Peach nac transcription factor ppnac050 gene and application thereof

Abstract

The application belongs to the field of plant molecular biology, and discloses a peach NAC class transcription factor PpNAC050A gene of peach pulp monosaccharide transporter PpERDL16 and application of the gene. The amino acid sequence of the protein encoded by the gene is shown as SEQ ID NO: 2. The application proves that the gene can be combined with the monosaccharide transporter PpERDL16 promoter, and through regulating the activity, the accumulation of fructose is affected. Further, the overexpression system of the gene is successfully constructed in the peach pulp, the activity of the monosaccharide transporter PpERDL16 can be effectively inhibited, the accumulation of fructose in the pulp is increased, and the sweetness of the pulp is increased. Therefore, the quality of the peach pulp can be improved by overexpressing the gene, important theoretical basis for cultivating high-quality fruits is provided, and the application has a wide application prospect.

Description

Technical Field

[0001] This invention belongs to the field of plant molecular biology, and in particular relates to the peach NAC transcription factor PpNAC050A gene, which is a peach pulp monosaccharide transporter PpERDL16, and its application. Background Technology

[0002] Peach (Prumuspersica (L.) Batsch.) is a diploid fruit tree belonging to the Rosaceae family and the Prunus genus. Originating in my country, the sweetness of ripe peach fruit is primarily determined by the content of soluble sugars stored in vacuoles. Soluble sugars are a fundamental component of peach's edible quality, mainly contributing to its sweetness and being one of the main attributes influencing consumer satisfaction. Different proportions of soluble sugars in the fruit also result in different taste sensations for consumers. As peaches grow and develop, they accumulate different types of soluble sugars and sugar alcohols, primarily sucrose, glucose, fructose, and sorbitol. Fructose has the highest sweetness, being 1.73 times sweeter than sucrose and 2.34 times sweeter than glucose. Monosaccharide transporter proteins (ERDLs) located on the vacuolar membrane regulate 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 peach pulp, playing a crucial role in fruit quality formation.

[0003] The yeast two-hybrid system (Y2H) involves cloning (fusing) two proteins to be studied separately into the DNA-binding domain (DNA-BD) and transcription activation domain (AD) of a transcription activator (such as GAL4) on a yeast expression plasmid, constructing a fusion expression vector, and analyzing the interaction between the two proteins based on the expression product and colorimetric reaction. The yeast two-hybrid system can effectively screen for interacting proteins of PpERDL16.

[0004] Transient overexpression is a technique that uses a specific vector to induce a temporary high expression of a target gene, reflecting gene function and finding wide applications in promoter analysis, protein-protein interaction analysis, and gene function analysis. We artificially construct regulatory elements and add them upstream of the target gene, enabling the gene to be transcribed and translated 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 PpNAC050 in peach pulp. Therefore, transient overexpression of the target gene PpNAC050 in peach pulp using Agrobacterium transformation is particularly important for its gene function analysis and application.

[0005] The technical problem to be solved by the present invention is to provide a method and application for cloning the peach NAC transcription factor PpNAC050 gene, which positively regulates the monosaccharide transport protein PpERDL16 in peach pulp and participates in sugar accumulation.

[0006] The technical solution adopted by the present invention to solve the above-mentioned technical problems is as follows:

[0007] This invention provides a peach NAC transcription factor PpNAC050 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 NAC transcription factor, which is a protein encoded by the peach NAC transcription factor PpNAC050 gene.

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

[0010] This invention provides an expression vector containing the PpNAC050 gene, a NAC transcription factor from peach fruit, and a recombinant host cell.

[0011] This invention also provides a method for cloning the PpNAC050 gene, a NAC-type transcription factor from peach fruit, comprising the following steps:

[0012] (1) Total RNA was extracted from peach fruits and reverse transcribed into cDNA as a template;

[0013] (2) Primers were designed based on the PpNAC050 gene sequence: upstream primer sequence: 5`-ATGAGTATGGCCAATGCCATG-3`, downstream primer sequence: 5`-TCAAGAACTTTGTGGGAAAAGAGTCGC-3`;

[0014] (3) PCR amplification: The amplified product of the PpNAC050 gene of NAC transcription factor in peach fruit was obtained by PCR amplification.

