Transcription factor PpcAPRR and its application in promoting anthocyanin synthesis in pear peel

Abstract

This invention discloses the transcription factor PpcAPRR and its application in promoting anthocyanin synthesis in pear peel. The 'Yunhong 1' pear contains the transcription factor PpcAPRR, which regulates anthocyanin biosynthesis in pear peel. Its nucleotide sequence is shown in SEQ ID No. 1, and its encoded amino acid sequence is shown in SEQ ID No. 2. Transient overexpression in pear fruit confirmed that PpcAPRR can promote the accumulation of anthocyanins in the peel; in the callus tissue of transgenic pear fruit overexpressing PpcAPRR, the anthocyanin content significantly increased after light treatment. This demonstrates that the PpcAPRR gene participates in regulating the biosynthesis of anthocyanins in pear peel. Furthermore, dual-luciferase assays demonstrate that PpcAPRR has the advantage of simultaneously regulating multiple genes, providing a more efficient approach for molecular breeding.

Description

Technical Field

[0001] This invention belongs to the field of plant genetic engineering and relates to the pear transcription factor PpcAPRR and its application in promoting anthocyanin synthesis in pear peel. Specifically, it involves the isolation and cloning of a member of the ARR-B family, PpcAPRR, that regulates anthocyanin accumulation in pear peel from the 'Yunhong No. 1' pear. Background Technology

[0002] Pears originated in my country and are an important fruit tree species in the Rosaceae family, with a cultivation history of over 3,000 years. Pears possess abundant germplasm resources, with at least 22 known species in the genus *Pyrus*, and over 5,000 species cataloged or preserved worldwide. China is a major pear producer, accounting for 71.40% of global pear production and supplying approximately 17.60% of the export pear market. Currently, pears are divided into two main categories: Eastern pears, mainly including white pear (*P. bretschneideri*), sand pear (*P. pyrifolia*), autumn pear (*P. ussuriensis*), and Xinjiang pear (*P. sinkiangensis*); and Western pears, primarily European pears (*P. communis*). Red-skinned pear varieties are relatively scarce in Dongfang pears, and their skin coloring is highly susceptible to environmental factors such as sunlight, leading to unstable coloring and consequently affecting the quality and commercial value of the pears. Therefore, cultivating and promoting superior red pear varieties has significant economic and social value for increasing income in the pear industry.

[0003] The color of pear peel is mainly determined by the content and ratio of three pigments: carotenoids, anthocyanins, and chlorophyll. The red color of the peel is primarily determined by the type and content of anthocyanins. Anthocyanin synthesis is the result of the collaboration of multiple structural genes and transcription factors that regulate these genes. Currently, structural genes related to pear anthocyanin synthesis have been cloned, and corresponding transcriptional patterns and functional analyses have been conducted. Feng Wenting et al. (2015) isolated three key structural genes in the anthocyanin biosynthesis pathway—PbCHS, PbDFR, and PbUFGT—from the bud mutation variety 'Hongzaosu', finding that their expression levels were significantly higher than in the 'Zaosu' pear variety. YANG et al. (2015) considered ANS and UFGT to be the decisive genes for anthocyanin synthesis, and their expression trends were consistent with anthocyanin content.

[0004] Transcription factors can regulate the expression of structural genes, either alone or in synergy, in the anthocyanin pathway, thereby affecting anthocyanin accumulation. Feng et al. (2008) cloned PyMYB10 from red sand pear and verified its function in regulating anthocyanin accumulation. Yao et al. (2017) used 102 F1 hybrids of 'August Red' × 'Dangshan Crisp Pear' to identify a new key anthocyanin synthesis gene, PyMYB114, through QTL mapping, and proposed a working model in which PyMYB114, PyERF3, and PybHLH3 form a complex to jointly regulate anthocyanin synthesis. In addition, Liu et al. (2019) identified a light-responsive transcription factor, PybZIPa, by bagging and unbagging the 'Mantianhong' variety. They found that PybZIPa can not only activate the anthocyanin synthesis gene UFGT, but also activate the expression of upstream key genes PyMYB114 and PyMYB10, thereby promoting light-induced anthocyanin synthesis. In addition, transcription factors such as BBX16 / 18 / 21 (Bai et al., 2019a and 2019b), WRKY26 / 44 (Li et al., 2020; Alabd et al., 2022), ERF22 / 24 / 96 / 105 / 4.1 / 4.2 (Wu et al., 2020; Ni et al., 2019 and 2021; Sun et al., 2023), MYB140 / 9 (Ni et al., 2021; Zhai et al., 2016), and bHLH64 (Tao et al., 2020) have also been shown to be involved in the regulation of anthocyanin synthesis in red pear. However, in the study of red-skinned pear, there are still no reports of ARR-B transcription factors participating in the regulation of anthocyanin synthesis. Summary of the Invention

