CN106754989A - The hydroxylase of strophanthus divaricatus flavanones 2 and its encoding gene and application - Google Patents

Abstract

The invention discloses a kind of hydroxylase of strophanthus divaricatus flavanones 2 and its encoding gene and application.As shown in SEQ ID NO.3, its coding gene sequence is as shown in SEQ ID NO.1 or 2 for the amino acid sequence of the hydroxylase of strophanthus divaricatus flavanones 2.Clone obtains the hydroxylase of strophanthus divaricatus flavanones 2 to the present invention from strophanthus divaricatus first（MpF2H）There is gene, MpF2H catalysis naringenin to generate the activity of 2 hydroxyl naringenins.The content of the flavone c-glycoside constituents such as Vitexina, Saponaretin and Wei Cai peaceful 2 in the plants such as strophanthus divaricatus can be improved by technique for gene engineering using the present invention, or the flavone c-glycoside constituents such as Vitexina, Saponaretin and Wei Cai peaceful 2 are prepared by biosynthesis technology, for further research strophanthus divaricatus ACGs biological relations are laid a good foundation, also will be with extremely important scientific meaning and application value to strophanthus divaricatus Secondary Metabolic Regulation of Callus and germplasm innovation research.

Description

Strophanthus divaricatus flavanones -2- hydroxylases and its encoding gene and application

Technical field

The invention belongs to gene engineering technology field.More particularly, to a kind of strophanthus divaricatus flavanones -2- hydroxylases and Its encoding gene and application.

Background technology

Plant flavonoids are the micromolecular compounds that a class is widely present, and are existed with glycoside forms mostly in plant, Its O- glycosides is too numerous to enumerate, and C- glycosides is clearly noticed.The sugared aglucon of flavones C- glycosides is connected directly between C-6/C-8 of its A ring mostly, Because parent nucleus is different, its species is also not quite similar.For a long time, plant flavonoids are eaten with its extensive physiologically active and good medicine Dual-purpose value causes that its application study is like a raging fire, wherein substantial amounts of work has mainly concentrated the separation of various flavonoids compositions pure In change, Structural Identification, activity rating and product development.But the basic research of correlation, such as plant flavonoids Secondary Metabolic Regulation of Callus Research, the especially Secondary Metabolic Regulation of Callus research of flavones C- glycosides, related research report is very few.

In recent years, deepening continuously with research, flavones C- glycosides and its bioactivity cause increasing concern. Knowable to document report, its aglycon is relatively conventional with apiolin or cyanidenon, and its sugared aglucon is with glucose, rhamnose, xylose Or arabinose etc. is in the majority, such as Vitexina, Saponaretin, Wei Caining -2, Schaftoside, Isoschaftoside.Research finds, yellow Ketone carbon glycosides generally has preferable anti-inflammatory, anti-oxidant, antitumor, hypoglycemic, hypotensive, protect liver isoreactivity.

Flavones C- glycosides chemical property is compared with O- glycosides stabilization, and its chemical synthesis difficulty is also larger.So far, the master of flavones C- glycosides is obtained It is still to extract to separate from plant material to want approach.With the development of modern biotechnology, Secondary Metabolism of Plant is increasingly becoming One of study hotspot.Now, by regulating and controlling the expression of corresponding biosynthesis key gene, to improve specific metabolite Accumulation, the application on plant genetic engineering is more and more ripe, such as qinghaosu, ginsenoside etc..Found in early-stage Study, Flavones C- glycosides contents are higher in guangdong herbal tea plant strophanthus divaricatus, and component is more, and most of is all 4',5,7-trihydroxyflavone-C- glycosides （Apigenin C-glycosides, ACGs）.Therefore, the research of exploration strophanthus divaricatus ACGs biosynthesis relevant enzymes has important Meaning.

The content of the invention

The technical problem to be solved in the present invention is to overcome grinding for existing strophanthus divaricatus ACGs biosynthesis relevant enzyme and its gene Study carefully deficiency, there is provided a kind of key gene related to its ACGs biosynthesis, and carried out prokaryotic expression and functional verification, be Further research strophanthus divaricatus ACG biological relations are laid a good foundation.

It is an object of the invention to provide a kind of strophanthus divaricatus flavanones -2- hydroxylases MpF2H and its encoding gene.

Another object of the present invention is to provide the application of the strophanthus divaricatus flavanones -2- hydroxylases and its encoding gene.

Still a further object of the present invention is to provide the preparation method of the strophanthus divaricatus flavanones -2- hydroxylases.

Above-mentioned purpose of the present invention is achieved through the following technical solutions：

A kind of DNA molecular for encoding strophanthus divaricatus flavanones -2- hydroxylases（I.e. strophanthus divaricatus flavanones -2- hydroxylases encode base CauseMpF2H）, its nucleotide sequence is as shown in SEQ ID NO.1 or SEQ ID NO.2（SEQ ID NO.1 are DNA full length sequences, SEQ ID NO.2 are cDNA sequence, i.e. ORFs）.

