Rubus chingii uv response transcription factor rcwrky51 and application thereof

Abstract

The application discloses a Rubus chingii Hu UV response transcription factor RcWRKY51 and application. The application provides a transcription factor RcWRKY51 for positively regulating biosynthesis of kaempferol-3-O-rutinoside, and the nucleotide sequence is shown as SEQ ID NO. 1. In the application, the transcription factor RcWRKY51 of Rubus chingii Hu is up-regulated and expressed by UV induction, the expression of flavonoid glycoside metabolic pathway genes PAL, C4H, CHI, CHS and FLS is up-regulated, and then the biosynthesis of kaempferol-3-O-rutinoside of Rubus chingii Hu is improved. The content of kaempferol-3-O-rutinoside in flavonoid glycosides of Rubus chingii Hu can be obviously increased by using genetic transformation technology to overexpress the transcription factor RcWRKY51 gene of Rubus chingii Hu. Therefore, it is shown that the RcWRKY51 can obviously promote the biosynthesis of kaempferol-3-O-rutinoside in flavonoid glycosides, and the medicinal value of flavonoid glycosides of Rubus chingii Hu is improved.

Description

Technical fields:

[0001] This invention belongs to the field of medicinal plant genetic engineering technology, specifically relating to a UV-responsive WRKY transcription factor RcWRKY51 of Raspberry palmatum and its application. Background technology:

[0002] Rubus chingii Hu, the medicinal plant of the Chinese raspberry family, is traditionally produced in Chun'an County, Zhejiang Province. The unripe, dried fruit is used medicinally. Rubus chingii Hu fruit is rich in flavonoid glycosides, such as kaempferol-3-O-rutinoside, tiliroside, hypericin-3-O-galactoside, and quercitrin. These compounds are listed in various editions of the Pharmacopoeia of the People's Republic of China, and kaempferol-3-O-rutinoside is used as one of the standards for evaluating the quality of raspberries. Modern pharmacological studies have shown that kaempferol-3-O-rutinoside has lipid-lowering, anti-glycation, and neuroprotective effects (Sheng J, Wang S, Liu K, Zhu B, Zhang Q, Qin L, Wu J. Rubus chingii Hu: an overview of botany, traditional uses, phytochemistry, and pharmacology. Chin J Nat Med. 2020, 18(6): 401-416; Yu G, Luo Z, Wang W, Li Y, Zhou Y, Shi Y. Rubus chingii Hu: a review of the phytochemistry and pharmacology. Front Pharmacol. 2019, 10: 799). Therefore, discovering transcription factors that regulate the biosynthesis of kaempferol-3-O-rutinoside has important health-preserving and medicinal value.

[0003] Kaempferol-3-O-rutin is a flavonoid glycoside compound, and is a pale yellow crystal. The biosynthesis of flavonoid glycosides originates from the phenylpropane metabolic pathway. Phenylalanine is converted to 4-coumaryl-CoA by phenylalanine ammonialyase (PAL), cinnamate 4-hydroxylase (C4H), and 4-coumarate CoA ligase (4CL). Then, under the catalysis of chalcone synthase (CHS), it combines with CoA to synthesize chalcone. Finally, under the catalysis of chalcone isomerase (CHI), it synthesizes the precursor of flavanone glycosides—naringenin. This precursor can then enter the metabolic pathways of flavanones, flavones, flavonols, and anthocyanins under the action of enzymes such as flavanone-3-hydroxylase (F3H) and flavonol synthase (FLS). AE,MühlemannJK,Muday GK.Flavonols modulate plant development,signaling,and stressresponses.Curr Opin Plant Biol.2023,72:102350;Dong NQ,Lin HX.Contribution ofphenylpropanoid metabolism to plant development and plant-environmentinteractions.J Integr Plant Biol. 2021, 63 (1): 180-209; Shen N, Wang T, Gan Q, Liu S, Wang L, Jin B. Plant flavonoids: classification, distribution, biosynthesis, and antioxidant activity. Food Chem. 2022, 383: 132531).

