CN109486806A - A kind of phenylalanine lyase and its encoding gene, recombinant vector, recombination engineering and application of anoectochilus formosanus - Google Patents

Abstract

The present invention provides a kind of phenylalanine lyase of anoectochilus formosanus and its encoding gene, recombinant vector, recombination engineering and applications, belong to protein engineering field.The phenylalanine lyase of anoectochilus formosanus provided by the invention, its sequence is a kind of new Phenylalanine Ammonia-Lyase Gene sequence, it is the key gene in roxburgh anoectochilus terminal bud primary medicinal component Flavonoid substances synthesis process, plays an important role in flavone compound metabolic pathway of synthesizing.

Description

The phenylalanine lyase and its encoding gene of a kind of anoectochilus formosanus, recombinant vector, Recombination engineering and application

Technical field

The present invention relates to protein engineering fields, in particular to a kind of phenylalanine solution of anoectochilus formosanus Adnosine deaminase and its encoding gene, recombinant vector, recombination engineering and application.

Background technique

Anoectochilus formosanus (Anoectochilus formosanus) is herbaceos perennial, is grown in Taiwan, Belong to orchid family Anoectochilus Blume roxburgh anoectochilus terminal bud kind.The active substance of valuable pharmacological is main in anoectochilus formosanus are as follows: flavone compound, steroidal Class compound, triterpene compound, carbohydrate content, alkaloid, Cardiac glycosides, esters, taurine, a variety of amino acid, micro member Element and inorganic elements etc..The laudatory titles such as " king of medicine ", " gold grass ", " god grass ", " bird ginseng " are known as civil.Wherein, anoectochilus formosanus In polysaccharide, flavone compound, steroidal etc. be considered being important active material, the synthesis of these substances directly affects platform The medical value of gulf roxburgh anoectochilus terminal bud.

Phenylalanine lyase (Phenylalanine ammonialyase, PAL) is Flavonoid substances anabolism way Crucial enzyme gene in diameter.In flavone compound metabolic pathway of synthesizing, the enzyme being catalyzed at first is exactly phenylalanine lyase (Phenylalanine ammonialyase, PAL), it is anti-that it can be catalyzed L-phenylalanine (L-phenylalanine) production Formula cinnamic acid (Trans-cinnamic acid).The trans-cinnamic acid of generation, in oxygen and nicotinamide-adenine dinucleotide phosphate Under conditions of (Nicotinamide adenine dinucleotide phosphate, NADPH) is existed simultaneously, by cortex cinnamomi Sour 4- hydroxyl enzyme (Cinnamic acid 4-hydroxy enzyme, C4H) catalysis, can be generated 4- hydroxyl coumaric acid (4- hydroxycinnamate acid).Then 4- hydroxyl coumaric acid-CoA ligase (4-hydroxycinnamate acid- Coenzyme A ligase, 4CL) catalysis connection, 4- hydroxyl tonka-bean is converted by the 4- hydroxyl coumaric acid that previous step generates The thioesters such as acyl-CoA, this step need ATP to provide energy.The 4- hydroxyl coumaric acyl-CoA of generation can be with malonyl-CoA in Cha Er It reacts, generates chalcone (Chalcone) under the action of ketone synthase.Enter the anabolic 5 branch (isoflavones of flavones later Branch, aurones branch, flavones branch, anthocyanidin branch and flavonols branch).Therefore, phenylalanine lyase is plant connection The important rate-limiting enzyme of main life and secondary metabolism, plays an important role in flavone compound metabolic pathway of synthesizing, active and each The anabolism of kind flavone compound and accumulation are closely related.

Summary of the invention

The first object of the present invention is to provide a kind of phenylalanine lyase of anoectochilus formosanus.

The second object of the present invention is to provide a kind of encoding gene of the phenylalanine solution enzyme of anoectochilus formosanus.

The third object of the present invention is to provide a kind of encoding gene of phenylalanine solution enzyme containing anoectochilus formosanus Recombinant vector.

The fourth object of the present invention is to provide a kind of weight of the encoding gene of phenylalanine solution enzyme containing gulf roxburgh anoectochilus terminal bud Group engineering bacteria.

The fifth object of the present invention is that the encoding gene for providing a kind of phenylalanine solution enzyme of anoectochilus formosanus is being expressed Application in phenylalanine lyase.

In order to realize above-mentioned purpose of the invention, the following technical scheme is adopted:

A kind of phenylalanine lyase of anoectochilus formosanus, the amino acid sequence of the enzyme is as shown in SEQ ID NO.1.

