CN105002193A

CN105002193A - Flavonol 3-O-glucosyltransferase CsUGT78A14 gene and coding protein and application thereof

Info

Publication number: CN105002193A
Application number: CN201510253488.3A
Authority: CN
Inventors: 夏涛; 崔利兰; 赵贤倩; 蒋晓岚; 高丽萍; 刘亚军
Original assignee: Anhui Agricultural University AHAU
Current assignee: Anhui Agricultural University AHAU
Priority date: 2015-05-18
Filing date: 2015-05-18
Publication date: 2015-10-28
Anticipated expiration: 2035-05-18
Also published as: CN105002193B

Abstract

The invention discloses a flavonol 3-O-glucosyltransferase CsUGT78A14 gene separated from fresh tea leaves. The gene has the nucleotide sequence shown as SEQ ID NO:1. Coding protein of the gene has the amino acid sequence shown as SEQ ID NO:2. Functions of the flavonol 3-O-glucosyltransferase CsUGT78A14 gene related to formation of the gentle astringent taste of tea drinks are cloned and verified for the first time. The invention further provides recombinant plasmid, transgenic engineering bacteria and recombinant protein which contain the GsUGT78A14 genes, and the solid foundation is laid for developing enzymes or engineering microorganisms capable of improving the taste of the tea leaves, deepening the tea drink processing and developing tea drinks with different tastes.

Description

A kind of flavonol 3-O-glucanotransferase CsUGT78A14 gene and proteins encoded thereof and application

Technical field

The present invention relates to biology field, in particular a kind ofly from fresh leaves of tea plant, be separated the flavonol 3-O-glucanotransferase CsUGT78A14 gene and proteins encoded thereof and application that obtain.

Background technology

Polyphenolic substance is secondary metabolite main in tea tree, with tea leaf quality and body-care closely related, wherein, catechin (flavane 3-alcohol), flavonol and their derivatize product such as ester catechin and flavonol 3-O-glucosides are the important composition compositions determining tea leaf quality and quality.In 2005, in the research of Scharbert and Hofmann, they pick some standard substance compounds and comprise 15 seed amino acids, 14 kinds of flavonol glycosides, 8 kinds of flavane 3-alcohol, 5 kinds of theoflavin, 5 kinds of organic acids, 3 kinds of sugar and caffeine, as the research object of tealeaves astringent taste, by the real content of these compounds in black tea infusion, simulate the mouthfeel similar with black tea with standard substance mixed aqueous solution.Find the caffeine comprising bitter taste, the flavonol 3-O-glucosides of 9 kinds of soft astringent tastes, the mixed aqueous solution of catechin and pained ester catechin EGCG is consistent with tea drink aqueous solution mouthfeel, therefore determines that these 12 kinds of compounds are compounds of very important decision black tea mouthfeel in tealeaves.Further, additional sensory review's research shows that flavonol 3-O-glucosides is not only given the mouthfeel of the soft astringent taste of tea drink but also added the sense of millet paste hardship by the bitter taste strengthening caffeine.Therefore, these compounds are considered to the compound than thearubigins and theoflavin more typical decision black tea infusion mouthfeel, and flavonol glycosides compound gives tealeaves soft astringent sense as the phenolic compound being only second to catechin content with very low threshold value, the glycosyltransferase (UDP-glycosyltransferases, UGTs) that uridine diphosphate (UDP) sugar relies on is the crucial enzyme participating in flavonol glycosides synthesis in tealeaves.Therefore, a kind of flavonol 3-O-glucanotransferase has potential using value improving in tea drink flavour.

Summary of the invention

The object of the invention is to overcome the deficiencies in the prior art, provide and a kind ofly from fresh leaves of tea plant, be separated the flavonol 3-O-glucanotransferase CsUGT78A14 gene and proteins encoded thereof and application that obtain, to provide a kind of can encode tea tree flavonol 3-O-glucosyl transferase gene and proteins encoded thereof newly.