[0004] The biosynthesis of plant flavonoid glycosides is accomplished collaboratively by a series of structural and regulatory genes. The expression levels of these genes are regulated by transcription factors from the MYB, bHLH, WD40, WRKY, AP2 / ERF, and NAC families (Shi Y, Li BJ, Su G, Zhang M, Grierson D, Chen KS. Transcriptional regulation of fruit texture. J Integr Plant Biol. 2022, 64:1649-1672; Wen W, Alseekh S, Fernie AR. Conservation and diversification of flavonoid metabolism in the plant kingdom. Curr Opin Plant Biol. 2020, 55:100-108; Xu W, Dubos C, Lepiniec L. Transcriptional control of flavonoid biosynthesis by MYB-bHLH-WDR complexes. Trends Plant). (Sci. 2015, 20(3): 176-85). Transcription factors regulate the differential accumulation of plant flavonoid glycosides by binding to cis-regulatory elements on the promoters of key structural enzymes in the flavonoid metabolic pathway, such as PAL, C4H, CHS, CHI, F3H, DFR, and FLS. Currently, research on the regulation of plant flavonoid glycoside biosynthesis by transcription factors is still limited. Gerbera hybrida GhMYB1a can regulate the accumulation of anthocyanins and flavonols by directly binding to the promoters of the upstream anthocyanin synthesis gene CHS and the flavonol synthesis gene FLS (Zhong C, Tang Y, Pang B, Li X, Yang Y, Deng J, Feng C, Li L, Ren G, Wang Y, Peng J, Sun S, Liang S, Wang X. The R2R3-MYB transcription factor GhMYB1a regulates flavonoland anthocyanin accumulation in Gerbera hybrida. Hortic Res. 2020, 7:78).The ERF-VII transcription factor SmERF73, induced by MeJA in Salvia miltiorrhiza, can specifically bind to GCC-box cis elements in vitro / in vivo, thereby activating the transcription of DXR1, CPS1, KSL and CYP76AH3 genes and promoting the accumulation of tanshinone (Zheng H, Jing L, Jiang X, Pu C, Zhao S, Yang J, Guo J, Cui G, Tang J, Ma Y, Yu M, Zhou X, Chen M, Lai C, Huang L, Shen Y. The ERF-VII transcription factor SmERF73 coordinately regulates tanshinone biosynthesis in response to stresselicitors in Salvia miltiorrhiza. New Phytol. 2021, 231(5):1940-1955). To date, the upstream transcription factors for the biosynthesis of kaempferol-3-O-rutinoside in Raspberry palmatum are unclear, and no transcription factors regulating the biosynthesis of kaempferol-3-O-rutinoside have been identified. Summary of the Invention:

[0005] The purpose of this invention is to provide a UV-responsive WRKY transcription factor RcWRKY51 for Raspberry palmatum, which can promote the biosynthesis of kaempferol-3-O-rutin.

[0006] To achieve the above-mentioned objective, the present invention provides a transcription factor RcWRKY51 that positively regulates the biosynthesis of kaempferol-3-O-rutin, wherein RcWRKY51 has the nucleotide sequence shown in SEQ ID NO.1.

[0007] The present invention provides a protein encoded by the above-mentioned palmate raspberry transcription factor RcWRKY51, the protein having an amino acid sequence as shown in SEQ ID NO.2.

[0008] The present invention also provides a recombinant vector containing the above-mentioned palmate raspberry transcription factor RcWRKY51, wherein the recombinant vector is pCAMBIA1301-RcWRKY51.

[0009] The present invention also provides a recombinant microorganism containing the recombinant vector, wherein the microorganism is Agrobacterium GV3101 (pSoup-p19) strain and the recombinant vector is pCAMBIA1301-RcWRKY51.

