Pear transcription factor PyHAT5 and application thereof in synthesis of red-flesh pear anthocyanin

Abstract

The invention discloses a pear transcription factor PyHAT5 and application of the pear transcription factor PyHAT5 in synthesis of red-flesh pear anthocyanin. The transcription factor PyHAT5 capable of regulating and controlling pulp anthocyanin biosynthesis in the red-pulp pears belongs to an HD-ZIP family member, the nucleotide sequence of the transcription factor PyHAT5 is as shown in SEQ ID No.1, and the amino acid sequence coded by the transcription factor PyHAT5 is as shown in SEQ ID No.2 in a sequence table. Through instantaneous overexpression in pear fruits, the PyHAT5 is proved to be capable of inhibiting accumulation of anthocyanin in the pear fruits; and the accumulation of the anthocyanin in the pear fruits can be promoted by instantaneously silencing the PyHAT5. The discovery of the gene supplements and perfects an anthocyanin synthesis regulation mechanism of HD-ZIP transcriptional regulation in pears, and the effect of the PyHAT5 gene on inhibiting accumulation of the flesh anthocyanin can be relieved through technologies such as gene editing and RNAi in the future, so that synthesis of the flesh anthocyanin of the pears is promoted, and a gene resource and a theoretical basis are provided for breeding of the red-flesh pears.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of plant genetic engineering, and relates to a pear transcription factor PyHAT5 and application thereof, in particular to an HD-ZIP family member regulating accumulation of pear flesh anthocyanin isolated and cloned from red-fleshed pear PyHAT5 gene. BACKGROUND

[0002] Pear is one of the important fruit tree species in the world, and its fruit is deeply loved by consumers. Compared with other species in Rosaceae family (such as apple, strawberry and peach), the flesh of pear is usually white, and red-fleshed varieties are very rare. In the two major pear groups in Asia and Europe, a small amount of red-fleshed germplasm is found in European pear, mainly in wild Pyrus calleryana, while Asian pear lacks red-fleshed germplasm and only a few varieties show red skin. It is well known that some consumers will choose to eat peeled fruit, so red-fleshed fruit provides more edible healthy nutrients. Therefore, red-fleshed fruit becomes an important trait in fruit tree breeding, including pear. The red color of red-fleshed pear fruit mainly comes from anthocyanin (Chang et al., 2023). The biosynthetic pathway of anthocyanin is a branch of plant secondary metabolism, which starts from phenylalanine and is catalyzed by a series of enzymes (such as PAL, CHS, CHI, F3H, DFR, ANS and UFGT) to ultimately lead to the formation of anthocyanin (Koes et al., 2005; Xu et al., 2015). The MYB-bHLH-WD40 (MBW) complex is a key activating complex in the anthocyanin biosynthetic pathway of many species, which regulates the expression of structural genes of the anthocyanin synthesis pathway, including apple (Espley et al., 2007; Honda and Moriya, 2018), peach (Rahim et al., 2014), strawberry, bayberry (Liu et al., 2013; Xue et al., 2024), kiwifruit (Liu et al.).

[0003] So far, the molecular mechanism of red-skin pear anthocyanin synthesis has been well studied. MYB transcription factors (TFs) were reported to play a crucial role in pear skin coloration, and MYB10 and MYB114 were identified as key transcription factors in pear anthocyanin biosynthesis (Feng et al., 2010; Yao et al., 2017). Subsequently, other transcription factors (TFs) have also attracted attention, such as bZIP (Liu et al., 2019), PySPL (Qian et al., 2017; Liu et al., 2021), ERF (Ni et al., 2023; Yang et al., 2024), PIF (Liu et al.), NAC (Zhang et al., 2024), and WRKY (Li et al., 2020; Alabd et al., 2022), which play a positive or negative regulatory role in the pear skin anthocyanin biosynthesis pathway. In recent years, it has been found that the BBX gene family plays an important role in pear skin anthocyanin synthesis. BBX16 and BBX18 can interact with HY5, activate the expression of MYB10, and thus promote the synthesis of anthocyanins in pear skin (Bai et al., 2019a, 2019b). The 14-nucleotide deletion mutation in the coding region of PpBBX24 gene is associated with the red-skin ‘Hongzaocu’ pear, which is a bud mutation of ‘Zaocu’ pear (Yang et al., 2023). In addition to transcription factors affecting anthocyanin synthesis, protein modification also plays an important role. MYB10 protein undergoes sulfhydrylation at Cys218 to respond to H2S signals, which will disrupt the formation of the MYB10-MYB73-MYB6 complex, thereby inhibiting the accumulation of anthocyanins in pear skin (Yao et al., 2023). ERF9 interacts with TPL1, leading to histone deacetylation of the MYB114 and RAP2.4 promoters, thereby reducing anthocyanin synthesis in pear skin (Ni et al., 2023). In addition, COP1 ubiquitinates and degrades bHLH64 under dark conditions, inhibiting anthocyanin synthesis in pear (Tao et al., 2020).

