CN110791486B

CN110791486B - Tobacco acyl glycosyltransferase gene NtASAT1 and encoding protein and application thereof

Info

Publication number: CN110791486B
Application number: CN201911217348.5A
Authority: CN
Inventors: 常爱霞; 屈亚芳; 陈铭; 于卫松; 王元英; 杨爱国; 罗成刚; 张玉; 刘旦; 冯全福
Original assignee: Tobacco Research Institute of CAAS
Current assignee: Tobacco Research Institute of CAAS
Priority date: 2019-12-03
Filing date: 2019-12-03
Publication date: 2024-02-09
Anticipated expiration: 2039-12-03
Also published as: CN110791486A

Abstract

The invention discloses a tobacco acyl glycosyltransferase gene NtASAT1, an encoded protein thereof and application thereof in biosynthesis of sucrose esters, wherein the encoded protein can regulate and control synthesis of sucrose monoesters in safer ammonium acetate buffer. The whole sucrose ester biocatalytic reaction was performed in ammonium acetate buffer with no addition of other substances than sucrose, acyl-COA and catalytic NtASAT1 protein. Compared with the prior method for synthesizing the sucrose ester by using a substrate or a solvent, the whole catalytic reaction system has high safety, simple reaction substrate, relatively single system product and easy subsequent separation and utilization because all the products are sucrose ester.

Description

Tobacco acyl glycosyltransferase gene NtASAT1 and encoding protein and application thereof

Technical Field

The invention relates to a tobacco acyl glycosyltransferase gene and a coded protein thereof and application thereof in biosynthesis of sucrose esters.

Background

Sucrose esters are important biosurfactants, and have strong surface activity and good emulsifying effect on oil and water because the molecular structure of the sucrose esters contains sucrose groups with strong hydrophilicity and fatty acid groups with lipophilicity. Since sucrose esters have a hydrophilic-lipophilic balance (HLB) that does not vary with temperature, their HLB value ranges are relatively broad, and can be used as both a W/O type emulsifier and an O/W type emulsifier. Sucrose ester has good emulsifying, dispersing, solubilizing, penetrating, foaming, viscosity regulating, ageing preventing, antibacterial and other performances, and is nontoxic, especially safe to human body, non-irritating, degradable and free from causing environmental pollution, so that the sucrose ester is widely applied to industries of food, medicine, chemical industry, cosmetics, petroleum exploitation, fruit preservation, textile, agriculture and the like. Sucrose esters are recommended food additives and pharmaceutical raw materials by the United nations grain and agricultural organization (FAO) and the World Health Organization (WHO), and are approved for use in China in the 80 th century.

The synthesis method of sucrose ester mainly comprises 4 methods such as an acyl chloride esterification method, a direct dehydration method, an ester exchange method and a microorganism method. The acyl chloride esterification method uses nitrogen-containing organic compounds with high toxicity as solvents, so that the product produced by the method is difficult to meet the standard requirements of food, cosmetics and medicines, and is more used for producing sucrose ester pesticides at present. The direct dehydration method is carried out under the condition that strong acid (p-toluenesulfonic acid) is used as a catalyst and a polar aprotic organic solvent DMF is used as a solvent, the method has simple process and easy operation, but DMF has high toxicity, the product is difficult to purify, and sucrose is easy to decompose under the acidic condition, so that the yield is low. The transesterification method includes a solvent method and a solvent-free method, wherein the solvent method is basically eliminated in industry so far because the solvent is toxic and is easy to remain in the finished product, the investment of equipment for refining into food grade is large, and the production cost is high. The solvent-free method is easy to generate coking because reactants are required to reach a molten state at a very high temperature, so that the quality of products is difficult to be ensured. Along with the development of bioengineering technology, it has been found that certain microorganisms catalyze the synthesis of sucrose esters, and enzyme catalysis also mainly comprises both solvent and solvent-free processes. Compared with the traditional chemical method, the enzyme catalysis method has the advantages of high catalytic activity, mild reaction, strong selectivity and the like. The sucrose ester synthesized by the enzyme method not only has the surface activity of emulsification wetting, capacity increasing and the like, but also has the anti-tumor performance. However, there is a solvent enzyme catalysis method, which still has the problem of toxicity of the solvent, and the application of the solvent in the fields of food, cosmetics and the like is limited. As the sugar ester has various medical and health care functions, the intensive research and development has wide market prospect and development potential. And its application prospect in the food industry is known. Therefore, solvent-free enzyme catalysis and the search for new enzyme-catalyzed processes have become one of the development directions for sucrose ester synthesis.

Disclosure of Invention

The invention aims to provide a tobacco acyl glycosyltransferase gene NtASAT1, a coded protein thereof and application thereof in biosynthesis of sucrose esters, wherein the coded protein can regulate and control synthesis of sucrose monoesters in safer ammonium acetate buffer.