[0015] The present invention also provides the application of the peach fruit NAC transcription factor PpNAC050 gene in the preparation of an activity promoter of peach pulp monosaccharide transporter protein PpERDL16.

[0016] The present invention further provides the application of the PpNAC050 gene, a NAC transcription factor from peach fruit, in the cultivation of peach varieties overexpressing the PpNAC050 gene.

[0017] This invention further provides the application of the aforementioned peach fruit NAC transcription factor PpNAC050 gene in the breeding of peach varieties with improved flesh quality. Preferably, the improvement in peach flesh quality is to increase the sweetness of the peach flesh.

[0018] This invention is the first to demonstrate that the peach fruit NAC transcription factor PpNAC050 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 PpNAC050 gene in peach pulp. Comparative analysis showed that peach pulp with significantly increased PpNAC050 expression effectively inhibited the activity of the monosaccharide transporter PpERDL16, increasing fructose accumulation in the pulp and thus enhancing the sweetness of the pulp. In summary, this invention provides a key gene, PpNAC050, that regulates the peach fruit monosaccharide transporter PpERDL16. It confirms the protein-DNA interaction between PpNAC050 and the PpERDL16 promoter, and dual-luciferase assays demonstrate that PpNAC050 negatively regulates the transcriptional activity of PpERDL16. This indicates that the PpNAC050 gene plays an effective role in the fructose transport mediated by PpERDL16. Therefore, overexpression of this gene can improve peach flesh quality, providing an important theoretical basis for cultivating high-quality fruit and showing broad application prospects. Attached Figure Description

[0019] Figure 1 To detect PpNAC050 gene expression during fruit development using qRT-PCR;

[0020] Figure 2 To confirm that PpNAC050 can bind to the promoter of the PpERDL16 gene in the Y1H system;

[0021] Figure 3 The results of dual-luciferase assays verified the inhibitory effect of PpNAC050 on the transcriptional activity of PpERDL16.

[0022] Figure 4 To suppress the expression of the PpERDL16 gene and the accumulation of fructose in peach pulp by overexpressing PpNAC050 in peach pulp using Agrobacterium transient transformation, the study included: (A) analysis of PpNAC050 expression level after Agrobacterium transient transformation; (B) analysis of PpERDL16 expression level after Agrobacterium transient transformation; and (C) the effect of successful Agrobacterium transient transformation on fructose accumulation in peach pulp. Detailed Implementation

[0023] The present invention will be further described in detail below with reference to the accompanying drawings and embodiments.

[0024] Example 1: Discovery of the Peach NAC-like Transcription Factor PpNAC050 Gene

[0025] Fruits from the peach (Prunus persica L.) cultivar “Ruiyoupan 1” at different developmental stages—the first rapid growth stage (S1), the pit hardening stage (S2), and the second rapid growth stage (S3)—were collected. Three fruits of uniform size, maturity, and free from disease, pests, and mechanical damage were selected from each stage. The transcriptional level of PpNAC050 during fruit development under normal peach growth conditions was analyzed using qRT-PCR. The results showed that PpNAC050 expression was significantly higher in the second rapid growth stage (S3) than in the previous two stages. This indicates that PpNAC050 may play an important role in the later stages of fruit development. Figure 1 ).

[0026] Example 2: Cloning and Sequence Analysis of Peach NAC Transcription Factor PpNAC050 Gene

[0027] 1. Total RNA was extracted from the fruit of the 'Ruiyoupan No. 1' peach and reverse-transcribed into cDNA as a PCR template. Specifically: RNA was extracted from 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.

[0028] 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 PpNAC050 gene (Gene ID:Prupe.7G014100) were designed. The upstream primer sequence was 5`-ATGAGTATGGCCAATGCCATG-3`, and the downstream primer sequence was 5`-TCAAGAACTTTGTGGGAAAAGAGTCGC-3`.

