CN109321543A

CN109321543A - The oxygen transmethylase and its encoding gene of participation orange peel flavones synthesis and application

Info

Publication number: CN109321543A
Application number: CN201811310003.XA
Authority: CN
Inventors: 孙崇德; 刘晓娟; 赵晨拧; 王岳; 曹锦萍; 李鲜; 陈昆松
Original assignee: Zhejiang University ZJU
Current assignee: Zhejiang University ZJU
Priority date: 2018-11-02
Filing date: 2018-11-02
Publication date: 2019-02-12
Anticipated expiration: 2038-11-02
Also published as: CN109321543B

Abstract

The present invention provides the oxygen transmethylase for participating in the synthesis of orange peel flavones and its encoding gene and application, the expression quantity of CitOMT1 are positively correlated in bowl mandarin orange (Citrus reticulata cv.Suavissima) Fruit Development Process with the accumulation of polymethoxyflavone；External enzyme activity is experiments have shown that the oxygen transmethylase can be applied in catalysis flavone compound specific site methylation, it is particularly possible to be catalyzed Quercetin and generate 3 ', 4 ' dimethyl ester of Quercetin；It is catalyzed luteolin and generates chrysoeriol；It is catalyzed eriodictyol and generates hesperetin, eriodictyonone, catalysis baicalein generates qroxylin A；It is catalyzed 7,8- dihydroxyflavone and generates 7- hydroxyl -8- methoxy flavone.The enzyme activity reaction condition is simple, and does not need additionally to add metal ion, application value with higher and Research Prospects.

Description

The oxygen transmethylase and its encoding gene of participation orange peel flavones synthesis and application

Technical field

The invention belongs to plant molecular biotechnology and gene engineering technology fields, and in particular to a kind of participation orange peel The oxygen transmethylase CitOMT1 and its encoding gene of flavones synthesis and application.

Background technique

Flavone compound is one of secondary metabolite important in plant, in plant growth, development and degeneration-resistant middle hair Wave important role.Existing document shows that the flavones of oxygen methylation is cardiovascular in anti-oxidant, antitumor, hypoglycemic and prevention Disease etc. has stronger activity, and it is active because the site of methoxyl group and the variation of quantity are different.However from plant It is isolated and purified in object or the flavones process flow of chemical synthesis oxygen methylation is complex.There are oxygen transmethylases in plant (O-methyltransferase, OMT) can be methylated with the flavone compound of catalytic hydroxylation in specific site, therefore It is with high application prospect that oxygen methylation flavones synthesis is carried out by OMT enzyme process.

There are the synthesis that a variety of OMT take part in polymethoxyflavone in orange peel (PMFs) in citrus.There is document report There are a kind of OMT in citrus, can be catalyzed 3,5,6,7 hydroxyls of flavones, and can be reacted by Bacillus coli cells The 3 of system catalytic Quercetin, 3 ', 5,7 sites methylation, ultimately generate 3,3 ', 5,7 four methyl esters of Quercetin.But from plant Separation identification obtain being catalyzed simultaneously 3 ' position of flavones, 4 ' positions, 6,8 oxygen transmethylases have not been reported.

Summary of the invention

The object of the present invention is to provide the oxygen transmethylase for participating in the synthesis of orange peel flavones, which comes Derived from Rutaceae citrus plant bowl mandarin orange, belong to II class oxygen transmethylase, but independent of metal ion.And external enzyme activity examination It tests and shows that CitOMT1 can be catalyzed Quercetin and most generate 3 ', 4 ' methyl esters of Quercetin eventually；It is catalyzed luteolin and generates chrysoeriol； It is catalyzed eriodictyol and generates hesperetin, eriodictyonone；It is catalyzed baicalein and generates qroxylin A；7,8- dihydroxyflavone is catalyzed to generate 7- hydroxyl -8- methoxy flavone.Its enzyme activity reaction condition is simple, and does not need additionally to add metal ion, easily operated.

To achieve the above object, the present invention adopts the following technical scheme:

One provided by the invention participates in the oxygen transmethylase CitOMT1 of orange peel flavones synthesis, amino acid sequence It is classified as following one:

(1) amino acid sequence shown in SEQ:No.1；

(2) or by amino acid sequence shown in SEQ:No.1 by one or several amino acid residues substitution, lack, add Add and the identical protein of function.

