A palm raspberry glycosyltransferase RcUGT78D1 and its encoding gene and application

Abstract

The present invention discloses a palm leaf raspberry glycosyltransferase RcUGT78D1 and its encoding gene and application. The present invention provides a glycosyltransferase RcUGT78D1 that can promote the accumulation of total flavonoids, kaempferol-3-O-rutinoside and anthocyanins in plants, and is significantly upregulated by low temperature, drought, salt stress, ABA and MeJA induction, and enhances the drought resistance, salt tolerance and low temperature tolerance of plants. The nucleotide sequence is shown in SEQ ID NO.1, and the amino acid sequence is shown in SEQ ID NO.2. Overexpression of the RcUGT78D1 gene by genetic engineering technology has good application prospects in cultivating high flavonoids, high anthocyanins, drought resistance, salt tolerance and low temperature tolerance plants.

Description

Technical Field

[0001] The invention belongs to the technical field of medicinal plant gene engineering and discloses a raspberry palmatum glycosyltransferase RcUGT78D1 and a coding gene and application thereof. Background Art

[0002] Rubus idaeus (RubuschingiiHu) is the original plant of the Chinese medicinal material raspberry, and its unripe dried fruit is used as medicine. Rubus idaeus fruits contain 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, MaY, 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 palmate, Tetrastigma hemsleyanum, Lycium chinense, Hibiscus mutabilis, and Carthamus tinctorius. Although flavonoids are diverse, 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, anthocyanins, 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] Plant uridine diphosphate glycosyltransferase (UGT) belongs to the glycosyltransferase family 1 (GT1), using UDP-sugar as glycosyl donor and small molecule compounds such as flavonoids, phenolic acids, terpenes or hormones as glycosyl acceptors. UGT catalyzes a wide variety of substrates and has a large difference in product activity. The glycosylation reaction catalyzed by UGT plays an important role in plant growth and development, stress response and metabolic regulation (Yu Anton, Liu Lin, Long Ruicai, Kang Junmei, Chen Lin, Yang Qingchuan, Li Mingna. Functions and application prospects of plant UDP-glycosyltransferase (UGT). Acta Physiologica Sinica, 2022, 58(4): 631-642.). However, there are few reports on glycosyltransferases in Raspberry palmata, and even less is known about its function. Summary of the invention

[0005] The first object of the present invention is to provide a Raspberry palmatum glycosyltransferase RcUGT78D1, whose amino acid sequence is shown in SEQ ID NO.2.

[0006] The second object of the present invention is to provide a gene encoding the Raspberry palmata glycosyltransferase RcUGT78D1.

[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 glycosyltransferase RcUGT78D1 gene into the EcoR I and Hind III restriction sites of the pCAMBIA1301 vector.

[0010] The fourth object of the present invention is to provide a genetically engineered bacterium containing the recombinant vector.

[0011] The fifth object of the present invention is to provide the use of overexpressing the gene in promoting the accumulation of flavonoids in Rubus idaeus.

[0012] Preferably, the flavonoids are kaempferol-3-O-rutinoside and anthocyanidins.

[0013] The sixth object of the present invention is to provide an application of overexpressing the gene in improving the tolerance of Rubus palmatus to drought, salt stress and low temperature.

[0014] The seventh object of the present invention is to provide the application of the gene in the improvement and breeding of palm-leaved raspberry, and the application is the application of the glycosyltransferase RcUGT78D1 gene in genetic engineering breeding for increasing the flavonoid content of palm-leaved raspberry and improving the tolerance of palm-leaved raspberry to drought, salt stress and low temperature.

[0015] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0016] The palm raspberry glycosyltransferase gene RcUGT78D1 of the glycosyltransferase UGT78 family cloned by the present invention promotes the accumulation of total flavonoids and kaempferol-3-O-rutinoside and participates in the response to adverse stresses such as salinity and drought. Through genetic transformation technology, overexpression of the RcUGT78D1 gene significantly increases the content of total flavonoids and polysaccharide kaempferol-3-O-rutinoside in palm raspberry. In addition, RcUGT78D1 can also improve the tolerance of palm raspberry to salinity and drought stress. Palm raspberry glycosyltransferase RcUGT78D1 has very important theoretical and application value in cultivating high-flavonoid, drought-resistant and salt-tolerant plants, especially new palm raspberry varieties. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 This is the gel electrophoresis image of the Raspberry palm leaf glycosyltransferase RcUGT78D1 gene; on the left is the nucleic acid molecular weight standard, and the bands are 2000, 1000, 750, 500, 250, and 100 bp from top to bottom; on the right is the RcUGT78D1 gene.