[0005] The purpose of this invention is to provide a PpcAPRR gene that regulates the accumulation of anthocyanins in pear pericarp.

[0006] Another object of the present invention is to provide a recombinant expression vector containing the gene.

[0007] Another object of the present invention is to provide the application of the gene and the recombinant expression vector.

[0008] The objective of this invention can be achieved through the following technical solutions:

[0009] A transcription factor, PpcAPRR, isolated from the 'Yunhong No. 1' pear, promotes the biosynthesis of anthocyanins in pear pericarp. It belongs to the ARR-B family and its nucleotide sequence is shown in SEQ ID No. 1, containing a 1728 bp open reading frame; it encodes 575 amino acids, and its encoded amino acid sequence is shown in SEQ ID No. 2.

[0010] A recombinant expression vector containing the PpcAPRR gene described in this invention.

[0011] The recombinant expression vector uses pCAMBIA1301 as the starting vector, and the insertion site of the PpcAPRR gene is between Xba I and BamH I.

[0012] Genetically engineered bacteria containing the PpcAPRR gene described in this invention.

[0013] Primer pairs for cloning the PpcAPRR gene cDNA sequence described in this invention are shown in SEQ ID No. 3 for the upstream primer PpcAPRR-F1 and SEQ ID No. 4 for the downstream primer PpcAPRR-R1.

[0014] The application of the PpcAPRR gene in anthocyanin synthesis described in this invention is preferably applied to the regulation of anthocyanin synthesis in pear peel and / or pear pulp callus.

[0015] The application of the recombinant expression vector described in this invention in anthocyanin synthesis is preferably in the regulation of anthocyanin synthesis in pear peel and / or pear pulp callus.

[0016] The application of the genetically engineered bacteria described in this invention in anthocyanin synthesis is preferably applied in regulating anthocyanin synthesis in pear peel and pear pulp callus tissue.

[0017] Beneficial effects

[0018] The inventors bagged 'Yunhong No. 1' pears 35 days after flowering and removed the bags 10 days before harvest. Transcriptome data were collected from peel samples on days 4, 8, and 10 after bag removal. Combined with phenotypic data from anthocyanin determination in the peels, Hi-C data were measured on day 8. Through Hi-C and transcriptome data analysis, differentially expressed transcription factors were predicted at the boundaries of significant interaction regions. Combined with co-expression network analysis, the applicant identified an ARR-B transcription factor, PpcAPRR. The expression level of this gene in the peels of 'Yunhong No. 1' pears after bagging and after bag removal showed a significant positive correlation with anthocyanin content and the expression levels of anthocyanin synthesis-related genes. Transient overexpression of PpcAPRR in pear fruit confirmed that it promotes the accumulation of anthocyanins in pear peel. In transgenic pear flesh callus tissue overexpressing PpcAPRR, light treatment significantly increased anthocyanin content and the expression levels of anthocyanin synthesis-related genes. Dual-luciferase assays verified that PpcAPRR significantly activated the promoter activities of PpcMYB10, PpcDFR, PpcANS, and PpcUFGT genes. This demonstrates that the PpcAPRR gene participates in regulating anthocyanin accumulation in pear peel. The discovery of this gene supplements and refines the ARR-B transcriptional regulation mechanism of anthocyanin synthesis in pear, providing a theoretical basis for improving anthocyanin content in pear fruit.