The technical scheme of the invention is realized as follows:

a nucleotide sequence of the tobacco acyl glycosyltransferase gene NtASAT1 is shown as SEQ ID NO. 1.

Further, the amino acid sequence of the protein encoded by the tobacco acyl glycosyltransferase gene NtASAT1 is shown as SEQ ID NO. 2.

Further, the sequence of a cloning front primer of the tobacco acyl glycosyltransferase gene NtASAT1 is shown as SEQ ID NO. 3, and the sequence of a cloning rear primer of the gene is shown as SEQ ID NO. 4.

Further, the preparation method of the monoclonal recombinant plasmid carrying the gene NtASAT1 comprises the following steps: PCR amplification is carried out by taking common tobacco (N.tabacum) glandular wool cDNA as a template, cloning primers of a gene NtASAT1 are utilized, a target strip of the gene is recovered by utilizing an agarose gel recovery kit, a gene fragment is connected to a cloning vector, a connection product is added into escherichia coli competence, after ice bath and ice bath are completed, the mixture is placed into a water bath kettle for heat shock, then liquid culture medium shaking table is added for incubation after ice bath is carried out again, partial supernatant is removed by centrifugation, the residual bacterial liquid is coated with a solid culture medium, culture is carried out in an incubator, white monoclonal bacteria are selected, amplification verification is carried out by utilizing a vector primer, positive clones are verified to be sent to sequencing, and the monoclonal recombinant plasmid vector with the gene NtASAT1 and correct sequencing is obtained.

Further, the primers P-NtASAT1_F and P-NtASAT1_R used for constructing the gene NtASAT1 expression vector, wherein the sequence of the P-NtASAT1_F is SEQ ID NO. 5; the sequence of the P-NtASAT1_R is SEQ ID NO. 6.

Further, the construction method of the gene NtASAT1 expression vector is as follows: amplifying the obtained positive monoclonal plasmid vector of the NtASAT1 by taking the P-NtASAT1_F and the P-NtASAT1_R as primers, recovering target gene fragments, mixing the target gene fragments with homologous recombinant enzyme, converting connection products into competence, culturing, identifying colony PCR, respectively selecting 1-3 positive clones for sequencing after identifying the PCR products by agarose gel electrophoresis, culturing single colony strains with correct sequencing overnight, centrifugally collecting thalli, and extracting plasmids.

Further, the protein expression method of the gene NtASAT1 is as follows: transforming the plasmid into colibacillus competent, activating strain, inducing expression in small system and screening condition, selecting optimal inducing expression condition according to the expression condition of small system, amplifying expression in large system and purifying protein.

Further, the application of the protein coded by the tobacco acyl glycosyltransferase gene NtASAT1 in regulating and controlling the synthesis of sucrose esters.

Further, the NtASAT1 protein uses sucrose as an acceptor substrate, uses acyl-COA as a donor substrate, and modulates synthesis of sucrose monoester in an ammonium acetate buffer.

Further, a method for regulating and controlling sucrose ester synthesis by using a protein coded by a tobacco acyl glycosyltransferase gene NtASAT1 is characterized in that: adding NtASAT1 protein into an ammonium acetate buffer solution, adding acyl-COA, adding sucrose for reaction, adding acetonitrile, isopropanol and formic acid mixed solution for stopping reaction, centrifuging, and passing the supernatant through an organic filter membrane to obtain sucrose monoester.

The beneficial effects of the invention are as follows: the tobacco acyl glycosyltransferase gene NtASAT1, the coded protein and the application thereof in biosynthesis of sucrose esters can regulate and control synthesis of sucrose monoesters in safer ammonium acetate buffer. The whole sucrose ester biocatalytic reaction was performed in ammonium acetate buffer with no addition of other substances than sucrose, acyl-COA and catalytic NtASAT1 protein. Compared with the prior method for synthesizing the sucrose ester by using a substrate or a solvent, the whole catalytic reaction system has high safety, simple reaction substrate, relatively single system product and easy subsequent separation and utilization because all the products are sucrose ester. Therefore, the NtASAT1 gene and the coded protein thereof have good application value and prospect in sucrose ester biosynthesis.

Drawings

In order to more clearly illustrate the embodiments of the invention or the technical solutions of the prior art, the drawings which are used in the description of the embodiments or the prior art will be briefly described, it being obvious that the drawings in the description below are only some embodiments of the invention, and that other drawings can be obtained according to these drawings without inventive faculty for a person skilled in the art.

FIG. 1 shows the NtASAT1 fragment of the gene of the invention, marker is DL5000;

FIG. 2 shows a subclone fragment of the gene NtASAT1, and Marker is DL10000;

FIG. 3 identification of recombinant expression vector NtASAT1 (positive monoclonal with correct number 2) Marker DL10000;

FIG. 4 is a screen of expression conditions of the NtASAT1 protein encoded by the gene NtASAT 1;

FIG. 5 shows the results of sample detection collected during the elution and elution of the NtASAT1 protein.

