Tea tree rutin: UDP-glucosyltransferase gene and its application

Abstract

The present invention belongs to the field of tea tree genetic engineering; specifically, the present invention relates to the separation and analysis of tea tree rutin: uridine diphosphate-glucosyltransferase (RUG) gene, and also relates to the in vitro synthesis of quercetin 3-O-glucosyl rhamnosyl glucoside (Q-Glu-Rha-Glu) using the gene. The present invention discloses a tea tree rutin: uridine diphosphate-glucosyltransferase RUG gene, and the nucleotide sequence of the tea tree RUG gene is shown in SEQ ID No: 1. The present invention also discloses the use of the above-mentioned tea tree RUG gene: the in vitro controlled synthesis of quercetin 3-O-glucosyl rhamnosyl glucoside Q-Glu-Rha-Glu is achieved by prokaryotic expression of the tea tree RUG gene.

Description

Technical Field

[0001] The present invention belongs to the field of tea tree genetic engineering; specifically, the present invention relates to the separation and analysis of tea tree rutin:uridine diphosphate-glucosyltransferase (RUG) gene, and also relates to the in vitro synthesis of quercetin 3-O-glucosyl rhamnosyl glucoside (Q-Glu-Rha-Glu) by using the gene. Background Art

[0002] Flavonol glycosides are important secondary metabolites of tea, and they contribute significantly to the growth and development of tea plants and their resistance to adversity. At the same time, flavonol glycosides are often yellow or yellow-green powders with a mild bitter taste, which can enhance the bitterness of caffeine and have a significant impact on the color and taste of tea soup. Flavonol glycosides have many active hydroxyl groups and strong antioxidant capacity, and have many physiological effects such as anti-inflammatory, anti-cancer, anti-mutation and prevention of cardiovascular diseases.

[0003] Flavonol glycosides are glycoside phenolic compounds formed by the combination of flavonol aglycones and glycosides. The aglycones have a 2-phenylchromone (C6-C3-C6) basic skeleton. The A ring comes from acetyl CoA metabolism and has 5- and 7-hydroxyl groups. The B ring comes from the shikimic acid pathway and usually has 4′- or 3′, 4′- or 3′, 4′, 5′-hydroxyl groups. The C ring is a heterocyclic ring composed of 3 carbon atoms and one oxygen atom and has a 3-hydroxyl group. The 3-, 5-, and 7-hydroxyl groups of flavonols can all combine with glycosides to form glycosides, but the 3-glycoside is the main one. The common flavonol aglycones in tea are myricetin, quercetin, and kaempferol, and the common glycosides are glucose, galactose, and rhamnose. Due to the different aglycones, the number of sugar groups, and their binding order, there are many types of flavonol glycosides. Tea flavonol glycosides can be divided into myricetin glycosides, quercetin glycosides and kaempferol glycosides according to the aglycones; and can be divided into flavonol monoglycosides, disaccharides and triglycosides according to the number of bound sugar groups. The content of flavonol triglycosides and flavonol disaccharides in tea is higher than that of flavonol monoglycosides and flavonol aglycones, but the abundance of each flavonol glycoside monomer is less than 2%, which makes it difficult and costly to prepare flavonol glycosides from tea.

[0004] Glycosylation can improve the stability and solubility of flavonols, and help their transport and storage in the body, which plays an important role in the biological function of flavonols. Plant flavonol aglycones can undergo primary, secondary and tertiary transglycosylation reactions to generate corresponding flavonol monosaccharides, disaccharides and trisaccharides. For example, quercetin undergoes a 3-O transglucosylation reaction to generate quercetin-3-O-glucoside, and the monosaccharide is then subjected to a rhamnosylation reaction to generate rutin (i.e., quercetin 3-O-rhamnosyl glucoside), and the disaccharide is then subjected to a transglucosylation reaction to generate quercetin 3-O-glucosyl rhamnosyl glucoside. Studies have shown that the transglycosylation reaction of flavonols and flavonol glycosides is mainly catalyzed by glycosyltransferase family 1. When catalyzing the transglycosylation reaction, uridine diphosphate (UDP) activated sugars such as UDP-glucose, UDP-rhamnose and UDP-galactose are mainly used as sugar donors. Therefore, this type of enzyme is also called uridine diphosphate-glycosyltransferase (UGTs). Although scientists have started to study the characteristics of UGTs very early, the progress has been slow. The main reason is that there are many members in the UGTs family and it is difficult to separate and purify them. In recent years, with the continuous advancement of molecular biology technology, the research on plant UGTs has made great progress. Up to now, genes such as UGT75E1, UGT75E2, UGT75E3, UGT73A17, UGT84A22, UGT78A14, UGT78A15, UGT73A20, UGT72AM1, UGT75L12, UGT79B28 and F3GalT in tea trees have been isolated and functionally analyzed. Among all the genes whose functions have been identified, most of the genes encode proteins that can catalyze the transglycosylation reaction of flavonols to produce flavonol monosides, and a few genes (such as UGT79B28) encode proteins that can catalyze the transglycosylation reaction of flavonol monosides to produce flavonol disaccharides. The flavonol glycosides in tea trees are mainly flavonol disaccharides (such as rutin and kaempferol-3-O rhamnosyl-glucoside) and flavonol trisaccharides (such as Q-Glu-Rha-Glu). To date, there has been no report on the isolation, functional identification and application of genes that catalyze the transglycosylation reaction of flavonol disaccharides to produce flavonol trisaccharides in tea trees. Summary of the invention