A second object of the present invention is to provide the encoding gene of the oxygen transmethylase CitOMT1, nucleotides sequences It is classified as following one:

(1) nucleotide sequence shown in SEQ:No.2；

(2) or with nucleotide sequence shown in SEQ:No.2 with 90% and the above homology and coding have identical function The nucleotide sequence of protein.

The present invention is provided to expand the primer pair of above-mentioned encoding gene, nucleotide sequence be respectively SEQ:No.3 and Shown in SEQ:No.4.

The present invention provides the expression vector of above-mentioned encoding gene, and the expression vector is the expression for being inserted into above-mentioned encoding gene Carrier pET32a.

The present invention provides the recombinant cell or transformant for containing above-mentioned expression vector, and the recombinant cell is BL21 (DE3) pLysS(Promega)。

Third object of the present invention is to provide the oxygen transmethylases to express realization catalysis flavonoids in vitro Application of the object in the methylation of specific site is closed, specially provides the oxygen transmethylase CitOMT1 in catalysis Quercetin Ultimately generate 3 ', 4 ' dimethyl ester of Quercetin；It is catalyzed luteolin and generates chrysoeriol；It is catalyzed eriodictyol and generates hesperetin, Gao Sheng Careless phenol；It is catalyzed baicalein and generates qroxylin A；Catalysis 7,8- dihydroxyflavone generates answering in 7- hydroxyl -8- methoxy flavone With.

Beneficial effects of the present invention:

The present invention isolates one from citrus genome database (www.citrusgenomedb.org) for the first time can 3 ' positions of catalytic hydroxylation flavones, 4 ' positions, 6,8 oxygen transmethylase CitOMT1.By constructing pET32a expression vector And it converts expression strain b- L21 bacterial strain and obtains destination protein.External enzyme activity is experiments have shown that CitOMT1 can be catalyzed Quercetin eventually most Generate 3 ', 4 ' dimethyl ester of Quercetin；It is catalyzed luteolin and generates chrysoeriol；It is catalyzed eriodictyol and generates hesperetin, eriodictyonone； It is catalyzed baicalein and generates qroxylin A；It is catalyzed 7,8- dihydroxyflavone and generates 7- hydroxyl -8- methoxy flavone.Enzyme activity reaction Condition is simple, and does not need additionally to add metal ion, application value with higher and Research Prospects.

Detailed description of the invention

Fig. 1: three kinds of the bowl mandarin orange stage of development main PMFs variations (A, B, C) and CitOMT1 table during its stage of development Expression patterns (D).

Fig. 2: CitOMT1 protein SDS-PAGE electrophoretogram；Wherein: M: protein molecular quality standard；U: upper final proof after induction Product；P: the CitOMT1-pET recombinant protein of purifying.

Fig. 3: the HPLC identification to reaction product；Wherein reaction substrate is Quercetin；QUE Quercetin, ISOR Isorhamnetin, TAM tamarixetin, 3 ', 4 ' dimethyl ester of QUE-diMF Quercetin.

Fig. 4: the HPLC identification to reaction product；Wherein reaction substrate is respectively luteolin (A), eriodictyol (B), radix scutellariae Element (C), 7,8- dihydroxyflavone (D)；LUC luteolin, CHR chrysoeriol, ERI eriodictyol, HOM eriodictyonone, HES orange peel Element, BAI baicalein, ORO qroxylin A, 7,8-DHF 7,8- dihydroxyflavone, 7,8-MF 7- hydroxyl -8- methoxy flavone.

The influence of Fig. 5: pH (A) and temperature (B) to enzyme reaction speed.

Fig. 6: CitOMT1 subcellular localization figure.

Specific embodiment

The present invention is further illustrated in the following with reference to the drawings and specific embodiments, but the present embodiment does not limit the present invention Protection scope.Conventional genetic manipulation method is referring to " Molecular Cloning:A Laboratory guide " (third edition) in following embodiments

The clone of embodiment 1:CitOMT1 gene

(1) experimental method

Gene cloning: by reported arabidopsis OMT gene and citrus genome database (http: // Www.citrusgenomedb.org/ it) carries out BLAST analysis and obtains CitOMT1, nucleotides sequence is classified as SEQ:No.2.In conjunction with drawing Object is to SEQ:No.3 and SEQ:No.4, using bowl mandarin orange fruit oil vacuole layer cDNA as template, referring to FastStart High Fidelity PCR System (Roche) carries out PCR amplification, wherein PCR system are as follows: 10 × Buffer, 3 2.4 μ L of μ L, dNTP, upstream and downstream are drawn Object (10 μM) each 1.2 μ L, 21.3 0.6 μ L of μ L, cDNA of water of enzyme 0.3 μ L, DEPC processing；PCR program are as follows: 95 DEG C of initial denaturations 5min；95 DEG C of denaturation 30sec, 58 DEG C of annealing 30sec, 72 DEG C of extension 1min, 35 thermal cycles；72 extend 1min, 4 DEG C of preservations. PCR product is connected to pGEM-T Easy vector (Promega) after recycling after 1% agarose gel electrophoresis, be sequenced Analysis.