[0004] Due to the scarcity of red-fleshed pear germplasm resources, only a few publications have declared the mechanisms regulating red flesh formation. Recently, Italian researchers used SSR marker technology to cluster 33 local red-fleshed pear resources in Emilia-Romagna to elucidate the genetic relationships (Bergonzoni et al., 2023). In addition, Chang et al. (2023) identified an ethylene response factor, PcERF5, through transcriptome analysis of red- and white-fleshed pear germplasm. PcERF5 was expressed at a higher level in fresh-eating pears and could regulate anthocyanin biosynthesis through multiple pathways. First, PcERF5 activated the expression of flavonoid biosynthesis genes (PcDFR, PcANS, and PcUFGT) and key transcription factors PcMYB10 and PcMYB114. Second, PcERF5 interacted with PcMYB10 to form a complex, thereby enhancing its transcriptional activation of target genes (Chang et al., 2023). These results differ from the regulatory relationships in red-skinned pears, indicating that the regulatory mechanisms differ between red-fleshed pears. In contrast, more progress has been made in the study of other red-fleshed fruits. In red-fleshed apples, MdMYB10 and MdMYB110a control two different types of fruit coloring (type I has a red skin and flesh, and type II has a red flesh) (Chagné et al., 2007 2013; Espley et al., 2007). MdNAC interacts with MdbZIP23 to activate the transcription of MdMYB10 and MdUFGT, thereby promoting anthocyanin synthesis in red-fleshed apples (Liu et al., 2023). In strawberries, insertion of a CACTA-like transposon (FaEnSpm-2) into the FaMYB10-2 promoter affects the expression of FaMYB10.2 and anthocyanin accumulation in strawberry fruits (Castilejo et al., 2020). In red-fleshed peaches, BLOOD forms a heterodimer with PpNAC1 and PpbHLH3, activates the promoter and gene expression of PpMYB10.1, and thereby promotes anthocyanin synthesis in peach flesh (Zhou et al., 2015). PpMYB75 binds to the CCGTTG cis-regulatory element in the PpDFR promoter, enhancing anthocyanin accumulation in the red-fleshed peach variety “RMHC” (Xu et al., 2024).

[0005] In addition, with the development of gene editing, RNAi and other technologies, the accumulation of flesh anthocyanins can also be promoted by relieving the inhibition of anthocyanin synthesis by inhibitory genes. In apples, RNAi-mediated MdHB1 silencing can promote the accumulation of anthocyanins in the flesh of white-fleshed apples (Jiang et al., 2017); compared with white-fleshed apples, the expression level of mdm-miR858 in red-fleshed apples is significantly reduced, and transient silencing of mdm-miR858 significantly promotes the accumulation of anthocyanins in white-fleshed callus (Li et al., 2023). In strawberries, RNAi-mediated FcMYB1 silencing significantly promotes the accumulation of anthocyanins in red-fleshed strawberries (Salvatierra et al., 2013). In grapes, the accumulation of anthocyanins is significantly increased after knocking out one allele of VvbZIP36 using CRISPR / Cas9 technology (Tu et al., 2022). In addition to this, research on the molecular mechanisms behind red-fleshed color has also made progress in other fruit species, such as blood oranges (Huang et al., 2019; Wang et al., 2023), waxberries (Liu et al., 2013; Xue et al., 2024), and kiwifruits (Liu et al., 2017; Liu et al., 2022). In summary, the mechanism of anthocyanin formation in red-fleshed pears is not yet clear and still needs to be further understood in order to find key genes for future genetic improvement of pear flesh color.

[0006] Using dynamic development transcriptome data of red-fleshed pears and white-fleshed pears, among the differential transcription factors, combined with the correlation analysis between their expression levels and anthocyanin contents, the applicant identified an HD-ZIP transcription factor PyHAT5, the expression level of which in red-fleshed pears and white-fleshed pears was significantly negatively correlated with the expression levels of anthocyanin synthesis-related genes. Through transient overexpression in pear fruits, it was confirmed that PyHAT5 can inhibit the accumulation of anthocyanins in pear fruits, while transient silencing of PyHAT5 can promote the accumulation of anthocyanins in pear fruits; in pear flesh callus overexpressing PyHAT5, the anthocyanin content after light treatment was significantly lower than that of wild type, and the expression level of anthocyanin synthesis-related genes was significantly reduced; overexpression of PyHAT5 significantly inhibited the accumulation of anthocyanins in red-fleshed peach flesh, and the expression level of anthocyanin synthesis-related genes was significantly reduced. It is proved that the PyHAT5 gene negatively regulates the accumulation of anthocyanins in pear fruits, and transient silencing of PyHAT5 can promote the accumulation of anthocyanins in pear fruits. The discovery of this gene complements the anthocyanin synthesis regulation mechanism of HD-ZIP transcriptional regulation in pears, and in the future, PyHAT5 gene can be used to relieve the inhibition of anthocyanin accumulation in pear flesh through gene editing, RNAi and other technologies, thereby promoting the synthesis of pear flesh anthocyanins and providing gene resources and theoretical basis for red-fleshed pear breeding. SUMMARY

[0007] The purpose of the present application is to provide a PyHAT5 gene for regulating the accumulation of anthocyanins in pear fleshPyHAT5 gene.

