A blackberry flavonol synthase gene RuFLS2 and its application

Abstract

The present invention discloses a blackberry flavonol synthase gene RuFLS2 and its application, which belongs to the field of genetic engineering technology. The present application successfully identified and isolated the RuFLS2 gene, whose ORF is 996bp long and encodes a protein composed of 331 amino acids. Spatiotemporal expression analysis showed that RuFLS2 is highly expressed in blackberry fruits, and the expression level reaches a peak when the fruit develops to reddish purple. Heterologous overexpression of tobacco found that the synthesis content of flavonol substances such as naringenin-7-O-glucoside, cyanidin, apigenin-7-glucoside, kaempferol-3-O-rutinoside, astragalin and quercetin increased, indicating that RuFLS2 is a key enzyme gene for regulating the synthesis of blackberry flavonol substances, and can increase the synthesis of flavonols in transgenic plants, and has broad application prospects in the field of plant medicine preparation technology.

Description

Technical Field

[0001] The present invention belongs to the technical field of genetic engineering, and more specifically relates to a blackberry flavonol synthase gene RuFLS2 and an application thereof. Background Art

[0002] Blackberry (Rubus spp.) is a perennial shrub of the genus Rubus in the Rosaceae family. It is native to North America and has a long history of cultivation in Europe and the United States. Blackberry was first introduced to China and promoted in 1986 by the Institute of Botany, Chinese Academy of Sciences, Jiangsu Province. It boasts advantages such as drought resistance, vigorous growth, and easy propagation. Its fruit is rich in nutrients such as carbohydrates, vitamin C, vitamin E, polyphenols, and flavonoids, and has high nutritional and medicinal value, including antibacterial, anti-inflammatory, antioxidant, and anti-aging properties. It has broad application prospects in the medical and healthcare fields.

[0003] Flavonoids are a class of polyphenolic compounds found in plant secondary metabolites, with a typical C6-C3-C6 carbon skeleton. Flavonoids can be divided into six subclasses: flavonoids, isoflavones, dihydroflavonoids, flavonols, flavanones, flavanols, and anthocyanidins. Flavonols can undergo modifications at multiple sites, including acylation, hydroxylation, and glycosylation. These modifications can coexist, resulting in diverse flavonol structures and a variety of biological activities, including antioxidant, antiviral, and anticancer properties. Furthermore, they protect against UV radiation damage and enhance the resistance of plant roots to drought stress. They play a crucial role in plant growth and development, serving as a key component in plant defense against external biotic and abiotic stresses.

[0004] Flavonol synthase (FLS) is a key enzyme in the flavonoid biosynthesis pathway, catalyzing the conversion of dihydroflavonols to flavonols such as quercetin and kaempferol, playing a crucial role in the flavonoid biosynthesis pathway. Dihydroflavonol 4-reductase (DFR) competes with FLS for the production of anthocyanidins, a substrate used for the synthesis of dihydroflavonols. This enzyme significantly influences the accumulation of anthocyanidins and flavonols in plants. DFR participates in the metabolic process that synthesizes colored anthocyanidins, while FLS catalyzes the production of colorless flavonols. Therefore, FLS expression significantly influences plant coloration. The FLS gene plays a key role in the white or yellow coloration of plant organs. Overexpression of the FLS gene in grape hyacinth (Muscari aucheri) results in white flower coloration. In plants, FLS is often encoded by multiple copies of genes, with varying copy numbers in different plants. Their expression levels vary significantly across tissues, metabolic periods, and developmental stages, affecting flavonol content. FLS genes have been identified and their functions studied in various plant species. While some FLS genes have similar functions, such as OcFLS1 and OcFLS2 in Ornithogalum caudatum, which both catalyze the conversion of dihydroflavonols to flavonols and possess flavanone 3-hydroxylase (F3H) activity, others have distinct functions. In Arabidopsis thaliana, FLS1 participates in flavonoid biosynthesis, while FLS3 catalyzes the production of avonols. Therefore, while the involvement of FLS in flavonol biosynthesis has been investigated, further research is needed to characterize their expression patterns and specific regulatory mechanisms in blackberry. Summary of the Invention

[0005] In view of the above problems existing in the prior art, the present invention aims to provide a blackberry flavonol synthase gene RuFLS2. Another technical problem to be solved by the present invention is to provide a specific application of the blackberry flavonol synthase gene RuFLS2.

[0006] In order to solve the above technical problems, the technical solutions adopted by the present invention are as follows:

[0007] A blackberry flavonol synthase gene RuFLS2, the nucleotide sequence of which is shown in SEQ ID NO.1.