(2) experimental result

By sequence verification, the CitOMT1-T carrier comprising SEQ:No.2 is obtained.

Expression pattern analysis of the embodiment 2:CitOMT1 in the bowl mandarin orange stage of development

(1) experimental method

1. fruit material acquires

Using different developmental phases bowl mandarin orange fruit as material.(S1), 60 (S2), 80 (S3), 100 30 after full blossom (S4), 120 (S5), 140 (S6), 170 (S7), the picking of 200 days (S8), are divided into 3 biology and repeat, each to repeat picking 8 Fruit；Bowl mandarin orange pericarp is divided into two positions of oil vacuole layer and white cortex, is placed in liquid nitrogen rapidly after being cut into small pieces, saves later At -80 DEG C.

2. flavone compound high performance liquid chromatography (HPLC) is analyzed

The sample powder 0.5g for weighing bowl mandarin orange pericarp oil vacuole layer and white cortex respectively, with 80% ethyl alcohol 3mL ultrasound 30min, Extracting solution is centrifuged (4000rpm, 10min), in triplicate, merges supernatant (about 9mL), the measurement for flavonoid component.Flavonoids The detection of compound uses 1260 VWD detector of Agilent, ODS C18 column (5 μm of Sunfire, 4.6 × 250mm), HPLC Analysis condition: the ultrapure water (solution B) with chromatography acetonitrile (solution A) and containing 0.1% formic acid is mobile phase, is washed using gradient De-, gradient is 0~5min, solution A 20%；5~10min, solution A 20%~27%；10~15min, solution A 27%；15~25min, solution A 27%~40%；25~35min, solution A 40%~60%；35~40min, solution A 60%~80%；40~42min, solution A 80%~100%；42~45min, solution A 100%~20%；45~50min, Solution A 20%.Detection wavelength is 330nm (PMFs), and temperature is room temperature (25 DEG C), and sample volume is 10 μ L.

3.RNA is extracted

The sample powder for weighing 0.3g bowl mandarin orange pericarp oil vacuole layer and white cortex respectively is added to 65 DEG C of 4mL preheatings In the 10mL centrifuge tube of CTAB/ beta -mercaptoethanol extracting solution, sufficient vortex is put into 65 DEG C of cracking 5min after mixing；It is added later 4mL chloroform: isoamyl alcohol (24:1/V:V) is centrifuged (15 DEG C, 10000rpm, 10min) after sufficient vortex, carefully draws supernatant To new 10mL centrifuge tube (being sure not touching precipitating), it is repeated twice, the supernatant merged；Final concentration is added into supernatant solution For the lithium chloride of 2M, 4 DEG C of overnight precipitation RNA；It second day, is centrifuged supernatant (4 DEG C, 10000rpm, 30min), carefully abandons net supernatant； The SSTE of 65 DEG C of 400 μ L preheatings is added, pats dissolution precipitating；Add 400 μ L chloroforms: isoamyl alcohol (24:1/V:V) fills Divide after being vortexed and be centrifuged (15 DEG C, 10000rpm, 10min), careful supernatant of drawing is into new 1.5mL centrifuge tube；To supernatant solution The dehydrated alcohol of middle -20 DEG C of pre-coolings that two volumes are added, is put into -80 DEG C of precipitating 30min after being mixed by inversion；It is centrifuged (4 later DEG C, 10000rpm, 30min), abandon net supernatant；It is deposited in draught cupboard and dries 5~dissolution of 20 μ L DEPC water is added after ten minutes Precipitating, obtains total serum IgE sample；Obtained RNA sample integrality is detected with 1% agarose gel electrophoresis, testing result is shown Sample has two bands, and the band brightness ratio of upper and lower two band is 2:1, shows that RNA sample is complete, can be used for subsequent analysis；It inhales 1 μ L RNA is taken, with UV spectrophotometer measuring OD₂₆₀And OD₂₈₀, OD₂₆₀/OD₂₈₀=1.8~2.0 show that RNA purity reaches mark It is quasi-.According to formula OD₂₆₀× 40 (RNA concentration factors) calculate the concentration (ng/ μ L) of sample RNA, for studying in next step.

