CN107177566A

CN107177566A - A kind of protective plant protecting agent glycosyl transferase and its encoding gene and application

Info

Publication number: CN107177566A
Application number: CN201710445349.XA
Authority: CN
Inventors: 黄璐琦; 周良云; 郭兰萍; 李佳兴; 王升; 蒋靖怡; 刘亚辉
Original assignee: Institute of Materia Medica of CAMS
Current assignee: Institute of Materia Medica of CAMS
Priority date: 2017-06-13
Filing date: 2017-06-13
Publication date: 2017-09-19
Anticipated expiration: 2037-06-13
Also published as: CN107177566B

Abstract

The invention discloses a kind of protective plant protecting agent glycosyl transferase and its encoding gene and application.Albumen provided by the present invention be it is following a) or b) or c)：A) albumen shown in sequence 1；B) by substitution and/or missing and/or addition of the sequence 1 by one or several amino acid residues, and quickbeam and the albumen as derived from sequence 1 with protective plant protecting agent glycosyl transferase activity are derived from；C) and a) or b) limiting sequence has 99%, 95%, 90%, 85% or more than 80% homology, and from quickbeam and the albumen with protective plant protecting agent glycosyl transferase activity.Present protein has protective plant protecting agent glycosyl transferase activity, can glycosylate the hydroxyl of the diverse location of biphenyl protective plant protecting agent, and then obtain diversified glucoside compound.The invention provides a kind of method for synthesizing biphenyl glycoside protective plant protecting agent, the disease resistance research and the seed selection of resistant variety for Maloideae plant are significant.

Description

A kind of protective plant protecting agent glycosyl transferase and its encoding gene and application

Technical field

The invention belongs to biological technical field, it is related to a kind of protective plant protecting agent glycosyl transferase and its encoding gene and application.

Background technology

Plant produces a large amount of non-necessary small molecular organic compounds that grow by secondary metabolism, and species is various, Chemical constitution is totally different.Currently known about 100,000 kinds secondary metabolites, including glucosides, terpene, phenols, flavonoids, cumarin, Lignanoid, alkaloid, steroid, saponin(e, polyacetylene, organic acid etc., many secondary metabolites all have important pharmacological activity And economic value.Secondary metabolite be structure basic framework formation after, then by hydroxylating, methylate, be acylated Or the modification such as small molecule is combined, ultimately generate various end-products.Glycosylation is a kind of compound-modified side being widely present Formula, is also conversion reaction important in organism --- and the final step of many metabolite synthesis, is also very important one Step, often all occurs glycosylation.The enzyme for being catalyzed glycosylation is glycosyl transferase, and it is catalyzed the glycoside formed Compound structure type is various, and is the important sources of drug leads with multiple biological activities.

In the secondary metabolism of plant, there is polytype glycosyl transferase to participate, more glycosyl is studied at present Transferase has plant hormone class glycosyl transferase, flavonoids glycosyl transferase and terpene glycosyl transferase etc., and protective plant protecting agent class Glycosyl transferase not yet have been reported that.

The content of the invention

It is an object of the invention to provide a kind of protective plant protecting agent glycosyl transferase and its encoding gene from quickbeam with Using.

Protein shown in following (a) or (b) or (c) is claimed in the present invention first：

(a) protein being made up of the amino acid sequence shown in sequence in sequence table 1；

(b) by the amino acid sequence of sequence 1 is by the substitution of one or several amino acid residues and/or missing and/or adds Plus, and derive from quickbeam and the protein as derived from sequence 1 with protective plant protecting agent glycosyl transferase activity；

(c) amino acid sequence limited with (a) or (b) have more than 99%, more than 95%, more than 90%, 85% with Upper or more than 80% homology, and derive from quickbeam and the protein with protective plant protecting agent glycosyl transferase activity.

, can be as follows in amino terminal or the carboxyl terminal connection of the protein for the ease of the protein purification Label shown in table.

Table：The sequence of label

The nucleic acid molecules of code for said proteins fall within protection scope of the present invention.

The nucleic acid molecules can be DNA, such as cDNA, genomic DNA or recombinant DNA；The nucleic acid molecules can also be RNA, such as mRNA, hnRNA or tRNA.

In one embodiment of the invention, the nucleic acid molecules are specially the gene of code for said proteins, the base Because concretely it is following it is any shown in DNA molecular：

1) DNA molecular in sequence table shown in sequence 2；

2) DNA molecular hybridization under strict conditions with 1) restriction and the DNA molecular of code for said proteins；

1) or 2) 3) have with the DNA sequence dna that limits more than 99%, more than 95%, more than 90%, more than 85% or More than 80% homology, and the DNA molecular of code for said proteins.

