the same function.

[0022] As described in the above-described applications or methods, the full-length coding sequence of the PpERF2 gene is shown in SEQ ID No. 1.

[0023] The present invention also provides a PpERF2 gene, wherein the amino acid sequence of the protein encoded by the PpERF2 gene is as shown in SEQ ID No. 2, or has 85%, 90%, 95%, 98%, or 99% or more of the same amino acid sequence as shown in SEQ ID No. 2 and has the same function.

[0024] The full-length coding sequence of the PpERF2 gene, as described above, is shown in SEQ ID No. 1.

[0025] This invention confirms that the peach fruit ERF transcription factor PpERF2 gene can bind to the promoter of the monosaccharide transporter PpERDL16 and affect fructose accumulation by regulating its activity. Furthermore, this invention successfully constructed an overexpression system of the PpERF2 gene in peach pulp, effectively increasing the expression level of PpERF2 in the pulp. This effectively inhibited the activity of the monosaccharide transporter PpERDL16, increasing fructose accumulation in the pulp and thus increasing the sweetness of the pulp. In summary, this invention provides a key gene, PpERF2, that regulates the monosaccharide transporter PpERDL16 in peach fruit. It confirms the protein-DNA interaction between PpERF2 and the PpERDL16 promoter, and dual-luciferase assays confirm that PpERF2 negatively regulates the transcriptional activity of PpERDL16. This indicates that the PpERF2 gene has an effective function in the PpERDL16-mediated fructose transport process. Therefore, overexpressing this gene can improve the quality of peach pulp, providing an important theoretical basis for cultivating high-quality fruit and showing broad application prospects. Attached Figure Description

[0026] Figure 1 Correlation analysis of PpERF2 and PpERDL16 gene expression levels; Figure 2 To confirm that PpERF2 can bind to the promoter of the PpERDL16 gene in the Y1H system; Figure 3 The results of dual-luciferase assays verified the inhibition of PpERF2 on the transcriptional activity of PpERDL16. Figure 4To investigate the effects of Agrobacterium-mediated transient transformation on the expression of PpERF2 in peach pulp, thereby inhibiting the expression of the PpERDL16 gene and promoting fructose accumulation in the pulp. (A) Analysis of PpERF2 expression level after Agrobacterium-mediated transient transformation; (B) Analysis of PpERDL16 expression level after Agrobacterium-mediated transient transformation; (C) Effect of successful Agrobacterium-mediated transient transformation on fructose accumulation in the pulp. Detailed Implementation

[0027] The present invention will be further described in detail below with reference to the accompanying drawings and embodiments.

[0028] Example 1: Discovery of the Peach ERF-like Transcription Factor PpERF2 Gene RNA-seq data from nine time points from the initial flowering stage to the early fruit ripening stage of peach were used to analyze the correlation between the expression levels of transcription factors PpERF2 and PpERDL16. The results showed a significant negative correlation between the expression levels of the two genes. Figure 1 The expression levels of the two genes in samples at different time points were detected by qRT-PCR experiments, validating the results of the transcriptome data correlation analysis. Figure 1 (B)