[0018] Figure 2 This is an analysis of the transmembrane region of the glycosyltransferase RcUGT78D1 protein from Raspberry palmatum.

[0019] Figure 3 This is the secondary structure of the Raspberry palmata glycosyltransferase RcUGT78D1 protein; blue represents α-helix, purple represents the extended main chain, and yellow represents random coil.

[0020] Figure 4 The tertiary structure of Raspberry palmata glycosyltransferase RcUGT78D1 protein. The arrow indicates the UGT catalytic active site.

[0021] Figure 5 Phylogenetic tree analysis of Raspberry palmata glycosyltransferase RcUGT78D1 protein and 121 Arabidopsis homologous proteins.

[0022] Figure 6is the evolutionary tree of the palm raspberry glycosyltransferase RcUGTs members and the expression levels of their homologous genes in different parts (roots, stems, leaves, flowers and fruits); A is the evolutionary tree of the palm raspberry glycosyltransferase RcUGTs members, and B is the expression levels of RcUGT78D1 and its 30 closely related homologous genes in different parts (roots, stems, leaves, flowers and fruits).

[0023] Figure 7 The expression levels of R. idaeus glycosyltransferase RcUGT78D1 gene in different parts (roots, stems, leaves, flowers and fruits). The bar of each group of data represents ± standard deviation (n≥10); different letters indicate that the expression levels of RcUGT78D1 gene in different parts are significantly different (p<0.05).

[0024] Figure 8 The expression levels of glycosyltransferase RcUGT78D1 gene in Raspberry palmata 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 RcUGT78D1 gene in different maturity stages of fruits (p<0.05).

[0025] Fig. 9 Figure 2 The expression levels of glycosyltransferase RcUGT78D1 gene in Raspberry palmate under normal conditions (control), low temperature (LT), drought (DT), salt treatment (ST), abscisic acid (ABA) and methyl jasmonate (MeJA) treatments. The bar for each group of data represents ± standard deviation (n≥6). Different letters indicate that the expression levels of RcUGT78D1 gene under different treatments are significantly different (p<0.05).

[0026] Fig.10 Schematic diagram of pCABIA1301-RcUGT78D1 overexpression vector. The accession number of pCABIA1301 is AF234297.

[0027] Fig.11Phenotypic analysis of R. palmatum callus overexpressing RcUGT78D1 gene; (A) is the expression level of RcUGT78D1 gene; (B) is the content of total flavonoids; (C) is the content of kaempferol-3-O-rutinoside; (D) is the content of anthocyanins; 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 overexpression (OE-RcUGT78D1) callus at p<0.01.

[0028] Fig.12 Determination of fresh weight of R. idaeus plants overexpressing RcUGT78D1 gene under normal conditions (Control), drought conditions (Drought), salt stress conditions (Salinity) and low temperature conditions (Low-temperature); the bar for 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 overexpression (OE-RcUGT78D1) plants at p<0.01.

[0029] Fig.13 The superoxide anion content (O2 ·- ) content determination; 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 overexpression (OE-RcUGT78D1) plants at p<0.01. DETAILED DESCRIPTION

[0030] The following examples are provided to further illustrate the present invention, rather than to limit the present invention.

[0031] 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 TMRT 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); HDCloning Kit was purchased from TaKaRa (Cat. No. 639648); pMD18-TVector 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.

[0032] Example 1: Cloning and bioinformatics analysis of the Raspberry palmata glycosyltransferase RcUGT78D1 gene

[0033] (1) Total RNA extraction and first-strand cDNA synthesis from immature fruits of Rubus idaeus

[0034] 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℃.