FIG. 6 shows the results of purification of the NtASAT1 protein;

FIG. 7 shows the catalysis of sucrose and various acyl-COAs by the NtASAT1 protein to sucrose monoesters.

Detailed Description

The present invention will be further specifically illustrated by the following examples, which are not to be construed as limiting the invention, but rather as falling within the scope of the present invention, for some non-essential modifications and adaptations of the invention that are apparent to those skilled in the art based on the foregoing disclosure.

Example 1

1.1 Cloning primers

NtASAT1 clone pre-primer F: ATGGCTGCCTCAGCTCTAGTT

post-NtASAT 1 clone primer R: TCAACATCGAGCTTCCATTTTAAT

1.2 Gene cloning method procedure

1.2.1 PCR amplification was performed using flue-cured tobacco red glandular wool cDNA as template and cloning primer of gene NtASAT 1. The PCR reaction system was 25. Mu.L, including 0.5. Mu.L of cDNA template, 1. Mu.L of each of forward and reverse primers, 2 XPimerstar MAX 12.5. Mu.L, and ddH2O 10. Mu.L.

Setting a PCR program: 98 ℃ for 2min;98 ℃,10sec,55 ℃,15sec,72 ℃,30sec,35 cycles; storing at 72deg.C for 8min at 4deg.C.

After completion of PCR, the gene fragment was detected by 1% agarose gel electrophoresis, as shown in FIG. 1.

1.2.2.2 recovery of the target bands of the genes using agarose gel recovery kit, after concentration detection the gene fragments were ligated into cloning vector pEASY-Blunt Simple Cloning Vector. The carrier ligation reaction system was 5. Mu.L, including 2. Mu.L of PCR product, pEASY-Blunt Simple Cloning Vector. Mu.L, and ddH2O 2. Mu.L, and was ligated in a metal bath at 25℃for 15min after the reaction system was added.

(1) The ligation product was added to 30. Mu.L of the just thawed Trans-T1 E.coli competence, and then gently mixed by pipetting with a gun head, and ice-bathed for 30min.

(2) After the ice bath is completed, the mixture is placed into a water bath kettle at 42 ℃ for heat shock for 30sec, and then the ice bath is immediately carried out for 2min.

(3) 600. Mu.L of LB liquid medium without antibiotics was added, and the mixture was placed on a shaking table at 37℃for 220 revolutions and incubated for 1 hour.

(4) Centrifuging at 5000 rpm for 1min, pouring out part of the supernatant, sucking and beating about 200 microliters of the supernatant by using a pipetting gun, uniformly mixing, spreading the bacterial liquid on a solid flat plate of LB (containing 50mg/L kan) by using a coating rod, and culturing in an incubator at 37 ℃ overnight in an inverted manner.

(5) White monoclonal bacteria are selected and verified by using carrier primer amplification. And (3) sending the clones with positive verification to sequencing, and obtaining a monoclonal recombinant plasmid vector with correct sequencing and carrying the gene NtASAT1 for preservation.

1.3 Sequence of the NtASAT1 Gene

The NtASAT1 gene sequence:

ATGGCTGCCTCAGCTCTAGTTTCTTTATCCAAGAAAATCATCAAACCATTCTCTCCAACCCCTTTTTCTGAAAGAATTTACAAGCTTTCCTTCATTGATCAATTCAATAGTACACAATATTGCCCCTTAGTCTTCTTCTATCCCAAGAATAAGGGCAATGTAGTAACACCCTCAATTGAACCAAGTGATATGTGTAAGGTTATTGAGAATTCCCTTTCAAAAACCTTAGCTGCTTATTATCCTTTTGCTGGAACATTAAGAGACAATGTTCATGTCGAATGCAACGATATAGGTGCTGATTTTTATAAGGCTCGATTCGATTGTCCCATGTCTGAAATTGTTAAAAGTCCTGATAGAAATGTCAAAGAAATGGTATATCCTAAGGGTATACCATGGAATATTGTTACATCTAATAGAAAGTTGGTCACGGTTCAATTTAACCAATTTGATTGTGGAGGAATAGCTCTAAGTACATGTGTGTCACATAAAATTGGAGATATGTGCACAATTTCTAAATTTTTACAAGACTGGGCTACAATTGCCCGTGATCCGAATTTAAAATTGTGTCCTCAATTTATTGGATCGTCAATCTTTCCACCTACTAATGAACCTGTGAATGAGCCACCTATTCAAAAATGTGTTACAAGAAGGCTAGTCTTTTCAAATCATACATTAAAATCACTCCTCTCTGAACCATCACAAGTGAAAAATCCAACTCGGGTAGAATTACTCACAGCACTTCTTTATAAATGTGGTATGAAAGCGAATTCGAGTTCATTGAAGCCATCCATTTTGTTCCAAACAGTGAATTTAAGATCTTTTATTCCTCTGCCAGATAATACTGCTGGAAATTTTAGTTCTTCCCTTTTTGTACCTACATATAATGAAGAAGAAATGATGTTATCAAGATTGGTTAGTCAGCTAAGAAAGGAAAAAGAACAACTTGTAGCTAACTACAAAAATTGTAAAGGGGGTCAAGATTTGGTTTCAACAACAATGAGACCATTTCAAGAAATAAGAAAGTTGTTTAAGGACATGGATTTTGATATGTATAGGTGTAGTAGTTTGGCTAATTATCCATTATATGATGTAGACTTTGGATGGGGTAAGCCTAATAAAATAAGTATTGCGGAAGGTGTATTTAGAAATGTTTTCCTGCTGTATGATAACAAGACAGGGGATGAAGTAGAAGCTTCTGTATGTTTGGATGAGGAAAGTACAATGTCTGCATTTTTGAGAGAGATGGAGCAGTTTCTTCAATTTGAAATTTCCTCTGAAGAAATTAAAATGGAAGCTCGATGTTGA