[0019] Compared with the prior art, the present invention has the following advantages and effects:

[0020] 1. The discovery of the PpcAPRR gene provides a new genetic resource for molecular breeding to promote anthocyanin accumulation in pear peel and a new genetic resource for implementing green agriculture. The development and utilization of this genetic resource is conducive to reducing agricultural costs and achieving environmental friendliness.

[0021] 2. The PpcAPRR gene was functionally verified in pear fruit and pear flesh callus tissue using Agrobacterium-mediated genetic transformation. The results showed that the PpcAPRR gene cloned in this invention has the advantage of simultaneously regulating multiple genes encoding structural genes in the anthocyanin synthesis pathway, providing a more efficient approach for molecular breeding. Attached Figure Description

[0022] Figure 1 This is a sample image of 'Yunhong No. 1' pear fruit after the bagging and unbagging processes of this invention.

[0023] Wherein: D1, D2, and D3 represent the fruits after bagging was removed 10 days before harvest, and on the 4th, 8th, and 10th days of sunlight exposure, respectively; B1, B2, and B3 are the control treatments (bagging) of D1, D2, and D3, respectively.

[0024] Figure 2 This study analyzed the expression levels of the PpcAPRR gene and anthocyanin synthesis-related genes in the peel of 'Yunhong No. 1' pears after bagging and after loading, in accordance with the present invention.

[0025] Figure 3 This is a schematic diagram of the carrier in Embodiment 2 of the present invention.

[0026] Figure 4 Functional analysis of transient injection of the PpcAPRR gene into 'Zaosu' pear fruit according to the present invention.

[0027] Where: A, Phenotypic image of fruit transiently transformed with the PpcAPRR overexpression vector, EV represents the site of transformation with the empty pCAMBIA1301 vector; PpcAPRR-OE represents the site of transformation with the 35S-PpcAPRR-GFP recombinant vector. B, Anthocyanin content in the pericarp of the injected portion. C, Expression level of PpcAPRR in the pericarp of the injected portion. D, Expression level of anthocyanin synthesis-related genes in the pericarp of pears injected with the 35S-PpcAPRR-GFP recombinant vector. NS. Not significant, *significant, **very significant, ***extremely significant.

[0028] Figure 5 Functional analysis of the PpcAPRR gene in transgenic pear flesh callus tissue for the present invention.

[0029] Wherein: A, Phenotypic diagrams of transgenic pear callus tissue under dark and light treatments, WT represents wild-type pear callus tissue, and PpcAPRR-OE represents transgenic callus transformed with the 35S-PpcAPRR-GFP recombinant vector. B, Determination of anthocyanin content in transgenic pear callus tissue. C, Expression levels of anthocyanin synthesis-related genes in pear callus tissue after dark treatment. D, Expression levels of anthocyanin synthesis-related genes in pear callus tissue after light treatment. NS. Not significant, *significant, **very significant, ***extremely significant.

[0030] Figure 6 This invention demonstrates that the PpcAPRR gene promotes the activity of promoters of genes related to anthocyanin synthesis. ***Extremely significant. Detailed Implementation

[0031] The present invention will now be described in detail with reference to specific embodiments. Based on the following description and these embodiments, those skilled in the art can determine the basic features of the present invention, and various changes and modifications can be made to the present invention to adapt it to various uses and conditions without departing from the spirit and scope of the invention.