4.cDNA synthesis

The RNA sample PrimeScript of extraction^TMRT reagent Kit with gDNA Eraser (Takara, day This) removal genomic DNA, and cDNA dilution is used for subsequent gene expression analysis for 10 times at cDNA by reverse transcription.

5. gene expression analysis

Use NCBI/Primer-BLAST online software (https: //www.ncbi.nlm.nih.gov/tools/ Primer-blast/index.cgi? LINK_LOC=BlastHome QPCR primer) is designed, primer specificity is through solubility curve Analysis, agarose gel electrophoresis analysis and QPCR product sequence verification again；Using Real-time quantitative PCR, with citrus β-actin It (SEQ:No.5) is internal reference, primer is SEQ:No.6 and SEQ:No.7.CitOMT1 QPCR primer pair is SEQ:No.8 and SEQ: No.9, QPCR reaction system be 20 μ L, the Ssofast EvaGreen Supermix (Bio-Rad, the U.S.) including 10 μ L, on Downstream primer (10 μM) each 1 μ L, 2 μ L dilute 10 times of cDNA, 6 μ L DEPC water, and each QPCR includes the feminine gender without cDNA Control, QPCR use CFX96 real-time fluorescence quantitative PCR instrument (Bio-Rad, the U.S.), and program is 95 DEG C of 3min；95 DEG C of 10s, 60 DEG C 30s, 45 circulations；95 DEG C of 10s, from 65 DEG C to 95 DEG C, 0.5 DEG C of acquisition first order fluorescence value of every increase.QPCR data analytical calculation Using △ Ct, the expression pattern of CitOMT1 in different citrus samples is analyzed.

(2) experimental result

Result of study shows that in bowl mandarin orange Fruit Development Process, the PMFs specificity of bowl mandarin orange pericarp is accumulated in oil vacuole layer Tired, content is very low (attached drawing 1A, B, C) in white cortex.CitOMT1 gene is specific expressed in the oil vacuole layer in pericarp, S1, S3 stage expression quantity is lower, reaches highest in S5 stage content, on a declining curve later；CitOMT1 gene expression in white cortex It measures very low.CitOMT1 totality expression trend and the PMFs content of bowl mandarin orange fruit development stage are in during the bowl mandarin orange stage of development Preferable correlation；Initial guess CitOMT1 may play more important role in the synthesis of bowl mandarin orange fruit PMFs.

The external enzyme activity Function Identification of embodiment 3:CitOMT1

(1) experimental method

1. recombinant vector and recombinant cell building

Using the primer pair SEQ:No.3 and SEQ:No.4 having verified that, and restriction enzyme site BamHI, XhoI of pET32a with And protection base constitutes new primer pair SEQ:No.10 and SEQ:No.11.The CitOMT1-T carrier crossed using sequence verification is mould Plate is carried on pET32a carrier in conjunction with pcr clone by the open reading frame of CitOMT1, is built into CitOMT1-pET Recombinant expression carrier, and be conducted into E. coli expression strains BL21 (DE3) pLysS (Promega) by heat shock method, it will Bacterium solution, which is spread evenly across on the LB plate containing 100 μ g/mL ampicillins (Amp), screens positive single colonie, is protected with 20% glycerol Be stored in -80 DEG C it is spare.