[0010] Another object of the present invention is to provide the application of the above-mentioned Raspberry transcription factor RcWRKY51 in promoting the biosynthesis of kaempferol-3-O-rutin in Raspberry.

[0011] Another objective of this invention is to provide the application of the above-mentioned Raspberry palmatum transcription factor RcWRKY51 in responding to UV-induced treatment to upregulate its expression and promote the expression of genes PAL, C4H, CHI, CHS, and FLS in the flavonoid metabolism pathway.

[0012] Another object of the present invention is to provide the application of the above-mentioned Raspberry transcription factor RcWRKY51 in the breeding improvement of Raspberry, wherein the application is preferably the application of Raspberry transcription factor RcWRKY51 in the genetic engineering breeding to increase the content of flavonoid glycosides in Raspberry.

[0013] This invention provides a UV-responsive WRKY transcription factor RcWRKY51 from *Raspberry palmatum*, belonging to the field of medicinal plant genetic engineering technology. The nucleotide sequence of the *Raspberry palmatum* transcription factor RcWRKY51 is shown in SEQ ID NO.1. In this invention, the *Raspberry palmatum* transcription factor RcWRKY51 can be upregulated in response to UV-induced treatment, activating the expression of flavonoid metabolism pathway genes PAL, C4H, CHI, CHS, and FLS, thereby promoting the biosynthesis of kaempferol-3-O-rutinoside in *Raspberry palmatum*. Verification analysis showed that the expression level of the RcWRKY51 gene was significantly positively correlated with the content of kaempferol-3-O-rutinoside at different developmental stages of *Raspberry palmatum* fruit. Overexpression of the *Raspberry palmatum* transcription factor RcWRKY51 gene using genetic transformation technology can significantly increase the content of kaempferol-3-O-rutinoside in flavonoid glycosides. This indicates that RcWRKY51 can significantly promote the biosynthesis of kaempferol-3-O-rutin in flavonoid glycosides, thereby enhancing the medicinal value of flavonoid glycosides in Raspberry palmatum. Attached image description:

[0014] Figure 1 This is an electrophoresis image of the RcWRKY51 gene amplification in *Rubus palmatus*. The left side shows the molecular weight standards for nucleic acids, with the bands from top to bottom being 2000, 1500, 1000, 750, 500, 250, and 100 bp; the right side represents the RcWRKY51 gene.

[0015] Figure 2 This is the secondary structure of the RcWRKY51 protein, a transcription factor from Raspberry palmatum. Blue indicates α-helices, red indicates extended backbones, green indicates β-turns, and purple indicates random coils.

[0016] Figure 3The image shows the tertiary structure of the palmate raspberry transcription factor RcWRKY51 protein, with red circles indicating WRKY binding sites.

[0017] Figure 4 Phylogenetic analysis of RcWRKY51 from Raspberry palmatum and reported Arabidopsis WRKY family members. RcWRKY51 clusters in subfamily IIIa and is most closely related to the Arabidopsis AtWRKY70 protein.

[0018] Figure 5 The expression levels of the RcWRKY51 gene in different parts and different developmental stages of the fruit of *Raspberry palmatum* are shown. Different parts include root, stem, leaf, flower, and fruit; different developmental stages of the fruit include small green fruit (LG), large green fruit (BG), yellow fruit (YG), and red fruit (RG). The bar for each data set represents ± standard error (n≥10).

[0019] Figure 6 The expression level of the RcWRKY51 gene in Rubus palmatus under UV irradiation treatment is given. The bar for each data set represents ± standard error (n≥10). ** indicates that the Student's t-test showed a significant difference between the control group and the UV-treated group at p<0.01.

[0020] Figure 7 The content of kaempferol-3-O-rutin in Raspberry palmatum at different fruit development stages is given. The different fruit development stages include small green fruit (LG), large green fruit (BG), yellow fruit (YG), and red fruit (RG). The bar for each data set represents ± standard error (n≥10).