It is a kind of to have with sequence hybridization shown in SEQ ID NO.1 or SEQ ID NO.2 and coding under strict conditions Identical function protein DNA molecule, also should be within protection scope of the present invention.

Preferably, above-mentioned stringent condition can be：In 6 × SSC, the solution of 0.5% SDS, hybridize at 65 DEG C, Ran Houyong 2 × SSC, 0.1% SDS, and 1 × SSC, 0.1% SDS respectively wash film once.

It is a kind of at least to have 80% with sequence shown in SEQ ID NO.1 or SEQ ID NO.2, at least have 85%, at least have Have 90%, at least have 95%, at least have 96%, at least have 97%, at least have 98% or at least there is 99% homology and volume Code has identical function protein DNA molecule, also should be within protection scope of the present invention.

A kind of strophanthus divaricatus flavanones -2- hydroxylases, its amino acid sequence is as shown in SEQ ID NO.3.

It is a kind of by amino acid sequence shown in SEQ ID NO.3 by the substitution of one or several amino acid residues and/or lack Lose and/or addition protein of the gained with identical function, also should be within protection scope of the present invention.

The present invention early-stage Study find strophanthus divaricatus in flavones C- glycosides contents it is higher on the basis of, meanwhile, comprehensive strophanthus divaricatus Transcript profile and metabolism group data, through differential gene expression and trend analysis, screening is found that related to its ACGs biosynthesis Key gene F2H（Flavanone 2-hydroxylase, MpF2H）, and MpF2H has been cloned from strophanthus divaricatus first Gene cDNA sequence, and prokaryotic expression and functional verification have been carried out, it is the flavones carbon such as Vitexina, Saponaretin and Wei Caining -2 The preparation of methods of glycosides provides a kind of new approach.

In addition, a kind of recombinant vector containing above-mentioned DNA molecular, expression cassette, transgenic cell line or recombinant bacterium, also should be Within protection scope of the present invention.

Preferably, the recombinant vector is that sequence shown in SEQ ID NO.1 or SEQ ID NO.2 is inserted into pET30a（+） CarrierBam HI andHind The carrier obtained between III digestion site, is named as pET30- MpF2H.

A kind of primer pair for expanding above-mentioned DNA molecular total length or its any fragment, also should protection scope of the present invention it It is interior.

Preferably, a kind of primer pair of sequence shown in amplification SEQ ID NO.1 or SEQ ID NO.2, its forward and reverse primer Sequence is respectively as shown in SEQ ID NO.4 and SEQ ID NO.5.

In addition, above-mentioned strophanthus divaricatus flavanones -2- hydroxylases as or in terms of preparing flavanones -2- hydroxylase preparations Application, the strophanthus divaricatus flavanones -2- hydroxylases, above-mentioned DNA molecular or above-mentioned recombinant vector, expression cassette, transgenosis are thin The application of born of the same parents system or recombinant bacterium in terms of catalysis naringenin generation 2- hydroxyl naringenins, and above-mentioned DNA molecular is preparing paniculata Application in leaf flavanones -2- hydroxylases, also all should be within protection scope of the present invention.

Specifically, can be by the recombinant vector containing the DNA molecular, expression cassette, transgenic cell line or recombinant bacterium Prepare strophanthus divaricatus flavanones -2- hydroxylases.

A kind of method for preparing strophanthus divaricatus flavanones -2- hydroxylases, comprises the following steps：Culture contains strophanthus divaricatus flavane Ketone -2- hydroxylation enzyme genesMpF2HRecombinant bacterium, collect culture, obtain strophanthus divaricatus flavanones -2- hydroxylases.

More specifically, the method for preparing strophanthus divaricatus flavanones -2- hydroxylases, comprises the following steps：

S1. recombinant vector is built

By strophanthus divaricatus flavanones -2- hydroxylase encoding genesMpF2HSequence be inserted into pET30a（+）PlasmidBam HI andHind Between III digestion site, recombinant vector pET30a-MpF2H is obtained；

S2. the expression of MpF2H albumen

Recombinant vector pET30a-MpF2H is imported into competent cell, recombinant bacterium is obtained；Added in the cultivating system of recombinant bacterium Isopropylthio-β-D-galactoside（IPTG）, vibrate Fiber differentiation；

S3. the purifying of MpF2H albumen

S31. Fiber differentiation is vibrated described in step S2 and collects culture after 6~8 hours, be collected by centrifugation bacterial sediment, add Tris- HCl, ultrasonication；

S32. the product centrifugation after step S31 is broken, takes supernatant, as MpF2H albumen CE, is obtained by purifying MpF2H albumen.