The induction and purifying of 2.CitOMT1 albumen

After the glycerol stock inoculation for depositing in -80 DEG C is activated, picking single bacterium is fallen within containing 20mL LB (100 μ g/mL Amp) 50mL centrifuge tube in, 37 DEG C, 150rpm is incubated overnight (12-14h)；10mL bacterium solution is drawn to 500mL LB (100 μ g/mL Amp), 37 DEG C, 150rpm cultivates 2-3h to OD₆₀₀=0.5~0.6；The IPTG of final concentration of 1mM is added into bacterium solution, 18 DEG C, 150rpm cultivates the expression of 20h inducible protein；Bacterium solution is dispensed into centrifugation (4 DEG C, 4000rpm, 10min) in 50mL centrifuge tube, Supernatant is abandoned, precipitating is resuspended in 1 × PBS of 50mL and is stored in -80 DEG C；Re-suspension liquid was melted at 30 DEG C in second day, later with super Sound cell crushing instrument smudge cells (ultrasonic 3.0S is spaced 2.0S, ultrasonic 5min, power 28%)；Centrifugation (4 DEG C, 10000rpm, 30min), supernatant is crossed 0.45 filter membrane (water system) and is placed on and prepare to purify on ice；Select gravity purification column HisTALON^TM Gravity Column Purification Kit (Takara, Japan) purifying Supernatant samples, the CitOMT1 weight purified Histone；The CitOMT1 recombinant protein of purifying is replaced to Tris-HCl using PD-10 desalting column (GE Healthcare, UK) Buffer (50mM Tris-HCl, pH8.0,2mM DTT, 10% glycerol) draws 1 μ L and carries out protein SDS-PAGE electrophoresis detection, Remaining be stored in -80 DEG C it is spare；With 10% pre-prepared colloid buffer (not moral biology, China) preparative separation glue and concentration glue；To electricity Appropriate 1X running buffer is added in swimming slot, by loading after the albumen of purifying and Supernatant samples denaturation, 200V constant pressure is (about 50min), until bromophenol blue stops electrophoresis to the lower edge of glue；Remove protein adhesive, be put into coomassie brilliant blue R_250 (not moral biology, China) in dye (15min, 70rpm)；With the dyeing liquor on distilled water flushing protein adhesive surface, boiled in water until protein adhesive Clean background takes pictures and saves result and analyzed.

The Function Identification 3. albumen vitro enzyme is lived

The identification of 3.1 vitro enzyme life birth objects

Five kinds of Quercetin, luteolin, eriodictyol, baicalein, 7,8- dihydroxyflavone substrates are chosen to recombinate CitOMT1 Albumen carries out the test of enzyme activity regionality selection function.Enzyme activity reaction system 200 μ L Tris-HCl (pH8.0), 25 μ of recombinant protein 200 μM of L, S-adenosylmethionine 1mM, flavones substrate.Isometric acetic acid second is added in 37 DEG C of reaction temperature reactions after a certain period of time Ester extraction, repetitive operation is primary, merges supernatant, and rotation is resuspended with 200 μ L methanol after being evaporated, is used for after crossing 0.22 μm of organic filter membrane HPLC analysis.Chromatographic condition is consistent with flavonoids in embodiment 2 and object HPLC analysis condition.Wherein Quercetin, luteolin, When baicalein, 7,8- dihydroxyflavone are substrate, Detection wavelength 350nm, Detection wavelength is 280nm when eriodictyol is substrate.

The influence of 3.2pH and temperature to enzyme reaction speed

Selection luteolin is substrate, measuring temperature and the pH influence living to CitOMT1 albumen vitro enzyme.

Optimal pH measurement: two kinds of buffers: kaliumphosphate buffer (pH are selected according to effective buffering range of different buffers 5.5, pH 6.0, pH 6.5, pH 7.0, pH 7.5, pH 8.0) and Tris-HCl buffer (pH 7.0, pH 7.5, pH 8.0, PH 8.5, pH 9.0), in 37 DEG C of reaction 30min；Optimum temperature measurement: setting 25 DEG C, 30 DEG C, 35 DEG C, 40 DEG C, 45 DEG C, 50 DEG C, 55 DEG C, 60 DEG C, 65 DEG C, 70 DEG C, 75 DEG C, 80 DEG C of 12 gradients, react 30min in Tris-HCl (pH 8.0) buffer. 2 times of volumes methanols are added into reaction solution and terminate reaction, filter membrane (0.22 μm of organic film) is analyzed for HPLC loading afterwards, measurement Product formation, chromatographic condition is consistent with flavonoids in embodiment 2 and object HPLC analysis condition, and wherein Detection wavelength is 350nm。

(2) experimental result

1.SDS-PAGE electrophoresis detection the result shows that, CitOMT1-pET fusion protein molecule amount is about 46kDa (attached drawing 2), It is consistent with its calculated value 46.89033KDa, and it is single to purify band.Show the amino acid sequence SEQ:No.1 of CitOMT1 CitOMT1-pET fusion recombinant protein is together constituted with 6 × histidine tag, can be used for the enzyme activity analysis of next step.

