The transcription factor Pbr3AP2-8 of the pear and the gene PbrC3apos are shown in the specification; application of H1 in regulation and control of accumulation of chlorogenic acid in pear fruits

Abstract

The invention discloses application of a pear transcription factor Pbr3AP2-8 and a gene PbrC3 'H1 in regulation and control of accumulation of chlorogenic acid in pear fruits. The amino acid sequence of the pear AP2 / ERF family transcription factor Pbr3AP2-8 is as shown in SEQ ID NO. 2, and the nucleotide sequence of the encoding gene of the pear AP2 / ERF family transcription factor Pbr3AP2-8 is as shown in SEQ ID NO. 1. The transcription factor Pbr3AP2-8 for regulating and controlling the PbrC3 'H1 gene expression of the pear fruit is disclosed for the first time by analyzing the distribution of cis-acting elements in a PbrC3' H1 promoter and the expression profile of PbrERFs after postharvest storage and ethylene / 1-MCP treatment. The action mechanism of interaction of AP2 binding sites (P: (-466)-(-457)) in Pbr3AP2-8 and PbrC3 'H1 gene promoters in regulation and control of pear fruit chlorogenic acid synthesis is clarified, and a theoretical and practical basis is provided for directional improvement of postharvest pear fruit quality characters.

Description

Technical Field

[0001] The present invention belongs to the fields of molecular biology and genetic engineering technology, and specifically relates to the use of the pear transcription factor Pbr3AP2-8 and the gene PbrC3′H1′ in regulating the accumulation of chlorogenic acid in pear fruit. The pear transcription factor Pbr3AP2-8 regulates the expression of the key gene PbrC3′H1 for chlorogenic acid metabolism, thereby regulating chlorogenic acid synthesis. Background Art

[0002] Pears, the king of fruits, are rich in nutrients and are highly sought after by consumers worldwide. Polyphenols, a major class of secondary metabolites, possess diverse structural and physicochemical properties, contributing significantly to the taste, color, and nutritional profile of pear fruit. Chlorogenic acid, also known as 3-O-caffeoylquinic acid (3-CQA), is the primary polyphenol and a major antioxidant. 3-CQA is a bioactive metabolite derived from the phenylpropanoid biosynthetic pathway that plays a key role in fruit ripening and plant responses to biotic stresses.

[0003] In plants, phenylalanine is converted into p-coumaric acid through the catalysis of phenylalanine ammonia-lyase (PAL) and cinnamate 4-hydroxylase (C4H), which can produce chlorogenic acid through four pathways. In pathways 1 and 2, p-coumaric acid is converted to caffeoyl skikimic acid by 4-hydroxycinnamoyl-CoA ligase (4CL), hydroxycinnamoyl-CoA:shikimate / quinatehydroxycinnamoyltransferase (HCT) / hydroxycinnamoyl-CoA:quinate hydroxycinnamoyl-transferase (HQT), and p-coumarate 3′-hydroxylase (C3′H). Caffeoyl skikimic acid is then converted to caffeoyl skikimic acid by caffeoyl shikimate esterase (C3′H). Chlorogenic acid is generated by the catalysis of hydroxylamine esterase (CSE), 4CL, and HQT / HCT (Pathway 1), or by the catalysis of HCT / HQT and HQT / HCT (Pathway 2). In some plants that do not contain CSE, p-coumaric acid can be converted to caffeoyl-CoA by catalysis of p-coumarate 3-hydroxylase (C3′H) and 4CL, which is then catalyzed by HQT / HCT to produce chlorogenic acid (Pathway 3). Pathway 4 is the most direct synthesis pathway, in which p-coumaric acid is converted to p-coumaroylquinic acid by 4CL and HQT / HCT, which is then catalyzed by C3′H to produce chlorogenic acid.

[0004] Transcription factors (TFs) bind to cis-acting elements in the promoters of downstream structural genes and regulate their expression. They play a crucial role in fruit ripening and postharvest storage. These factors include genes from the AP2 / ERF, WRKYs, and MYB families. In carrot cells cultured in vitro, DcMYB3 and DcMYB5 promote DcPAL3 transcription, thereby increasing chlorogenic acid levels. In Salvia miltiorrhiza, transient overexpression of ERF115 transiently increases chlorogenic acid content, while silencing this gene reduces chlorogenic acid content. To date, little has been reported on the C3'H gene family members involved in chlorogenic acid biosynthesis in pear fruit and their regulatory mechanisms. Summary of the Invention

[0005] The object of the present invention is to provide an application of the PbrC3′H1 gene or a biological material related to the PbrC3′H1 gene in regulating the accumulation of chlorogenic acid in the fruit of Rosaceae fruit trees.

[0006] Another object of the present invention is to provide the use of the AP2 / ERF family transcription factor Pbr3AP2-8 or biomaterials related to the AP2 / ERF family transcription factor Pbr3AP2-8 for regulating chlorogenic acid accumulation in the fruit of Rosaceae fruit trees. Studies have shown that the AP2 / ERF family transcription factor Pbr3AP2-8 interacts with the PbrC3′H1 gene promoter to regulate chlorogenic acid accumulation in pear fruit. The transcription factor Pbr3AP2-8 specifically binds to the AP2 binding site (P: (-466)-(-457)) in the upstream promoter of the PbrC3′H1 gene, promoting transcription of the PbrC3′H1 gene.

[0007] Another object of the present invention is to provide a method for promoting the accumulation of chlorogenic acid in the fruits of Rosaceae fruit trees.

[0008] The purpose of the present invention is achieved through the following technical solutions:

[0009] In a first aspect, the present invention seeks to protect the use of a PbrC3′H1 gene having a nucleotide sequence as shown in SEQ ID NO. 3 or a biological material related to the PbrC3′H1 gene in regulating the accumulation of chlorogenic acid in the fruit of a Rosaceae fruit tree.

[0010] Furthermore, the biological material related to the PbrC3′H1 gene is at least one of (a1) to (a7):

[0011] (a1) a PbrC3H'1 protein encoded by the PbrC3'H1 gene, the amino acid sequence of which is shown in SEQ ID NO. 4;

[0012] (a2) an expression cassette containing the PbrC3′H1 gene;

[0013] (a3) a recombinant vector containing the PbrC3′H1 gene, or a recombinant vector containing the expression cassette described in (a2);

[0014] (a4) a recombinant microorganism containing the PbrC3′H1 gene, or a recombinant microorganism containing the expression cassette described in (a2), or a recombinant microorganism containing the recombinant vector described in (a3);

[0015] (a5) a transgenic plant cell line containing the PbrC3′H1 gene, or a transgenic plant cell line containing the expression cassette of (a2), or a transgenic plant cell line containing the recombinant vector of (a3);

[0016] (a6) transgenic plant tissue containing the PbrC3′H1 gene, or transgenic plant tissue containing the expression cassette described in (a2), or transgenic plant tissue containing the recombinant vector described in (a3);

[0017] (a7) A transgenic plant organ containing the PbrC3′H1 gene, or a transgenic plant organ containing the expression cassette described in (a2), or a transgenic plant organ containing the recombinant vector described in (a3).

[0018] Furthermore, the above application is to overexpress the PbrC3′H1 gene to promote the accumulation of chlorogenic acid in fruits.

[0019] In a second aspect, the present invention seeks to protect the use of the AP2 / ERF family transcription factor Pbr3AP2-8 with an amino acid sequence as shown in SEQ ID NO. 2 or a biological material related to the AP2 / ERF family transcription factor Pbr3AP2-8 in regulating the accumulation of chlorogenic acid in the fruit of Rosaceae fruit trees.

