Application of pear transcription factor PbrDDI2 and PbrAPX14 genes in regulation and control of maturation and senescence of pear fruits

Abstract

The invention discloses an application of interaction of a pear transcription factor PbrDDI2 and a PbrAPX14 gene promoter in regulation and control of maturation and senescence of pear fruits. The nucleotide sequence of the pear transcription factor PbrDDI2 is as shown in SEQ ID NO.1 in a sequence table, and the amino acid sequence of the gene coded protein is as shown in SEQ ID NO.2. The invention discloses the transcription factor PbrDDI2 for regulating and controlling the PbrAPX14 gene expression of the pear fruit for the first time. The invention clarifies an action mechanism of interaction of cis-acting elements (TTTGTCG / CGACAAA) in PbrDDI2 and PbrAPX14 gene promoters in clearing endogenous H2O2 accumulation and further inhibiting synthesis of ethylene so as to inhibit fruit ripening and senescence, and provides a theoretical and practical basis for cultivating anti-senescence high-quality pear varieties.

Description

Technical Field

[0001] The invention belongs to the technical fields of molecular biology and genetic engineering, and particularly relates to an application of a pear transcription factor PbrDDI2 in inhibiting fruit ripening and senescence by regulating the expression of a key gene PbrAPX14 in the ascorbic acid-glutathione cycle (AsA-GSH) to eliminate endogenous H2O2 and regulate the synthesis of ethylene. Background Art

[0002] The ripening and senescence of climacteric fruits, such as tomatoes and pears, are developmental processes regulated by genetic programs, in which climacteric ethylene plays a central role. Ethylene is synthesized from methionine stepwise via the "System 2" pathway: first, S-adenosylmethionine synthase (SAMS) catalyzes the production of S-adenosylmethionine (SAM), which is then catalyzed by ACC synthase (ACS) and ACC oxidase (ACO) to complete the biosynthesis using ACC as an intermediate (Alba et al., 2005). Ethylene reacts with copper ions (Cu + ) synergistically binds to the ethylene receptor (ETR), inhibiting CTR1 kinase activity, triggering cleavage of the carboxyl terminus of EIN2 and its entry into the nucleus, thereby blocking EBF1 / 2-mediated ubiquitination and degradation of EIN3 / EIL1. Accumulated EIN3 / EIL1 further activates the transcription of downstream ethylene response factors (ERFs), driving changes in fruit quality and physiological and biochemical characteristics (Shan et al., 2022). Currently, multiple key ethylene biosynthesis genes (such as PbrACS1b and PbrACO54) and their upstream regulatory factors (such as PbrERF24 and PbrERF114) have been identified in the pear genome.

[0003] The effects of ethylene on fruit ripening are closely related to reactive oxygen species (ROS) metabolism. During storage, ethylene accumulation and ROS levels in 'Xingshui' pears increase synchronously, reaching a peak on the 12th day. Similarly, in 'Ailsa Craig' tomatoes, H2O2 and ethylene accumulate explosively during veraison and three days after veraison, respectively. H2O2 levels are significantly reduced in ethylene synthesis-deficient mutants (rin, nor, and Nr). The ethylene perception inhibitor 1-MCP simultaneously inhibits ethylene and ROS production, delaying fruit ripening, while ethephon treatment accelerates the process. In 'Tainong' mangoes, exogenous ethylene promotes, while 1-MCP inhibits, H2O2 accumulation, leading to differential ripening progression.

[0004] Plant cells eliminate H2O2 through the ascorbate-glutathione (AsA-GSH) cycle, which is widely distributed in subcellular organelles such as chloroplasts and mitochondria. Its core steps are as follows: APX reduces H2O2 to H2O using AsA, generating MDHA; MDHA is reduced to AsA or spontaneously converted to DHA by MDHAR; DHA is regenerated to AsA by DHAR (using GSH as a donor); and oxidized GSSG is reduced to GSH by GR using NAD(P)H. Overexpression of tomato APX genes inhibits H2O2 accumulation, while knockout of rice APX1 / 2 exacerbates oxidative stress. Fourteen PbrAPXs and eight PbrMDHARs, including genes involved in the AsA-GSH cycle, have been identified in the pear genome. Heterologous expression of PbDHAR2 enhances AsA regeneration and mitigates salt and chilling damage in tomatoes, suggesting that this cycle may participate in ethylene-dependent ripening by regulating H2O2 levels.

[0005] The metabolism of reactive oxygen species, particularly H2O2, is thought to play a crucial role in the ethylene-dependent ripening and senescence of horticultural fruits. Consistent with observations in 'Tainong' mango fruit with and without 1-MCP / ethephon treatment, a mutation in the ethylene receptor 3 (Nr) in 'Ailsa Craig' tomato also inhibits H2O2 production.

[0006] In horticultural crops, the activation or repression of downstream structural gene expression is regulated by transcription factors (TFs), which bind to cis-acting elements in gene promoters. For example, PbrbZIP15 in the 'Cuiguan' pear activates glucose isomerase expression by binding to the G-box of the PbrXylA1 promoter; PubZIP914 in the 'Nanguo' pear promotes ester synthesis by regulating PuLOX3.1 transcription; and PbrWRKY62 in the 'Dangshansuli' pear promotes proline accumulation by activating PbrADC1 expression. Furthermore, PbMYB5 enhances PbDHAR2 expression by binding to the MYB site (CAACTG) in its promoter, reducing H₂O₂ levels and cold sensitivity.

[0007] To date, the role of the AsA-GSH cycle in the ripening and senescence of 'Korla Fragrant Pear' and its regulatory mechanisms remain largely unexplained. This study identified and further validated the role of PbrAPX14 and its upstream regulator, PbrDDI2, in scavenging H2O2 in vivo, inhibiting fruit ripening and senescence by suppressing ethylene evolution, softening, and color development. Summary of the Invention

[0008] The object of the present invention is to provide an application of the PbrAPX14 gene or a biological material related to the PbrAPX14 gene in participating in the removal of H2O2 and regulating the synthesis of ethylene to regulate the ripening and aging of fruits.

[0009] Another object of the present invention is to provide an application of the transcription factor PbrDDI2 or a biological material related to PbrDDI2 in participating in the removal of H2O2 and regulating the synthesis of ethylene to regulate the ripening and aging of fruits.

[0010] Another object of the present invention is to provide a method for inhibiting fruit ripening and senescence. The transcription factor PbrDDI2 specifically binds to the cis-acting element TTTGTCG / CGACAAA in the promoter of the PbrAPX14 gene, promoting the transcription of the PbrAPX14 gene.

[0011] The purpose of the present invention is achieved through the following technical solutions:

[0012] In a first aspect, the present invention claims protection for at least one use of the PbrAPX14 gene or a biological material related to the PbrAPX14 gene in the following (a1) to (a6):

[0013] (a1) Regulate the accumulation of endogenous H2O2 in fruits;

[0014] (a2) preparing a product for regulating endogenous H2O2 accumulation in fruits;

[0015] (a3) Regulates ethylene synthesis in fruits;

[0016] (a4) preparing products for regulating ethylene synthesis in fruits;

[0017] (a5) regulating fruit ripening and senescence;

[0018] (a6) preparing products for regulating ripening and senescence of fruits;

[0019] The PbrAPX14 gene is any one of the following DNA molecules (b1)-(b4):

[0020] (b1) a DNA molecule whose coding region includes the nucleotide sequence shown in SEQ ID NO.1;

[0021] (b2) a DNA molecule with a nucleotide sequence as shown in SEQ ID NO. 1;

[0022] (b3) a DNA molecule that hybridizes under stringent conditions with the DNA sequence defined in (b1) or (b2) and encodes a protein with the same function;

[0023] (b4) A DNA molecule having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% homology to the DNA sequence defined in (b1) or (b2) and encoding a protein having the same function. In the present invention, the above 70% homology can be at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity.

[0024] The present invention found that the PbrAPX14 gene regulates fruit ripening and aging by participating in the removal of H2O2 and regulating the synthesis of ethylene.

[0025] Furthermore, the biological material related to the PbrAPX14 gene is at least one of (c1) to (c10):

[0026] (c1) PbrAPX14 protein encoded by the PbrAPX14 gene;

[0027] (c2) an expression cassette containing the PbrAPX14 gene;

[0028] (c3) a recombinant vector containing the PbrAPX14 gene, or a recombinant vector containing the expression cassette described in (c2);

[0029] (c4) a recombinant microorganism containing the PbrAPX14 gene, or a recombinant microorganism containing the expression cassette described in (c2), or a recombinant microorganism containing the recombinant vector described in (c3);

[0030] (c5) a transgenic plant cell line containing the PbrAPX14 gene, or a transgenic plant cell line containing the expression cassette described in (c2), or a transgenic plant cell line containing the recombinant vector described in (c3);

[0031] (c6) transgenic plant tissue containing the PbrAPX14 gene, or transgenic plant tissue containing the expression cassette described in (c2), or transgenic plant tissue containing the recombinant vector described in (c3);

[0032] (c7) a transgenic plant organ containing the PbrAPX14 gene, or a transgenic plant organ containing the expression cassette described in (c2), or a transgenic plant organ containing the recombinant vector described in (c3);

[0033] (c8) A transgenic plant containing the PbrAPX14 gene, or a transgenic plant containing the expression cassette described in (c2), or a transgenic plant containing the recombinant vector described in (c3).

[0034] (c9) tissue cultures produced from regenerable cells of the transgenic plant described in (c8);

[0035] (c10) Protoplasts produced from the tissue culture described in (c9).

