A pear transcription factor PyERF4.1 gene that inhibits anthocyanin biosynthesis, its recombinant expression vector and application

Abstract

A pear transcription factor that inhibits anthocyanin biosynthesis PyERF4.1 gene, its recombinant expression vector and application, wherein the PyERF4.1 nucleotide sequence of the gene is shown in SEQ ID No.1. In the present invention, the transcription factor PyERF4.1 was overexpressed and knocked out in pear callus and tomato fruits to verify its function of inhibiting anthocyanin biosynthesis, and PyERF4.1 was PyERF3, PyMYB114, and PybHLH3 co-transformed with the PyERF4.1 gene into pear fruits and strawberry fruits, resulting in reduced anthocyanin accumulation in pear fruits and strawberry fruits. Through biological function verification, it is shown that the PyERF3, PyMYB114, and PybHLH3 gene cloned in the present invention interacts with the PyERF4.1 gene to inhibit the function of anthocyanin biosynthesis in pear peel.

Description

Technical Field

[0001] The present invention belongs to the field of plant genetic engineering and relates to a pear transcription factor PyERF4.1 gene that inhibits anthocyanin biosynthesis, a recombinant expression vector thereof, and an application thereof. Specifically, the present invention relates to a PyERF4.1 gene, a member of the AP2 / ERF family related to inhibiting anthocyanin biosynthesis in pear peel, isolated and cloned from 'Hongqie' pear, and an application thereof. Background Art

[0002] Pear (Pyrus L.) is one of the most popular fruits worldwide, with red-skinned pears favored by consumers for their attractive color and rich anthocyanin content (Li et al., 2020). Anthocyanins are widely found in plants. They not only enhance plant reproduction but also play a vital role in stress response, such as protecting plants from ultraviolet (UV) damage, scavenging free radicals, and increasing antioxidant activity (Peng et al., 2017; Valenta et al., 2017). Furthermore, anthocyanins have been shown to protect humans from neurological and cardiovascular diseases (Poracova et al., 2011; Yang et al., 2017).

[0003] Anthocyanins are biosynthesized through the flavonoid pathway (Koes et al., 2005; Hichri et al., 2011), in which key enzymes include phenylalanine ammonia lyase (PAL), chalcone synthase (CHS), chalcone isomerase (CHI), flavonoid-3-hydroxylase (F3H), dihydroflavonol 4-reductase (DFR), anthocyanidin synthase (ANS), and UDP-glucose:flavonoid 3-glucosyltransferase (UFGT) (Tanaka et al., 2008; Zhao et al., 2009). Numerous transcription factors are involved in regulating anthocyanin biosynthesis in plants. Currently, MYBs, bHLHs, and WDRs have been found to regulate the anthocyanin biosynthesis pathway (Hichri et al., 2010; Xu et al., 2014). Generally speaking, anthocyanin biosynthesis is primarily regulated by a complex consisting of three transcription factors: MYB, bHLH, and WD40 (MBW) (Ramsay & Glover et al., 2005; Liu et al., 2021). As core regulators of the MBW complex, MYBs have been widely reported to co-regulate anthocyanin biosynthesis with bHLHs. In apple, MdMYB10 interacts with MdbHLH3 and MdbHLH33 to promote anthocyanin biosynthesis (Espley et al., 2007). In pear, PyMYB114, PyMYB10, or PyMYB10b interacts with PybHLH3 to significantly promote anthocyanin biosynthesis (Zhai et al., 2016; Yao et al., 2017). In addition, peach PpMYB18 and red pear PpMYB140 act as repressors to compete with anthocyanin-related MYB activators for binding to bHLHs to regulate anthocyanin accumulation (Ni et al., 2021; Zhou et al., 2019).

[0004] Accumulating evidence indicates that, in addition to the MBW complex, the AP2 / ERF family also participates in regulating anthocyanin biosynthesis (An et al., 2020a; Ma et al., 2021). AP2 / ERFs are a large family of plant-specific transcription factors with a conserved AP2 domain. They have been found to participate in plant development, regulate the synthesis of secondary metabolites, and regulate various stress responses by binding to short cis-acting elements such as the GCC-box and dehydration-responsive elements (DRE) / C-repeat elements (CRT) in target gene promoters (Liu et al., 1998; Fujimoto et al., 2000; Li et al., 2018). Numerous studies have demonstrated that AP2 / ERFs interact with the MBW complex and participate in the regulation of anthocyanin biosynthesis. They exhibit diverse regulatory patterns in anthocyanin biosynthesis induced under different conditions and in different plant species. In phytohormone-induced anthocyanin biosynthesis, MdERF1B and MdERF3 directly activate the expression of MdMYB11 and MdMYB1, respectively, thereby promoting ethylene-induced anthocyanin biosynthesis in apple fruit (An et al., 2018; Zhang et al., 2018). Jasmonic acid and ethylene-regulated PbERF22 enhance the activation of the PbUFGT promoter by PbMYB10 and PbMYB10b to promote lanolin-induced anthocyanin biosynthesis in 'Zaosu' pear fruit (Wu et al., 2020). Ethylene-activated PpERF105 induces the expression of the repressor PpMYB140, inhibiting anthocyanin biosynthesis in red pear fruit (Ni et al., 2021). In light-induced anthocyanin biosynthesis, Pp4ERF24 and Pp12ERF96 interact with PpMYB114 in ‘Hongzaosu’ pear fruit to promote blue light-induced anthocyanin biosynthesis (Ni et al., 2019). In drought-induced anthocyanin biosynthesis, MdERF38 interacts with MdMYB1 to promote drought-induced anthocyanin biosynthesis in apple fruit (An et al., 2020b). Furthermore, PyERF3, identified from a green mutant of ‘Hongqie’ pear, interacts with PyMYB114 and forms a novel complex with PybHLH3 to co-regulate anthocyanin biosynthesis (Yang et al., 2015; Yao et al., 2017). While many AP2 / ERFs have been reported to positively regulate anthocyanin biosynthesis, negative regulation of anthocyanin biosynthesis by AP2 / ERFs has not been reported. In addition, whether AP2 / ERFs participate in anthocyanin biosynthesis by interacting with the reported PyERF3, PyMYB114, PyMYB10 and PybHLH3 is still unclear and requires further study. Summary of the Invention

[0005] The purpose of the present invention is to provide a pear transcription factor PyERF4.2 gene for inhibiting anthocyanin biosynthesis, a recombinant expression vector and an application thereof.