[0020] Furthermore, the biomaterial related to the transcription factor Pbr3AP2-8 is at least one of the following (b1) to (b7):

[0021] (b1) gene Pbr3AP2-8 encoding the transcription factor Pbr3AP2-8;

[0022] (b2) an expression cassette containing the gene Pbr3AP2-8 described in (b1);

[0023] (b3) a recombinant vector containing the gene Pbr3AP2-8 described in (b1) and a recombinant vector containing the expression cassette described in (b2);

[0024] (b4) a recombinant microorganism containing the gene Pbr3AP2-8 described in (b1), or a recombinant microorganism containing the expression cassette described in (b2), or a recombinant microorganism containing the recombinant vector described in (b3);

[0025] (a5) a transgenic plant cell line containing the gene Pbr3AP2-8 described in (b1), or a transgenic plant cell line containing the expression cassette described in (b2), or a transgenic plant cell line containing the recombinant vector described in (b3);

[0026] (a6) transgenic plant tissue containing the gene Pbr3AP2-8 described in (b1), or transgenic plant tissue containing the expression cassette described in (b2), or transgenic plant tissue containing the recombinant vector described in (b3);

[0027] (a7) A transgenic plant organ containing the gene Pbr3AP2-8 described in (b1), or a transgenic plant organ containing the expression cassette described in (b2), or a transgenic plant organ containing the recombinant vector described in (b3).

[0028] Furthermore, the nucleotide sequence of the gene Pbr3AP2-8 encoding the transcription factor Pbr3AP2-8 in (b1) is shown as SEQ ID NO.1.

[0029] Furthermore, the above application is to overexpress the gene Pbr3AP2-8 encoding the transcription factor Pbr3AP2-8 or increase the activity or content of the transcription factor Pbr3AP2-8 protein to promote the accumulation of chlorogenic acid in the fruit; the AP2 / ERF family transcription factor Pbr3AP2-8 interacts with the above PbrC3′H1 gene to regulate the accumulation of chlorogenic acid in pear fruit.

[0030] Furthermore, the aforementioned application involves the AP2 / ERF family transcription factor Pbr3AP2-8 promoting the expression of the PbrC3'H1 gene by binding to the AP2 binding site in the PbrC3'H1 gene promoter, thereby promoting the accumulation of chlorogenic acid in fruit. Research in the present invention has shown that the AP2 / ERF family transcription factor Pbr3AP2-8 promotes the expression of the PbrC3'H1 gene and the accumulation of chlorogenic acid in fruit by binding to the AP2 binding site (P: (-466)-(-457)) in the PbrC3'H1 gene promoter. The P binding site sequence is TTCTCGTCCA.

[0031] In a third aspect, the present invention claims a method for promoting the accumulation of chlorogenic acid in the fruit of a Rosaceae fruit tree, the method comprising at least one of the following (c1) to (c3):

[0032] (c1) overexpressing the gene Pbr3AP2-8 encoding the transcription factor Pbr3AP2-8;

[0033] (c2) increasing the activity or content of the transcription factor Pbr3AP2-8 protein;

[0034] (c3) overexpressing the PbrC3′H1 gene;

[0035] Among them, the amino acid sequence of the transcription factor Pbr3AP2-8 is shown in SEQ ID NO.2; the nucleotide sequence of the gene Pbr3AP2-8 encoding the transcription factor Pbr3AP2-8 is shown in SEQ ID NO.1; and the nucleotide sequence of the PbrC3′H1 gene is shown in SEQ ID NO.3.

[0036] The Rosaceae fruit tree described in the present invention is pear, but is not limited thereto.

[0037] Research process of the technical solution of the present invention:

[0038] All PbrC3′H1 genes (six gene family members) were identified based on the pear genome database. Furthermore, by analyzing the expression profile of PbrC3′H1 genes at different growth and development stages of 'Fengshui' pear fruit and its correlation with chlorogenic acid content, the family member PbrC3′H1 that may be involved in regulating chlorogenic acid synthesis was screened out.

[0039] The recombinant plasmid pBI221-PbrC3′H1-GFP was constructed and co-transformed with the endoplasmic reticulum marker protein ER-rk-mcherry into Arabidopsis protoplasts. GFP fluorescence was observed using a laser confocal microscope, and it was found that PbrC3′H1 was located in the endoplasmic reticulum.

[0040] The pCAMBIA1300-PbrC3′H1 overexpression vector was constructed and introduced into Agrobacterium GV3101 to transform pear callus tissue. After positive tissue was obtained by PCR identification, the positive transgenic tissue and its control (callus tissue transformed with the pCAMBIA1300 empty vector) were transferred to MS medium with sorbitol and sucrose (1:1, w:w) as carbon sources for growth.

[0041] Using qPT-PCR detection technology and high-performance liquid chromatography (HPLC), the expression level of PbrC3′H1 gene and the content of chlorogenic acid in pear PbrC3′H1-positive transgenic tissues were significantly increased.

[0042] By analyzing the distribution of cis-acting elements in the PbrC3′H1 promoter, overexpressing the PbrACO1 gene, and the expression profiles of PbrERFs during postharvest storage, we identified the transcription factor Pbr3AP2-8 as a potential regulator of PbrC3′H1 expression. Because Pbr3AP2-8 had the highest correlation with PbrC3′H1 expression levels, we selected it for further investigation.

[0043] The recombinant plasmid pSAK277-Pbr3AP2-8 was constructed, and the reporter vector (pGreenII 0800-PbrC3′H1-LUC (full-length promoter)) containing the PbrC3′H1 gene promoter sequence containing different numbers of AP2 binding sites or its mutants was constructed and transformed into Agrobacterium GV3101 and GV3101 (psoup). Tobacco leaves were co-infected with Agrobacterium carrying pSAK277-PbrC3′H1 and the reporter vector, 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-Pbr3AP2-8 was significantly higher than that of the control group.

[0044] The prey vector Pbr3AP2-8-AD was constructed, and the bait vector (PbrC3′H1pro-pAbAi) was constructed using the PbrC3′H1 promoter fragment containing the AP binding site (P: (-466)-(-457)). The Matchmaker Gold Yeast One-Hybrid Library Screening System was used to detect that Pbr3AP2-8 can bind to the promoter fragment containing the AP2 binding site (PbrC3′H1pro).

[0045] The recombinant plasmid pBI221-Pbr3AP2-8-GFP was constructed and co-transformed with the nuclear marker protein AtH2B-mcherry into Arabidopsis protoplasts. GFP fluorescence was observed using a laser confocal microscope, and it was found that Pbr3AP2-8 was located in the cell nucleus.

[0046] The pCAMBIA1300-Pbr3AP2-8 overexpression vector was constructed and transformed into Agrobacterium GV3101 to infect pear callus. After positive tissue was obtained by PCR identification, the positive transgenic tissue and its control (callus transformed with pCAMBIA1300 empty vector) were transferred to MS medium with sorbitol and sucrose (1:1, w:w) as carbon sources for growth.

[0047] Using qPT-PCR detection technology and high-performance liquid chromatography (HPLC), the expression level of Pbr3AP2-8 gene and the content of chlorogenic acid in pear Pbr3AP2-8 positive transgenic tissues were significantly increased.

[0048] Pbr3AP2-8-AD and Pbr3AP2-8-BK vectors were constructed (primer sequences are shown in Table 3 of the attached table) and transformed into Y2H competent cells. The ability of Pbr3AP2-8 to interact with itself was tested using a yeast two-hybrid library screening system.