[0035] (2) Amplification and sequence analysis of the R. idaeus glycosyltransferase gene RcUGT78D1

[0036] 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 glycosyltransferase RcUGT78D1 gene, and 2×Hieff PCR Master Mix (YEASEN, Shanghai, catalog number: 10136ES03) was used for nested PCR amplification to obtain the gene sequence of Raspberry palmata glycosyltransferase RcUGT78D1, 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 HiPure Gel 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 On LB plates with ampicillin antibiotics, overnight at 37°C. Single colonies were selected as templates, and colony PCR verification was performed using the above upstream and downstream sequences (SEQ ID NO.3 and SEQ ID NO.4) as primers. Positive clones were sent to Zhejiang Shangya Biotechnology Co., Ltd. for bacterial liquid sequencing. The nucleotide sequence of Raspberry palmata glycosyltransferase RcUGT78D1 was obtained as shown in SEQ ID NO.1.

[0037] The coding region of Raspberry palmata glycosyltransferase RcUGT78D1 is 1479 bp ( Figure 1 ), encoding 492 amino acids (amino acid sequence as shown in SEQ ID NO.2), with leucine (Leu) accounting for the largest proportion, followed by valine (Val) and serine (Ser), containing 55 acidic amino acids (Asp+Glu) and 49 basic amino acids (Arg+Lys), with a protein molecular weight of 54.93 kDa and a molecular formula of C 2493 H 3873 N 653 O 706 S 20The theoretical isoelectric point (Theoretical pI) was 6.09, the aliphatic amino acid index (Aliphatic index) was 93.92, the instability index (instability index) was 54.34, and the grand average of hydropathicity was 0.021. It was speculated that the palm raspberry glycosyltransferase RcUGT78D1 was an unstable non-hydrophilic protein.

[0038] The 313-457 positions of the protein sequence of R. palmatum glycosyltransferase RcUGT78D1 contain the conserved functional domain of UDP-glucoronosyl and UDP-glucosyl transferase (PF00201, E-value is 4.5×e -24 ). According to the pLoc-mPlant subcellular localization prediction software, the palm leaf raspberry glycosyltransferase RcUGT78D1 protein is predicted to be located on the cell membrane. The biological software TMHMM analysis found that there is a transmembrane region at the 176-193 site ( Figure 2 ).

[0039] Analysis of the SOPMA online biology website showed that the secondary structure of the palm raspberry glycosyltransferase RcUGT78D1 protein included alpha helix, random coil and extended strand, accounting for 40.04%, 45.93% and 14.02% respectively. Figure 3 ). The tertiary structure of the palm leaf raspberry glycosyltransferase RcUGT78D1 protein was predicted using the SWISS-MODEL online biological software. The homology with the UGT78D1 sequence of the Arabidopsis thaliana UGT78 subfamily reached 76.23%, the GMQE value was 0.87, and the arrow pointed to the UGT catalytic active site ( Figure 4 ). Further, using the plant UGT database website platform, it was found that the palm leaf raspberry glycosyltransferase RcUGT78D1 protein was clustered in the UGT78 subfamily, and the phylogenetic tree of RcUGT78D1 protein and 121 proteins in the Arabidopsis UGT78 subfamily was constructed ( Figure 5 ), the palm leaf raspberry glycosyltransferase RcUGT78D1 protein was clustered in the same branch as Arabidopsis UGT78D1 and UGT78D3. In view of the above results, it was named RcUGT78D1.

[0040] Example 2: Analysis of the expression pattern of the Raspberry palmata glycosyltransferase RcUGT78D1 gene

[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 upstream primers (SEQ ID NO.5) and downstream primers (SEQ ID NO.6) designed according to the RcUGT78D1 gene, and real-time fluorescence quantitative PCR amplification was performed using 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 to calculate the relative expression data of each sample using the 2-ΔΔCT method. The internal reference was the palm leaf rubus β-actin gene, the upstream primer was shown in SEQ ID NO.7, and the downstream primer was shown in SEQ ID NO.8.

[0042] By comparing the Raspberry genome at the chromosome level, it was found that the Raspberry glycosyltransferase RcUGT78D1 has 116 homologous proteins. Phylogenetic tree analysis showed that the glycosyltransferase RcUGT78D1 was clustered on the same small branch with LG04.3958, LG04.3361, LG07.63, and LG02.2782, indicating that they are closely related ( Figure 6 A in the figure). Gene expression analysis showed that among the five genes in the same branch, only the glycosyltransferase RcUGT78D1 gene was highly expressed in Raspberry fruit ( Figure 6 B).

