Raspberry palmatum transcription factor RcWRKY33 and its application

Abstract

The present invention discloses a palm leaf raspberry transcription factor RcWRKY33 and its application. The present invention provides a transcription factor RcWRKY33 that can promote the accumulation of plant total flavonoids and kaempferol-3-O-rutinoside, and is induced by hormones ABA, MeJA, and GA, and the nucleotide sequence is shown in SEQ ID NO.1. The transcription factor RcWRKY33 can bind to the W-box site (TTGACC) in the promoter region of the flavonoid synthesis pathway gene RcFLS1, activate the expression of the RcFLS1 gene, and then promote the biosynthesis of palm leaf raspberry total flavonoids and kaempferol-3-O-rutinoside. RcWRKY33 can significantly promote the biosynthesis of palm leaf raspberry total flavonoids and kaempferol-3-O-rutinoside, and improve the medicinal quality of palm leaf raspberry flavonoids and kaempferol-3-O-rutinoside.

Description

Technical Field

[0001] The invention belongs to the technical field of medicinal plant gene engineering, and specifically relates to a Rubus idaeus transcription factor RcWRKY33 and an application thereof. Background Art

[0002] Rubus chingii Hu is the original plant of the Chinese medicinal material raspberry, and its unripe dried fruit is used as medicine. Rubus chingii Hu fruit contains a large amount of metabolites such as kaempferol-3-O-rutinoside, ellagic acid, linaloside, quercetin and hyperoside, which are included in the "Pharmacopoeia of the People's Republic of China". Kaempferol-3-O-rutinoside is one of the quality evaluation standards for raspberries. Pharmacological studies have shown that kaempferol-3-O-rutinoside has lipid-lowering, anti-atherosclerotic, anti-cancer, anti-diabetic and cardio-cerebral protective effects (Zhou P, Ma Y, Peng J, Hua F. Kaempferol-3-O-rutinoside: A natural flavonoid glycosides with multifaceted therapeutic potential. Neurochemical Journal, 2023, 17: 247-252.). Therefore, it is of great value to identify the key structural genes that regulate the biosynthesis of kaempferol-3-O-rutinoside.

[0003] Kaempferol-3-O-rutinoside is a flavonoid widely found in traditional Chinese herbal medicines such as Raspberry palm leaf, Tetrastigma hemsleyanum, Lycium chinense, Hibiscus mutabilis, and Carthamus tinctorius. Although flavonoids are numerous, complex in structure, and diverse in function, their biosynthetic pathways are relatively conservative (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.). Flavonoids are metabolized through the phenylpropanoid pathway to produce 4-coumaryl-CoA under the catalysis of phenylalanine ammonia lyase (PAL), cinnamate 4-hydroxylase (C4H), and 4-coumarate CoA ligase (4CL), followed by chalcone synthase (CHS) and chalcone isomerase (CHI) to produce naringenin, and then enter the metabolic branches of lignin, anthocyanidins, flavonols, isoflavones, flavanones, and proanthocyanidins under the catalysis of flavone synthase (FNS), flavone-3-hydroxylase (F3H), and flavonol synthase (FLS) (Daryanavard H, Postiglione AE, Mühlemann JK, Muday et al., 2013). GK.Flavonols modulate plant development,signaling,and stress responses.Curr Opin Plant Biol.2023,72:102350.) Under the action of various glycosyltransferases, characteristic metabolic compounds with complex structures and diverse functions are generated.

