CN116515787B - Vietnam camellia oleifera glycosyltransferase CvUM7 and application thereof - Google Patents

Abstract

The invention relates to the technical field of bioengineering, in particular to a vietnam camellia glycosyltransferase CvUM7 and application thereof. The amino acid sequence of the Vietnam camellia glycosyltransferase CvUM7 is shown in a sequence table SEQ ID No. 1; the nucleotide sequence of the coding gene of the vietnam sasanqua glycosyltransferase CvUM7 is shown in a sequence table SEQ ID No. 2; a recombinant vector is obtained by inserting a gene shown in a sequence table SEQ ID No.2 into a pET-28a vector; provides the application of the vietnam camellia glycosyltransferase CvUM7 in the aspect of preparing tea saponin. The advantages are that: the coding gene of the Vietnam camellia glycosyltransferase CvUM7 can catalyze oleanolic acid to carry out glycosylation reaction to synthesize tea saponin, and support is provided for research such as breeding of new varieties of Vietnam camellia and industrialized synthesis of tea saponin.

Description

Vietnam camellia oleifera glycosyltransferase CvUM7 and application thereof

Technical Field

The invention relates to the technical field of bioengineering, in particular to a vietnam camellia glycosyltransferase CvUM7 and application thereof.

Background

Glycosyltransferases (GTs) are a highly divergent family of multimeric polygenes that are widely found in animals, plants, fungi, bacteria and viruses and are responsible for the glycosylation reaction, i.e., coupling of a glycoside residue from a donor to an acceptor molecule (Mackenzie et al, 1997). Based on the similarity between amino acid sequences, a total of 114 GT supergene families are currently included in CAZy databases (khororagchaa et al, 2014), along with the conserved sequences and catalytic mechanisms. Among them, the GT1 family is the most bulky and has 44 conserved amino acid sequences at the C-terminus of the protein, which is called PSPG-box (Hughes and Hughes, 1994). One class of this family that catalyzes the transfer of UDP-sugars to a variety of substrates, including secondary metabolites, is known as UGTs. Different folded forms exist in over 120,000 GT proteins. The scientists classified 288 GT proteins with definite structural information into three classes GT-A/B/C, while the remaining GT-101/26 proteins with unknown structure were classified as GT-D/E (Hassan et al, 2022), respectively. In folded form, most UGTs are classified as GT-B, comprising two sugar donor-binding C-terminal domains with a substrate acceptor-recognizing N-terminal domain (Guo Fang et al, 2021). UGTs activate sugar donors typically predominantly UDP-Glucose (UDP-Glucose) while UDP-Rhamnose (UDP-Rhamnose), UDP-Galactose (UDP-Galactose), UDP-Xylose (UDP-Xylose) and UDP-glucuronic acid (UDP-GlcUA) are both characterized as being involved in the transfer reaction.

From the public database, among various plants producing triterpenes, there were 438, 416, 265 UGT genes in alfalfa, ginseng and licorice, respectively, while there were only 162 in arabidopsis. After amino acid sequence alignment, arabidopsis thaliana is divided into 65 families, 14 phylogenetic groups, each designated by capital letters (A-N) (Li et al, 2001; brazier-Hicks et al, 2018). Subsequent studies have found that there are also O, P classes of UGTs found in plants such as apples, whereas these two classes of UGTs that are lost in arabidopsis are presumed to be largely lost in plant evolution (Caputi et al 2012). Khorolragchaa et al (2014) 12 UGTs most likely involved in triterpene compound biosynthesis were screened in Korean ginseng (P. Ginseng Meyer) and these candidate UGTs were divided into 6 groups (A, E, G, L, N, O). UGTs currently characterized as being involved in triterpene compound biosynthesis belong to family members of UGTs 71, 73, 74, 85, 91, 94, with the number of members of the UGT73 family involved in modification of pentacyclic triterpene compounds being greatest (Yao et al 2020).