[0029] 3. PCR amplification: The PpNAC050 gene amplification product was obtained by PCR amplification. The 50 μL reaction system for PCR amplification was: 25 μL of 2×PhantaMax Master Mix, 2 μL each of forward and reverse primers, and 2 μL of template cDNA. The PCR amplification program was as follows: pre-denaturation: 95℃ for 3 min; denaturation: 95℃ for 15 sec, annealing: 58℃ for 15 s, extension: 72℃ for 2 min, 35 cycles; final extension: 72℃ for 5 min.

[0030] 4. Colony PCR and Sequencing Alignment: 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 PpNAC050 sequence (SEQ ID NO: 1) matching the peach genome data.

[0031]

[0032] The Peach NAC transcription factor PpNAC050 gene encodes a protein, the amino acid sequence of which is (SEQ ID NO: 2):

[0033] MFPDSPYQPRAWFSFSRPDYKYANSSRCNRATGKGFWKITGKPRQVKSRQLPKSVTCKKRTLTFHEGRVSKSRNTGWVRQEYYLTDPGSNPNQLSGFVLCRMKNKSTDNESDDKKQQDVSICVESADPGIAGCMASNSELDQAAGIHLIPEAEEHLTYKELEHVLLGSGNQNDGEPNGFVSSDFDDMIQELCAKGGEYLDSP SPPERPHQHQLPQLGNVPVTGDYVGSNSNNQAAAINHMIPHPEDFLTYKELEHVLGTCNPDAGEPGGCVSSNMLQKLCAQLGEELDSPIPPPGPPELGNATYVYTDECSSWPSPIGDNDSSLPNKNSIPTNYESKPVSYDSKPVSNTASNFENQTRISEVYSQAEENLESFFRQFELEGDFLHANNYIGCNESQSATLFPQSS.

[0034] Example 3: Using the yeast single-hybrid system (Y1H), it was confirmed that PpNAC050 can bind to the promoter of the PpERDL16 gene.

[0035] 1. Construction and identification of the bait recombinant vector pAbAi-PpERDL16pro-A and the prey recombinant vector Pgadt7-PpNAC050 containing the truncated PpERDL16 promoter (PpERDL16pro-A).

[0036] The PpERDL16 promoter contains abundant transcription factor binding sites. Since the cloned PpNAC050 promoter sequence is relatively long, and the front end of the promoter sequence contains a potential PpNAC050 binding site CACG, 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 PpNAC050 transcription factor.

[0037] Specific amplification primers for the promoter truncated versions of the PpNAC050 and PpERDL16 genes in peach fruit (Gene IDs: Proppe.7G014100 and Proppe.1G144800, 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 sites and protective bases at both ends (Table 1).

[0038] Table 1 Primer sequences for constructing the recombinant vectors Pgadt7-PpNAC050 and pAbAi-PpERDL16pro-A

[0039]

[0040] Note: Bold text indicates enzyme cleavage sites: KpnI, SA1I, EcoRI in that order.

[0041] The PCR amplification reaction system was 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 PpNAC050 and PpERDL16pro-A was as follows: pre-denaturation: 95℃ for 3 min; denaturation: 95℃ for 15 sec, annealing: 58℃ for 15 s, extension: 72℃ for 2 min, 35 cycles; final extension: 72℃ for 5 min.

[0042] 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 on LB-Amp plates, and incubated overnight at 37°C. 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.

[0043] The extracted plasmids and the vectors to be ligated were double-digested with the appropriate 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 PpNAC050 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). According to the requirements of DH5aCompetent Cell (CWBIO, Beijing, China), the 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-PpNAC050, respectively.

[0044] 2. Detection of bait plasmid self-activation and interaction identification

[0045] 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-PpNAC050 was transformed into these competent cells to obtain a yeast expression strain co-transformed with a promoter truncated and PpNAC050 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.

[0046] like Figure 1 As shown, SD / -Leu / AbA 600 Yeast strains containing pAbAi-PpERDL16pro-A and pGADT7-PpNAC050 grew well on the defect-specific medium, while the negative control on SD / -Leu / AbA... 600 No colonies grew on the defective culture medium, proving that PpNAC050, as a transcription factor, can recognize the truncated promoter PpERDL16pro-A of the peach fruit monosaccharide transporter PpERDL16 and undergo DNA-protein interaction.