A kind of encoding gene of the coding such as phenylalanine solution enzyme of above-mentioned anoectochilus formosanus.

A kind of recombinant vector of the encoding gene of the phenylalanine solution enzyme containing above-mentioned anoectochilus formosanus.

A kind of recombination engineering of the encoding gene of the phenylalanine solution enzyme containing above-mentioned anoectochilus formosanus.

Application of the encoding gene of the phenylalanine solution enzyme of above-mentioned anoectochilus formosanus in expression phenylalanine lyase.

Compared with prior art, the invention has the benefit that the phenylalanine solution of anoectochilus formosanus provided by the invention Adnosine deaminase, sequence are a kind of new Phenylalanine Ammonia-Lyase Gene sequences, are that roxburgh anoectochilus terminal bud primary medicinal component Flavonoid substances close At key gene in the process, play an important role in flavone compound metabolic pathway of synthesizing.

Detailed description of the invention

In order to illustrate the technical solution of the embodiments of the present invention more clearly, below will be to needed in the embodiment attached Figure is briefly described, it should be understood that the following drawings illustrates only certain embodiments of the present invention, therefore is not construed as pair The restriction of range for those of ordinary skill in the art without creative efforts, can also be according to this A little attached drawings obtain other relevant attached drawings.

Fig. 1 is gene C DS sequence electrophoresis detection result figure；

Fig. 2 is the albumen tertiary structure figure of anoectochilus formosanus PAL prediction；

Fig. 3 is related for anoectochilus formosanus PAL albumen and sets analysis；

Fig. 4 is gene different tissues expression pattern figure；

Fig. 5 is expression pattern figure of the gene at 4mg/L Phe in 12h；

Fig. 6 is expression pattern figure of the gene at 100mM NaCl in 12h；

Fig. 7 is expression pattern figure of the gene under the ultraviolet stress of 253.7nm in 12h；

Fig. 8 is expression pattern figure of the gene under feux rouges stress in 12h；

Fig. 9 is transient expression vector pC2300-35S-PAL-eGFP；

Figure 10 is the subcellular localization of anoectochilus formosanus PAL albumen；

Figure 11 is anoectochilus formosanus PAL gene southern botting result figure；

Figure 12 is the screening figure for turning anoectochilus formosanus PAL gene arabidopsis；

Figure 13 is that the PCR of transgenic arabidopsis detects figure；

Figure 14 is the general flavone enrichment condition of transgenic arabidopsis.

Specific embodiment

Embodiment of the present invention is described in detail below in conjunction with embodiment, but those skilled in the art will Understand, the following example is merely to illustrate the present invention, and is not construed as limiting the scope of the invention.It is not specified in embodiment specific Condition person carries out according to conventional conditions or manufacturer's recommended conditions.Reagents or instruments used without specified manufacturer is The conventional products that can be obtained by commercially available purchase.

Feature and performance of the invention are described in further detail with reference to embodiments.

Embodiment

One, the extraction and purifying of anoectochilus formosanus total blade RNA, Total RNAs extraction use the precious biological Cable companies in Dalian Trizol extracts kit, includes the following steps.

(1) powder after taking about 100mg to grind respectively is packed into 1.5mL centrifuge tube, and 1mL RNAiso is added immediately after Plus is mixed by inversion.

(2) homogenate mixed well is being stored at room temperature 5min, then 12000g, 4 DEG C of centrifugation 5min.

(3) Aspirate supernatant 800ml is transferred in new centrifuge tube, is sure not to draw precipitating, supernatant volume is then added 200ml chloroform, and shake vigorously and mix well to homogenate emulsification and be creamy white, then it is stored at room temperature 5min.

(4) by mixed liquor in 12000g, 4 DEG C of centrifugation 15min, homogenate is divided into three layers at this time.It is respectively from top to bottom: on Clear liquid (containing RNA), intermediate white layer (most of is DNA) and coloured lower layer's organic phase.

(5) Aspirate supernatant 400ml is transferred in new centrifuge tube, is sure not to touch middle layer.Then the different of 400ml is added Propyl alcohol is simultaneously mixed by inversion, and then stands 10min at room temperature.

(6) by the solution after standing in 12000g, 4 DEG C of centrifugation 10min, the cotton-shaped RNA of visible white at this time.Carefully discard Simultaneously 75% ethyl alcohol of 1mL is added in supernatant, turns upside down and discards supernatant after washing RNA.

(7) after opening centrifuge tube lid drying at room temperature RNA a few minutes, the RNase-free water that 30 μ l are added dissolves RNA.