Wherein it is preferred to, the specific method of step S1 is：

With strophanthus divaricatus leaf cDNA as template, using forward primer MpF2H-F（Sequence is as shown in SEQ ID NO.4）With reversely draw Thing MpF2H-R（Sequence is as shown in SEQ ID NO.5）Enter performing PCR amplification geneMpF2HFragment, then use restriction enzymeBam HI and Hind III carries out double digestion to pcr amplification product and pET-30a (+) plasmid, is connected by ligase（T4 DNA 18 DEG C of connections of ligase are overnight）After obtain recombinant plasmid pET30a-MpF2H.

Wherein it is preferred to, the reaction system of the PCR amplifications is：CDNA templates 1 μ L, MpF2H-F 1 μ L, MpF2H-R The μ L of 1 μ L, 10 × ExTaq buffer, 5 μ L, 2.5mmol/L dNTP, 4 μ L, ExTaq enzymes 0.25, add water to 50 μ L.

Preferably, the response procedures of the PCR amplifications are（Touchdown PCR）：95 DEG C of min of predegeneration 5,98 DEG C in circulation 10s is denatured, from 65 DEG C of each cycle downs, 1 DEG C of annealing, 72 DEG C of extension 2min, 10 circulations；In 55 DEG C of annealing, carry out 20 and follow Ring；72 DEG C of 7 min of extension after PCR reaction cycles, then 4 DEG C of preservations.

Furthermore it is preferred that competent cell described in step S2 isE.coliTrans1 T1 competent cells, are recombinated BacteriumE.coli trans1 T1-pET30a-MpF2H。

Preferably, it is containing 50~100 μ g/ml recombinant bacterium used medium to be cultivated in step S2（More preferably 50 μ g/ml） The LB culture mediums of kalamycin.

Preferably, isopropylthio-β-D-galactoside in step S2（IPTG）Final concentration of 0.5 μM.

Preferably, in step S2, the incubation of recombinant bacterium is：First behind 37 DEG C of shaken cultivation to OD600=0.6~0.8, Add IPTG, 28 DEG C of vibration Fiber differentiations.

It is highly preferred that in step S2, the incubation of recombinant bacterium is：Recombinant bacterium is inoculated in containing 50 μ g/ml kalamycins LB culture mediums in, 37 DEG C of shaken cultivations overnight, with 1：100 ratios are inoculated in the fresh culture medium containing antibiotic kalamycin In, 37 DEG C of 2~3h of shaken cultivation, after behind OD600=0.6~0.8, add isopropylthio-β-D-galactoside（IPTG, eventually Concentration is 0.5 μM）, Fiber differentiation is vibrated at 28 DEG C.

Preferably, the condition being centrifuged described in step S31 is 12000r/min, 4 DEG C, 1min is centrifuged.

Preferably, after bacterial sediment described in step S31 also needs to be resuspended in PBS (pH 7.5) buffer solution washing 2 times, then add Enter Tris-HCl.The concentration of Tris-HCl described in step S31 is 0.1mM, pH7.5.

Preferably, the condition of ultrasonication is described in step S31：Ultrasonication on trash ice is placed in, 20KHz, 120W open 2 Second, stop 10 seconds, circulate 10 times.

Preferably, the condition being centrifuged described in step S32 is 12000r/min, 4 DEG C, 30min is centrifuged.

Preferably, purified described in step S32 is carried out using His-Bind protein purifications QIAquick Gel Extraction Kit.

The invention has the advantages that：

Clone obtains strophanthus divaricatus flavanones -2- hydroxylase (MpF2H) gene, experiment in vitro to the present invention from strophanthus divaricatus first Show, there is MpF2H catalysis naringenin to generate the activity of 2- hydroxyl naringenins, be a kind of flavanones -2- hydroxylases, to enter one Step research strophanthus divaricatus ACGs biological relations are laid a good foundation；Result of study also will to its Secondary Metabolic Regulation of Callus and germplasm innovation research With extremely important scientific meaning and application value.

Using the present invention, can be based on MpF2H genes, in improving the plants such as strophanthus divaricatus by technique for gene engineering The content of the flavone c-glycoside constituents such as Vitexina, Saponaretin and Wei Caining -2, or by biosynthesis technology prepare Vitexina, The flavone c-glycoside constituents such as Saponaretin and Wei Caining -2, for the preparation synthesis of such flavone c-glycoside constituents provides one kind newly Approach.

Brief description of the drawings

Fig. 1 is the collection of illustrative plates of recombinant expression carrier pET30a-MpF2H.

Fig. 2 is the SDS- polyacrylamide gel electrophoresis results of MpF2H protein.

Specific embodiment

The present invention, but embodiment are further illustrated below in conjunction with Figure of description and specific embodiment not to the present invention Limit in any form.Unless stated otherwise, reagent, the method and apparatus that the present invention is used are for the art is routinely tried Agent, method and apparatus.

Unless stated otherwise, agents useful for same of the present invention and material are purchased in market.