Glycosylation is the last step in plant secondary metabolite synthesis, whereas enzymes involved in glycosylation are typically UGTs. The plant triterpene UGTs catalyze triterpene sapogenins to form glycosidic bonds by subjecting UDP-sugar donors to form triterpene saponins. The number, composition and position of the triterpene saponin sugar chain on the triterpene skeleton are considered to have a great influence on the storage and transportation inside and outside the plant cells, the bioactivity, stability, absorption performance and the like (Seki et al 2015). Glycosylation of triterpenes can provide plant defense mechanisms (Mugford and Osbourn, 2012) for the insecticidal and antifungal activity of triterpene saponins. The resistance of triterpenoid saponins to fungal pathogens is due to glycosylation at specific carbon positions. For example, wheat holothuria infects plant roots and is induced to produce beta-glucosidase to hydrolyze C-3 chains, thereby releasing saponins for detoxification purposes (Wang and Hou, 2009). Glycosylation reactions can be directly/indirectly involved in phytohormone homeostasis regulation by altering the recognition between receptors and hormones as well as hormonal properties. In addition, glycosylation is involved in biological processes such as cell detoxification, storage and transport of secondary metabolites, synthesis, etc. (Wang and Hou, 2009).

When triterpenoid saponins are produced in medicinal plants such as ginseng, liquorice and the like, improvement of production efficiency by utilizing the related characteristics of glycosyltransferase is also a research hot spot of students. Under the combined action of UGTPG45 and UGTPn50, the yield of ginsenoside produced by ginseng yeast factory reaches 2.25 g/L. In addition, a plurality of UGTs enzymes with substrate hybridization that are extracted from microorganisms such as Saccharomyces cerevisiae can also form ginsenosides (Li Nuonan and Li Chun, 2019). The regional specificity and the omnipotence of the triterpene UGT in the liquorice also provide a guiding direction for the industrialized biosynthesis of the triterpene saponin. UGT73F17 only catalyzes the carboxylation reaction at the C29/30 position on the pentacyclic triterpene backbone, while there is a high degree of heterogeneity in the choice of UDP-sugar donors. In addition, UGT in arabidopsis thaliana (Barbarea vulgaris) can also catalyze glycyrrhetinic acid to form its own triterpene saponins (Li Nuonan and Li Chun, 2019).

Vietnam tea-oil treeCamellia vietnamensisHuang) is a member of the genus camellia of the family camelliaAnd firstly, the plant is also a characteristic woody oil crop widely planted in China. Tea saponin (teasaponin) is one of important functional components of Tea-oil camellia, and has various effects of reducing blood lipid, sterilizing, inhibiting bacteria, resisting tumor, etc. However, as the eight-ploidy plant of the Vietnam tea-oil tree, the research difficulty is high, and genome data is not published temporarily, so that the cloning of UGT genes in the Vietnam tea-oil tree and the functional verification of the UGT genes have very important significance.

Disclosure of Invention

The invention provides a vietnam camellia glycosyltransferase CvUM7 and application thereof for solving the problems.

The first aim of the invention is to provide a vietnam camellia glycosyltransferase CvUM7, and the amino acid sequence of the vietnam camellia glycosyltransferase CvUM7 is shown in a sequence table SEQ ID No. 1.

The coding gene of the Vietnam camellia glycosyltransferase CvUM7 has a nucleotide sequence shown in a sequence table SEQ ID No. 2.

The second object of the present invention is to provide a recombinant vector, which is to insert a gene shown in a sequence table SEQ ID No.2 into a pET-28a vector.

The third purpose of the invention is to provide an application of vietnam camellia glycosyltransferase CvUM7 in preparation of tea saponin.

Preferably, oleanolic acid and UDP glucose are used as substrates, and the Vietnam camellia glycosyltransferase CvUM7 is used as a catalyst for catalytic reaction to obtain tea saponin.

Preferably, the catalytic reaction temperature is 28-32 ℃ and the time is 0.5-1.5 h.

Compared with the prior art, the invention has the following beneficial effects:

the coding gene of the vietnam camellia glycosyltransferase CvUM7 can catalyze oleanolic acid to carry out glycosylation reaction to synthesize tea saponin; the invention explores the structure and the expression characteristics of the CvUGT gene of the Vietnam tea-oil tree from the molecular level, further knows the synthesis mechanism of the tea saponin in the Vietnam tea-oil tree body, provides theoretical support for research such as breeding of new varieties and industrialized synthesis of the tea saponin, and provides important theoretical and practical support for promoting the development of the Hainan tea-oil tree industry.

Drawings

Fig. 1 is a diagram of a step of gel cutting recovery of a PCR product according to an embodiment of the present invention.