(8) concentration that total serum IgE is calculated using the value of ultramicrospectrophotometer (Bio-Rad, the U.S.) measurement A260, is read The value of OD260/OD280 is taken to estimate total serum IgE purity and integrality.With 1.2% agarose gel electrophoresis, 135V is quickly detected RNA mass.

Two, the transcript profile sequencing of anoectochilus formosanus, the method is as follows:

The total serum IgE of anoectochilus formosanus blade is extracted, detection RNA extracts quality, meets requirement for construction data base (RNA concentration > 250ng/ μ L, total amount > 20 μ g, OD₂₆₀/OD₂₈₀Between 1.8~2.2, integrality is good, RIN > 6.5).Then, with enrichment with magnetic bead poly (A) mRNA is broken into a section segment, as template, the 1st article of cDNA chain and the 2nd article of cDNA chain is successively synthesized, by purifying, washing De-, end is repaired plus poly (A) connects sequence measuring joints afterwards.It selects the segment of 200bp~700bp size to carry out PCR amplification, builds Vertical cDNA sequencing library, is sequenced with IIIumina HiSeq 2000.Part Experiment commission beauty is because of the wired public affairs of science service Department completes.

De novo is using short reads composite software Trinity (v2.4.0) to assemble, and is obtained the Contig without N and is assembled After segment, de-redundancy is carried out using tgicl (v2.1), removes that quality in sequence is low, uncertain sequence.Remain larger than 200bp Sequence carry out subsequent analysis.Prediction of gene structure is carried out to above-mentioned splicing result using transdecoder (v2.0.1), in advance Subsequent analysis is used for after geodesic structure.

Three, the first chain of anoectochilus formosanus cDNA synthesizes, the method is as follows:

Using the roxburgh anoectochilus terminal bud blade total serum IgE of above-mentioned one resulting purifying as template, drawn with oligo (dT) 18 for reverse transcription Object, using PrimeScriptTM Reverse Transcriptase (TakaRa China) according to SMART^TM PCR cDNA The synthesis of Synthesis Kit (Clontech USA) operating instruction progress the first chain of cDNA.Reaction system total volume is 20 μ L.

(1) reverse transcription mixed liquor 1 (table 1) is prepared in 0.2mL PE pipe；

(2) following reverse transcription mixed liquor 2 (table 2) is prepared in another 0.2mL PE pipe；

(3) by chilling 2min on ice rapid after 165 DEG C of heat preservation 5min of reverse transcription mixed liquor of first step configuration, it is centrifuged number Second the mixed liquor of template ribonucleic acid, primer etc. is made to be gathered in PE bottom of the tube；

(4) the reverse transcription mixed liquor 2 by second step configuration is added in the reaction solution of first step configuration, is gently mixed with liquid-transfering gun 42 DEG C of reaction 90min after conjunction；

Cooled on ice after (5) 80 DEG C of heat preservation 5min, obtained cDNA solution are used directly for subsequent experimental.

1 reverse transcription mixed liquor 1 of table

2 reverse transcription mixed liquor 2 of table

Four, the extraction and purifying of anoectochilus formosanus total leaf DNA, Genome DNA extraction use CTAB method, include the following steps.

(1) tissue about 100mg is taken, liquid nitrogen is added and sufficiently mills.The RNase A of 400 μ L buffer FP1 and 6 μ L is added (10mg/mL), vortex vibrate 1min, are placed at room temperature for 10min.

(2) 130 μ L buffer FP2 are added, mix well, vortex oscillation 1min.

(3) 12000r/min is centrifuged 5min, supernatant is transferred in new centrifuge tube.

(4) step 3 is repeated, to remove the precipitated impurities in supernatant, keeps extraction genomic DNA purity higher.

(5) isopropanol of 0.7 times of volume pre-cooling is added into supernatant and mixes well, will appear cotton-shaped genome at this time DNA.Then 12000r/min is centrifuged 2min, abandons supernatant and retains precipitating.

(6) 70% ethyl alcohol of 1mL is added, after vortex oscillation 10s, 12000r/min is centrifuged 2min, abandons supernatant.

(7) step 6 is repeated.

(8) it uncaps inversion, appropriate elution buffer TE is added after remaining ethyl alcohol thoroughly volatilizees in 5~10min of room temperature Dissolving DNA is mixed by inversion for several times therebetween, finally obtains DNA solution.

Five, the clone of anoectochilus formosanus PAL gene open reading frame and code area, method are as follows.