construction of expression vector for NtASAT1 Gene

2.1 Primer for constructing NtASAT1 gene expression vector

P-NtASAT1_F：GAACAGATTGGTGGCCAAGGATCCATGGCTGCCTCAGCTCTAGTTTCTTTA

P-NtASAT1_R：TCAGTGGTGGTGGTGGTGGTGCTCGAGTCAACATCGAGCTTCCATTTTAATTTC

2.2 Construction process of NtASAT1 gene expression vector

2.2.1 PCR amplification was performed using the positive monoclonal plasmid vector of NtASAT1 obtained in 1.2.2 above as a template, and the primers of the genes listed in 2.1.

PCR amplification was performed twice, the first 10ul system, including 0.5. Mu.L of plasmid template, 0.4. Mu.L each of forward and reverse primers (10. Mu.M), 2 XPimerstar MAX 5. Mu.L, ddH2O 3.7. Mu.L.

Then, a 50ul system PCR amplification was performed using 10ul system PCR amplification product as a template, the system comprising 10ul system PCR amplification product 0.5. Mu.L, forward and reverse primers (10. Mu.M) each 2. Mu.L, 2 XPmerstar MAX 25. Mu.L, ddH2O 20.5. Mu.L.

Setting a PCR program: 95 ℃ for 20sec;58 ℃,20sec;72 ℃,70sec;30 cycles; preserving at 16 ℃. After completion of PCR, the target fragment was detected by agarose gel electrophoresis, as shown in FIG. 2.

2.2.2 And (3) recovering the target gene fragment by using an OMEGA bio-tek gel recovery kit. The recovered target gene fragment, bamHI and XhoI were digested and linearized pATX-sumo vector fragment, and the homologous recombinases were mixed. The reaction system was 10ul (wherein the target gene fragment was 2ul, vector was 1ul, homologous recombinase was 2ul, ddH2O 5 ul) and reacted at 50℃for 30min.

2.2.3 The ligation product was transformed into DH 5. Alpha. Competent, plated and incubated overnight at 37 ℃. The method comprises the following specific steps:

(1) Adding 10 mu L of the ligation product into 50 mu L of escherichia coli DH5 alpha competent, gently mixing, and carrying out ice bath for 30min;

(2) Placing the transformed bacterial liquid in a water bath kettle at 42 ℃, carrying out heat shock for 90s, rapidly taking out and placing on ice for 3-5min;

(3) Then adding 800 mu L of liquid LB culture medium, and resuscitating for 40-50min at 37 ℃ with a shaking table at 150 rpm;

(4) Centrifuging at 3000rpm for 5min at room temperature, leaving 100 μl of supernatant in the tube, and re-suspending the thallus;

(5) The cells were transferred to LB solid plates containing 50mg/L Kan, spread with sterilized spreading bars, and after the bacterial liquid was dried, the cells were inverted and cultured overnight in an incubator at 37 ℃.

2.2.4 Colony PCR identification and sequencing: 4-8 monoclone are selected for colony PCR identification. The PCR reaction was carried out using 1. Mu.L of template, 0.4. Mu.L of forward and reverse primers (10. Mu.M), 2X Fast Taq Mastermix. Mu.L of each, and 3.2. Mu.L of ddH 2O. Setting a PCR program: 95 ℃ for 5min;95 ℃ for 30sec;55 ℃,30sec;72 ℃ for 1min;30 cycles; 72 ℃ for 5min; preserving at 16 ℃. After the PCR products are identified by agarose gel electrophoresis, 1-3 positive clones are respectively selected for sequencing, and single colony strains with correct sequencing are cultured overnight.

2.3 4mL of the bacterial liquid cultured overnight with the cloned strain is taken, and after centrifugation at 12,000rpm for 1min, bacterial cells are collected, and plasmids are extracted according to the steps of the Endo-free plasmid Mini Kit I (50) plasmid extraction kit instruction.

