A pear transcription factor PyERF4.2 gene for inhibiting anthocyanin biosynthesis and its recombinant expression vector and application

Abstract

A pear transcription factor that inhibits anthocyanin biosynthesis PyERF4.2 Gene and its recombinant expression vector and application, the PyERF4.2 The nucleotide sequence of the gene is shown in SEQ ID No. 1. PyERF4.2 pear callus to verify its function of inhibiting anthocyanin biosynthesis and PyERF4.2 and PyERF3, PyMYB114, and PybHLH3 Gene co-transformation of pear and strawberry fruits resulted in reduced anthocyanin accumulation in pear and strawberry fruits, and biological function verification showed that the cloned PyERF4.2 and PyERF3, PyMYB114, and PybHLH3 Genetic interactions 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 PyERF4.2 gene for inhibiting anthocyanin biosynthesis, a recombinant expression vector and an application thereof, and specifically relates to a PyERF4.2 gene, a member of the AP2 / ERF family related to inhibiting anthocyanin biosynthesis in pear peel, obtained by separation and cloning from 'Hongqie' pear, and an application thereof. Background Art

[0002] Pear (Pyrus L.) is one of the most popular fruits in the world. Red-skinned pears are favored by consumers for their attractive color and rich anthocyanins (Li et al., 2020). Anthocyanins are widely present in plants. They not only enhance the reproductive capacity of plants, but also play an important 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). In addition, anthocyanins can protect humans from the effects of 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 aminolyase (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). Many transcription factors are involved in regulating plant anthocyanin biosynthesis. At present, MYBs, bHLHs and WDRs have been found to regulate the anthocyanin biosynthesis pathway (Hichri et al., 2010; Xu et al., 2014). In general, anthocyanin biosynthesis is mainly regulated by a complex consisting of three transcription factors, MYB-bHLH-WD40 (MBW) (Ramsay & Glover et al., 2005; Liu et al., 2021). As the core regulator of the MBW complex, MYBs have been widely reported to regulate anthocyanin biosynthesis together with bHLHs. In apple, MdMYB10 interacts with MdbHLH3 and MdbHLH33 to promote anthocyanin biosynthesis (Espley et al., 2007). In pear, PyMYB114, PyMYB10 or PyMYB10b interact 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] There is increasing evidence that, in addition to the MBW complex, the AP2 / ERF family is also involved 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 developmental processes, regulate the synthesis of plant secondary metabolites, and respond to various stresses by binding to short cis-acting elements such as the GCC-box and dehydration response elements (DRE) / C-repeat elements (CRT) in the promoters of target genes (Liu et al., 1998; Fujimoto et al., 2000; Li et al., 2018). A large number of studies have shown that AP2 / ERFs interact with the MBW complex and participate in the regulation of anthocyanin biosynthesis. They have diverse regulatory patterns in anthocyanin biosynthesis induced under different conditions and in different plant species. In the 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); PbERF22 regulated by jasmonic acid and ethylene enhances 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 promote blue light-induced anthocyanin biosynthesis by interacting with PpMYB114 in ‘Hongzaosu’ pear fruit (Ni et al., 2019). In drought stress-induced anthocyanin biosynthesis, MdERF38 interacts with MdMYB1 to promote drought stress-induced anthocyanin biosynthesis in apple fruit (An et al., 2020b). In addition, PyERF3 screened from the green mutant of ‘Hongqie’ pear interacts with PyMYB114 and forms a new complex with PybHLH3 to jointly regulate anthocyanin biosynthesis (Yang et al., 2015; Yao et al., 2017). Many AP2 / ERFs have been reported to positively regulate anthocyanin biosynthesis, but AP2 / ERFs negatively regulating anthocyanin biosynthesis have 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 needs 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 and a recombinant expression vector and application thereof.

[0006] To achieve the above purpose and other related purposes, the technical solution provided by the present invention is: a pear transcription factor PyERF4.2 gene that inhibits anthocyanin biosynthesis, and the nucleotide sequence of the PyERF4.2 gene is shown in SEQ ID No.1.

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

[0008] To achieve the above purpose and other related purposes, the technical solution provided by the present invention is: an overexpression vector containing the pear transcription factor PyERF4.2 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 PyERF4.2 gene that inhibits anthocyanin biosynthesis is obtained by using pCAMBIA1300 as the starting vector and inserting the pear transcription factor PyERF4.2 gene that inhibits anthocyanin biosynthesis between the BamHI and SacI sites.