[0049] Pbr3AP2-8-YNE and Pbr3AP2-8-YCE vectors were constructed (primer sequences are shown in Table 3 of the attached table) and transformed into Agrobacterium tumefaciens GV3101. Tobacco leaves were co-infected with the Agrobacterium strains carrying both Pbr3AP2-8-YNE and Pbr3AP2-8-YCE. Laser confocal microscopy revealed fluorescence in tobacco leaves co-transformed with Pbr3AP2-8-YNE and Pbr3AP2-8-YCE.

[0050] The room temperature described in the present invention is 25±5°C.

[0051] Compared with the prior art, the present invention has the following beneficial effects:

[0052] 1. For the first time, the transcription factor Pbr3AP2-8 that regulates the expression of the PbrC3′H1 gene in pear fruit was revealed.

[0053] 2. The present invention clarifies the mechanism of interaction between Pbr3AP2-8 and the AP2 binding site (P: (-466)-(-457)) in the promoter of the PbrC3′H12 gene in regulating the synthesis of chlorogenic acid in pear fruit, providing a theoretical and practical basis for the targeted improvement of pear fruit quality traits. BRIEF DESCRIPTION OF THE DRAWINGS

[0054] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments.

[0055] Figure 1 Correlation between PbrC3′H1 gene and chlorogenic acid and ethylene content (A) Heat map of the correlation between chlorogenic acid pathway genes and ethylene and chlorogenic acid, the red line represents extremely significant correlation, and the green line represents negative correlation; (B) Fluorescence quantitative verification.

[0056] Figure 2 PbrC3′H1 is localized in the cytoplasm of Arabidopsis protoplasts and tobacco.

[0057] Figure 3 Transient function verification of PbrC3′H1 (A) Expression level of PbrC3′H1 after transient expression; (B) Chlorogenic acid content in pear after transient expression; (C) Expression level of PbrC3′H1 after silencing; (D) Chlorogenic acid content in pear after silencing.

[0058] Figure 4 Stable transformation of PbrC3′H1 into pear callus promotes chlorogenic acid production. (A) Phenotypic image of pear callus; (B) Positive identification of callus; (C) Expression levels of PbrC3′H1 (OE1-OE3) after stable transformation; (D) Changes in chlorogenic acid content after stable transformation. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001; ns, not significant.

[0059] Figure 5 Screening of PbrC3′H1 transcriptional regulators (A) Effects of ethylene and 1-MCP treatments on PbrC3′H1 expression in postharvest 'Fengshui' pear fruit (B) Correlation analysis between PbrERFs and ethylene, chlorogenic acid, and PbrC3′H1, with red lines representing extremely significant correlations and green representing negative correlations; (C) Effects of overexpression of PbrACO1 on the ERF and chlorogenic acid biosynthesis pathway gene families.

[0060] Figure 6 Pbr3AP2-8 is localized in the nuclei of Arabidopsis protoplasts and tobacco.

[0061] Figure 7 Functional validation of Pbr3AP2-8. (A) Expression level of Pbr3AP2-8 after transient overexpression; (B) Chlorogenic acid content in pears after transient expression; (C) Expression level of Pbr3AP2-8 after silencing; (D) Chlorogenic acid content in pears after silencing, *p < 0.05. (E) Expression level of PbrC3′H1 in pear fruit after transient overexpression of Pbr3AP2-8. (F) Expression level of PbrC3′H1 in pear fruit after transient silencing of Pbr3AP2-8.

[0062] Figure 8 Stable transformation of Pbr3AP2-8 pear callus promotes chlorogenic acid synthesis. (A) Phenotypic image of pear callus; (B) Positive identification of callus; (C) Expression levels of Pbr3AP2-8 (OE1-OE3) after stable transformation; (D) Changes in chlorogenic acid content after stable transformation; (E) Changes in PbrC3′H1 expression after overexpression of Pbr3AP2-8. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001; ns, not significant.

[0063] Figure 9 Pbr3AP2-8 interaction map (a) Pbr3AP2-8 self-interaction yeast two-hybrid map; (b) Pbr3AP2-8 dual fluorescence complementation experiment (BiFC).

[0064] Figure 10 The mechanism of action of the Pbr3AP2-8-PbrC3′H1 module in regulating chlorogenic acid metabolism in postharvest pear fruit in response to ethylene. DETAILED DESCRIPTION

[0065] 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.

[0066] Example 1 PbrC3′H1 gene expression profile and its correlation with chlorogenic acid content:

[0067] 1. Collection of tissue samples

[0068] The 'Fengshui' pears with similar tree age and vigor were planted in the orchard in Gaoyou, Yangzhou as experimental materials. Water (control), 0.5 mL·L -1 vinyl ester and 1.5 μL·L -1 Pear fruits (3 per group) were sprayed with 1-methylcyclopropene (1-MCP) and stored at 25° C. When the decay rate of the samples exceeded 30%, the pulp under the peel was collected for analysis.

[0069] 2. Determination of chlorogenic acid content

[0070] Ultra-performance liquid chromatography (UPLC) was used to determine the chlorogenic acid content in 'Hōsui' pear tissue. Fruit samples were thoroughly ground in a mortar and pestle, and 0.1 g of sample was accurately placed in a 5 ml sample tube. 5 ml of methanol (80% by volume) was added. The tube was then sonicated at 100 Hz and 30°C for 20 minutes (inverted every 5 minutes for 30 seconds). The tube was allowed to rest at room temperature for 10 minutes, followed by centrifugation at 12,000 rpm for 15 minutes. After centrifugation, the supernatant was filtered through a 0.22 μm aqueous filter membrane and placed in a small brown bottle for analysis. Chlorogenic acid content was measured using a Thermo high-performance chromatograph.

[0071] 3 Transcriptome sequencing

[0072] Total RNA was extracted from pear peel and flesh 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 NEBNext Ultra™ RNA Library Prep Kit for Illumina (E7770, NEB, USA). The constructed libraries were sequenced using the HiSeq X-ten sequencing platform. After removing low-quality reads, clean reads were obtained for subsequent data analysis. HISAT2 software was used for alignment with the Dangshan Pear genome database (http: / / peargenome.njau.edu.cn / ), and gene expression levels (FPKM) were calculated. Transcriptome sequencing and analysis were performed by Biomarker Biotechnology Co., Ltd. (Beijing, China). Based on transcriptome annotation, we discovered five C3'H-encoding genes in the pear genome (see Table 1 in the Supplementary Table).

[0073] 4qRT-PCR detection

[0074] Total RNA was extracted from 'Fengshui' pear tissue using a plant total RNA extraction kit (RE-05011, Chengdu Fuji Biotechnology Co., Ltd.) according to the kit instructions. All centrifuge tubes and pipette tips used during the extraction process were RNase-free. The extracted total RNA was converted to cDNA using the Quanshijin AE301-02 EasyScript cDNA First-Strand Synthesis Kit (AE301-02, Beijing Quanshijin Biotechnology). Finally, PrimeScript TM The relative expression of PbrC3′H1 at different developmental stages was determined using an RT-PCR Kit (RR014A, Bio-Rad Biotechnology (Beijing) Co., Ltd.). The pear Tublin gene (PbrTub) was used as a reference gene. Primer sequences for the PbrC3′H1 gene are shown in Table 2, SEQ ID NOs. 6 to 9.

[0075] 5. Screening of genes involved in pear chlorogenic acid metabolism

[0076] The correlation between the expression levels of genes related to the chlorogenic acid synthesis pathway and the chlorogenic acid and ethylene contents during the growth and development of 'Fengshui' pear fruit was analyzed, and the key genes involved in chlorogenic acid synthesis during the postharvest storage of pear fruit were screened.