Protein expression of the NtASAT1 Gene

3.1 Plasmid transformation E.coli competence

(1) 100 μl of competent cells (BL 21 (DE 3), C41 strain) thawed on ice bath was added to the above-extracted plasmid, gently mixed, and placed in ice bath for 30min.

(2) The tube was then quickly transferred to an ice bath for 2min without shaking the centrifuge tube by heat shock in a 42℃water bath for 45 s.

(3) 500. Mu.l of sterile LB medium (without antibiotics) was added to the centrifuge tube, and after mixing, the mixture was incubated at 37℃for 45min at 200rpm to resuscitate the cells.

(4) A50 ul volume of the transformed competent cells was pipetted onto LB agar medium containing kanamycin antibiotic (50 ug/ml) and the cells were spread evenly. The plate was placed at 37℃until the liquid was absorbed, the plate was inverted, and incubated overnight at 37 ℃.

3.2 Activating strain, inducing expression by small system and screening condition. The method comprises the following specific steps:

(1) Single colonies containing the recombinant plasmid were picked from the plates and inoculated into 5mL LB medium containing kanamycin antibiotic (50 ug/mL) for overnight culture at 37 ℃.

(2) 200. Mu.L of the culture medium was inoculated into 20mL of LB medium containing the resistance, and cultured at 37℃until the OD600 = 0.6.

(3) 1mL of the uninduced bacterial solution was aspirated and placed in a sterilized 2mL centrifuge tube as an uninduced control. IPTG was added to the remaining bacterial liquid to a final concentration of 1mM, and the bacterial liquid after the addition of IPTG was cultured under the following different conditions: BL21 (strain No. 1) and C41 (strain No. 2) were cultured at 16℃overnight or 37℃for 4 hours.

(4) Samples of different expression conditions were collected, and cells were collected by centrifugation at 12,000rpm for 1 min. Cells were sonicated with 200ul of PBS for 2s at intervals of 2s for a total of 4min. Supernatant (native) and pellet (condensed) were centrifuged at 12000rpm in 2min and pellet was dissolved in 8 Murea+PBS. 12ul of the samples were taken and subjected to SDS-PAGE (12%, 80v gel concentrate, 20min,120v gel isolate, 45 min.) to verify expression. The expression is shown in FIG. 4.

3.3 Large scale expression and protein purification

According to the expression condition of the small system, selecting the optimal induction expression condition: expression of strain C41,1mM IPTG, overnight treatment at 16℃200ml of cells were cultivated, cells after induction of expression were collected by centrifugation, 10ml of PBS buffer was added, and the sonication was carried out for 2s at intervals of 2s for a total of 30min. After centrifugation, the supernatant was collected and protein purification was started. The method comprises the following steps of

(1) Preparing a chromatographic column: the resin is packed into a suitable column with an upper spacer over the resin.

(2) Washing resin: a10-fold resin volume Binding buffer wash (Binding buffer:50mM Tris-HCl pH 8.0,150mM NaCl,10%Glycerol) was selected based on the resin usage.

(3) Loading: the collected supernatant was passed through the column and the process could not be accelerated. The speed must be controlled for 1-2 seconds, one drop if acceleration is desired.

(4) Leaching: the binding solution must be allowed to drop down by gravity itself, and the speed (1-2 seconds-one drop) is controlled to strictly disable the drop-line formation.

W1: 10ml of the solution was rinsed with binding buffer (pH 7.5);

w2: 5ml of binding buffer (pH 7.5) containing 30mM imidazole;

w3: 5ml of binding buffer (pH 7.5) containing 50mM imidazole was rinsed.

(5) Eluting: disabling acceleration

Binding buffer (pH: 7.5) containing 200mM imidazole eluted from 0.5ml X2 tube (E1-E2);

binding buffer (pH: 7.5) containing 400mM imidazole was eluted into 0.5 ml.times.7 tubes (E3-E9).

Each eluted sample was run from SDS-PAGE gel (IN: input and FT: flow through loading was 10ul, the other loading was 20 ul). The detection results are shown in FIG. 5.

(6) Desalting and replacing buffer: the purified samples were placed in dialysis bags and placed in a displacement buffer (TBS) for 4 degrees of dialysis overnight until imidazole was substantially removed.

(7) Concentrating: the dialyzed protein samples were loaded into 10kDa ultrafiltration tubes (Millipore) and centrifuged at 4000rpm for 30min. The filtrate was discarded, and a proper volume of TBS was added to continue the centrifugation to concentrate the protein. The protein was concentrated to the appropriate concentration after three replicates.

(8) The trapped final protein samples were split and run for SDS-PAGE detection at 2ug, loading 2.5ul, as shown in FIG. 6.