[0032] Example 1: Analysis of PpcAPRR gene expression patterns

[0033] 'Yunhong No. 1' pear samples were collected from the Anning Experimental Station of the Yunnan Academy of Agricultural Sciences. Young 'Yunhong No. 1' pears, 35 days after flowering, were bagged (yellow outer paper bag, black inner paper bag). The bags were removed 10 days before harvest. Fruits were collected on days 4 (D1), 8 (D2), and 10 (D3) after bag removal. Fruits without bags (B1, B2, and B3) served as a control. Figure 1 Immediately after fruit peeling, the peel was flash-frozen in liquid nitrogen and stored at -80 °C for subsequent experiments. Total RNA was extracted using the CTAB method. The quality of the extracted samples was determined using a spectrophotometer and agarose gel electrophoresis. 3 µg of extracted total RNA was reverse transcribed using a one-step gDNA removal and cDNA synthesis kit (Transgen, China), following the manufacturer's instructions. Gene-specific primer pairs were used for quantitative real-time PCR, with GAPDH as the internal control gene. The quantitative real-time PCR kit was purchased from Roche. The instrument used for real-time PCR was a Roche 480 quantitative PCR instrument. The reaction mixture consisted of: 10 μL of 2×SYBR GreenI Master Mix, 0.4 μL of forward and reverse primers (10 μM), 2 μL of cDNA, and 7.2 μL of PCR-grade water. The reaction conditions were: denaturation at 95℃ for 5 min; pre-denaturation at 95℃ for 5 s, annealing at 60℃ for 5 s, and extension at 72℃ for 10 s, repeated for 45 cycles. Melting curve analysis was performed at 65℃ to 95℃, increasing by 1℃ every 5 s.

[0034] qRT-PCR results from samples of 'Yunhong No. 1' pear after bagging and after bagging showed that PpcAPRR was highly expressed in the samples after bagging, significantly higher than in the samples after bagging. Furthermore, with the accumulation of anthocyanins, the expression level reached its highest point in pears 10 days after bagging, and was positively correlated with the expression levels of anthocyanin biosynthesis-related genes PpcMYB10, PpcMYB114, PpcbHLH3, PpcDFR, PpcANS, and PpcUFGT. Figure 2 The results indicate that PpcAPRR may be involved in the biosynthesis of anthocyanins in pear fruit.

[0035] Example 2: Isolation, cloning, and construction of the overexpression vector for the PpcAPRR gene

[0036] 3 μg of RNA from the peel of 'Yunhong No. 1 pear' was reverse transcribed using a one-step gDNA removal and cDNA synthesis kit (Transgen, China), following the manufacturer's instructions. Based on the multiple cloning site of the pCAMBIA-1301 vector and the restriction enzyme sites in the coding region of the PpcAPRR gene, Xba I and BamHI were selected as restriction enzymes. Primers with restriction sites (SEQ ID NO. 3 and SEQ ID NO. 4) were designed using Snapgene software according to general primer design principles. The 50 μL reaction mixture included 200 ng cDNA, 1× buffer (TransStart FastPfu Buffer), 10 mM dNTPs, 1 U Taq polymerase (TransStart FastPfu DNA Polymerase) (the aforementioned buffer and Taq polymerase were purchased from TRANS), and 500 nM of the aforementioned primers. The PCR reaction was performed on an Eppendorf amplification instrument according to the following program: 95°C pre-denaturation for 2 minutes, 95°C denaturation for 20 seconds, 60°C annealing for 20 seconds, 72°C extension for 1 minute, 35 thermal cycles, 72°C extension for 10 minutes, and storage at 4°C. A single PCR band was produced.

[0037] After PCR products were detected by 1% agarose gel electrophoresis, DNA fragments were recovered using a small-volume gel recovery kit (purchased from Kangwei Century, and the operation was performed according to the kit's instructions). The total volume of the double digestion system for the pCAMBIA1301 vector was 50 μL, containing 10 μL of the pCAMBIA1301 vector plasmid obtained from plasmid extraction, 5 μL of 10× Buffer (purchased from NEB), 1 μL of Xba I, 1 μL of BamHI, and 33 μL of water. Digestion was performed at 37°C for 3 hours, followed by recovery. The expression vector pCAMBIA1301, digested with restriction endonucleases, was ligated to the PpcAPRR gene using recombinase Exnase II (purchased from Vazyme) at 37°C for 30 minutes. The total reaction volume was 20 μL, containing 4 μL of 5 × CE II Buffer, 2 μL of Exnase II, 2 μL of the PCR product of the PpcAPRR gene, 6 μL of the double digestion product of the pCAMBIA1301 vector, and 6 μL of water. 10 μL of the ligation product was transformed into competent E. coli DH5α cells. Positive clones were screened on LB agar plates containing 50 mg / L kanamycin. Plasmids were extracted, digested, and identified by PCR. The recombinant plasmid samples were sent to a biotechnology company for sequencing. Sequencing results showed that the full-length PpcAPRR gene is 1728 bp, and its nucleotide sequence is shown in SEQ ID NO.1. It encodes a protein with 575 amino acid residues, the sequence of which is shown in SEQ ID NO.2. The recombinant vectors were named 35S-PpcAPRR-GFP. The constructed vector map is shown below. Figure 3 As shown, the recombinant vector was introduced into Agrobacterium GV3101 using the freeze-thaw method.