Above-mentioned stringent condition can be that with 6 × SSC, 0.5%SDS solution hybridizes at 65 DEG C, then with 2 × SSC, 0.1%SDS and 1 × SSC, 0.1%SDS respectively wash film once.

Recombinant vector, expression cassette, transgenic cell line or recombinant bacterium containing above-mentioned nucleic acid molecules fall within the guarantor of the present invention Protect scope.

The recombinant vector can be recombinant expression carrier, or recombinant cloning vector.

The recombinant expression carrier can use existing expression vector establishment.The expression vector can also include foreign gene 3 ' ends untranslated region, i.e., the DNA fragmentation comprising polyadenylation signals and any other participation mRNA processing or gene expression.Institute State the 3 ' ends that the bootable polyadenylic acid of polyadenylation signals is added to mRNA precursor.Use the gene constructed recombinant expression carrier When, any enhanced, composing type, organizing specific type or inducible promoter can be added before its transcription initiation nucleotides, They can be used alone or are used in combination with other promoters；In addition, using the gene constructed recombinant expression carrier of the present invention When, enhancer, including translational enhancer or transcriptional enhancer are it is also possible to use, these enhancer regions can be ATG initiation codons Son or neighboring region initiation codon etc., but must be identical with the reading frame of coded sequence, to ensure that the correct of whole sequence turns over Translate.The source of the translation control signal and initiation codon is extensive, can be natural or synthesis.Turn over Translate initiation region and can come from transcription initiation region or structural gene.

In one embodiment of the invention, the recombinant vector be pET-28a (+) carrier multiple cloning sites (such as BamH I and Not I) between insert the recombinant plasmid that the gene is obtained.

The expression cassette is by that can start the promoter of the gene expression, the gene, and transcription terminator group Into.

In one embodiment of the invention, the recombinant bacterium is the Escherichia coli containing the recombinant vector；It is described big Enterobacteria is specific such as Transetta (DE3).

Application of the protein in as glycosyl transferase falls within protection scope of the present invention.

The protein or the nucleic acid molecules or the recombinant vector, expression cassette, transgenic cell line or recombinant bacterium exist The application prepared in the product with glycosyl transferase activity falls within protection scope of the present invention.

In above two application, specifically glycosylated receptor substrate can be used as using protective plant protecting agent；More specific, it is described to plant It is biphenyl protective plant protecting agent to protect element.

In the present invention, the biphenyl protective plant protecting agent is specially the compound as shown in following formula I, Formula II or formula III：

Further, the protein is produced as glycosylation compound of formula I ' shown in glycosyl transferase catalysis type I Suo Shi Thing；The protein is used as glycation product shown in compound of formula II ' shown in glycosyl transferase catalysis type II；The albumen Matter is used as glycation product shown in compound of formula III ' shown in glycosyl transferase catalysis type III.

Secondly, (A) as follows or (B) or the method shown in (C) is also claimed in the present invention.

(A) prepare Formulas I above ' shown in glycation product method, comprise the following steps：Turn by glycosyl of the protein Enzyme is moved, using compound shown in Formulas I above as glycosylated receptor substrate, using UDPG as glycosyl donor, enzymatic is carried out Reaction.

(B) prepare hereinbefore Formula II ' shown in glycation product method, comprise the following steps：Using the protein as sugar Based transferase, using compound shown in Formula II above as glycosylated receptor substrate, using UDPG as glycosyl donor, is carried out Enzymatic reaction.

(C) prepare formula III above ' shown in glycation product method, comprise the following steps：Using the protein as glycosyl Transferase, using compound shown in formula III above as glycosylated receptor substrate, using UDPG as glycosyl donor, is carried out Enzymatic reaction.

Further, in methods described, the mol ratio of the receptor substrate and the donor can be 1：2；Shifted as glycosyl The protein of enzyme is enough.

In the process, the temperature for carrying out the enzymatic reaction is 30 DEG C；Reaction time is 16h.

It is demonstrated experimentally that the protein shown in the sequence 1 provided by the present invention from quickbeam has protective plant protecting agent sugar Based transferase activity, can glycosylate the hydroxyl of the diverse location of three shown in Formulas I, Formula II and formula III kind biphenyl protective plant protecting agent, And then diversified glucoside compound can be obtained.The invention provides it is a kind of synthesize biphenyl glycoside protective plant protecting agent method, for The disease resistance research and the seed selection of resistant variety of Maloideae plant are significant.