[0006] To achieve the above-mentioned and other related purposes, the present invention provides a technical solution: a pear transcription factor PyERF4.1 gene that inhibits anthocyanin biosynthesis, wherein the nucleotide sequence of the PyERF4.1 gene is shown in SEQ ID No.1.

[0007] To achieve the above objectives and other related objectives, the present invention provides a technical solution: a protein encoded by the pear transcription factor PyERF4.1 gene that inhibits anthocyanin biosynthesis, the amino acid sequence of which is shown in SEQ ID No. 2.

[0008] To achieve the above-mentioned purpose and other related purposes, the technical solution provided by the present invention is: an overexpression vector containing the pear transcription factor PyERF4.1 gene that inhibits anthocyanin biosynthesis.

[0009] To achieve the above-mentioned purpose and other related purposes, the technical solution provided by the present invention is: the overexpression vector of the pear transcription factor PyERF4.1 gene that inhibits anthocyanin biosynthesis is obtained by using pCAMBIA1300 as the starting vector and inserting the pear transcription factor PyERF4.1 gene that inhibits anthocyanin biosynthesis between the BamHI and SacI sites.

[0010] To achieve the above-mentioned purpose and other related purposes, the technical solution provided by the present invention is: use of the overexpression vector in inhibiting the biosynthesis of anthocyanins in pear peel.

[0011] To achieve the above-mentioned purpose and other related purposes, the technical solution provided by the present invention is: a transient expression vector containing the pear transcription factor PyERF4.1 gene that inhibits anthocyanin biosynthesis.

[0012] To achieve the above-mentioned purpose and other related purposes, the technical solution provided by the present invention is: the transient expression vector of the pear transcription factor PyERF4.1 gene that inhibits anthocyanin biosynthesis is characterized in that: pSAK277 is used as the starting vector, and the pear transcription factor PyERF4.1 gene that inhibits anthocyanin biosynthesis is inserted between the EcoRI and XbaI sites.

[0013] To achieve the above-mentioned and other related purposes, the present invention provides a technical solution: application of a transient expression vector in inhibiting anthocyanin biosynthesis in pear peel.

[0014] To achieve the above-mentioned purpose and other related purposes, the technical solution provided by the present invention is: a host bacteria containing the pear transcription factor PyERF4.1 gene that inhibits anthocyanin biosynthesis.

[0015] To achieve the above-mentioned purpose and other related purposes, the technical solution provided by the present invention is: a primer pair for cloning the pear transcription factor PyERF4.1 gene that inhibits anthocyanin biosynthesis, the upstream primer PyERF4.1-F1 sequence is shown as SEQ ID No. 3, the PyERF4.1-F2 sequence is shown as SEQ ID No. 4, the downstream primer PyERF4.1-R1 sequence is shown as SEQ ID No. 5, and the PyERF4.1-R2 sequence is shown as SEQ ID No. 6.

[0016] To achieve the above-mentioned purpose and other related purposes, the technical solution provided by the present invention is: an application of the pear transcription factor PyERF4.1 gene that inhibits anthocyanin biosynthesis in inhibiting the biosynthesis of anthocyanins in pear peel, and an application of the pear transcription factor PyERF4.1 gene that inhibits anthocyanin biosynthesis alone or in combination with genes PyERF3, PyMYB114 and PybHLH3 in inhibiting the biosynthesis of anthocyanins in pear peel; the nucleotide sequence of the PyERF4.1 gene is shown in SEQ ID No.1, the nucleotide sequence of the PyERF3 gene is shown in SEQ ID No.7, the nucleotide sequence of the PyMYB114 gene is shown in SEQ ID No.9, and the nucleotide sequence of the PybHLH3 gene is shown in SEQ ID No.11.

[0017] To achieve the above-mentioned purpose and other related purposes, the technical solutions provided by the present invention are: an application of the pear transcription factor PyERF4.1 gene that inhibits anthocyanin biosynthesis in inhibiting the biosynthesis of anthocyanins in pear peel, and an application of a transient expression vector containing the PyERF4.1 gene that inhibits anthocyanin synthesis combined with a recombinant vector containing the PyERF3, PyMYB114 and PybHLH3 genes in inhibiting the biosynthesis of anthocyanins in pear peel.

[0018] To achieve the above-mentioned purpose and other related purposes, the technical solutions provided by the present invention are: an application of the pear transcription factor PyERF4.1 gene that inhibits anthocyanin biosynthesis in inhibiting the biosynthesis of anthocyanins in pear peel, and an application of an overexpression vector containing the PyERF4.1 gene that inhibits anthocyanin synthesis in combination with a recombinant vector containing the PyERF3, PyMYB114 and PybHLH3 genes in inhibiting the biosynthesis of anthocyanins in pear peel.

[0019] Due to the application of the above technical solution, the present invention has the following advantages compared with the prior art:

[0020] 1. The application of the PyERF4.1 gene in inhibiting the biosynthesis of anthocyanins in pear peel provides a new genetic resource for molecular breeding of anthocyanin accumulation in pear peel and a new genetic resource for the implementation of green agriculture. The development and utilization of this genetic resource is conducive to reducing agricultural costs and achieving environmental friendliness.

[0021] 2. The present invention overexpressed and knocked out the transcription factor PyERF4.1 in pear callus and tomato fruit to verify its function of inhibiting anthocyanin biosynthesis, and co-transformed PyERF4.1 with PyERF3, PyMYB114 and PybHLH3 genes into pear fruit and strawberry fruit, resulting in reduced anthocyanin accumulation in pear fruit and strawberry fruit. The biological function verification showed that the cloned PyERF4.1 of the present invention interacts with PyERF3, PyMYB114 and PybHLH3 genes to inhibit the biosynthesis of anthocyanins in pear peel. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 Bioinformatics analysis of candidate AP2 / ERF genes screened from transcriptome data of 'Hongqie' pear and its green mutant. (a) Heatmap of differentially expressed AP2 / ERF genes at different developmental stages in the peel of 'Hongqie' pear and its green mutant. R1, R2, and R3 represent samples of 'Hongqie' pear at 40, 55, and 85 days after anthesis; G1, G2, and G3 represent samples of the 'Hongqie' pear green mutant at 40, 55, and 85 days after anthesis, respectively. Blue boxes indicate downregulated AP2 / ERFs screened in red-skinned pear. (b) Phylogenetic analysis of the inhibitory profiles of AP2 / ERFs in different plant species. The C-terminus of the ERF domain was analyzed using the neighbor-joining method using the MEGA7 program. (c) Protein sequence alignment of candidate AP2 / ERFs. Among them, Pbr000398.1 was named PyERF4.1; the protein sequence of the AP2 domain is in the green box; the protein sequence and position of the EARmotif are in the red box.