[0021] Figure 8 The chromatogram of kaempferol-3-O-rutin standard was obtained for HPLC-MS analysis.

[0022] Figure 9 This is a schematic diagram of the pCABIA1301-RcWRKY51 overexpression vector. The accession number for pCABIA1301 is AF234297.

[0023] Figure 10 To promote the increase of kaempferol-3-O-rutin content among flavonoid glycosides by overexpressing the RcWRKY51 gene, the expression of genes involved in the flavonoid glycoside metabolism pathway in *Raspberry palmatum* was upregulated by overexpressing the RcWRKY51 gene. The bar represents ± standard error (n≥10) for each data set. ** indicates a significant difference between the control group and the UV treatment group as found by Student's t-test (p<0.01). Detailed Implementation

[0024] The following embodiments are further illustrations of the present invention, but not limitations thereof.

[0025] Unless otherwise specified in the following examples, all experimental methods can be performed using conventional methods, such as those described in J. Sambrook et al.'s *Molecular Cloning: A Laboratory Manual*, F. Osborne et al.'s *A Concise Laboratory Manual of Molecular Biology*, or the manufacturer's instructions for use of the products employed.

[0026] The *Rubus chingii* Hu used in this example was cultivated at the Medicinal Botanical Garden of Zhejiang University of Traditional Chinese Medicine (N30°5′, E119°53′; Hangzhou, China); the polysaccharide and polyphenol plant RNA extraction kit was purchased from Huayueyang Biotechnology Co., Ltd. (catalog number: 0416-50); the reverse transcriptase M-MLV kit was purchased from Promega (catalog number: M1701); *Escherichia coli* DH5α was purchased from Shanghai Weidi Biotechnology Co., Ltd. (catalog number: DL1001); the HiPure Gel Pure Micro Kit was purchased from Guangzhou Meiji Technology Co., Ltd. (catalog number: D2110-02); 2× Hieff The PCR Master Mix was purchased from YEASEN (product number: 10136ES03); The HD Cloning Kit was purchased from Takara (item number: 639648); the pMD18-TVector was purchased from Takara (item number: D101A); and the SYBR Premix Ex Taq... TM Kit purchased from Takara (item number: DRR420A); TaKaRa LA Purchased from Takara (item number: RR52A); The ReporterAssaySystem kit (Promega, catalog number: E1910); LB, MS, and SD media are commonly used media in this field, and their formulations are based on J. Sambrook et al., *Molecular Cloning: A Laboratory Manual*. Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0027] Example 1: Cloning and Bioinformatics Analysis of the RcWRKY51 Gene Transcription Factor from Raspberry Palmarosa

[0028] (1) Extraction of total RNA and synthesis of the first strand of cDNA from Raspberry fruit

[0029] 100 mg of freshly harvested *Raspberry palmatum* fruit was ground into powder under liquid nitrogen. Total RNA was extracted from the fruit using a polysaccharide and polyphenol plant RNA extraction kit (Huayueyang Biotechnology Co., Ltd., catalog number: 0416-50). The RNA was analyzed using a 1.0% agarose gel electrophoresis system (Biorad, California, USA) and NanoDrop gel electrophoresis. TM The content and purity of total RNA were determined using a 2000°C ultra-micro spectrophotometer (Thermo Scientific, Wisconsin, USA). Two μg of purified total RNA from *Raspberry palmatum* fruit was used to synthesize the first strand of cDNA according to the instructions of the reverse transcriptase M-MLV kit (Promega, catalog number: M1701). The reaction product was diluted to the required concentration and stored at -80°C.

[0030] (2) Amplification and sequence analysis of the RcWRKY51 gene transcription factor from Rubus palmatus.