[0010] To achieve the above 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 purpose and other related purposes, the technical solution provided by the present invention is: a transient expression vector containing the pear transcription factor PyERF4.2 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 PyERF4.2 gene that inhibits anthocyanin biosynthesis, characterized in that: pSAK277 is used as the starting vector, and the pear transcription factor PyERF4.2 gene that inhibits anthocyanin biosynthesis is inserted between the EcoRI and XbaI sites.

[0013] To achieve the above purpose and other related purposes, the technical solution provided by the present invention is: application of 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 PyERF4.2 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 PyERF4.2 gene that inhibits anthocyanin biosynthesis, the upstream primer PyERF4.2-F1 sequence is shown in SEQ ID No.3, the PyERF4.2-F2 sequence is shown in SEQ ID No.4, the downstream primer PyERF4.2-R1 sequence is shown in SEQ ID No.5, and the PyERF4.2-R2 sequence is shown in SEQ ID No.6.

[0016] To achieve the above-mentioned purpose and other related purposes, the technical scheme provided by the present invention is: an application of a pear transcription factor PyERF4.2 gene that inhibits anthocyanin biosynthesis in inhibiting the biosynthesis of anthocyanins in pear peel, and an application of a pear transcription factor PyERF4.2 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 PyERF4.2 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 IDNo.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 scheme provided by the present invention is: an application of a pear transcription factor PyERF4.2 gene that inhibits anthocyanin biosynthesis in inhibiting the biosynthesis of anthocyanins in pear peel, and an application of a transient expression vector containing the PyERF4.2 gene that inhibits anthocyanin synthesis combined with a recombinant vector containing 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 scheme provided by the present invention is: an application of the pear transcription factor PyERF4.2 gene that inhibits anthocyanin biosynthesis in inhibiting the biosynthesis of anthocyanins in pear peel, and an overexpression vector containing the PyERF4.2 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.

[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 PyERF4.2 gene in inhibiting the biosynthesis of anthocyanins in pear peel provides new genetic resources for molecular breeding of anthocyanins accumulation in pear peel and new genetic resources 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 overexpresses the transcription factor PyERF4.2 in pear callus to verify its function of inhibiting anthocyanin biosynthesis, and co-transforms PyERF4.2 with PyERF3, PyMYB114 and PybHLH3 genes into pear and strawberry fruits, resulting in reduced anthocyanin accumulation in pear and strawberry fruits. The biological function verification shows that the cloned PyERF4.2 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 / ERFs genes screened from transcriptome data of ‘Hongqie’ pear and its green mutant. (a) Heat map of differentially expressed AP2 / ERFs genes at different developmental stages of the fruit skin of ‘Hongqie’ pear and its green mutant. R1, R2 and R3 are samples of ‘Hongqie’ pear at 40, 55 and 85 days after flowering; G1, G2 and G3 represent samples of ‘Hongqie’ pear green mutant at 40, 55 and 85 days after flowering, respectively. Blue boxes represent down-regulated AP2 / ERFs screened in red-skinned pear. (b) The inhibitory types of AP2 / ERFs in different plant species were analyzed in the phylogenetic tree. The C-terminus of the ERF domain was analyzed by the neighbor-joining method using the MEGA7 program. (c) Protein sequence alignment analysis of candidate AP2 / ERFs. Among them, Pbr038280.1 was named PyERF4.2; 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 PyERF4.2 inhibits anthocyanin biosynthesis in pear callus. Wherein: (a) Phenotype of pear callus with overexpression of PyERF4.2 after 15 days of light treatment. WT, wild type; OE, overexpression. (b) The difference in color is represented by the values ​​of L*, a*, and b*. L* represents brightness; a* represents the range from green to magenta; b* represents the range from yellow to blue. (c) Total anthocyanin content in pear callus with overexpression of PyERF4.2. (d) Expression level of PyERF4.2 in transgenic pear callus. (f) Expression levels of PyERF3, PyMYB114, PyDFR, PyANS, and PyUFGT in transgenic pear callus.