FIG. 2 is a diagram showing cloning vector ligation and transformation steps according to an embodiment of the present invention.

FIG. 3 is a schematic illustration of an embodiment of the present inventionCvUM7Double enzyme cutting electrophoresis diagram of recombinant vector; 1: pET28a plasmid; 2:CvUM7a PCR product; 3: recombinant plasmidNdelAndBamH1double enzyme cutting.

FIG. 4 is a diagram showing experimental procedures for transforming E.coli DE3 with plasmids according to the examples of the present invention.

FIG. 5 is a schematic illustration of an embodiment of the present inventionCvUM7SDS-PAGE identification result of recombinant plasmid strain induced expression products; 1: recombinant bacterial liquid pET-28a-CvUM7 induces the expressed supernatant protein; 2: an empty strain induced protein.

Fig. 6 is a CvUM7 chromatogram provided according to an embodiment of the present invention.

Fig. 7 shows SDS-PAGE identification results of CvUM7 purified and concentrated proteins provided in accordance with an embodiment of the present invention.

Fig. 8 is a CvUM7 chromatogram provided according to an embodiment of the present invention.

Fig. 9 is a CvUM7 secondary mass spectrum provided according to an embodiment of the present invention.

Detailed Description

Hereinafter, embodiments of the present invention will be described with reference to the accompanying drawings. In the following description, like modules are denoted by like reference numerals. In the case of the same reference numerals, their names and functions are also the same. Therefore, a detailed description thereof will not be repeated.

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention will be further described in detail with reference to the accompanying drawings and specific embodiments. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not to be construed as limiting the invention.

Experimental materials: vietnam oil tea.

Example 1 tea saponin correlationUGTsCloning of genes

1. Extraction of RNA

The RNA extraction process was performed with reference to the rapid universal plant RNA extraction kit (beijing hua vietnamese biotechnology limited) instructions. Detecting the extracted RNA by 1% agarose gel electrophoresis, detecting the concentration of the RNA by a nucleic acid tester, and placing the RNA sample with qualified quality in a refrigerator with the temperature of-80 ℃ for standby.

2. Reverse transcription to obtain cDNA

cDNA selection MonScript ^TM RTIII Super Mix with dsDNase (Two-Step) kit.

3. Gene full-length CDS amplification

According to the MeJA transcriptome annotation result of Vietnam camellia oleifera, 7 glycosyltransferase gene fragments with identity more than 40 and the largest variation of expression quantity are screened by local blast, and glycosyltransferase gene sequences with verified functions in other species are compared, searched and downloaded in NCBI, primers are designed based on the glycosyltransferase gene sequences (Table 1), and PrimeSTAR series high-fidelity PCR enzymes are used for amplifying cDNA full-length sequences, wherein the reaction system is shown in Table 2.

TABLE 1 Vietnam tea-oil treeCvUGTsList of Gene cloning primers

Table 2 Vietnam tea-oil treeCvUGTsGene amplification reaction system

Screening for triterpene glycosyltransferase genes with similarity to other species of greater than 40 based on local blastUGTsAs candidate genes, primers are designed according to the gene sequences corresponding to BLAST comparison results, and the camellia oleifera glycosyltransferase genes with the full lengths of 1850, 1539, 1430, 1640, 1552, 1673 and 1527 and bp are obtained through amplification, and the camellia oleifera glycosyltransferase genes have bright and clear single specific bands through gel electrophoresis detection and are respectively named asCvUM1-7。

4. Cut gel recovery of PCR products

After the amplification is finished, gel electrophoresis observation results are carried out, and target bands are purified and recovered, wherein the specific steps are carried out according to the specification of a general DNA purification gel recovery kit (shown in figure 1).

5. Cloning vector ligation and sequencing

Ligation of cloning vectors was performed strictly according to the pBM16A Toposmart cloning kit and the E.coli DH 5. Alpha. Competent cell instructions (as shown in FIG. 2). The monoclonal was picked up and subjected to colony PCR identification according to the reaction system and procedure shown in Table 3, and the bacterial liquid carrying company which succeeded in connecting the cloning vector was subjected to sequencing analysis by M13F/R. The bacterial liquid with successful sequencing is extracted from the plasmid, and the operation method is according to the instruction of General Plasmid Mini Kit. The plasmid was kept at-20℃until use.