[0023] Figure 2 Overexpression of PyERF4.1 inhibits anthocyanin biosynthesis in pear calli. (a) Phenotype of pear calli overexpressing PyERF4.1 after 15 days of light treatment. WT, wild type; OE, overexpressed. (b) Color differences are represented by L*, a*, and b* values. L* represents lightness; a* represents a range from green to magenta; and b* represents a range from yellow to blue. (c) Total anthocyanin content in pear calli overexpressing PyERF4.1. (d) Expression level of PyERF4.1 in transgenic pear calli. (e) Expression levels of PyERF3, PyMYB114, PyDFR, PyANS, and PyUFGT in transgenic pear calli.

[0024] Figure 3 Figure 1. Effects of PyERF4.1-RNAi on anthocyanin biosynthesis in pear fruit. Figures: (a) Phenotype of 'Zaosu' pear peel injected with RNAi-induced gene silencing: a, pSAK277; b, PyERF4.1; c, PyERF4.1-RNAi. (b) Color differences are represented by L*, a*, and b* values. L* represents lightness; a* represents a range from green to magenta; and b* represents a range from yellow to blue. (c) Total anthocyanin content in pear peel after injection. (d) Expression level of PyERF4.1. (e) Expression levels of PyERF3 and PyMYB114. (f) Expression levels of PyDFR, PyANS, and PyUFGT.

[0025] Figure 4 PyERF4.1 inhibits anthocyanin biosynthesis in tomato fruit. (a) Phenotypes of transgenic tomato fruits at the red ripening stage after overexpression and knockout of ERF4.1. Scale bar = 1 cm. WT, wild type; OE, overexpression; erf4.1, ERF4.1 knockout. (b) Total anthocyanin content in transgenic tomato fruits. (cd) Expression levels of PyERF4.1 and SlERF4.1 in transgenic tomato fruits. (e) Expression levels of SlERF3, SlMYB114, SlDFR, SlANS, and SlUFGT in transgenic tomato fruits.

[0026] Figure 5 PyERF4.1 co-transformed with PyERF3, PyMYB114, and PybHLH3 inhibits anthocyanin biosynthesis in pear fruit. (a) Phenotypes of 'Zaosu' pear peel after injection: a, pSAK277; b, PyMYB114+PybHLH3; c, PyERF4.1+PyMYB114+PybHLH3; d, PyERF3+PyMYB114+PybHLH3; e, PyERF4.1:PyERF3 (1:1)+PyMYB114+PybHLH3; f, PyERF4.1:PyERF3 (2:1)+PyMYB114+PybHLH3; g, PyERF4.1:PyERF3 (1:2)+PyMYB114+PybHLH3. (b) Color differences are represented by L*, a*, and b* values. L* represents lightness; a* represents the range from green to magenta; and b* represents the range from yellow to blue. (c) Total anthocyanin content in pear peel after injection. (d) Expression levels of PyDFR, PyANS, and PyUFGT.

[0027] Figure 6PyERF4.1 co-transformed with PyERF3, PyMYB114, and PybHLH3 inhibits anthocyanin biosynthesis in strawberry fruit. (a) Phenotypes of strawberry fruit after injection: a, pSAK277; b, PyMYB114 + PybHLH3; c, PyERF4.1 + PyMYB114 + PybHLH3; d, PyERF3 + PyMYB114 + PybHLH3; f, PyERF4.1 + PyERF3 + PyMYB114 + PybHLH3. 1, Overall view. 2, Cross-sectional view. (b) Color differences are indicated by L*, a*, and b* values. L* represents lightness; a* represents the range from green to magenta; and b* represents the range from yellow to blue. (c) Total anthocyanin content in strawberry fruit after injection. (d) Expression levels of FvDFR, FvANS, and FvUFGT.

[0028] Figure 7 Figure 2: Interaction between PyERF4.1 and PyERF3. (a) Models of NLuc, CLuc, and NLuc / CLuc constructs. (b) Firefly luciferase complementation assay confirming the interaction between PyERF4.1 and PyERF3. (c) Segmented protein sequence of PyERF4.1. P1-P5, parts 1 to 5, represent distinct amino acid residues in PyERF4.1. (d) Yeast two-hybrid assay confirming the interaction between PyERF4.1 and PyERF3. (e) Pull-down assay confirming the interaction between PyERF4.1 and PyERF3. α-MBP, MBP antibody; α-HIS, HIS antibody.

[0029] Figure 8 PyMYB114 binds to the PyERF4.1 promoter and regulates the transcription of anthocyanin biosynthesis genes. (a) Schematic diagram of the PyERF4.1 promoter. Cis-acting elements in the promoter region were predicted using the PlantPAN 3.0 database and segmented (S1). Pro, promoter. MYB, MYBs cis-acting element. (b) Yeast one-hybrid assay confirms that PyMYB114 binds to the PyERF4.1 promoter. Yeast colonies were screened on SD / -Ura / -Leu / AbA800 plates. (c) Dual-luciferase reporter assay in tobacco leaves confirms that PyMYB114 activates the PyERF4.1 promoter. (df) Dual-luciferase reporter assay confirms the effect of co-transformation of PyERF4.1 with PyERF3, PyMYB114, and PybHLH3 on the transcriptional activity of PyDFR, PyANS, and PyUFGT in tobacco leaves. DETAILED DESCRIPTION

[0030] The following describes the implementation of the present invention through specific embodiments. People skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification.

[0031] See also Figure 1-8 . It should be noted that the structures, proportions, sizes, etc. illustrated in the drawings of this specification are only used to match the contents disclosed in the specification for people familiar with this technology to understand and read, and are not used to limit the limiting conditions for the implementation of the present invention. Therefore, they have no substantive technical significance. Any modification of the structure, change in the proportional relationship or adjustment of the size should still fall within the scope of the technical content disclosed by the present invention without affecting the efficacy and purpose that can be achieved by the present invention. At the same time, the terms such as "upper", "lower", "left", "right", "middle" and "one" quoted in this specification are only for the convenience of description, and are not used to limit the scope of the implementation of the present invention. Changes or adjustments in their relative relationships should also be regarded as the scope of the implementation of the present invention without substantially changing the technical content.