The present invention is achieved by the following technical solutions:

The invention provides a kind of flavonol 3-O-glucanotransferase CsUGT78A14 gene, this gene is separated and obtains from fresh leaves of tea plant, has the nucleotide sequence as shown in SEQ ID NO:1.

Present invention also offers above-mentioned flavonol 3-O-glucanotransferase CsUGT78A14 gene and improve the application in tea drink flavour.

Present invention also offers a kind of proteins encoded of above-mentioned flavonol 3-O-glucanotransferase CsUGT78A14 gene, described proteins encoded has the aminoacid sequence as shown in SEQ ID NO:2.

The proteins encoded that present invention also offers above-mentioned flavonol 3-O-glucanotransferase CsUGT78A14 gene is improving the application in tea drink flavour.

Present invention also offers a kind of recombinant plasmid containing above-mentioned flavonol 3-O-glucanotransferase CsUGT78A14 gene.

Described recombinant plasmid is be connected to by above-mentioned flavonol 3-O-glucanotransferase CsUGT78A14 gene in the multiple clone site of pMal-c2X carrier to build to obtain, called after pMal-c2X-CsUGT78A14.

Present invention also offers a kind of transgenic engineered bacteria, described transgenic engineered bacteria contains above-mentioned recombinant plasmid, or is integrated with the flavonol 3-O-glucanotransferase CsUGT78A14 gene order of external source in its genome.

Described transgenic engineered bacteria for containing above-mentioned recombinant plasmid, or is integrated with intestinal bacteria Novablue (DE3) bacterial strain of flavonol 3-O-glucanotransferase CsUGT78A14 gene order of external source in its genome.

The present invention has the following advantages compared to existing technology: the invention provides a kind of flavonol 3-O-glucanotransferase CsUGT78A14 gene and proteins encoded thereof and application, clone first and demonstrate and form the relevant flavonol 3-O-glucosyl transferase gene CsUGT78A14 function of the soft astringent taste of tea drink, present invention also offers the recombinant plasmid containing CsUGT78A14 gene, transgenic engineered bacteria and recombinant protein, for exploitation has the enzyme or engineered microbes that improve tealeaves flavour, the processing of in-depth tea drink, develop different flavour teabag drink, establish solid foundation.

Accompanying drawing explanation

Fig. 1 is the plasmid map of pMal-c2X carrier;

Fig. 2 is the albumin crystal model of the CsUGT78A14 created for masterplate with the crystal model 2c9z of VvGT1;

Fig. 3 is the SDS-PAGE protein electrophoresis analysis chart of CsUGT78A14 recombinant protein (rCsUGT78A14); Wherein, M is albumen Marker; 1 for before recombinant plasmid induction; 2 for after recombinant plasmid induction; 3 is rear supernatant broken after induction; 4 is broken postprecipitation after induction; 5 is albumen after purifying.

Fig. 4 is the enzyme life birth thing result figure that HPLC analyzes rCsUGT78A14 catalysis; Wherein, Fig. 4-A ~ 4-C is be saccharide donor with UDPG respectively, and with flavonol compound kaempferide, Quercetin and ampelopsin are as the HPLC collection of illustrative plates of saccharide acceptor;

Fig. 5 is first mass spectrometric and the second mass analysis collection of illustrative plates that rCsUGT78A14 catalyzes and synthesizes flavonol 3-O-glucoside product; Wherein, Fig. 5-A is first mass spectrometric and the second mass analysis collection of illustrative plates of kaempferide 3-O-glucoside; The first mass spectrometric of Fig. 5-B Quercetin 3-O-glucoside and second mass analysis collection of illustrative plates; Fig. 5-C is first mass spectrometric and the second mass analysis collection of illustrative plates of ampelopsin 3-O-glucoside.

Embodiment

Elaborate to embodiments of the invention below, the present embodiment is implemented under premised on technical solution of the present invention, give detailed embodiment and concrete operating process, but protection scope of the present invention is not limited to following embodiment.Unreceipted actual conditions person in embodiment, the condition of conveniently conditioned disjunction manufacturers suggestion is carried out.Agents useful for same or the unreceipted production firm person of instrument, being can by the conventional products of commercial acquisition.