[0036] Furthermore, the PbrAPX14 protein is the protein described in (d1) or (d2) or (d3) below:

[0037] (d1) a protein with an amino acid sequence as shown in SEQ ID NO. 2;

[0038] (d2) a protein derived from SEQ ID NO. 2 obtained by substituting and / or deleting and / or adding amino acid residues in the amino acid sequence of SEQ ID NO. 2;

[0039] (d3) A protein derived from SEQ ID NO. 2 having 80% or greater identity with the protein shown in (d1) or (d2). In the present invention, the 80% or greater identity may be at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity.

[0040] Furthermore, the above application is to overexpress the PbrAPX14 gene to eliminate endogenous H2O2 accumulation and thereby inhibit the synthesis of ethylene to inhibit the ripening and aging of the fruit; or to silence or inhibit the PbrAPX14 gene to promote endogenous H2O2 accumulation and thereby promote the synthesis of ethylene to accelerate the ripening and aging of the fruit.

[0041] In a second aspect, the present invention claims protection for at least one use of the transcription factor PbrDDI2 or a biomaterial related to the transcription factor PbrDDI2 in the following (a1)-(a6):

[0042] (a1) Regulate the accumulation of endogenous H2O2 in fruits;

[0043] (a2) preparing a product for regulating endogenous H2O2 accumulation in fruits;

[0044] (a3) Regulates ethylene synthesis in fruits;

[0045] (a4) preparing products for regulating ethylene synthesis in fruits;

[0046] (a5) regulating fruit ripening and senescence;

[0047] (a6) preparing products for regulating ripening and senescence of fruits;

[0048] The transcription factor PbrDDI2 is the protein described in (e1), (e2), or (e3):

[0049] (e1) a protein with an amino acid sequence as shown in SEQ ID NO. 4;

[0050] (e2) a protein derived from the amino acid sequence of SEQ ID NO. 4 obtained by substitution and / or deletion and / or addition of amino acid residues;

[0051] (e3) A protein derived from SEQ ID NO. 4 that is 80% or greater identical to the protein of (e1) or (e2). In the present invention, the 80% or greater identity may be at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity.

[0052] Furthermore, the biological material related to the transcription factor PbrDDI2 is at least one of (f1) to (f10):

[0053] (f1) gene PbrDDI2 encoding the transcription factor PbrDDI2;

[0054] (f2) an expression cassette containing the gene PbrDDI2 described in (f1);

[0055] (f3) a recombinant vector containing the gene PbrDDI2 described in (f1), or a recombinant vector containing the expression cassette described in (f2);

[0056] (f4) a recombinant microorganism containing the gene PbrDDI2 described in (f1), a recombinant microorganism containing the expression cassette described in (f2), or a recombinant microorganism containing the recombinant vector described in (f3);

[0057] (f5) a transgenic plant cell line containing the gene PbrDDI2 described in (f1), or a transgenic plant cell line containing the expression cassette described in (f2), or a transgenic plant cell line containing the recombinant vector described in (f3);

[0058] (f6) transgenic plant tissue containing the gene PbrDDI2 described in (f1), or transgenic plant tissue containing the expression cassette described in (f2), or transgenic plant tissue containing the recombinant vector described in (f3);

[0059] (f7) a transgenic plant organ containing the gene PbrDDI2 described in (f1), or a transgenic plant organ containing the expression cassette described in (f2), or a transgenic plant organ containing the recombinant vector described in (f3);

[0060] (f8) a transgenic plant containing the gene PbrDDI2 described in (f1), or a transgenic plant containing the expression cassette described in (f2), or a transgenic plant containing the recombinant vector described in (f3);

[0061] (f9) tissue cultures produced from regenerable cells of the transgenic plant described in (f8);

[0062] (f10) Protoplasts produced from the tissue culture described in (f9).

[0063] Furthermore, the gene PbrDDI2 encoding the transcription factor PbrDDI2 in (f1) is any one of the following DNA molecules (g1)-(g4):

[0064] (g1) a DNA molecule whose coding region includes the nucleotide sequence shown in SEQ ID NO. 3;

[0065] (g2) a DNA molecule with a nucleotide sequence as shown in SEQ ID NO. 3;

[0066] (g3) a DNA molecule that hybridizes with the DNA sequence defined in (g1) or (g2) under stringent conditions and encodes a protein with the same function;

[0067] (g4) A DNA molecule having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% homology to the DNA sequence defined in (g1) or (g2) and encoding a protein having the same function. In the present invention, the above 70% homology can be at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity.

[0068] Furthermore, the above application is that the transcription factor PbrDDI2 interacts with the aforementioned PbrAPX14 gene to regulate endogenous H2O2 accumulation and then regulate the synthesis of ethylene and then regulate the ripening and aging of the fruit; specifically, overexpressing the gene PbrDDI2 encoding the transcription factor PbrDDI2 or / and increasing the protein level or / and activity of the transcription factor PbrDDI2 to eliminate endogenous H2O2 accumulation and then inhibit the synthesis of ethylene to inhibit the ripening and aging of the fruit; or silencing or inhibiting the gene PbrDDI2 encoding the transcription factor PbrDDI2 or / and reducing the protein level or / and activity of the transcription factor PbrDDI2 to promote endogenous H2O2 accumulation and then promote the synthesis of ethylene to accelerate the ripening and aging of the fruit.

[0069] Furthermore, the transcription factor PbrDDI2 promotes the transcription of the PbrAPX14 gene by binding to the cis-acting element TTTGTCG / CGACAAA in the PbrAPX14 gene promoter, clearing H2O2 accumulation and thereby inhibiting ethylene synthesis to inhibit fruit ripening and aging; the nucleotide sequence of the PbrAPX14 gene promoter is shown in SEQ ID NO.5.

[0070] In a third aspect, the present invention claims a method for inhibiting fruit ripening and senescence, which comprises removing endogenous H2O2 accumulation and inhibiting ethylene synthesis to inhibit fruit ripening and senescence by using any one of the following methods (h1) to (h2) or a combination of two methods;

[0071] (h1) overexpressing the gene PbrDDI2 encoding the transcription factor PbrDDI2 and / or increasing the protein level and / or activity of the transcription factor PbrDDI2;

[0072] (h2) overexpressing the PbrAPX14 gene or / and increasing the protein level and / or activity of the PbrAPX14 protein encoded by the PbrAPX14 gene;

[0073] The nucleotide sequence of the gene PbrDDI2 encoding the transcription factor PbrDDI2 is shown in SEQ ID No.3; the amino acid sequence of the transcription factor PbrDDI2 is shown in SEQ ID No.4; the nucleotide sequence of the PbrAPX14 gene is shown in SEQ ID No.1; the amino acid sequence of the PbrAPX14 protein encoded by the PbrAPX14 gene is shown in SEQ ID No.2.

[0074] The fruit of the present invention is a fruit of a fruit tree of the Rosaceae family or a fruit of a vegetable of the Solanaceae family. The plant is a plant of the Rosaceae family or the Solanaceae family.

[0075] Research process of the technical solution of the present invention:

[0076] The study clarified the involvement of endogenous H2O2 levels in regulating the ethylene-dependent ripening and senescence of Korla Fragrant Pear fruit. Further analysis of metabolites, enzyme activity, ethylene content, and gene expression profiles in the AsA-GSH cycle during ripening and senescence identified PbrAPX14 as a gene that may regulate the ethylene-dependent ripening and senescence of Korla Fragrant Pear fruit through ethylene-mediated H2O2 accumulation.

[0077] The recombinant plasmid pBI221-PbrAPX14-GFP was constructed and transformed into Arabidopsis protoplasts. AtDHAR2-mcherry was used as a marker. Laser confocal microscopy revealed that PbrAPX14 was located in the cytoplasm.

[0078] The pCAMBIA1301-PbrAPX14-GFP overexpression vector and the pTRV2-PbrAPX14 silencing vector were constructed and transformed into Agrobacterium tumefaciens GV3101. Positive strains were collected and injected into pear peels. Separately, tomato explants were infected with the PbrAPX14 gene overexpression vector (pCAMBIA1301-PbrAPX14-GFP). Positive homozygous strains were obtained by hygromycin resistance testing and PCR analysis. Tomato fruits from the F2 generation of positive homozygous strains were harvested (35 days after anthesis), refrigerated at 4°C for 10 days, and then placed at 20°C for 7 days.

[0079] The relative expression level of the PbrAPX14 gene in pear peel tissue was detected using RT-qPCR technology. It was found that the expression level of this gene was significantly increased in the peel of the pear with transient overexpression of the PbrAPX14 gene, while the expression level of this gene was significantly decreased in the peel of the pear with transient silencing of PbrAPX14.

[0080] High-performance liquid chromatography (HPLC) revealed that APX activity was significantly increased in the peel tissue of pear and tomato fruits transiently overexpressing the PbrAPX14 gene, while the opposite phenomenon was observed in the peel of fruits in which the PbrAPX14 gene was transiently silenced. H2O2 in the cortical tissue was measured using the H2O2-1-Y detection kit (Suzhou Keming Biotechnology Co., Ltd., Suzhou, China). Fruit ethylene concentration was measured using a gas chromatograph (GC, HP 5890A, Hewlett-Packard, Avondale, PA) equipped with a GSQ column and a flame ionization detector (FID). Further studies revealed that overexpression of the PbrAPX14 gene significantly increased APX activity in pear and tomato fruits, reduced H2O2 content and ethylene concentration, and thus inhibited fruit ripening and senescence.

[0081] Transcriptome analysis revealed 456 differentially expressed TFs; 40 of these were strongly positively correlated with PbrAPX14 during cold storage in 'Korla Fragrant Pear' fruit (fold change ≥ 2.0, FDR < 0.05, correlation coefficient ≥ 0.8). Because Pbr006167.1 had the highest expression correlation with PbrAPX14, it was designated PbrDDI2 and further investigated. Using the PlantRegMap database, PbrDDI2 was shown to potentially interact with a cis-acting element within the PbrAPX14 promoter (a sequence 2000 bp upstream of the transcription start site).