[0043] Real-time fluorescence quantitative PCR verification showed that the palm leaf raspberry glycosyltransferase RcUGT78D1 gene was expressed to varying degrees in the roots, stems, leaves, flowers and fruits of palm leaf raspberry, with the lowest expression in the roots and the highest expression in the fruits. The expression level in the fruits was 3.17 times that in the roots ( Figure 7 ).

[0044] Furthermore, the expression levels of the glycosyltransferase RcUGT78D1 gene of R. palmatum at different fruit maturity stages (small green fruit, large green fruit, yellow fruit, and red fruit) were analyzed. The results showed that as the R. palmatum fruit gradually matured, the expression level of the glycosyltransferase RcUGT78D1 gene showed a trend of first increasing and then decreasing. The expression level was lowest in red fruit and highest in large green fruit. The expression level in large green fruit was 2.73 times that in red fruit ( Figure 8 ).

[0045] Example 3: Expression analysis of Raspberry palmata glycosyltransferase RcUGT78D1 gene under adverse stress

[0046] Through the PlantCare online software, it was found that there were multiple stress response elements in the promoter region of the glycosyltransferase RcUGT78D1 gene of Raspberry palm leaf, such as stress-response element (STRE), antioxidant response element (ARE), drought stress response element (MYBbinding site involved in drought-inducibility, MBS), low temperature responsive element (LTR), ABA response element (ABRE, ABA-responsive element), JA response element (TGACG motif), etc. It is speculated that the glycosyltransferase RcUGT78D1 gene is induced by adverse stresses such as drought and low temperature, and is involved in stress resistance physiological response.

[0047] Six-month-old Raspberry palm leaf tissue culture seedlings were treated in the culture medium, and normal (control group, CK) ABA (100 μM), drought (10% PEG), exogenous low temperature (4°C), salt stress (200mM NaCl), MeJA (100 μM) treatments were set respectively. After 6 hours of treatment, the tissue culture seedling samples were uniformly collected and quickly frozen in liquid nitrogen. Real-time fluorescence quantitative PCR was performed using upstream primers (SEQ ID NO.5) and downstream primers (SEQ ID NO.6) to detect the expression level of the glycosyltransferase RcUGT78D1 gene. The internal reference was the Raspberry 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.

[0048] Real-time fluorescence quantitative PCR detection found that the expression levels of glycosyltransferase RcUGT78D1 gene after treatment with ABA (100 μM), drought (10% PEG), exogenous low temperature (4°C), salt stress (200 mM NaCl), and MeJA (100 μM) increased by 11.09 times, 7.84 times, 2.41 times, 5.97 times, and 3.65 times, respectively, compared with the normal treatment (CK), indicating that the palm leaf raspberry glycosyltransferase RcUGT78D1 gene was significantly upregulated by adverse stress and stress-related hormone induction ( Fig. 9 ).

[0049] Example 4: Overexpression of glycosyltransferase RcUGT78D1 gene promotes the accumulation of total flavonoids and kaempferol-3-O-rutinoside

[0050] (1) Construction of overexpression vector of Raspberry palmata glycosyltransferase RcUGT78D1

[0051] According to the coding sequence of R. idaeus glycosyltransferase RcUGT78D1 (SEQ ID NO.1), upstream primers (SEQ ID NO.9) and downstream primers (SEQ ID NO.10) were set in the EcoR I and Hind III restriction sites of the plant expression vector pCAMBIA1301. The RcUGT78D1 coding region was amplified using the cDNA of immature fruit of R. idaeus foliata as a template, and the plant expression vector pCAMBIA1301 (Abcam, USA, catalog number: ab275753) was double-digested with EcoR I and Hind III. HD Cloning Kit (Takara, Dalian, Cat. No. 639648) was used to construct the RcUGT78D1 overexpression vector, and the overexpression recombinant vector obtained above was named pCAMBIA1301-RcUGT78D1 ( Fig.10 ).

[0052] (2) Transformation of recombinant plasmid pCAMBIA1301-RcUGT78D1 into Agrobacterium GV3101 (pSoup-p19)

[0053] The pCAMBIA1301-RcUGT78D1 was transformed into GV3101 (pSoup-p19) Agrobacterium using the heat shock method. The steps are as follows: 1 μg of pCAMBIA1301-RcUGT78D1 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. Add 700 μL of LB liquid medium without antibiotics and rotate at 100 rpm min. -1 , 28°C for 120 min. Spread LB medium (containing 50 mg mL -1 Kanamycin). Invert and culture at 28°C until colonies grow (about 2 days), and select positive clones for subsequent experiments.