[0008] The amino acid sequence of the expressed protein of the blackberry flavonol synthase gene RuFLS2 is shown in SEQ ID NO.2.

[0009] A vector containing the blackberry flavonol synthase gene RuFLS2.

[0010] The vector containing the blackberry flavonol synthase gene RuFLS2 is a plant expression vector.

[0011] Furthermore, the plant expression vector is PB1121-RuFLS2.

[0012] The blackberry flavonol synthase gene RuFLS2 is used to increase the amount of flavonols in plants.

[0013] Furthermore, the application of the blackberry flavonol synthase gene RuFLS2 in increasing the amount of flavonols in plants comprises the following steps:

[0014] 1) Construction of a vector encoding the blackberry flavonol synthase gene RuFLS2;

[0015] 2) transforming the constructed blackberry flavonol synthase gene RuFLS2 vector into plants or plant cells;

[0016] 3) Cultivate and screen transgenic plants with increased flavonol content.

[0017] In the application, the flavonol substances are one or more of naringenin-7-O-glucoside, cyanidin, apigenin-7-glucoside, kaempferol-3-O-rutinoside, astragaloside, and quercetin.

[0018] Compared with the prior art, the present invention has the following beneficial effects:

[0019] This application successfully identified and isolated the RuFLS2 gene. Its ORF is 996 base pairs long and encodes a hydrophobic, acidic protein composed of 331 amino acids. It belongs to the 2OG-Fe(II)_Oxygenase superfamily. Spatiotemporal expression analysis showed that RuFLS2 is highly expressed in blackberry fruit, reaching a peak expression level when the fruit develops to a reddish-purple color. Heterologous overexpression of RuFLS2 in tobacco revealed increased synthesis of flavonols, including naringenin-7-O-glucoside, cyanidin, apigenin 7-glucoside, kaempferol-3-O-rutinoside, astragalin, and quercitrin. Furthermore, overexpression of RuFLS2 increased the expression levels of NtF3H, NtFLS, NtDFR, and NtANS genes in transgenic tobacco, indicating that RuFLS2 is a key enzyme gene regulating the synthesis of flavonols in blackberry and plays an important role in the synthesis of flavonols. RuFLS2 is expected to be used in the cultivation of transgenic plants with high levels of flavonoids and phenolic compounds, and has broad application prospects in the field of plant drug preparation technology. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 This is a phenotypic diagram of blackberry 'Chester' fruit at different developmental stages;

[0021] Figure 2 Prediction diagram of RuFLS protein hydrophilicity and domain structure: (a) hydrophilicity of RuFLS1; (b) hydrophilicity of RuFLS2; (c) domain structure of RuFLS1; (d) domain structure of RuFLS2;

[0022] Figure 3 is a map of conserved domains or motifs in RuFLSs;

[0023] Figure 4 Figure 2 shows the expression patterns of RuFLS2 gene in different tissues of blackberry (a) and different fruit development stages (b). Different letters indicate significant differences (p < 0.05).

[0024] Figure 5 The expression patterns of flavonoid biosynthesis pathway genes in WT and RuFLS2 overexpressing tobacco, including: (a) RuFLS2; (b) NtFLS; (c) NtF3H; (d) NtDFR; (e) NtANS; (f) NtLAR;

[0025] Figure 6 The diagram shows the biosynthetic pathway of blackberry flavonoids;

[0026] Figure 7 Analysis diagram of differentially expressed metabolites (DEMs) in tobacco of the control group and FLS group: (a) the number of DEMs in positive and negative ion modes; (b): KEGG enrichment analysis diagram of DEMs in different pathways. DETAILED DESCRIPTION

[0027] The present invention will be further described below with reference to specific embodiments.

[0028] Example 1: Cloning and analysis of blackberry flavonol synthase gene

[0029] 1. Experimental Materials

[0030] The experimental material is the blackberry 'Chester' variety, which comes from the Baima Experimental Base in Lishui District, Nanjing (119°11′E, 31°36N). The area belongs to the subtropical monsoon climate zone, with an average annual temperature of 15.6°C, an annual rainfall of 1031.9mm, and an average sunshine of 50%. Among them, the average temperature from June to August is 27.1°C, the rainfall is 435.6mm, the soil is acidic clay, pH 5.52, containing 18.67g / kg of organic matter, 1.25g / kg of total nitrogen, 4.83mg / kg of available phosphorus, and 94.21mg / kg of available potassium. The roots, stems, leaves, flowers, fruiting branches and fruits of blackberries were picked separately as plant materials for expression analysis in different tissue parts. In addition, the sampling period of the fruit development stage was distinguished according to the color of the fruit, and green fruit, green-red fruit, red fruit, red-purple fruit and purple fruit ( Figure 1 All experiments were repeated at least three times. After collection, the samples were stored in liquid nitrogen and brought back to the laboratory and stored in a -80°C refrigerator for future use.