On the basis of the sequencing splicing of anoectochilus formosanus transcript profile obtains the CDS sequence of PAL gene, using Primer 5.0 software Design primers (PALF/PALR:5'-ATGGACCATGCTAGGGAGAACG-3'/5'- of Premier CTAGCAAATAGGGAGAGGAGCTTCA-3'), the specificity of designed primer analyte primer in Oligo6.0.Reaction Program are as follows: 95 DEG C of initial denaturation 3min；38 circulations are expanded by 95 DEG C of denaturation 30s, 52 DEG C of annealing 30s, 72 DEG C of extension 1min later. PCR reaction uses 50 μ L systems (table 3).

Table 3PCR reaction system

Take gained amplified production 50ul through 1% non denatured Ago-Gel 180V electrophoresis 30min, after GoLdenView dyeing Ultraviolet inspection amplified fragments, as a result as shown in Figure 1.Wherein, A is the amplification of open reading class, and B is gene coding region (containing including Son) amplification, M marker, 1 is anoectochilus formosanus, and 2 be Anoectochilus roxburghii.Illustrate that Anoectochilus roxburghii and anoectochilus formosanus are open Reading frame is similar with coding domain segment size.

The recycling of target fragment:

The specific band come will be amplified to be dug out in clean blade in the UV lamp fast and standard slave agarose, be placed in In 1.5ml centrifuge tube, recycled with gel reclaims kit (the E.Z.N.A.TM Gel Extraction Kit of OMEGA company) DNA fragmentation in glue.

Connection reaction:

It will be in the Pmd19-T carrier of the DNA fragmentation clone of recycling and TaKaRa company.Connection reaction is using connection kit (TaKaRa company), linked system total volume 10ul, 16 DEG C of connections are overnight.

The preparation of competent cell:

The E.coLiDH5a single colonie newly activated from picking on LB plate, is inoculated in 3-5ml LB liquid medium, 37 DEG C, 225r/min shaken cultivation 12h or so, until the logarithmic growth later period.By the bacterium solution suspension with the ratio of 1::10-1:50 It is inoculated in 100ml LB liquid medium, 37 DEG C of shaken cultivation 2-3h to OD₆₀₀=0.35-0.5 or so.

Culture solution is transferred to centrifuge tube, places 10min on ice, then 3000r/min is centrifuged 10min. at 4 DEG C

It discards supernatant, with the CaCl of the 0.05mol/L of pre-cooling₂Solution 10ml gently suspension cell, places 15- on ice After 30min, 3000r/min is centrifuged 10min at 4 DEG C.

Supernatant is abandoned, the CaCl of 0.05mol/L of the 4ml pre-cooling containing 15% glycerol is added₂Solution, gently suspension cell, on ice Place a few minutes, competent cell suspension.

Competent cell is distributed into the aliquot of 100ul, is stored in -70 DEG C of storable half a year.

The conversion of Plasmid DNA:

A pipe competent escherichia coli cell DH5a is taken out from -70 DEG C of refrigerators, is placed on ice to melt.

10ml connection reaction solution is aseptically added, after gently concussion mixes, places 30min on ice.

42 DEG C of water-bath thermal shock 90s, not shake, and be put into the cooling 2-3min. of ice bath rapidly later

LB liquid medium of the 890ul without Amp is added, after mixing, 37 DEG C, the shaking culture of 150r/min shaking table incubate 1h.

The bacterium solution for taking 100ul to convert is applied on the culture plate of a LB Amp, face-up places 30min, to bacterium After liquid is cultured base absorption completely, with ParafilmTM, culture dish is then reversed, 37 DEG C are protected from light culture 12-16h；Then screening Positive bacterium colony.

Recombinate the identification and preservation of bacterium colony

From the appearance point of view, the bacterium colony of general white and circle is the positive, further identified by bacterium solution PCR.

Several white colonies are taken with the lancet choicest of sterilizing on the plate being incubated overnight, dibbling is in 0.1g/L Amp respectively LB liquid medium 1.5ml centrifuge tube in, 37 DEG C, 150r/min shaking culture 4-6h.

It takes 1ul bacteria suspension as template, carries out PCR amplification, pyrolysis time in PCR reaction condition with primer PALF/PALR 5min is extended to, detects amplified production with 1% non denatured agarose gel electrophoresis.If product is purpose band, this bacterium It falls as positive colony, is otherwise feminine gender.The negative control that bacteria suspension is not added is set simultaneously.

The bacterium colony suspension 750ul for taking identified positive colony, is added the sterile glycerol of 250ul, after mixing, uses liquid Nitrogen is quick-frozen, is placed in -70 DEG C of refrigerators and saves backup.

Handsome biotech company is finally sent to be sequenced, gained sequence is compared in the BLastn of NCBI, verifying gram Grand segment is correct.