Brief description of the drawings

When Fig. 1 is using compound 1 and UDPG as glycosyl acceptor and glycosyl donor, SaUGT7 catalysis glycosyls Change the UPLC-UV detection collection of illustrative plates of reaction product.

When Fig. 2 is using compound 2 and UDPG as glycosyl acceptor and glycosyl donor, SaUGT7 catalysis glycosyls Change the UPLC-UV detection collection of illustrative plates of reaction product.

When Fig. 3 is using compound 3 and UDPG as glycosyl acceptor and glycosyl donor, SaUGT7 catalysis glycosyls Change the UPLC-UV detection collection of illustrative plates of reaction product.

Fig. 4 is that UPLC-UV/ESI-MS detection restructuring glycosyl transferase SaUGT7 catalytic cpds 1 carry out glycosylation. (A) glycosylation of SaUGT7 catalytic cpds 1, compound 1 and product 1a, 1b UPLC spectrograms and compound 1UV absorption spectras Figure；(B) product 1a UV absorbs spectrogram and in the negative ion mode MS spectrograms, and its molecular ion peak is m/z 407.1289 [M-H]^-；(C) product 1b UV absorbs spectrogram and in the negative ion mode MS spectrograms, and its molecular ion peak is m/z 407.1288[M-H]^-。

Fig. 5 is that UPLC-UV/ESI-MS detection restructuring glycosyl transferase SaUGT7 catalytic cpds 2 carry out glycosylation. (A) glycosylation of SaUGT7 catalytic cpds 2, compound 2 and product 2a, 2b UPLC spectrograms and compound 2UV absorption spectras Figure；(B) product 2a UV absorbs spectrogram and in the negative ion mode MS spectrograms, and its molecular ion peak is the [M of m/z 453.1360 +HCOO]^-；(C) product 2b UV absorbs spectrogram and in the negative ion mode MS spectrograms, and its molecular ion peak is m/z 453.1357[M+HCOO]^-。

Fig. 6 is that UPLC-UV/ESI-MS detection restructuring glycosyl transferase SaUGT7 catalytic cpds 3 carry out glycosylation. (A) glycosylation of SaUGT7 catalytic cpds 3, compound 3 and product 3a and 3b UPLC spectrograms absorb spectrogram with (3) UV； (B) product 3a UV absorbs spectrogram and in the negative ion mode MS spectrograms, and its molecular ion peak is the [M- of m/z 377.1170 H]^-；(C) product 3b UV absorbs spectrogram and in the negative ion mode MS spectrograms, and its molecular ion peak is m/z 377.1169 [M-H]^-。

Fig. 7 is catalyzed the glycation product structure of substrate structure and correspondence product for restructuring glycosyl transferase SaUGT7.

Embodiment

Experimental method used in following embodiments is conventional method unless otherwise specified.

Material, reagent used etc., unless otherwise specified, are commercially obtained in following embodiments.

Quickbeam suspension cell：It is recorded in " the such as Huang Lei, Xiao Wenjuan, Yang Guang yeast extracts induction quickbeam suspension The mechanism of cell synthesis secondary metabolite probes into CHINA JOURNAL OF CHINESE MATERIA MEDICAs, 2014,39 (11):The texts of 2019-2023 " one, Gong Zhongke Obtained at applicant, can only be used to repetition present invention experiment and use.

The clone of embodiment 1, quickbeam protective plant protecting agent glycosyltransferase gene

Total serum IgE is extracted from quickbeam suspension cell, and reverse transcription obtains cDNA.Using gained cDNA as template, use Primer 1 and primer 2 enter performing PCR reaction.

Primer 1：5’-GAGGGATCCATGAAGAGACCAGTACAACT-3’；

Primer 2：5’-GAGGCGGCCGCTTAAATTTGATTAATAAAAC-3’。

PCR reaction systems are as follows：KOD-Plus-Neo 2μL；2mM dNTPs 10μL；25mM MgSO₄6μL；10×PCR buffer for KOD-Plus-Neo 10μL；Primer 1 and each 3 μ L of primer 2；The μ L of cDNA templates 2；ddH₂O complements to 100 μ L.

PCR reaction conditions：94℃10min；94 DEG C of 30s, 55 DEG C of 30s, 72 DEG C of 1min, 35 circulations；72℃10min；4℃ Maintain.

PCR carries out 1.5% agarose gel electrophoresis detection and sequencing after terminating to gained PCR primer.Sequencing result shows, The sequence of PCR primer is " 5 '-GAGGGATCC+ sequence 2+GCGGCCGCCTC”.It is SaUGT7 by unnamed gene shown in sequence 2. Protein in the polynucleotide of sequence 2 shown in sequence 1, the protein is named as SaUGT7.