Embodiment 1

One, material

1, tea tree breed: agriculture anti-morning (Camellia sinensis (L.) O.Kuntze.var.sinensis cultivar Nongkangzao), gathers fresh leaves of tea plant, uses liquid nitrogen freezing rapidly, be stored in-80 DEG C of refrigerators for subsequent use;

2, pMal-c2X carrier: its plasmid map as shown in Figure 1;

3, intestinal bacteria Novablue (DE3) expressive host bacterium: be purchased from Bei Nuo bio tech ltd, Shanghai;

4, LB substratum: the NaCl taking 10g, the yeast extract of 5g, the Tryptones of 10g, add 950mL and go ultrapure water stirring and dissolving, adjust pH to 7.0 with the NaOH of 1mol/L, add water and be settled to 1000mL, high pressure steam sterilization 15min, namely obtain LB liquid nutrient medium, LB solid medium is the agar powder adding 15g in LB liquid nutrient medium;

5, mass ratio is the glucose solution of 40%: take 40g glucose, adds ultrapure water dissolving and stirs, be settled to 100mL, 110 DEG C of sterilizing 10min;

6, penbritin mother liquor (Amp ⁺, 100mg/mL): take 1g penbritin Amp, be dissolved in 10mL aqua sterilisa, filtration sterilization, packing tubule ,-20 DEG C of preservations;

7, the IPTG (isopropylthio-β-D-galactoside) of 1mol/L: take 2.383g IPTG, be dissolved in sterilizing ultrapure water, be settled to 10mL, filtration sterilization, packing in-20 DEG C of preservations;

8, protein purification damping fluid: comprise column-loading buffer and elution buffer:

Column-loading buffer: the DTT of the Tris of the NaCl of the EDTA taking 0.37g, 11.67g, 2.42g, 0.15g, in enough pure water, stirs and makes it fully mix; Adjust its PH to 7.4 with dilute hydrochloric acid, be settled to 1L, obtain column-loading buffer;

Elution buffer: add 3.60g maltose in 1L column-loading buffer, dissolving stirs;

9, the Tris-HCL buffered soln of the pH7.5 of 100mM: take 1.1214gTris and add water to 90mL stirring and dissolving evenly, add rare HCL and adjust pH to 7.5, moisturizing is settled to 100mL;

10, volume ratio is the acetic acid of 1%: measure 10mL chromatographic grade acetic acid solution in 1L volumetric flask with transfer pipet, be settled to 1L with ultrapure water.

Two, the clone of CsUGT78A14 gene:

1, design the special primer of the polyclone restriction enzyme site with expression vector pMal-c2X carrier, its primer sequence is as shown in SEQ ID NO:3 and SEQ ID NO:4:

SEQ ID NO:3: forward primer: 5 '- tCTAGAaTGAACGGTGACTCCCAACAACACC-3 '

SEQ ID NO:4: reverse primer: 5 '- gTCGACtTAAGGGTGCTTACAAGCTTTGATTACC-3 ';

2, according to TaKaRa RNAiso test kit and RNAiso Plus test kit specification sheets, extract tea tree breed agriculture fresh leaf RNA anti-morning, and reverse transcription is cDNA;

3, with reverse transcription product cDNA for template, increase with SEQ ID NO:3 and SEQ ID NO:4 primer, amplification program is 94 DEG C of denaturation 30s, 94 DEG C of sex change 10s, 62 DEG C of annealing 20s, 72 DEG C extend 50s, 30 circulations, 72 DEG C are continued to extend 10min, and the PCR primer of acquisition is placed in 16 DEG C of preservations.

4, PCR primer is utilized PCR Purification Kit, and carry out bacterium colony PCR checking after being connected to pMD19-T Simple Vector, obtain positive bacterium colony, extract bacterium colony plasmid, obtain the carrier T containing CsUGT78A14 gene, bacterium liquid is delivered to Shenzhen Hua Da company simultaneously and check order.