[0038] Example 3: Transient transformation and functional analysis of pear fruit

[0039] (1) Instantaneous transformation of pear fruit

[0040] The bacterial culture containing the plasmids 35S-PpcAPRR-GFP and pCAMBIA1301 was injected into nearly mature 'Zaosu Pear' using the Agrobacterium-mediated transformation method. The specific method is as follows:

[0041] 1. Agrobacterium containing 35S-PpcAPRR-GFP and pCAMBIA1301 plasmids was activated on solid medium (containing 50 μM K+ and 50 μM R+) and cultured in a bacterial incubator at 28℃ for 36 hours.

[0042] 2. Add 15 mL of LB liquid medium (containing 50 μM K+ and 50 μM R+) to a 50 mL sterile centrifuge tube, pick up the activated Agrobacterium colony with a pipette tip, and incubate for 10 hours in a shaker at 28℃ and 180 rpm.

[0043] 3. Centrifuge the cultured bacterial solution at 5000 r·min⁻¹ for 10 minutes and collect all bacterial cells;

[0044] 4. Resuspend the precipitate with an appropriate amount of induction solution with pH = 5.8 [containing 10 mM MES, 10 mM MgCl2, and 200 mM acetylsylgenone (AS)], adjust the OD600 of the bacterial culture to 0.8, and induce for 3 hours at room temperature using a shaker at 180 rpm in the dark.

[0045] 5. Using a sterile syringe, inject the induced infection solution into nearly mature 'Zaosu Pear' fruits. Each experiment involves injecting 15 fruits, with three biological replicates.

[0046] 6. After injection, the pear fruits were cultured in a 20°C incubator in the dark for 24 hours, and then placed in a light incubator at 20°C with 24 hours of light for 10 days.

[0047] (2) Determination of total anthocyanin content

[0048] 0.1 g of pear peel material was ground with liquid nitrogen and then mixed with a methanol solution containing 0.1% hydrochloric acid. The mixture was incubated at 4°C in the dark for 24 hours. After centrifugation at 12000 rpm for 10 minutes, the supernatant was diluted to a volume of 4 mL. OD values ​​at 520, 630, and 650 nm were measured using a spectrophotometer. OD values ​​were standardized using the formula A = (A530 - A650) - 0.1 * (A650 - A620). Three biological replicates were performed for each sample.

[0049] Anthocyanin content determination revealed that the anthocyanin content was significantly increased in the region where PpcAPRR was overexpressed, while there was no significant difference between the region injected with pCAMBIA1301 vector and the uninjected region. Figure 4 A&B).

[0050] (3) Detection of relative expression levels of anthocyanin synthesis-related genes at the injection site

[0051] RNA extraction, cDNA synthesis, and the system and steps for quantitative real-time PCR were as described in Example 1. In the pericarp of the fruit where PpcAPRR was overexpressed, the expression levels of anthocyanin biosynthesis-related genes PpcDFR, PpcANS, PpcUFGT, PpcMYB10, and PpcMYB114 were significantly increased. Figure 4(C&D). In summary, the above results indicate that overexpression of PpcAPRR promotes the synthesis of anthocyanins in pear pericarp.