[0024] Figure 3Effects of PyERF4.2-RNAi on anthocyanin biosynthesis in pear fruit. (a) Phenotypes of pear peels injected with RNAi-induced gene silencing: a, pSAK277; b, PyERF4.2; c, PyERF4.2-RNAi. (b) Color differences are represented by the values ​​of L*, a*, and b*. L* represents brightness; a* represents the range from green to magenta; and b* represents the range from yellow to blue. (c) Total anthocyanin content in pear peels after injection. (d) Expression level of PyERF4.2. (e) Expression levels of PyERF3 and PyMYB114. (f) Expression levels of PyDFR, PyANS, and PyUFGT.

[0025] Figure 4 PyERF4.2 was co-transformed with PyERF3, PyMYB114 and PybHLH3 to inhibit the biosynthesis of anthocyanins in pear fruit. Among them: (a) Phenotypes of 'Zaosu' pear skin after injection: a, pSAK277; b, PyMYB114+PybHLH3; c, PyERF4.2+PyMYB114+PybHLH3; d PyERF3+PyMYB114+PybHLH3; e, PyERF4.2:PyERF3(1:1)+PyMYB114+PybHLH3; f, PyERF4.2:PyERF3(2:1)+PyMYB114+PybHLH3; g, PyERF4.2:PyERF3(1:2)+PyMYB114+PybHLH3. (b) The color difference is represented by the values ​​of L*, a*, and b*. L* represents lightness; a* represents the range from green to magenta; and b* represents the range from yellow to blue. (c) The total anthocyanin content in pear peel after injection. (d) The expression levels of PyDFR, PyANS, and PyUFGT.

[0026] Figure 5 PyERF4.2 was co-transformed with PyERF3, PyMYB114 and PybHLH3 to inhibit the biosynthesis of anthocyanins in strawberry fruit. Wherein: (a) Phenotype of strawberry fruit after injection: a, pSAK277; b, PyMYB114+PybHLH3; c, PyERF4.2+PyMYB114+PybHLH3; d, PyERF3+PyMYB114+PybHLH3; f, PyERF4.2+PyERF3+PyMYB114+PybHLH3. 1, is the overall view. 2, is the cross-sectional view. (b) The difference in color is represented by the values ​​of L*, a* and b*. L* represents brightness; a* represents the range from green to magenta; b* represents the range from yellow to blue. (c) The total anthocyanin content of strawberry fruit after injection. (d) Expression levels of FvDFR, FvANS and FvUFGT.

[0027] Figure 6 Figure 2. The interaction between PyERF4.2 and PyERF3. (a) Models of NLuc, CLuc and NLuc / CLuc constructs. (b) Firefly luciferase complementation assay to verify the interaction between PyERF4.2 and PyERF3. (c) PyERF4.2 protein sequence segmentation. P1-P5, part1 to part5, different amino acid residues of PyERF4.2. (d) Yeast two-hybrid assay to verify the interaction between PyERF4.2 and PyERF3. (e) Pull-down assay to verify the interaction between PyERF4.2 and PyERF3. α-MBP, MBP antibody; α-HIS, HIS antibody.

[0028] Figure 7 PyMYB114 binds to the PyERF4.2 promoter and regulates the transcription of anthocyanin biosynthesis genes. Wherein: (a) Schematic diagram of the PyERF4.2 promoter. The cis-acting elements in the promoter region were predicted using the PlantPAN 3.0 database and segmented (S1-S3). Pro, promoter. MYB is the MYBs cis-acting element. (b) Yeast one-hybrid assay verifies that PyMYB114 binds to the PyERF4.2 promoter. Yeast colonies were screened on SD / -Ura / -Leu / AbA800 plates. (c) Dual luciferase reporter assay in tobacco leaves verifies that PyMYB114 activates the PyERF4.2 promoter. (df) Dual luciferase reporter assay verifies the effect of co-transformation of PyERF4.2 with PyERF3, PyMYB114 and PybHLH3 on the transcriptional activity of PyDFR, PyANS and PyUFGT in tobacco leaves. DETAILED DESCRIPTION

[0029] The following is a description of the implementation of the present invention by means of specific embodiments. Those skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification.

[0030] See also Figure 1-7. It should be noted that the structures, proportions, sizes, etc. illustrated in the drawings in 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, so they have no substantial 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 effects and purposes 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.