Three, the function prediction analysis of CsUGT78A14 gene

By online software Jpred ( http:// www.compbio.dundee.ac.uk/www-jpred) secondary structure prediction is carried out to the glycosyltransferase gene sequence in tea tree transcript profile database, find the grape uridine diphosphoglucose of CsUGT78A14 gene and known function: anthocyanidin 3-O-glycosyltransferase (VvGT1, accession number:P51094.2, crystal model 2c9z_A) comparison of coherence high, reach the consistence of 56%, be predicted to be and have flavonoid 3-O-glycosyltransferase functionally active, the albumin crystal model of described CsUGT78A14 gene as shown in Figure 2;

Four, the prokaryotic expression of CsUGT78A14 gene and functional verification

Prokaryotic expression used in the present embodiment and and functional verification technique means commonly use for those of ordinary skill in the art or be appreciated that technique means completely.

1, the carrier T Xba I containing CsUGT78A14 gene and Sal I is carried out double digestion, digestion products is connected in the multiple clone site of pMal-c2X carrier, obtains pMal-c2X-CsUGT78A14 recombinant plasmid;

2, by pMal-c2X-CsUGT78A14 recombinant plasmid transformed in intestinal bacteria Novablue (DE3) expressive host bacterium, be inoculated into the LB liquid nutrient medium of 100 μ L, 37 DEG C, under 180r/min cultivate 45 ~ 60min; The bacterium liquid getting 100 μ L is coated containing 100 μ g/mL Amp ⁺lB flat board on, 37 DEG C be inverted cultivate;

3, verify through bacterium colony PCR, the positive bacterium colony of picking, is seeded in the LB liquid nutrient medium of the sterilizing of the 100mL containing 2g/L, 28 DEG C, and under 200r/min, concussion is cultivated, until OD ₆₀₀be about 0.6, obtain genetically modified engineering bacteria;

4, in above-mentioned genetically modified engineering bacteria, adding IPTG is 1mmol/L to final concentration, 37 DEG C of incubated overnight, collect thalline, add 10mL upper prop buffered soln, abundant suspension thalline, is placed in-20 DEG C and spends the night, and is placed in by thalline and thaws on ice, wait to thaw and be placed in Ultrasonic Cell Disruptor with 15% power ultrasonic broken 10min, 12000rpm collected by centrifugation supernatant liquor; Utilize amylose resin affinity column purification of recombinant proteins (affinity chromatography on an amylase resin, New England Biolabs, MA, USA), the SDS-PAGE method utilizing this area conventional detects Protein expression and purification effect, and result as shown in Figure 3.

Can find out in Fig. 3, pMal-c2X-CsUGT78A14 recombinant plasmid transformed expressive host bacterium Novablue (DE3), after abduction delivering, compared with (swimming lane 1) before induction, this gene (swimming lane 2) after induction has the expression of recombinant protein, and the size of recombinant protein band and the consistent of prediction, add that 42.5kDa maltose binding protein (MBP) is recombinated after label, between 70kd to 100kd, have obvious recombinant protein band; After induction, thalline is after ultrasonication is centrifugal, has soluble recombinant protein (swimming lane 3), can be used for being further purified analysis in supernatant; Supernatant protein is after amylose resin column purification, and obtain purer recombinant protein (swimming lane 4), the albumen of purifying can be used for further enzymatic analysis.

Five, the enzyme activity of CsUGT78A14 recombinant protein detects and analyzes:

For the Enzyme activity assay of flavonoid substrate, in 50 μ L reaction systems, the Tris-HCL buffered soln of 100mM pH7.5 comprises 5mM UDPG or UDP-semi-lactosi as glycosyl donor, 200 μMs of potential flavonoidss (kaempferol, Quercetin, ampelopsin, kaempferide, naringenin, eriodictyol, apigenin, catechin and cyanidin) as the recombinant protein after the purifying of glycosyl acceptor, 5-10 μ g and 0.1% beta-mercaptoethanol.