[0082] The recombinant plasmid pSAK277-PbrDDI2 was constructed, and the reporter vectors (pGreenII0800-PbrAPX14pro-LUC (full-length promoter), pGreenII ... frag1 -LUC, pGreenII 0800-PbrAPX14pro frag2 -LUC, pGreenII 0800-PbrAPX14pro mut -LUC), were transformed into Agrobacterium GV3101 and GV3101 (psoup), respectively. Agrobacterium carrying pSAK277-PbrDDI2 and the reporter vector were co-infected with tobacco leaves, and the activities of firefly luciferase and Renilla luciferase were detected. It was found that the Luc / Ren ratio of tobacco leaves co-transformed with pSAK277-PbrDDI2&pGreenII 0800-PbrAPX14pro-LUC was significantly higher than that of the control, while the Luc / Ren ratio of tobacco leaves co-transformed with pSAK277-PbrDDI2&pGreenII 0800-PbrAPX14pro frag1 -LUC(or pSAK277-PbrDDI2&pGreenII 0800-PbrAPX14pro mut There was no significant difference in the Luc / Ren ratio between tobacco leaves co-transformed with WT-LUC and those of the control group.

[0083] The prey vector PbrDDI2-AD was constructed, and the bait vector (PbrAPX14pro-pAbAi) was constructed using a PbrAPX14 promoter fragment containing a wild-type binding element (core motif: TTTGTCG) and a mutant element (TTTGTCG→AAAAAAA). The Matchmaker Gold Yeast One-Hybrid Library Screening System was used to detect that PbrDDI2 could bind to the promoter fragment (PbrAPX14pro-pAbAi) containing the cis-acting element.

[0084] The recombinant plasmid pCAMBIA1301-PbrDDI2-GFP was constructed and transformed into pear callus. Pear callus carrying pCAMBIA1301-GFP served as a control. Chromatin immunoprecipitation-quantitative PCR (ChIP-qPCR) analysis of a PbrAPX14 promoter fragment containing a cis-acting element (TTTGTCG / CGACAAA) revealed that PbrDDI2 binds to the promoter fragment containing the cis-acting element.

[0085] Combined with further electrophoretic mobility shift assay (EMSA) results, it was concluded that PbrDDI2 activated the expression of PbrAPX14 gene by binding to the cis-acting element (TTTGTCG / CGACAAA) in the upstream promoter.

[0086] The recombinant plasmid pBI221-PbrDDI2-GFP was constructed and transformed into tobacco leaves and Arabidopsis protoplasts. AtH2B-mcherry was used as a nuclear marker. Laser confocal microscopy revealed that PbrDDI2 was located in the cell nucleus.

[0087] The pCAMBIA1301-PbrDDI2-GFP overexpression vector and the pTRV2-PbrDDI2 silencing vector were constructed and transformed into Agrobacterium tumefaciens GV3101. Positive strains were collected and injected into pear peels. Separately, tomato explants were infected with the PbrDDI2 overexpression vector (pCAMBIA1301-PbrDDI2-GFP). Positive homozygous strains were obtained by hygromycin resistance testing and PCR analysis. Tomato fruits from the F2 generation of positive homozygous strains were harvested (35 days after anthesis) and refrigerated at 4°C for 10 days followed by 7 days at 20°C.

[0088] The relative expression levels of PbrDDI2 and PbrAPX14 genes in pear peel tissue were detected using RT-qPCR technology. It was found that the expression levels of PbrDDI2 and PbrAPX14 genes in the peel of pears with transient overexpression of PbrDDI2 gene were significantly increased, while the expression levels of PbrDDI2 and PbrAPX14 genes in the peel of pears with transient silencing of PbrDDI2 gene were significantly decreased.

[0089] High-performance liquid chromatography (HPLC) revealed a significant increase in APX activity in the peel tissue of pear and tomato fruits transiently overexpressing the PbrDDI2 gene, while the opposite phenomenon was observed in the peel of fruits in which the PbrDDI2 gene was transiently silenced. Hydrogen ions (H2O2) in the cortical tissue were detected using a H2O2-1-Y detection kit (Suzhou Keming Biotechnology Co., Ltd., Suzhou, China). Fruit ethylene concentration was measured using a gas chromatograph (GC, HP5890A, Hewlett-Packard, Avondale, PA) equipped with a GSQ column and a flame ionization detector (FID). Further studies revealed that overexpression of the PbrDDI2 gene significantly increased APX activity in pear fruit and decreased H2O2 content and ethylene concentration, thereby inhibiting fruit ripening and senescence.

[0090] Compared with the prior art, the present invention has the following beneficial effects:

[0091] 1. For the first time, the transcription factor PbrDDI2 that regulates the expression of the pear PbrAPX14 gene was revealed.

[0092] 2. The present invention clarifies the mechanism by which the interaction between PbrDDI2 and the cis-acting element (TTTGTCG / CGACAAA) in the PbrAPX14 gene promoter clears endogenous H2O2 accumulation and thereby inhibits fruit ripening and aging, providing a theoretical and practical basis for the cultivation of high-quality anti-aging pear varieties. BRIEF DESCRIPTION OF THE DRAWINGS

[0093] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments.

[0094] Figure 1 Effects of 1-MCP treatment on the visual quality of 'Korla Fragrant Pear' during cold storage.

[0095] Figure 2 Effects of 1-MCP treatment on ethylene biosynthesis in 'Korla Fragrant Pear' during cold storage.

[0096] Among them, (a) metabolite content. (b) enzyme activity. (c) gene expression profile. Before 180 days of storage at 0℃, 'Kulla Fragrant Pear' pear fruits were randomly divided into two treatments: H2O (control) and 1.0μL L -11-MCP fumigation was performed for 24 h, and samples were collected every 60 days. Data are the means of three biological replicates. The differences were statistically significant (*p<0.05; **p<0.01; ***p<0.001) as determined by one-way analysis of variance.

[0097] Figure 3 This study aimed to investigate the effect of 1-MCP treatment on the content of metabolites in the AsA-GSH cycle during cold storage of 'Korla Fragrant Pear'.

[0098] Before storage at 0°C for 180 days, 'Korla Fragrant Pear' pear fruits were randomly divided into two treatments: H2O (control) and 1.0 μL L-11-MCP fumigation for 24 h. Samples were collected every 60 days. Data are the means of three biological replicates. Statistically significant differences were found (*p < 0.05; **p < 0.01; ***p < 0.001) as determined by one-way analysis of variance.

[0099] Figure 4 Effects of 1-MCP treatment on the enzyme activities of the AsA-GSH cycle during cold storage of 'Korla Fragrant Pear'.

[0100] Before storage at 0℃ for 180 days, the 'Korla Fragrant Pear' pear fruits were randomly divided into two treatments: H2O (control) and 1.0μLL - 1 1-MCP fumigation was performed for 24 h, and samples were collected every 60 days. Data are the means of three biological replicates. The differences were statistically significant (*p<0.05; **p<0.01; ***p<0.001) as determined by one-way analysis of variance.

[0101] Figure 5 Effects of 1-MCP treatment on the gene expression profile of AsA-GSH cycle in 'Korla Fragrant Pear' during cold storage.

[0102] The color scale represents the normalized log2 transformation (mean + 1), where red indicates high levels, blue indicates low levels, and white indicates intermediate levels.

[0103] Figure 6 The subcellular localization of PbrAPX14 was detected using a TCS SP5 confocal microscope (Leica Microsystems, Wetzlar, Germany).

[0104] Figure 7 To verify the function of PbrAPX14 gene in 'Korla Fragrant Pear' fruit.

[0105] (a) Transient overexpression of PbrAPX14. Overexpression (OE) pears were compared with 'Korla Fragrant Pear' fruit transformed with the empty vector pCAMBIA1301. (b) Transient silencing of PbrAPX14. 'Korla Fragrant Pear' fruit co-transformed with empty pTRV2 and pTRV1 served as controls for silenced pears. Each treatment had three biological replicates, with eight fruits per replicate. qRT-PCR was performed, setting the expression level of PbrAPX14 in control pears to 1.0. Data are the means of three biological replicates; statistically significant differences (*p < 0.05; **p < 0.01; ***p < 0.001) were determined by one-way analysis of variance.

[0106] Figure 8 To verify the function of PbrAPX14 gene in pear callus and tomato fruit.

[0107] (a) Functional validation of the PbrAPX14 gene in pear callus. (b) Functional validation of the PbrAPX14 gene in tomato fruit. qRT-PCR was performed, with the expression level of PbrAPX14 in the control pear set as 1.0. Data are the means of three biological replicates; statistically significant differences (*p < 0.05; **p < 0.01; ***p < 0.001) were determined by one-way analysis of variance.

[0108] Figure 9 The data are for the differentially expressed transcription factors (TFs) upstream of the PbrAPX14 gene.

[0109] (a) Venn diagram showing the number of differentially expressed TFs in each stage and their intersection with other stages. Differentially expressed TFs were identified based on the following criteria: fold change ≥ 2.0, paddj < 0.05. (b) Information on 456 differentially expressed TFs whose mRNA abundance was consistently higher or lower in 1-MCP-treated fruit. 'Kulla Fragrant Pear' pear fruit was randomly assigned to two treatments before being stored at 0°C for 180 days: H2O (control) and fumigated with 1.0 μL L-11-MCP for 24 hours. Fruit samples were collected every 60 days.

[0110] Figure 10 Expression profiles of 456 differentially expressed TFs in 'Korla Fragrant Pear' fruit under 1-MCP treatment and their correlation with PbrAPX14.

[0111] Data are from transcriptome analysis and represent the average of three biological replicates. The color scale represents the normalized log2 transformation (mean FPKM + 1), with red indicating high levels, blue indicating low levels, and white indicating moderate levels. Spearman correlations between different attributes are visualized in the heatmap, where red (or light red) lines indicate very strong (or strong) positive correlations, while blue (or light blue) lines indicate very strong (or strong) negative correlations.