[0031] 2. Total RNA Extraction and cDNA Conversion

[0032] Total RNA was extracted using a universal plant total RNA rapid extraction kit (BioTeKe, Beijing, China) according to the manufacturer's instructions. Then, total RNA from different tissues and fruit development stages of blackberry was reverse transcribed into cDNA using a PrimeScript RT Master Mix (Perfect Real Time) kit (TaKaRa, Dalian, China). The total RNA was diluted to a concentration of approximately 300 ng / μL. -1 The cDNA was used as a template for subsequent expression analysis.

[0033] 3. Cloning of the Blackberry RuFLS Gene

[0034] First, based on the differentially expressed RuFLS unigene sequence screened from the blackberry transcriptome data, its open reading frame (ORF) and three-generation full-length transcript set were obtained by analysis using BioXM2.6 software for correction. Primers were designed using Oligo 6.0 software, and its ORF sequence was cloned using the high-fidelity PCR enzyme PrimeSTAR Max DNA Polymerase (TaKaRa, Dalian, China).

[0035] 50 μL PCR reaction system: Primer Star Max 25 μL, forward and backward primers 1 μL each, cDNA template 1 μL, ddH2O 22 μL. PCR program: 98°C 3 min; 35 cycles of 98°C 10 s, 55°C 5 s, 72°C 15 s; 72°C 3 min, hold at 4°C.

[0036] The amplified product was ligated with the vector according to the pClone007 Blunt Vector Kit (TSINGKE, Nanjing, China) and transformed into competent Escherichia coli. After incubation at 37°C, 250 rpm, for 6–8 h, potential positive bacterial cultures were verified by PCR using 2×T5 Super PCR Mix (Colony) (TSINGKE, Nanjing, China). Positive bacterial cultures were sent to Qingke Biotechnology Co., Ltd. for first-generation Sanger sequencing. Primer sequences are listed in Table 1.

[0037] Table 1 PCR primer information related to blackberry FLS2 gene and transgenic tobacco

[0038]

[0039]

[0040] Three genes potentially encoding FLSs proteins were identified from transcriptome data from blackberry at different developmental stages (https: / / doi.org / 10.1007 / s00468-022-02291-3). One of these genes had no ORF identified and verified, while the remaining two were identified and named RuFLS1 and RuFLS2. Structural analysis revealed that the ORFs of RuFLS1 and RuFLS2 were 1017 and 996 bp long, respectively (SEQ ID NO. 1), encoding polypeptides consisting of 338 and 331 amino acids (SEQ ID NO. 2), with stop codons of TAA and TAG, respectively.

[0041] 4. Bioinformatics Analysis

[0042] The protein sequence encoded by the blackberry FLSs gene was analyzed using DNAMAN software, and its physical and chemical properties and hydrophilicity were predicted using ExPASy software (https: / / web.expasy.org). The RuFLSs protein motif was predicted using the MEME online tool (https: / / meme-suite.org / meme / tools / meme). The RuFLSs protein domain structure, including transmembrane domains, functional domains, and conserved domains, was analyzed using the SMART online tool (http: / / smart.embl-heidelberg.de / ) and the NCBI online tool CDART (Conserved Domain Architecture Retrieval Tool) (https: / / www.ncbi.nlm.nih.gov / Structure / cdd / wrpsb.cgi#opennewwindow). Furthermore, the protein secondary structure was analyzed using the SOPMA online tool, and a three-dimensional model was constructed and verified using SWISS-MODEL (https: / / swissmodel.expasy.org / ). The RuFLSs protein sequences were aligned (similarity) using MEGAX and a phylogenetic tree was constructed using the neighbor-joining (NJ) method with 1000 bootstrap tests.

[0043] The physicochemical properties of RuFLS1 and RuFLS2 proteins were analyzed using ProtParam software, as shown in Table 2. The protein molecular weights were 39.03 kDa and 37.30 kDa, the theoretical isoelectric points were 5.72 and 5.44, the total number of negatively charged residues (Asp+Glu) was 42 and 44, the total number of positively charged residues (Arg+Lys) was 32 and 33, the instability index was 36.45 and 34.70, and the instability coefficients were both less than 40, indicating that they were stable proteins. The fat index was 81.36 and 88.91.