All enzyme reaction systems, add isopyknic methyl alcohol termination reaction after 30 DEG C of water-bath 30min, and Minor centaury is the reaction system exception of substrate, need add the 5% hydrochloric acid termination reaction of 20 μ L, and reaction all with unloaded albumen in contrast, obtains enzyme reaction product.

Enzyme reaction product is identified in conjunction with HPLC-MS through product standard substance, and described HPLC-MS testing conditions is as follows: Wei Tesi HSS T3 chromatographic column (Waters ACQUITY UPLC HSS T3,150mm × 2.1mm, 1.7tzm); Column temperature is 30 DEG C; Flow velocity is 1mL/min; Sampling volume is 5 μ L; Mobile phase A is for containing 1% (v/v) acetic acid solution; Mobile phase B is 100% acetonitrile solution; For the detection of flavonoids, HPLC Gradient program is set to: 0 ~ 5min, 10 ~ 15%B; 5 ~ 15min, 15 ~ 40%B; 15 ~ 20min, 40 ~ 60%B; 20 ~ 25min, 60 ~ 80%B; 25 ~ 30min, 80 ~ 10%B; Spectral detection wavelength scanning range is 200 ~ 550nm.In the MS qualitative recognition of compound, adopt ESI electric spray ion source, negative ion mode; Capillary voltage is 3.5kV, and ion source temperature is 350 DEG C, and atomization gas (nitrogen) flow velocity is 6L/min, and compound test scanning mass charge ratio range is set to m/z 100 ~ 1000, and collision voltage is 45V.

By HPLC and flavonol glycosides standard substance enzyme analysis reaction product, result as shown in Figure 4 and Figure 5, can find in figure that rCsUGT78A14 can specific catalysis flavonol substrate, any activity all do not detected to other flavonoid and compound of phenolic acid; Compare with flavonol glycosides standard substance, catalysis flavonol (the kaempferol of rCsUGT78A14 regioselectivity can also be found, Quercetin and ampelopsin) glycosylation on 3-OH, and using glucose and semi-lactosi as saccharide donor, can illustrate that CsUGT78A14 has glucosyl transferase and the bifunctional activity of galactosyltransferase.

Claims

1. a flavonol 3-O-glucanotransferase CsUGT78A14 gene, is characterized in that, this gene has the nucleotide sequence as shown in SEQ ID NO:1.

2. a flavonol 3-O-glucanotransferase CsUGT78A14 gene as claimed in claim 1 is improving the application in tea drink flavour.

3. a proteins encoded for flavonol 3-O-glucanotransferase CsUGT78A14 gene as claimed in claim 1, it is characterized in that, described proteins encoded has the aminoacid sequence as shown in SEQ ID NO:2.

4. the proteins encoded of a flavonol 3-O-glucanotransferase CsUGT78A14 gene as claimed in claim 3 is improving the application in tea drink flavour.

5. a recombinant plasmid, is characterized in that, described recombinant plasmid contains flavonol 3-O-glucanotransferase CsUGT78A14 gene as claimed in claim 1.

6. a kind of recombinant plasmid according to claim 5, it is characterized in that, described recombinant plasmid is be connected to by flavonol 3-O-glucanotransferase CsUGT78A14 gene in the multiple clone site of pMal-c2X carrier to build to obtain, called after pMal-c2X-CsUGT78A14.

7. a transgenic engineered bacteria, it is characterized in that, described transgenic engineered bacteria containing, for example recombinant plasmid according to claim 5, or is integrated with the flavonol 3-O-glucanotransferase CsUGT78A14 gene order as claimed in claim 1 of external source in its genome.

8. transgenic engineered bacteria according to claim 7, it is characterized in that, described transgenic engineered bacteria is containing, for example recombinant plasmid according to claim 5, or is integrated with intestinal bacteria Novablue (DE3) bacterial strain of flavonol 3-O-glucanotransferase CsUGT78A14 gene order as claimed in claim 1 of external source in its genome.