[0031] Example 1: The present invention screens candidate AP2 / ERF genes through transcriptome data and bioinformatics analysis

[0032] In order to study the regulation of AP2 / ERF genes on anthocyanin biosynthesis in red-skinned pear, the transcriptome data of red-skinned pear 'Hongqie' and its green mutant at 40, 55 and 85 days after flowering were deeply explored, and 114 AP2 / ERFs were found to have differentially expressed genes. Through heat map analysis, 33 differentially expressed ERFs were downregulated in the critical period of fruit peel coloration in 'Hongqie' pear ( Figure 1 Next, these 33 down-regulated ERFs were compared with negative regulators 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, Pbr038280.1 is most closely related to EjERF11, MdERF4, MaERF11 and AdERF9. In addition, protein sequence alignment analysis showed that these genes all have AP2 domains ( Figure 1 c). At different developmental stages, the expression level of Pbr038280.1 in ‘red eggplant’ pear was significantly downregulated compared with that in the green mutant, indicating that it may be involved in anthocyanin biosynthesis as a repressor. Therefore, Pbr038280.1 was selected for further study and named PyERF4.2.

[0033] Example 2: Cloning and overexpression of the PyERF4.2 gene of the present invention, and construction of a transient expression vector

[0034] RNA was extracted from the peel of 'Hongqie' pear using the Plant Total RNA Isolation Kit Plus (Foregene, RE-05022) kit according to the operating instructions provided by the kit, specifically: 500 mg of freeze-dried 'Hongqie' pear peel was placed in a mortar, and liquid nitrogen was added to fully grind into fine powder; 500 μL of Buffer PSL1 was aspirated into a 2 mL centrifuge tube, 10 μL of β-mercaptoethanol was added, and mixed; an appropriate amount (about 50 mg) of the ground powder was scraped with a blue pipette tip cooled by liquid nitrogen and transferred into Buffer PSL1, and vortexed to mix; after standing at room temperature for 5 min, 100 μL of Buffer PS was added and gently mixed; all liquids were transferred to the DNA-Cleaning Column, and the filter column was removed after centrifugation at 12000 rpm for 2 min, 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 PSL2, gently mix; take 500μL of the mixture and transfer it to the RNA-only Column, centrifuge at 12000rpm for 1min and discard the waste liquid; add 500μL Buffer PRW1 to the RNA-only Column, centrifuge for 1min and discard the waste liquid; add 700μL anhydrous ethanol, centrifuge for 1min and discard the waste liquid; add 700μL Buffer PRW2 to the RNA-only Column, centrifuge at 12000rpm for 1min, discard the waste liquid, and repeat this step once; centrifuge at 12000rpm for 2min and discard the collection tube; transfer the RNA-only Column to a new 2mL centrifuge tube, drop 60μL RNase-Free ddH2O preheated at 65℃ into the center of the membrane, leave it at room temperature for 2min, centrifuge at 12000rpm for 1min and collect RNA. Take 2μL of RNA and run agarose gel electrophoresis, estimate the RNA concentration based on the band brightness, and store it in a -80℃ refrigerator for later use.

[0035] Total RNA was reverse transcribed into first-strand cDNA, and the first-strand cDNA was synthesized using Prime Script TM RT MasterMix (Takara) reverse transcription kit (follow the instructions provided by the kit). The reverse transcription system is 10μL: 5× Prime Script RT Master Mix 2μL, total RNA 5μL, RNase-Free ddH2O 5μL. The reaction conditions are 37℃, 15min; 85℃, 5s; 4℃, ∞. The obtained cDNA can be stored in a -20℃ refrigerator.