[0032] The present invention screens candidate AP2 / ERF genes through transcriptome data and bioinformatics analysis

[0033] To investigate the regulation of anthocyanin biosynthesis by AP2 / ERF genes in red-skinned pear, we conducted an in-depth study of transcriptome data from the red-skinned pear 'Hongqie' and its green mutant at 40, 55, and 85 days after anthesis. We found 114 differentially expressed genes in the AP2 / ERFs domain. Heat map analysis revealed that 33 differentially expressed ERFs were downregulated during the critical period of fruit peel pigmentation in 'Hongqie' pear ( Figure 1 Next, these 33 down-regulated ERFs were compared with negative regulatory factors of other species (SlAP2a, SlERF6, MdERF1, MdERF2, AdERF9, MaERF11, MdERF4, and EjERF11), and a phylogenetic tree was constructed using the neighbor-joining method and bootstrap analysis (1000 replicates) using MEGA7 software. Figure 1 As shown in (b), Pbr000398.1 is most closely related to EjERF11, MdERF4, MaERF11, and AdERF9. In addition, protein sequence alignment analysis showed that these genes all have AP2 domains, and Pbr000398.1, EjERF11, MdERF4, MaERF11, and AdERF9 contain a complete EAR motif (DLNLxP) ( Figure 1c). At different developmental stages, the expression level of Pbr000398.1 in the 'Red Egg' pear was significantly downregulated compared to the green mutant, suggesting that it may act as a repressor in anthocyanin biosynthesis. Therefore, Pbr000398.1 was selected for further study and named PyERF4.1.

[0034] Example 2: Cloning and overexpression of the PyERF4.1 gene and construction of transient expression vectors of the present invention

[0035] RNA was extracted from the peel of 'Hongqie' pear using the Plant Total RNA Isolation Kit Plus (Foregene, RE-05022) according to the instructions provided with the kit. Specifically, 500 mg of freeze-dried 'Hongqie' pear peel was placed in a mortar and ground thoroughly with liquid nitrogen until fine powder was obtained. 500 μL of Buffer PSL1 was pipetted into a 2 mL centrifuge tube, 10 μL of β-mercaptoethanol was added, and the mixture was mixed. An appropriate amount (about 50 mg) of the ground powder was scraped with a blue pipette tip cooled with liquid nitrogen and transferred to Buffer PSL1, which was vortexed to mix thoroughly. After standing at room temperature for 5 min, 100 μL of Buffer PS was added and the mixture was gently mixed. All the liquid was transferred to a DNA-Cleaning Column, centrifuged at 12,000 rpm for 2 min, the filter column was removed, and the supernatant in the collection tube was retained. 300 mL of the supernatant was carefully transferred to a new 2 mL centrifuge tube and 450 mL of Buffer was added. Gently mix PSL2; transfer 500 μL of the mixture to the RNA-only column, centrifuge at 12,000 rpm for 1 minute, and discard the waste liquid; add 500 μL of Buffer PRW1 to the RNA-only column, centrifuge for 1 minute, and discard the waste liquid; add 700 μL of anhydrous ethanol, centrifuge for 1 minute, and discard the waste liquid; add 700 μL of Buffer PRW2 to the RNA-only column, centrifuge at 12,000 rpm for 1 minute, and discard the waste liquid. Repeat this step once; centrifuge at 12,000 rpm for 2 minutes and discard the collection tube. Transfer the RNA-only column to a new 2 mL centrifuge tube, add 60 μL of RNase-free ddH2O preheated at 65°C to the center of the membrane, let it stand at room temperature for 2 minutes, and centrifuge at 12,000 rpm for 1 minute to collect the RNA. Run 2 μL of RNA on agarose gel electrophoresis, estimate the RNA concentration based on band brightness, and store at -80°C until further use.

[0036] Total RNA was reverse transcribed into first-strand cDNA, and the first-strand cDNA was synthesized using Prime Script TMRT MasterMix (Takara) Reverse Transcription Kit (follow the kit's instructions). The reverse transcription system (10 μL) consists of 2 μL of 5× Prime Script RT Master Mix, 5 μL of total RNA, and 5 μL of RNase-Free ddH2O. The reaction conditions are 37°C for 15 min, 85°C for 5 s, and 4°C for ∞. The resulting cDNA can be stored in a -20°C refrigerator.

[0037] The first-strand cDNA obtained by reverse transcription was used to amplify the full-length coding region of the PyERF4.1 gene. The primer pairs for amplification were PyERF4.1-F1: 5'-acgggggactctagaggatccATGGCGCCGAGAGAGAAGA-3' (SEQ ID No. 3); PyERF4.1-R1: 5'-cgatcggggaaattcgagctcTCAAGCGAGCTCCGGTGG-3' (SEQ ID No. 4);

[0038] PyERF4.1-F2: 5'-actagtggatccaaagaattcATGGCGCCGAGAGAGAAGA-3' (SEQ ID No. 5); PyERF4.1-R2: 5'-tcattaaagcaggactctagaTCAAGCGAGCTCCGGTGG-3' (SEQ ID No. 6). Ultra-fidelity DNA polymerase Super-Fidelity DNA Polymerase (P505-d1) was purchased from Novagen Biotech. The amplification reaction system consisted of 50 μL of a 50-μL amplification system containing 200 ng of cDNA, 25 μL of 2× Phanta Max Buffer, 1 μL of 10 mM dNTPs, 1 μL of Phanta Max Super-Fidelity DNA Polymerase (1 U / μL), 2 μL of the above primers at 10 μM concentration, and ddH2O to 50 μL. PCR reactions were performed in an Eppendorf thermal cycler using the following protocol: 95°C initial denaturation for 3 minutes, 95°C denaturation for 15 seconds, 60°C annealing for 15 seconds, 72°C extension for 90 seconds, 35 cycles of thermal cycling, 72°C extension for 5 minutes, and storage at 4°C. A single PCR product band was generated. The PCR product was analyzed by 1% agarose gel electrophoresis, and the DNA fragments were recovered using an agarose gel recovery kit.

[0039] The purified DNA solution was recovered and ligated with the double-enzyme-digested (BamHI / SacI) linear pCAMBIA1300 vector and the double-enzyme-digested (EcoRI / XbaI) linear pSAK277 vector. IIOne StepCloning Kit was purchased from Novozymes Biotech, and the ligation reaction was performed according to the instructions. The total volume of the system was 10 μL, including 2 μL 5×CE II Buffer, 50-200 ng linearized cloning vector, 50-200 ng insert amplification product, and 1 μL II. Ligate at 37°C for 30 minutes. After the reaction is complete, immediately cool the reaction in an ice-water bath for 5 minutes. The reaction product can be directly transformed. The transformation was carried out using the heat shock method (refer to the third edition of "Molecular Cloning Laboratory Manual", Science Press, 2002) to transform Escherichia coli DH5α. Positive clones were screened on LB solid plates containing 50 mg / L spectinomycin, and 6 positive clones were selected for sequencing (performed by Sangon Biotechnology Co., Ltd.). The sequencing results showed that the full length of the PyERF4.1 gene coding region is 759 bp, and its nucleotide sequence is shown in SEQ ID NO.1. The constructed recombinant vectors were named pSAK277-PyERF4.1 and pCAMBIA1300-PyERF4.1, and the constructed recombinant vectors were transformed into Agrobacterium tumefaciens strain GV3101 using chemical transformation.