[0077] Experimental results: During the growth and development of 'Fengshui' pear fruit, the correlation between the expression of the PbrC3′H1 gene, chlorogenic acid, and ethylene content was analyzed at 0, 6, 12, 18, and 24 days of storage after harvest. With the increase in storage time, the expression of the PbrC3′H1 gene and the chlorogenic acid content gradually decreased, while the ethylene content gradually increased; there was a high correlation between the expression of the PbrC3′H1 gene and the chlorogenic acid content (correlation coefficient = 0.98). qRT-PCR verified the transcriptome expression pattern of PbrC3′H1. Therefore, we selected the PbrC3′H1 gene for further study ( Figure 1 Example 2 Functional Verification of PbrC3′H1 Gene

[0078] Determination of subcellular localization of protein encoded by 1PbrC3′H1 gene

[0079] Using 'Hōsui' pear fruit cDNA as a template, the coding sequence of the PbrC3'H1 gene was amplified (primer sequences are shown in SEQ ID NO. 10 and SEQ ID NO. 11 in Table 3 of the Appendix). The recombinant plasmid pBI221-PbrC3'H1-GFP was constructed by inserting the recombinant plasmid into the pBI221-GTP vector containing the restriction enzyme sites Xbal and BamHI. The recombinant plasmid was then co-transformed with the endoplasmic reticulum marker ER-rk-mcherry into Arabidopsis protoplasts and tobacco plants. Fluorescence signals were observed using a laser confocal microscope (Leica Microsystems, Germany), revealing that PbrC3'H1 was located in the endoplasmic reticulum ( Figure 2 The CDS sequence of the PbrC3′H1 gene is shown in SEQ ID NO.3; the protein sequence encoded by the PbrC3′H1 gene is shown in SEQ ID NO.4.

[0080] 2. Determination of transient overexpression or silencing of PbrC3′H1 gene in pear fruit

[0081] Using 'Hōsui' pear fruit cDNA as a template, the coding sequence of the PbrC3′H1 gene was amplified (primer sequences are shown in Table 3 of the Appendix, SEQ ID NO. 30 and SEQ ID NO. 31). This was then inserted into the pCAMBIA1300 vector containing the restriction enzyme sites Xbal and BamHI to construct the pCAMBIA1300-PbrC3′H1 overexpression vector. The vector was then transformed with Agrobacterium tumefaciens GV3101 and infected with pear fruit. The fruit was placed in an incubator in the dark for 1 day and then in the light for 2 days. Pear pulp was extracted for qPCR and chlorogenic acid content determination ( Figure 3 ).

[0082] Using 'Hōsui' pear fruit cDNA as a template, the coding sequence of the PbrC3′H1 gene was amplified (primer sequences are shown in SEQ ID NO. 32 and SEQ ID NO. 33 in Table 3 of the Appendix). The vector was inserted into the TRV vector containing the restriction sites Xbal and SmalI to construct the PbrC3′H1-TRV expression vector. The vector was then transformed with Agrobacterium tumefaciens GV3101 and infected with pear fruit. The fruit was placed in an incubator in the dark for 1 day and in the light for 2 days. Pear pulp was extracted for qPCR and chlorogenic acid content determination ( Figure 3 ).

[0083] Obtaining callus tissue from pear overexpressing PbrC3′H1 gene

[0084] Using 'Hōsui' pear fruit cDNA as a template, the coding sequence of the PbrC3′H1 gene was amplified (primer sequences are shown in SEQ ID NO. 30 and SEQ ID NO. 31 in Table 3 of the Supplementary Table). This sequence was then inserted into the pCAMBIA1300 vector containing the restriction enzyme sites Xbal and BamHI to construct the pCAMBIA1300-PbrC3′H1 overexpression vector. The vector was then transformed with Agrobacterium tumefaciens GV3101 and 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 (carbon source: 30 g L-1) containing hygromycin (Hyg). -1 After PCR identification, the positive tissues and the control (transformed empty pCAMBIA1300) were transferred to a sorbitol / sucrose (15 g·L -1 , 1:1) as the carbon source on MS medium (containing Hyg).

[0085] 4 Analysis of PbrC3′H1 gene expression in callus tissue

[0086] Total RNA was extracted from callus using a plant total RNA extraction kit (RE-05011, Chengdu Fuji Biotechnology Co., Ltd.) and reverse transcribed to synthesize cDNA. Real-time qRT-PCR was used to detect PbrC3′H1 gene expression. Primer sequences for the PbrC3′H1 gene are shown in Table 2 as SEQ ID NOs. 6 and 7. Using the pear Tublin gene (PbrTub) as a reference gene, primer sequences are shown in Table 2 as SEQ ID NOs. 8 and 9.

[0087] 5. Determination of chlorogenic acid content in callus tissue

[0088] Fruit samples were thoroughly ground in a mortar and pestle. Accurately remove 0.1 g of sample and place in a 5 ml sample tube. Add 5 ml of methanol (80% by volume). Ultrasonication was then performed at 100 Hz and 30°C for 20 min (inverting every 5 min for 30 s). The tubes were allowed to rest at room temperature for 10 min, followed by centrifugation at 12,000 rpm for 15 min. After centrifugation, the supernatant was filtered through a 0.22 μm aqueous filter membrane and placed in a small brown bottle for analysis. Chlorogenic acid content was measured using a Thermo high-performance chromatograph.

[0089] Experimental results: PbrC3′H1 is located in the endoplasmic reticulum of Arabidopsis protoplasts (e.g. Figure 2 ). Using qPT-PCR detection technology and high-performance liquid chromatography, it was found that the expression level of PbrC3′H1 gene and the content of chlorogenic acid in pear PbrC3′H1 positive transgenic callus and pear fruit were significantly increased. Overexpression of PbrC3′H1 gene in pear callus and pear fruit can increase the accumulation of chlorogenic acid (such as Figure 3 and Figure 4 ).

[0090] Example 3 Screening of PbrC3′H1 Upstream Regulatory Genes and Their Regulatory Mechanism

[0091] Screening of upstream regulatory genes of 1PbrC3′H1

[0092] Using 'Fengshui' pear fruit DNA as a template, a 1956-bp sequence upstream of the PbrC3′H1 gene promoter (SEQ ID NO. 5) was cloned and submitted to the PlantCARE database (http: / / bioinformatics.psb.ugent.be / webtools / plantcare / html / ) to predict cis-regulatory elements. By overexpressing the PbrACO1 gene (a key factor in ethylene biosynthesis) and analyzing the expression profiles of PbrERFs during postharvest storage (using a screening threshold of >0.8 or <-0.8), we identified the transcription factor Pbr3AP2-8 as a potential regulator of PbrC3′H1 expression. Because Pbr3AP2-8 had the highest correlation with PbrC3′H1 expression levels, we selected it for further study. Figure 5 ).

[0093] 2 Dual luciferase reporter assay

[0094] Using 'Fengshui' pear fruit cDNA as a template, the coding sequence of the Pbr3AP2-8 gene was cloned (primer sequences are shown in Table 3, SEQ ID NOs. 16 and 17) and inserted into the pSAK277 vector to construct the pSAK277-Pbr3AP2-8 recombinant vector. The CDS sequence of the Pbr3AP2-8 gene is shown in SEQ ID NO. 1; the protein sequence encoded by the Pbr3AP2-8 gene is shown in SEQ ID NO. 2.

[0095] Using the fruit DNA of 'Fengshui' pear as a template, the promoter sequence of the PbrC3′H1 gene with a possible AP2 binding site (P: (-466)-(-457)) was cloned (primer sequences are shown in SEQ ID NO.14 and SEQ ID NO.15 in Table 3 of the Appendix) and inserted into the pGreenII 0800-LUC vector digested with HindIII and BamHI to construct a reporter vector.