Following examples used kit material source：

Homologous recombination Cloning Kit (pEASY-Uni Seamless Cloning andAssembly Kit), albumen Marker, E. coli competent Trans1-T1, pET30a（+）Carrier and DNA gel QIAquick Gel Extraction Kit are purchased from the full formula in Beijing Golden Bioisystech Co., Ltd；RNA reverse transcription reagent box, BamH I restriction enzymes are purchased from precious biology (Dalian) engineering finite Company (TaKaRa) company.Primer synthesizes and is sequenced by the completion of Guangzhou Sheng Gong Co., Ltds.Naringenin and 2- hydroxyls naringenin are purchased From Chemface (Wuhan Tian plant and biology Technology Co., Ltd.).

Strophanthus divaricatus flavanones -2- the hydroxylation enzyme genes of embodiment 1MpF2HClone

1st, the total serum IgE of strophanthus divaricatus blade is extracted using plant total serum IgE extracts kit, reverse transcription obtains cDNA for template, uses Forward primer MpF2H-F（Sequence is as shown in SEQ ID NO.4）With reverse primer MpF2H-R（Sequence such as SEQ ID NO.5 institutes Show）Enter performing PCR amplification.

Forward primer：MpF2H-F（Sequence is as shown in SEQ ID NO.4）

5’-CGGGATCCATGATGCCCATGATACTTGAAT-3’；

Reverse primer：MpF2H-R（Sequence is as shown in SEQ ID NO.5）

5’-CCCAAGCTTTTAGGTTGCAAAGAGAGTGGGA3’。

Wherein, the reaction system of the PCR amplifications is：

111 μ L, 10 × ExTaq buffer of μ L, MpF2H-R of μ L, MpF2H-F of cDNA templates 5 μ L, 2.5mmol/L dNTP The μ L of 4 μ L, ExTaq enzyme 0.25, add water to 50 μ L.

Described PCR reaction conditions are（Touchdown PCR）：95 DEG C of min of predegeneration 5,98 DEG C of 10s denaturation in circulation, from 65 The 1 DEG C of annealing of DEG C each cycle down, 72 DEG C of extension 2min, 10 circulations；In 55 DEG C of annealing, 20 circulations are carried out；PCR reaction cycles 72 DEG C extend 7 min afterwards, then 4 DEG C of preservations.

2nd, PCR products are tested and analyzed with 1% agarose gel electrophoresis, are purified with PCR Product Purification Kits.With limitation Property restriction endonucleaseBam HI andHind III carries out double digestion, T4 DNA to PCR purified products and pET-30a (+) plasmid 16 DEG C of connections of ligase overnight convert Escherichia coli Trans1-T1 competent cells afterwards, are sieved in OK a karaoke club resistant panel Choosing, through bacterium solution PCR and electrophoresis detection after, select positive colony sequencing.

3rd, by sequencing analysis, strophanthus divaricatus flavanones -2- hydroxylation enzyme genes are obtainedMpF2HSequence, such as SEQ ID Shown in NO.1（1826bp）, its ORFs is as shown in SEQ ID NO.2（1557bp）, the albumen of coding is named as strophanthus divaricatus Flavanones -2- hydroxylase MpF2H, the amino acid sequence of the albumen is as shown in SEQ ID NO.3（518aa）.

Strophanthus divaricatus flavanones -2- the hydroxylases of embodiment 2MpF2HExpression and purifying

1st, the structure of recombinant vector

Strophanthus divaricatus blade total serum IgE is extracted, reverse transcription obtains cDNA for template, with using forward primer MpF2H-F（Sequence such as SEQ ID Shown in NO.4）With reverse primer MpF2H-R（Sequence is as shown in SEQ ID NO.5）Enter performing PCR amplification.

PCR reaction systems are：11 μ L, 10 × ExTaq buffer of μ L, MpF2H-R of μ L, MpF2H-F of cDNA templates 1 The μ L of 5 μ L, 2.5mmol/L dNTP, 4 μ L, ExTaq enzymes -0.5, add water to 50 μ L.

Reaction condition is（Touchdown PCR）：95 DEG C of min of predegeneration 5,98 DEG C of 10s denaturation in circulation, from 65 DEG C each follow 1 DEG C of annealing, 72 DEG C of extension 2min, 10 circulations drop in ring；In 55 DEG C of annealing, 20 circulations are carried out；72 DEG C are prolonged after PCR reaction cycles 7 min are stretched, then 4 DEG C of preservations.

PCR primer Purification Kit.Use restriction enzymeBam HI and Hind III is purified to PCR and produced Thing and pET-30a (+) plasmid carry out double digestion, and the 18 DEG C of connections of T4 DNA ligases overnight obtain recombinant plasmid afterwards, the restructuring Carrier is that MpF2H gene orders are inserted into pET30a（+）PlasmidBam HI andHind The load obtained between III digestion site Body, is named as pET30a-MpF2H（As shown in Figure 1）.