[0008] Another object of the present application is to provide the use of the gene.

[0009] The object of the present application can be achieved by the following technical solutions. A transcription factor PyHAT5 inhibiting anthocyanin biosynthesis in pear fruit is separated from red-fleshed pear, belonging to the HD-ZIP family member, the nucleotide sequence of which is shown in SEQ ID No. 1, containing an open reading frame of 996 bp; encoding 332 amino acids, and the amino acid sequence encoded by the gene is shown in SEQ ID No. 2.

[0010] The recombinant transient silencing vector containing the gene of the present application. PyHAT5 The recombinant transient silencing vector containing the gene of the present application.

[0011] The recombinant transient silencing vector, taking pTRV2 as the starting vector, and the 363 bp specific coding sequence (SEQ ID No. 3) of the PyHAT5 gene is inserted between BamH I and Kpn I.

[0012] The host bacteria of the recombinant transient silencing vector containing the gene of the present application. PyHAT5 The host bacteria of the recombinant transient silencing vector containing the gene of the present application.

[0013] The primer pair for cloning the 363 bp coding sequence of the gene of the present application, the sequence of the upstream primer PyHAT5-VIGS-F1 is shown in SEQ ID No. 4, and the sequence of the downstream primer PyHAT5-VIGS-R1 is shown in SEQ ID No. 5. PyHAT5 The use of the expression of the gene in inhibiting or silencing anthocyanin synthesis.

[0014] The use of the recombinant transient silencing vector in promoting anthocyanin accumulation in pear fruit.

[0015] The use of the host bacteria in promoting anthocyanin accumulation in pear fruit.

[0016] The use of the host bacteria in promoting anthocyanin accumulation in pear fruit.

[0017] Beneficial effects

[0018] The present application utilizes the dynamic development transcriptome data of red-fleshed pear and white-fleshed pear, in the differential transcription factors, combined with the correlation analysis between the expression amount and the anthocyanin content, the applicant identifies an HD-ZIP transcription factor PyHAT5, the expression amount change of the gene in red-fleshed pear and white-fleshed pear is significantly negatively correlated with the expression amount of the anthocyanin synthesis-related gene. Through the transient overexpression in pear fruit, it is confirmed that PyHAT5 the anthocyanin accumulation in pear fruit can be inhibited, while the transient silencing PyHAT5It can promote the accumulation of anthocyanins in pear fruit; overexpression PyHAT5 In transgenic pear flesh callus, light treatment significantly reduced anthocyanin content compared to the wild type, and significantly decreased expression levels of anthocyanin synthesis-related genes; overexpression PyHAT5 It significantly inhibited the accumulation of anthocyanins in the pulp of red-fleshed peaches, and significantly reduced the expression levels of genes related to anthocyanin synthesis. This proves... PyHAT5 Genes negatively regulate the accumulation of anthocyanins in pear fruit, transiently silencing them. PyHAT5 This gene can promote the accumulation of anthocyanins in pear fruit. The discovery of this gene supplements and improves the regulatory mechanism of anthocyanin synthesis under HD-ZIP transcriptional regulation in pear, and it may be possible to unlock its potential through gene editing, RNAi, and other technologies in the future. PyHAT5 The gene inhibits the accumulation of anthocyanins in the pulp, thereby promoting the synthesis of anthocyanins in pear pulp, providing genetic resources and a theoretical basis for the breeding of red-fleshed pears.

[0019] Compared with the prior art, the present invention has the following advantages and effects: 1. PyHAT5 The discovery of the gene provides new genetic resources for molecular breeding of anthocyanin accumulation in pear pulp and new genetic resources for implementing green agriculture. The development and utilization of this genetic resource is conducive to reducing agricultural costs and achieving environmental friendliness.

[0020] 2. Using Agrobacterium-mediated genetic transformation, [the following method was used] to transform [the organism]... PyHAT5 Functional verification of the gene in pear fruit, pear flesh callus, and red-fleshed peach showed that the gene cloned in this invention... PyHAT5 The gene has the function of inhibiting the accumulation of anthocyanins in the pulp, and is transiently silenced. PyHAT5 It can promote the accumulation of anthocyanins in pear fruit. In the future, this can be addressed through gene editing, RNAi, and other technologies. PyHAT5 The gene inhibits the accumulation of anthocyanins in the pulp, thereby promoting the synthesis of anthocyanins in pear pulp, providing a more efficient approach for molecular breeding of red-fleshed pears. Attached Figure Description

[0021] Figure 1 These are fruit samples of red-fleshed pears and white-fleshed pears, as presented in this invention.