[0036] The first-strand cDNA obtained by reverse transcription was used to amplify the full-length coding region of the PyERF4.2 gene. The primer pair for amplifying the gene was PyERF4.2-F1: 5'-acgggggactctagaggatccATGGCTCCGACGACGGAG-3' (SEQ ID No. 3); PyERF4.2-R1: 5'-cgatcggggaaattcgagctcTTACATAAAAAGGAACAAACTTGCACC-3' (SEQ ID No. 4); PyERF4.2-F2: 5'-actagtggatccaaagaattcATGGCTCCGACGACGGAG-3' (SEQ ID No. 5); PyERF4.2-R2: 5'-tcattaaagcaggactctagaTTACATAAAAAGGAACAAACTTGCACC-3' (SEQ ID No. 6). Ultra-fidelity DNA polymerase Super-Fidelity DNA Polymerase (P505-d1) was purchased from Novagen Biotech. The amplification reaction system was 50 μL, including 200 ng cDNA, 25 μL 2×Phanta Max Buffer, 1 μL 10 mM dNTP, 1 μL Phanta Max Super-Fidelity DNA Polymerase (1U / μl), 2 μL 10 μM of the above primers, and ddH2O was added to 50 μL. The PCR reaction was completed on an eppendorf amplification instrument according to the following program: 95°C, pre-denaturation for 3 minutes, 95°C denaturation for 15 seconds, 60°C annealing for 15 seconds, 72°C extension for 90 seconds, 35 thermal cycles, 72°C extension for 5 minutes, and storage at 4°C. A single PCR band product was generated. After the PCR product was detected by 1% agarose gel electrophoresis, the DNA fragment was recovered using an agarose gel recovery kit.

[0037] 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 Novozyme 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. Connect at 37℃ for 30min. After the reaction is completed, immediately place the reaction in an ice water bath to cool for 5min, and the reaction product can be directly transformed. The transformation adopts the heat shock method (refer to the third edition of "Molecular Cloning Experiment Manual", Science Press, 2002) to transform Escherichia coli DH5α, screen positive clones in LB solid plates containing 50mg / L spectinomycin, and pick 6 positive clones for sequencing (completed by Sangon Biotechnology Co., Ltd.). The sequencing results show that the full length of the coding region of the PyERF4.2 gene is 642bp, and its nucleotide sequence is shown in SEQ ID NO.1. The constructed recombinant vectors are named pSAK277-PyERF4.2 and pCAMBIA1300-PyERF4.2, and the constructed recombinant vectors are transformed into Agrobacterium strain GV3101 by chemical transformation.

[0038] 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), other steps and systems are the same as described above, sequencing results show that the full length of the PyERF3 coding region gene is 1200bp, and its nucleotide sequence is shown in SEQ ID NO.7, and the constructed recombinant vector is named pSAK277-PyERF3; the full length of the PyMYB114 coding region gene is 687bp, and its nucleotide sequence is shown in SEQ ID NO.8, and the constructed recombinant vector is named pSAK277-PyMYB114; the full length of the PybHLH3 coding region gene is 2130bp, and its nucleotide sequence is SEQ ID As shown in Figure 9, the constructed recombinant vector was named pSAK277-PybHLH3, and the constructed recombinant vector was transformed into Agrobacterium strain GV3101 by chemical transformation.

[0039] Example 3: The PyERF4.2 gene of the present invention inhibits the biosynthesis of anthocyanins in pear fruit

[0040] To verify the regulatory function of PyERF4.2 in anthocyanin synthesis, PyERF4.2 was overexpressed in pear callus. The transgenic pear callus and wild-type (WT) pear callus obtained were treated with light, and the total anthocyanin content was determined. After 15 days of light treatment, WT pear callus produced a large amount of anthocyanins, while PyERF4.2-OE pear callus only accumulated a small amount of anthocyanins ( Figure 2Compared with WT callus, the anthocyanin content and a* value of PyERF4.2-OE pear callus were significantly reduced ( Figure 2 RT-qPCR analysis showed that PyERF4.2 was overexpressed in transgenic pear callus ( Figure 2 d). Compared with WT callus, the expression levels of PyERF3, PyMYB114, PyDFR, PyANS, and PyUFGT were significantly decreased in PyERF4.2-OE pear callus ( Figure 2 e). In summary, PyERF4.2 has a negative regulatory effect on the biosynthesis of anthocyanins in pear callus.