[0004] Transcription factors WRKY, MYB, NAC, bHLH, bZIP, AP2 / ERF, TCP, etc., regulate the biosynthesis of flavonoids by binding to cis-regulatory elements on the promoters of key enzymes in the flavonoid synthesis pathway, such as PAL, C4H, 4CL, CHS, CHI, FNS, F3H, FLS, etc. (Cao Y, Mei Y, Zhang R, Zhong Z, Yang X, Xu C, Chen K, Li X. Transcriptional regulation of flavonol biosynthesis in plants. Hortic Res, 2024, 11(4): uhae043; Wen W, Alseekh S, Fernie AR. Conservation and diversification of flavonoid metabolism in the plant kingdom. Curr Opin Plant Biol, 2020, 55: 100-108.). Grape (Vitis vinifera) VvWRKY70 transcriptionally inhibits β-carotene hydroxylase 2 (VvBCH2) and chalcone synthase 3 (VvCHS3), thereby hindering the accumulation of norisoprenoid and flavonols. Light and high temperature downregulate the expression of VvWRKY70, promote the accumulation of norisoprenoid and flavonols, and then regulate the aroma and color of grapes (Wei Y, Meng N, Wang Y, Cheng J, Duan C, Pan Q. Transcription factor VvWRKY70 inhibits both norisoprenoid and flavonol biosynthesis in grape. Plant Physiol, 2023, 193(3): 2055-2070.). Sweet potato (Ipomoea batatas) IbBBX29 is highly expressed in leaves and is significantly induced by auxin.Overexpression of IbBBX29 promoted the increase of sweet potato leaf biomass by 21.37%-70.94%, and the accumulation of flavonoids by 31.33%-63.03%, activated and targeted the key genes IbCHS, IbCHI1 and IbF3'H for IAA signaling and flavonoid biosynthesis, and regulated leaf development and flavonoid biosynthesis (Gao XR, Zhang H, Li X, Bai YW, Peng K, Wang Z, Dai ZR, Bian XF, Zhang Q, Jia LC, Li Y, Liu QC, Zhai H, Gao SP, Zhao N, He SZ. The B-box transcription factor IbBBX29 regulates leaf development and flavonoid biosynthesis in sweet potato. Plant Physiol, 2023, 191(1): 496-514.). However, the transcription factors that regulate the synthesis of Raspberry palm flavonoids and kaempferol-3-O-rutinoside are still unclear, and their functions are even less understood. Summary of the invention

[0005] The first object of the present invention is to provide a Raspberry palmatum transcription factor RcWRKY33 protein, the amino acid sequence of which is shown in SEQ ID NO.2.

[0006] The second object of the present invention is to provide a gene encoding the Raspberry palmatum transcription factor RcWRKY33 protein.

[0007] Preferably, the nucleotide sequence of the gene is as shown in SEQ ID NO.1.

[0008] The third object of the present invention is to provide a recombinant vector containing the gene.

[0009] Preferably, the recombinant vector is a recombinant vector obtained by inserting the gene into the EcoR I and HindIII restriction sites of the pCAMBIA1301 vector.

[0010] The fourth object of the present invention is to provide an application of overexpressing the gene in promoting the accumulation of total flavonoids and kaempferol-3-O-rutinoside from Rubus idaeus.

[0011] The fifth object of the present invention is to provide the use of overexpressing the gene in promoting the expression of flavonoid synthesis genes in Rubus palmatus.

[0012] Preferably, the flavonoid biosynthesis genes are Rc4CL1, RcCHI, RcFLS1, and RcF3H.

[0013] The sixth object of the present invention is to provide the application of the gene in the improvement and breeding of Rubus idaeus, wherein the application is the application in genetic engineering breeding for increasing the biosynthesis of total flavonoids and kaempferol-3-O-rutinoside in Rubus idaeus.

[0014] The seventh object of the present invention is to provide a method for promoting the expression of genes in the flavonoid biosynthesis pathway in Rubus idaeus, comprising the step of treating Rubus idaeus with ABA, MeJA or GA3.

[0015] Beneficial effects of the present invention:

[0016] The present invention provides a transcription factor RcWRKY33 involved in the regulation of palm leaf raspberry kaempferol-3-O-rutinoside, belonging to the field of plant genetic engineering technology, and the nucleotide sequence of the palm leaf raspberry RcWRKY33 is shown in SEQ ID NO.1. In the present invention, the palm leaf raspberry transcription factor RcWRKY33 can bind to the W-box site (TTGACC) on the promoter of the flavonoid synthesis gene RcFLS1, activate the expression of the RcFLS1 gene, and then promote the biosynthesis of palm leaf raspberry total flavonoids and kaempferol-3-O-rutinoside. Verification shows that the transcription factor RcWRKY33 gene is highly expressed in palm leaf raspberry fruits, is significantly positively correlated with the content of kaempferol-3-O-rutinoside, and responds to hormone induction such as ABA. Genetic transformation technology was used to overexpress the R. idaeus transcription factor RcWRKY33 gene, which significantly increased the content of total flavonoids and kaempferol-3-O-rutinoside in R. idaeus, and upregulated the expression of flavonoid synthesis pathway genes Rc4CL1, RcCHI, RcFLS1, and RcF3H. This shows that the RcWRKY33 can significantly promote the biosynthesis of total flavonoids and kaempferol-3-O-rutinoside in R. idaeus, and improve the medicinal quality of total flavonoids and kaempferol-3-O-rutinoside in R. idaeus. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 This is the gel electrophoresis of the transcription factor RcWRKY33 gene of Raspberry palmate. On the left is the nucleic acid molecular weight standard, and the bands from top to bottom are 2000, 1000, 750, 500, 250, and 100 bp; on the right is the RcWRKY33 gene.

[0018] Figure 2 Genetic phylogenetic tree analysis of the transcription factor RcWRKY33 protein in Raspberry.