[0044] The total hydrophilicity of RuFLS1 and RuFLS2 was predicted by ProtScale online software to be -0.494 and -0.341 ( Figure 2 a, b), it is speculated that they are both hydrophobic acidic proteins.

[0045] Functional domain prediction of RuFLS1 and RuFLS2 revealed that both contain a DIOX_N functional domain and a 2OG-FeII_Oxy functional domain ( Figure 2c, d), indicating that it belongs to the α-ketoglutarate-dependent dioxygenase family. The DIOX_N domain of RuFLS1 is located between amino acids 38 and 152, and the 2OG-FeII_Oxy domain is located between amino acids 190 and 287. In contrast, the DIOX_N domain of RuFLS2 is located between amino acids 40 and 148, and the 2OG-FeII_Oxy domain is located between amino acids 192 and 292. The locations of these domains vary between RuFLS1 and RuFLS2. Both RuFLS1 and RuFLS2 lack transmembrane regions and are extramembrane proteins.

[0046] Table 2 Physicochemical properties of RuFLSs

[0047]

[0048] Amino acid alignment of FLS protein sequences from multiple plants using DNAMAN software revealed that RuFLS1 and RuFLS2 share high amino acid sequence identity with FLSs from almond (Prunus dulcis, XM_034342566.1), peach (Prunus persica, XM_007222519.2), peony (Paeonia lactifiora, KM259902.3), alpine strawberry (Fragaria vesca subsp., XM_011460471.1), and tea (Camellia sinensis, DQ198089.1). All of these proteins share key amino acid residues required for binding the substrates dihydroquercetin and ferrous ions. Mutations in RuFLS1 at multiple substrate binding sites suggest that some protein functions may have been lost during blackberry evolution. Differential gene expression analysis based on blackberry transcriptome data revealed no significant differential expression of the RuFLS1 gene, likely due to changes in amino acid residues at key sites in the RuFLS1 gene protein, suggesting that it may not be a key gene regulating the production of important secondary metabolites such as flavonoids. However, the RuFLS2 gene was a significantly differentially expressed enzyme gene, suggesting that it may play a key role in the accumulation of flavonoids during blackberry ripening. Therefore, the following examples further examined the function of the RuFLS2 gene to reveal its role in the synthesis and metabolism of flavonoid-related secondary metabolites in blackberry.

[0049] The conserved motifs of the two RuFLSs proteins were predicted. The results showed that both RuFLS1 and RuFLS2 had eight identical conserved motifs, and their positions in the sequences were basically the same ( Figure 3Motif 3 belongs to the DIOX_N superfamily and is a highly conserved N-terminal region of proteins with 2-oxoglutarate / Fe(II)-dependent dioxygenase activity. Motif 2 belongs to the Fe(II)-dependent oxygenase superfamily, and Motif 1 belongs to the 2OG superfamily. The combination of the latter two forms the 2OG-Fe(II)_Oxygenase superfamily, which is consistent with the domain structure prediction results and the conserved domain structure of the FLS protein family in plants. Two conserved motifs, "IGTKMN" and "YRNEGLR," are positioned differently in RuFLS1 and RuFLS2, which may contribute to the functional differences between RuFLS1 and RuFLS2.

[0050] The phylogenetic tree was constructed using MEGA using the NJ method. The results showed that RuFLS1 and RuFLS2 were closely related to F3H of Rosaceae species, including alpine strawberry (Fragaria vesca subsp.), sweet cherry (Prunus avium), almond (Prunus dulcis), peach (Prunus persica), and plum (Prunus mume). The FLS of Brassicaceae clustered into one branch, and the FLS of Liliaceae and Vitaceae clustered into one branch, which was consistent with the evolutionary relationship of species ( Figure 4 ), and the FLS proteins of apple (Malus domestica), which are also Rosaceae plants, are distantly related to RuFLS1 and RuFLS2 proteins. It is speculated that RuFLS1 and RuFLS2 are not single-function FLS enzymes, but may be bifunctional enzymes with both FLS and F3H activities.

[0051] Example 2: Detection of RuFLS2 expression levels in blackberry and transgenic tobacco seedlings

[0052] 1. Construction of plant overexpression vector

[0053] An overexpression vector was constructed using GATEWAY technology. The target fragment from the entry vector (linker-7) was transferred to the overexpression vector pBI121-des-3HA, driven by the strong 35S promoter, via LR reaction. First, the entry vector was linearized. The reaction system used the 50 μL PCR reaction for cloning the blackberry RuFLS gene described in Example 1. The DNA template was a positive bacterial suspension of FLS2, and Topo-F / R primers were commonly used linearization primers. The PCR product was then purified and recovered. An LR reaction was then performed to construct the target gene vector. The LR reaction system consisted of 50 ng of the entry vector, 75 ng of the expression vector, and 0.5 μL of LR Clonase II Plus enzyme mix. The total reaction volume was brought to 2.5 μL with ultrapure water. The reaction was incubated at 25°C for 1.5 hours. The reaction product was transformed into Escherichia coli strain Top10, picked, shaken, and sent to the company for sequencing verification. Positive clones were obtained and expanded in LB containing kanamycin for plasmid extraction.