2nd, the expression of MpF2H albumen

（1）Target plasmid pET30a-MpF2H is importedE.coliTrans1 T1 competent cells, obtain recombinant bacteriumE.coli trans1 T1-pET30a-MpF2H。

Empty carrier pET30a is importedE.coliTrans1 T1 competent cells, obtain compareing bacteriumE.coli trans1 T1-pET30a。

（2）By recombinant bacteriumE.coliTrans1 T1-pET30a-MpF2H are inoculated in the LB trainings containing 50 μ g/ml kalamycins Support base in, 37 DEG C of shaken cultivations overnight, with 1:100 ratios are inoculated in the fresh culture medium containing antibiotic kalamycin, and 37 DEG C 2~3h of shaken cultivation, the control before 1ml bacterium solutions behind OD600=0.6~0.8, are taken as induction, is subsequently adding isopropyl sulphur Generation-β-D- galactosides (IPTG, final concentration of 0 .5 μM), Fiber differentiations are vibrated at 28 DEG C.WithE.coli trans1 T1- PET30a is negative control.

（3）After Fiber differentiation 6~8 hours, culture is collected, is placed in ultrasonication on trash ice, 20KHz, 120W are opened 2 seconds, Stop 10 seconds, circulate 10 times, supernatant is collected by centrifugation.Supernatant is carried out into SDS- polyacrylamide gel electrophoresises, as a result such as Fig. 2 institutes Show, swimming lane M is Protein Marker (17-180kDa), it can be seen that be about at 70 kD in molecular weight, occur one substantially Specific protein band of expression, it is consistent with theoretical value, show that expression obtains albumen MpF2H（As shown in Figure 2）.

3rd, MpF2H protein purifications

（1）By recombinant bacteriumE.coliTrans1 T1-pET30a-MpF2H are inoculated in the LB culture mediums containing 50 μ g/ml kalamycins In, 37 DEG C of shaken cultivations overnight, with 1:100 ratios are inoculated in the fresh culture medium containing antibiotic kalamycin, and 37 DEG C are shaken 2~3h of culture is swung, after behind OD600=0.6~0.8, isopropylthio-β-D-galactoside (IPTG, final concentration of 0 is added .5 μM), Fiber differentiations are vibrated at 28 DEG C, culture is collected after 6~8 hours, 12000r/min 4 DEG C, is centrifuged 1min, collects bacterium Body is precipitated, and is resuspended in PBS (pH 7.5) buffer solution, is washed 2 times, adds 0.1mM Tris-HCl (pH 7.5), is placed in broken Ultrasonication on ice, 20KHz, 120W are opened 2 seconds, are stopped 10 seconds, are circulated 10 times, 12000r/min, 4 DEG C, 30min are centrifuged, in absorption Clear liquid puts standby on ice as MpF2H albumen CEs.

（2）Above-mentioned crude extract is purified according to His-Bind protein purification QIAquick Gel Extraction Kit specifications, obtains MpF2H Albumen.

The functional study of the strophanthus divaricatus flavanones -2- hydroxylases MpF2H of embodiment 3 and its gene

1st, according to the method for embodiment 2 by strophanthus divaricatus flavanones -2- hydroxylation enzyme genesMpF2HGive expression to strophanthus divaricatus flavanones- 2- hydroxylase MpF2H, study its function.

MpF2H protein functions are verified：

Vitro enzyme functional verification is carried out by reaction substrate of naringenin.Include in 200 μ L reaction systems：0.1 M glycine （KOH, pH 7.9）, 0.1 mM naringenins, the glutathione of 0.5 mM reproducibilities, 1 mM NADPH, 50 μ g MpF2H albumen are pure Change product.Mix, 30 DEG C of water-bath, be incubated 6~12h, the methyl alcohol terminating reaction for adding 400 μ L ice-cold, 12000rpm centrifugations 10min, takes supernatant, crosses 0.22 μm of filter membrane, carries out LC-ESI-QTOF-MS analyses.With naringenin and 2- hydroxyl naringenin reference substances （Purchased from Chemface）It is control, target product is carried out according to accurate molecular weight, MS/MS fragments mass-to-charge ratio and chromatographic retention etc. Thing is identified.

UPLC-ESI-QTOF-MS analysis conditions are as follows：

Liquid-phase condition：Mobile phase is 0.1% formic acid-acetonitrile (A), 0.1% formic acid-water (B), the mL/min of flow velocity 0.2, gradient elution Program is 0min 7%B, 3min 18%B, 3.5min 7%B, 5.5min 7%B；

Mass Spectrometry Conditions：Ion gun：ESI+, ionizes pattern：Positive ion mode；Ion source temperature：150℃；Desolventizing temperature 450 ℃；Capillary voltage：2500V Desolvention gas velocity 900L/hr, taper hole gas velocity 50L/hr, cracking can 20-30eV；High-resolution Mass spectral analysis parameter value is as shown in table 1 below.