[0041] To further verify the inhibitory effect of PyERF4.2 on anthocyanin biosynthesis in red-skinned pears, the RNAi-induced gene silencing method was used to transiently silence PyERF4.2 in ‘Zaosu’ pears. In order to avoid silencing other genes with similar sequences, a specific coding fragment was selected from the 3’ region of the transcription factor and inserted into the vector to generate a recombinant vector: PyERF4.2-RNAi. Figure 3 As shown in a, no pigmentation was observed in the peel transformed with empty vector pSAK277 and PyERF4.2, while obvious pigmentation was observed in the peel transformed with PyERF4.2-RNAi. In addition, after PyERF4.2-RNAi transformation, the total anthocyanin content and a* value also increased significantly ( Figure 3 RT-qPCR analysis showed that the expression level of PyERF4.2 was significantly decreased in the pear peel after PyERF4.2-RNAi transformation compared with the control, while the expression levels of PyERF3, PyMYB114, PyDFR, PyANS, and PyUFGT were significantly increased ( Figure 3 df). Therefore, these results indicate that PyERF4.2 inhibits anthocyanin biosynthesis in pear.

[0042] Example 4: Co-transformation of the PyERF4.2 gene of the present invention with the PyERF3-PyMYB114-PybHLH3 complex inhibits the biosynthesis of anthocyanins in pear fruit

[0043] To investigate the molecular mechanism by which PyERF4.2 inhibits anthocyanin biosynthesis in pears, PyERF4.2 was transiently transformed with related transcription factors (PyERF3, PyMYB114, and PybHLH3), which are reported activators of anthocyanin biosynthesis, in pear peels. PyERF4.2 was co-transformed with PyERF3 at different ratios (1:1, 2:1, 1:2) and PyMYB114-PybHLH3 into mature ‘Zaosu’ pear fruits, and their phenotypes were observed 6 days after injection. Figure 4 As shown in a, no pigmentation was observed in the peel transformed with the empty vector pSAK277. Some pigmentation was observed when PyMYB114-PybHLH3 was co-transformed, whereas significant pigmentation was observed when PyERF3-PyMYB114-PybHLH3 was co-transformed. In addition, when the transformation ratio of PyERF4.2 to PyERF3 increased from 1:1 to 2:1, pigment deposition gradually decreased, whereas pigment accumulation increased again when the transformation ratio of PyERF3 increased. The total anthocyanin content and a* value decreased significantly with the increase in the transformation ratio of PyERF4.2, which is consistent with the phenotypic changes in the pear peel after injection ( Figure 4 bc). 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 PyERF4.2 with PyERF3-PyMYB114-PybHLH3. In addition, this inhibitory effect was enhanced with the increase in the amount of PyERF4.2 transformation, while increasing the amount of PyERF3 transformation could alleviate this inhibitory effect to a certain extent ( Figure 4 d). Therefore, co-transformation of PyERF4.2 with the PyERF3-PyMYB114-PybHLH3 complex can inhibit anthocyanin biosynthesis in pear fruit.

[0044] Example 5: Co-transformation of the PyERF4.2 gene of the present invention with the PyERF3-PyMYB114-PybHLH3 complex inhibits the biosynthesis of anthocyanins in strawberry fruit

[0045] To further explore how PyERF4.2 regulates anthocyanin biosynthesis together with PyERF3, PyMYB114, and PybHLH3, it was 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, e.g. Figure 5 As shown in a, no pigmentation was observed in the fruits 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 PyERF4.2 was co-transformed, a significant decrease in pigmentation was observed. In addition, the L*, a*, and b* values ​​were also affected by the color differences of strawberry fruits ( Figure 5b). When PyERF3-PyMYB114-PybHLH3 were co-transformed, the anthocyanin content in strawberry fruit was significantly higher than that when PyMYB114-PybHLH3 was co-transformed, while when PyERF4.2 was co-transformed with them, the anthocyanin content was significantly reduced ( Figure 5 c). In conclusion, the co-transformation of PyERF4.2 with PyERF3-PyMYB114-PybHLH3 significantly reduced the biosynthesis of anthocyanins in strawberry fruit. Next, RT-qPCR analysis showed that the expression levels of anthocyanin biosynthesis-related genes (FvDFR, FvANS, and FvUFGT) in strawberry were significantly reduced when PyERF4.2 was co-transformed with PyMYB114-PybHLH3 or PyERF3-PyMYB114-PybHLH3 ( Figure 5 d). In conclusion, co-transformation of PyERF4.2 and PyERF3-PyMYB114-PybHLH3 complex can inhibit the biosynthesis of anthocyanins in strawberry fruit.