[0047] Example 4: Verification of the results of PpNAC050 inhibiting the transcriptional activity of PpERDL16 using a dual-luciferase assay.

[0048] 1. The full-length PpERDL16 promoter was constructed into pGreen II800-LUC as a reporter vector, and the CDS sequence of PpNAC050 was constructed into pGreen II62-sk as a control and effector vector.

[0049] Primers for specific amplification of the promoters of the PpNAC050 and PpERDL16 genes in peach fruit were designed using the online website NCBI-PRIMER (https: / / www.ncbi.nlm.nih.gov / tools / primer-blast / ). Appropriate restriction sites and vector homologous sequences were added to both ends of the primers (Table 2).

[0050] Table 2 Primer sequences for constructing the recombinant vectors pGreen II62-sk-PpNAC050 and pGreen II800-PpERDL16pro

[0051]

[0052] Note: Underlined lines indicate vector homology dummy columns; bold lines indicate restriction enzyme sites, in the following order: HindIII, BamHI, SalI.

[0053] The PCR amplification reaction system consisted of 50 μL of 2×Phanta Max Master Mix (25 μL), forward and reverse primers (2 μL each), and template cDNA (2 μL). The PCR amplification program for the target fragment PpNAC050 was: 95℃ for 3 min, 95℃ for 15 sec, 58℃ for 15 s, 72℃ for 2 min, and 72℃ for 5 min, with 35 cycles for steps 2 through 4. The PCR amplification program for the target fragment PpERDL16pro was: 95℃ for 3 min, 95℃ for 15 sec, 58℃ for 15 s, 72℃ for 2 min, and 72℃ for 5 min, with 35 cycles for steps 2 through 4. The instrument used was a BIO-RAD MJMINI PCR instrument.

[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 from the vector and the target fragment, and the recombination reaction was completed. The PpERDL16 promoter was ligated into the expression vector pGreen II0800, and the recombination vector was named pGreen II0800-PpERDL16pro.

[0055] 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. PpNAC050 was ligated into the expression vector pGreen62-sk, and the recombination vector was named pGreenII-62-sk-PpNAC050.

[0056] 2. Dual-luciferase assay after Agrobacterium infection of tobacco.

[0057] The recombinant vectors pGreen II0800-PpERDL16pro and pGreenII-62-sk-PpNAC050 were dedicated to GV3101Soup (CWBIO, Beijing, China) Agrobacterium and cultured extensively. The Agrobacterium was collected and resuspended in a prepared infection solution (containing Gen, MES, MgCl2, and AS), and the OD600 was adjusted to 0.5. The infection solutions of pGreen II62-sk-NAC050 and pGreen II0800-PpERDL16pro were mixed at a 1:1 ratio as the experimental group, and the infection solutions of pGreen-sk and pGreenII 0800-ERDL16 were mixed at a 1:1 ratio as the control group.

[0058] 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 infected leaf sites by punching holes 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 2 Based on the obtained ratio, we can determine that PpNAC050 inhibits the transcriptional activity of PpERDL16.

[0059] Example 5: Transient overexpression of PpNAC050 inhibits the expression of PpERDL16 and increases the fructose and glucose content of fruit pulp.

[0060] Transient overexpression of PpNAC050 in peach pulp via Agrobacterium transient transformation significantly inhibited the expression of PpERDL16 and increased the fructose and glucose content of the pulp.

[0061] The tested variety "Wanmi" 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.

[0062] 1. Construction and identification of recombinant vector pGreen0029-PpNAC050-62SK: The PpNAC050 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).

[0063] Table 3 Primer sequences for constructing the recombinant vector pGreen0029-PpNAC050-62SK

[0064]

[0065] Note: Underlined lines indicate homologous sequences of the vector; lines indicate restriction enzyme sites, namely BamHI and SalI.

[0066] The PCR amplification reaction system consisted of 50 μL of the following reaction system: 25 μL of 2×Phanta Max Master Mix, 2 μL each of forward and reverse primers, 2 μL of template cDNA, and the PCR amplification program for the target fragment PpNAC050 was: 95℃ for 3 min, 95℃ for 15 sec, 58℃ for 15 s, 72℃ for 2 min, and 72℃ for 5 min. Steps 2 to 4 were performed for 35 cycles.