[0005] The technical problem to be solved by the present invention is to provide a gene sequence of tea tree rutin:uridine diphosphate-glucosyltransferase (RUG) and the protein encoded by it, and the in vitro controlled synthesis of quercetin 3-O-glucosyl rhamnosyl glucoside (Q-Glu-Rha-Glu) can be achieved through the prokaryotic expression of the gene.

[0006] In order to solve the above technical problems, the present invention provides a tea plant RUG gene having a nucleotide sequence shown in SEQ ID No: 1.

[0007] The present invention also provides a protein encoded by the above RUG gene, which has an amino acid sequence shown in SEQ ID NO: 2 (the terminator is not listed).

[0008] The present invention also provides the use of the RUG gene: the in vitro controlled synthesis of quercetin 3-O-glucosyl rhamnosyl glucoside (Q-Glu-Rha-Glu) is achieved through the prokaryotic expression of the tea tree RUG gene.

[0009] As a further improvement of the use of the tea tree RUG gene of the present invention: the exogenously expressed RUG gene encoding product is used to catalyze the glycosyl transfer reaction between rutin and UDP-glucose, and quercetin 3-O-glucosyl rhamnosyl glucoside Q-Glu-Rha-Glu is prepared by in vitro synthesis.

[0010] That is, the gene expression product of the present invention can catalyze the transglucosylation reaction of rutin and UDP-glucose to achieve the controlled synthesis of Q-Glu-Rha-Glu in vitro.

[0011] The present invention also provides a method for synthesizing Q-Glu-Rha-Glu by expressing RUG using prokaryotic engineering bacteria, comprising transforming Escherichia coli with the tea tree RUG gene (SEQ ID No: 1), and using the induced expressed RUG to catalyze the reaction of rutin and UPD-glucose to achieve the synthesis of Q-Glu-Rha-Glu.

[0012] As an improvement on the in vitro synthesis method for preparing Q-Glu-Rha-Glu of the present invention: after the tea tree RUG gene (SEQ ID No: 1) is expressed and purified in an Escherichia coli prokaryotic expression system, rutin and UDP-glucose are catalyzed to synthesize Q-Glu-Rha-Glu at 30°C, a phosphate buffer system, and pH 8.0, and the Q-Glu-Rha-Glu with a purity of ≥80% is prepared by extraction with ethyl acetate and freeze-drying.

[0013] The present invention mainly aims at the practical problems such as the difficulty in preparing flavonol trisaccharide and the lack of reports on genes related to catalyzing the synthesis of flavonol trisaccharide. Through the screening and cloning of differentially expressed genes in the transcriptomes of different tea leaves with obvious differences in flavonol trisaccharide content, and the analysis of the activity of enzymes expressed by prokaryotic expression of exogenous genes, the role of the RUG gene encoding product of the present invention in catalyzing the synthesis of Q-Glu-Rha-Glu is clarified, and a method for catalyzing the synthesis of Q-Glu-Rha-Glu by expressing exogenous proteins using transgenic engineering bacteria is developed.

[0014] The present invention is realized by adopting the following technical scheme: transcriptome differential gene screening and flavonol glycoside metabolite association analysis of tea tree varieties with higher flavonol triglycoside abundance at different leaf positions are adopted to obtain a candidate gene RUG related to Q-Glu-Rha-Glu synthesis, and the RUG gene is cloned by reverse transcription-polymerase chain reaction (RT-PCR) and sequencing analysis, and the RUG gene has a nucleotide sequence shown in SEQ ID No: 1, and can encode a RUG protein, which has a sequence shown in SEQ ID No: 2.

[0015] The present invention also provides the catalytic properties of the prokaryotic expression product of the RUG gene of tea tree, proving that the RUG gene encoding product of the present invention can catalyze rutin and UDP-glucose to synthesize Q-Glu-Rha-Glu, and provides an optimized method for synthesizing Q-Glu-Rha-Glu in vitro using RUG expressed by prokaryotic bacteria.