[0052] Table 1. Primers for real-time fluorescence quantitative analysis of pear

[0053]

[0054] Example 4: Stable transformation and functional analysis of pear pulp callus

[0055] (1) Stable transformation of pear pulp callus

[0056] Agrobacterium containing the PpcAPRR overexpression vector was used to infect pear pulp callus tissue via Agrobacterium-mediated infection, as follows:

[0057] 1. Agrobacterium containing 35S-PpcAPRR-GFP and pCAMBIA1301 plasmids was activated on solid culture medium (containing 50 μM kanamycin and 50 μM rifampin) and cultured in a bacterial incubator at 28℃ for 36 hours.

[0058] 2. Add 30 mL of LB liquid medium (containing 50 μM kanamycin and 50 μM rifampin) to a 100 mL Erlenmeyer flask, pick up the activated Agrobacterium colonies with a pipette tip, and incubate for 12 hours in a shaker at 28℃ and 180 rpm.

[0059] 3. Transfer the bacterial culture to a 50 mL sterile centrifuge tube and centrifuge at 5000 r·min. -1 Centrifuge for 10 minutes and collect all bacterial cells;

[0060] 4. Resuspend the precipitate with an appropriate amount of induction solution [10 mM MES, 10 mM MgCl2, 200 mM acetylsuccinone (AS), pH = 5.8] and adjust the bacterial concentration OD. 600 = 0.6, and induced in a shaker at 28℃ and 180 rpm for 4 hours in the dark;

[0061] 5. In MS liquid medium, induce for 20 minutes at room temperature in a shaker at 200 rpm in the dark;

[0062] 6. Pear callus infected with Agrobacterium was evenly spread on symbiotic MS solid medium and co-cultured at 25°C in the dark for 2 days.

[0063] 7. Spread the co-cultured callus tissue evenly on the screening MS solid medium and culture it at 25°C in the dark for 2 months. The callus tissue that can continue to grow on the screening MS solid medium is the transgenic callus.

[0064] (2) Identification and light treatment of callus in transgenic pear pulp

[0065] RNA extraction, cDNA synthesis, and the system and procedures for quantitative real-time PCR were performed as described in Example 1. We analyzed normally growing callus tissue on a culture medium containing hygromycin using quantitative real-time PCR and found that the relative expression level of the PpcAPRR gene in these callus tissues was higher than that in wild-type (WT) callus tissues. Therefore, this type of callus tissue was selected for subsequent research.

[0066] Transgenic callus and WT were cultured in the dark at 25°C for 3 days on MS medium containing 50 mM methyl jasmonate (MJ). The callus was then divided into two parts, one part was cultured in the dark and the other part was placed in a light incubator for 24 hours of light, and the phenotype was observed.

[0067] (3) Analysis of anthocyanin content in transgenic callus

[0068] Phenotypic observation revealed that under dark conditions, there were no significant changes in the callus of WT and transgenic PpcAPRR-OE, both remaining milky white. Under light conditions, the PpcAPRR-OE callus accumulated a large amount of anthocyanins, and its surface turned red. Figure 5 A). Anthocyanin content determination revealed no significant difference in anthocyanin content between WT and transgenic PpcAPRR-OE callus under dark conditions, but after light treatment, the anthocyanin content of transgenic PpcAPRR-OE callus was significantly higher than that of WT. Figure 5 B). This indicates that PpcAPRR promotes anthocyanin synthesis in pear callus tissue and is light-dependent.

[0069] (4) Detection of relative expression levels of anthocyanin synthesis-related genes in transgenic callus

[0070] RNA extraction, cDNA synthesis, and the system and steps for quantitative real-time PCR were as described in Example 1. The expression levels of anthocyanin synthesis-related genes in transgenic callus tissue were analyzed using quantitative real-time PCR. The results showed that, compared with WT pear pulp callus, the expression levels of anthocyanin synthesis-related genes PpcMYB10, PpcDFR, PpcANS, and PpcUFGT in pear pulp callus tissue overexpressing PpcAPRR were significantly increased. Figure 5 (C&D). In summary, the above results indicate that overexpression of PpcAPRR promotes anthocyanin synthesis in pear pulp callus tissue.