Table 3 Vietnam tea-oil treeCvCYP450Gene colony PCR reaction system and program

6. Bioinformatics analysis

Analysis of the relevant information was performed according to table 4.

Table 4 analysis software information

7. qRT-PCR reaction

RNA extraction and reverse transcription cDNA operation are the same as in the first step and the second step; the primers used in the procedure are shown in Table 5. Fluorescent quantitative PCR reactions were performed using Monamp ^TM The chemoshs qPCR Mix kit was completed and the reaction system and procedure are shown in table 6.

TABLE 5 real-time fluorescent quantitative PCR primer list

TABLE 6 qPCR reaction System

Example 2 CvUM1-7 bioinformatics analysis

Through relevant bioinformatics analysis and according to phylogenetic tree results, the CvUM1, 6 and 7 and triterpene related glycosyltransferases in soybean, medicago tribulus and European arabidopsis thaliana are gathered into a group; the CvUM3 and the triterpene glycosyltransferase of the ginseng and the soybean in the group A are gathered into one branch, and the relationship is nearest; cvUM2, 4, 5 and Grosvenor momordica, avena sativa, cyanopsis glauca and Panax ginseng are grouped together in group L. Wherein, the homology of the SgUGT74AC1 glycosyltransferase of the CvUM4 and the Momordica grosvenori is highest, and the homology of the CvUM6 and the CvUM7 and the soybean GmUGT73P2 glycosyltransferase is highest.

Example 3CvUM7Plasmid extraction, recombinant vector construction and enzyme digestion identification

Recombinant plasmid construction was performed using homologous recombination double restriction enzyme method. In the form of vectors to be constructedCvUM7The CDS region was primer designed and introduced with a sequence complementary to the pET-28a vector (Table 7).CvUM7Gene PCR amplification was performed by the method of full-length CDS amplification of the gene in reference example 1. The bacterial liquid obtained after the successfully sequenced escherichia coli with the recombinant plasmid is further amplified and cultured is referenced to a General Plasmid Mini Kit plasmid extraction kit (Chengdu Fuji Biotechnology Co., ltd.) instruction for extracting the plasmid. Then according to the designed enzyme cutting site, adoptingNdelAndBamH1double-digested empty pET-28a vector. The double enzyme digestion system is as follows:NdelandBamH1each 2 mu L,10 Xbuffer M5 mu L, plasmid DNA 1 mu g and make-up water until the total reaction volume is 50 mu L. The metal bath reaction is carried out for 5 to 15 minutes at 37 ℃. And recovering the carrier after the reaction is finished. The fragment of interest was ligated with the recovered pET-28a vector using a homologous recombination kit (Tolo Biotech.).

Table 7 tea-oil camelliaCvUM7Prokaryotic expression primer list

Results: large intestine obtained by cloningThe bacillus plasmid is used as a template, and containsNdel1、BamH1Specific primers for cleavage sitesCvUM7The PCR amplification of the gene is carried out according to the instruction of the plasmid extraction kit after the correct sequencing identification resultCvUM7And (3) extracting the target fragment plasmid.

By endonucleaseNdel1、BamH1Double enzyme digestion is carried out on the pET-28a empty vector, the empty vector plasmid is digested into a linearization vector, and then the pET-28a linearization vector and the linearization vector are subjected to homologous recombination technologyCvUM7The target fragments are connected to obtain the recombinant vector. After transformation of E.coli DE3, positive recombinants are identified, and the positive bacterial liquid is used again after plasmid extractionNdel1、BamH1The results of the double enzyme digestion and gel electrophoresis are shown in FIG. 3. The result shows that two bands are obtained after double enzyme digestion, one band is between 1500bp and 2000bp, and the other band is more than 5000bp, which indicates that the gene fragment about 1500bp is successfully inserted into the pET-28a vector. Sequencing results showedCvUM7The gene is successfully connected to the vector, and the prokaryotic expression vector is constructed.