[0016] The specific technical steps for implementing the present invention are as follows:

[0017] 1. Cloning and sequence analysis of RUG gene in tea plant

[0018] In spring, the first, second, third and fourth leaves under the buds of small and medium-leaf tea varieties with high abundance of flavonol trisaccharides were collected, and the samples from each leaf position were divided into two parts. One part was prepared as a fixed sample by steaming at 100°C for 2 minutes and drying at 80°C until the moisture content of the sample was ≤5%. The composition and content of flavonol glycosides were analyzed by UPLC-DAD-MS / MS to obtain phenotypic data of different samples; the other part was placed in liquid nitrogen and fully ground, and the total RNA was extracted by TRIZOL method / total RNA was extracted by Polysaccharide and Polyphenol Plant RNA Extraction Kit (Hangzhou Easyside Biotechnology Co., Ltd.) (refer to the kit manual for operation), and the total RNA of each leaf position sample was obtained. The RNA quality and concentration were confirmed by electrophoresis and ultraviolet analysis to meet the requirements of subsequent experiments. Using the total RNA of each leaf position as the starting material, the Ultra TM Directional RNA Library Prep Kit for The kit (New England Biolabs, Inc.) was used to separate mRNA and fragment it, and the transcriptome library was constructed through double-stranded cDNA synthesis, cDNA end repair and A addition, NEBNext Adaptor adapter ligation, selection of specific length fragment recovery, PCR amplification and enrichment. The specific operation refers to the relevant instructions of the kit. The constructed transcriptome library was entrusted to a sequencing company for sequence determination, assembly and gene differential expression analysis using the Illumina HiSeq 2500 platform.

[0019] The differentially expressed genes were clustered by weighted correlation network analysis (WGCNA) and associated with phenotypic data such as the content of flavonol trisaccharides in each sample to screen out RUG candidate genes involved in Q-Glu-Rha-Glu synthesis. Primers for cloning the full-length open reading frame (ORF) of the gene were designed based on the sequence of the RUG candidate gene obtained by transcriptome sequencing (NO1 in Table 1, with an expected product length of 1410 bp); RT-PCR was used to amplify the sequence of the candidate gene ORF ( Figure 1 ), amplified product inserted 18-T vector construction containing RUG gene recombinant vector 18-T-RUG was used to transform JM109 competent Escherichia coli. After blue-white spot screening, white spots were picked for amplification, and colony PCR was performed with M13 universal primers (NO2 in Table 1). The positive clones that met the growth conditions on ampicillin-resistant medium were entrusted to Shanghai Shenggong Biotechnology Co., Ltd. for sequencing using the Sanger sequencing method. EditSeq in the DNAStar software package was used to deduce the RUG amino acid sequence encoded by the ORF, and the nucleotide sequence and amino acid sequence were compared with the GenBank database of the American Center for Biotechnology Information using Blast (www.blast.ncbi.nlm.nih.gov / Blast.cgi). It was found that the RUG gene encoding product cloned by the present invention is a member of the glycosyltransferase family, and has the highest homology with a tea tree UDP-glycosyltransferase 91Q1 gene (Camellia sinensis UDP-glycosyltransferase 91Q1, CsUGT91Q1) with unknown function in the database, that is, the nucleotide sequence (number KP682355.1) and amino acid sequence (number ALO19883.1) of the present invention have 98.37% and 98.51% homology with CsUGT91Q1, respectively.

[0020] 2. RUG gene function analysis

[0021] According to the RUG gene sequence, a prokaryotic expression primer containing the complete ORF was designed (NO3 in Table 1, with an estimated product length of 1410 bp) to construct a recombinant vector containing the RUG gene. 18-T-RUG was used as a template to amplify the complete RUG ORF by PCR reaction, and the pCold-TF vector was purchased (TF refers to the trigger factor, i.e. Factor, a prokaryotic ribosome-binding partner protein that promotes the co-translational folding of nascent peptide chains), the pCold-TF vector was linearized by restriction endonuclease Eco53KI, the RUG complete ORF amplification product and the linearized pCold-TF vector were exchanged and recombined with the homologous recombination ligase to form a circular pCold-TF-RUG, and then transformed into competent Escherichia coli DH5α. After ampicillin resistance screening, positive clones were picked for colony PCR using pCold-TF universal primers (NO4 in Table 1), and the positive clones were entrusted to Shanghai Shenggong Biotechnology Co., Ltd. for sequencing verification; the recombinant plasmid pCold-TF-RUG after sequencing verification was extracted and transformed into competent Escherichia coli Origami (DE3), the positive clones after sequencing verification were expanded, and the recombinant protein expression was induced by isopropyl-β-D-thiogalactoside (IPTG) under low temperature conditions. SDS-PAGE analysis showed that the target gene RUG was highly expressed and highly soluble ( Figure 2 ). The fusion recombinant protein was purified by nickel column adsorption using a His tag protein purification kit, and TF was removed by HRV 3C protease digestion to obtain purified RUG protein. For specific operations, refer to the instructions of the relevant plasmids and kits. Using quercetin, quercetin 3-O-glucoside, rutin, kaempferol, kaempferol-3-O-glucoside, kaempferol-3-O-rhamnose-glucoside, etc. as acceptors and UDP-glucose, UDP-rhamnose, UDP-galactose, etc. as donors, the catalytic specificity analysis of RUG protein was carried out, and it was found that RUG could only catalyze the glycosyl transfer reaction between rutin and UDP-glucose to generate Q-Glu-Rha-Glu( Figure 3 ). At the same time, the study also found that the prokaryotic expressed RUG recombinant protein can effectively catalyze rutin and UDP-glucose to synthesize Q-Glu-Rha-Glu regardless of whether it has been digested with HRV 3C protease to remove the TF factor.