[0029] Example 2: Cloning and Sequence Analysis of Peach ERF Transcription Factor PpERF2 Gene 1. Total RNA was extracted from the fruit of the "Longhua Honey" peach and reverse-transcribed into cDNA as a PCR template. Specifically: RNA was extracted from the peach pulp using a plant RNA extraction kit (Accurate Biology AG21019), and the concentration was measured using a Nanodrop 2000 micro spectrophotometer. Reverse transcription was performed using a reverse transcription kit (Accurate Biology AG11745), transcribing 1 μg of RNA to obtain cDNA, which was then used as a template for the PCR reaction. 2. Using the online website NCBI-PRIMER (https: / / www.ncbi.nlm.nih.gov / tools / primer-blast / ), specific amplification primers for the CDS region of the peach PpERF2 gene (Gene ID: Pp.LH.02G04640) were designed. The upstream primer sequence was 5`-ATGGACTCTAGTATTCTATGTCC-3`, and the downstream primer sequence was 5`-GAGGACAACAAGGGGGTCTG-3`. 3. PCR Amplification: The PpERF2 gene amplification product was obtained by PCR amplification. The 50μL reaction system for PCR amplification consisted of: 25μL of 2×PhantaMax Master Mix, 2μL each of forward and reverse primers, and 2μL of template cDNA. The PCR amplification program was as follows: pre-denaturation: 95℃ for 3 min; denaturation: 95℃ for 15 sec, annealing: 56℃ for 15 s, extension: 72℃ for 2 min, 30 cycles; final extension: 72℃ for 5 min. 4. Colony PCR and Sequencing Comparison: After PCR amplification, the product was recovered, purified, and ligated into the pBM23 vector. This vector was then transformed into *E. coli* DH5α (Sangon Biotech B528413), plated on LB-Amp plates, and incubated overnight at 37°C. After PCR gene verification, positive clones were sent to Beijing Biotechnology Co., Ltd. for sequencing, yielding the PpERF2 sequence (SEQ ID NO: 1) matching the peach genome data.

[0030] The PpERF2 gene, a transcription factor for peach ERF, encodes a protein with the following amino acid sequence (SEQ ID NO: 2): MDSSILCPIKYTEHRNTTKKLTKPRKKPTTESTSSSIPRIVRISMTDPDATDSSSGEEDEFFGRQRVKHYVDEIIIETDFRNTLVLPNNGRKRSTVQPPARRRTVKNSVSQTNGSRKFRGVRQRPWGKWAAEIRDPARRVRLWLGTFETAEEAAMVYDNAAIKLRGPDALTNFV SPLPKEEEKPELPVEPLIEKAEPVIESLESHNNLSSPTSVLHFRTQPKMEEAHEPQKLQVFDQVMQECEDETCTNFVSDDLGNYLPLDLPYLDDVFSFPAPDSPLFFDTPMFFDDDATAAAATLPECLLKEDFSDMFRDTYDTSLSPTTSSMCQGGEDYFQDILFGSDPLVVL.

[0031] Example 3: Using the yeast single-hybrid system (Y1H), it was confirmed that PpERF2 can bind to the promoter of the PpERDL16 gene. 1. Construction and identification of the bait recombinant vector pAbAi-PpERDL16pro-A and the prey recombinant vector Pgadt7-PpERF2 containing the truncated PpERDL16 promoter (PpERDL16pro-A). The PpERDL16 promoter contains abundant transcription factor binding sites. Since the cloned PpERF2 promoter sequence is long and contains a potential PpERF2 binding site CCGCC at the front end of the promoter sequence, it is planned to truncate the PpERDL16 promoter, take the front part of the promoter and name it PpERDL16prp-A to verify whether it can bind to the PpERF2 transcription factor.

[0032] Specific amplification primers for the promoter truncated versions of the PpERF2 and PpERDL16 genes in peach fruit (Gene IDs Pp.LH.02G04640 and Pp.LH.01G01754, respectively) were designed using the online website NCBI-PRIMER (https: / / www.ncbi.nlm.nih.gov / tools / primer-blast / ). The primers were equipped with appropriate restriction enzyme sites and protective bases at both ends (Table 1).

[0033] Table 1. Primer sequences for constructing the recombinant vectors Pgadt7-PpERF2 and pAbAi-PpERDL16pro-A

[0034] Note: Bold text indicates enzyme cleavage sites: KpnI, SA1I, EcoRI in that order.

[0035] All PCR amplification reaction volumes were 50 μL. The reaction volume consisted of: 25 μL of 2×Phanta Max Master Mix, 2 μL each of forward and reverse primers, and 2 μL of template cDNA. The PCR amplification program for PpERF2 and PpERDL16pro-A was as follows: pre-denaturation: 95℃ for 3 min; denaturation: 95℃ for 15 sec, annealing: 56℃ for 15 s, extension: 72℃ for 2 min, 35 cycles; final extension: 72℃ for 5 min.