[0022] Wherein: A, Images of red and white fleshed pear fruit samples, WF indicates white fleshed pear, RF indicates red fleshed pear; B, Anthocyanin content of red and white fleshed pears.

[0023] Figure 2 For the present invention PyHAT5 Analysis of the expression levels of genes and anthocyanin synthesis-related genes in red and white-fleshed pears.

[0024] Figure 3 This is a schematic diagram of the transient overexpression vector in Embodiment 2 of the present invention.

[0025] Figure 4 Schematic diagram of transient silencing vector for Example 2 of the present application.

[0026] Figure 5 Schematic diagram of transient silencing vector for Example 2 of the present application. PyHAT5 Functional analysis of PyHAT5 gene in transient injection of‘Hongzaoqu’ and‘Zaoqu’ pear fruits.

[0027] Wherein: A, transient transformation of PyHAT5 overexpression vector; B, determination of anthocyanin content in the injected part of the pear fruit; C, determination of the expression level of PyHAT5 in the injected part of the pear fruit; D, determination of the expression level of anthocyanin synthesis-related genes in the injected part of the pear fruit; and E, determination of the expression level of anthocyanin synthesis-related genes in the injected part of the pear fruit. PyHAT5 Fruit phenotype of overexpression vector and transient silencing vector, EV is the part transformed with empty pCAMBIA1301 vector; PyHAT5-OE is the part transformed with 35S-PyHAT5-GFP recombinant vector, pTRV2 represents empty vector, and pTRV2-PyHAT5 represents the part transformed with transient silencing vector. B, anthocyanin content in the fruit peel of the injected part; C, expression level of anthocyanin synthesis-related genes in the fruit peel of the injected part; D, expression level of anthocyanin synthesis-related genes in the fruit peel of the part injected with 35S-PyHAT5-GFP recombinant vector; and E, expression level of anthocyanin synthesis-related genes in the fruit peel of the part injected with pTRV2-PyHAT5 recombinant vector. **Very significant. PyHAT5

[0028] Figure 6 Schematic diagram of transient silencing vector for Example 2 of the present application. PyHAT5 Functional analysis of PyHAT5 gene in transgenic pear flesh callus.

[0029] Wherein: A, phenotype of transgenic pear callus under light treatment, WT is wild-type pear callus, and PyHAT5-OE is transgenic callus transformed with 35S-PyHAT5-GFP recombinant vector. B, determination of anthocyanin content in the transgenic pear callus; C, determination of the expression level of anthocyanin synthesis-related genes in the pear callus after light treatment. NS, not significant; and ** very significant.

[0030] Figure 7 Functional analysis of PyHAT5 gene in transient injection of red-fleshed peach.

[0031] Wherein: A, phenotype of red-fleshed peach transiently transformed with PyHAT5 overexpression vector, EV is the part transformed with empty pCAMBIA1301 vector; and PyHAT5-OE is the part transformed with 35S-PyHAT5-GFP recombinant vector. B, determination of anthocyanin content in the peach fruit flesh of the injected part; C, determination of the expression level of PyHAT5 in the fruit peel of the injected part; and D, determination of the expression level of anthocyanin synthesis-related genes in the peach fruit flesh of the part injected with 35S-PyHAT5-GFP recombinant vector. **Very significant. DETAILED DESCRIPTION

[0032] ​The application will be described in detail below with specific embodiments. Based on the following description and these embodiments, those skilled in the art can determine the essential features of the application, and make various changes and modifications to the application without departing from the spirit and scope of the application, so that the application can be applied to various uses and conditions.

[0033] Example 1 PyHAT5 Gene expression pattern analysis Red and white flesh pear samples were collected from elite plants in P265R237T006 (white flesh pear) and P427R349T078 (red flesh pear) hybrid populations Figure 1 ). After fruit collection, freeze-drying was performed, and the samples were stored at -80 ℃ for subsequent experiments. Total RNA was extracted using the CTAB method (Porebski et al., 1997), and the quality of the extracted samples was detected by spectrophotometer and agarose gel. 3 μg of extracted total RNA was reverse transcribed using one-step gDNA removal and cDNA synthesis kit (Transgen, China), and the method was referred to the instruction manual. The primers used for fluorescence quantitative PCR were gene-specific primer pairs (Table 1), and GAPDH was used as the internal reference gene. The fluorescence quantitative reagent kit was purchased from Roche Company. The instrument used for real-time PCR was Roche 480 quantitative PCR instrument, and the reaction system was as follows: 2×SYBR Green I Master Mix 10 μL, upstream and downstream primers (10 μM) 0.4 μL, 2 μL cDNA, 7.2 μL PCR-grade water. The reaction conditions were as follows: 95 ℃ denaturation for 5 min; 95 ℃ pre-denaturation for 5 s, 60 ℃ annealing for 5 s, 72 ℃ extension for 10 s, repeated for 45 cycles; melting curve analysis from 65 ℃ to 95 ℃, every 5 s increase by 1 ℃.