[0019] Figure 3 This is the secondary structure of the Raspberry transcription factor RcWRKY33 protein; blue represents α-helix, purple represents the extended main chain, and yellow represents random coil.

[0020] Figure 4 The tertiary structure of the Raspberry transcription factor RcWRKY33 protein. The arrow points to the WRKY binding site.

[0021] Figure 5 The expression levels of the transcription factor RcWRKY33 gene in different parts (fruit, root, stem, leaf and flower) of Raspberry. The bar of each group of data represents ± standard deviation (n≥10). Different letters indicate that the expression levels of RcWRKY33 gene in different parts are significantly different (p<0.05).

[0022] Figure 6 The expression levels of the transcription factor RcWRKY33 gene in Raspberry (Italy) at different maturity stages of fruits (small green fruit, large green fruit, yellow fruit and red fruit); the bar of each group of data represents ± standard deviation (n≥10), and different letters indicate that there are significant differences in the expression levels of the RcWRKY33 gene in different maturity stages of fruits (p<0.05).

[0023] Figure 7 The content of kaempferol-3-O-rutinoside in fruits of Rubus idaeus at different maturity stages (small green fruit, large green fruit, yellow fruit and red fruit); the bar of each group of data represents ±standard deviation (n≥10), and different letters indicate that the content of kaempferol-3-O-rutinoside in fruits at different maturity stages is significantly different (p<0.05).

[0024] Figure 8 The expression levels of the transcription factor RcWRKY33 gene in Raspberry callus under abscisic acid (ABA), methyl jasmonate (MeJA) and gibberellin (GA3) induction. The bar for each group of data represents ± standard deviation (n≥6). ** indicates that the expression levels of the RcWRKY33 gene under the induction of ABA, MeJA, and GA3 were significantly different from those of CK at p<0.01.

[0025] Fig. 9 Schematic diagram of pCABIA1301-RcWRKY33 overexpression vector. The accession number of pCABIA1301 is AF234297.

[0026] Fig.10Expression level and flavonoid content analysis of R. palmatum callus overexpressing RcWRKY33 gene; A is the expression level of RcWRKY33 gene, B is the total flavonoid content, and C is the kaempferol-3-O-rutinoside content; the bar of each group of data represents ± standard error (n≥6), and ** indicates that Student's t-test statistics showed that there was a significant difference between the control (EV) and the overexpression callus at p<0.01.

[0027] Fig.11 The expression levels of flavonoid biosynthesis pathway genes Rc4CL1, RcCHI, RcFLS1 and RcF3H in R. idaeus callus overexpressing RcWRKY33 gene; the bar of each group of data represents ± standard error (n≥6), ** indicates that Student's t-test statistics showed that there was a significant difference between the control (EV) and the overexpression callus at p<0.01.

[0028] Fig.12 The dual luciferase system shows that the transcription factor RcWRKY33 binds to the RcFLS1 promoter through the W-box and activates the expression of the RcFLS1 gene; the bar for each group of data represents ± standard error (n≥6), ** indicates that Student's t-test statistics show that there is a significant difference between the control and treatment groups at p<0.01; RcFLS1p indicates that the W-box element in the RcFLS1 promoter region has not changed, and RcFLS1pm indicates that the W-box element in the RcFLS1 promoter region has changed to TTGATC. DETAILED DESCRIPTION

[0029] The following examples are provided to further illustrate the present invention, rather than to limit the present invention.

[0030] Rubus chingii Hu used in the examples was planted in the medicinal plant garden of Zhejiang Chinese Medical University (N30°5′, E119°53′; Hangzhou, China) and was identified as Rubus chingii Hu by Professor Shen Xiaoxia of Zhejiang Chinese Medical University. Polysaccharide and polyphenol plant RNA extraction kit was purchased from Huayueyang Biotechnology Co., Ltd. (Cat. No.: 0416-50); reverse transcription PrimeScript TM RT Reagent Kit was purchased from TaKaRa (Cat. No. RR014A); Escherichia coli DH 5α was purchased from Shanghai Weidi Biotechnology Co., Ltd. (Cat. No. DL1001); HiPure Gel Pure Mini Kit was purchased from Guangzhou Meiji Technology Co., Ltd. (Cat. No. D2110-02); 2×Hieff PCR Master Mix was purchased from YEASEN (Cat. No. 10136ES03); In- HD Cloning Kit was purchased from TaKaRa (Cat. No. 639648); pMD18-T Vector was purchased from TaKaRa (Cat. No. D101A); SYBR Premix Ex Taq TM Kit was purchased from TaKaRa (catalog number: DRR420A); LB and MS media are commonly used media in this field, and their formulas refer to the "Molecular Cloning Experiment Guide" by J. Sambrook and MR Green.