[0054] 2. Transformation of Competent Agrobacterium

[0055] Take 150 ng of the plasmid to be transformed, add it to 100 μL of Agrobacterium GV3101 competent cells, mix gently, put it in an ice bath for 20 minutes, and quickly freeze it in liquid nitrogen for 60 seconds; then heat shock it at 37°C for 4 minutes and quickly put it in an ice bath for 2 minutes; add 800 μL of LB liquid medium (with rifampicin) and shake it at 100 rpm at 28°C for 3 hours; finally, centrifuge it at 4000 rpm for 3 minutes, keep a small amount of supernatant, mix it by pipetting, spread it on a plate, and incubate it at 28°C for 48 hours; select positive clones by PCR detection and store them at 4°C for later use.

[0056] 3. Tobacco infection and positive strain screening

[0057] The successfully verified bacterial solution was further expanded to 50 mL and the OD value of the bacterial solution was measured, preferably 0.5-0.8. The bacterial solution was transferred to a 50 mL centrifuge tube and centrifuged at 5000 rpm for 10 min. The supernatant was discarded and the bacterial solution was resuspended in 40 mL of MS0 (4.43 g / L MS, pH 5.8). Acetosyringone (AS) was added to a concentration of 20 mM. Select the vigorously growing wild-type tobacco tissue culture seedlings, select the leaves in the middle part, cut off the leaf margins and veins, and cut the rest into small pieces of about 1-1.5 cm. Immerse the leaves in the prepared bacterial solution for 6-12 minutes, shake them gently several times during the period, take them out, dry the bacterial solution, and spread them on MS1 ​​(4.43 g / L MS + 25 g / L sucrose + 5.6 g / L agar + 2.0 mg / L 6-BA + 0.1 mg / L NAA + 100 μmol / L AS, pH 5.8) medium, culture them in the dark at 25 ° C for 2-3 days, then light for 16 hours and dark for 8 hours, and continue to culture at 25 ° C. After callus tissue is formed, transfer them to MS2 medium (4.43 g / L MS + 25 g / L sucrose + 5.6 g / L agar + 2.0 mg / L 6-BA + 0.1 mg / L NAA + 100 mg / L Timentin + 100 mg / L Kanara, pH 5.8). 5.8). When shoots form on the callus, excise them and transfer them to MS rooting medium (4.43 g / L MS + 25 g / L sucrose + 5.6 g / L agar + 100 mg / L timentin, pH 5.8). After mature, leaves from independent lines are taken for RNA extraction and testing to verify and select positive clones.

[0058] 4. qRT-PCR Analysis Method

[0059] TB Green Premix Taq II (Tli RNA SEH Plus) (Takara, Dalian, China) was used for qRT-PCR. The 15 μL reaction system included 7.5 μL of TB Green Premix Taq II fluorescent dye, 1 μL of cDNA template, 0.6 μL of upstream and downstream primers, and 5.3 μL of ddH2O. The qRT-PCR program was as follows: 95°C for 2 min, 95°C for 10 s, 60°C for 10 s, 72°C for 15 s, Melt for 6 s, and 40 cycles. Real-time fluorescence quantitative PCR was performed using 18S as the internal reference gene for blackberry (Table 1) and NtActin as the internal reference gene for transgenic tobacco seedlings (Table 1). Finally, 2 -ΔΔCT Quantitative data analysis was performed.

[0060] 1. Expression levels of RuFLS2 in roots, stems, leaves, flowers, fruiting branches and fruits of blackberry

[0061] qRT-PCR analysis of the expression levels of RuFLS2 in roots, stems, leaves, flowers, fruiting branches and fruits of blackberry showed that the expression level of RuFLS2 was higher in fruits, while the expression levels in stems, flowers and leaves were generally lower. It is speculated that RuFLS2 may play a major role in blackberry fruit. Figure 4 a). Based on this, the expression level of RuFLS2 gene was detected at different fruit development stages. It was found that the expression level of RuFLS2 gene showed significant differences during fruit development. The expression level was low in the early stage of fruit development, increased sharply in the red-purple fruit stage, and then decreased rapidly, but it was still higher than that in the early stage of fruit development ( Figure 4 b).