The high resolution mass spectrum assay value of table 1

Result finds that naringenin and 2- hydroxyl naringenin standard items retention times are respectively 4.24min and 3.52min, above-mentioned MpF2H enzymatic reaction systems are at 4.24min detection 2- hydroxyl naringenin individual features peak, and control reaction system is not detected Corresponding characteristic peak.

Characteristic peak to MpF2H enzymatic reaction systems carries out ms fragment analysis, corresponding molecular ion accurate molecular weight And fragment ion accurate molecular weight (m/z) is consistent with standard items MS and MS/MS (m/z)；So far after enzymatic reaction being determined 2- hydroxyls naringenin is truly had to generate.

Result shows, although corresponding induced expression condition, the purification condition of albumen and enzymatic reaction system can Further to optimize, but there is MpF2H fusion proteins catalysis naringenin to synthesize the activity of 2- hydroxyl naringenins really, illustrate the base Because flavanones -2- hydroxylase encoding genes.Therefore, it can based on MpF2H genes, carried by technique for gene engineering The content of the flavone c-glycoside constituents such as Vitexina, Saponaretin and Wei Caining -2 in the plants such as strophanthus divaricatus high, or closed by biology The flavone c-glycoside constituents such as Vitexina, Saponaretin and Wei Caining -2 are prepared into technology.

SEQUENCE LISTING

<110>Guangdong pharmaceutical university

<120>Strophanthus divaricatus flavanones -2- hydroxylases and its encoding gene and application

<130>

<160> 5

<170> PatentIn version 3.3

<210> 1

<211> 1826

<212> DNA

<213>The DNA full length sequences of strophanthus divaricatus flavanones -2- hydroxylation enzyme genes MpF2H

<400> 1

ttccctctag aataattttg tttaacttta agaaggagat atacatatgc accatcatca 60

tcatcattct tctggtctgg tgccacgcgg ttctggtatg aaagaaaccg ctgctgctaa 120

attcgaacgc cagcacatgg acagcccaga tctgggtacc gacgacgacg acaaggccat 180

ggctgatatc ggatccatga tgcccatgat acttgaattc ctatcatatc tagctccctt 240

agttgtttca tttcttctcg ttaagagaat actcatctct accaacaagc aaggtcctaa 300

tgccaagcag cttcttcccc caggcccaat cgccttaccg atcattggcc acctccatct 360

cctgggcccc ttccttcatc aaaccttccg caagctctcc tcgcgctacg gtcccttaat 420

gtatcttcgt cttggctctg ttggctgcgt ggtggcttcc aatccagagt ttgccaaaga 480

gcttctcaaa acttacgagc tcgccttcgc tgcccgcatg cacaccgctg ccatcaccca 540

cctgacctac aactcgtcct ttgccttcgc accgtacgga acgtactgga aattcatcag 600

aaagttcagc acgtacgagc tcctaggcaa ccgtactctc gcccagtttc ttcccgttcg 660

aaccaaggaa ttgcaccatc tccttcagtt tgttcttggc aaggctaaag caggggaaag 720

cgtgaattta acccaagagc tgttgaaact aaccaacaac accatatcgc agatgatgct 780

gagcatgcgc tgttcgggga caggaaaccc tgccgactcg gttagaactc ttgtgcgaga 840

ggtgacagag atcttcggag agttcaacat ctcagatagc atatggtttt tgaaaaactg 900

ggacttgcag ggattcagaa agagattcga agacctgcat aggaggtttg atgcattgtt 960

ggagatgatt atgagagaac gtgagcgagt aagatcagaa agcaagcaaa agaaaggcga 1020

caatgttaac aaggtaaaag atttcctgga catcatgctt gacgttttgg aaaaggataa 1080

ctcggagtcg gaggtagatt ttaccagaaa tcacatcaag gccttaattt tggatttctt 1140

cacagccgct acagatacaa cggcaattgt acttgaatgg gcaatggcag agttgatcaa 1200

ccacccgaag ctactaaaaa tagctcagca agaaattgat caagttgtgg gaaaaggcag 1260

gttggtggaa gaatctgaca gcccacatct ccattacatc caagccatta ttaaagaaac 1320

atttcggctt cacccaccag tcccaatgat caataggaaa tcaatccaaa catgccaggt 1380

taagggatac acaatccctg ctgaatgtat ggtgtttgtg aacgtttggg ctatcggaag 1440

agatcccaag gtttggacag atccattgaa gtttcagcct gagagattcc tgaaatccga 1500

tgattctata gatgttagag ggcaacatta cgagctgttg cctttcgggt cagggaggag 1560

gggctgccct ggcgcttctt tggcgttgca ggagctgccc accactttgg ctgccatgat 1620

tcagtgcttt gactggaagg tggcgaatgg cgttgttgac atggttgaaa ggcctggact 1680

tacggctccc agggctaagg atctcgagtg tgttcctgtt gtacgcttca ctcccactct 1740

ctttgcaacc taaaagcttg cggccgcact cgagcaccac caccaccacc actgagatcc 1800

ggctgctaac aaagcccgaa agaagc 1826

<210> 2

<211> 1557

<212> DNA

<213>The cDNA sequence of strophanthus divaricatus flavanones -2- hydroxylation enzyme genes MpF2H