[0040] The template for cloning the auxiliary factors PyERF3, PyMYB114, and PybHLH3 was the 'Hongqie' pear cDNA, and the primer pairs for PCR amplification were: PyERF3-F: 5'-actagtggatccaaagaattcATGTTTTTGGGGTACAGTCGGG-3' (SEQ ID No. 13); PyERF3-R: 5'-tcattaaagcaggactctagaTCAACTGGATGAGGATGGATTGTTGC-3' (SEQ ID No. 14); PyMYB114-F: 5'-actagtggatccaaagaattcATGAGGAAGGGTGCCTGG-3' (SEQ ID No. 15); PyMYB114-R: 5'-tcattaaagcaggactctagaCTAAATCTTAGTTATCTCTTCTTCTAGATTCCA-3' (SEQ ID No.16); PybHLH3-F: 5'-actagtggatccaaagaattcATGGCTGCACCGCCGCCAAG-3' (SEQ ID No.17); PybHLH3-R: 5'-tcattaaagcaggactctagaTTAAGAGTCAGATTGGGGTATAATTTGATTTATC-3' (SEQ ID No.18). The other steps and systems were the same as described above. Sequencing results showed that the PyERF3 coding region gene was 1200 bp in length, and its nucleotide sequence was shown in SEQ ID NO.7. The recombinant vector was named pSAK277-PyERF3. The PyMYB114 coding region gene was 687 bp in length, and its nucleotide sequence was shown in SEQ ID NO.8. The recombinant vector was named pSAK277-PyMYB114. The PybHLH3 coding region gene was 2130 bp in length, and its nucleotide sequence was shown in SEQ ID As shown in Figure 9, the constructed recombinant vector was named pSAK277-PybHLH3, and the constructed recombinant vector was transformed into Agrobacterium tumefaciens strain GV3101 by chemical transformation.

[0041] Example 3: The PyERF4.1 gene of the present invention inhibits the biosynthesis of anthocyanins in pear and tomato fruits

[0042] To verify the regulatory function of PyERF4.1 in anthocyanin synthesis, PyERF4.1 was overexpressed in pear calli. The resulting transgenic pear calli and wild-type (WT) pear calli were light-treated, and the total anthocyanin content was measured. After 15 days of light treatment, WT pear calli produced a large amount of anthocyanins, while PyERF4.1-OE pear calli did not accumulate anthocyanins ( Figure 2Compared with WT callus, the anthocyanin content and a* value of PyERF4.1-OE pear callus were significantly reduced ( Figure 2 RT-qPCR analysis showed that PyERF4.1 was overexpressed in transgenic pear calli ( Figure 2 d). Compared with WT callus, the expression levels of PyERF3, PyMYB114, PyDFR, PyANS, and PyUFGT were significantly decreased in PyERF4.1-OE pear callus ( Figure 2 In summary, PyERF4.1 negatively regulates anthocyanin biosynthesis in pear callus.

[0043] To further verify the inhibitory effect of PyERF4.1 on anthocyanin biosynthesis in red-skinned pears, we used RNAi-induced gene silencing to transiently silence PyERF4.1 in 'Zaosu' pears. To avoid silencing other genes with similar sequences, we selected a specific coding fragment from the 3' region of the transcription factor and inserted it into a vector to generate a recombinant vector: PyERF4.1-RNAi. Figure 3 As shown in a, no pigmentation was observed in the peels transformed with the empty vector pSAK277 and PyERF4.1, while obvious pigmentation was observed in the peels transformed with PyERF4.1-RNAi. In addition, the total anthocyanin content and a* value were significantly increased after PyERF4.1-RNAi transformation ( Figure 3 ), which is consistent with the phenotype. RT-qPCR analysis showed that the expression level of PyERF4.1 was significantly decreased in the pear peel after PyERF4.1-RNAi transformation compared with the control, while the expression levels of PyERF3, PyMYB114, PyDFR, PyANS, and PyUFGT were significantly increased ( Figure 3 Therefore, these results indicate that PyERF4.1 inhibits anthocyanin biosynthesis in pear.

[0044] Because stable transformation in pear is difficult, we screened the tomato homolog of PyERF4.1, SlERF4.1, for overexpression and knockout, and obtained T2 transgenic lines. Compared with WT tomatoes, ERF4.1-OE fruits are smaller, have rough skin, and are darker in color, while erf4.1 fruits are larger, have smooth skin, and are lighter in color during the ripening period ( Figure 4 Since it is not possible to directly observe the changes in anthocyanins in transgenic tomato fruits, the total anthocyanin content of transgenic tomato fruits was determined. RT-qPCR analysis showed that PyERF4.1 was significantly overexpressed in ERF4.1-OE tomato fruits ( Figure 4 c), while the expression of SlERF4.1 was significantly reduced in erf4.1 tomato fruits ( Figure 4 d). Compared with WT tomato fruits, the anthocyanin content of ERF4.1-OE fruits was significantly reduced, while the anthocyanin content of erf4.1 fruits was significantly increased ( Figure 4 b). The expression levels of SlERF3, SlMYB114, and anthocyanin biosynthesis-related genes SlDFR, SlANS, and SlUFGT in ERF4.1-OE fruits were significantly lower than those in WT fruits, but significantly higher than those in erf4.1 fruits ( Figure 4 In summary, ERF4.1 negatively regulates anthocyanin biosynthesis in tomato fruit.