[0031] Using the obtained *Rubus palmatus* fruit cDNA as a template, the upstream primer ATGGGTACGACTTGGCTCGA and the downstream primer TTACAAGGGGCCTAAAGGAA for the *Rubus palmatus* transcription factor RcWRKY51 gene were designed using Primer Premier 5.0 (Premier Biosoft, California, USA). The 2×Hieff The RcWRKY51 gene sequence of *Rubus palmatus* was obtained by PCR amplification using PCR Master Mix (YEASEN, catalog number: 10136ES03), as shown in SEQ ID NO.1. PCR reaction system: 1.0 μL template, 2× Hieff PCR Master Mix 12.5 μL, forward primer 1 μL, reverse primer 1 μL, and sterile ddH2O were added to bring the total volume to 25 μL. PCR reaction program: 98℃ pre-denaturation for 3 min, followed by 35 cycles (98℃ 10 s, 60℃ 30 s, 72℃ 1 min), and final extension at 72℃ for 5 min. PCR products were detected using a 1.0% agarose gel electrophoresis system (Biorad, California, USA). The target fragment was recovered using the HiPure GelPure Micro Kit (Guangzhou Meiji Technology Co., Ltd., catalog number: D2110-02) and ligated into pMD18-TVector (Takara, catalog number: D101A). The ligation product was transformed into *E. coli* DH5α (Shanghai Weidi Biotechnology Co., Ltd., catalog number: DL1001) using the heat shock method and plated on a plate containing 100 μg / mL of agarose gel. -1The ampicillin antibiotic was incubated overnight at 37°C on LB agar plates. Single colonies were picked as templates, and colony PCR was performed using the aforementioned upstream and downstream sequences as primers for verification. Positive clones were sent to Zhejiang Shangya Biotechnology Co., Ltd. for bacterial sequencing. The nucleotide sequence of *Raspberry palmatum* RcWRKY51 was obtained as shown in SEQ ID NO. 1.

[0032] The open reading frame of the RcWRKY51 transcription factor in Raspberry palmatum is 1020 bp. Figure 1 The protein, encoding 339 amino acids (amino acid sequence shown in SEQ ID NO.2), has a predicted molecular weight of 38.05 kDa and a molecular formula of C2. 1646 H 2540 N 468 O 542 S 14 The theoretical isoelectric point (pI) is 5.46, the aliphatic index is 58.67, the instability index is 43.77, and the grand average of hydropathicity is -0.827. The WRKY DNA-binding domain (PF03106; E-value 1×e) is conserved at positions 146-206 of the RcWRKY51 transcription factor protein in *Rubus palmatus*. -22 According to the pLoc-mPlant subcellular localization prediction software, the RcWRKY51 transcription factor protein of Raspberry palmatum was predicted to be located in the cell nucleus.

[0033] Analysis using the biological software SOPM showed that the secondary structure of the RcWRKY51 protein in *Rubus palmatus* contains 22.71% α-helix, 8.55% β-turn, 66.37% random coil, and 2.36% extended strand. Figure 2 The tertiary structure of the RcWRKY51 protein from *Rubus palmatus* was deduced using SWISS-MODEL software, as shown below. Figure 3 As shown, its GMQE value is 0.58, and the sequence homology reaches 77.48%.

[0034] Further phylogenetic analysis showed that the RcWRKY51 protein in *Raspberry palmatum* belongs to the WRKY family, clusters in subfamily IIIa, and is most closely related to the AtWRKY70 protein in *Arabidopsis thaliana*. Figure 4 ).