[0112] Figure 11 Detailed information on the PbrIDD2 binding element in the PbrAPX14 promoter.

[0113] The PbrIDD2-binding element was identified from the PbrAPX14 promoter using the PlantRegMap database.

[0114] Figure 12 This is a dual-luciferase reporter gene assay (Dual-LUC).

[0115] The open reading frame (ORF) of PbrIDD2 was cloned into the pSAK277 vector, and the vectors containing different numbers of potential PbrIDD2 binding elements (TTTGTCG; PbrAPX14pro and PbrAPX14pro) were also cloned into the pSAK277 vector. frag1 ) or mutated binding elements (TTTGTCG→AAAAAAA, PbrAPX14pro mut ) were inserted into the pGreenII 0800-LUC reporter vector. Transformants co-transfected with empty pSAK277 and each reporter vector served as controls. Data are the mean of three biological replicates; statistically significant differences (*p < 0.05; **p < 0.01; ***p < 0.001) were determined by one-way analysis of variance.

[0116] Figure 13 Yeast one-hybrid (Y1H) analysis.

[0117] The PbrIDD2 ORF was constructed into the prey vector pGADT7, and the vector containing the wild-type PbrIDD2 binding element (TTTGTCG; PbrAPX14pro) or the mutant element (TTTGTCG→AAAAAA, PbrAPX14pro) was also constructed. mut A 200 bp PbrAPX14 promoter fragment of ) was inserted into the bait vector pAbAi. Yeast cells co-transfected with AD-p53 and p53-AbAi served as positive controls, and yeast cells co-transfected with the empty AD vector and each bait vector served as negative controls.

[0118] Figure 14 Chromatin immunoprecipitation PCR (ChIP-PCR) analysis.

[0119] Callus overexpressing the empty vector served as a negative control. Data are the means of three biological replicates; statistically significant differences (*p<0.05; **p<0.01; ***p<0.001) were determined by one-way ANOVA.

[0120] Figure 15 Electrophoretic mobility shift assay (EMSA)

[0121] An unlabeled PbrAPX14 promoter fragment containing the PbrIDD2 binding element was used as a competitive probe. "+" and "-" indicate the presence or absence of His-tagged protein, His-PbrIDD2 fusion protein, labeled probe, or competitive probe, respectively. The competitive probe concentration was 50x and 100x that of the labeled probe.

[0122] Figure 16 The subcellular localization of PbrIDD2 was shown. AtH2B-mcherry was used as a nuclear localization marker.

[0123] Fluorescence signals were detected using a TCS SP5 confocal microscope (Leica Microsystems, Wetzlar, Germany).

[0124] Figure 17 To verify the function of PbrIDD2 gene in 'Korla Fragrant Pear' fruit.

[0125] (a) Transient overexpression of PbrIDD2. Fruits of 'Korla Fragrant Pear' transformed with the empty pCAMBIA1301 vector served as the overexpression (OE) control. (b) Transient silencing of PbrIDD2. Fruits of 'Korla Fragrant Pear' cotransformed with the empty pTRV2 and pTRV1 vectors served as the silenced (SE) control. Three biological replicates were performed for each treatment, with eight fruits per replicate.

[0126] Figure 18 To verify the function of PbrIDD2 gene in pear callus and tomato fruit.

[0127] (a) Functional validation of the PbrIDD2 gene in pear callus. In qRT-PCR analysis, the expression level of each gene in the control callus was set to 1.0. (b) Functional validation of the PbrIDD2 gene in tomato fruit. In qRT-PCR analysis, the expression level of each gene in overexpression line 4 was set to 1.0.

[0128] Figure 19 The mechanism of action of the PbrDDI2-PbrAPX14 module during ethylene-dependent ripening and senescence of 'Korla Fragrant Pear' fruit. DETAILED DESCRIPTION

[0129] The present invention will be described in detail below in conjunction with specific embodiments. The examples are provided for a better understanding of the present invention, but are not intended to limit the present invention. The experimental methods in the following embodiments are all conventional methods, and the experimental reagents involved are all conventional biochemical reagents.

[0130] Example 1: Identification of the key gene PbrAPX14 involved in the AsA-GSH cycle and the fruit ripening and aging process

[0131] 1. Collection of Tissue Samples

[0132] In this study, the 'Korla Fragrant Pear' fruits of uniform size, free of mechanical damage and pests and diseases were used as experimental materials. The harvested 'Korla Fragrant Pear' (Alaer, Xinjiang, 2024) were randomly divided into H2O (control) and 1.0 μL L -1 Two treatments were conducted using 1-MCP fumigation for 24 hours, with three biological replicates per treatment and 60 fruits per replicate. After treatment, all samples were dried / ventilated, packed in plastic bags, and then stored at 0°C for 180 days. Sampling was performed every 60 days. At each sampling stage, the first layer of skin (below the peel) of five fruits per replicate was quickly removed with a sharp peeler, immersed in nitrogen liquid, broken into pieces, and then stored at -80°C for analysis of metabolites, enzyme activity, gene expression profiles, etc.; on the other hand, an additional eight fruits per replicate were used to measure ethylene release, firmness, color, etc.

[0133] 2. Ethylene content determination

[0134] Five fruits were placed at 20°C for 12 h, weighed, and then placed in a 2-L glass jar with a rubber septum lid. After incubation in the glass jar at 20°C for 30 min, 5.0 ml of headspace gas was withdrawn using a gas-tight syringe. Ethylene concentration was determined using a gas chromatograph (GC, HP 5890A, Hewlett Packard, Avondale, PA) equipped with a GSQ column and a flame ionization detector (FID). The flow rates of helium, hydrogen, and air were 10, 35, and 350 mL min, respectively. -1 The qualitative and quantitative analysis of ethylene was performed by comparing the retention time and peak area with those in gas standards.

[0135] 3. ACC Content Determination

[0136] 5.0 g of tissue was homogenized with 4 mL of 5% (w / v, g / 100 ml) sulfosalicylic acid. Oscillate at 4°C for 30 min, centrifuge at 4°C for 10 min, take 1.5 mL of the supernatant, and add 10 mmol L -1HgCl2 was mixed with NaOCl and saturated NaOH (2:1, v / v). The ACC content was calculated by monitoring the formation of ethylene in the headspace of the mixture using a gas chromatograph.

[0137] 4. Analysis of AsA-GSH Circulating Metabolites

[0138] H2O2 in cortical tissue was detected according to the instructions of the corresponding detection kit (H2O2-1-Y, Suzhou Keming Biotechnology Co., Ltd., Suzhou, China). Briefly, the samples were homogenized with extraction buffer and then centrifuged at 12000g for 20 min at 4°C, and the supernatant was collected for H2O2 determination.

[0139] AsA, GSH and GSSG were analyzed according to the method of López-Huertas and Palma (2020). 0.5 g of cortical tissue was homogenized in 2 mL of cooled HCl (0.1 M) and centrifuged at 12000 g for 20 min at 4 °C. After collecting the supernatant, the sample was loaded into an Oasis MAX solid phase extraction cartridge (3 cc / 60 mg) (Waters, Milford, USA), eluted with 2 mL of 5% (v / v) NH4OH, and then filtered through a 0.45 μm nylon filter for analysis. AsA, GSH and GSSG were determined using liquid chromatography-electrospray ionization / mass spectrometry (LC-ES / MS) equipped with a Quattro Micro triple quadrupole mass spectrometer. 20 mg L -1 After incubation in the dark for 15 min, total AsA (T-AsA) was detected by HPLC, and DHA content was calculated as the difference between T-AsA and AsA. On the other hand, total GSH (T-GSH) was calculated as the sum of GSH and GSSG.

[0140] 5. ACS and ACO Activity Assay

[0141] Ten grams of cortical tissue was homogenized, filtered, and centrifuged at 18,000 g for 20 minutes at 4°C. The supernatant was purified using a Sephadex G-25 column (pre-equilibrated with 5 mM Tricine buffer, pH 8.0, 1 mM DTT, and 2 μM pyridoxal 5-phosphate). 1.5 mL of the eluate was incubated with 200 mM Tricine buffer, pH 8.0, and 100 μM SAM at 25°C for 2 hours. The reaction was terminated by the addition of 100 mM HgCl₂. Finally, a 1 mL sample was mixed with 100 μL of NaOCl (2:1, v / v) and saturated NaOH. ACS activity was calculated by monitoring ethylene formation in the headspace of the mixture by gas chromatography.

[0142] A 10 g sample was homogenized, filtered, and centrifuged at 16,000 g for 20 min at 4°C. The supernatant was purified using a Sephadex G-25 column (pre-equilibrated with 20 mM Tris-HCl, pH 7.4, 10% (v / v) glycerol, 3 mM Na-AsA, and 1 mM DTT). 0.5 mL of the eluate was mixed with 10 M FeSO₄, 3 mM sodium bicarbonate, and 50 μM AC and incubated at 25°C for 20 min. Ethylene production in the headspace of the mixture was monitored by gas chromatography to calculate ACO activity.

[0143] 6. Enzyme Activity Assay

[0144] 0.2 g of sample was homogenized in the corresponding extraction buffer and then centrifuged at 50,000 g for 15 min. The supernatant was collected and assayed for ascorbate catalase, glutathione reductase, dehydroascorbate reductase, and dehydroascorbate reductase according to the respective assay kits (APX-1-W, GR-1-W, DHAR-1-W, and MDHAR-1-W, Suzhou Keming Biotechnology Co., Ltd., Suzhou, China). Protein concentration in the supernatant was determined using a bicinchoninic acid protein assay kit (A045-4, Nanjing Jiancheng Bioengineering Institute, Nanjing, China).