[0054] (3) Agrobacterium-mediated genetic transformation of Raspberry palmata glycosyltransferase RcUGT78D1 gene

[0055] Transfer the positive clones of Raspberry palmata pCAMBIA1301-RcUGT78D1 into 100 mL LB liquid medium (containing 50 mg mL -1 Kanamycin, 180 rpm·min -1, 28 ℃ overnight culture (about 16h) until OD600 reaches 0.8. Centrifuge at 5000×g for 5min at room temperature, discard the supernatant to obtain the precipitate. Resuspend in 100mL infiltration buffer (10mM MES, 10mM MgCl2, 0.2mM acetosyringone, the balance is water, pH 5.7), centrifuge at 5000×g for 5min at room temperature, discard the supernatant to obtain the precipitate, adjust the OD600 to 0.8 with the above infiltration buffer again, stand at room temperature in the dark for 2h, and obtain RcUGT78D1 activation infiltration solution. Select palm leaf rubus callus with relatively consistent growth, culture in the dark for 2d, use the above RcUGT78D1 activation infiltration solution to infect the callus for 10min, culture in the dark at 23℃ for 24h, culture for 2d under alternating conditions of 12h light-12h dark (light intensity of 4000lx), and use 50mg mL -1 Positive callus tissues were screened by hygromycin B combined with fluorescence quantitative PCR, and Raspberry palm leaf callus tissues infected with pCAMBIA1301 empty vector (not connected to RcUGT78D1 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 palm leaf callus tissue powder to determine the contents of total flavonoids and kaempferol-3-O-rutinoside.

[0056] (4) Determination of the expression level of R. idaeus RcUGT78D1 in transgenic callus tissue

[0057] RNA was extracted from the transformed Raspberry palm leaf callus and reverse transcribed into cDNA for quantitative PCR detection of RcUGT78D1. The upstream primer for quantitative PCR of RcUGT78D1 gene 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.

[0058] (5) Determination of total flavonoids and anthocyanins in callus tissue of Rubus palmatus

[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.vmL 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] Take 0.2g of Rubus palmatus callus powder (passed through No. 3 sieve) in a conical flask and add 10mL of 0.1mol·L -1 After the solution of hydrochloric acid and ethanol was added, it was placed in a dark place at 60℃ for extraction. When the callus tissue turned completely white with naked eyes, 0.1 mol·L -1 15mL of hydrochloric acid ethanol solution was used to obtain the extraction solution. -1 Hydrochloric acid ethanol solution was used as a blank control, and the absorbance values ​​at wavelengths of 530nm, 620nm and 650nm were measured using a spectrophotometer according to the pH differential method. Calculation of anthocyanin absorbance OD λ =(OD 530nm -OD 620nm )-0.1×(OD 650nm -OD 620nm ), anthocyanin content (nmol·g -1 )=[(OD λ / ε)×(V / m)]×10 6 ; where ε is the molar extinction coefficient of anthocyanin 4.62×10 6 ; V is the volume of the extract (mL); m is the mass of the sample (g).

[0061] (6) Determination of kaempferol-3-O-rutinoside content in callus of Rubus palmatus

[0062] Take 1.0g palm leaf rubus callus powder (pass through No. 3 sieve), add 50mL of 70% methanol solution by volume, heat and reflux to extract for 1h, cool, make up the lost weight with 70% methanol solution by volume, shake well, 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.

[0063] 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).