2. external enzyme activity experiments have shown that, CitOMT1 recombinant protein can be catalyzed Quercetin and methylate in 3 ', 4 ' positions, respectively Isorhamnetin, tamarixetin are generated, and with the increase in reaction time, ultimately generates 3 ', 4 ' dimethyl ester of Quercetin (attached drawing 3)； Being catalyzed luteolin, methylation generates chrysoeriol in 3 ' sites；Catalysis eriodictyol methylates in 3 ', 4 ' positions, generates respectively high holy Careless phenol, hesperetin, catalysis baicalein generate qroxylin A in 6 methylations；7,8- dihydroxyflavone is catalyzed to methylate at 8 It generates 7- hydroxyl -8- methoxy flavone (attached drawing 4).

3. when using luteolin as substrate, CitOMT1 recombinant protein is in pH 8.0 (Tris-HCl buffer) and 40 DEG C Reaction rate highest (attached drawing 5).

Embodiment 4: gene subcellular localization

(1) experimental method

1. construction of recombinant vector

Using CE design (promise is only praised, China) design single slice cloning primer to SEQ:No.12 and SEQ:No.13.With The CitOMT1-T carrier that sequence verification is crossed is template, and the open reading frame of CitOMT1 (is removed end in conjunction with recombinant clone technology It is only sub), it is carried on 35S-eGFP carrier, is built into CitOMT1-eGFP recombinant expression carrier.And it will by electric robin CitOMT1-eGFP recombinant vector and 35S-eGFP empty carrier are directed respectively into Agrobacterium GV3101:pSoup, and bacterium solution is uniformly applied It is distributed on the LB plate containing 50 μ g/mL kanamycins (Kan) and 25 μ g/mL gentamicins (Get) and screens positive single colonie, use 20% glycerol be stored in -80 DEG C it is spare.

2. injecting tobacco

Will deposit in -80 DEG C agrobacterium strains inoculation activation, thalline were collected by centrifugation, with permeabilization buffer be resuspended floating to OD₆₀₀=1.0, the agrobacterium strains containing CitOMT1-eGFP recombinant vector and 35S-eGFP empty carrier are injected at two plants respectively In Ben Shi cigarette, every plant of injection three pieces leaf (about 4 weeks sizes) uses confocal laser scanning microscope GFP fluorescence after 2 days.

Permeabilization buffer 10mM MgCl₂, 10mM MES-NaOH (pH5.6), 150 μM of acetosyringones

(2) experimental result

GFP fluorescence localization is in core and cytoplasm in the tobacco leaf of the injection Agrobacterium of empty carrier containing 35S-eGFP, and injects GFP fluorescence is distributed mainly on cytoplasm in the tobacco leaf of the Agrobacterium of recombinant vector containing CitOMT1-eGFP, weakens core significantly Interior distribution shows that CitOMT1 is primarily targeted for cytoplasm (attached drawing 6) in plant.

Sequence table

<110>Zhejiang University

<120>the oxygen transmethylase and its encoding gene of the synthesis of participation orange peel flavones and application

<160> 13

<170> SIPOSequenceListing 1.0

<210> 1

<211> 249

<212> PRT

<213>citrus (Citrus reticulata cv. Suavissima)