[0071] Example 5: Dual-luciferase assay to verify the expression of genes related to PpcAPRR promoting anthocyanin synthesis.

[0072] (1) Dual-luciferase test

[0073] The construction of the transient expression and dual-luciferase activity detection vectors followed the method of Hellens et al. (Hellens et al., 2005). To detect the transcriptional activation effect of PpcAPRR on anthocyanin synthesis-related genes, the promoter sequences of PpcMYB10, PpcDFR, PpcANS, and PpcUFGT were inserted into the pGreen 0800-LUC vector as a reporter vector, and the PpcAPRR overexpression vector was used as the effector vector. The correctly sequenced recombinant plasmid was transformed into Agrobacterium GV3101 (pSoup). The Agrobacterium culture carrying the effector and reporter vectors was co-transformed into leaves of Nicotiana benthamiana (Xue et al., 2018). Leaves from the injection site were collected 72 h after injection for enzyme activity assay. LUC (Firefly luciferase) and REN (Renilla luciferase) activities were measured using the Dual-Luciferase® Reporter Assay System (E1910, Promega, Madison, USA).

[0074] (2) PpcAPRR can enhance the promoter activity of PpcDFR, PpcANS and PpcUFGT.

[0075] Dual-luciferase assay results showed that PpcAPRR could simultaneously enhance the promoter activities of PpcDFR, PpcANS, and PpcUFGT, with the highest enhancement efficiency on the PpcUFGT promoter. Figure 6 This indicates that PpcAPRR promotes the expression of anthocyanin synthesis-related genes and affects anthocyanin accumulation.

[0076] Main References Feng Shouqian, Chen Xuesen, Zhang Chunyu, Liu Xiaojing, Liu Zunchun, Wang Haibo, Wang Yanling, Zhou Chaohua (2008). Study on anthocyanin synthesis and related enzyme activities in sand pear variety 'Mantianhong' and its bud mutation 'Aoguan'. Chinese Journal of Agricultural Science 41(10): 3184-3190.

[0077] Feng Wenting, Zhai Rui, Wang Zhigang (2015). Cloning and expression analysis of genes related to the synthesis of pear anthocyanins in 'Hongzaosu' pear. Northwest Agriculture Journal 24(06):75-83.

[0078] Ahmed Alabd, Mudassar Ahmad, Xiao Zhang, Yuhao Gao, Lin Peng, LuZhang, Junbei Ni, Songling Bai, Yuanwen Teng (2022). Light-responsivetranscription factor PpWRKY44 induces anthocyanin accumulation by regulatingPpMYB10 expression in pear. Horticulture Research 9,uhac199.

[0079] Bai S, Tao R, Tang Y, Yin L, Ma Y, Ni J, Yan X, Yang Q, Wu Z, Zeng Y,Teng Y (2019). BBX16, a B-box protein, positively regulates light-inducedanthocyanin accumulation by activating MYB10 in red pear. Plant Biotechnol J17(10):1985-1997.

[0080] Bai S, Tao R, Yin L, Ni J, Yang Q, Yan X, Yang F, Guo X, Li H, Teng Y(2019). Two B-box proteins, PpBBX18 and PpBBX21, antagonistically regulateanthocyanin biosynthesis via competitive association with Pyrus pyrifoliaELONGATED HYPOCOTYL 5 in the peel of pear fruit. Plant J 100(6):1208-1223.

[0081] Bao L, Chen K, Zhang D, Cao Y, Yamamoto T, Teng Y. 2007. Geneticdiversity and similarity of pear (Pyrus L.) cultivars native to East Asiarevealed by SSR (simple sequence repeat) markers. Genetic Resources and CropEvolution 54(5): 959.

[0082] Gaifang Yao, Meiling Ming, Andrew C. Allan, Chao Gu, Leiting Li, XiaoWu, Runze Wang, Yaojun Chang, Kaijie Qi, Shaoling Zhang, Jun Wu (2017). Map-based cloning of the pear gene MYB114 identifies an interaction with othertranscription factors to coordinately regulate fruit anthocyaninbiosynthesis. The Plant Journal 92, 437–451.