[0031] Example 1: Cloning and bioinformatics analysis of the transcription factor RcWRKY33 gene from Raspberry palmata

[0032] (1) Total RNA extraction and first-strand cDNA synthesis from immature fruits of Rubus idaeus

[0033] Unripe fruits of Raspberry palmate were collected from the Medicinal Plant Garden of Zhejiang Chinese Medical University and ground into powder under liquid nitrogen. Total RNA of unripe fruits of Raspberry palmate was extracted using a plant RNA extraction kit (polysaccharide and polyphenol series, Huayueyang Biotechnology Co., Ltd., catalog number: 0416-50). Agarose gel electrophoresis (Bio-Rad, USA) and NanoDrop TM The total RNA content and purity of immature fruit were determined by 2000c ultra-micro spectrophotometer (Thermo Scientific, USA). 1 μg of total RNA from immature fruit of Raspberry palmate was collected and analyzed by PrimeScript TM RT Reagent Kit (TaKaRa, Dalian, Cat. No.: RR014A) was used to synthesize the first-strand cDNA and the reaction product was diluted to 50 ng·μL -1 , stored at -80℃.

[0034] (2) Amplification and sequence analysis of the transcription factor RcWRKY33 gene from Raspberry

[0035] Using the cDNA of immature fruit of Raspberry palmate leaf as template, Primer Premier 5.0 (Premier Biosoft, USA) was used to design upstream primer SEQ ID NO.3 and downstream primer SEQ ID NO.4 of Raspberry palmate leaf transcription factor RcWRKY33 gene, and 2×Hieff PCR Master Mix (YEASEN, Shanghai, catalog number: 10136ES03) was used for nested PCR amplification to obtain the gene sequence of R. idaeus transcription factor RcWRKY33, as shown in SEQ ID NO. 1. PCR reaction system: template 1.0 μL, 2×Hieff PCR Master Mix 25 μL, upstream primer 1 μL, downstream primer 1 μL, sterile ddH2O to make up to a total volume of 50 μL. PCR reaction program: 98 ° C pre-denaturation 3min, 35 cycles of reaction (98 ° C 10s, 60 ° C 30s, 72 ° C 1min), 72 ° C final extension 5min. PCR products were detected by agarose gel electrophoresis, and the target fragment was recovered using the HiPureGel Pure Micro Kit (Guangzhou Meiji Technology Co., Ltd., Catalog No.: D2110-02), connected to pMD18-T Vector (Takara, Dalian, Catalog No.: D101A), and the ligation product was transformed into Escherichia coli DH5α competent (Shanghai Weidi Biotechnology Co., Ltd., Catalog No.: DL1001) by heat shock method and coated with 100 μg mL -1 The culture was carried out on an LB plate containing ampicillin antibiotics at 37°C overnight. A single colony was selected as a template, and the upstream and downstream primers (SEQ ID NO.3 and SEQ ID NO.4) were used for colony PCR verification, and the positive clones were sent to Zhejiang Shangya Biotechnology Co., Ltd. for bacterial liquid sequencing. The nucleotide sequence of the palm leaf raspberry transcription factor RcWRKY33 was obtained as shown in SEQ ID NO.1.

[0036] The coding region of R. idaeus transcription factor RcWRKY33 is 1389 bp ( Figure 1 ), encoding 462 amino acids (amino acid sequence as shown in SEQ ID NO.2), the largest proportion of which is serine (Ser), threonine (Thr) and glutamine (Gln), containing 47 acidic amino acids (Asp+Glu) and 47 basic amino acids (Arg+Lys), the protein molecular weight is 51.16kDa, and the molecular formula is C 2212 H 3415 N 629 O 749 S 11 The theoretical isoelectric point (Theoretical pI) was 7.02, the aliphatic amino acid index (Aliphatic index) was 47.32, the instability index (instability index) was 65.30, and the grand average of hydropathicity was -0.933. It was speculated that the transcription factor RcWRKY33 was an unstable hydrophilic protein.

[0037] The 213-268 and 380-400 sites of the transcription factor RcWRKY33 protein of Raspberry palmata contain WRKY conserved functional domains (PF03106, E-values ​​are 4.9×e -26 and 6.7×e -7 ). According to the pLoc-mPlant subcellular localization prediction software, the transcription factor RcWRKY33 protein of Raspberry palmata is predicted to be localized in the nucleus. According to the classification of the Arabidopsis WRKY family, Raspberry palmata RcWRKY33 is most closely related to Arabidopsis WRKY33 and belongs to the WRKY I subfamily ( Figure 2 ).