The heterogenous expression of embodiment 2, quickbeam protective plant protecting agent glycosyl transferase

1st, the structure of recombinant expression carrier

By PCR primer " the 5 '-GAG of embodiment 1GGATCC+ sequence 2+GCGGCCGCAfter CTC " purifying is reclaimed, with restricted Restriction endonuclease BamH I and Not I carry out double digestion, with pET-28a (+) plasmid by same double digestion after digestion products recovery Skeleton large fragment be connected, obtain recombinant plasmid.

It will show through sequencing, between restriction enzyme site BamH I and the Not I of pET-28a (+) plasmid shown in insetion sequence 2 Recombinant plasmid after DNA fragmentation is named as pET-28a-UGT7.

2nd, expressive host system constructing

The recombinant expression carrier pET-28a-UGT7 that step 1 is built is transformed into Escherichia coli Transetta (DE3) senses By state cell, select positive colony and enter after performing PCR checking, send the correctness of sequence verification expression system.

3rd, induced expression and destination protein SaUGT7 extraction

(a) aspiration step 2 correctly expresses the 200mL LB liquid training that bacterial strain goes to the Kan containing 50mg/L through sequence verification Support in base, 37 DEG C of shaken cultivations to OD₆₀₀To 0.6-1.0；

(b) appropriate IPTG derivants (final concentration is about 0.4mM), 30 DEG C, 200rpm Fiber differentiations 5h are added；

(c) 200mL bacterium solutions, 4 DEG C, 5000g centrifugation 10min abandon bacterium solution, collect thalline；

(d) bacterium solution is cleaned with the pure water of precooling 2 times, 4 DEG C, 5000g centrifugation 10min abandon bacterium solution, collect thalline；

(e) with the buffer A of precooling【Formula：Tris-HCl (pH7.4,1M) 2.5mL；EDTA(0.5M)100μL；Glycerine (50%V/V) 10 μ L；PMSF(100mM)500μL；ddH₂O 36.9mL】It is resuspended (8mL), inhaling to beat with pipette tips dissolves thalline；

(f) with the ultrasonication of ultrasonic cell-break machine (30% power, ultrasonic 5s are spaced 5s, continue 5min)；

(g) 4 DEG C, 13000rpm centrifugations 15min；

(h) supernatant is taken to dispense -80 DEG C of refrigerators of (200 μ L/ pipes) placement standby.

Experiment is provided with the control that pET-28a-UGT5 is substituted with pET-28a (+) plasmid simultaneously, obtains in unloaded control Clearly.

The activity checking of embodiment 3, quickbeam protective plant protecting agent glycosyl transferase

First, experimental method

1st, the screening of heterogenous expression glycosyl transferase catalytic activity

In glycosyl transferase activity preliminary screening, screening experiment is carried out with SaUGT7 crude enzyme liquid.Respectively with Rhaphiolepsin (compound 1, shown in formula I), 2'-Hydroxyaucuparin (compound 2, as shown in Formula II) and Noraucuparin (compound 3, as shown in formula III) these three biphenyl protective plant protecting agents are glycosylated receptor substrate, with UDP- Portugals Grape sugar carries out enzymatic reaction as donor.

Glycosyl transferase activity screens reaction system：SaUGT7 crude enzyme liquids (supernatant i.e. in the step 3 (h) of embodiment 2) 194μL；The μ L of UDPG (40mM) 4；The μ L of receptor substrate (40mM) 2.

Experiment is used as control using unloaded control supernatant prepared by embodiment 2 simultaneously.

Glycosyl transferase activity screens reaction condition：Under the conditions of 30 DEG C, 16h is reacted, 400 μ L methanol is added and terminates instead Should, shaking is mixed, Isosorbide-5-Nitrae 000g centrifugation 20min, takes supernatant to cross 0.22 μm of filter membrane, to be measured.

2nd, enzymatic preparation UPLC is detected

Chromatographic condition：Chromatographic column is Waters ACQUITY UPLC BEH C18 ultra high efficiency liquid phase posts (Waters Corporation, Milford, MA, USA, 2.1 × 100mm, 1.7 μm), 35 DEG C of column temperature.Mobile phase is 0.1% formic acid-water-soluble Liquid (A) -0.1% formic acid-acetonitrile (B) (% represents volumn concentration), gradient elution program is (0-4) min, 10%-40% B；(4.0-5.0) min, 40%-52%B；(5.0-8.0) min, 52%-72%B；(8.0-8.2) min, 72%-95%B (% Represent volumn concentration).Flow velocity 0.5mL/min.Sample size：1μL.