[0036] After PCR amplification, the product was recovered, purified, and ligated into a pBM23 vector. This vector was then transformed into E. coli DH5α (Sangon Biotech B528413), plated onto LB-Amp plates, and incubated overnight at 37°C inverted. Positive clones identified by PCR and sequencing were sent to Beijing Biotechnology Co., Ltd. for sequencing. Plasmid extraction was performed according to the instructions of the high-purity plasmid DNA miniature extraction kit (PE707-200) from Beijing Jinsha Biotechnology Co., Ltd.

[0037] The extracted plasmids and the vectors to be ligated were double-digested with the corresponding restriction endonucleases and then recovered using a DNA gel extraction kit (GE706-200). The promoter cloning plasmid PpERDL16pro-A, purified by enzyme digestion, was ligated to the yeast one-hybrid bait expression vector pAbAi using the Uniclone one-step seamless cloning kit (SC612) from Beijing Jinsha Biotechnology Co., Ltd. The PpERF2 cloning plasmid was ligated to the yeast single-hybrid protein expression vector pGADT7 using the Uniclone one-step seamless cloning kit (SC612) from Beijing Jinsha Biotechnology Co., Ltd. The ligation method was in accordance with the instructions of the Uniclone one-step seamless cloning kit (SC612). Following the requirements of DH5aCompetent Cell (CWBIO, Beijing, China), recombinant plasmids were transformed into E. coli DH5a and evenly spread on LB-Amp / Kana plates. The plates were incubated overnight at 37°C with the plates inverted. Single clones were picked for colony PCR identification. Positive clones containing the target fragment were sent to Beijing Biotechnology Co., Ltd. for sequencing. After confirming the accuracy of the results, the bacterial culture was amplified and preserved, and plasmids were extracted to obtain the positive recombinant bait plasmid pAbAi-PpERDL16pro-A and the recombinant protein expression plasmid pGADT7-PpERF2, respectively.

[0038] 2. Detection of bait plasmid self-activation and interaction identification The yeast recombinant bait plasmid pAbAi-PpERDL16pro-A was linearized with BBSI restriction endonuclease. Y1H competent cells were prepared according to the Matchmaker Gold One-Hybrid System instructions, and the linearized pAbAi-PpERDL16pro-A vector was transformed into them. The optimal resistance concentration for the yeast bait strain AbA was screened according to the instructions. Competent cells of the obtained yeast bait strain Y1H [pAbAi-proERD16A] were prepared according to the Matchmaker Gold yeast one-hybrid system instructions. The yeast recombinant protein expression plasmid pGADT7-PpERF2 was transformed into these competent cells to obtain a yeast expression strain co-transformed with a promoter truncated and PpERF2 protein. The empty pGADT7 plasmid was transformed into the competent cells of the bait strain Y1H [pAbAi-proERD16A] as a negative control. Each co-transformed strain was plated on SD / -Leu / AbA* deficient medium (* represents the optimal AbA resistance background concentration for the bait strain) and incubated at 30°C for 3-5 days.

[0039] like Figure 2As shown, SD / -Leu / AbA 1000 Yeast strains containing pAbAi-PpERDL16pro-A and pGADT7-PpERF2 grew well on the defect-prone medium, while the negative control on SD / -Leu / AbA... 1000 No colonies grew on the defective culture medium, proving that PpERF2, as a transcription factor, can recognize the truncated promoter PpERDL16pro-A of the peach fruit monosaccharide transporter PpERDL16 and undergo DNA-protein interaction.