[0083] Hainan Liu, Jun Su, Yangfan Zhu, Gaifang Yao, Andrew C. Allan,Charles Ampomah-Dwamena, Qun Shu, Kui Lin-Wang, Shaoling Zhang, and Jun Wu(2019). The involvement of PybZIPa in light-induced anthocyanin accumulationvia the activation of PyUFGT through binding to tandem G-boxes in itspromoter. Horticulture research 6(1), 1-13.

[0084] Hellens RP, Allan AC, Friel EN, Bolitho K, Grafton K, Templeton MD,Karunairetnam S, Gleave AP, Laing WA (2005). Transient expression vectors forfunctional genomics, quantification of promoter activity and RNA silencing inplants. Plant Methods 1:13.

[0085] Li C, Wu J, Hu KD, Wei SW, Sun HY, Hu LY, Han Z, Yao GF, Zhang H(2020). PyWRKY26 and PybHLH3 cotargeted the PyMYB114 promoter to regulateanthocyanin biosynthesis and transport in red-skinned pears. HorticultureResearch 15;7:37.

[0086] Ni J, Premathilake AT, Gao Y, Yu W, Tao R, Teng Y, Bai S (2021).Ethylene-activated PpERF105 induces the expression of the repressor-typeR2R3-MYB gene PpMYB140 to inhibit anthocyanin biosynthesis in red pear fruit.Plant J 105(1):167-181.

[0087] Tao R, Yu W, Gao Y, Ni J, Yin L, Zhang X, Li H, Wang D, Bai S, Teng Y(2020). Light-Induced Basic / Helix-Loop-Helix64 Enhances AnthocyaninBiosynthesis and Undergoes CONSTITUTIVELY PHOTOMORPHOGENIC1-MediatedDegradation in Pear. Plant Physiol 184(4):1684-1701.

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[0089] YANG Yanan,YAO Gaifang,ZHENG Danman,ZHANG Shaoling ,WANG Chao,ZHANGMingyue,WU Jun (2015). Expression differences of anthocyanin biosynthesisgenes reveal regulation patterns for red pear coloration. Plant Cell Reports34 (2):189-198.

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Claims

1. Isolated from 'Yunhong No. 1' pear, it has the ability to regulate the accumulation of anthocyanins in pear pericarp. PpcAPRR The application of genes in promoting the synthesis of piridin is characterized by, PpcAPRR The CDS sequence of the gene is shown in SEQ ID No. 1, and the sequence of the encoded protein is shown in SEQ ID No.

2.

2. The application according to claim 1, characterized in that, The aforementioned PpcAPRR Application of genes in promoting anthocyanin synthesis in pear peel and / or pear pulp callus.

3. Containing the contents of claim 1 PpcAPRR Application of recombinant gene expression vectors in promoting the synthesis of anthocyanins.

4. The application according to claim 3, characterized in that, The application of the recombinant expression vector in promoting anthocyanin synthesis in pear peel and / or pear pulp callus.

5. The application according to claim 3 or 4, characterized in that... The recombinant expression vector uses pCAMBIA1301 as the starting vector, and the recombinant expression vector is used to express the recombinant expression vector. PpcAPRR Gene insertion starting vector Xba I and BamH What I obtained between I.

6. Containing the contents of claim 1 PpcAPRR Application of genetically engineered bacteria in promoting anthocyanin synthesis.

7. The application according to claim 6, characterized in that, Agrobacterium GV3101 was used as the host bacterium.

8. The application according to claim 7, characterized in that... The genetically engineered bacteria contain the aforementioned PpcAPRR The gene was obtained by transfecting Agrobacterium GV3101 with a recombinant expression vector.

9. The application according to claim 7, characterized in that, The application of the genetically engineered bacteria in promoting anthocyanin synthesis in pear peel and / or pear pulp callus.

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