<400> 2

atgatgccca tgatacttga attcctatca tatctagctc ccttagttgt ttcatttctt 60

ctcgttaaga gaatactcat ctctaccaac aagcaaggtc ctaatgccaa gcagcttctt 120

cccccaggcc caatcgcctt accgatcatt ggccacctcc atctcctggg ccccttcctt 180

catcaaacct tccgcaagct ctcctcgcgc tacggtccct taatgtatct tcgtcttggc 240

tctgttggct gcgtggtggc ttccaatcca gagtttgcca aagagcttct caaaacttac 300

gagctcgcct tcgctgcccg catgcacacc gctgccatca cccacctgac ctacaactcg 360

tcctttgcct tcgcaccgta cggaacgtac tggaaattca tcagaaagtt cagcacgtac 420

gagctcctag gcaaccgtac tctcgcccag tttcttcccg ttcgaaccaa ggaattgcac 480

catctccttc agtttgttct tggcaaggct aaagcagggg aaagcgtgaa tttaacccaa 540

gagctgttga aactaaccaa caacaccata tcgcagatga tgctgagcat gcgctgttcg 600

gggacaggaa accctgccga ctcggttaga actcttgtgc gagaggtgac agagatcttc 660

ggagagttca acatctcaga tagcatatgg tttttgaaaa actgggactt gcagggattc 720

agaaagagat tcgaagacct gcataggagg tttgatgcat tgttggagat gattatgaga 780

gaacgtgagc gagtaagatc agaaagcaag caaaagaaag gcgacaatgt taacaaggta 840

aaagatttcc tggacatcat gcttgacgtt ttggaaaagg ataactcgga gtcggaggta 900

gattttacca gaaatcacat caaggcctta attttggatt tcttcacagc cgctacagat 960

acaacggcaa ttgtacttga atgggcaatg gcagagttga tcaaccaccc gaagctacta 1020

aaaatagctc agcaagaaat tgatcaagtt gtgggaaaag gcaggttggt ggaagaatct 1080

gacagcccac atctccatta catccaagcc attattaaag aaacatttcg gcttcaccca 1140

ccagtcccaa tgatcaatag gaaatcaatc caaacatgcc aggttaaggg atacacaatc 1200

cctgctgaat gtatggtgtt tgtgaacgtt tgggctatcg gaagagatcc caaggtttgg 1260

acagatccat tgaagtttca gcctgagaga ttcctgaaat ccgatgattc tatagatgtt 1320

agagggcaac attacgagct gttgcctttc gggtcaggga ggaggggctg ccctggcgct 1380

tctttggcgt tgcaggagct gcccaccact ttggctgcca tgattcagtg ctttgactgg 1440

aaggtggcga atggcgttgt tgacatggtt gaaaggcctg gacttacggc tcccagggct 1500

aaggatctcg agtgtgttcc tgttgtacgc ttcactccca ctctctttgc aacctaa 1557

<210> 3

<211> 518

<212> PRT

<213>Strophanthus divaricatus flavanones -2- hydroxylase MpF2H amino acid sequences