[0064] The results showed that compared with the control group (EV), the expression level of RcUGT78D1 gene in Raspberry palm leaf callus overexpressing the glycosyltransferase RcUGT78D1 gene (OE-RcUGT78D1) was upregulated by 5.11 times ( Fig.11 (A)), the total flavonoid content in the control plant was 7.73 mg g -1 The total flavonoid content in R. palmatum overexpressing the RcUGT78D1 gene was 12.72 mg·g -1 , the total flavonoid content was 1.65 times that of the control ( Fig.11 In addition, the theoretical plate number of the kaempferol-3-O-rutinoside peak was 14715, which was baseline-separated from the adjacent impurity peaks, meeting the detection requirements of the Pharmacopoeia of the People's Republic of China (2020 Edition). The content of kaempferol-3-O-rutinoside in the control plant was 6.01 μg·g -1 Compared with the control group (EV), the content of kaempferol-3-O-rutinoside in R. idaeus callus overexpressing RcUGT78D1 gene was 19.46 μg·g -1 , which is 3.24 times that of the control ( Fig.11 (C)). The anthocyanin content in the control plant was 0.48 mg g -1 Compared with the control group (EV), the anthocyanin content in R. idaeus callus overexpressing RcUGT78D1 gene was 0.88 mg·g -1 , which is 1.81 times that of the control ( Fig.11 The above results showed that overexpression of glycosyltransferase RcUGT78D1 gene significantly promoted the accumulation of total flavonoids, kaempferol-3-O-rutinoside and anthocyanin in Raspberry fruit.

[0065] Example 5: Overexpression of the glycosyltransferase RcUGT78D1 gene improves the drought resistance, salt tolerance and low temperature tolerance of Rubus palmatus

[0066] There was no significant difference in the fresh weight of Raspberry callus overexpressing the glycosyltransferase RcUGT78D1 gene under normal conditions compared with the control group (EV); under drought conditions (20% PEG), the fresh weight of Raspberry callus overexpressing the glycosyltransferase RcUGT78D1 gene was 2.06 times higher than that of the control group; under salt stress conditions (150μM NaCl), the fresh weight of Raspberry callus overexpressing the glycosyltransferase RcUGT78D1 gene was 1.83 times higher than that of the control group. Under low temperature conditions (4℃), the fresh weight of Raspberry callus overexpressing the glycosyltransferase RcUGT78D1 gene was 1.48 times higher than that of the control group. The above results show that compared with the control group, the callus overexpressing the glycosyltransferase RcUGT78D1 gene can still maintain a higher fresh weight content (EV) under drought, salt stress and low temperature conditions. Fig.12 ), therefore, the drought resistance, salt tolerance and low temperature tolerance of plants transformed with RcUGT78D1 gene were significantly higher than those of wild plants.

[0067] Superoxide anion (O2 ·- ) content was not significantly different from that of the control (EV) under normal conditions; under drought conditions (20% PEG), the O2 ·- The content was 28.01% lower than that of the control; under salt stress (150 μM NaCl), the callus O2 of Raspberry palm leaf overexpressing glycosyltransferase RcUGT78D1 gene ·- The content was 37.03% lower than that of the control; under low temperature conditions (4°C), the callus O2 of Raspberry palm leaf overexpressing glycosyltransferase RcUGT78D1 gene ·- The above results show that compared with the control, the callus overexpressing the glycosyltransferase RcUGT78D1 gene can still maintain a lower O2 under drought, salt stress and low temperature conditions. ·- content( Fig.13 ), which helps to remove the damage caused by reactive oxygen species (ROS). Therefore, transgenic plants with the RcUGT78D1 gene can reduce O2 by enhancing the ability to remove ROS. ·- content, thereby promoting the transgenic plants to have stronger resistance to drought, salt and low temperature.

Claims

1. A palm raspberry glycosyltransferase RcUGT78D1, characterized in that Its amino acid sequence is shown in SEQ ID NO.

2.

2. A gene encoding the Raspberry glycosyltransferase RcUGT78D1 described in 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 glycosyltransferase RcUGT78D1 gene into the EcoR I and Hind III restriction sites of the pCAMBIA1301 vector. A genetically engineered bacterium containing the recombinant vector according to claim 4.

7. Use of overexpressing the gene described in claim 2 or 3 in promoting the accumulation of flavonoids in Rubus idaeus, wherein the flavonoids in Rubus idaeus are kaempferol-3-O-rutinoside and anthocyanidins.

8. Use of overexpressing the gene according to claim 2 or 3 in improving the tolerance of Rubus palmatus to drought, salt stress and low temperature.

9. Application of the gene according to claim 2 or 3 in improved breeding of Rubus idaeus, wherein the application is application of the glycosyltransferase RcUGT78D1 gene in genetic engineering breeding for increasing the content of flavonoids in Rubus idaeus and improving the tolerance of Rubus idaeus to drought, salt stress and low temperature, and the flavonoids in Rubus idaeus are kaempferol-3-O-rutinoside and anthocyanidins.

Images