[0022] Furthermore, the antisense oligonucleotide gene silencing experiment proved that the RUG gene encoding product catalyzed the synthesis of Q-Glu-Rha-Glu in tea plants. Specifically, the primers RUG.expF and RUG.expR (NO5 in Table 1) and the actin ACT reference gene primers ACT.expF and ACT.expR (NO6 in Table 1) for RUG gene expression analysis were designed using PrimerSelection software, and the RUG gene-specific antisense oligonucleotide AsODN and the control sense oligonucleotide sODN (NO7 in Table 1) were designed using RNAfold software (https: / / sfold.wadsworth.org / cgi-bin / index.pl). The second leaf under the bud of 'Fuding Dabaicha' with the same tenderness was harvested, and the AsODN and the control sODN were respectively prepared with sterile water. Each leaf was injected with the AsODN or control sODN solution using a sterile syringe, and then immersed in a solution containing the corresponding oligonucleotide (NO5 in Table 1) and rutin and UDP-glucose, and cultured at 25°C under light for 24-36h. After the culture, the expression of RUG was detected by fluorescence quantitative PCR, and the composition of flavonol glycosides was analyzed by UPLC-DAD-MS / MS. It was found that compared with the injection of control sODN, the expression of RUG gene in the leaves of tea plants was significantly downregulated after the RUG gene was silenced by AsODN, and the synthesis of Q-Glu-Rha-Glu was significantly inhibited ( Figure 4 ). It was proved that the isolated tea tree RUG encoding product can catalyze rutin and UDP-glucose to synthesize the corresponding quercetin trisaccharide in the tea tree.

[0023] It can be seen that the RUG gene is the key functional gene for the synthesis of Q-Glu-Rha-Glu flavonol trisaccharide in tea trees, and has an important influence on the synthesis of tea flavonol glycoside flavor substances.

[0024] The primers mentioned above are specifically shown in Table 1 below:

[0025] Table 1

[0026]

[0027]

[0028] 3. Application of RUG Gene

[0029] After the Origami (DE3) engineered bacteria containing the recombinant plasmid pCold-TF-RUG were propagated, the recombinant protein expression was induced by IPTG at low temperature. The engineered bacteria were broken by lysozyme ultrasound, and RUG was purified by nickel column. After mixing with rutin and UDP-glucose, the product formation under different temperature, buffer system and acidity conditions was compared. It was found that the catalytic efficiency was highest under the conditions of temperature 30°C, phosphate buffer system and pH 8.0 ( Figure 5 ). After reacting for 4-8 hours under this condition, the reaction was terminated by heating at 90°C, and an equal volume of ethyl acetate was added to the mixture for extraction twice. The ethyl acetate layers were combined, and the ethyl acetate was removed by reduced pressure distillation at 45°C. The paste was transferred to a culture dish and freeze-dried to obtain a dry powder, in which the Q-Glu-Rha-Glu content exceeded 80%. At the same time, the tea tree CsUGT91Q1 gene in the database was induced to express using the same vector and engineering bacteria, and induced to express under the same low-temperature induction conditions, and subjected to the same protein purification and enzymatic reaction under optimized conditions, and then subjected to the same extraction, distillation and drying operations, and the Q-Glu-Rha-Glu content in the obtained dry powder was less than 38%.

[0030] It should be noted that:

[0031] The use of the gene CsUGT91Q1, which has a high homology to the RUG gene of the present invention, has not been clearly reported. The present invention has proved through experiments that although CsUGT91Q1 can catalyze the generation of Q-Glu-Rha-Glu, its catalytic efficiency is far lower than that of the tea tree RUG gene of the present invention, and there is no report on the specific and efficient catalytic function of CsUGT91Q1 on other substances, and rutin and UDP-glucose are not its optimal reaction substrates.

[0032] The gene PgUGT94Q4 is used for catalyzing the glycosylation reaction between kaempferol and UDP-glucose to generate kaempferol-3-O-glucoside, which is completely different from the catalytic substrate of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 This is the RT-PCR electrophoresis diagram of the open reading frame of the RUG gene of tea plant;

[0034] Note: M stands for 2000bp DNAMaker, and lane 1 is the tea tree RUG gene.