[0040] Example 4: Verification of the results of PpERF2 inhibiting the transcriptional activity of PpERDL16 using a dual-luciferase assay. 1. The PpERDL16 promoter (the first 2000 bp of the start codon ATG) was constructed into pGreen II800-LUC as a reporter vector, and the PpERF2 CDS sequence was constructed into pGreen II62-sk as a control and effector vector. Specific amplification primers for the PpERF2 and PpERDL16 gene promoters in peach fruit were designed using the online website NCBI-PRIMER (https: / / www.ncbi.nlm.nih.gov / tools / primer-blast / ). Appropriate restriction enzyme sites and vector homologous sequences were added to both ends of the primers (Table 2).

[0041] Table 2 Primer sequences for constructing the recombinant vectors pGreenII-62sk-PpERF2 and pGreenII800-PpERDL16pro

[0042] Note: Underlined lines indicate vector homology dummy columns; bold lines indicate restriction enzyme sites, in the following order: HindIII, BamHI, SalI.

[0043] All PCR amplification reaction volumes were 50 μL. The reaction volume consisted of: 25 μL of 2×Phanta Max Master Mix, 2 μL each of forward and reverse primers, and 2 μL of template cDNA. The PCR amplification program for the target fragment PpERF2 was: 95℃ for 3 min, 95℃ for 15 sec, 56℃ for 15 s, 72℃ for 2 min, and 72℃ for 5 min, with 35 cycles for steps 2 to 4. The PCR amplification program for the target fragment PpERDL16pro was: 95℃ for 3 min, 95℃ for 15 sec, 56℃ for 15 s, 72℃ for 2 min, and 72℃ for 5 min, with 30 cycles for steps 2 to 4. A BIO-RAD MJMINI PCR instrument was used.

[0044] Following the Uniclone one-step seamless cloning kit recombination reaction system, the amounts of each component used in the recombination reaction were calculated based on the concentrations of the recovered products from the vector and the target fragment, and the recombination reaction was completed. The PpERDL16 promoter was ligated into the expression vector pGreenII0800, and the recombination vector was named pGreenII0800-PpERDL16pro.

[0045] Following the Uniclone one-step seamless cloning kit recombination reaction system, the amounts of each component used in the recombination reaction were calculated based on the concentrations of the recovered products from the vector and the target fragment, and the recombination reaction was completed. PpERF2 was ligated into the expression vector pGreen-62-sk, and the recombination vector was named pGreenII-62sk-PpERF2.

[0046] 2. Dual-luciferase assay after Agrobacterium infection of tobacco. The recombinant vectors pGreenII0800-PpERDL16pro and pGreenII-62sk-PpERF2 were transformed into *Agrobacterium* zhGV3101Soup (CWBIO, Beijing, China) and cultured. The *Agrobacterium* were collected and resuspended in a prepared infection solution (containing Gen, MES, MgCl2, and AS), and the OD was adjusted. 600 =0.5. The pGreenII-62sk-ERF2 and pGreenII0800-PpERDL16pro infection solutions were mixed at a ratio of 1:1 as the experimental group, and the pGreen-sk and pGreenII0800-ERDL16 infection solutions were mixed at a ratio of 1:1 as the control group.

[0047] Tobacco seedlings approximately 30 days old were injected, with 3-5 leaves injected per plant, and 4 biological replicates per group. Infected tobacco plants were cultured normally for 72 hours before dual-luciferase assay. Samples were taken from the infection sites on the leaves and analyzed using the dual-luciferase reporter gene assay kit (RG027), following the kit instructions. With Renida luciferase as an internal control, the Rlu value obtained using firefly luciferase was divided by the Rlu value obtained using Renida luciferase. Figure 3 Based on the obtained ratio, we can determine that PpERF2 inhibits the transcriptional activity of PpERDL16.

[0048] Example 5: Transient overexpression of PpERF2 inhibits the expression of PpERDL16 and increases the fructose content of fruit pulp. Transient overexpression of PpERF2 in peach pulp via Agrobacterium-mediated transient transformation significantly inhibited the expression of PpERDL16 and increased the fructose content of the pulp.

[0049] The tested variety "Zhongyou 8" was harvested from an orchard in Pinggu District, Beijing. Peach fruits of uniform size, free from pests, diseases, and mechanical damage were selected and subjected to Agrobacterium infection.