[0062] 2. RuFLS2 expression level in transgenic tobacco and its effects on upstream and downstream genes

[0063] Three relatively vigorous transgenic tobacco lines were randomly selected for expression level detection, and it was found that RuFLS2 was highly expressed in all transgenic plants ( Figure 5 a). To explore the effects of RuFLS2 heterologous expression on key enzyme genes in tobacco flavonoid biosynthesis pathway, we detected the expression levels of tobacco NtFLS and the upstream gene NtF3H, as well as NtDFR, NtANS (Anthocyanidin synthase, ANS) and NtLAR (Leucoanthocyanidin reductase, LAR) on another branch ( Figure 5 The results showed that the expression level of NtFLS gene in transgenic tobacco was significantly increased ( Figure 5 b), the expression level change trend of the F3H gene in the three transgenic tobacco lines is consistent with that of FLS ( Figure 5 c). DFR and FLS guide different branches of the flavonoid biosynthesis pathway and are in a competitive relationship. The expression level of DFR changes in different transgenic lines in the opposite direction to that of FLS. The NtFLS gene was significantly upregulated in RuFLS-1 and RuFLS-2 lines than in RuFLS-3, while the NtDFR gene expression was significantly upregulated in RuFLS-1 and RuFLS-2 lines than in RuFLS-3. Figure 5 b, d), ANS is a downstream gene of DFR, and its expression level is also significantly upregulated in transgenic tobacco ( Figure 5 e). LAR is also the downstream gene of DFR ( Figure 5 f), but the expression levels of NtLAR varied greatly among transgenic lines. This suggests that heterologous expression of RuFLS2 upregulates the expression of tobacco NtFLS genes, NtDFR competes with NtFLS, and LAR gene expression is unaffected by RuFLS2.

[0064] Example 3: Determination of total phenolic and flavonoid contents during blackberry development

[0065] The total phenolic content was determined according to the method of Cheok et al. (Cheok, CY; Chin, NL; Yusof, YA; Law, CL Extraction of total phenolic content from Garcinia mangostana Linn. hull. I. Effects of solvents and UV-Vis spectrophotometer absorbance method. Food Bioprocess Technol. 2012, 5, 2928-2933. DOI: 10.1007 / s11947-011-0627-2) using a commercial Plant total phenol test kit (A143-1-1, Nanjing Jiancheng Bioengineering Institute). Fresh samples were ground into powder with liquid nitrogen, 0.1 g was weighed, and 2 mL of extraction solution (60% ethanol in water) was added. The samples were vortexed and extracted for 3 min at 60°C for 30 min and at 4000 rpm / min. -1 Centrifuge for 10 minutes, take the supernatant and place it in a centrifuge tube, add reagents as required, mix well, let it stand at room temperature for 10 minutes, measure the absorbance of each tube at a wavelength of 760nm, repeat the experiment three times, and calculate the total phenol content.

[0066] The flavonoid content (TFC) was determined by the aluminum chloride calorimetry (Mohamed, AF; Abeer, AA; Ahlam, AH; Sharifa, AB Color, flavonoids, phenolics and antioxidants of Omanihoney. Heliyon. 2018, 4, e00874. DOI: 10.1016 / j.heliyon.2018.e00874.) using a commercial Plant flavonoids test kit (A142-1-1, Nanjing Jiancheng Bioengineering Institute). 0.05 g of sample was weighed, washed with saline, dried, and then ground into powder with liquid nitrogen. 2 mL of 60% ethanol was added and the mixture was shaken at 60°C for 2 h at 10,000 rpm / min. -1 Centrifuge at room temperature for 10 minutes and collect the supernatant for analysis. Repeat the standard curve procedure for each sample three times, taking the mean value and substituting it into the standard curve to calculate the flavonoid content.

[0067] Anthocyanin content (TAC) was determined using the pH differential method (Cheng, FR; Cui, HX; Fang, JL; Yuan, K.; Jin, SH; Zhu, XT; Xu, Y. Content determination of functional composition and antioxidant activity from six purple plants. Pharmacogn. Mag. 2021, 17, 342-347; DOI: 10.4103 / PM.PM_203_20.) with slight modifications: 50 g of blackberry fruit was homogenized, 3 g of the homogenate was added to 30 mL of 50% ethanol extract, shaken well, ultrasonically treated at 60 Hz for 20 min at 35°C, centrifuged at 5000 rpm for 5 min, 0.5 mL of the supernatant was placed in a 10 mL centrifuge tube, 4.5 mL of pH 1.0 buffer was added, reacted at room temperature for 20 min, zeroed with double distilled water, and the absorbance was measured at 510 nm to calculate the anthocyanin content.