3.4 NtASAT1 protein sequence

(1) NtASAT1 protein sequence

MAASALVSLSKKIIKPFSPTPFSERIYKLSFIDQFNSTQYCPLVFFYPKNKGNVVTPSIEPSDMCKVIENSLSKTLAAYYPFAGTLRDNVHVECNDIGADFYKARFDCPMSEIVKSPDRNVKEMVYPKGIPWNIVTSNRKLVTVQFNQFDCGGIALSTCVSHKIGDMCTISKFLQDWATIARDPNLKLCPQFIGSSIFPPTNEPVNEPPIQKCVTRRLVFSNHTLKSLLSEPSQVKNPTRVELLTALLYKCGMKANSSSLKPSILFQTVNLRSFIPLPDNTAGNFSSSLFVPTYNEEEMMLSRLVSQLRKEKEQLVANYKNCKGGQDLVSTTMRPFQEIRKLFKDMDFDMYRCSSLANYPLYDVDFGWGKPNKISIAEGVFRNVFLLYDNKTGDEVEASVCLDEESTMSAFLREMEQFLQFEISSEEIKMEARC

NtASAT1 protein regulates sucrose ester synthesis

The NtASAT1 protein can regulate the synthesis of sucrose monoester in 50mM pH6.0 ammonium acetate buffer with sucrose as acceptor substrate and acyl-COA as donor substrate.

4.1 NtASAT1 regulates sucrose monoester synthesis

In 60ul 50mM ammonium acetate buffer (pH 6.0), 2ug of purified NtASAT1 protein was added, acyl-COA (three acyl-CoA were used as acyl donor chains in this experiment, respectively, including acetyl-COA, isobutyryl-COA and isovaleryl-COA) was added to give a final concentration of 100. Mu.M, sucrose was added to give a final concentration of 1mM, and then the reaction system was incubated at 30℃for 30min (the reaction time may be lengthened in the case of a large system to obtain more product). Then, 120ul of the solution was added at a volume ratio of 1:1: the reaction was terminated with a mixture of acetonitrile, isopropanol and formic acid of 0.001, and after centrifugation at 12000rpm for 10 minutes, the supernatant was passed through an organic filter of 0.22. Mu.m, and then, the product was detected by LC-QTOF-HRMS.

The detection results are shown in FIG. 7. The retention time in A is 0.46, which is sucrose acetyl monoester (S1: 2); b, wherein 0.80 and 0.92 are two isomers of sucrose isobutyl monoester (S1:4); the retention times 1.44 and 1.71 in C are two isomers of sucrose isovaleryl monoester (S1:5).

As can be seen, the NtASAT1 protein is capable of catalyzing sucrose and various acyl-COAs to produce the corresponding sucrose monoesters (S1: 2, S1:4 and S1:5. Note: sn: m represents sucrose, n represents the number of acyl chains, m represents the total number of all acyl chain carbon atoms, and the same applies below). Meanwhile, when isobutyryl-COA and isovaleryl-COA are used as substrates, sucrose monoesters have the generation of isomers. The reaction system of sucrose and isovaleryl-COA is expanded to 60ml, the reaction time is prolonged to 4 hours, the main peak product of sucrose isovaleryl monoester is separated and purified, nuclear Magnetic Resonance (NMR) identification is carried out, and isovaleryl is found to be connected at the R2 position of the sucrose pyran ring.

As can be seen from the above examples of the present study, the entire sucrose ester biocatalytic reaction was performed in 50mM pH6.0 in ammonium acetate buffer, with no addition of other substances than sucrose, acyl-COA and catalytic NtASAT1 protein. Compared with the prior method for synthesizing the sucrose ester by using a substrate or a solvent, the whole catalytic reaction system has high safety, simple reaction substrate, relatively single system product and easy subsequent separation and utilization because all the products are sucrose ester. Therefore, the NtASAT1 gene and the coded protein thereof have good application value and prospect in sucrose ester biosynthesis.

The foregoing description of the preferred embodiments of the invention is not intended to be limiting, but rather is intended to cover all modifications, equivalents, alternatives, and improvements that fall within the spirit and scope of the invention.

Sequence listing

<110> tobacco institute of national academy of agricultural sciences

<120> tobacco acyl glycosyltransferase gene NtASAT1 and encoded protein and application thereof

<130> 1

<160> 6

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1305

<212> DNA

<213> tobacco (Nicotiana acuminata)