[0035] Figure 2 It is the effect of tea tree RUG gene induction expression and nickel column purification in the prokaryotic expression system;

[0036] Figure 2 middle:

[0037] A is the unpurified SDS-PAGE image of the protein induced by RUG gene in tea plant, and B is the purified image of the protein induced by RUG gene in tea plant;

[0038] In A, M is the 250 kD protein Maker, lane 1 is the soluble protein after induction, lane 2 is the total protein after induction, and lane 3 is the uninduced protein;

[0039] In B, lane 1 is the unpurified soluble protein, lane 2 is the wash solution during the purification process, and lane 3 is the purified soluble protein (the target protein is about 105 kDa in size, which contains a 53 kDa stimulating factor that promotes the co-translational folding of the nascent peptide chain and a 52 kDa tea tree RUG gene encoding product).

[0040] Figure 3 It is the UPLC-DAD-MS / MS detection result of the recombinant RUG catalytic reaction product;

[0041] Figure 4 is the result of RUG gene expression and Q-Glu-Rha-Glu content detection after antisense oligonucleotide treatment of tea leaves;

[0042] Figure 4 middle:

[0043] A is the RUG gene expression level in tea leaves after treatment with antisense oligonucleotide AsODN and sense oligonucleotide control sODN; B is the Q-Glu-Rha-Glu content in tea leaves after treatment with antisense oligonucleotide AsODN and sense oligonucleotide control sODN; different letters represent significant differences when p<0.05.

[0044] Figure 5 It is the result of optimization of catalytic temperature and acidity of recombinant RUG;

[0045] Figure 5 middle:

[0046] A is the effect of pH and buffer system on enzyme activity, where Buffer 1 is 50 mM citrate buffer, Buffer 2 is 50 mM phosphate buffer, and Buffer 3 is 50 mM Tris-HCl buffer; B is the effect of temperature on enzyme activity. DETAILED DESCRIPTION

[0047] Example 1

[0048] On April 20, the first, second, third and fourth leaves under the bud of the tea variety 'Zhe Nong 901' with a high abundance of flavonol triglycosides were harvested, and three biological replicates were set for each leaf position sample, each replicate sample weighed about 30 g and was divided into two portions. One portion (about 28 g) was used to prepare fixed samples, which were steam-sterilized at 100°C for 2 minutes and dried at 80°C until the sample moisture content was ≤5%. The flavonol glycoside composition and content of samples from different leaf positions were obtained by UPLC-DAD-MS / MS analysis. The other portion (about 2 g) was removed from the leaf veins and fully ground in liquid nitrogen. The total RNA was extracted using the TRIZOL method / the polysaccharide and polyphenol plant RNA extraction kit (Hangzhou Easyside Biotechnology Co., Ltd.) was used to extract the total RNA (refer to the kit manual for operation). The total RNA samples of each leaf position were obtained. After 1.5% agarose electrophoresis (100 v, 30 min), the RNA was found to be intact and not degraded. Ultraviolet analysis showed that the RNA OD260 / OD280 extracted from the samples of each leaf position was 1.9-2.0 and the concentration was about 800 μg / ml, which met the requirements of subsequent experiments. Using the total RNA of each leaf position as the starting material, the Ultra TM Directional RNA Library Prep Kit for The kit (New England Biolabs, Inc.) was used to separate mRNA and fragment it, and the transcriptome library was constructed through double-stranded cDNA synthesis, cDNA end repair and A addition, NEBNext Adaptor adapter ligation, selection of specific length fragment recovery, PCR amplification and enrichment. The specific operation refers to the relevant instructions of the kit. The constructed transcriptome library was entrusted to a sequencing company for sequence determination, assembly and gene differential expression analysis using the Illumina HiSeq2500 platform.

[0049] The differentially expressed genes were clustered by weighted correlation network analysis (WGCNA) and correlated with the content of flavonol trisaccharides in each sample. The expression of more than 50 genes was significantly correlated with the Q-Glu-Rha-Glu content of each sample, with a correlation coefficient of 0.95. The above 50 genes were compared and annotated, and a glycosyltransferase gene numbered 3991 was found. This gene was listed as a RUG candidate gene that catalyzes the synthesis of Q-Glu-Rha-Glu. Primers for cloning the open reading frame (ORF) were designed based on the RUG candidate gene sequence obtained by transcriptome sequencing (NO1 in Table 1, with an expected product length of 1410 bp). The first chain of cDNA was synthesized using a reverse transcription kit using the extracted total RNA as a template, and the candidate gene ORF ( Figure 1 ), for specific operations, refer to the instructions of the reverse transcription kit.