[0096] The transcription factor recombinant plasmid (pSAK277-Pbr3AP2-8) and reporter vector (pGreenII0800-PbrC3′H1-LUC) that were sequenced correctly were transformed into Agrobacterium GV3101 and GV3101 (psoup) respectively. The positive strains were selected and expanded to OD 600 Bacteria were harvested when the luciferase concentration (pSAK277) was 0.6-0.8 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 were infected. Before injection, Agrobacterium containing pSAK277-Pbr3AP2-8 and the reporter vector were mixed at a 9:1 (v / v) ratio. Tobacco leaves co-transfected with Agrobacterium carrying both the empty pSAK277 vector and the corresponding reporter vector served as controls. 60-72 hours after infection, leaves were harvested and assayed for firefly and Renilla luciferase activities using the Dual-Luciferase Reporter Assay System kit (Promega, USA) (see the manufacturer's instructions for specific procedures). The results showed that the Luc / Ren ratio in tobacco leaves co-transformed with pSAK277-Pbr3AP2-8 and pGreenII 0800-PbrC3′H1-LUC was significantly higher than that in the control.

[0097] 3. Yeast one-hybrid assay (Y1H)

[0098] Using 'Hōsū' pear fruit cDNA as a template, the Pbr3AP2-8 gene coding sequence was cloned (primer sequences are shown in SEQ ID NOs. 18 and 19 in Table 3 of the Supplementary Table) and inserted into pGADT7 with restriction sites EcoRI and BamHI to create the prey vector Pbr3AP2-8-AD. Using 'Hōsū' pear fruit DNA as a template, a 300-bp PbrC3′H1 gene promoter fragment containing the AP2 binding site (P: (-466)-(-457)) was inserted into the pAbAi vector with restriction sites KpnI and XhoI (primer sequences are shown in SEQ ID NOs. 20 and 21 in Table 3 of the Supplementary Table) to create the bait vector (PbrC3′H1pro-pAbAi).

[0099] The bait and prey vectors were co-transformed into yeast strains, and Y1H assays were performed using the Matchmaker Gold Yeast One-Hybrid Library Screening System (Weidi, Shanghai, China) (see the manufacturer's instructions for specific procedures). PbrC3′H1pro-pAbAi self-activation assays were performed on SD / -Ura-deficient medium supplemented with aurobacitin A (AbA), and appropriate AbA concentrations were selected for subsequent experiments. Yeast strains co-transformed with the bait and prey vectors were then cultured on SD / -Leu-deficient medium supplemented with appropriate concentrations to test the binding ability of Pbr3AP2-8 to the PbrC3′H1 promoter. Yeast strains co-transformed with pGAD7-p53 and p53-AbAi served as positive controls, while yeast strains co-transformed with pGADT7-AD and PbrC3′H1pro (a 300-bp fragment of the PbrC3′H1 gene promoter containing the AP2 binding site) served as negative controls. The Matchmaker Gold Yeast One-Hybrid Library Screening System was used to detect that Pbr3AP2-8 could bind to the promoter fragment (PbrC3′H1pro) containing the AP2 binding site.

[0100] Example 4 Functional Verification of Pbr3AP2-8

[0101] Determination of subcellular localization of protein encoded by 1Pbr3AP2-8 gene

[0102] The coding sequence of the Pbr3AP2-8 gene was amplified using the fruit cDNA of the 'Hōsui' pear (primer sequences are shown in SEQ ID NO. 12 and SEQ ID NO. 13 in Table 3 of the Appendix) and inserted into the pBI221-GTP vector to construct the recombinant plasmid pBI221-Pbr3AP2-8-GFP. This recombinant plasmid was then co-transformed with the nuclear marker AtH2B-mcherry into Arabidopsis protoplasts and tobacco plants. Fluorescence signals were observed using a confocal microscope (Leica Microsystems, Germany). Figure 6 ).

[0103] 2. Determination of transient overexpression or silencing of Pbr3AP2-8 gene in pear fruit

[0104] Using 'Hōsui' pear fruit cDNA as a template, the coding sequence of the Pbr3AP2-8 gene was amplified (primer sequences are shown in Table 3 of the Appendix, SEQ ID NO. 34 and SEQ ID NO. 35). This was then inserted into the pCAMBIA1300 vector with restriction enzyme sites Xbal and BamHI to construct the pCAMBIA1300-Pbr3AP2-8 overexpression vector. The vector was then transformed with Agrobacterium tumefaciens GV3101 and infected with pear fruit. The fruit was placed in an incubator in the dark for 1 day and then in the light for 2 days. Pear pulp was extracted for qPCR and chlorogenic acid content determination (e.g., Figure 7 ).

[0105] Using 'Hōsui' pear fruit cDNA as a template, the coding sequence of the Pbr3AP2-8 gene was amplified (primer sequences are shown in Table 3 of the Appendix, SEQ ID NO. 36 and SEQ ID NO. 37). The vector was inserted into the TRV vector containing the restriction sites Xbal and SmalI to construct the Pbr3AP2-8-TRV expression vector. The vector was then transformed with Agrobacterium tumefaciens GV3101 and infected with pear fruit. The fruit was placed in an incubator in the dark for 1 day and in the light for 2 days. Pear pulp was extracted for qPCR and chlorogenic acid content determination (e.g., Figure 7 ).

[0106] Obtaining Pear Callus Overexpressing Pbr3AP2-8 Gene

[0107] Using 'Hōsui' pear fruit cDNA as a template, the coding sequence of the Pbr3AP2-8 gene was amplified (primer sequences are shown in SEQ ID NO. 34 and SEQ ID NO. 35 in Table 3 of the Supplementary Table). This was then inserted into the pCAMBIA1300 vector containing the restriction enzyme sites Xbal and BamHI to construct the pCAMBIA1300-Pbr3AP2-8 overexpression vector. The vector was then used to infect pear calli (induced from small fruit of P. communis cv. 'Clapp's Favorite'). The infected calli were then transferred to MS medium (carbon source: 30 g L-1) containing hygromycin (Hyg).-1 After PCR identification, the positive tissues and the control (transformed empty pCAMBIA1300) were transferred to a sorbitol / sucrose (15 g·L -1 , 1:1) as the carbon source on MS medium (containing Hyg).

[0108] 3 Analysis of Pbr3AP2-8 and PbrC3′H1 gene expression in callus tissue

[0109] Total RNA was extracted from callus using a plant total RNA extraction kit (RE-05011, Chengdu Fuji Biotechnology Co., Ltd.) and reverse transcribed to synthesize cDNA. Real-time qRT-PCR was used to measure the expression levels of the Pbr3AP2-8 and PbrC3′H1 genes. The primer sequences for the PbrC3′H1 gene are shown in Table 2, and the primer sequences for the Pbr3AP2-8 gene are shown in Table 3, SEQ ID NOs. 22 and 23. The pear Tublin gene (PbrTub) was used as a reference gene.

[0110] 4 Determination of chlorogenic acid content in callus tissue

[0111] Take the fruit sample and grind it thoroughly in a mortar. Accurately take 0.1g of sample in a 5ml sample tube and add 5ml of methanol solution (volume coefficient is 80%). Then ultrasonicate at 100HZ and 30℃ for 20min (invert once every 5min, each time for 30s), let it stand at room temperature for 10min, and then centrifuge at 12000rpm for 15min. After centrifugation, filter the supernatant with a 0.22um water filter membrane and put it into a small brown bottle for testing. The instrument used is a high performance chromatograph (Thermo) to measure the chlorogenic acid content (such as Figure 8 ).