<400> 1

atggctgcct cagctctagt ttctttatcc aagaaaatca tcaaaccatt ctctccaacc 60

cctttttctg aaagaattta caagctttcc ttcattgatc aattcaatag tacacaatat 120

tgccccttag tcttcttcta tcccaagaat aagggcaatg tagtaacacc ctcaattgaa 180

ccaagtgata tgtgtaaggt tattgagaat tccctttcaa aaaccttagc tgcttattat 240

ccttttgctg gaacattaag agacaatgtt catgtcgaat gcaacgatat aggtgctgat 300

ttttataagg ctcgattcga ttgtcccatg tctgaaattg ttaaaagtcc tgatagaaat 360

gtcaaagaaa tggtatatcc taagggtata ccatggaata ttgttacatc taatagaaag 420

ttggtcacgg ttcaatttaa ccaatttgat tgtggaggaa tagctctaag tacatgtgtg 480

tcacataaaa ttggagatat gtgcacaatt tctaaatttt tacaagactg ggctacaatt 540

gcccgtgatc cgaatttaaa attgtgtcct caatttattg gatcgtcaat ctttccacct 600

actaatgaac ctgtgaatga gccacctatt caaaaatgtg ttacaagaag gctagtcttt 660

tcaaatcata cattaaaatc actcctctct gaaccatcac aagtgaaaaa tccaactcgg 720

gtagaattac tcacagcact tctttataaa tgtggtatga aagcgaattc gagttcattg 780

aagccatcca ttttgttcca aacagtgaat ttaagatctt ttattcctct gccagataat 840

actgctggaa attttagttc ttcccttttt gtacctacat ataatgaaga agaaatgatg 900

ttatcaagat tggttagtca gctaagaaag gaaaaagaac aacttgtagc taactacaaa 960

aattgtaaag ggggtcaaga tttggtttca acaacaatga gaccatttca agaaataaga 1020

aagttgttta aggacatgga ttttgatatg tataggtgta gtagtttggc taattatcca 1080

ttatatgatg tagactttgg atggggtaag cctaataaaa taagtattgc ggaaggtgta 1140

tttagaaatg ttttcctgct gtatgataac aagacagggg atgaagtaga agcttctgta 1200

tgtttggatg aggaaagtac aatgtctgca tttttgagag agatggagca gtttcttcaa 1260

tttgaaattt cctctgaaga aattaaaatg gaagctcgat gttga 1305

<210> 2

<211> 434

<212> PRT

<213> tobacco (Nicotiana acuminata)

<400> 2

Met Ala Ala Ser Ala Leu Val Ser Leu Ser Lys Lys Ile Ile Lys Pro

1 5 10 15

Phe Ser Pro Thr Pro Phe Ser Glu Arg Ile Tyr Lys Leu Ser Phe Ile

20 25 30

Asp Gln Phe Asn Ser Thr Gln Tyr Cys Pro Leu Val Phe Phe Tyr Pro

35 40 45

Lys Asn Lys Gly Asn Val Val Thr Pro Ser Ile Glu Pro Ser Asp Met

50 55 60

Cys Lys Val Ile Glu Asn Ser Leu Ser Lys Thr Leu Ala Ala Tyr Tyr

65 70 75 80

Pro Phe Ala Gly Thr Leu Arg Asp Asn Val His Val Glu Cys Asn Asp

85 90 95

Ile Gly Ala Asp Phe Tyr Lys Ala Arg Phe Asp Cys Pro Met Ser Glu

100 105 110

Ile Val Lys Ser Pro Asp Arg Asn Val Lys Glu Met Val Tyr Pro Lys

115 120 125

Gly Ile Pro Trp Asn Ile Val Thr Ser Asn Arg Lys Leu Val Thr Val

130 135 140

Gln Phe Asn Gln Phe Asp Cys Gly Gly Ile Ala Leu Ser Thr Cys Val

145 150 155 160

Ser His Lys Ile Gly Asp Met Cys Thr Ile Ser Lys Phe Leu Gln Asp

165 170 175

Trp Ala Thr Ile Ala Arg Asp Pro Asn Leu Lys Leu Cys Pro Gln Phe

180 185 190

Ile Gly Ser Ser Ile Phe Pro Pro Thr Asn Glu Pro Val Asn Glu Pro

195 200 205

Pro Ile Gln Lys Cys Val Thr Arg Arg Leu Val Phe Ser Asn His Thr

210 215 220

Leu Lys Ser Leu Leu Ser Glu Pro Ser Gln Val Lys Asn Pro Thr Arg

225 230 235 240

Val Glu Leu Leu Thr Ala Leu Leu Tyr Lys Cys Gly Met Lys Ala Asn

245 250 255

Ser Ser Ser Leu Lys Pro Ser Ile Leu Phe Gln Thr Val Asn Leu Arg

260 265 270

Ser Phe Ile Pro Leu Pro Asp Asn Thr Ala Gly Asn Phe Ser Ser Ser

275 280 285

Leu Phe Val Pro Thr Tyr Asn Glu Glu Glu Met Met Leu Ser Arg Leu

290 295 300

Val Ser Gln Leu Arg Lys Glu Lys Glu Gln Leu Val Ala Asn Tyr Lys

305 310 315 320

Asn Cys Lys Gly Gly Gln Asp Leu Val Ser Thr Thr Met Arg Pro Phe

325 330 335

Gln Glu Ile Arg Lys Leu Phe Lys Asp Met Asp Phe Asp Met Tyr Arg

340 345 350

Cys Ser Ser Leu Ala Asn Tyr Pro Leu Tyr Asp Val Asp Phe Gly Trp

355 360 365

Gly Lys Pro Asn Lys Ile Ser Ile Ala Glu Gly Val Phe Arg Asn Val

370 375 380

Phe Leu Leu Tyr Asp Asn Lys Thr Gly Asp Glu Val Glu Ala Ser Val

385 390 395 400

Cys Leu Asp Glu Glu Ser Thr Met Ser Ala Phe Leu Arg Glu Met Glu

405 410 415

Gln Phe Leu Gln Phe Glu Ile Ser Ser Glu Glu Ile Lys Met Glu Ala

420 425 430

Arg Cys

<210> 3

<211> 21

<212> DNA

<213> Artificial sequence (ordo artificialis)