[0050] Insert the PCR amplification product into 18-T vector construction containing RUG gene recombinant vector 18-T-RUG, and transformed JM109 competent Escherichia coli, and streaked, picked white spots and amplified them in LB liquid medium for 12h, and colony PCR was performed with M13 universal primers (NO2 in Table 1) (the formula and reaction parameters are shown in Table 2), and the positive recombinant bacteria that met the growth conditions on ampicillin resistance medium were entrusted to Shanghai Shenggong Biotechnology Co., Ltd. for sequencing by Sanger sequencing. The results showed that the gene ORF was 1410bp long (SEQ ID No: 1). EditSeq in the DNAStar software package was used to deduce the RUG amino acid sequence encoded by the ORF, which contained 469 amino acid residues (SEQ ID No: 2) and had a relative molecular weight of about 52kDa. The Blast (www.blast.ncbi.nlm.nih.gov / Blast.cgi) tool was used to compare the RUG amino acid sequence with the GenBank database of the American Center for Biotechnology Information, proving that the cloned RUG gene encoding product was a member of the glycosyltransferase family. The comparison also shows that the sequence of the present invention has the highest homology with a tea tree UDP-glycosyltransferase 91Q1 gene (Camellia sinensis UDP-glycosyltransferase 91Q1, CsUGT91Q1) with unknown function in the database, that is, the homology of the present invention with the CsUGT91Q1 nucleotide sequence (number KP682355.1) and amino acid sequence (number ALO19883.1) is 98.37% and 98.51%, respectively.

[0051] Table 2

[0052]

[0053]

[0054] Example 2

[0055] According to the RUG gene sequence, a prokaryotic expression primer containing the complete ORF was designed (NO3 in Table 1, with an estimated product length of 1410 bp). The recombinant vector containing the RUG gene obtained in Example 1 was used. 18-T-RUG was used as a template, and the complete RUG ORF with an expression vector restriction site linker was amplified by PCR reaction (the formula and reaction parameters are shown in Table 3). The pCold-TF vector was purchased (TF refers to the trigger factor, which is a prokaryotic ribosome binding partner protein that can promote the co-translational folding of the nascent peptide chain). The pCold-TF vector was linearized by digestion with the single-cutting endonuclease Eco53KI (for specific operations, refer to the endonuclease instructions). The RUG complete ORF amplification product and the pCold-TF vector were exchanged and recombined with the pCold-TF vector under the action of homologous recombination ligase using the In-Fusion HD Cloning Kit one-step cloning kit to form a circular pCold-TF-RUG. For specific operations, refer to the relevant instructions of the kit. The ligation product was transformed into competent Escherichia coli DH5α, and then streaked on an LB solid plate containing ampicillin, and the positive clones that met the growth conditions on the ampicillin resistance medium were picked and propagated in LB liquid medium for 12 hours. Colony PCR was performed with the universal primers of the pCold-TF vector (NO4 in Table 1), and the positive clones were entrusted to Shanghai Shenggong Biotechnology Co., Ltd. for sequencing verification; the positive recombinant bacteria verified by sequencing were propagated in LB liquid medium, the recombinant plasmid pCold-TF-RUG was extracted, transformed into competent Origami (DE3), and streaked on an LB solid plate containing ampicillin, and the positive clones that met the growth conditions on the ampicillin resistance medium were picked and propagated in LB liquid medium. The positive clones were selected and the inserted genes were sequenced by Sanger sequencing using the universal primers for the pCold-TF vector (NO4 in Table 1). The positive colonies with correct sequencing were the Origami (DE3) engineering bacteria containing the recombinant plasmid pCold-TF-RUG. The Origami (DE3) engineering bacteria containing the recombinant plasmid pCold-TF-RUG were propagated in LB liquid medium and induced to express the recombinant protein by isopropyl-β-D-thiogalactoside (IPTG) at 16°C. SDS-PAGE analysis showed that the target gene RUG fused with the TF factor was highly expressed and highly soluble ( Figure 2 ).

[0056] Description: The bacterial solution of Origami (DE3) engineered bacteria containing the recombinant plasmid pCold-TF-RUG that was expanded in LB liquid medium but not induced by IPTG was used as the uninduced protein, the bacterial solution induced by IPTG after expansion was used as the induced total protein, the supernatant after the total protein was broken on ice with lysozyme and centrifuged at low temperature was the induced soluble protein (i.e., the unpurified soluble protein), the unpurified soluble protein was adsorbed on a nickel column using a His tag protein purification kit (MCE Company), other proteins could not bind to the nickel column and were therefore removed in the washing step, the liquid flowing out of the washing step was the washing liquid in the purification process, and the recombinant protein obtained after eluting the nickel column was the purified soluble protein (for specific operations, refer to the kit instructions).