Example 4 transformation competence

Rapidly adding a vector plasmid (pET-28 a-CvUM 7) connected with a target fragment into the DH5a competent cells which are just melted by using a pipetting gun, standing on ice for 30 min after gentle mixing, converting the competent cells by adopting a heat shock method (water bath heat shock at 42 ℃ for 45 sec), preparing LB liquid culture medium to absorb 500 mu L, fully mixing with a conversion solution, shaking for 60 min at 37 ℃ and 200rpm, absorbing a certain volume, coating the mixture onto a culture medium plate with kana resistance, inverting overnight at 37 ℃ in a constant temperature incubator, selecting single bacterial colonies growing on the next day for bacterial liquid PCR verification, and carrying out sequencing inspection. The positive clone which is successfully detected extracts high-quality plasmid and then transfers the plasmid into Rosetta (DE 3) competent cells (figure 4), so that the transformation efficiency is higher.

Example 5 CvUM7 recombinant protein induced expression

Inducible expression is performed according to the method of Xu Guojie (2017). Supernatant 16 and uL and bacterial pellet were mixed with loading buffer, and SDS-PAGE was performed.

E.coli DE3 strain containing pET-28a-CvUM7 recombinant vector was used as a control with a final concentration of 0And 5mM IPTG is subjected to induction expression, and the supernatant proteins and precipitated insoluble proteins after induction expression are subjected to SDS-PAGE protein electrophoresis to detect whether protein expression exists. FIG. 5 shows that recombinant proteins produced large amounts of protein around 60kDa, indicating candidate genesCvUM7The expression is smoothly induced in the escherichia coli.

Example 6 in vitro enzymatic detection of CvUM7 crude enzyme

The ultrasonic disruption bacterial supernatant protease liquid obtained in the research is subjected to in vitro enzymatic reaction, the catalytic substrate is oleanolic acid, and the catalytic product is detected by high performance liquid chromatography mass spectrometry. The results in fig. 6 show that CvUM7 catalysis produces a new peak. The results indicate thatCvUM7May be the target gene of the study.

EXAMPLE 7 purification and Activity detection of CvUM7 recombinant protein

Following the procedure of Sun Rong (2017) with minor modifications, the following reaction solutions were added sequentially to a sterile, enzyme-free centrifuge tube: 25 mug oleanolic acid, 1 mM DTT,5 mM UDP-Glc and crude protein lysate, wherein the total reaction system is 500 mugL. The reaction conditions were 30℃and the time was 1 h. Then 1mL ethyl acetate is fully extracted, after nitrogen is blown to volatilize the ethyl acetate, 1mL methanol is added for dissolution, and high-fraction mass spectrometry (AB SCIEX 6500 QTRAP LC/MS/MS) is adopted to detect whether tea saponin monomers are generated. Adding water into the reaction system to serve as CK, and repeating for 3 times.

Results: to further verify the CvUM7 enzyme activity of glycosyltransferases belonging to group D we will containCvUM7BL21 (DE 3) E.coli of the recombinant plasmid was propagated in large quantities and purified (FIG. 7). The purified protein is catalyzed by oleanolic acid and UDP glucose as substrates, the temperature of the catalysis is 30 ℃, the catalytic reaction time is 1h, and the catalytic activity is verified. The results in FIGS. 8 and 9 show that a new peak of oleanolic acid plus glycoside appears at a retention time of 10.42min compared to the Control (CK), indicating that the CvUM7 protein can transfer UDP-Glc glycosyl to oleanolic acid, indicating that the CvUM7 has catalytic activity and can cause the oleanolic acid to undergo glycosylation.

It should be appreciated that various forms of the flows shown above may be used to reorder, add, or delete steps. For example, the steps described in the present disclosure may be performed in parallel, sequentially, or in a different order, provided that the desired results of the technical solutions of the present disclosure are achieved, and are not limited herein.

The above embodiments do not limit the scope of the present invention. It will be apparent to those skilled in the art that various modifications, combinations, sub-combinations and alternatives are possible, depending on design requirements and other factors. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the scope of the present invention.

Claims (1)

1. The application of vietnam camellia glycosyltransferase CvUM7 in preparing tea saponin is characterized in that: the oleanolic acid and UDP glucose are used as substrates, and the Vietnam camellia glycosyltransferase CvUM7 is used as a catalyst for catalytic reaction to obtain tea saponin; the amino acid sequence of the Vietnam camellia glycosyltransferase CvUM7 is shown in a sequence table SEQ ID No. 1; the catalytic reaction temperature is 28-32 ℃ and the time is 0.5-1.5 h.