<400> 3

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

1 5 10 15

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

20 25 30

Gly Pro Asn Ala Lys Gln Leu Leu Pro Pro Gly Pro Ile Ala Leu Pro

35 40 45

Ile Ile Gly His Leu His Leu Leu Gly Pro Phe Leu His Gln Thr Phe

50 55 60

Arg Lys Leu Ser Ser Arg Tyr Gly Pro Leu Met Tyr Leu Arg Leu Gly

65 70 75 80

Ser Val Gly Cys Val Val Ala Ser Asn Pro Glu Phe Ala Lys Glu Leu

85 90 95

Leu Lys Thr Tyr Glu Leu Ala Phe Ala Ala Arg Met His Thr Ala Ala

100 105 110

Ile Thr His Leu Thr Tyr Asn Ser Ser Phe Ala Phe Ala Pro Tyr Gly

115 120 125

Thr Tyr Trp Lys Phe Ile Arg Lys Phe Ser Thr Tyr Glu Leu Leu Gly

130 135 140

Asn Arg Thr Leu Ala Gln Phe Leu Pro Val Arg Thr Lys Glu Leu His

145 150 155 160

His Leu Leu Gln Phe Val Leu Gly Lys Ala Lys Ala Gly Glu Ser Val

165 170 175

Asn Leu Thr Gln Glu Leu Leu Lys Leu Thr Asn Asn Thr Ile Ser Gln

180 185 190

Met Met Leu Ser Met Arg Cys Ser Gly Thr Gly Asn Pro Ala Asp Ser

195 200 205

Val Arg Thr Leu Val Arg Glu Val Thr Glu Ile Phe Gly Glu Phe Asn

210 215 220

Ile Ser Asp Ser Ile Trp Phe Leu Lys Asn Trp Asp Leu Gln Gly Phe

225 230 235 240

Arg Lys Arg Phe Glu Asp Leu His Arg Arg Phe Asp Ala Leu Leu Glu

245 250 255

Met Ile Met Arg Glu Arg Glu Arg Val Arg Ser Glu Ser Lys Gln Lys

260 265 270

Lys Gly Asp Asn Val Asn Lys Val Lys Asp Phe Leu Asp Ile Met Leu

275 280 285

Asp Val Leu Glu Lys Asp Asn Ser Glu Ser Glu Val Asp Phe Thr Arg

290 295 300

Asn His Ile Lys Ala Leu Ile Leu Asp Phe Phe Thr Ala Ala Thr Asp

305 310 315 320

Thr Thr Ala Ile Val Leu Glu Trp Ala Met Ala Glu Leu Ile Asn His

325 330 335

Pro Lys Leu Leu Lys Ile Ala Gln Gln Glu Ile Asp Gln Val Val Gly

340 345 350

Lys Gly Arg Leu Val Glu Glu Ser Asp Ser Pro His Leu His Tyr Ile

355 360 365

Gln Ala Ile Ile Lys Glu Thr Phe Arg Leu His Pro Pro Val Pro Met

370 375 380

Ile Asn Arg Lys Ser Ile Gln Thr Cys Gln Val Lys Gly Tyr Thr Ile

385 390 395 400

Pro Ala Glu Cys Met Val Phe Val Asn Val Trp Ala Ile Gly Arg Asp

405 410 415

Pro Lys Val Trp Thr Asp Pro Leu Lys Phe Gln Pro Glu Arg Phe Leu

420 425 430

Lys Ser Asp Asp Ser Ile Asp Val Arg Gly Gln His Tyr Glu Leu Leu

435 440 445

Pro Phe Gly Ser Gly Arg Arg Gly Cys Pro Gly Ala Ser Leu Ala Leu

450 455 460

Gln Glu Leu Pro Thr Thr Leu Ala Ala Met Ile Gln Cys Phe Asp Trp

465 470 475 480

Lys Val Ala Asn Gly Val Val Asp Met Val Glu Arg Pro Gly Leu Thr

485 490 495

Ala Pro Arg Ala Lys Asp Leu Glu Cys Val Pro Val Val Arg Phe Thr

500 505 510

Pro Thr Leu Phe Ala Thr

515

<210> 4

<211> 30

<212> DNA

<213>Forward primer MpF2H-F

<400> 4

cgggatccat gatgcccatg atacttgaat 30

<210> 5

<211> 31

<212> DNA

<213>Reverse primer MpF2H-R

<400> 5

cccaagcttt taggttgcaa agagagtggg a 31

Claims (10)

1. it is a kind of encode strophanthus divaricatus flavanones -2- hydroxylases DNA molecular, it is characterised in that its nucleotide sequence such as SEQ Shown in ID NO.1 or SEQ ID NO.2.

2. one kind under strict conditions can with sequence hybridization shown in SEQ ID NO.1 or SEQ ID NO.2 and coding have phase Congenerous protein DNA molecule.

3. it is a kind of at least to have 80% with sequence shown in SEQ ID NO.1 or SEQ ID NO.2, at least have 85%, at least have 90%th, at least have 95%, at least have 96%, at least have 97%, at least have 98% or at least there is 99% homology and coding With identical function protein DNA molecule.

4. a kind of strophanthus divaricatus flavanones -2- hydroxylases, it is characterised in that its amino acid sequence is as shown in SEQ ID NO.3.

5. it is a kind of by amino acid sequence shown in SEQ ID NO.3 by one or several amino acid residues substitution, missing and/ Or addition gained has the protein of identical function.

6. a kind of recombinant vector containing any DNA molecular of claims 1 to 3, expression cassette, transgenic cell line or restructuring Bacterium.

7. it is a kind of to expand any DNA molecular total length of claims 1 to 3 or the primer pair of its any fragment.

8. a kind of primer pair for expanding DNA molecular described in claim 1, it is characterised in that forward and reverse primer sequence is respectively such as Shown in SEQ ID NO.4 and SEQ ID NO.5.

9. strophanthus divaricatus flavanones -2- hydroxylases described in claim 4 as or prepare flavanones -2- hydroxylases preparation side The application in face.

10. application of any DNA molecular of claims 1 to 3 in strophanthus divaricatus flavanones -2- hydroxylases are prepared.