<400> 3

atggctgcct cagctctagt t 21

<210> 4

<211> 24

<212> DNA

<213> Artificial sequence (ordo artificialis)

<400> 4

tcaacatcga gcttccattt taat 24

<210> 5

<211> 51

<212> DNA

<213> Artificial sequence (ordo artificialis)

<400> 5

gaacagattg gtggccaagg atccatggct gcctcagctc tagtttcttt a 51

<210> 6

<211> 54

<212> DNA

<213> Artificial sequence (ordo artificialis)

<400> 6

tcagtggtgg tggtggtggt gctcgagtca acatcgagct tccattttaa tttc 54

Claims

1. A method for regulating and controlling synthesis of sucrose ester by using protein coded by tobacco acyl glycosyltransferase gene NtASAT1, wherein the sucrose ester is sucrose monoester, is characterized in that the protein NtASAT1 uses sucrose as an acceptor substrate, uses acyl-COA as a donor substrate and regulates and controls synthesis of sucrose monoester in an ammonium acetate buffer solution; the amino acid sequence of the protein is shown as SEQ ID NO.2, ntASAT1 protein is added into an ammonium acetate buffer solution, acyl-COA is added, sucrose is added for reaction, then acetonitrile, isopropanol and formic acid mixed solution are added for stopping reaction, and the reaction is centrifugated, and supernatant fluid is filtered through an organic filter membrane to obtain sucrose monoester;

the acyl-COA comprises acetyl-COA and isobutyryl-COA, and an acetyl chain and an isobutyryl chain are correspondingly connected at the R2 position of the saccharopyran ring;

the nucleotide sequence of the tobacco acyl glycosyltransferase gene NtASAT1 is shown as SEQ ID NO.1, the sequence of a primer before cloning of the gene is shown as SEQ ID NO. 3, the sequence of a primer after cloning of the gene is shown as SEQ ID NO. 4, and the sequence of a primer P-NtASAT1_F and a primer P-NtASAT1_R used for constructing a gene NtASAT1 expression vector are shown as SEQ ID NO. 5; the sequence of the P-NtASAT1_R is SEQ ID NO. 6.

2. The method for regulating sucrose ester synthesis by using a protein encoded by the tobacco acyl glycosyltransferase gene NtASAT1 according to claim 1, wherein the method comprises the steps of: the preparation method of the monoclonal recombinant plasmid carrying the gene NtASAT1 comprises the following steps: prepared from common tobaccoN.tabacum) The method comprises the steps of taking glandular hair cDNA as a template, carrying out PCR amplification by using clone primers of a gene NtASAT1, recovering a target strip of the gene by using an agarose gel recovery kit, connecting a gene fragment to a cloning vector, adding a connection product into the competent E.coli, carrying out ice bath, after the ice bath is completed, putting into a water bath for heat shock, then carrying out ice bath again, adding a liquid culture medium for shaking table incubation, centrifuging to remove part of supernatant, coating a solid culture medium on the rest bacterial liquid, culturing in an incubator, selecting white monoclonal bacteria, carrying out amplification verification by using a vector primer, and carrying out sequencing by verifying positive clones to obtain the monoclonal recombinant plasmid vector carrying the gene NtASAT1 with correct sequencing.

3. The method for regulating sucrose ester synthesis by using a protein encoded by the tobacco acyl glycosyltransferase gene NtASAT1 according to claim 2, wherein the method comprises the steps of: the construction method of the gene NtASAT1 expression vector is as follows: amplifying the obtained positive monoclonal plasmid vector of the NtASAT1 by taking the P-NtASAT1_F and the P-NtASAT1_R as primers, recovering target gene fragments, mixing the target gene fragments with homologous recombinant enzyme, converting connection products into competence, culturing, identifying colony PCR, respectively selecting 1-3 positive clones for sequencing after identifying the PCR products by agarose gel electrophoresis, culturing single colony strains with correct sequencing overnight, centrifugally collecting thalli, and extracting plasmids.

4. A method of regulating sucrose ester synthesis by a protein encoded by the tobacco acyl glycosyltransferase gene NtASAT1 as claimed in claim 3 wherein: the protein expression method of the gene is as follows: transforming the plasmid into colibacillus competent, activating strain, inducing expression in small system and screening condition, selecting optimal inducing expression condition according to the expression condition of small system, amplifying expression in large system and purifying protein.