[0045] Example 4: Co-transformation of the PyERF4.1 gene and the PyERF3-PyMYB114-PybHLH3 complex of the present invention inhibits anthocyanin biosynthesis in pear fruit

[0046] To investigate the molecular mechanism by which PyERF4.1 inhibits anthocyanin biosynthesis in pears, PyERF4.1 was transiently transformed with related transcription factors (PyERF3, PyMYB114, and PybHLH3), which are reported activators of anthocyanin biosynthesis, into pear peels. PyERF4.1 and PyERF3 were co-transformed with PyMYB114-PybHLH3 at different ratios (1:1, 2:1, and 1:2) into mature 'Zaosu' pear fruits, and their phenotypes were observed 6 days after injection. Figure 5 As shown in a, no pigmentation was observed in the peel transformed with the empty vector pSAK277. When PyMYB114-PybHLH3 was co-transformed, some pigmentation was observed, while when PyERF3-PyMYB114-PybHLH3 was co-transformed, significant pigmentation was observed. In addition, when the transformation ratio of PyERF4.1 to PyERF3 increased from 1:1 to 2:1, pigment deposition gradually decreased, while when the transformation ratio of PyERF3 increased, pigment accumulation increased again. As the transformation ratio of PyERF4.1 increased, the total anthocyanin content and a* value decreased significantly, which is consistent with the phenotypic changes in the pear peel after injection ( Figure 5 Next, the expression levels of anthocyanin biosynthesis genes in transformed pear fruit were analyzed by RT-qPCR. Consistent with the phenotypic results, the expression levels of PyDFR, PyANS, and PyUFGT were significantly reduced after co-transformation of PyERF4.1 with PyERF3-PyMYB114-PybHLH3. Furthermore, this inhibitory effect was enhanced with increasing the amount of PyERF4.1 transformed, while increasing the amount of PyERF3 transformed could alleviate this inhibitory effect to a certain extent ( Figure 5d). Therefore, co-transformation of PyERF4.1 with the PyERF3-PyMYB114-PybHLH3 complex can inhibit anthocyanin biosynthesis in pear fruit.

[0047] Example 5: Co-transformation of the PyERF4.1 gene of the present invention with the PyERF3-PyMYB114-PybHLH3 complex inhibits anthocyanin biosynthesis in strawberry fruit

[0048] To further explore how PyERF4.1 regulates anthocyanin biosynthesis together with PyERF3, PyMYB114, and PybHLH3, they were transiently co-transformed into yellow strawberry 'Yellow wonder' 5AF7 (YW5AF7) fruit (2 weeks after anthesis). Pigmentation appeared at the infiltration site 6 days after injection, as shown in Figure 6 As shown in a, no pigmentation was observed in the fruit transformed with the empty vector pSAK277. When PyMYB114-PybHLH3 was co-transformed, slight pigmentation was observed, while when PyERF3-PyMYB114-PybHLH3 was co-transformed, significant pigmentation was observed. In contrast, when PyERF4.1 was co-transformed, a significant decrease in pigmentation was observed. In addition, L*, a*, and b* values ​​were also affected by the color difference of strawberry fruit ( Figure 6 b). When PyERF3-PyMYB114-PybHLH3 were co-transformed, the anthocyanin content in strawberry fruit was significantly higher than when PyMYB114-PybHLH3 was co-transformed, while when PyERF4.1 was co-transformed with them, the anthocyanin content was significantly decreased ( Figure 6 c). In conclusion, co-transformation of PyERF4.1 with PyERF3-PyMYB114-PybHLH3 significantly reduced anthocyanin biosynthesis in strawberry fruit. Next, RT-qPCR analysis showed that co-transformation of PyERF4.1 with PyMYB114-PybHLH3 or PyERF3-PyMYB114-PybHLH3 significantly reduced the expression levels of anthocyanin biosynthesis-related genes (FvDFR, FvANS, and FvUFGT) in strawberry ( Figure 6 In conclusion, co-transformation of PyERF4.1 and the PyERF3-PyMYB114-PybHLH3 complex can inhibit the biosynthesis of anthocyanins in strawberry fruit.

[0049] Example 6: Verification of the interaction between PyERF4.1 and PyERF3

[0050] To explore the potential interactions between PyERF4.1 and PyERF3, PyMYB114, and PybHLH3, firefly luciferase complementation assays were performed in tobacco leaves. PyERF4.1 was inserted into the pCAMBIA1300-Nluc vector, while PyERF3 was inserted into the pCAMBIA1300-Cluc vector ( Figure 7 a). Co-expression of PyERF4.1-Nluc and PyERF3-CLuc showed strong luciferase activity. In contrast, no significant luciferase activity was observed in the control groups including PyERF4.1-Nluc and Cluc, and PyERF3-CLuc and Nluc ( Figure 7 b). Thus, PyERF4.1 and PyERF3 showed interaction. Next, the interaction between PyERF4.1 and PyERF3 was verified by yeast two-hybrid assay (Y2H). PyERF4.1 was cloned into pGBKT7 vector as full-length cDNA or C-terminal or N-terminal deletion. Figure 7 c), the full-length cDNA of PyERF3 was inserted into the pGADT7 vector. Figure 7 As shown in d, PyERF4.1 and PyERF3 had no self-activation phenomenon, but when the full-length PyERF4.1 (P3) and the two C-terminal parts of PyERF4.1 75-253 (P4), PyERF4.1 164-253 (P5) Co-transformed with PyERF3 can grow in both SD / Trp / Leu and SD / Trp / Leu / His / Ade media. Therefore, there is an interaction between PyERF4.1 and PyERF3, and these results are consistent with the firefly luciferase complementation experiment. Pull-down assays were used to further verify the interaction between PyERF4.1 and PyERF3. PyERF4.1 was inserted into the pMAL-c2x vector with an MBP protein tag, and PyERF3 was inserted into the pCold-TF vector with a HIS protein tag. Figure 7 As shown in Figure ​e, the MBP-tagged PyERF4.1 protein can be pulled down by the HIS-tagged PyERF3 protein, indicating that there is a protein interaction between PyERF4.1 and PyERF3, which is consistent with the results of firefly luciferase complementation and Y2H assays.

[0051] Example 7: PyMYB114 activates the PyERF4.1 promoter and regulates the transcription of pear anthocyanin biosynthesis structural genes

[0052] Transient expression experiments in pear and strawberry fruit showed that co-transformation of PyERF4.1 with PyMYB114-PybHLH3 inhibited pigment deposition, but PyERF4.1 did not interact with PyMYB114 and PybHLH3. Therefore, it was hypothesized that PyMYB114 binds to the PyERF4.1 promoter, and this hypothesis was tested using a yeast one-hybrid assay (Y1H). PlantPAN 3.0 was used to predict cis-acting elements in the PyERF4.1 promoter sequence. Figure 8 a. It was found that the 2kb promoter region upstream of PyERF4.1 contained a cis-acting element of the MYB transcription factor. Then, the constructed recombinant vector pGADT7-PyMYB114 was introduced into the Y1HGold yeast strain. The results showed that PyMYB114 could bind to the S1 fragment of PyERF4.1 ( Figure 8 b) The binding of PyMYB114 to the PyERF4.1 promoter was further verified using a dual luciferase reporter assay in tobacco leaves. Figure 8 As shown in Figure c, compared with the empty vector pSAK277, the transformation of PyMYB114 has a significant activation effect on the PyERF4.1 promoter. In summary, PyMYB114 binds to the PyERF4.1 promoter and activates its transcription.