3rd, the preparation of SaUGT7 catalysis glycosylation iodine and product is with separating

SaUGT7 crude enzyme liquid is amplified enzymatic reaction and glycation product is prepared.Reaction system is 30mL, including SaUGT7 crude enzyme liquids (supernatant i.e. in the step 3 (h) of embodiment 2), UDPG (40mM) 0.5mL, receptor substrate (80mM) 0.125mL, is reacted in 30 DEG C of water-baths and stays overnight, and is extracted 5 times with 2 times of volume of ethylacetate, and organic phase pressurization is used about after being evaporated 2mL methanol is dissolved, and crosses 0.22 μm of filter membrane, and half is carried out using acetonitrile-water as mobile phase and is prepared.Liquid phase systems：Shimadzu preparation solution Chromatography LC-20AR；Preparing post is：YMC-Pack ODS-A(20×250mm,5.0μm).Isolated each product is entered Row high resolution mass spectrum and¹H-NMR,¹³C-NMR, H-H COSY, HSQC and HMBC are analyzed and identified.

2nd, experimental result

In preliminary screening is carried out to glycosyl transferase activity, saccharide donor, 3 kinds are used as using the UDPG that market is bought Biphenyl protective plant protecting agent (Formulas I, Formula II and formula III) carries out screening active ingredients experiment as acceptor.Detect that polarity becomes big by UPLC-UV Glycation product, testing result shows that, relative to zero load, heterogenous expression SaUGT7 crude enzyme liquid can be catalyzed 3 kinds of each shapes of biphenyl substrate Into two kinds of new products, as shown in Figure 1, Figure 2 and Figure 3.

2nd, restructuring glycosyl transferase SaUGT7 is catalyzed the research of glycation product

Fig. 4 shows that the UV absorption spectrograms of the product 1a and 1b in Fig. 1 and receptor substrate 1 are basically identical, and 1a and 1b molecules Quasi-molecular ions is respectively m/z 407.1289 [M-H]^-With m/z 407.1288 [M-H]^-, it is big by 162 compared with the molecular weight of receptor substrate 1.Cause This, product 1a and 1b are respectively to add 1 molecule glucose on the different hydroxyls position of receptor substrate 1.

Fig. 5 shows that the UV absorption spectrograms of the product 2a and 2b in Fig. 2 and receptor substrate 2 are basically identical, and 2a and 2b molecules Quasi-molecular ions is respectively m/z 453.1360 [M+HCOO]^-With m/z 453.1357 [M+HCOO]-, it is big compared with the molecular weight of receptor substrate 2 162.Therefore, product 2a and 2b is respectively to add 1 molecule glucose on the different hydroxyls position of receptor substrate 2.

Fig. 6 shows that the UV absorption spectrograms of the product 3a and 3b in Fig. 3 and receptor substrate 3 are basically identical, and 3a and 3b molecules Quasi-molecular ions is respectively m/z 377.1170 [M-H]^-With m/z 377.1169 [M-H]^-, it is big by 162 compared with the molecular weight of receptor substrate 3.Cause This, product 3a and 3b are respectively to add 1 molecule glucose on the different hydroxyls position of receptor substrate 3.

3rd, glycosylation modified product is prepared using recombinating glycosyl transferase SaUGT7

In order to further determine that restructuring glycosyl transferase SaUGT7 is catalyzed to form the glycosylation positions of different compounds, and then Systematicness evaluates SaUGT7 catalysis, and the present invention has carried out enzymatic to the glycation product of the SaUGT7 with bioactivity The preparation of iodine.The glycation product preparation condition of each catalytic receptor substrate is shown in Table 1.All compounds prepared are sharp With MS,¹H-NMR,¹³C-NMR, H-H COSY, HSQC and HMBC carry out the confirmation of glycosylation position, and its structure is shown in Fig. 7.