[0145] 7. Transcriptome Analysis

[0146] Total RNA was extracted from pear peel tissue using the EASYspin Plus Plant RNA Rapid Extraction Kit (RN3802, Aidelai, Beijing). Sample RNA purity, concentration, and integrity were assessed using a NanoDrop 2000 spectrophotometer (Thermo Scientific, USA) and an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). Transcriptome libraries were constructed using the NEB Next Ultra™ RNA Library Prep Kit for Illumina (E7770, NEB, USA). The constructed libraries were then sequenced using the Illumina NovaSeq 6000 sequencing platform. After removing low-quality reads, clean reads were obtained for subsequent data analysis. HISAT2 software was used for alignment with the Korla Fragrant Pear genome database (http: / / peargenome.njau.edu.cn / ), and gene expression levels (FPKM) were calculated. Transcriptome sequencing and analysis were performed by Biomike Biotechnology Co., Ltd. (Beijing, China).

[0147] 8. Screening of key candidate genes involved in the AsA-GSH cycle in fruit ripening and aging

[0148] Transcriptome annotation was used to identify genes involved in the AsA-GSH cycle. Furthermore, the expression profiles of genes involved in the AsA-GSH cycle during fruit ripening and senescence in the 'Korla Fragrant Pear' were analyzed, along with their correlations with H2O2, enzyme activity, and ethylene. This approach allowed the identification of genes involved in the AsA-GSH cycle during fruit ripening and senescence.

[0149] Experimental results: The visual quality of 'Korla Fragrant Pear' during cold storage is as follows: Figure 1 As shown in Figure 2, ethylene accumulation in 'Korla Fragrant Pear' pears during cold storage reached its peak on the 120th day ( Figure 2 Consistent with this, the transcripts of PbrACS1b and PbrACO54, the activities of ACO and ACS, and the content of ACC in their biosynthesis pathways all peaked at 60, 120, and 180 days. Meanwhile, the expression levels of their upstream regulatory factors, PbrERF24 and PbrERF114, also showed an upward trend throughout the period ( Figure 2 Exogenous 1-MCP fumigation significantly inhibited the mRNA abundance of PbrERF24, PbrERF114, PbrACS1b, and PbrACO54, the activities of ACO and ACS, and the content of ACC throughout the storage period, resulting in lower ethylene release compared with control fruits ( Figure 2 ).

[0150] like Figure 3 As shown in the figure, H2O2 accumulated gradually and reached a peak on the 120th day, which was associated with the decrease of AsA, T-AsA, GSH, T-GSH, AsA / DHA and GSH / GSSG ratios and the increase of DHA and GSSG. Ethylene accumulated gradually and reached a peak on the 120th day, which was associated with the expression profile of genes (PbrERF24, PbrERF114, PbrACS1b and PbrACO54) in its biosynthesis pathway. Figure 5 ), enzyme (ACO and ACS) activities ( Figure 4 ) and changes in metabolite content ( Figure 3 ). Exogenous 1-MCP fumigation promoted the production of AsA, T-AsA, GSH, and T-GSH, but inhibited the production of H2O2, DHA, and GSSG, resulting in increased AsA / DHA and GSH / GSSG ratios. For enzymes in the AsA-GSH cycle, GR activity steadily accumulated, while APX, DHAR, and MDHAR activities showed a downward trend during storage ( Figure 4 Further studies have shown that exogenous 1-MCP treatment upregulated the activities of these four enzymes ( Figure 4 ). Transcriptome results showed that 31 genes in the pear AsA-GSH cycle were transcribed throughout the storage period, but with different expression patterns ( Figure 5 Among them, 1-MCP treatment before storage continuously upregulated the expression of PbrAPX14 and PbrMDHAR2 / 7 during the entire storage period ( Figure 5 These results suggest that PbrAPX14 and PbrMDHAR2 / 7 may be candidate genes involved in the scavenging of endogenous H2O2, thereby contributing to the ripening and senescence of 'Korla Fragrant Pear' fruit. Because APX is a direct enzyme that catalyzes the reduction of H2O2, we selected PbrAPX14 for further investigation.

[0151] Example 2 Functional verification of the PbrAPX14 gene

[0152] 1. Determination of subcellular localization of the protein encoded by the PbrAPX14 gene

[0153] Using 'Korla Fragrant Pear' fruit cDNA as a template, the coding sequence of the PbrAPX14 gene (excluding the stop codon) was amplified (primer sequences are shown in Table 1 in the attached table). The primers were inserted into the XbaI restriction site of the pBI221-GFP vector to construct the recombinant plasmid pBI221-PbrAPX14-GFP, which was then transformed into Arabidopsis thaliana protoplasts. Fluorescence signals were observed using a confocal microscope (Leica Microsystems, Germany) using AtDHAR2-mcherry as a marker. The CDS sequence of the PbrAPX14 gene is shown in SEQ ID No. 1; the protein sequence encoded by the PbrAPX14 gene is shown in SEQ ID No. 2.

[0154] 2. Transient overexpression of the PbrAPX14 gene in pear peel

[0155] Using the fruit cDNA of 'Korla Fragrant Pear' as a template, the coding sequence of the PbrAPX14 gene was amplified (primer sequences are shown in Table 1 in the attached table), inserted into the XbaI restriction site of the pCAMBIA1301 plasmid, and transformed into Agrobacterium GV3101. After PCR identification, positive strains were selected for propagation and cultured until OD 600 When the OD value is 0.6-0.8, the cells are collected; the cells are resuspended in infection solution (10 mM MgCl2, 10 mM MES and 200 mM acetosyringone), and after induction for 4 h under dark conditions, the OD value is adjusted. 600 The bacterial solution was injected slowly and evenly into the pericarp of 'Korla Fragrant Pear' fruit using a syringe. The fruit was protected from light for 24 hours after injection. Three days after injection, pericarp tissue from the injection site was collected for subsequent testing. Similarly, fruits of similar size, free of pests, diseases, and mechanical damage, were selected and injected with Agrobacterium carrying an empty pCAMBIA1301 vector as a control.

[0156] 3. Transient silencing of the PbrAPX14 gene in pear peel

[0157] Using cDNA from 'Korla Fragrant Pear' fruit as a template, the approximately 250-bp coding sequence of the PbrAPX14 gene was amplified (primer sequences are shown in Table 1 in the appendix). The primers were inserted into the XbaI restriction site of the pTRV2 vector and transformed into Agrobacterium tumefaciens GV3101. After PCR identification, positive strains were selected for propagation and harvested when the OD600 reached 0.6-0.8. The cells were resuspended in an infection solution (10 mM MgCl2, 10 mM MES, and 200 mM acetosyringone) and incubated in the dark for 4 hours before adjusting the OD600 to 1.0. The bacterial suspensions carrying the recombinant plasmids pTRV2-PbrAPX14 and pTRV1 were mixed at a 1:1 volume ratio and injected into the pericarp of 'Korla Fragrant Pear' fruit. The mixture was protected from light for 24 hours. Three days after injection, the flesh of the injected fruit was collected for subsequent analysis. The pericarp tissue co-injected with bacterial solution carrying pTRV2 empty vector and pTRV1 was used as a control.

[0158] 4. Obtaining Pear Callus Overexpressing the PbrAPX14 Gene

[0159] Using the fruit cDNA of 'Korla Fragrant Pear' as a template, the coding sequence of the PbrAPX14 gene was amplified and inserted into the pCAMBIA1301 vector to construct the pCAMBIA1301-PbrAPX14 overexpression vector (primers are shown in Table 1 in the Appendix). The vector was then used to infect pear callus (induced from small fruit of P. communis cv. 'Clapp's Favorite'). The infected callus was then transferred to MS medium containing hygromycin (Hyg) (carbon source: 30 g L -1 After PCR identification, the positive tissues and the control (transformed empty pCAMBIA1301) were transferred to a sorbitol / sucrose (15 g L -1 , 1:1) as the carbon source on MS medium (containing Hyg).

[0160] 5. Obtaining transgenic tomato fruits

[0161] The constructed pCAMBIA1301-PbrAPX14 overexpression vector was transformed into Agrobacterium tumefaciens GV3101 using Agrobacterium-mediated genetic transformation. Tomato cotyledons were then infected. Tissue culture seedlings were obtained through callus induction, resistance differentiation, and rooting. T2 generation overexpression seeds were tested for hygromycin resistance, and positive transgenic homozygous lines were verified using PCR. Seeds from the T2 homozygous lines were planted, and fruits were harvested 35 days after flowering. They were refrigerated at 4°C for 10 days and then stored at 25°C for 7 days.

[0162] 6. Analysis of PbrAPX14 Gene Expression in Tissues (qRT-PCR)

[0163] Total RNA was extracted from tissues using a plant total RNA extraction kit (RE-05011, Chengdu Fuji Biotechnology Co., Ltd.) and reverse transcribed into cDNA. The expression of the PbrAPX14 gene was detected using the Light Cycler 480 SYBR Green IMaster (4887352001, Roche) reagent (primer sequences are shown in Table 1 in the attached table). PbrTub was used as an internal reference gene.

[0164] 7. Enzyme Activity Assay

[0165] 0.2 g of sample was homogenized in the corresponding extraction buffer and then centrifuged at 50,000 g for 15 min. The supernatant was collected and assayed for ascorbate catalase (APX-1-W, Suzhou Keming Biotechnology Co., Ltd., Suzhou, China) according to the protocol of the relevant assay kit. Protein concentration in the supernatant was determined using the Bicinchoninic Acid Protein Assay Kit (A045-4, Nanjing Jiancheng Bioengineering Institute, Nanjing, China).

[0166] 8. Determination of H2O2 content

[0167] H2O2 in cortical tissue was detected according to the instructions of the corresponding detection kit (H2O2-1-Y, Suzhou Keming Biotechnology Co., Ltd., Suzhou, China). Briefly, the samples were homogenized with extraction buffer and then centrifuged at 12000g for 20 min at 4°C, and the supernatant was collected for H2O2 determination.