[0067] 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. PpNAC050 was ligated into the expression vector pGreen0029-62SK, and the recombination vector was named pGreen0029-PpNAC050-62SK.

[0068] Following the requirements of DH5a Competent Cell (CWBIO, Beijing, China), the ligation solution pGreen0029-PpNAC050-62SK was transformed into E. coil DH5a, and then evenly spread on LB / kana plates. The plates were incubated overnight at 37°C with the plates inverted. 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 sequence of PpNAC050, matching the peach fruit genome data, was obtained.

[0069] 2. Recombinant plasmids were transformed into Agrobacterium and used to infect peach pulp.

[0070] The constructed pGreen0029-PpNAC050-62SK recombinant vector and pGreen0029-62SK control vector were transformed into Agrobacterium GV3101 using a freeze-thaw method. The vectors were activated on LB solid medium (kana, 50 μg / ml; Rif, 50 μg / ml) at 28°C for 12–16 h, and then cultured 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 (digestive activity) was achieved. 600 =0.8-1. Twelve undamaged peaches from the S3 stage were selected. After cleaning, four 1cm thick slices of peach flesh were cut from each peeled peach in a laminar flow hood and pre-cultured on MS medium at 25℃ 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 (-70kPa) for 15 minutes. After vacuum penetration, the slices were rinsed 6-7 times with sterile water and then cultured on MS medium at 25℃ for 48 hours. After culture, 0.32g-0.42g of peach flesh was placed in test tubes (for sugar content detection). The remaining peach flesh tissue was wrapped in aluminum foil, rapidly frozen in liquid nitrogen, and stored at -80℃ (for gene expression detection).

[0071] 3. Analysis of the basic expression levels of PpNAC050 and PpERDL16 in peach pulp after transient transformation by Agrobacterium tumefaciens

[0072] qPCR analysis showed that after peach pulp was infected and stored at 25℃ for 48 h, the expression level of PpNAC050 in the pGreen0029-PpNAC050-62SK group was significantly higher than that in the control group pGreen0029-62SK. The expression level of PpERDL16 in the pGreen0029-PpNAC050-62SK group was significantly lower than that in the control group pGreen0029-62SK. Specifically, the gene expression level of PpNAC050 was 6.5 times higher than that in the control group, while the gene expression level of PpERDL16 was 0.3 times lower, indicating effective overexpression of the PpNAC050 gene in peach pulp. A system for overexpressing PpNAC050 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 PpNAC050 further demonstrates that PpNAC050 inhibits the transcriptional activity of PpERDL16.

[0073] 4. Detection of fructose and glucose content in peach pulp with transient overexpression of PpNAC050 by Agrobacterium tumefaciens

[0074] like Figure 4 As shown, compared to the control group, the experimental group overexpressing PpNAC050 showed a 25% increase in fructose and a 15% increase in glucose, indicating that effectively increasing PpNAC050 in peach pulp can significantly improve the fructose and glucose content. Overexpression of the PpNAC050 gene leads to a large accumulation of fructose and glucose, which are related to fruit sweetness, thereby increasing the sweetness of the pulp. The discovery of the PpNAC050 gene provides a theoretical basis for obtaining high-quality peach varieties through molecular breeding techniques. Molecular breeding techniques can be used to overexpress the PpNAC050 gene in the pulp, increasing the fructose and glucose content of peach pulp, thus facilitating the selection of high-sweetness peach varieties.

[0075] 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 the peach fruit NAC transcription factor PpNAC050 gene in breeding peach varieties with improved flesh quality, characterized in that... The amino acid sequence encoded by the PpNAC050 gene, a NAC transcription factor in peach fruit, is shown in SEQ ID NO: 2; the improvement of peach flesh quality is to increase the sweetness of peach flesh.

2. The application as described in claim 1, characterized in that, The nucleotide sequence of the peach fruit NAC transcription factor PpNAC050 gene is shown in SEQ ID NO: 1.

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