[0068] The results showed that the change trends of total phenols and flavonoids were similar, with an overall trend of a sharp decline in the early stage and a gentle trend in the later stage. There was a slight increase in the purple fruit stage, and the content in the green fruit stage was significantly higher than in other stages, with total phenols reaching 40.68 mg·g -1 FW, flavonoids reached 11.50 mg g -1 Anthocyanin accumulation is low in the early stage of fruit development, approaching zero, and increases significantly from red-purple fruit to purple fruit, reaching a maximum of 2.60 mg·g during the purple fruit stage. -1 FW, indicating that anthocyanins are mainly synthesized and accumulated in the late stage of fruit development (Table 3).

[0069] Table 3 Changes in total phenolic, flavonoid and anthocyanin contents in blackberry fruits at different developmental stages

[0070] Fruit color <![CDATA[Total phenols (mg·g -1 FW)]]> <![CDATA[Flavonoids (mg·g -1 FW)]]> <![CDATA[Anthocyanin (mg·g -1 FW)]]> Green fruit 40.68±1.00a 11.50±0.37a 0.02±0.002d Green and red fruit 11.91±1.05b 6.97±0.11b 0.05±0.01d Red fruit 3.28±0.03c 1.81±0.01c 0.39±0.01c Red-purple fruit 2.35±0.01c 1.80±0.12c 0.93±0.02b Purple fruit 3.10±0.01c 1.95±0.10c 2.60±0.03a

[0071] Note: Different letters indicate significant differences among different developmental stages of blackberry (p<0.05).

[0072] Data related to blackberry are presented as mean ± standard deviation (SD) using SPSS 24.0 statistical software (SPSS, Chicago, IL, USA). Mean values ​​were compared using one-way ANOVA, followed by Duncan's post hoc multiple comparisons to determine significant differences (P < 0.05) in flavonoid, anthocyanin, and total phenolic content and RuFLS expression levels among different blackberry tissues (flowers, roots, stems, leaves, and branches) and fruit at different stages.

[0073] Example 4: Metabolome Analysis of Wild-Type and Transgenic Tobacco

[0074] 1. LC-MS / MS Analysis of Wild-Type and Transgenic Tobacco

[0075] Six tobacco samples (three wild-type and three transgenic strains) were thawed at 4°C and added to pre-cooled methanol / acetonitrile / water solution (2:2:1, v / v). Low-temperature ultrasonic extraction was performed, and the supernatant was vacuum-dried. For mass spectrometry analysis, 100 μL of acetonitrile-water solution (acetonitrile:water = 1:1, v / v) was added for re-dissolution, and the supernatant was sampled and analyzed.

[0076] Samples were separated using an Agilent 1290 Infinity LC ultra-high performance liquid chromatography (UHPLC) system with a HILIC column. Samples were placed in an autosampler at 4°C throughout the analysis. Sequential analysis of samples was performed in a randomized order to minimize the effects of instrument signal fluctuations. Primary and secondary spectra were acquired using an AB Triple TOF 6600 mass spectrometer.

[0077] 2. Statistical Analysis

[0078] Regarding the metabolomic analysis of transgenic tobacco, unsupervised dimensionality reduction principal component analysis (PCA) was applied to all samples using the r package models (Warnes, GR; Bolker, B.; Lumley, T.; Johnson, RCGmodels: Various R Programming Tools for ModelFitting. Available online: https: / / CRAN.R-project.org / package=gmodels.) to preliminarily show the differences between samples in different groups. Partial least squares discriminant analysis (PLS-DA) was applied to the control group using the r package ropls (http: / / www.r-project.org / ) to better distinguish the metabolomic characteristics of the two groups by screening variables related to classification membership. Orthogonal projection discriminant analysis of latent structures (OPLS-DA) was applied to the control group using the r package models (http: / / www.r-project.org / ) and further validated by cross-validation and permutation test. The predictive ability (Q 2 ) value to verify the OPLS-DA model in metabolomics. 2 When the value is greater than 0.4, it is considered to be an acceptable prediction model. 2 When the value is greater than 0.9, it is considered a good prediction model. 2’ Value and Q 2’ The distribution of values.