[0035] Example 2: Analysis of the expression pattern of the RcWRKY51 gene in Raspberry palmatum

[0036] Different tissues (roots, stems, leaves, flowers, and fruits) of *Raspberry palmatum* were collected, and total RNA was extracted and reverse transcribed according to the method in Example 1-(1). Real-time quantitative PCR was performed using upstream primers (CTCTGTTCTGACTGACCATTCC) and downstream primers (TCTGCCTTCACCTCTCCATA) designed based on the RcWRKY51 gene. A SYBR Premix ExTaq Kit (Takara, catalog number: DRR420A) was used for real-time quantitative PCR amplification. The reaction program was 95℃ denaturation for 2 min, followed by 40 cycles (95℃ for 15 s, 60℃ for 1 min). Data were obtained after running a 480 Instrument real-time quantitative PCR test (Roche Diagnostics, Mannheim, Germany), and then analyzed using 2... -ΔΔCT The relative expression levels of each sample were calculated. The internal control was the β-actin gene of *Rubus palmatus*, with upstream primers such as ATCCACGAGACTACATACAACTCC and downstream primers such as CTGTCTGCAATACCAGGGAAC.

[0037] The results showed that the RcWRKY51 gene of *Rubus palmatus* was expressed in the roots, stems, leaves, flowers, and fruits of *Rubus palmatus*, with the highest expression in the fruits. Figure 5 Furthermore, we analyzed the expression level of the RcWRKY51 gene in *Rubus palmatus* at different fruit development stages (small green fruit, large green fruit, yellow fruit, and red fruit). The results showed that the expression level of the RcWRKY51 gene gradually decreased as the fruit matured, with the highest expression level observed in small green fruit. Figure 5 ).

[0038] Furthermore, PlantCare online software revealed multiple light-responsive elements (3-AF1 binding site, G-box, GT1-motif, GATA-motif, Sp1) in the promoter region of the RcWRKY51 gene in *Rubus palmatus*. qRT-PCR analysis showed that the RcWRKY51 gene exhibited significantly upregulated expression after UV-induced treatment. Figure 6 ).

[0039] Example 3: Analysis of the content of kaempferol-3-O-rutin in the fruit of Raspberry palmatum.

[0040] The content of kaempferol-3-O-rutin in the fruit of *Rubus palmatus* was determined according to the *Rubus* section of the *Pharmacopoeia of the People's Republic of China*. 1.0 g of *Rubus palmatus* fruit powder at different developmental stages (passed through a No. 4 sieve) was accurately weighed and placed in a stoppered conical flask. 50 mL of 70% methanol was accurately added, and the mixture was weighed. The mixture was heated under reflux for 1 hour, cooled, and weighed again. The lost weight was replenished with 70% methanol, and the mixture was shaken well and filtered. 25 mL of the subsequent filtrate was accurately measured, evaporated to dryness, and the residue was dissolved in 20 mL of water. The mixture was then extracted three times with 20 mL of petroleum ether, and the petroleum ether solution was discarded. The mixture was then extracted three times with 20 mL of water-saturated n-butanol, and the n-butanol solutions were combined and evaporated to dryness. The residue was dissolved in an appropriate amount of methanol, transferred to a 5 mL volumetric flask, and methanol was added to the mark. The mixture was shaken well, filtered, and the subsequent filtrate was used for HPLC analysis.

[0041] The column was packed with octadecylsilane-bonded silica gel; the mobile phase was acetonitrile-0.2% phosphoric acid solution (15:85); the detection wavelength was 344 nm; the column temperature was 30℃; and the flow rate was 1 mL / min. The theoretical plate number, calculated based on the kaempferol-3-O-rutin peak, should be no less than 3000. 10 μL each of the kaempferol-3-O-rutin standard solution (Sigma-Aldrich, catalog number: PHL80700) and the test solution from fruits at different developmental stages were accurately injected into the liquid chromatograph for determination.

[0042] The results showed that the theoretical plate number of the kaempferol-3-O-rutin peak was 15237, and it achieved baseline separation from adjacent impurity peaks, meeting the detection requirements of the *Pharmacopoeia of the People's Republic of China*. The content of kaempferol-3-O-rutin varied significantly among different developmental stages of *Raspberry palmatum* fruit (small green fruit, large green fruit, yellow fruit, and yellow fruit), with the highest content found in the small green fruit. Figure 7 This is significantly positively correlated with the expression level of the transcription factor RcWRKY51 gene (R, 8). 2 =0.96, p<0.01). Therefore, the RcWRKY51 gene in *Raspberry palmatum* is very likely to regulate the differential accumulation of kaempferol-3-O-rutin at different developmental stages of the fruit.