[0053] PyERF4.1 interacts with PyERF3, PyMYB114, and PybHLH3 to inhibit anthocyanin biosynthesis, but the downstream target genes they act on remain to be determined. Therefore, we used a dual-luciferase reporter assay in tobacco leaves to investigate the effects of co-transformation of PyERF4.1 with PyERF3-PyMYB114-PybHLH3 on the promoter activity of the downstream target genes PyDFR, PyANS, and PyUFGT for anthocyanin biosynthesis. Compared with transformation with the empty vector pSAK277, co-transformation with PyERF3, PyMYB114, and PybHLH3 significantly activated the PyDFR, PyANS, and PyUFGT promoters, whereas co-transformation with PyERF4.1 significantly weakened the activation effect ( Figure 8 df). Therefore, PyERF4.1 reduces the activation of anthocyanin biosynthesis gene promoters by the PyERF3-PyMYB114-PybHLH3 complex, thereby inhibiting anthocyanin accumulation.

[0054] References

[0055] 1、An JP,Wang XF,Zhang XW,Xu HF,Bi SQ,You CX,Hao YJ.2020a.An apple MYBtranscription factor regulates cold tolerance and anthocyanin accumulationand undergoes MIEL1-mediated degradation.Plant Biotechnology Journal 18(2):337-353.https: / / doi.org / 10.1111 / pbi.13201。

[0056] 2、An JP,Zhang XW,Bi SQ,You CX,Wang XF,Hao YJ.2020b.The ERFtranscription factor MdERF38 promotes drought stress-induced anthocyaninbiosynthesis in apple.The Plant Journal101(3):573-589.https: / / doi.org / 10.1111 / tpj.14555。

[0057] 3、An JP,Wang XF,Li YY,Song LQ,Zhao LL,You CX,Hao YJ.2018.EIN3-LIKE1,MYB1,and ETHYLENE RESPONSE FACTOR3 act in a regulatory loop thatsynergistically modulatesethylene biosynthesis and anthocyaninaccumulation.Plant Physiology 178(2):808-823.https: / / doi.org / 10.1104 / pp.18.00068。

[0058] 4、Espley RV,Hellens RP,Putterill J,Stevenson DE,Kutty-Amma S,AllanAC.2007.Redcolouration in apple fruit is due to the activity of the MYBtranscription factor,MdMYB10.ThePlant Journal 49(3):414-27.https: / / doi.org / 10.1111 / j.1365-313X.2006.02964.x。

[0059] 5、Fujimoto SY,Ohta M,Usui A,Shinshi H,Ohme-TakagiM.2000.Arabidopsisethylene-responsive element binding factors act astranscriptional activators or repressors of GCCbox-mediated geneexpression.The Plant Cell 12(3):393-404.https: / / doi.org / 10.1105 / tpc.12.3.393。

[0060] 6、Hichri I,Heppel SC,Pillet J,Léon C,Czemmel S,Delrot S etal.2010.The basichelix-loop-helix transcription factor MYC1 is involved inthe regulation of the flavonoidbiosynthesis pathway in grapevine.MolecularPlant 3(3):509-23.https: / / doi.org / 10.1093 / mp / ssp118。

[0061] 7、Hichri I,Barrieu F,Bogs J,Kappel C,Delrot S,LauvergeatV.2011.Recent advances in thetranscriptional regulation of the flavonoidbiosynthetic pathway.Journal of Experimental Botany62(8):2465-2483.https: / / doi.org / 10.1093 / jxb / erq442。

[0062] 8、Koes R,Verweij W,Quattrocchio F.2005.Flavonoids:a colorful modelfor the regulationand evolution of biochemical pathways.Trends in PlantScience 10(5):236-42.https: / / doi.org / 10.1016 / j.tplants.2005.03.002。

[0063] 9、Li C,Wu J,Hu KD,Wei SW,Sun HY,Hu LY,Han Z,Yao GF,ZhangH.2020.PyWRKY26and PybHLH3 cotargeted the PyMYB114 promoter to regulateanthocyanin biosynthesis andtransport in red-skinned pears.HorticultureResearch 7:37.https: / / doi.org / 10.1038 / s41438-020-0254-z。

[0064] 10、Li X,Tao S,Wei S,Ming M,Huang X,Zhang S,Wu J.2018.The mining andevolutionaryinvestigation of AP2 / ERF genes in pear(Pyrus).BMC Plant Biology18(1):46.https: / / doi.org / 10.1186 / s12870-018-1265-x。

[0065] 11、Liu H,Liu Z,Wu Y,Zheng L,Zhang G.2021.Regulatory mechanisms ofanthocyaninbiosynthesis in apple and pear.International Journal of MolecularSciences 22(16):8441.https: / / doi.org / 10.3390 / ijms22168441。

[0066] 12、Liu Q,Kasuga M,Sakuma Y,Abe H,Miura S et al.1998.Two transcriptionfactors,DREB1 and DREB2,with an EREBP / AP2 DNA binding domain separate twocellular signaltransduction pathways in drought-and low-temperature-responsive gene expression,respectively,inArabidopsis.The Plant Cell 10(8):1391-406.https: / / doi.org / 10.1105 / tpc.10.8.1391。

[0067] 13、Ma H,Yang T,Li Y,Zhang J,Wu T,Song T,Yao Y,Tian J.2021.The longnoncoding RNAMdLNC499 bridges MdWRKY1 and MdERF109 function to regulateearly-stage light-inducedanthocyanin accumulation in apple fruit.The PlantCell 33(10):3309-3330.https: / / doi.org / 10.1093 / plcell / koab188。

[0068] 14、Ni J,Bai S,Zhao Y,Qian M,Tao R,Yin L,Gao L,Teng Y.2019.Ethyleneresponse factorsPp4ERF24 and Pp12ERF96 regulate blue light-induceddanthocyanin biosynthesis in'Red Zaosu'pear fruits by interacting withMYB114.Plant Molecular Biology 99(1-2):67-78.

[0069] https: / / doi.org / 10.1007 / s11103-018-0802-1

[0070] 15、Ni J,Premathilake AT,Gao Y,Yu W,Tao R,Teng Y,Bai S.2021.Ethylene-activatedPpERF105 induces the expression of the repressor-type R2R3-MYB genePpMYB140 to inhibitanthocyanin biosynthesis in red pear fruit.The PlantJournal 105(1):167–181.https: / / doi.org / 10.1111 / tpj.15049 .