[0079] PLS-DA can maximize the distinction between groups and is conducive to finding differential metabolites. This experiment shows that the metabolites of WT control group and FLS transgenic tobacco group in positive ion mode 2 X=0.678,R 2 Y=0.999,Q 2 Y=0.763; in negative ion mode R 2 X=0.427,R 2 Y=0.998,Q 2 The Y value was 0.688, consistent with our expectations for the experimental data model, indicating that the parameters of the PLS-DA model established in this study are reasonable and stable, suitable for metabolomics analysis. OPLS-DA is a derivative algorithm of PLS-DA. Both supervised pattern recognition multivariate statistical analyses found that FLS fell to the left of the confidence interval, while WT fell to the right, indicating that both models were able to effectively distinguish metabolites from transgenic and non-transgenic tobacco. Furthermore, permutation validation of the OPLS-DA model revealed good reliability. Subsequent model testing and differential metabolite screening were analyzed using OPLS-DA results.

[0080] The VIP value of multivariate statistical analysis OPLS-DA and the P value of univariate statistical analysis T test were combined to screen the differential metabolites between different comparison groups. The results showed that there were 47 extremely significant differential metabolites in the FLS transgenic tobacco group under positive ion mode. Compared with wild-type tobacco, 16 metabolites were upregulated and 31 metabolites were downregulated in RuFLS2 gene overexpression tobacco. There were 37 differential metabolites in the negative ion control mode, of which 18 metabolites were upregulated and 19 metabolites were downregulated ( Figure 7 a).

[0081] The Kyoto Encyclopedia of Genes and Genomes (KEGG) was used to analyze the metabolic pathways of the differential metabolites between the WT group and the transgenic tobacco FLS group, and 40 KEGG pathways were found to be enriched. The differential metabolites were mainly enriched in four categories ( Figure 7b) Metabolism (including 10 subcategories), genetic information processing (including 2 subcategories), environmental information processing (including 2 subcategories), and human diseases (including 2 subcategories). Among these, 19 differentially expressed metabolites were distributed in metabolic pathways (ko01110, Metabolic pathways), 6 in the biosynthesis of secondary metabolites (ko01110, Biosynthesis of secondary metabolites), and 5 in microbial metabolism in diverse environments (ko01120, Microbial metabolism in diverse environments).

[0082] To investigate the effects of RuFLS2 overexpression on flavonoid-related metabolites, we focused on analyzing differentially expressed metabolites related to flavonoid biosynthesis in transgenic tobacco plants (FLS) and WT plants. The results showed that six differentially expressed metabolites were detected in the flavonoid biosynthesis pathway of transgenic tobacco plants. Except for naringenin-7-O-glucoside, the levels of the other five metabolites were downregulated. One differentially expressed metabolite, cyanidin, was detected in the anthocyanin biosynthesis pathway, and its level was upregulated. A total of seven differential metabolites were detected in the flavonoid and flavonol biosynthesis pathways. Among them, the levels of apigenin 7-glucoside, kaempferol-3-O-rutinoside, astragalin, and quercetin were significantly upregulated, while the levels of luteolin 7-glucoside, quercetin 3-glucoside, and ligustroflavone were downregulated. This suggests that overexpression of the RuFLS2 gene consumes upstream flavonoids and significantly increases the flavonol content in the plants (Table 4).

[0083] Table 4 Major differential metabolites related to flavonoid biosynthesis in transgenic tobacco

[0084]

Claims

1. A blackberry flavonol synthase gene RuFLS2, the nucleotide sequence of which is shown in SEQ ID NO.

1.

2. The expressed protein of the blackberry flavonol synthase gene RuFLS2 according to claim 1, whose amino acid sequence is shown in SEQ ID NO.

2.

3. A vector containing the blackberry flavonol synthase gene RuFLS2 according to claim 1.

4. The vector containing the blackberry flavonol synthase gene RuFLS2 according to claim 3, characterized in that: The vector is a plant expression vector.

5. The vector containing the blackberry flavonol synthase gene RuFLS2 according to claim 4, characterized in that: The plant expression vector is PBI121-RuFLS2.

6. Use of the blackberry flavonol synthase gene RuFLS2 according to claim 1 in increasing flavonols in plants, wherein the plant is tobacco, and the flavonols are one or more of kaempferol-3-O-rutinoside, quercetin, and astragaloside.

7. The use of the blackberry flavonol synthase gene RuFLS2 according to claim 6 in increasing flavonols in plants, characterized in that: The following steps are involved: 1) Construction of a vector encoding the blackberry flavonol synthase gene RuFLS2; 2) transforming the constructed blackberry flavonol synthase gene RuFLS2 vector into plants or plant cells; 3) Cultivate and screen transgenic plants with increased flavonol content.

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