[0034] The qRT-PCR results of red and white flesh pear samples showed that PyHAT5 was highly expressed in white flesh pear samples, which was significantly higher than that in red flesh pear samples, and was negatively correlated with the expression levels of anthocyanin content and anthocyanin biosynthesis-related genes PyMYB10, PyMYB114, PyDFR, PyANS and PyUFGT , indicating that Figure 2 may be involved in the biosynthesis of anthocyanin in pear fruit. PyHAT5

[0035] Table 1 Real-time fluorescence quantitative primers of pear

[0036] Example 2 PyHAT5 Gene isolation and cloning, construction of overexpression vector and transient silencing vector ​The 3 μg of'red flesh pear' pulp RNA was used for reverse transcription with one-step gDNA removal and cDNA synthesis kit (Transgen, China) according to the instructions. According to the analysis of the multiple cloning site of pCAMBIA-1301 vector and the restriction enzyme cutting site on the coding region sequence of PyHAT5 gene, Xba I and BamH I were selected as endonucleases. The primers with enzyme cutting sites were designed with Snapgene software according to the general design principle of primers. The 50 μL reaction system included 200 ng of cDNA, 1×buffer (TransStart FastPfu Buffer), 10 mM dNTP, 1 U of Taq polymerase (TransStart FastPfu DNA Polymerase) (the aforementioned buffer and Taq polymerase were purchased from TRANS company), and 500 nM of the above-mentioned primers. The PCR reaction was completed on an eppendorf amplifier according to the following program: 95℃, pre-denaturation for 2 minutes, 95℃ denaturation for 20 seconds, 60℃ annealing for 20 seconds, 72℃ extension for 1 minute, 35 thermal cycles, 72℃ extension for 10 minutes, and 4℃ preservation. A single PCR band product was produced.

[0037] The PCR product was detected by 1% agarose gel electrophoresis and recovered by a small amount of gel recovery kit (purchased from Kangwei Century, according to the operation instruction provided by the kit). The total volume of the double enzyme digestion system of pCAMBIA1301 vector was 50 μL, which contained 10 μL of pCAMBIA1301 vector plasmid obtained by plasmid extraction, 5 μL of 10× Buffer (purchased from NEB company), 1 μL of Xba I, 1 μL of BamH I and 33 μL of water. After enzyme digestion at 37°C for 3 hours, the product was recovered. The expression vector pCAMBIA1301 and PyHAT5 gene digested by restriction enzymes were connected by recombinant enzyme Exnase II (purchased from Vazyme company) at 37°C for 30 minutes. The total volume of the reaction was 20 μL, which contained 4 μL of 5 × CE II Buffer, 2 μL of Exnase II, 2 μL of PCR recovery product of PyHAT5 gene, 6 μL of double enzyme digestion recovery product of pCAMBIA1301 vector and 6 μL of water. 10 μL of the connection product was transformed into E. coli competent DH5α, and positive clones were screened on LB solid plates containing 50 mg / L kanamycin. The plasmid was extracted, enzyme-digested and PCR-identified, and the recombinant plasmid sample was sent to a biological company for sequencing. The sequencing results showed that the full length of PyHAT5 gene was 996 bp, the nucleotide sequence was shown as SEQ ID NO. 1, and it could encode a protein of 332 amino acid residues, the sequence of which was shown as SEQ ID NO. 2. The recombinant vector was named 35S-PyHAT5-GFP, and the vector map of the constructed vector was shown in FIG. 1. Figure 3 The recombinant vector was introduced into Agrobacterium GV3101 by freeze-thaw method.