It is prepared by the glycation product of table 1

The MS of glycation product,¹H-NMR,¹³C-NMR data are specific as follows：

Rhaphiolepsin 4-O-β-D-glucopyranoside(1b)

HR-ESI-MS(neg)m/z 407.1288[M-H]^-(Calcd.for C₂₀H₂₃O₉,407.1342),ESI-MS (neg)m/z 407[M-H]^-,245[M-H-162]^-.¹H-NMR(600MHz,CD₃OD) δ 7.39 (2H, dd, J=8.6,2.0Hz, H-2 ', 6 '), 6.87 (2H, d, J=8.6,2.0Hz, H-3 ', 5 '), 6.62 (2H, s, H-2,6), 4.61 (1H, d, J=7.8Hz, glc-H-l),3.25-3.72(6H,m,glc-H-2,3,4,5,6),3.80(3H,s,3-OCH ₃),3.72(3H,s,4′-OCH ₃) ；¹³C-NMR(150MHz,CDCl₃)δ138.3(C-1),105.5(C-2),150.7(C-3),133.0(C-4),152.9(C-5), 107.4(C-6),133.3(C-1′),127.5(C-2′,6′),113.7(C-3′,5′),159.4(C-4′),55.4(3- OCH₃),54.3(4′-OCH₃),102.1(glc-C-1),74.0(glc-C-2),77.0(glc-C-3),69.5(glc-C-4), 76.3(glc-C-5),60.7(glc-C-6).

2'-Hydroxyaucuparin 2'-O-β-D-glucopyranoside(2b)

HR-ESI-MS(negative)m/z 407.1288[M-H]^-(Calcd.for C₂₀H₂₃O₉,407.1342),ESI- MS(neg)m/z 407[M-H]^-,245[M-H-162]^-.¹H-NMR(600MHz,CD₃OD):7.27-7.30(2H,m,H-4′, 6 '), 7.04-7.05 (2H, m, H-3 ', 5 '), 6.78 (2H, s, H-2,6), 4.83 (1H, d, J=7.8Hz, glc-H-l), 3.32-3.94(6H,m,glc-H-2,3,4,5,6),3.78(6H,s,3,5-OCH₃)；¹³C-NMR(150MHz,CDCl₃):δ 130.7(C-1),107.3(C-2,6),152.4(C-3,5),135.5(C-4),130.7(C-1′),153.9(C-2′),115.7 (C-3′),128.6(C-4′),119.5(C-5′),130.2(C-6′),55.7(3,5-OCH₃),101.8(glc-C-1),73.7 (glc-C-2),77.0(glc-C-3),69.9(glc-C-4),76.1(glc-C-5),63.0(glc-C-6).

Noraucuparin 4-O-β-D-glucopyranoside(3b)

HR-ESI-MS(negative)m/z 377.1169[M-H]^-(Calcd.for C₁₉H₂₁O₈,377.1236),ESI- MS(neg)m/z 377[M-H]^-,215[M-H-162]^-.¹H-NMR(600MHz,CD₃OD) δ 7.61 (2H, d, J=7.4Hz, H- 2 ', 6 '), 7.43 (1H, t, J=7.6Hz, H-3 ', 5 '), 7.33 (1H, t, J=7.4Hz, H-4 '), 6.76 (1H, d, J= 1.9Hz, H-6), 6.74 (1H, d, J=1.9Hz, H-2), 4.63 (1H, d, J=7.7Hz, glc-H-l), 3.14-3.67 (6H, m,glc-H-2,3,4,5,6),3.83(3H,s,3-OCH ₃)；¹³C-NMR(150MHz,CD₃OD)δ133.1(C-1),104.5(C- 2),152.5(C-3),136.2(C-4),150.1(C-5),107.1(C-6),139.5(C-1′),126.0(C-2′,6′), 128.2(C-3′,5′),126.8(C-4′),55.7(3-OCH₃),102.4(glc-C-1),73.3(glc-C-2),76.6 (glc-C-3),69.0(glc-C-4),75.6(glc-C-5),60.1(glc-C-6).

<110>Institute Of Chinese Materia Medica Of China Academy of Chinese Medical Sciences

<120>A kind of protective plant protecting agent glycosyl transferase and its encoding gene and application