[0050] 1. Recombinant vector pGreen0029-PpERF2 - Construction and identification of 62SK: The PpERF2 gene and specific amplification primers in peach fruit were designed using the online website NCBI-PRIMER (https: / / www.ncbi.nlm.nih.gov / tools / primer-blast / ). The primers were equipped with appropriate restriction sites and vector homologous sequences at both ends (Table 3).

[0051] Table 3 Construction of the recombinant vector pGreen0029-PpERF2 - 62SK primer sequence

[0052] Note: Underlined sequences indicate homologous sequences of the vector; bolded sequences indicate restriction enzyme sites, namely BamHI and SalI.

[0053] All PCR amplification reaction systems were 50 μL. The reaction system consisted of: 25 μL of 2×Phanta Max Master Mix, 2 μL each of forward and reverse primers, and 2 μL of template cDNA. The PCR amplification program for the target fragment PpERF2 was: 95℃ for 3 min, 95℃ for 15 sec, 56℃ for 15 s, 72℃ for 2 min, and 72℃ for 5 min. Steps 2 to 4 were performed for 30 cycles.

[0054] Following the Uniclone one-step seamless cloning kit recombination reaction system, the amounts of each component used in the recombination reaction were calculated based on the concentrations of the recovered products of the vector and the target fragment, and the recombination reaction was completed. PpERF2 was ligated into the expression vector pGreen0029. - 62SK, the recombinant vector was named pGreen0029-PpERF2 - 62SK.

[0055] According to the requirements of DH5a Competent Cell (CWBIO, Beijing, China), the linker solution pGreen0029-PpERF2 was used. -After transforming E. coil DH5α with 62SK, the cells were evenly spread on LB / kana plates and incubated overnight at 37°C upside down. Single clones were selected for colony PCR identification. Positive clones containing the target fragment were sent to Beijing Biotechnology Co., Ltd. for sequencing. The full-length PpERF2 sequence matching the peach fruit genome data was obtained.

[0056] 2. Recombinant plasmids were transformed into Agrobacterium and used to infect peach pulp. The constructed pGreen0029-PpERF2 was prepared using a freeze-thaw method. - The 62SK recombinant vector and the pGreen0029-62SK control vector were transformed into Agrobacterium GV3101, respectively. After activation on LB solid medium (Kana, 50 µg / ml; Rif, 50 µg / ml) at 28°C for 12-16 h, the cells were incubated on LB liquid medium (Kana, 50 µg / ml; Rif, 50 µg / ml; MES, 10 Mm; AS, 40 mM) at 28°C on a shaker at 200 rpm for 16-24 h until OD (Organic Growth Rate) was reached. 600 =0.8-1. Twelve undamaged peaches at near maturity were selected, cleaned, and four 1cm thick slices of peach flesh were cut from each fruit in a laminar flow hood and pre-cultured on MS medium at 25°C for 24 hours. The slices were then immersed in Agrobacterium infection solution containing the appropriate carrier in the laminar flow hood and placed under vacuum (-70 kPa) for 15 minutes. After vacuum penetration, the slices were rinsed 6-7 times with sterile water and then cultured on MS medium at 25°C for 48 hours. After culture, 0.32g-0.42g of peach flesh was placed in test tubes (for sugar content detection), and the remaining peach flesh tissue was wrapped in aluminum foil, rapidly frozen in liquid nitrogen, and stored at -80°C (for gene expression detection).