[0038] Analysis of the SOPMA online biology website showed that the secondary structure of the palm raspberry transcription factor RcWRKY33 protein includes alpha helix, random coil and extended strand, accounting for 4.98%, 86.80% and 8.23% respectively. Figure 3 ). The tertiary structure of the transcription factor RcWRKY33 protein of Raspberry palmata was predicted by SWISS-MODEL online software, and the sequence homology reached 87.99% and the GMQE value was 0.61 ( Figure 4 ).

[0039] Example 2: Analysis of the expression pattern of the R. idaeus transcription factor RcWRKY33 gene

[0040] (1) Real-time fluorescence quantitative PCR detection of RcWRKY33 gene expression

[0041] Different parts of Rubus palmatus (roots, stems, leaves, flowers, fruits) and different maturity stages of fruits (small green fruit, large green fruit, yellow fruit, red fruit) were collected, and total RNA was extracted and reverse transcribed according to the method of Example 1-(1). Real-time fluorescence quantitative PCR was performed using the upstream primers (SEQ ID NO.5) and downstream primers (SEQ ID NO.6) designed according to the RcWRKY33 gene, and the real-time fluorescence quantitative PCR amplification was performed using the SYBR Premix Ex Taq Kit (Takara, Dalian, Cat. No.: DRR420A). The reaction procedure was: denaturation at 95°C for 2 min, and 40 cycles of amplification reaction (95°C for 15 s, 60°C for 1 min). The reaction was performed at 480Instrument real-time fluorescence quantitative PCR (Roche Diagnostics, Germany) was used for -ΔΔCTMethods The relative expression data of each sample were calculated. The internal reference was the β-actin gene of Rubus idaeus, the upstream primer was shown in SEQ ID NO.7, and the downstream primer was shown in SEQ ID NO.8.

[0042] (2) Determination of kaempferol-3-O-rutinoside content in Rubus idaeus plants

[0043] Take 1.0g of fruit powder (passed through No. 3 sieve) of palm leaf raspberry at different growth stages, add 50mL of 70% methanol by volume, heat and reflux to extract for 1h, cool, make up the lost weight with 70% methanol by volume, shake well, and filter. Take 25mL of the filtrate, evaporate to dryness, add 20mL of water to dissolve the residue, shake and extract 3 times with petroleum ether, 20mL each time, discard the petroleum ether liquid, shake and extract 3 times with water-saturated n-butanol, 20mL each time, combine the n-butanol liquid, evaporate to dryness, dissolve the residue with 5mL of pure methanol, filter, take the filtrate, and use it for HPLC detection.

[0044] A common C18 column was used with acetonitrile-0.2% phosphoric acid (volume ratio of the two was 15:85) as the mobile phase, injection volume of 10 μL, detection wavelength of 344 nm, column temperature of 30 °C, flow rate of 1 mL min -1 The theoretical plate number calculated based on the kaempferol-3-O-rutinoside peak should be no less than 3000. The content was calculated using the kaempferol-3-O-rutinoside standard (Sigma-Aldrich, USA, catalog number: PHL80700).

[0045] The results showed that the transcription factor RcWRKY33 gene of R. palmata was expressed in the roots, stems, leaves, flowers and fruits of R. palmata, with the highest expression in the fruits, followed by the roots, leaves and stems, and the lowest expression in the flowers ( Figure 5 ).

[0046] Furthermore, the expression level of the transcription factor RcWRKY33 gene of Raspberry palmate leaf at different maturity stages of the fruit (small green fruit, large green fruit, yellow fruit, and red fruit) was analyzed. The results showed that as the fruit of Raspberry palmate leaf gradually matured, the expression level of the transcription factor RcWRKY33 gene showed a gradually decreasing trend, with the highest expression level in small green fruit, followed by large green fruit and yellow fruit, and the lowest expression level in red fruit ( Figure 6 In the palm leaf rubus fruit, as the fruit gradually matures, the content of kaempferol-3-O-rutinoside shows a trend of gradually decreasing ( Figure 7 ), which is consistent with the expression trend of transcription factor RcWRKY33 gene. The Pearson correlation coefficient R 2 =98.91 (p<0.01), indicating that the transcription factor RcWRKY33 is involved in the accumulation of kaempferol-3-O-rutinoside in Raspberry fruit.