<130> GNCLN171140

<160> 2

<170> PatentIn version 3.5

<210> 1

<211> 471

<212> PRT

<213>Quickbeam（Sorbus aucuparia L.）

<400> 1

Met Lys Arg Pro Val Gln Leu Val Leu Val Pro Ala Pro Val Ile Gly

1 5 10 15

His Ile Val Ser Ala Ile Glu Ile Ala Glu Gln Leu Val Ala Arg Asp

20 25 30

Asp Gln Leu Phe Ile Thr Val Leu Val Met Lys Leu Pro Tyr Asp Gln

35 40 45

Pro Phe Thr Asn Thr Asp Ser Ser Ile Ser His Arg Ile Asn Phe Val

50 55 60

Asn Leu Pro Glu Ala Gln Gln Asp Lys Lys Glu Ala Ile Pro Lys Pro

65 70 75 80

Gly Ser Leu Ile Arg Ile Leu Val Glu Asn His Lys Thr His Ile Arg

85 90 95

Asp Ala Val Val Asn Leu Leu Ser Glu Ser Asp Gln Ser Glu Ser Met

100 105 110

Ser Lys Pro Arg Leu Ala Gly Phe Val Leu Asp Phe Phe Cys Ala Thr

115 120 125

Leu Val Asp Val Ala Asp Glu Phe Met Val Pro Ser Tyr Val Phe Phe

130 135 140

Thr Ser Asn Ala Ser Leu Leu Ala Leu Leu Phe His Phe Gln Leu Leu

145 150 155 160

Arg Asp Glu Gly Gly Ile Asp Ile Thr Glu Phe Thr Arg Ser Ser Ala

165 170 175

Glu Phe Ala Val Pro Ser Phe Ile Asn Pro Tyr Pro Ala Ala Val Leu

180 185 190

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

195 200 205

Thr Ser Arg Tyr Arg Gln Ile Lys Gly Ile Leu Val Asn Thr Phe Leu

210 215 220

Glu Leu Asp Leu His Ala Leu His Tyr Leu Asp Phe Asp Asp Lys Ile

225 230 235 240

Pro Pro Val Tyr Pro Val Gly Pro Leu Leu Asn Leu Lys Ser Ser Asp

245 250 255

Glu Asn Lys Gly Ser Asp Ile Leu Arg Trp Leu Asp Asp Gln Pro Pro

260 265 270

Phe Ser Val Leu Phe Leu Cys Phe Gly Ser Met Gly Ser Phe Gly Glu

275 280 285

Ala Gln Val Lys Glu Ile Ala Cys Ala Leu Glu His Ser Gly Gln Arg

290 295 300

Phe Leu Trp Ser Leu Arg Arg Pro Pro Pro Lys Gly Glu Met Ala Met

305 310 315 320

Pro Ser Asp Tyr Ala Asp Leu Asn Ala Val Leu Pro Glu Gly Phe Leu

325 330 335

Asn Arg Thr Ala Pro Ile Gly Lys Val Ile Gly Trp Ala Pro Gln Ala

340 345 350

Ala Ile Leu Ala His Pro Ala Thr Gly Gly Phe Val Ser His Cys Gly

355 360 365

Trp Asn Ser Thr Leu Glu Ser Leu Trp Asn Gly Val Pro Ile Ala Thr

370 375 380

Trp Pro Met Tyr Ala Glu Gln Asn Leu Asn Ala Phe Gln Leu Val Lys

385 390 395 400

Glu Leu Gly Leu Ala Val Glu Ile Lys Met Asp Tyr Arg Lys Asp Ser

405 410 415

Asp Val Val Val Ser Ala Glu Asp Ile Glu Arg Gly Ile Arg Gln Val

420 425 430

Met Glu Leu Asp Gly Asp Val Arg Lys Arg Val Lys Glu Met Ser Glu

435 440 445

Lys Ser Lys Lys Thr Leu Val Lys Gly Gly Ser Ser Tyr Ser Ser Leu

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Gly Arg Phe Ile Asn Gln Ile

465 470

<210> 2

<211> 1416

<212> DNA

<213>Quickbeam（Sorbus aucuparia L.）

<400> 2

60

gcgatcgaga ttgcagagca actcgttgct cgggacgacc aactgtttat cacagtcctc 120

gtcatgaagc ttccctacga ccaaccattc accaacactg actcttcaat ctctcaccgc 180

atcaacttcg tcaacctccc cgaagcccaa caggacaaaa aagaagccat ccccaaaccc 240

gggtccttga tcagaatcct cgtcgaaaat cacaaaactc acatcagaga cgccgttgtc 300

aacttactct ctgagtcaga tcagtctgag tcgatgtcga agcctcggct tgccgggttc 360

gtgctcgact ttttttgcgc aaccctagtt gacgtggctg acgaatttat ggttccctcc 420

tacgtgttct tcacttccaa cgcttcgctg ctcgcacttt tgttccattt tcaattgctt 480

cgcgacgagg gcggcataga tataactgag tttacgcgct cgagcgctga gtttgccgtc 540

ccgagtttca tcaaccctta ccctgccgca gtcttgcccg gtacgtttgc ggacaaagag 600

atcaccgaat caatcctcaa cactaccagc aggtatagac aaatcaaggg tattttggta 660

aatacattct tggagctgga tttgcatgct cttcattatc ttgattttga tgataaaatc 720

ccacccgtgt atccagtggg gcccttgttg aacctcaaaa gtagtgatga aaataagggc 780

tcggatatct tgaggtggct tgatgatcag ccccctttct cggtgctgtt cctatgtttt 840

gggagcatgg gaagtttcgg tgaggcccag gtgaaagaga tagcttgtgc gctggagcat 900

agcgggcagc ggtttttgtg gtccctacgc cggcccccac ctaagggcga gatggctatg 960

ccaagcgact atgcggatct aaacgcggtc ttgcccgaag ggtttcttaa tcggacagct 1020

ccgattggga aggtgatagg ttgggccccg caagcggcca tcctggccca tccggcgacc 1080

ggagggtttg tatcacattg cgggtggaat tctacgttgg aaagtttatg gaatggggtg 1140

ccgattgcca cgtggccaat gtacgcggag caaaacctga atgcctttca actggtgaag 1200

gaattgggat tggcagtgga gattaagatg gattatagga aggacagtga cgtggtggtg 1260