[0112] 5 Yeast two-hybrid assay

[0113] Using 'Hōsui' pear fruit cDNA as a template, the coding sequence of the Pbr3AP2-8 gene was amplified (primer sequences are shown in Table 3, SEQ ID NOs. 24 and 25, and SEQ ID NOs. 26 and 27). The primers were inserted into the pGADT7 bait vector (with EcoRI and BamHI restriction sites) and the pGBDT7 prey vector (with EcoRI and BamHI restriction sites), respectively, to construct the Pbr3AP2-8-AD and Pbr3AP2-8-BK expression vectors.

[0114] The bait vector and prey vector were co-transformed into yeast strains, and the Y2H experiment was carried out using the yeast two-hybrid library screening system (see the instructions for specific steps). The yeast strains co-transformed with the bait vector and the prey vector were cultured on SD / -Leu / Trp and SD / -Leu / -Trp / -His / -Ura deficient medium to detect the binding ability of Pbr3AP2-8 itself. The yeast strain co-transformed with pGADT7-AD and pGBDT7-p53 was used as a positive control, and the yeast strain co-transformed with pGADT7-AD and pGBDT7-Lam was used as a negative control. The yeast two-hybrid library screening system detected that Pbr3AP2-8 can bind to itself ( Figure 9 ).

[0115] 6 Bimolecular fluorescence complementation (BiFC)

[0116] Using 'Hōsui' pear fruit cDNA as a template, the coding sequence of the Pbr3AP2-8 gene was amplified (primer sequences are shown in Table 3, Appendix, SEQ ID NO. 28 and SEQ ID NO. 29). These primers were inserted into the YCE and YNE expression vectors, which have restriction enzyme sites of XbaI and BamHI, respectively, to construct the Pbr3AP2-8-YNE and Pbr3AP2-8-YCE expression vectors, which were then co-transformed into tobacco plants. Fluorescence signals were observed using a laser confocal microscope (Leica Microsystems, Germany), revealing that Pbr3AP2-8 can interact with itself, with the interaction site located in the cell nucleus. Figure 9 ).

[0117] Experimental results: Pbr3AP2-8 is located in the cell nucleus (e.g. Figure 6 Pbr3AP2-8 can interact with itself to form dimers. Overexpression of the Pbr3AP2-8 gene in pear callus and pear fruit can increase the expression of the PbrC3′H1 gene and the accumulation of chlorogenic acid (e.g. Figure 7 and 8 ).

[0118] In summary, the present invention is based on a transcription factor Pbr3AP2-8, which can bind to the AP2 binding site in the PbrC3′H1 gene promoter to regulate its transcription level (such as Figure 10 ), affecting the accumulation of chlorogenic acid and flavor quality of pear fruit, and has great market application potential for breeding high / low chlorogenic acid pear varieties.

[0119] 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.