[0043] Example 4: Overexpression of RcWRKY51 in Raspberry promotes the biosynthesis of kaempferol-3-O-rutin.

[0044] (1) Construction of an overexpression vector for the RcWRKY51 transcription factor from Raspberry palmatum

[0045] Based on the nucleotide sequence of transcription factor RcWRKY51 (SEQ ID NO.1), upstream primers (CGGTACCCGGGGATCCATGGGTACGACTTGGCTCGAAGCA) and downstream primers (GGCCAGTGCCAAGCTTTTACAAGGGGCCTAAAGGAAATGC) were designed at the BamHI and HindIII restriction sites, respectively. Using *Rubus palmatus* fruit cDNA as a template, the RcWRKY51 sequence was amplified with high fidelity. Simultaneously, the plant binary expression vector pCAMBIA1301 was digested with BamHI and HindIII. The HD Cloning Kit (Takara, catalog number: 639648) was used to construct the RcWRKY51 overexpression vector. This plant recombinant expression vector was named pCAMBIA1301-RcWRKY51. Figure 9 ).

[0046] (2) The recombinant plasmid pCAMBIA1301-RcWRKY51 was transformed into Agrobacterium GV3101 (pSoup-p19).

[0047] The pCAMBIA1301-RcWRKY51 was transformed into Agrobacterium GV3101 (pSoup-p19) using a heat shock method. The specific steps are as follows: 1 μg of recombinant plasmid pCAMBIA1301-RcWRKY51 was mixed with 100 μL of Agrobacterium GV3101 (pSoup-p19) competent cells, incubated on ice for 5 min, flash-frozen in liquid nitrogen for 5 min, and then rapidly transferred to a 37°C water bath for 5 min, followed by incubation on ice for 5 min. 700 μL of antibiotic-free LB medium was added, and the mixture was incubated on a shaker at 28°C and 100 rpm for 2-3 h. The culture was then plated onto 25 mL LB agar plates (containing 50 mg / mL of the medium). -1 Kanamycin). Invert the plate and incubate it in a 28°C incubator until colonies grow (about 2 days). Select positive clones for subsequent experiments.

[0048] (3) Agrobacterium-mediated genetic transformation of RcWRKY51 gene in Rubus palmatus callus

[0049] Positive clones containing *Raspberry palmatum* pCAMBIA1301-RcWRKY51 were selected and placed in 100 mL LB liquid medium (containing 50 mg / mL). -1 Kanamycin), incubate overnight (approximately 16 hours) at 28°C with shaking at 180 rpm until OD reaches 100%. 600=0.6~0.8. Collect the bacterial culture by centrifugation at 5000×g for 5 min at room temperature. Resuspend the precipitated Agrobacterium in 100 mL of osmotic buffer (0.2 mM acetylsalicylic acid, 10 mM MgCl2, 10 mM MMES, pH 5.7), centrifuge at 5000×g for 5 min at room temperature, discard the supernatant, and adjust the OD with osmotic buffer. 600 ≈0.6, activated at 25℃ in the dark for 2 hours. Healthy *Rubus palmatus* callus tissue was selected, pre-cultured for 2 days, and then infected with activated bacterial solution containing the RcWRKY51 gene for 10 minutes. After 24 hours of dark culture in a tissue culture room at 23±2℃, it was cultured under light for 2 days. Positive plants were screened using semi-quantitative PCR for subsequent experiments. *Rubus palmatus* callus tissue not infected with the RcWRKY51 gene served as a control.