[0168] 9. Ethylene content determination

[0169] Five fruits were placed at 20°C for 12 h, weighed, and then placed in a 2-L glass jar with a rubber septum lid. After incubation in the glass jar at 20°C for 30 min, 5.0 ml of headspace gas was withdrawn using a gas-tight syringe. Ethylene concentration was determined using a gas chromatograph (GC, HP 5890A, Hewlett Packard, Avondale, PA) equipped with a GSQ column and a flame ionization detector (FID). The flow rates of helium, hydrogen, and air were 10, 35, and 350 mL min, respectively. -1 The qualitative and quantitative analysis of ethylene was performed by comparing the retention time and peak area with those in gas standards.

[0170] 10. Determination of peel color and hardness

[0171] The color of the peel was analyzed using a Minolta CR-400 colorimeter (Konica Minolta Sensing Co., Ltd., Osaka, Japan) (Wang et al., 2018a). On the other hand, the firmness of the cortex was measured using a hand penetrometer (GY-1, Zhejiang, China) (Xie et al., 2017).

[0172] Experimental results: Figure 6 As shown, PbrAPX14 is located in the cytoplasm. Compared with the control, transient overexpression of PbrAPX14 in 'Kuerle Xiangli' pear, pear callus, and 'MicroTom' tomato cortex tissue upregulated APX activity, thereby reducing H2O2 levels, which is associated with the inhibition of ethylene synthesis, softening process, and color development in the fruit ( Figure 7 a, 8); the opposite phenomenon was observed in the peel of the fruit in which the PbrAPX14 gene was transiently silenced (e.g. Figure 7 b).

[0173] Example 3 Screening of PbrAPX14 Upstream Regulatory Genes and Their Regulatory Mechanism

[0174] 1. Screening of upstream regulatory genes of PbrAPX14

[0175] Transcriptome analysis revealed 456 differentially expressed TFs; 40 of these showed strong positive correlations with PbrAPX14 expression (fold change ≥ 2.0, FDR < 0.05, correlation coefficient ≥ 0.8). Because Pbr006167.1 showed the highest correlation with PbrAPX14 gene expression, it was designated PbrDDI2 and further investigated. Based on results from the PlantRegMap database, PbrDDI2 may interact with a cis-acting element (TTTGTCG / CGACAAA) in the PbrAPX14 promoter, and therefore was selected for further investigation.

[0176] 2. Dual luciferase reporter assay

[0177] Using 'Korla Fragrant Pear' fruit cDNA as a template, specific primers were designed to clone the PbrDDI2 gene coding sequence (primer sequences are shown in Table 1 in the appendix). The sequence was then inserted into the XbaI restriction site of the pSAK277 vector (pSAK277-PbrDDI2). The CDS sequence of the PbrDDI2 gene is shown in SEQ ID No. 3; the protein sequence encoded by the PbrDDI2 gene is shown in SEQ ID No. 4.

[0178] Using the fruit DNA of 'Korla Fragrant Pear' as template, the promoters of the PbrAPX14 gene (PbrAPX14pro, PbrAPX14pro frag1) (primer sequences are shown in Table 1 in the attached table), inserted into the pGreenII 0800-LUC vector, and constructed the reporter vector (pGreenII 0800-PbrAPX14pro-LUC (promoter full length), pGreenII 0800-PbrAPX14pro frag1 On this basis, mutant primers were designed (primer sequences are shown in Table 1 in the attached table) to mutate the cis-acting element in the PbrAPX14 gene promoter (TTTGTCG→AAAAAAA) to obtain the mutant promoter sequence PbrAPX14pro mut , inserted into the pGreenII 0800-LUC vector to construct the reporter vector pGreenII 0800-PbrAPX14pro mut -LUC.

[0179] The transcription factor recombinant plasmid (pSAK277-PbrDDI2) and the reporter vector (pGreenII0800-PbrAPX14pro-LUC or pGreenII 0800-PbrAPX14pro frag1 -LUC or pGreenII 0800-PbrAPX14pro mut -LUC) were transformed into Agrobacterium GV3101 and GV3101 (psoup) respectively. The positive strains were selected and expanded to OD 600 When the luciferase concentration (pSAK277) reaches 0.6-0.8, the cells are harvested and resuspended in infection solution (10 mM MgCl2, 10 mM MES, 200 μM acetosyringone, pH 5.7). After induction in the dark for 3-5 hours, tobacco leaves are infected. Before injection, Agrobacterium containing pSAK277-PbrDDI2 and the reporter vector are mixed at a 9:1 (v / v) ratio. Tobacco leaves co-transfected with Agrobacterium carrying both the pSAK277 empty vector and the corresponding reporter vector serve as controls. 60-72 hours after infection, leaves are harvested and firefly and Renilla luciferase activities are measured using the Dual-Luciferase Reporter Assay System kit (Promega, USA) (see the manufacturer's instructions for specific steps).

[0180] 3. Yeast one-hybrid assay (Y1H)

[0181] Using the fruit cDNA of 'Korla Fragrant Pear' as a template, specific primers were designed to clone the PbrDDI2 gene coding sequence (primer sequences are shown in Table 1 in the appendix) and inserted into the XbaI restriction site of AD to form the prey vector PbrDDI2-AD. Using the fruit DNA of 'Korla Fragrant Pear' as a template, a 200-bp vector containing the wild-type PbrIDD2 binding element (core motif: TTTGTCG; PbrAPX14 promoter) and the mutant element (TTTGTCG→AAAAAAA; PbrAPX14pro mut ) was inserted into the XbaI restriction site of the pAbAi vector (primer sequences are shown in Table 1 in the attached table) to form the bait vector (PbrAPX14pro-pAbAi and PbrAPX14pro mut -pAbAi).

[0182] The bait vector and prey vector were co-transformed into yeast strains, and the Y1H assay was performed using the Matchmaker Gold Yeast One-Hybrid Library Screening System (Weidi, Shanghai, China) (see the instructions for specific steps). PbrAPX14pro-pAbAi and PbrAPX14pro were screened on SD / -Ura deficient medium supplemented with aurobasidin A (AbA). mut -pAbAi self-activation detection, and select the appropriate AbA concentration for subsequent experiments. The yeast strains co-transformed with the bait vector and the prey vector were cultured on SD / -Leu deficiency medium supplemented with appropriate concentrations to detect the binding ability of PbrDDI2 to the PbrAPX14 promoter. The yeast strains co-transformed with pGAD-p53 and p53-AbAi (refer to Jia L, Zhang X, Zhang Z, Luo W, Nambeesan SU, Li Q, Qiao X, Yang B, Wang L, Zhang S. PbrbZIP15 promotes sugar accumulation in pear via activating the transcription of the glucose isomerase gene PbrXylA1. Plant J. 2024, 117(5): 1392-1412.) were used as positive controls, and AD and PbrAPX14pro-pAbAi (or PbrAPX14pro mut A yeast strain co-transformed with pAbAi-pAbAi was used as a negative control.

[0183] 4. ChIP-qPCR Experiment

[0184] Callus tissue from the flesh of 'Clapp's Favorite' (CF) pears was used as the experimental material. Wild-type calli were grown on MS medium (30 g / L sucrose, 1 mg / L 2,4-D, and 0.5 mg / L 6-BA) and subcultured every two weeks to prepare for subsequent transgenic development. Calli were infected with Agrobacterium carrying the pCAMBIA1301-PbrDDI2-GFP vector (sequences of primers used to construct the pCAMBIA1301-PbrDDI2-GFP vector are shown in Table 1 in the appendix). Infected calli were cultured on MS medium supplemented with acetosyringone (AS) for two days and then transferred to MS medium supplemented with hygromycin (Hyg) (carbon source: 30 g / L sucrose) for 1.5-2 months. Regenerated calli were identified by DNA and RNA analysis, and positive lines were obtained and propagated. Subsequently, the positive calli transformed with the PbrDDI2 gene and the control (calli transformed with empty pCAMBIA1301-GFP) were co-transferred to MS medium (containing Hyg) with a mixture of sorbitol and sucrose (15 g / L each) or glucose (30 g / L) as the carbon source for growth for subsequent determination.

[0185] 10 g of callus tissue containing pCAMBIA1301-PbrDDI2-GFP was weighed, ground, and cross-linked in 1% (v / v) formaldehyde cross-linking buffer for 15-30 minutes. Cross-linking was terminated by the addition of glycine. The resulting cross-linked product was ground with liquid nitrogen and lysed to obtain chromatin, which was further fragmented by sonication to obtain soluble sheared chromatin (average DNA length 200-500 bp). A portion of the soluble sheared chromatin was used as input, and the remaining portion was washed and pre-cleared before immunoprecipitation (IP) with a GFP antibody. Pear callus carrying pCAMBIA1301-GFP was used as a control. Specific fluorescent quantitative primers were designed for the PbrAPX14 promoter region containing the cis-acting element (TTTGTCG / CGACAAA) (primers are shown in Table 1 in the appendix), and enrichment was determined by qPCR.

[0186] 5. EMSA Experiment

[0187] Using 'Korla Fragrant Pear' fruit cDNA as a template, the full-length CDS of PbrDDI2 was amplified and inserted into the pCold-TF expression vector (primer sequences are shown in Table 1 in the Supplementary Table). The correctly sequenced pCold-PbrDDI2 plasmid was transformed into Escherichia coli BL21(DE3) to produce the His-tagged recombinant protein His-PbrDDI2. Empty pCold-TF protein was also expressed as a control. The crude protein was purified and used in subsequent experiments. DNA probes of approximately 30-40 pb were synthesized based on the sequence of the cis-acting element (TTTGTCG / CGACAAA) within the promoter of the target gene PbrAPX14. These included a biotinylated probe carrying the unmutated (or mutated) cis-acting element and an unlabeled competitive probe carrying the cis-acting element. DNA probes were synthesized by Suzhou Jinweizhi Biotechnology Co., Ltd. (probe sequences are shown in Table 1 in the Supplementary Table). EMSA experiments were performed according to the protocol provided by the Chemiluminescent EMSA Kit (Beyond Sky, China).