[0038] A specific coding sequence (300-400 bp) of PyHAT5 gene was selected, and primers with enzyme cutting sites were designed by Snapgene software: agaaggcctccatggggatccGCTTTCGAGAGAGAAAGGAAACC (SEQ ID NO. 4) and gagacgcgtgagctcggtaccTAATCATAGCAGCAATCTTCAAGCT (SEQ ID NO. 5). The sequence was amplified using the PCR program. After the PCR product was detected by 1% agarose gel electrophoresis, the DNA fragment was recovered by a small amount of gel recovery kit (purchased from Kangwei Century and operated according to the operating instructions provided by the kit). The total volume of the double enzyme cutting system of the pTRV2 vector was 50 μL, which contained 10 μL of pTRV2 vector plasmid obtained by plasmid extraction, 5 μL of 10× Buffer (purchased from NEB company), 1 μL of KPnI, 1 μL of BamH I and 33 μL of water. After enzyme cutting at 37°C for 3 hours, it was recovered. The transient silencing vector pTRV2 and PyHAT5 gene specific sequence digested by restriction enzymes were connected using recombinant enzyme Exnase II (purchased from Vazyme company) at 37°C for 30 minutes. The total volume of the reaction was 20 μL, which contained 4 μL of 5 × CE II Buffer, 2 μL of Exnase II, 2 μL of PyHAT5 gene specific sequence PCR recovery product, 6 μL of double enzyme cutting recovery product of pTRV2 vector and 6 μL of water. 10 μL of the ligation product was transformed into E. coli competent DH5α, and positive clones were selected on LB solid plates containing 50 mg / L kanamycin. The plasmid was extracted for enzyme cutting and PCR identification, and the recombinant plasmid sample was sent to a biological company for sequencing. The sequencing results showed that the PyHAT5 gene specific sequence was 363 bp, and its nucleotide sequence was shown in SEQ ID NO. 3. The recombinant vector was named pTRV2-PyHAT5, and the vector map of the constructed vector is shown in Figure 4 The recombinant vector was introduced into Agrobacterium GV3101 by freeze-thaw method.

[0039] Example 3: Pear fruit transient transformation and functional analysis (1) Pear fruit transient transformation The bacteria liquid containing pTRV2-PyHAT5 or 35S-PyHAT5-GFP plasmid was injected into the near-mature bagged ‘Hongzaocu’ pear by Agrobacterium-mediated method, and the specific method was as follows: 1. The Agrobacterium containing pTRV2-PyHAT5 or 35S-PyHAT5-GFP plasmid was activated on solid medium (containing 50 μM K+, 50 μM R+) and cultured in a bacterial incubator at 28°C for 36 hours; 2. Add 15 mL LB liquid medium (containing 50 μM K+, 50 μM R+) into a 50 mL sterile centrifuge tube, pick the activated Agrobacterium colonies with a gun head, and cultivate in a shaker at a temperature of 28°C and a rotation speed of 180 rpm for 10 hours; 3. Centrifuge the cultivated bacterial liquid at 5000 r·min-1 for 10 minutes to collect all the bacterial bodies; 4. Resuspend the precipitate with an appropriate amount of pH = 5.8 induction liquid [containing 10 mM MES, 10 mM MgCl2, 200 mM acetosyringone (AS)], adjust the OD600 of the bacterial liquid to 0.8, and induce in a shaker at a rotation speed of 180 rpm at room temperature for 3 hours in the dark; 5. Inject the induced infection liquid into the nearly mature ‘Zaocui’ pears with a sterile syringe, 15 fruits per test, and three biological repeats; 6. Culture the injected pear fruits in a culture box at 20°C in the dark for 24 hours, and then culture in a light culture box at a temperature of 20°C and 24 hours of light for 10 days; (2) Total anthocyanin content determination Grind 0.1 g of pear peel material in liquid nitrogen, and add a 0.1% hydrochloric acid methanol solution, and place at 4°C in the dark for 24 hours. Then centrifuge at 12000 rpm for 10 minutes, take the supernatant, and dilute to a volume of 4 mL. Use a spectrophotometer to read the OD values at 520, 630, and 650 nm. Standardize the OD values according to the formula A = (A530-A650)-0.1*(A650-A620), and perform three biological repeats for each sample.

[0040] Anthocyanin content determination found that the anthocyanin content of the overexpression PyHAT5 site was significantly reduced, the anthocyanin content of the transiently silenced PyHAT5 site was significantly increased, and the site injected with the empty vector had no significant difference from the non-injected part ( Figure 5 ABC).

[0041] (3) Relative expression amount detection of anthocyanin synthesis related genes in the injection site The extraction of RNA, synthesis of cDNA, and system and steps of fluorescence quantitative PCR refer to Example 1. The anthocyanin biosynthesis related gene PyHAT5 expression amount in the overexpression PyDFR, PyANS, PyUFGT, PyMYB10 peel site was significantly reduced, while the anthocyanin biosynthesis related gene PyMYB114 expression amount in the transiently silenced PyHAT5 peel site was significantly increased ( PyDFR, PyANS, PyUFGT, PyMYB10 PyMYB114 Figure 5 ​​DE). In summary, the above results indicated that overexpression of PyHAT5 PyHAT5 would inhibit the synthesis of anthocyanins in pear fruit, while transient silencing of PyHAT5 PyHAT5 would promote the synthesis of anthocyanins in pear fruit. Example 4 Stable transformation of pear flesh callus and functional analysis