[0050] The results showed that, compared with the control (Mock), the expression level of the RcWRKY51 gene was upregulated by 5.47 times and the content of kaempferol-3-O-rutin increased by 2.07 times in callus tissue overexpressing RcWRKY51 (OE-RcWRKY51). Figure 10 Gene expression analysis revealed that RcWRKY51 of *Rubus palmatus* acts as a positive regulatory transcription factor, increasing the accumulation of kaempferol-3-O-rutinoside by upregulating the expression of genes involved in the flavonoid glycoside metabolism pathway, including PAL, C4H, CHI, CHS, and FLS. The specific sequence listing information is as follows:

[0051] >RcWRKY51 nucleotide sequence (SEQ ID NO.1)

[0052]

[0053] >RcWRKY51 amino acid sequence (SEQ ID NO.2)

[0054] MGTTWLEALPADHKSVIIKELVHGRKRATELQTLLNNRGDGSALLSPEELVKEILTSFTESLSVLTDHKSCGDDDHQYSGTGCGEVYPYGEVKAEPSHVDHSHCDDRSYEDSGESRKRSGCKDRRGCYKRRKNSESWIVVSPTIDDGQAWRKYGQKEILNAPYPRAYF RCTRKYDQGCQATKQVQQFQDTPKLYKITYIGKHTCRNIIKGPQMIMGTSGESHGPQATTVSSESGSTPIYDHGGRVSLSMIQSTPVKEEEYKEGNTSSDLTDNHNDTSDAWPDFKDHHRHDFGFTETAAALSNEDAVSNMQYLDMDLVKSIDFESDFPFDEVAFPLGPL

[0055] RcWRKY51 nucleotide sequence amplification primers

[0056] upstream primer

[0057] ATGGGTACGACTTGGCTCGA

[0058] Downstream primer

[0059] TTACAAGGGGCCTAAAGGAA

[0060] The real-time quantitative PCR (RT-qPCR) primers used by RcWRKY51

[0061] Upstream primer:

[0062] CTCTGTTCTGACTGACCATTCC

[0063] Downstream primer:

[0064] TCTGCCTTCACCTCTCCATA

[0065] Primers used for real-time quantitative PCR (RT-qPCR) of the internal reference gene β-actin

[0066] Upstream primer:

[0067] ATCCACGAGACTACATACAACTCC

[0068] Downstream primer:

[0069] CTGTCTGCAATACCAGGGAAC

[0070] Primers used in the construction of recombinant plasmid pCAMBIA1301-RcWRKY51

[0071] Upstream primer:

[0072] CGGTACCCGGGGATCCATGGGTACGACTTGGCTCGAAGCA

[0073] Downstream primer:

[0074] GGCCAGTGCCAAGCTTTTACAAGGGGCCTAAAGGAAATGC

Claims

1. A protein encoding a UV-responsive transcription factor RcWRKY51 from Raspberry palmatum, characterized in that, The amino acid sequence is shown in SEQ ID NO.

2.

2. The gene encoding the protein of the UV-responsive transcription factor RcWRKY51 of Raspberry palmatum as described in claim 1.

3. The gene according to claim 2, characterized in that, It has a nucleotide sequence as shown in SEQ ID NO.

1.

4. A recombinant vector containing the gene as described in claim 2 or 3.

5. The recombinant vector according to claim 4, characterized in that, The carrier is pCAMBIA1301.

6. A recombinant microorganism containing the gene of claim 2 or 3 in a recombinant vector.

7. The microorganism according to claim 6, characterized in that, The microorganism in question is Agrobacterium GV3101 (pSoup-p19) strain.

8. The use of overexpression of the gene according to claim 2 or 3 in enhancing the biosynthesis of kaempferol-3-O-rutin from Raspberry palmatum.

9. The application of the gene according to claim 2 or 3 in the breeding improvement of Raspberry palmatum, wherein the application is the application of overexpression of transcription factor RcWRKY51 in genetic engineering breeding to increase the content of kaempferol-3-O-rutin in Raspberry palmatum.

Images