[0188] Experimental results: Based on the results of transcriptome analysis, 456 of the 559 differentially expressed transcription factors (TFs) in the 1-MCP fumigated 'Korla Fragrant Pear' fruit showed a persistent high or low expression pattern throughout the storage period ( Figure 9 Screening criteria: expression fold change ≥ 2.0 and padj < 0.05 in at least one stage. Among them, 40 members showed extremely significant positive correlation with PbrAPX14 in 1-MCP-treated (or untreated) chilling-injured fragrant pear (correlation coefficient ≥ 0.8). Figure 10 ), suggesting that it may act as an upstream activator of PbrAPX14. Figure 10 ) has a high correlation with PbrAPX14, and PlantRegMap predicts that this gene may bind to the cis-acting element in the PbrAPX14 promoter ( Figure 11 ), so this gene was selected for in-depth study.

[0189] Further combined with dual luciferase reporter assay ( Figure 12 ), yeast one-hybrid ( Figure 13 )、ChIP-qPCR( Figure 14 ) and EMSA assays ( Figure 15 ) showed that the transcription factor PbrIDD2 specifically binds to the cis-acting element ((TTTGTCG / CGACAAA)) in the upstream promoter of the PbrAPX14 gene and activates the transcription of the PbrAPX14 gene.

[0190] Example 4 Functional Verification of PbrDDI2

[0191] 1. Subcellular localization of the protein encoded by the PbrDDI2 gene

[0192] The coding sequence of the PbrDDI2 gene (excluding the stop codon) was amplified using the fruit cDNA of 'Korla Fragrant Pear' as a template (primer sequences are shown in Table 1 in the Appendix), inserted into the pBI221-GFP vector, and transformed into tobacco leaves. AtH2B-mcherry was used as a nuclear marker, and the fluorescence signal was observed using a confocal microscope (Leica Microsystems, Germany).

[0193] 2. Transient overexpression of the PbrDDI2 gene in pear peel

[0194] Using the fruit cDNA of 'Korla Fragrant Pear' as a template, the coding sequence of the PbrDDI2 gene was amplified (primer sequences are shown in Table 1 in the attached table), inserted into the pCAMBIA1301 plasmid, and transformed into Agrobacterium GV3101. After PCR identification, positive strains were selected for propagation and cultured until OD 600 When the OD value is 0.6-0.8, the cells are collected; the cells are resuspended in infection solution (10 mM MgCl2, 10 mM MES and 200 mM acetosyringone), and after induction for 4 h under dark conditions, the OD value is adjusted. 600 The bacterial solution was injected slowly and evenly into the pericarp of 'Korla Fragrant Pear' fruit using a syringe. The fruit was protected from light for 24 hours after injection. Three days after injection, pericarp tissue from the injection site was collected for subsequent testing. Similarly, fruits of similar size, free of pests, diseases, and mechanical damage, were selected and injected with Agrobacterium carrying an empty pCAMBIA1301 vector as a control.

[0195] 3. Transient silencing of the PbrDDI2 gene in pear peel

[0196] Using the fruit cDNA of 'Korla Fragrant Pear' as a template, the coding sequence of the PbrDDI2 gene of about 250 bp was amplified (primer sequences are shown in Table 1 in the attached table), inserted into the pTRV2 vector, and transformed into Agrobacterium GV3101. After PCR identification, positive strains were selected for propagation and cultured to OD 660 The cells were collected when the OD value was 1.0; the cells were resuspended in infection solution (10 mM MgCl2, 10 mM MES and 200 mM acetosyringone), and after 4 h of dark induction, the OD value was adjusted. 660 to 1.0. Bacterial cultures carrying the recombinant plasmids pTRV2-PbrAPX14 and pTRV1 were mixed at a 1:1 volume ratio and injected into the pericarp of 'Korla Fragrant Pear' fruit. The mixture was protected from light for 24 hours. Three days after injection, the flesh of the fruit at the injection site was collected for subsequent analysis. Pericarp tissue co-injected with bacteria carrying the pTRV2 empty vector and pTRV1 served as a control.

[0197] 4. Obtaining Pear Callus Overexpressing the PbrDDI2 Gene

[0198] Using the fruit cDNA of 'Korla Fragrant Pear' as a template, the coding sequence of the PbrDDI2 gene was amplified and inserted into the pCAMBIA1301 vector to construct the pCAMBIA1301-PbrDDI2 overexpression vector (primers are shown in Table 1 in the Appendix). The vector was then used to infect pear callus (induced from small fruit of P. communis cv. 'Clapp's Favorite'). The infected callus was then transferred to MS medium containing hygromycin (Hyg) (carbon source: 30 g L -1 After PCR identification, the positive tissues and the control (transformed empty pCAMBIA1301) were transferred to a sorbitol / sucrose (15 g L -1 , 1:1) as the carbon source on MS medium (containing Hyg).

[0199] 5. Obtaining transgenic tomato fruits

[0200] The constructed pCAMBIA1301-PbrDDI2-GFP overexpression vector was transformed into Agrobacterium tumefaciens GV3101 using Agrobacterium-mediated genetic transformation. Tomato cotyledons were then infected. Tissue culture seedlings were obtained through callus induction, resistance differentiation, and rooting. T2 generation overexpressing seeds were tested for hygromycin resistance, and positive transgenic homozygous lines were verified using PCR. Seeds from the T2 homozygous lines were planted, and fruits were harvested 35 days after flowering. They were refrigerated at 4°C for 10 days and then stored at 20°C for 7 days.

[0201] 6. Analysis of PbrDDI2 and PbrAPX14 Gene Expression in Tissues (qRT-PCR)

[0202] Total RNA was extracted from tissues using a plant total RNA extraction kit (RE-05011, Chengdu Fuji Biotechnology Co., Ltd.) and reverse transcribed into cDNA. The expression levels of PbrDDI2 and PbrAPX14 genes were detected using the Light Cycler 480 SYBR Green I Master reagent (4887352001, Roche). Primer sequences for the PbrAPX14 gene are shown in Table 1 in the attached table, and primer sequences for the PbrDDI2 gene are shown in Table 1 in the attached table. PbrTub was used as an internal reference gene.

[0203] 7. Enzyme Activity Assay

[0204] 0.2 g of sample was homogenized in the corresponding extraction buffer and then centrifuged at 50,000 g for 15 min. The supernatant was collected and assayed for ascorbate catalase (APX-1-W, Suzhou Keming Biotechnology Co., Ltd., Suzhou, China) according to the protocol of the relevant assay kit. Protein concentration in the supernatant was determined using the Bicinchoninic Acid Protein Assay Kit (A045-4, Nanjing Jiancheng Bioengineering Institute, Nanjing, China).

[0205] 8. Determination of H2O2 content

[0206] H2O2 in cortical tissue was detected according to the instructions of the corresponding detection kit (H2O2-1-Y, Suzhou Keming Biotechnology Co., Ltd., Suzhou, China). Briefly, the samples were homogenized with extraction buffer and then centrifuged at 12000g for 20 min at 4°C, and the supernatant was collected for H2O2 determination.

[0207] 9. Ethylene content determination

[0208] Five fruits were placed at 20°C for 12 h, weighed, and then placed in a 2-L glass jar with a rubber septum lid. After incubation in the glass jar at 20°C for 30 min, 5.0 ml of headspace gas was withdrawn using a gas-tight syringe. Ethylene concentration was determined using a gas chromatograph (GC, HP 5890A, Hewlett Packard, Avondale, PA) equipped with a GSQ column and a flame ionization detector (FID). The flow rates of helium, hydrogen, and air were 10, 35, and 350 mL min, respectively. -1 The qualitative and quantitative analysis of ethylene was performed by comparing the retention time and peak area with those in gas standards.

[0209] 10. Determination of peel color and hardness

[0210] The color of the peel was analyzed using a Minolta CR-400 colorimeter (Konica Minolta Sensing Co., Ltd., Osaka, Japan) (Wang et al., 2018a). On the other hand, the firmness of the cortex was measured using a hand penetrometer (GY-1, Zhejiang, China) (Xie et al., 2017).

[0211] Experimental results: Figure 16 As shown in Figure 2, PbrDDI2 is localized in the nucleus of Arabidopsis protoplasts. Transient overexpression of the PbrDDI2 gene significantly increased the expression of the PbrAPX14 gene and APX activity in pear peel tissue, thereby reducing H2O2 content and inhibiting ethylene synthesis; while the opposite phenomenon was observed when the PbrAPX14 gene was transiently silenced (e.g. Figure 17Further studies have shown that the H2O2 content in the pear callus overexpressing the PbrDDI2 gene was significantly lower than that in the control, while the expression level of the PbrAPX14 gene and the APX activity were significantly higher than those in the control (e.g. Figure 18 ); the H2O2 content in the peel tissue of tomatoes overexpressing the PbrDDI2 gene was significantly lower than that of the control, the firmness was significantly higher, and the a* was significantly lower than that of the control. This indicates that overexpression of the PbrDDI2 gene significantly enhances the resistance of pear fruit to ripening and senescence.

[0212] In summary, the present invention is based on a transcription factor PbrDDI2, which can bind to the cis-acting element in the promoter of the PbrAPX14 gene to regulate its transcription level, affecting the regulation of H2O2 and ethylene levels in pears and thus regulating the ripening and senescence of pear fruits (such as Figure 19 ), which has important theoretical and practical significance for breeding high-quality anti-aging pear varieties.