[0042] (1) Stable transformation of pear flesh callus The Agrobacterium containing the overexpression vector was used to infect the pear flesh callus by Agrobacterium-mediated method, as follows: PyHAT5 1. The Agrobacterium containing 35S-PyHAT5-GFP and pCAMBIA1301 plasmid was activated on solid medium (containing 50 μM kanamycin, 50 μM rifampicin) and cultured in a bacterial incubator at 28°C for 36 hours; 2. 30 mL of LB liquid medium (containing 50 μM kanamycin, 50 μM rifampicin) was added to a 100 mL conical flask, and the activated Agrobacterium colonies were picked up with a gun head and cultured in a shaker at 28°C and 180 rpm for 12 hours; 3. The bacterial solution was transferred to a 50 mL sterile centrifuge tube and centrifuged at 5000 r·min -1 for 10 minutes to collect all the bacteria; 4. The precipitate was resuspended with an appropriate amount of induction solution [10 mM MES, 10 mM MgCl2, 200 mM acetosyringone (AS), pH = 5.8] to adjust the bacterial solution concentration OD 600 = 0.6, and the solution was induced in the dark at 28°C and 180 rpm for 4 hours; 5. The solution was induced in the dark at room temperature and 200 rpm for 20 minutes in MS liquid medium; 6. The pear callus infected by Agrobacterium was evenly spread on symbiotic MS solid medium and co-cultured in the dark at 25°C for 2 days. 7. The callus after co-culture was evenly spread on selection MS solid medium and cultured in the dark at 25°C for 2 months. The callus that continued to grow on the selection MS solid medium was the transgenic callus.

[0043] (2) Identification and light treatment of transgenic pear flesh callus

[0044] The extraction of RNA, synthesis of cDNA, system and steps of fluorescent quantitative PCR refer to Example 1. We performed fluorescent quantitative PCR analysis on the callus that normally grew on the medium containing hygromycin, and found that the callus had a higher expression level of PyHAT5 PyHAT5 ​The relative expression of genes in the calli was higher than that in the wild type (WT) calli, and thus the calli were selected for subsequent studies.

[0045] The transgenic calli and WT were cultured in MS medium containing 50 mM methyl jasmonate (MJ) at 25 °C in the dark for 3 days, and then the calli were divided into two parts, one of which was continuously cultured in the dark, and the other was placed in a light incubator for 24 hours of light to observe the phenotype.

[0046] (3) Analysis of anthocyanin content in transgenic calli It was found by observing the phenotype that under light conditions, the wild type calli accumulated a large amount of anthocyanin, and the surface was red, while the PyHAT5-OE calli did not accumulate anthocyanin, and the surface was milky yellow. Figure 6 A). It was found by anthocyanin content determination that the anthocyanin content of the transgenic PyHAT5-OE calli was significantly lower than that of the WT. Figure 6 B). Therefore, it was shown that PyHAT5 anthocyanin synthesis in pear calli was inhibited.

[0047] (4) Detection of relative expression of anthocyanin synthesis related genes in transgenic calli The extraction of RNA, the synthesis of cDNA, the system and steps of fluorescence quantitative PCR refer to Example 1. The expression of anthocyanin synthesis related genes in the transgenic calli was analyzed by fluorescence quantitative PCR. The results showed that compared with the WT pear flesh calli, the relative expression of anthocyanin synthesis related genes PyHAT5 , PpcMYB10 , PpcDFR , PpcANS , and PpcUFGT in the pear flesh calli overexpressing Figure 6 C). In summary, the above results showed that overexpression of PyHAT5 inhibited anthocyanin synthesis in pear flesh calli.

[0048] Example 5 Transient transformation and functional analysis of red-fleshed peach (1) Transient transformation of red-fleshed peach fruit The bacterial liquid containing 35S-PyHAT5-GFP, pCAMBIA1301 plasmid was injected into the nearly mature red-fleshed peach flesh by Agrobacterium-mediated method, and the specific method was as follows, which referred to Example 3.

[0049] (2) Determination of total anthocyanin content The specific method was as follows, which referred to Example 3. It was found by anthocyanin content determination that the anthocyanin content of the part overexpressing PyHAT5 was significantly reduced, the anthocyanin content of the part transiently silenced PyHAT5 was significantly increased, and there was no significant difference between the part injected with empty vector and the part not injected. Figure 7AB).

[0050] (3) Relative expression level detection of anthocyanin synthesis related genes in injection site RNA extraction, cDNA synthesis, fluorescence quantitative PCR system and steps refer to Example 1. Overexpression PyHAT5 of anthocyanin biosynthesis related genes in pericarp PpDFR, PpLPOX, PpUFGT, PpMYB10.1 , PpMYB10.2 and PpMYB10.3 significantly reduced expression level Figure 7 CD). In summary, the above results show that overexpression PyHAT5 will inhibit the synthesis of anthocyanin in red flesh peach fruit. References