agtgcagaag atatagaaag agggataagg caagtgatgg agctcgatgg tgatgtacgg 1320

aaaagagtga aggagatgag tgaaaagagc aagaaaacct tggtgaaagg tggttcttcc 1380

tactcttcat taggacgttt tattaatcaa atttaa 1416

Claims

1. protein, is following (a) or (b) or (c)：

(b) amino acid sequence of sequence 1 is passed through to the substitution and/or missing and/or addition of one or several amino acid residues, and From quickbeam and with protective plant protecting agent glycosyl transferase activity the protein as derived from sequence 1；

(c) amino acid sequence limited with (a) or (b) have more than 99%, more than 95%, more than 90%, more than 85% or The homology of person more than 80%, and derive from quickbeam and the protein with protective plant protecting agent glycosyl transferase activity.

2. encode the nucleic acid molecules of protein described in claim 1.

3. nucleic acid molecules according to claim 2, it is characterised in that：The nucleic acid molecules are described in coding claim 1 The gene of protein, the gene is following DNA molecular 1) or 2) or 3)：

1) DNA molecular in sequence table shown in sequence 2；

2) hybridize under strict conditions with the DNA molecular of 1) restriction and encode protein DNA molecule described in claim 1；

1) or 2) 3) have with the DNA sequence dna that limits more than 99%, more than 95%, more than 90%, more than 85% or 80% with Upper homology, and protein DNA molecule described in coding claim 1.

4. recombinant vector, expression cassette, transgenic cell line or recombinant bacterium containing nucleic acid molecules described in Claims 2 or 3.

5. application of the protein described in claim 1 in as glycosyl transferase.

6. the nucleic acid molecules described in protein or claim 3 or 4 described in claim 1 or 2 or the weight described in claim 5 Group carrier, the application of expression cassette, transgenic cell line or recombinant bacterium in the product with glycosyl transferase activity is prepared.

7. the application according to claim 5 or 6, it is characterised in that：In the application, using protective plant protecting agent as it is glycosylated by Body substrate；

Specifically, the protective plant protecting agent is biphenyl protective plant protecting agent.

8. application according to claim 7, it is characterised in that：The biphenyl protective plant protecting agent is such as following formula I, Formula II or formula III Shown compound：

9. application according to claim 8, it is characterised in that：In the application, protein is used as sugar described in claim 1 Glycation product shown in compound of formula I ' shown in based transferase catalysis type I；

In the application, protein described in claim 1 is used as compound of formula II ' institutes shown in glycosyl transferase catalysis type II Show glycation product；

In the application, protein described in claim 1 is used as compound of formula III ' shown in glycosyl transferase catalysis type III Shown glycation product；

10. method, is following (A) or (B) or (C)：

(A) prepare claim 9 in Formulas I ' shown in glycation product method, comprise the following steps：With egg described in claim 1 White matter is glycosyl transferase, and compound shown in Formulas I is made as glycosylated receptor substrate with UDPG using in claim 9 For glycosyl donor, enzymatic reaction is carried out；

(B) prepare claim 9 in Formula II ' shown in glycation product method, comprise the following steps：With described in claim 1 Protein is glycosyl transferase, and compound shown in Formula II is glycosylated receptor substrate using in claim 9, with UDPG As glycosyl donor, enzymatic reaction is carried out；

(C) prepare claim 9 in formula III ' shown in glycation product method, comprise the following steps：With described in claim 1 Protein is glycosyl transferase, and compound shown in formula III is glycosylated receptor substrate using in claim 9, with UDP- grapes Sugar carries out enzymatic reaction as glycosyl donor.