<400> 1

Met Ala Ala Asn Arg Glu Gly Gln Glu Glu Asn Lys Asp Arg Tyr Tyr

1 5 10 15

Lys Thr Leu His Gly Asn Lys Thr Leu Leu Gln Ser Glu Lys Leu Tyr

20 25 30

Glu Tyr Ile Leu Glu Thr Ser Val Tyr Pro Arg Glu Pro Gln Cys Leu

35 40 45

Lys Glu Ile Arg Glu Leu Thr Asp Lys His Pro Trp Ser Ala Met Met

50 55 60

Thr Ala Pro Asp Glu Ala Gln Phe Leu Ser Met Leu Leu Lys Leu Ile

65 70 75 80

Asn Ala Lys Asn Thr Met Glu Ile Gly Val Phe Thr Gly Tyr Ser Leu

85 90 95

Leu Ala Thr Ala Leu Ala Ile Pro Asp Asp Gly Lys Ile Leu Ala Leu

100 105 110

Asp Ile Thr Lys Glu His Tyr Glu Lys Gly Leu Pro Ile Ile Gln Lys

115 120 125

Ala Gly Val Ala His Lys Ile Asp Phe Arg Glu Gly Pro Ala Leu Pro

130 135 140

Leu Leu Asp Gln Leu Ile Gln Asp Glu Lys Tyr His Gly Thr Phe Asp

145 150 155 160

Phe Val Phe Val Asp Ala Asp Lys Asn Asn Tyr Ile Asn Tyr His Lys

165 170 175

Arg Leu Ile Glu Leu Val Lys Val Gly Gly Val Ile Gly Tyr Asp Asn

180 185 190

Thr Leu Trp Gly Gly Ser Val Val Ala Gln Pro Asp Ala Asp Leu Asp

195 200 205

Glu Leu Ile Leu Gly Trp Arg Asp Ser Val Gln Glu Leu Asn Lys Ala

210 215 220

Leu Ala Val Asp Pro Arg Ile Glu Ile Cys Gln Leu Ser Ile Ala Asp

225 230 235 240

Gly Val Thr Leu Cys Arg Arg Ile Gly

245

<210> 2

<211> 750

<212> DNA

<213>citrus (Citrus reticulata cv. Suavissima)

<400> 2

atggctgcga atcgggaagg tcaggaggaa aacaaggatc gttactacaa aactctacat 60

gggaacaaga cccttttaca gagtgaaaaa ctttatgagt atatacttga aaccagtgta 120

tatccaagag aaccacaatg cttgaaggag atccgcgagt tgacggacaa gcatccatgg 180

agcgccatga tgactgcacc tgatgaagca caatttttga gcatgctact caagcttatc 240

aatgccaaga ataccatgga aattggcgtt ttcactggtt actctctcct agccactgcc 300

ctcgccattc ccgacgatgg caagatactg gccctggaca tcaccaagga acactacgag 360

aagggtctcc ctataattca aaaagctggt gttgcccaca agattgattt cagagaaggc 420

cctgctcttc ctcttcttga tcaattgatc caagacgaaa aatatcatgg gacattcgac 480

ttcgtctttg ttgatgctga caagaataac tacattaatt accacaagag gcttattgag 540

cttgtgaagg ttgggggagt gattggctac gacaacacct tgtggggtgg atctgtggtg 600

gcacaacctg atgccgacct ggatgagcta atcttaggct ggagggattc tgtgcaggaa 660

ctcaacaagg ccctggccgt tgatccaagg attgagattt gccagctctc cattgctgat 720

ggggttacat tatgtcgtcg cattggctga 750

<210> 3

<211> 21

<212> DNA

<213>artificial sequence (Unknown)

<400> 3

atatggctgc gaatcgggaa g 21

<210> 4

<211> 18

<212> DNA

<213>artificial sequence (Unknown)

<400> 4

tcagccaatg cgacgaca 18

<210> 5

<211> 1777

<212> DNA

<213>citrus (Citrus reticulata cv. Suavissima)