[0051] Feng Shuqian, Chen Xuesen, Zhang Chunyu, Liu Xiaojing, Liu Zhunchun, Wang Haibo, Wang Yanling, Zhou Zhaohua (2008). Research on anthocyanin synthesis and related enzyme activity of sand pear variety ‘Mantianhong’ and bud mutation line ‘Ouguan’. Chinese Journal of Agricultural Science 41(10): 3184-3190 Feng Wenting, Zhai Rui, Wang Zhigang (2015). Cloning and expression analysis of anthocyanin synthesis related genes in ‘Hongzaosu’ pear. Acta Agronomica Sinica 24(06):75-83 Ahmed Alabd, Mudassar Ahmad, Xiao Zhang, Yuhao Gao, Lin Peng, Lu Zhang, Junbei Ni, Songling Bai, Yuanwen Teng (2022). Light-responsive transcription factor PpWRKY44 induces anthocyanin accumulation by regulating PpMYB10 expression in pear. Horticulture Research 9, uhac199 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-induced anthocyanin accumulation by activating MYB10 in red pear. Plant Biotechnol J 17(10):1985-1997 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 regulate anthocyanin biosynthesis via competitive association with Pyrus pyrifolia ELONGATED HYPOCOTYL 5 in the peel of pear fruit. Plant J 100(6): 1208-1223 Bao L, Chen K, Zhang D, Cao Y, Yamamoto T, Teng Y. 2007. Genetic diversity and similarity of pear (Pyrus L.) cultivars native to East Asia revealed by SSR (simple sequence repeat) markers. Genetic Resources and Crop Evolution 54(5): 959. Gaifang Yao, Meiling Ming, Andrew C. Allan, Chao Gu, Leiting Li, Xiao Wu, Runze Wang, Yaojun Chang, Kaijie Qi, Shaoling Zhang, Jun Wu (2017). Map-based cloning of the pear gene MYB114 identifies an interaction with other transcription factors to coordinately regulate fruit anthocyanin biosynthesis. The Plant Journal 92, 437–451 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 accumulation via the activation of PyUFGT through binding to tandem G-boxes in its promoter. Horticulture research 6(1), 1-13 Hellens RP, Allan AC, Friel EN, Bolitho K, Grafton K, Templeton MD, Karunairetnam S, Gleave AP, Laing WA (2005). Transient expression vectors for functional genomics, quantification of promoter activity and RNA silencing in plants. Plant Methods 1:13 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 regulate anthocyanin biosynthesis and transport in red-skinned pears. Horticulture Research 15;7:37 Ni J, Premathilake AT, Gao Y, Yu W, Tao R, Teng Y, Bai S (2021). Ethylene-activated PpERF105 induces the expression of the repressor-type R2R3-MYB gene PpMYB140 to inhibit anthocyanin biosynthesis in red pear fruit. Plant J 105(1):167-181 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 Anthocyanin Biosynthesis and Undergoes CONSTITUTIVELY PHOTOMORPHOGENIC1-Mediated Degradation in Pear. Plant Physiol 184(4):1684-1701 Tu M, Fang J, Zhao R, Liu X, Yin W, Wang Y, Wang X, Wang X, Fang Y (2022). CRISPR / Cas9-mediated mutagenesis of VvbZIP36 promotes anthocyanin accumulation in grapevine (Vitis vinifera). Hortic Res 19;9:uhac022 Wu J, Wang Z, Shi Z, Zhang S, Ming R, Zhu S, Khan MA, Tao S, Korban SS, Wang H. 2013. The genome of the pear (Pyrus bretschneideri Rehd.). Genome research 23(2): 396-408. YANG Yanan, YAO Gaifang, ZHENG Danman, ZHANG Shaoling, WANG Chao, ZHANG Mingyue, WU Jun (2015). Expression differences of anthocyanin biosynthesis genes reveal regulation patterns for red pear coloration. Plant Cell Reports 34 (2):189-198 Zhai R, Wang Z, Zhang S, Meng G, Song L, Wang Z, Li P, Ma F, Xu L (2015). Two MYB transcription factors regulate flavonoid biosynthesis in pear fruit (Pyrus bretschneideri Rehd.). J Exp Bot 67(5):1275-84

Claims

1. A PyHAT5 gene isolated from 'red-fleshed' pear that regulates the accumulation of anthocyanins in pear pulp, the CDS sequence of which is shown in SEQ ID No. 1, and the protein sequence encoded by which is shown in SEQ ID No.

2.

2. The recombinant transient silencing vector of the PyHAT5 gene as described in claim 1.

3. The recombinant transient silencing vector according to claim 3, characterized in that... Using pTRV2 as the starting vector, the 363 bp coding sequence of the PyHAT5 gene shown in SEQ ID No.3 was inserted between BamH I and Kpn I.

4. A host bacterium containing the gene recombination transient silencing vector of claim 1.

5. The use of suppressing or silencing the expression of the gene described in claim 1 in anthocyanin synthesis.

6. The application of the recombinant transient silencing vector according to claim 2 in promoting anthocyanin accumulation in pear fruit.

7. The use of the host bacteria as described in claim 4 in promoting anthocyanin accumulation in pear fruit.

Images