[0046] Example 6: Verification of the interaction between PyERF4.2 and PyERF3

[0047] To explore the potential interactions between PyERF4.2 and PyERF3, PyMYB114, and PybHLH3, firefly luciferase complementation assays were performed in tobacco leaves. PyERF4.2 was inserted into the pCAMBIA1300-Nluc vector, while PyERF3 was inserted into the pCAMBIA1300-Cluc vector ( Figure 6 a). The co-expression of PyERF4.2-Nluc and PyERF3-CLuc showed strong luciferase activity. In contrast, no obvious luciferase activity was observed in the control groups including PyERF4.2-Nluc and Cluc, and PyERF3-CLuc and Nluc ( Figure 6 b). It can be seen that PyERF4.2 and PyERF3 show interaction. Next, the interaction between PyERF4.2 and PyERF3 was verified by yeast two-hybrid assay (Y2H). PyERF4.2 was cloned into pGBKT7 vector as full-length cDNA or C-terminal or N-terminal deletion ( Figure 6 c), the full-length cDNA of PyERF3 was inserted into the pGADT7 vector. Figure 6As shown in d, neither PyERF4.2 nor PyERF3 has self-activation phenomenon, and when the full-length PyERF4.2 (P3) and PyERF3 are co-transformed, they can grow in SD / Trp / Leu and SD / Trp / Leu / His / Ade medium. Therefore, there is an interaction between PyERF4.2 and PyERF3, and these results are consistent with the firefly luciferase complementation experiment. Pull-down experiments were used to further verify the interaction between PyERF4.2 and PyERF3. PyERF4.2 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. As shown Figure 6 As shown in e, the MBP-tagged PyERF4.2 protein can be pulled down by the HIS-tagged PyERF3 protein, indicating that there is a protein interaction between PyERF4.2 and PyERF3, which is consistent with the results of firefly luciferase complementation and Y2H assays.

[0048] Example 7: PyMYB114 activates the PyERF4.2 promoter and regulates the transcription of pear anthocyanin biosynthesis structural genes

[0049] The results of transient expression experiments in pear and strawberry fruits showed that co-transformation of PyERF4.2 with PyMYB114-PybHLH3 inhibited pigment deposition, but PyERF4.2 did not interact with PyMYB114 and PybHLH3. Therefore, it was assumed that PyMYB114 binds to the PyERF4.2 promoter, and this hypothesis was verified by yeast one-hybrid assay (Y1H). PlantPAN 3.0 was used to predict the cis-acting elements on the PyERF4.2 promoter sequence, and the results are shown in Figure 7 a. It was found that the 2kb promoter region upstream of PyERF4.2 contained 6 cis-acting elements of MYB transcription factors. 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 PyERF4.2 ( Figure 7 b). The binding of PyMYB114 to the PyERF4.2 promoter was further verified using a dual luciferase reporter assay in tobacco leaves. Figure 7 As shown in c, compared with the empty vector pSAK277, the transformation of PyMYB114 has a significant activation effect on the promoter of PyERF4.2. In summary, PyMYB114 binds to the promoter of PyERF4.2 and activates its transcription.

[0050] PyERF4.2 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 study the effects of co-transformation of PyERF4.2 with PyERF3-PyMYB114-PybHLH3 on the promoter activity of the downstream target genes PyDFR, PyANS, and PyUFGT of anthocyanin biosynthesis. Compared with the empty vector pSAK277 transformation, it was found that co-transformation of PyERF3, PyMYB114, and PybHLH3 could significantly activate the promoters of PyDFR, PyANS, and PyUFGT, while the activation was significantly weakened when co-transformed with PyERF4.2 ( Figure 7 Therefore, PyERF4.2 reduces the activation of anthocyanin biosynthesis gene promoters by the PyERF3-PyMYB114-PybHLH3 complex, thereby inhibiting the accumulation of anthocyanins.

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[0082] 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. A pear transcription factor containing anthocyanin biosynthesis inhibitor PyERF4.2 The use of a gene overexpression vector in inhibiting anthocyanin biosynthesis in pear peel is characterized by: Said PyERF4.2 The nucleotide sequence of the gene is shown in SEQ ID No.

1.

2. A pear transcription factor containing anthocyanin biosynthesis inhibitor PyERF4.2 The application of a transient expression vector of a gene in inhibiting anthocyanin biosynthesis in pear peel is characterized by: Said PyERF4.2 The nucleotide sequence of the gene is shown as SEQ ID No.1.

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