[0057] The soluble fusion recombinant protein induced by IPTG was adsorbed on a nickel column using a His tag protein purification kit, and purified recombinant protein was obtained by elution. The recombinant protein was treated with HRV 3C protease to cut off the TF factor and RUG, and then removed the TF factor after nickel column adsorption to obtain purified RUG protein. The specific operation refers to the instructions of the relevant plasmid, protease and kit. The purified RUG protein was dissolved in 50mM phosphate buffer (pH8), and 1mM quercetin, quercetin 3-O-glucoside, rutin, kaempferol, kaempferol-3-O-glucoside, kaempferol-3-O-rhamnose-glucoside and other receptor solutions, as well as 10mM UDP-glucose, UDP-rhamnose, UDP-galactose and other donor solutions were prepared. The RUG protein was mixed with each receptor and donor solution, respectively, so that the RUG protein concentration in the system was 0.05mg / ml, the receptor concentration was 160μM, and the donor concentration was 2mM. The reaction system was shaken in a water bath shaker at 35°C for 2 h, and methanol was added to terminate the reaction. At the same time, the protein expressed by the pCold-TF empty plasmid without RUG was mixed with the receptor and donor and reacted in a water bath and added with the stop solution as a control. The catalytic specificity analysis of RUG protein was carried out. The results showed that RUG could only catalyze the glycosyl transfer reaction between rutin and UDP-glucose and generate Q-Glu-Rha-Glu( Figure 3 ). At the same time, the study also found that the prokaryotic expressed RUG recombinant protein can effectively catalyze rutin and UDP-glucose to synthesize Q-Glu-Rha-Glu regardless of whether it has been digested with HRV 3C protease to remove the TF factor.

[0058] Note: When the acceptor is quercetin, quercetin 3-O-glucoside, kaempferol, kaempferol-3-O-glucoside, and kaempferol-3-O-rhamnose-glucoside, no matter which of the above donors is, the results are that RUG protein cannot catalyze the glycosyl transfer reaction between the acceptor and the donor, no new glycoside is generated, and there is no enzyme activity for the above acceptors;

[0059] When the donor is UDP-rhamnose or UDP-galactose, no matter which of the above acceptors is used, the result is that the RUG protein cannot catalyze the glycosyl transfer reaction between the donor and the acceptor, no new glycoside is generated, and there is no enzyme activity for the above donors.

[0060] Table 3

[0061]

[0062] Furthermore, antisense oligonucleotide gene silencing experiments were performed to demonstrate that the RUG gene-encoded product catalyzes the synthesis of Q-Glu-Rha-Glu in tea plants. Specifically, PrimerSelection software was used to design primers RUG.expF and RUG.expR (NO5 in Table 1 ) for RUG gene expression analysis and actin ACT reference gene primers ACT.expF and ACT.expR (NO6 in Table 1 ), and RNAfold software (https: / / sfold.wadsworth.org / cgi-bin / index.pl) was used to design RUG gene-specific antisense oligonucleotides AsODN, and control sense oligonucleotides sODN (NO7 in Table 1 ) were also designed. The second leaf under the bud of 'Fuding Dabaicha' with the same tenderness was harvested, and asODN and sODN were prepared into 20 μM solutions with sterile water, and 50 μL of asODN or aODN was injected into each leaf using a sterile syringe. Then, the leaves were immersed in a solution containing 1 mL of the corresponding oligonucleotide (20 μM) and rutin (100 μM) and UDP-glucose (100 μM), respectively, and cultured at 25°C for 24-36 h under light (2000-10000 lux). After the culture, the total RNA in the treated leaves was extracted with TRIZOL, and the first chain of cDNA was synthesized with a reverse transcription kit. The primer pairs RUG.expF / RUG.expR and ACT.expF / ACT.expR were used to detect the expression levels of RUG and ACT genes, respectively (Table 4), and 2 -△△ct The relative expression of RUG was calculated, and the results showed that the expression of RUG in leaves after AsODN treatment decreased by more than 1 times compared with that after sODN treatment. At the same time, UPLC-DAD-MS / MS detection found that the expression of Q-Glu-Rha-Glu in leaves after AsODN treatment decreased by more than 1 times compared with that after sODN treatment ( Figure 4 ). This indicates that after the RUG gene of tea plant was silenced by antisense oligonucleotides, the expression of RUG gene in leaves was significantly downregulated, and the synthesis of Q-Glu-Rha-Glu was also significantly inhibited, which further proves that the RUG encoding product can catalyze rutin and UDP-glucose to synthesize the corresponding quercetin trisaccharide in tea plants.

[0063] Table 4

[0064]

[0065] It can be seen that the RUG gene is the key functional gene for the synthesis of Q-Glu-Rha-Glu flavonol trisaccharide in tea trees, and has an important influence on the synthesis of tea flavonol glycoside flavor substances.