[0120] sequence table

[0121] SEQ ID NO.1 1440

[0123] DNA

[0124] Pyrus bretschneideri

[0125] ATGTTGGATCTTAATCTGAACGTTGTCGTTTCGGGGCAAAACGACGTCCAATCGTGC

[0126] GGCACCCAAATGGACGAGTCGGGGACGTCCAACTCGTCCGTTGTCAATGCCGACGC

[0127] GTCCAGCACAACGACGACTCGTGCTCCGCACCGGCCCGCCAGAAATGACGCCGTCA

[0128] CGATCTTCAACTTCGGTATTCTCAAGGTCAGGGACGGAGAAGAAGAGGAAGACGAC

[0129] GACGTCGTCGTGACCAAGGAACTGTTTCCGGTCACTGGAGGCTTGAACAATTGGCCA

[0130] GGGCAGGGGGAGTCCTCTTCATCATCGCCGTTGGTGAGGAAGAATCTGATGGAGCT

[0131] TGGGTTCACTCGTGGCAGCGCCGGAGAGGTCAGGTTGGTTCAACAGAAGCAACCAC

[0132] CACAACCAGCTGCGCCCGCCGCAGCAGCAGGCGAAGAAGAGCAGGAGAGGACC

[0133] CAGGTCTCGGAGCTCTCAGTACAGAGGGTCACCTTCTACAGAAGAACTGGTAGAT

[0134] GGGAATCGCACATTTGGGACTGTGGGAAACAAGTGTATTTGGGTGGATTTGACACT

[0135] GCTCATGCTGCGGCTAGAGCCTACGACCGCGCTGCGATTAAGTTCAGAGGAGTTGAT

[0136] GCTGATATCAATTACAACCTCAGTGATTATGAGGATGATCTGAAACAGATGAAGAA

[0137] TTTGACTAAGGAAGAATTCGTGCACATACTGCGTCGGCAGAGTACTGGCTTCTCCGAG

[0138] GGGCAGCTCCAGATATAGAGGGGTTACACTCCACAAATGTGGTCGATGGGAAGCTC

[0139] GAATGGGACAGTTCCTCGGAAAAAAGTATATATATCTTGGGCTATTCGACAGCGAA

[0140] GTAGAAGCTGCAAGGGCTTATGACAAGACAGCAATCAAATAATGGAAGGGAAG

[0141] CGGTCACCAACTTTGAGCCAAGCACATATGAAGGGAAGATGATGTCTGAGGCTGGT

[0142] AATGAAGATGGCAATCACAATCTTGATCTAAATTTGGGGATATCTCCCCCCTCATTT

[0143] GGCACGGGTCAAAGGAAGGCGAAGGGCGACTTCAGTTCCACTCTGGCCCTTTTGA

[0144] CGTGCACAATGGAAAAAGGATGGAGCACAATGTGAACTTAACGATGAGTGATCCAC

[0145] CTTTTAAAGGGCTAGTAATGACATCAGATCACCATCCACCATTGTGGAACGGTGTAT

[0146] ATCCTAGTTATTTTTCCAATCAGGAAAAGCGCAACAGAGAAGAGCCTTGCATTAGGAT

[0147] CTCAAGGACACCCCAACTGGGCTTGGCAAATGCATGGGCAGGTTACCCCAATGCCA

[0148] CTGTTCCTACTGCAGCATCATCAGGATTCTCATTTTCAGCTAACGCTCCAGCCGCTG

[0149] CTGTCCACCCCTTACAACCGCCAAACCCAACAGCCCTCAATCTCTGTTTTACTTCACC

[0150] AGCCGCGGCCACCACTCGCACATCTCAATGASEQ ID NO.2 479

[0152] PRT

[0153] Pyrus bretschneideri

[0154] MLDLNLNVVVSGQNDVQSCGTQMDESGTSNSSVVNADASSTNDDSCSARAARNDAVT

[0155] IFNFGILKVRDGEEEDDDVVVTKELFPVTGGLNNWPGQGESSSSSPLVRKNLMELGFT

[0156] RGSAGEVRLVQQKQPPQPAAPPPQQQAKKSRRGPRSRSSQYRGVTFYRRTGRWESHIW

[0157] DCGKQVYLGGFDTAHAAARAYDRAAIKFRGVDADINYLSDYEDDLKQMKNLTKEEF

[0158] VHILRRQSTGFSRGSSRYRGVTLHKCGRWEARMGQFLGKKYIYLGLFDSEVEAARAYD

[0159] KTAIKYNGREAVTNFEPSTYEGKMMSEAGNEDGNHNLDLNLGISPPSFGTGQKEGEGRL

[0160] QFHSGPFDVHNGKRMEHNVNLTMSDPPFKGLVMTSDHHPPLWNGVYPSYFSNQENAT

[0161] EXSLALGSQGHPNWAWQMHGQVTPPMPLFSTAASSGFSANAPAAAVHPLQPPNPTAL

[0162] NLCFTSPAAATTRTSQSEQ ID NO.3 1539

[0164] DNA

[0165] Pyrus bretschneideri

[0166] ATGGCTCTCCCTGGACTACTTCTCCCCACTTCATCAATCATCATCTTCATCTTCCTTG

[0167] CATACAAGCTCTACCAACGGCTCAGATTCAAGCTCCCACCCGGTCCACGTCCATGGC

[0168] CCGTCGTCGGAAACCTCTACCAGATAAAGCCGGTGAGGTTCCGCTGCTACGCCGAGT

[0169] GGGCAGAGAAATACGGGTCCATCTTCTCAGTGTGGATTGGATCCACACTGAACGTC

[0170] GTAGTGAGCAACACAGAGCTTGCAAAGGAGGTGCTCAAGGAGAATGACCAGAAAC

[0171] TGGCTGATAGGCACAGGAACAGATCGACCGCAAGGTTTAGCAGGGACGGTCAGGAT

[0172] CTTATCTGGGCTGACTATGGGCCGCACTACGTGAAGGTGAGGAAGGTGTGCACTCTT

[0173] GAGCTTTTCTCTGCAAAGAGGCTTGAGGATCTCCGACCCATTAGAAGATGAGGTC

[0174] ACTGCTATGGTCGAGTCCATCTTCAACCACTGCACCAAACCTGAAAACCAAGGGAA

[0175] AAGTTTGGCAGTGAAGAAGTATTTGGGAAACGTGGCTTTCAACAACATAACAAGGC

[0176] TAGCATTTGGGAAACGTTTCATCAGCTCCGATGAAATAACTGATGAGCAAGGGAAA

[0177] CGATTCAGAGCTCTTATGGCCAACGGGGTGAAAATAAGTGGATCGCTCTCGGTGGCT

[0178] GAGCACATTTCATGGTTGCGTTGGGCATGCCCGTTTGACGAAGAGGCGTTTGCTCGA

[0179] CACGGAGCAAACAGAGACAGACTCACCAAGGAAATCATGGACGAACACGCTCAGG

[0180] CACGCTCCAAGGGCGGCGTTGCCAAGAGCCACTTTGTGGATGCATTGTTTACCTTGA

[0181] AGGAAAAGTATGACCTTAGCATGGACACTGTTATTGGACTTCTTTGGGATATGATGG

[0182] CTGCCGGTACAGACACCACAGCAATCTCAGTTGAATGGGCCATGGCTGAGCTGATT

[0183] AAGAACCCGAGGGTGCAACAAAAGGCTCAAGAGGAGCTAGATCGGGTTATTGGGC

[0184] ACGAACGGGTCATGACCGAAAATGATTTCTCAAACCTCCCTTACCTACAATGTGTAG

[0185] CAAAGGAAGCACTGCGGTTGCACCCTCCAACACCACTAATGCTCCCCCACCGAGCC

[0186] AATGCCGATGTCAAGATTGGTGGCTACGACATCCCCAAGGGTTCGATTGTTCATGTT

[0187] AATGTTTGGGCTGTAGCACGTGATCCCGCTGTCTGGAAAAACCCAAATGAATTTCGA

[0188] CCTGAAAGGTTCCTAGAGGAAGATGTTGACATGAAAGGCCATGATTTTAGACTACTT

[0189] CCATTTGGAGCAGGCAGGCGAGTATGTCCCGGAGCTCAACTCGGTATCAACTTGGTT

[0190] GCCTCCATGTTGGGTCATCTGTTGCACCATTTCAGTTGGGCTCCTCCTGAAGGGGTG

[0191] AAACCAGAGGAGATTGACTTGTCGGAAAATCCAGGGATAGTCACTTACATGGCAAC

[0192] CCCAGTACAAGTAGTGCCTACTCCAAGGCTCCCTTCACACTTGTACAAGCGTGTGGC

[0193] CGCGAATATGTAASEQ ID NO.4 512

[0195] PRT

[0196] Pyrus bretschneideri

[0197] MALPGLLLPTSSIIIIFIFLAYKLYQRLRFKLPPGPRPWPVVGNLYQIKPVRFRCYAEWAE

[0198] KYGSIFSVWIGSTLNVVVSNTELAKEVLKENDQKLADRHRNRSTARFSRDGQDLIWAD

[0199] YGPHYVKVRKVCTLELFSAKRLEDLRPIREDEVTAMVESIFNHCTCKPENQGKSLAVKKY

[0200] LGNVAFNNITRLAFGKRFISSDEITDEQGKRFRALMANGVKISGSLSVAEHISWLRWACP

[0201] FDEEAFARHGANRDRLTKEIMDEHAQARSKGGVAKSHFVDALFTLKEKYDLSMDTVIG

[0202] LLWDMMAAGTDTTAISVEWAMAELIKNPRVQQKAQEELDRVIGHERVMTENDFSNLP

[0203] YLQCVAKEALRLHPPTPLMLPHRANADVKIGGYDIPKGSIVHVNVWAVARDPAVWKN

[0204] PNEFRPERFLEEDVDMKGHDFRLLPFGAGRRVCPGAQLGINLVASMLGHLLHHFSWAPP

[0205] EGVKPEEIDLSENPGIVTYMATPVQVVPTPRLPSHLYKRVAANMSEQ ID NO.5 1956

[0207] DNA

[0208] Pyrus bretschneideri

[0209] GCCCTCAGTTGGAGATGGAGATAAATATGATCTTAATTGTTTATTAAAATATTAAT

[0210] ATCTTAAAAAACCAGAAGATTAAACGAGCTCCCTGATTAGTCCTTAATGGAAGAT

[0211] GACCTTACAAGCGGTGCCTGCTCCCAGGCTTTCATCACAGTTATACAAACGAGTGGC

[0212] TGCACCGCACACATGTAACTTGTCAGTTTCTTCTTTTTATTATTTTATTTCTTTTGA

[0213] GTTTTGATTTCTGCAAAGTCCTACTTATTAATCTTTTCAATTTAATCCATCATTAATTA

[0214] CAGACATGTTTAATTAAGATATGTTGGCGGCCCACCGTCCGTGTCGTTCCAAAAAAAG

[0215] TTGACGTCTGATGAATATGGACCGCTGGGACCAGAGCACTCGGATCATCTCTCATCC

[0216] TCACATTCAATAGGCCTTAAATAATTCACCGACCACCCTACCCTCTCTGTCTCTTTCT

[0217] CGTCCACTCATCCCACAAGCAAATAAATTCAAATCATGTGATGATATTTAGTTTC

[0218] AGAAAATGATTTTCGCCCATTCTTTAAACCTTTGTTTTTTTCATTATTTTTAACTCTCG

[0219] AAATGAATTAATAAAAATAAGAAACAAAAACTAAAGAGCGTGTGAAAACCAAA

[0220] AGAAATATGTAAAAATTATTACTCTCGTTCCAAACTCTGCAAAATTGACCGAATATA

[0221] TCACAACATTTGACGCTAGCAATGAAATGTTTGAATGTGAAAGTTCAGCATAGAGAT

[0222] TCGATGTGAAGTTGGAGCATCGGTTGTTGACGACTGACGCCCACATGTTCTTCTTTT

[0223] GTTTGGACTTGTAAATGGCAACGTAAGATAATGGGCAATGGCATATCAAACTCCCTC

[0224] CAGCCTCCATTCTTGCTTCAACCCCCATTCCTCCTCCATCTCCCGCTCTCATCCTACA

[0225] TTCTCCAACTTTTTTGTTTTTTTTTTTTTAAGGGTTTTTGTTTTGTAAAGAGTTTATTATTT

[0226] TTATCTTAAATCGGGCATAAATTACACTCATGACAAATTAGAACATGTTACGTCTT

[0227] TAAAGTGTTTATTGAAAAAAAATATCCCGTGTAATAGAGAGTTTGTCAACATATCCA