[0047] Example 3: Expression analysis of the transcription factor RcWRKY33 gene of Raspberry palmate under ABA, MeJA and GA3 treatments

[0048] Through the PlantCare online software, it was found that there were multiple exogenous hormone response elements in the promoter region of the transcription factor RcWRKY33 gene of Raspberry palm leaf, such as ABA responsive element binding protein (AREB), MeJA response elements (CGTCA motif and TGACG motif), GA response element (P-box), etc. It is speculated that the transcription factor RcWRKY33 gene is induced by hormones such as ABA, MeJA, GA, and is involved in plant growth and development and adversity response.

[0049] To investigate whether the RcWRKY33 gene is induced by ABA, MeJA, and GA hormones, calli of R. palmatum induced for 3 months were transferred to 1 / 2 Murashige and Skoog (MS) medium containing ABA (100 μM), MeJA (50 μM), and GA3 (50 μM) and placed at 25°C, 16 h light / 8 h dark alternation, and light intensity of 100 mmol·m -2 ·s -1 After 6 hours of treatment, callus tissue was collected, frozen by liquid nitrogen, and stored in a -80℃ refrigerator for analysis of the expression level of RcWRKY33 gene under ABA, MeJA, and GA3 treatments. The internal reference was the palm leaf β-actin gene, the upstream primer was shown in SEQ ID NO.7, and the downstream primer was shown in SEQ ID NO.8.

[0050] Real-time fluorescence quantitative PCR detection showed that the transcription factor RcWRKY33 gene was significantly upregulated after treatment with ABA (100 μM), MeJA (50 μM), and GA3 (50 μM), with the highest induction fold of ABA ( Figure 8 ).

[0051] Example 4: Overexpression of the transcription factor RcWRKY33 gene promotes the accumulation of total flavonoids and kaempferol-3-O-rutinoside

[0052] (1) Construction of an overexpression vector for the transcription factor RcWRKY33 gene from Raspberry

[0053] According to the coding region sequence of the transcription factor RcWRKY33 gene of Raspberry palmate leaf (SEQ ID NO.1), which does not contain the stop codon TAA, the upstream primer (SEQ ID NO.9) and the downstream primer (SEQ ID NO.10) were set in the EcoR I and Hind III restriction sites on the plant expression vector pCAMBIA1301. The RcWRKY33 coding region was amplified using the cDNA of immature fruit of Raspberry palmate leaf as a template, and the plant expression vector pCAMBIA1301 (Abcam, USA, catalog number: ab275753) was double-digested with EcoR I and Hind III, and then the protein was expressed by In- HD Cloning Kit (Takara, Dalian, Cat. No. 639648) was used to construct the RcWRKY33 overexpression vector, and the overexpression recombinant expression vector obtained above was named pCAMBIA1301-RcWRKY33 ( Fig. 9 ).

[0054] (2) Transformation of recombinant plasmid pCAMBIA1301-RcWRKY33 into Agrobacterium GV3101 (pSoup-p19)

[0055] pCAMBIA1301-RcWRKY33 was transformed into GV3101 (pSoup-p19) Agrobacterium using the heat shock method. The steps were as follows: 1 μg of pCAMBIA1301-RcWRKY33 recombinant plasmid was mixed with 100 μL of GV3101 (pSoup-p19) competent cells, placed on ice for 5 min, quickly frozen in liquid nitrogen for 5 min, transferred to 37 °C for 5 min, and then placed on ice for 5 min. 700 μL of LB liquid medium without antibiotics was added and the culture was incubated at 100 rpm min. -1 , 28℃ for 120min. Spread LB medium (containing 50mg·mL -1 Kanamycin). Invert and culture at 28°C until colonies grow (about 2 days), and select positive clones for subsequent experiments.

[0056] (3) Agrobacterium-mediated genetic transformation of the Raspberry transcription factor RcWRKY33 gene

[0057] Transfer the pCAMBIA1301-RcWRKY33 positive clones to 100 mL LB liquid medium (containing 50 mg mL -1 Kanamycin, 180 rpm·min -1, cultured overnight at 28°C (about 16h) until OD600 reached 0.8. Centrifuged at 5000×g for 5min at room temperature, discarded the supernatant to obtain the precipitate. Resuspended in 100mL infiltration buffer (10mM MES, 10mM MgCl2, 0.2mM acetosyringone, the balance is water, pH 5.7), centrifuged at 5000×g for 5min at room temperature, discarded the supernatant to obtain the precipitate, and adjusted the OD600 to 0.8 with the above-mentioned infiltration buffer again to obtain the RcWRKY33 activation infiltration solution, and placed it at room temperature in the dark for 2h. Selected palm leaf Rubus idaeus callus with relatively consistent growth, cultured in the dark for 2d, the above-mentioned RcWRKY33 activation infiltration solution was used to infect the callus for 10min, and then cultured in the dark at 23°C for 24h, cultured under alternating conditions of 12h light-12h dark (4000lx) for 2d, and used 50mg mL -1 Positive callus tissues were screened by hygromycin B combined with fluorescence quantitative PCR, and Raspberry palmate leaf callus tissues infected with pCAMBIA1301 empty vector (not connected to RcWRKY33 gene) were used as control (EV). The obtained callus tissues were collected and screened, part of which was used for quantitative PCR detection, and part of which was dried and ground into Raspberry palmate leaf callus tissue powder to determine the contents of total flavonoids and kaempferol-3-O-rutinoside.