[0066] Example 3

[0067] The Origami (DE3) engineered bacteria containing the recombinant plasmid pCold-TF-RUG were inoculated into liquid LB medium and propagated at 37°C until the bacterial solution OD600 was about 0.6. The culture solution was filtered out in a filter equipped with a 0.2 μm filter membrane and washed twice with an equal volume of 50 mM phosphate buffer (pH 8) as the engineered bacteria, or centrifuged at 2000 rpm for 20 min and washed twice with an equal volume of 50 mM phosphate buffer (pH 8) as the engineered bacteria. The washed engineered bacteria were collected and resuspended with no more than 5 times the volume of the engineered bacteria in 50 mM phosphate buffer (pH 8), and isopropyl-β-D-thiogalactoside (IPTG) was added at a final concentration of 1 mM, and induced at 16°C for 24 h to promote the expression of the recombinant protein. After adding lysozyme at a final concentration of 0.1 mg / ml to the above-mentioned engineered bacteria, the mixture was crushed with ultrasound for 15 min, and centrifuged at 8000 rpm for 20 min to remove the residue. The supernatant was passed through a nickel column at a flow rate of 4 column volumes / h, and 10 times the column volume of 10 mM imidazole buffer was added to wash and remove impurities. Then, the recombinant target protein RUG was eluted with 3 times the column volume of 250 mM imidazole buffer. The purified recombinant RUG was mixed with different concentrations of rutin and UDP-glucose, and the following reaction system was set up (Table 5):

[0068] Table 5

[0069]

[0070] Comparing the product formation under different temperature, buffer system and acidity conditions, the catalytic efficiency is highest under the conditions of reaction temperature 30℃, phosphate buffer system and pH 8.0 ( Figure 5 ). After reacting for 6 hours under this condition, the reaction was terminated by heat treatment at 90°C for 5 minutes, and then an equal volume of ethyl acetate was added to the reaction mixture for extraction, the ethyl acetate layer was separated, and then an equal volume of ethyl acetate was added to the reaction mixture for extraction, and the two ethyl acetates were combined and removed by reduced pressure distillation at 45°C. The paste was transferred to a culture dish and freeze-dried to obtain a dry powder, in which the Q-Glu-Rha-Glu content exceeded 80%.

[0071] At the same time, referring to the experimental method of the tea tree RUG gene of the present invention, the corresponding expression vector and engineered bacteria are used to insert the tea tree CsUGT91Q1 gene in the database into the vector and transform Origami (DE3), and the gene is expanded and expressed under the same low-temperature induction conditions under the above-mentioned equivalent culture conditions. The induced engineered bacteria are subjected to ultrasonic-assisted extraction and protein purification under the above-mentioned equivalent conditions, and the purified CsUGT91Q1 is subjected to enzymatic reaction under the same optimized conditions, and then subjected to the same extraction, distillation and drying operations. The Q-Glu-Rha-Glu content in the obtained dry powder is less than 38%.

[0072] Therefore, the tea tree RUG gene in the present invention and its most homologous tea tree CsUGT91Q1 can both catalyze the glycosyl transfer reaction between rutin and UDP-glucose to produce Q-Glu-Rha-Glu, but the catalytic efficiency of CsUGT91Q1 is much lower than that of the tea tree RUG gene in the present invention, and the specific catalytic function of CsUGT91Q1 on other substances has not been reported, and rutin and UDP-glucose are not its optimal reaction substrates.

[0073] Finally, it should be noted that the above examples are only some specific embodiments of the present invention. Obviously, the present invention is not limited to the above embodiments, and there are many variations. All variations that can be directly derived or associated with the content disclosed by a person skilled in the art should be considered as the protection scope of the present invention.

Claims

1. Tea tree rutin: UDP-glucosyltransferase RUG gene, characterized by: The nucleotide sequence of the tea plant RUG gene is shown in SEQ ID No:

1.

2. The protein encoded by the tea tree RUG gene as claimed in claim 1, characterized in that: The amino acid sequence of the protein is shown in SEQ ID NO:

2.

3. The use of the tea tree RUG gene as claimed in claim 1, characterized in that: In vitro controlled synthesis of quercetin 3-O-glucosylrhamnosyl glucoside Q-Glu-Rha-Glu was achieved through prokaryotic expression of the tea plant RUG gene.

4. The use of the tea tree RUG gene according to claim 3, characterized in that: The product encoded by the exogenously expressed RUG gene was used to catalyze the transglycosylation reaction between rutin and UDP-glucose, and quercetin 3-O-glucosyl rhamnosyl glucoside Q-Glu-Rha-Glu was synthesized in vitro.

5. A method for preparing Q-Glu-Rha-Glu by in vitro synthesis, characterized in that: The method comprises transforming Escherichia coli with the RUG gene of tea tree, and using the induced expressed RUG to catalyze the reaction of rutin and UPD-glucose to achieve the synthesis of Q-Glu-Rha-Glu; The nucleotide sequence of the tea plant RUG gene is shown in SEQ ID No:

1.

6. The method for preparing Q-Glu-Rha-Glu by in vitro synthesis according to claim 5, characterized in that: After the tea tree RUG gene is expressed and purified in an Escherichia coli prokaryotic expression system, it is then catalyzed to synthesize Q-Glu-Rha-Glu from rutin and UDP-glucose at 30°C, a phosphate buffer system, and pH 8.0, and then extracted with ethyl acetate and freeze-dried to prepare Q-Glu-Rha-Glu with a purity of ≥80%.

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