<400> 5

atatatattt atattccttg gctcttccgc ttctctcact actacatttt taatctctcg 60

gcagtaccaa atacagagcg atagagagaa tcgacactct cagctagact ccatcttcga 120

atttttctct cgtcgtctct tcaatctaac ttttataaaa aatggccgat gctgaggata 180

ttcaacccct tgtttgtgac aatggaactg gaatggtgaa ggctggattt gctggagatg 240

atgctccaag ggcagtattc cctagtattg ttggtaggcc tcgacacact ggagtcatgg 300

ttggaatggg gcagaaggat gcctatgttg gtgatgaggc ccaatcgaaa agaggtattc 360

ttactttgaa atacccaatt gagcacggta ttgtaagcaa ctgggatgat atggaaaaga 420

tctggcatca taccttctac aacgagcttc gtgttgctcc tgaagagcac ccagtgcttc 480

tcactgaggc acctcttaac cccaaggcca acagagagaa gatgacccaa attatgtttg 540

aaactttcaa tgtgccagcc atgtacgttg ccatccaggc tgttctatct ctttatgcca 600

gtggtcgtac aactggtatt gtgctggatt ctggtgatgg tgtgtctcat actgtaccaa 660

tttatgaggg atatgccctt cctcatgcca ttcttcgttt ggacttggct ggtcgtgatc 720

tcactgatgc attgatgaag attcttactg agagaggtta catgtttacc accacagccg 780

aacgggaaat tgtccgtgac atgaaggaga agcttgctta tgttgcccta gactatgagc 840

aagaacttga gactgccaag agcagctcat ctgttgagaa gaactatgag ctgcctgatg 900

gccagatcat caccattgga gctgaaagat tccgttgccc agaagttctg ttccagccat 960

ctctcatcgg aatggaagct gctggtattc atgagactac ctacaactcc atcatgaagt 1020

gtgatgtgga tattagaaag gatctgtatg gtaacattgt tctcagtggg ggttcaacta 1080

tgttccccgg tattgccgac cgtatgagca aggaaatcac agcacttgct ccaagcagca 1140

tgaagatcaa ggtcgtggct ccaccagaga gaaaatacag tgtttggatt ggaggatcaa 1200

tccttgcatc cctcagcacc ttccagcaga tgtggatctc aaagggcgag tatgatgagt 1260

ctggtccatc cattgtccac aggaaatgct tctaagttct ttaagtgctt gatgggtgag 1320

ttcttttttt ccatttagtt ggttttttgt gtctcgtttc gcgaactcaa gttggttgac 1380

atggagaagt gctaaggttg gggccattga aggaggcata tctcgatatt gatgttatta 1440

tacaagcttc ttacagcgtt tgaggggggc ttatttctgt actcaaccgt cttccttgcc 1500

ttgcattcat ctggtgttca accaagtctt ttttcttttt tttccttttt tttttttaaa 1560

gtaggatgtt tgtagctgga gatgaagagt ggttgtgatg cttttctatt tcattttcta 1620

aattttcata tttgaaggtt ttttttctcc ccctctcaaa aggaacatta atgttaatag 1680

ctattgtatg agaaaacttt aattagtgtc agcttgcagt cataaatttg gttattaaaa 1740

tatatttaat acttgtttga gagctttcaa tgatgaa 1777

<210> 6

<211> 18

<212> DNA

<213>artificial sequence (Unknown)

<400> 6

catccctcag caccttcc 18

<210> 7

<211> 19

<212> DNA

<213>artificial sequence (Unknown)

<400> 7

ccaaccttag cacttctcc 19

<210> 8

<211> 20

<212> DNA

<213>artificial sequence (Unknown)

<400> 8

atgccgacct ggatgagcta 20

<210> 9

<211> 20

<212> DNA

<213>artificial sequence (Unknown)

<400> 9

gagaggatca gccaatgcga 20

<210> 10

<211> 28

<212> DNA

<213>artificial sequence (Unknown)

<400> 10

tcaggatcca tggctgcgaa tcgggaag 28

<210> 11

<211> 29

<212> DNA

<213>artificial sequence (Unknown)

<400> 11

cgactcgagg ccaatgcgac gacataatg 29

<210> 12

<211> 35

<212> DNA

<213>artificial sequence (Unknown)

<400> 12

cggtacccgg ggatccatgg ctgcgaatcg ggaag 35

<210> 13

<211> 36

<212> DNA

<213>artificial sequence (Unknown)

<400> 13

cgactctaga ggatccgcca atgcgacgac ataatg 36

Claims

1. one participates in the oxygen transmethylase of orange peel flavones synthesis, which is characterized in that the oxygen transmethylase The amino acid sequence of CitOMT1 is one of the following:

(1) amino acid sequence shown in SEQ:No.1；

(2) or by amino acid sequence shown in SEQ:No.1 by the substitutions of one or several amino acid residues, missing, addition and The identical protein of function.

2. one according to claim 1 participates in the oxygen transmethylase of orange peel flavones synthesis, which is characterized in that institute The nucleotide sequence for stating oxygen transmethylase CitOMT1 encoding gene is one of the following:

(1) nucleotide sequence shown in SEQ:No.2；

(2) or with nucleotide sequence shown in SEQ:No.2 with 90% and the above homology and coding have identical function albumen The nucleotide sequence of matter.

3. the expression vector containing encoding gene described in claim 2, which is characterized in that the expression vector is that insertion right is wanted Seek the expression vector pET32a of 2 encoding genes.

4. oxygen transmethylase according to claim 1 answering in catalysis flavone compound specific site methylation With.

5. application according to claim 4, which is characterized in that generate 3 ', 4 ' dimethyl ester of Quercetin in catalysis Quercetin, urge To change luteolin and generates chrysoeriol, catalysis eriodictyol generates hesperetin, eriodictyonone, and catalysis baicalein generates qroxylin A, The application being catalyzed in 7,8- dihydroxyflavone generation 7- hydroxyl -8- methoxy flavone.