[0058] (4) Determination of total flavonoids content in callus tissue of Rubus idaeus

[0059] Take 1.0g of palm leaf Rubus idaeus callus powder (pass through No. 3 sieve), add 80mL of 80% ethanol solution, ultrasonicate for 60min, centrifuge and take the supernatant; add 80mL of 80% ethanol solution to the residue and extract again, combine the supernatant and make up to 100mL, which is the test solution. Take 1.0mL of the test solution and add 0.5mL of 0.05mg·mL -1 Sodium nitrite solution was allowed to stand at room temperature for 6 min, and 0.3 mL of 0.1 g mL -1 Aluminum nitrate solution, mix well, let stand at room temperature for 6 min, add 3 mL 0.04 g mL -1 Sodium hydroxide solution was added to 10 mL, and the solution was allowed to stand at room temperature for 15 min. The absorbance at 510 nm was measured. The total flavonoids content of Raspberry palmata was calculated using rutin as the standard.

[0060] (5) Determination of the expression levels of RcWRKY33 and key genes for flavonoid biosynthesis in Raspberry plants

[0061] RNA was extracted from the transformed Raspberry palm leaf callus and reverse transcribed into cDNA for quantitative PCR detection of RcWRKY33 and key genes of flavonoid biosynthesis. The upstream primer of RcWRKY33 quantitative PCR is shown in SEQ ID NO.5, and the downstream primer is shown in SEQ ID NO.6. The internal reference is the Raspberry palm leaf β-actin gene, the upstream primer is shown in SEQ ID NO.7, and the downstream primer is shown in SEQ ID NO.8. The quantitative PCR primers of key genes of flavonoid biosynthesis are shown in Table 1.

[0062] Table 1 Quantitative PCR primers for key genes in flavonoid biosynthesis

[0063] Gene name Upstream primer number Downstream primer number Rc4CL1 SEQ ID NO.11 SEQ ID NO.12 R SEQ ID NO.13 SEQ ID NO.14 RcFLS1 SEQ ID NO.15 SEQ ID NO.16 R SEQ ID NO.17 SEQ ID NO.18

[0064] The results showed that the expression level of RcWRKY33 gene was upregulated by 7.04-fold in OE-RcWRKY33 overexpressing RcWRKY33 gene compared with the control (EV). Fig.10 A); the total flavonoids content in the control Raspberry palm leaf callus was 8.18 mg g -1 The total flavonoid content in R. palmatum callus overexpressing RcWRKY33 gene reached 14.33 mg·g -1 , which is 1.75 times the total flavonoids content in the control Raspberry palm leaf callus ( Fig.10 B); the content of kaempferol-3-O-rutinoside in the callus of Raspberry palm leaf was 7.34 μg·g -1 The content of kaempferol-3-O-rutinoside in R. idaeus callus overexpressing RcWRKY33 gene reached 22.24 μg·g -1 , which is 3.03 times the content of kaempferol-3-O-rutinoside in callus of Raspberry palm leaf ( Fig.10 The above results indicate that the transcription factor RcWRKY33 gene significantly promotes the accumulation of total flavonoids and kaempferol-3-O-rutinoside in Raspberry fruit.

[0065] Compared with the control (EV), the expression levels of flavonoid biosynthesis pathway genes Rc4CL1, RcCHI, RcFLS1, and RcF3H were significantly upregulated in callus tissues of R. palmatum overexpressing the RcWRKY33 gene (OE-RcWRKY33), especially the RcFLS1 gene, which was upregulated by 13.81 times ( Fig.11 ). Therefore, RcWRKY33 in Raspberry palmatum promotes the accumulation of total flavonoids and increases the content of kaempferol-3-O-rutinoside by transcriptionally activating the expression of genes such as RcFLS1 in the flavonoid biosynthesis pathway.