[0071] 16、Peng M,Shahzad R,Gul A et al.2017.Differentially evolved glycosyltransferasesdetermine natural variation of rice flavone accumulationand UV-tolerance.Nature Communications8:1975.https: / / doi.org / 10.1038 / s41467-017-02168-x.

[0072] 17、Poracova J,Tkacikova L,Blascakova M,Muchanicova A.2011.Theimportance ofanthocyanins for human and animal health.Planta Medica 77:1447.https: / / doi.org / 10.1055 / s-0031-1282949。

[0073] 18、Ramsay NA,Glover BJ.2005.MYB-bHLH-WD40 protein complex and theevolution ofcellular diversity.Trends in Plant Science 10(2):63-70.https: / / doi.org / 10.1016 / j.tplants.2004.12.011

[0074] 19、Tanaka Y,Sasaki N,Ohmiya A.2008.Biosynthesis of plant pigments:anthocyanins,betalains and carotenoids.The Plant Journal 54(4):733-49.https: / / doi.org / 10.1111 / j.1365-313X.2008.03447.x。

[0075] 20、Valenta K,Nevo O,Martel C et al.2017.Plant attractants:integratinginsights frompollination and seed dispersal ecology.Evolutionary Ecology 31:249-267.https: / / doi.org / 10.1007 / s10682-016-9870-3。

[0076] 21、Wu T,Liu HT,Zhao GP,Song JX,Wang XL,Yang CQ,Zhai R etal.2020.Jasmonate andethylene-regulated ethylene response factor 22 promoteslanolin-induced anthocyanin biosynthesisin'Zaosu'pear(Pyrus bretschneideriRehd.)fruit.Biomolecules 10(2):278.https: / / doi.org / 10.3390 / biom10020278。

[0077] 22、Xu W,Grain D,Bobet S,Le Gourrierec J,Thévenin J,Kelemen Z etal.2014.Complexity and robustness of the flavonoid transcriptional regulatorynetwork revealed by comprehensive analyses of MYB-bHLH-WDR complexes andtheir targets in Arabidopsis seed.New Phytologist202(1):132-144.https: / / doi.org / 10.1111 / nph.12620。

[0078] 23、Yang LP,Ling WH,Du ZC,Chen YM,Li D et al.2017.Effects ofanthocyanins on cardiometabolic health:a systematic review and meta-analysisof randomized controlled trials.Advances in Nutrition 8(5):684-693.https: / / doi.org / 10.3945 / an.116.014852。

[0079] 24、Yang Y,Yao G,Yue W,Zhang S,Wu J.2015.Transcriptome profilingreveals differential gene expression in proanthocyanidin biosynthesisassociated with red / green skin color mutant of pear(Pyrus communis L.).Frontiers in Plant Science 6:795.https: / / doi.org / 10.3389 / fpls.2015.00795。

[0080] 25、Yao G,Ming M,Allan AC,Gu C,Li L,Wu X et al.2017.Map-based cloningof the pear gene MYB114 identifies an interaction with other transcriptionfactors to coordinately regulate fruit anthocyanin biosynthesis.The PlantJournal 92(3):437-451.https: / / doi.org / 10.1111 / tpj.13666。

[0081] 26、Zhai R,Wang Z,Zhang S,Meng G,Song L,Wang Z et al.2016.Two MYBtranscription factors regulate flavonoid biosynthesis in pear fruit(Pyrusbretschneideri Rehd.).Journal of Experimental Botany 67(5):1275-84.https: / / doi.org / 10.1093 / jxb / erv524。

[0082] 27. Zhang J, Xu H, Wang N, Jiang S, Fang H, Zhang Z, Yang G et al. 2018. Theethylene response factor MdERF1B regulates anthocyanin and proanthocyanidinbiosynthesis in apple.Plant Molecular Biology 98(3):205-218. https: / / doi.org / 10.1007 / s11103-018-0770-5.

[0083] 28. Zhao J, Dixon RA. 2009. MATE transporters facilitate vacuolar uptake of epicatechin3'-O-glucoside for proanthocyanidin biosynthesis in Medicagotruncatula and Arabidopsis. The Plant Cell 21(8):2323-40. https: / / doi.org / 10.1105 / tpc.109.067819.

[0084] 29. Zhou H, Lin-Wang K, Wang F, Espley RV, Ren F, Zhao J etal. 2019. Activator-type R2R3-MYB genes induce a repressor-type R2R3-MYB gene to balance anthocyanin and proanthocyanidin accumulation. New Phytologist 221(4):1919-1934. https: / / doi.org / 10.1111 / nph.15486.

[0085] The above description is only used to explain the preferred embodiments of the present invention and is not intended to limit the present invention in any form. Therefore, any modifications or changes made to the present invention under the same inventive spirit should still be included in the scope of protection intended by the present invention.

Claims

1. Application of an overexpression vector containing a pear transcription factor that inhibits anthocyanin biosynthesis in inhibiting anthocyanin biosynthesis in pear pericarp PyERF4.1 ​ Characterized in that: The pear transcription factor that inhibits anthocyanin biosynthesis PyERF4.1 The nucleotide sequence of the gene is shown in SEQ ID No.

1.

2. Use of an overexpression vector containing a pear transcription factor that inhibits anthocyanin biosynthesis according to claim 1 PyERF4.1 in inhibiting anthocyanin biosynthesis in pear peel Characterized in that: Using pCAMBIA1300 as the starting vector, insert the pear transcription factor that inhibits anthocyanin biosynthesis PyERF4.1 gene into Bam the HI and Sac I sites to obtain an overexpression vector containing the pear transcription factor PyERF4.1 gene that inhibits anthocyanin biosynthesis.

3. Application of a transient expression vector containing a pear transcription factor that inhibits anthocyanin biosynthesis PyERF4.1 in inhibiting anthocyanin biosynthesis in pear peel Characterized in that: The pear transcription factor that inhibits anthocyanin biosynthesis PyERF4.1 The nucleotide sequence of the gene is shown in SEQ ID No.

1.

4. Use of the transient expression vector containing the pear transcription factor that inhibits anthocyanin biosynthesis according to claim 3 PyERF4.1 in inhibiting anthocyanin biosynthesis in pear pericarp Characterized in that: Using pSAK277 as the starting vector, the pear transcription factor that inhibits anthocyanin biosynthesis PyERF4.1 gene was inserted EcoR between I and Xba I sites to obtain a transient expression vector containing the pear transcription factor PyERF4.1 gene that inhibits anthocyanin biosynthesis.

Images