[0213] The above-described specific embodiments of the present invention do not constitute a limitation on the scope of protection of the present invention. It should be pointed out that any improvements, equivalent substitutions, etc. made by those skilled in the art without departing from the technical principles of the present invention should be included in the scope of protection of the claims of the present invention.

[0214] Appendix

[0215] Table 1 Primer or probe sequences related to subcellular localization, dual-luciferase reporter assay, Y1H, ChIP-qPCR, EMSA, overexpression, and gene silencing

[0216]

[0217]

[0218] Sequence Listing SEQ ID No. 1PbrAPX14 CDS

[0219]

[0220] SEQ ID No.2PbrAPX14 Protein

[0221]

[0222]

[0223] SEQ ID No. 3

[0224] PbrDDI2

[0225] CDS

[0226]

[0227]

[0228] SEQ ID No.4PbrDDI2Protein

[0229]

[0230] SEQ ID No.5PbrAPX14 promoter

[0231]

[0232]

Claims

1. Use of the PbrAPX14 gene or a biological material related to the PbrAPX14 gene in at least one of the following (a1) to (a6): (a1) Regulate the accumulation of endogenous H2O2 in fruits; (a2) preparing a product for regulating endogenous H2O2 accumulation in fruits; (a3) Regulates ethylene synthesis in fruits; (a4) preparing products for regulating ethylene synthesis in fruits; (a5) regulating fruit ripening and senescence; (a6) preparing products for regulating ripening and senescence of fruits; The PbrAPX14 gene is any one of the following DNA molecules (b1)-(b4): (b1) a DNA molecule whose coding region includes the nucleotide sequence shown in SEQ ID NO.1; (b2) a DNA molecule with a nucleotide sequence as shown in SEQ ID NO. 1; (b3) a DNA molecule that hybridizes under stringent conditions with the DNA sequence defined in (b1) or (b2) and encodes a protein with the same function; (b4) A DNA molecule that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the DNA sequence defined in (b1) or (b2) and encodes a protein with the same function.

2. The use according to claim 1, characterized in that The biological material related to the PbrAPX14 gene is at least one of (c1) to (c10): (c1) PbrAPX14 protein encoded by the PbrAPX14 gene; (c2) an expression cassette containing the PbrAPX14 gene; (c3) a recombinant vector containing the PbrAPX14 gene, or a recombinant vector containing the expression cassette described in (c2); (c4) a recombinant microorganism containing the PbrAPX14 gene, or a recombinant microorganism containing the expression cassette described in (c2), or a recombinant microorganism containing the recombinant vector described in (c3); (c5) a transgenic plant cell line containing the PbrAPX14 gene, or a transgenic plant cell line containing the expression cassette described in (c2), or a transgenic plant cell line containing the recombinant vector described in (c3); (c6) transgenic plant tissue containing the PbrAPX14 gene, or transgenic plant tissue containing the expression cassette described in (c2), or transgenic plant tissue containing the recombinant vector described in (c3); (c7) a transgenic plant organ containing the PbrAPX14 gene, or a transgenic plant organ containing the expression cassette described in (c2), or a transgenic plant organ containing the recombinant vector described in (c3); (c8) A transgenic plant containing the PbrAPX14 gene, or a transgenic plant containing the expression cassette described in (c2), or a transgenic plant containing the recombinant vector described in (c3). (c9) tissue cultures produced from regenerable cells of the transgenic plant described in (c8); (c10) Protoplasts produced from the tissue culture described in (c9).

3. The use according to claim 2, characterized in that The PbrAPX14 protein is the protein described in (d1), (d2), or (d3): (d1) a protein with an amino acid sequence as shown in SEQ ID NO. 2; (d2) a protein derived from SEQ ID NO. 2 obtained by substituting and / or deleting and / or adding amino acid residues in the amino acid sequence of SEQ ID NO. 2; (d3) A protein derived from SEQ ID NO. 2 that is 80% or more identical to the protein shown in (d1) or (d2).

4. The use according to claim 1, characterized in that Overexpression of the PbrAPX14 gene eliminates endogenous H2O2 accumulation and thereby inhibits ethylene synthesis to inhibit fruit ripening and senescence; or silencing or inhibiting the PbrAPX14 gene promotes endogenous H2O2 accumulation and thereby promotes ethylene synthesis to accelerate fruit ripening and senescence.

5. Use of the transcription factor PbrDDI2 or a biomaterial related to the transcription factor PbrDDI2 in at least one of the following (a1) to (a6): (a1) Regulate the accumulation of endogenous H2O2 in fruits; (a2) preparing a product for regulating endogenous H2O2 accumulation in fruits; (a3) Regulates ethylene synthesis in fruits; (a4) preparing products for regulating ethylene synthesis in fruits; (a5) regulating fruit ripening and senescence; (a6) preparing products for regulating ripening and senescence of fruits; The transcription factor PbrDDI2 is the protein described in (e1), (e2), or (e3): (e1) a protein with an amino acid sequence as shown in SEQ ID NO. 4; (e2) a protein derived from the amino acid sequence of SEQ ID NO. 4 obtained by substitution and / or deletion and / or addition of amino acid residues; (e3) A protein derived from SEQ ID NO. 4 that is 80% or more identical to the protein shown in (e1) or (e2).

6. The use according to claim 5, characterized in that The biological material related to the transcription factor PbrDDI2 is at least one of (f1) to (f10): (f1) gene PbrDDI2 encoding the transcription factor PbrDDI2; (f2) an expression cassette containing the gene PbrDDI2 described in (f1); (f3) a recombinant vector containing the gene PbrDDI2 described in (f1), or a recombinant vector containing the expression cassette described in (f2); (f4) a recombinant microorganism containing the gene PbrDDI2 described in (f1), a recombinant microorganism containing the expression cassette described in (f2), or a recombinant microorganism containing the recombinant vector described in (f3); (f5) a transgenic plant cell line containing the gene PbrDDI2 described in (f1), or a transgenic plant cell line containing the expression cassette described in (f2), or a transgenic plant cell line containing the recombinant vector described in (f3); (f6) transgenic plant tissue containing the gene PbrDDI2 described in (f1), or transgenic plant tissue containing the expression cassette described in (f2), or transgenic plant tissue containing the recombinant vector described in (f3); (f7) a transgenic plant organ containing the gene PbrDDI2 described in (f1), or a transgenic plant organ containing the expression cassette described in (f2), or a transgenic plant organ containing the recombinant vector described in (f3); (f8) a transgenic plant containing the gene PbrDDI2 described in (f1), or a transgenic plant containing the expression cassette described in (f2), or a transgenic plant containing the recombinant vector described in (f3); (f9) tissue cultures produced from regenerable cells of the transgenic plant according to (f8); (f10) Protoplasts produced from the tissue culture described in (f9).

7. The use according to claim 6, characterized in that The gene PbrDDI2 encoding the transcription factor PbrDDI2 in (f1) is any one of the following DNA molecules (g1) to (g4): (g1) a DNA molecule whose coding region includes the nucleotide sequence shown in SEQ ID NO. 3; (g2) a DNA molecule with a nucleotide sequence as shown in SEQ ID NO. 3; (g3) a DNA molecule that hybridizes with the DNA sequence defined in (g1) or (g2) under stringent conditions and encodes a protein with the same function; (g4) A DNA molecule that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the DNA sequence defined in (g1) or (g2) and encodes a protein with the same function.

8. The use according to claim 5 or 6, characterized in that The transcription factor PbrDDI2 interacts with the PbrAPX14 gene described in claim 1 to regulate the accumulation of endogenous H2O2 and then regulate the synthesis of ethylene and then regulate the ripening and aging of the fruit; overexpressing the gene PbrDDI2 encoding the transcription factor PbrDDI2 or / and increasing the protein level or / and activity of the transcription factor PbrDDI2 eliminates the endogenous H2O2 accumulation and then inhibits the synthesis of ethylene to inhibit the ripening and aging of the fruit; or silencing or inhibiting the gene PbrDDI2 encoding the transcription factor PbrDDI2 and / or reducing the protein level or / and activity of the transcription factor PbrDDI2 promotes the accumulation of endogenous H2O2 and then promotes the synthesis of ethylene to accelerate the ripening and aging of the fruit.

9. The use according to claim 8, characterized in that The transcription factor PbrDDI2 eliminates H2O2 accumulation by binding to the cis-acting element TTTGTCG / CGACAAA in the PbrAPX14 gene promoter, thereby inhibiting fruit ripening and senescence; the nucleotide sequence of the PbrAPX14 gene promoter is shown in SEQ ID NO.

5.

10. A method for inhibiting fruit ripening and aging, characterized in that: Using any one of the following methods (h1)-(h2) or a combination of two methods to remove endogenous H2O2 accumulation and inhibit ethylene synthesis to inhibit fruit ripening and senescence; (h1) overexpressing the gene PbrDDI2 encoding the transcription factor PbrDDI2 and / or increasing the protein level and / or activity of the transcription factor PbrDDI2; (h2) overexpressing the PbrAPX14 gene or / and increasing the protein level and / or activity of the PbrAPX14 protein encoded by the PbrAPX14 gene; The nucleotide sequence of the gene PbrDDI2 encoding the transcription factor PbrDDI2 is shown in SEQ ID No.3; the amino acid sequence of the transcription factor PbrDDI2 is shown in SEQ ID No.4; the nucleotide sequence of the PbrAPX14 gene is shown in SEQ ID No.1; the amino acid sequence of the PbrAPX14 protein encoded by the PbrAPX14 gene is shown in SEQ ID No.

2.

11. The use according to any one of claims 1 to 9, the method according to claim 10, characterized in that: The fruit is the fruit of a Rosaceae fruit tree or the fruit of a Solanaceae vegetable; and the plant is a Rosaceae or Solanaceae plant.

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