[0057] 3. Analysis of the basic expression levels of PpERF2 and PpERDL16 in peach pulp after transient transformation by Agrobacterium tumefaciens qPCR analysis showed that after peach pulp was infected and stored at 25℃ for 48 hours, pGreen0029-PpERF2 - The expression level of PpERF2 in the 62SK group was significantly higher than that in the control group pGreen0029-62SK. -The expression level of PpERDL16 in the 62SK group was significantly lower than that in the control group pGreen0029-62SK. Specifically, the gene expression level of PpERF2 was 3.6 times higher than that in the control group, while the gene expression level of PpERDL16 was 0.5 times lower. This indicates that the PpERF2 gene was effectively overexpressed in peach pulp. A system for overexpressing PpERF2 in peach pulp using Agrobacterium transient transformation was successfully constructed. Furthermore, the change in the expression level of the PpERDL16 gene in peach pulp after overexpression of PpERF2 further demonstrates that PpERF2 inhibits the transcriptional activity of PpERDL16.

[0058] 4. Detection of fructose content in peach pulp with transient overexpression of PpERF2 by Agrobacterium tumefaciens like Figure 4 As shown, compared to the control group, the fructose content in the PpERF2 overexpression group increased by 13.5%, indicating that effectively increasing PpERF2 in peach pulp can significantly increase the fructose content. Overexpression of the PpERF2 gene leads to a large accumulation of fructose, which is related to fruit sweetness, thereby increasing the sweetness of the pulp. The discovery of the PpERF2 gene provides a theoretical basis for obtaining high-quality peach varieties through molecular breeding techniques. Molecular breeding techniques can be used to overexpress the PpERF2 gene in the pulp to increase the fructose content of peach pulp, thus facilitating the selection of high-sweetness peach varieties.

[0059] 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. The application of a PpERF2 gene and / or its encoded protein in regulating the synthesis of the monosaccharide transporter PpERDL16, characterized in that, The applications include overexpressing the PpERF2 gene in plants to attenuate the synthesis of the monosaccharide transporter PpERDL16, or knocking out or attenuating the expression of the PpERF2 gene in plants to increase the synthesis of the monosaccharide transporter PpERDL16.

2. A method for regulating the synthesis of the monosaccharide transporter PpERDL16 in plants, characterized in that, The method includes overexpressing the PpERF2 gene in plants to attenuate the synthesis of the monosaccharide transporter PpERDL16, or knocking out the PpERF2 gene or attenuating the expression of the PpERF2 gene in plants to increase the synthesis of the monosaccharide transporter PpERDL16.

3. The application of a PpERF2 gene and / or its encoded protein in improving the quality of plant fruit pulp, preferably, the improvement of plant fruit pulp quality is to change the sweetness of plant fruit pulp.

4. The application of a PpERF2 gene and / or the protein encoded therein in the breeding of plant varieties with improved fruit flesh quality compared to wild-type plants, preferably wherein the improvement in fruit flesh quality is an alteration of the sweetness of the plant flesh.

5. A method for regulating the quality of plant pulp or cultivating plant varieties whose pulp quality can be altered, characterized in that, The method includes altering the expression of the PpERF2 gene, preferably by overexpressing the PpERF2 gene in plants to increase the sweetness of plant flesh, or by knocking out the PpERF2 gene or weakening the expression of the PpERF2 gene in plants to reduce the sweetness of plant flesh.

6. The application according to any one of claims 1, 3-4, or the method according to claim 2 or 5, characterized in that, The plant in question is a peach or another plant that possesses the PpERF2 gene.

7. The application or method according to claim 6, characterized in that, The amino acid sequence of the protein encoded by the PpERF2 gene is shown in SEQ ID No. 2, or has 85% or more, 90% or more, or 99% or more of the same amino acid sequence as shown in SEQ ID No. 2 and has the same function, especially a homologous gene derived from peach.

8. The application or method according to claim 6, characterized in that, The full-length coding sequence of the PpERF2 gene is shown in SEQ ID No.

1.

9. A PpERF2 gene, characterized in that, The amino acid sequence of the protein encoded by the PpERF2 gene is shown in SEQ ID No. 2, or has 85%, 90%, 95%, 98%, or 99% or more of the same amino acid sequence as shown in SEQ ID No. 2 and has the same function.

10. A PpERF2 gene, characterized in that, The full-length coding sequence of the PpERF2 gene is shown in SEQ ID No. 1.

Images