[0228] ATTAAATACCACTACGATTTTTTTTTTTTGTCAAGCAAGAGAGCTTTCATTCCATT

[0229] GAAAACTAGTATAAGGCCAACATGAAATAGGTAAACACACACATAAAGTAAAA

[0230] GCCTCAAGTGAGATAAGAGCGGCAACTCAAGATATAACGGGAGGGCCACACAAA

[0231] CTTAGGCTCTAAGACATAATGAGAGCTGCAAAAACAAATAACAAAAAACTCTAATC

[0232] TCAAGCAGCATAAACTAGACCGTCATGCCGTCACATCCTCGAAGGAAGCCAAACTC

[0233] CGGACCAAGAGAGTAGACCAAATCCAAGTTCCTTTCACAAACCCCCGCATATAT

[0234] ATAAACTACAATTTCAAAAATAGAATTCCACAAGCTGGGTCCTAAAATAGATGCAT

[0235] AATAATTAAGGTTAATGCTAGCAACTTTCTCATTTACAATTCCTTTCTTA

[0236] TCTTACAATGTTACTTGATCCGAACAATTTACTTTATATCACTCATTAAAGAT

[0237] CAACTCTATAAAATCAATTAAATTAATCATTTAACTGTTGTTTAACATTTGTC

[0238] AAAATTTGGTGTTTAAGTGCGAAACATAATTTGTCTATTTTAGACAACATTTGAA

[0239] AGATAAGTACCAAAATCGAGAAGATTGCTAGTATTCTTTTAAGGATAATGTTAGAA

[0240] AGATAAATTTACAAACTAAATAACGTATTTTCAATAAGAATGAACATGTTTATCAA

[0241] CGTTTAAGTAATAAATTAATCATGAACTTATATATCATTTAGCTTTTAAAATTTTTTC

[0242] TAAAATTTAGTTTTCCTGGTAATATACCTCTTAATAATAACAGGTTTGTGGCAGTGA

[0243] GGAGTGAGGGGTAG

[0244]

[0245]

Claims

1. Use of the PbrC3'H1 gene having a nucleotide sequence as shown in SEQ ID NO. 3 or biological materials related to the PbrC3'H1 gene in regulating the accumulation of chlorogenic acid in the fruit of Rosaceae fruit trees.

2. The use according to claim 1, characterized in that: The biological material related to the PbrC3'H1 gene is at least one of (a1) to (a7): (a1) a PbrC3'H1 protein having an amino acid sequence as shown in SEQ ID NO.4 encoded by the PbrC3'H1 gene; (a2) an expression cassette containing the PbrC3'H1 gene; (a3) a recombinant vector containing the PbrC3'H1 gene, or a recombinant vector containing the expression cassette described in (a2); (a4) a recombinant microorganism containing the PbrC3'H1 gene, or a recombinant microorganism containing the expression cassette described in (a2), or a recombinant microorganism containing the recombinant vector described in (a3); (a5) a transgenic plant cell line containing the PbrC3'H1 gene, or a transgenic plant cell line containing the expression cassette of (a2), or a transgenic plant cell line containing the recombinant vector of (a3); (a6) transgenic plant tissue containing the PbrC3'H1 gene, or transgenic plant tissue containing the expression cassette described in (a2), or transgenic plant tissue containing the recombinant vector described in (a3); (a7) A transgenic plant organ containing the PbrC3'H1 gene, or a transgenic plant organ containing the expression cassette of (a2), or a transgenic plant organ containing the recombinant vector of (a3).

3. The use according to claim 1, characterized in that: Overexpression of the PbrC3'H1 gene promotes chlorogenic acid accumulation in fruits.

4. Application of the AP2 / ERF family transcription factor Pbr3AP2-8 having an amino acid sequence as shown in SEQ ID NO. 2 or a biological material related to the AP2 / ERF family transcription factor Pbr3AP2-8 in regulating the accumulation of chlorogenic acid in the fruit of Rosaceae fruit trees.

5. The use according to claim 4, characterized in that: The biological material related to the transcription factor Pbr3AP2-8 is at least one of the following (b1) to (b7): (b1) gene Pbr3AP2-8 encoding the transcription factor Pbr3AP2-8; (b2) an expression cassette containing the gene Pbr3AP2-8 described in (b1); (b3) a recombinant vector containing the gene Pbr3AP2-8 described in (b1) and a recombinant vector containing the expression cassette described in (b2); (b4) a recombinant microorganism containing the gene Pbr3AP2-8 described in (b1), or a recombinant microorganism containing the expression cassette described in (b2), or a recombinant microorganism containing the recombinant vector described in (b3); (b5) a transgenic plant cell line containing the gene Pbr3AP2-8 described in (b1), or a transgenic plant cell line containing the expression cassette described in (b2), or a transgenic plant cell line containing the recombinant vector described in (b3); (b6) transgenic plant tissue containing the gene Pbr3AP2-8 described in (b1), or transgenic plant tissue containing the expression cassette described in (b2), or transgenic plant tissue containing the recombinant vector described in (b3); (b7) A transgenic plant organ containing the gene Pbr3AP2-8 described in (b1), or a transgenic plant organ containing the expression cassette described in (b2), or a transgenic plant organ containing the recombinant vector described in (b3).

6. The use according to claim 5, characterized in that: The nucleotide sequence of the gene Pbr3AP2-8 encoding the transcription factor Pbr3AP2-8 in (b1) is shown as SEQ ID NO.

1.

7. The use according to claim 4 or 5, characterized in that: Overexpression of the gene Pbr3AP2-8 encoding the transcription factor Pbr3AP2-8 or increasing the activity or content of the transcription factor Pbr3AP2-8 protein promotes the accumulation of chlorogenic acid in the fruit; the AP2 / ERF family transcription factor Pbr3AP2-8 interacts with the PbrC3'H1 gene described in claim 1 to regulate the accumulation of chlorogenic acid in pear fruit.

8. The use according to claim 7, characterized in that: The AP2 / ERF family transcription factor Pbr3AP2-8 promotes the expression of the PbrC3'H1 gene by combining with the AP2 binding site in the PbrC3'H1 gene promoter, thereby promoting the accumulation of chlorogenic acid in the fruit.

9. A method for promoting the accumulation of chlorogenic acid in the fruit of a Rosaceae fruit tree, characterized in that: The method is at least one of the following (c1) to (c3): (c1) overexpressing the gene Pbr3AP2-8 encoding the transcription factor Pbr3AP2-8; (c2) increasing the activity or content of the transcription factor Pbr3AP2-8 protein; (c3) overexpressing the PbrC3'H1 gene; The amino acid sequence of the transcription factor Pbr3AP2-8 is shown in SEQ ID NO.2; the nucleotide sequence of the gene Pbr3AP2-8 encoding the transcription factor Pbr3AP2-8 is shown in SEQ ID NO.1; and the nucleotide sequence of the PbrC3'H1 gene is shown in SEQ ID NO.

3.

10. The use according to any one of claims 1 to 8, the method according to claim 9, characterized in that: The Rosaceae fruit tree is pear.

Images