[0066] Example 5: RcWRKY33 binds to the RcFLS1 promoter through the W-box and activates RcFLS1 gene expression

[0067] (1) Construction of pGreen II 62-SK-RcWRKY33 recombinant vector

[0068] According to primers SEQ ID NO.19 and SEQ ID NO.20, RcWRKY33 with the termination codon TAA removed was obtained by nested PCR amplification. The PCR reaction system and procedure were consistent with those in Example 4-(1). The Effector vector pGreen II62-SK was double-digested at the BamH I and Hind III sites, and In- HD Cloning Kit (Takara, Dalian, Cat. No.: 639648) was used to connect pGreen II 62-SK and transcription factor RcWRKY33 to obtain the pGreen II 62-SK-RcWRKY33 recombinant vector, and the positive clones were identified by sequencing.

[0069] (2) Construction of pGreen II 0800-RcFLSp recombinant vector

[0070] Referring to the chromosome-level annotation of the Raspberry palm leaf genome, the promoter sequence (RcFLSp) of the RcFLS gene was mined, and the RcFLSp sequence was analyzed by the promoter online software PlantCARE. The results showed that the W-box binding site TTGACC was found at positions -505 to -510. According to primers SEQ ID NO.21 and SEQ ID NO.22, the RcFLSp sequence containing Hind III and BamH I double enzyme digestion was obtained by nested PCR amplification. The PCR reaction system and procedure were consistent with Example 4-(1). The reporter vector pGreen II 0800-LUC was double-digested with Hind III and BamH I, and In- HD Cloning Kit (Takara, Dalian, Cat. No.: 639648) was used to connect pGreen II 0800 and RcFLSp to obtain the pGreen II 0800-RcFLSp recombinant vector, and the positive clones were identified by sequencing.

[0071] (3) Transformation of the recombinant plasmid into Agrobacterium GV3101 (pSoup-p19)

[0072] pGreen II 62-SK-RcWRKY33 and pGreen II 0800-RcFLSp were transformed into GV3101 (pSoup-p19) respectively by heat shock method according to the operating instructions. The method was consistent with Example 4-(2), and the positive clones were identified by sequencing.

[0073] (4) GV3101 Agrobacterium infection of tobacco leaves and LUC / REN fluorescence detection

[0074] GV3101 (pSoup-p19) Agrobacterium containing pGreen II 62-SK-RcWRKY33 and pGreen II 0800-RcFLSp was resuspended in infiltration buffer (10mM MES, 10mM MgCl2, 0.2mM acetosyringone, the balance was water, pH 5.7), OD600 was adjusted to 0.8, and the suspension was kept at room temperature in the dark for 2h to obtain a bacterial suspension. Tobacco leaves with relatively consistent growth were selected, and the bacterial suspensions of pGreen II 62-SK-RcWRKY33 and pGreen II 0800-RcFLSp were mixed in a volume ratio of 9:1, and the tobacco leaves were instantly transformed. More than 6 replicates were made and marked, and the mixed suspension (EV) of two empty bacterial cells of pGreen II 62-SK and pGreen II 0800 was used as the control group. After culturing for 3 days at 23°C and 16 h light / 8 h dark, the ratio of the two luciferases (LUC / REN) in tobacco leaves was detected using a Reporter Assay System kit (Promega, USA, Cat. No.: E1910).

[0075] The results of dual luciferase in tobacco leaves showed that RcWRKY33 could bind to the downstream target gene RcFLS1 through W-box and activate the expression of RcFLS1. The transcriptional activation factor was 6.83 times that of the control (EV). However, when the W-box site was mutated, RcWRKY33 could not bind to RcFLS1 ( Fig.12 ).

Claims

1. A transcription factor RcWRKY33 protein from Rubus idaeus, characterized in that: Its amino acid sequence is shown in SEQ ID NO.

2.

2. A gene encoding the RcWRKY33 protein of the Rubus idaeus transcription factor according to claim 1.

3. The gene according to claim 2, characterized in that Its nucleotide sequence is shown in SEQ ID NO.

1.

4. A recombinant vector containing the gene according to claim 2 or 3.

5. The recombinant vector according to claim 4, characterized in that The recombinant vector is obtained by inserting the gene described in claim 2 or 3 into the EcoR I and Hind III restriction sites of the pCAMBIA1301 vector.

6. Use of overexpressing the gene described in claim 2 or 3 in promoting the accumulation of total flavonoids and kaempferol-3-O-rutinoside in Rubus idaeus.

7. Use of overexpression of the gene described in claim 2 or 3 in promoting the expression of flavonoid synthesis genes in Rubus idaeus, wherein the flavonoid synthesis genes are Rc4CL1, RcCHI, RcFLS1, and RcF3H.

8. Use of the gene according to claim 2 or 3 in improving and breeding Rubus idaeus, characterized in that: The application is application in genetic engineering breeding for increasing the biosynthesis of total flavonoids and kaempferol-3-O-rutinoside in Rubus palmatus.

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