Expanding obtained product is ArPAL gene cDNA open reading frame sequence and coding region sequence, wherein amino sequence For SEQ ID NO.1, coding region nucleotide sequence is SEQ ID NO.2, and open reading frame nucleotides sequence is classified as SEQ ID NO.3。

Six, AfPAL protein biology bioinformatics analysis, the method is as follows:

Its physics and chemical property are carried out using ProtParam (http://web.expasy.org/protparam/) Analysis.Anoectochilus formosanus PAL full length gene 2148bp, coding region sequence are all 2733bp, and the 408th to being inserted into 1 between 993bp The introne of long 585bp encodes 715 amino acid.There are PAL albumen 2 PAL typically to guard domain, phenylalanine and histidine The region MIO that Structure and function domain (GTITASGDLVPLSYIA) and Ala-Ser-Gly active site are formed.In addition, strict conservation Site (Y109, L137, S202, N259, Q347, Y350, R353, F399 and Q487), deamination site (L205, V206, L255 and A256), active site (N259, G260, NDN 381-383aa, H395 and HNQDV 485-488aa) and Phosphorylation site (VAKRVLTF 542-549aa) can be found in anoectochilus formosanus PAL albumen corresponding position.Utilize ExPASy Proteomics Server provide online tool ProtParam the physicochemical property of PAL gene coded protein is predicted, Speculate that the albumen relative molecular mass is 77.4kDa, isoelectric point pI 6.18.

Do you use GOR IV (http://npsa-pbil.ibcp.fr/cgi-bin/npsa_automat.pl? page= npsa_gor4.html)；The secondary structure of PAL gene is predicted.Secondary structure on-line prediction the result shows that, alpha-helix (Alpha helix), accounts for the 48.53% of total amino acid, extended chain (Extended strand), Zhan Suoyou amino acid 10.07%, random coil (Random coil) accounts for the 41.40% of total amino acid.

By SWISS-MODEL (https: //swissmodel.expasy.org/) come the PAL for anoectochilus formosanus Gene carries out the prediction of albumen tertiary structure, is mapped by PyMOL Viewer, threedimensional model is shown in Fig. 2.

The PAL amino acid sequence for finally first finding the higher plant of homology carries out amino acid sequence using ClustalW method The comparison of column constructs Neighbor-joining systematic evolution tree such as Fig. 3 (for locating for anoectochilus formosanus in frame by MEGA7.0 Position).

Seven, the endogenous detection of expression of AfPAL, the method is as follows:

(1) material processing

By the anoectochilus formosanus tissue culture transplantation of seedlings of culture 4 months to the flowerpot that Nutrition Soil (Nutrition Soil: vermiculite=3:1) is housed In, in 200 μm of 25 DEG C of (daytime)/20 DEG C (night) of temperature, relative humidity 60~70% and illumination ol/m²S (14h)/dark (10h) Under conditions of cultivate.It adapting to 3~5 days, the uniform untreated seedling of growing way takes root, stem, leaf sample, and -70 DEG C of liquid nitrogen flash freezer It saves.The uniform seedling of remaining growing way is divided into two groups, one group of carry out ultraviolet light processing, at another group of carry out red light irradiation Reason.Ultraviolet light processing: the roxburgh anoectochilus terminal bud of potting being placed in ultraviolet and feux rouges incubator, ultraviolet radiation wave-lengths 253.7nm, Red light wavelength 650nm handles 0h, 0.5h, 1h, 2h, 4h, 8h and 12h respectively.Each each processing is repeated 3 times, and throughout manages the time Point samples -70 DEG C of liquid nitrogen flash freezer preservations immediately.

The anoectochilus formosanus tissue-cultured seedling cultivated 4 months is moved on plastic foam plate with holes, with Hoagland nutrient solution 3~5d is cultivated, the uniform seedling of growing way is selected to carry out phenylalanine and salt stress processing.Phenylalanine and salt stress processing are That Phe and NaCl are added to Hoagland nutrient solution to final concentration 4mg/L and 100mmol/L, handle respectively 0h, 0.5h, 1h, 2h, 4h, 8h and 12h.Each each processing is repeated 3 times, and throughout reason time point samples -70 DEG C of liquid nitrogen flash freezer preservations immediately.

(2) RNA is extracted

The blade of above-mentioned 3 kinds of Stress treatments and control group seedling is taken, liquid nitrogen flash freezer grinding, is tried using Total RNAs extraction respectively Agent box Trizol (TaKaRa, Dalian) simultaneously extracts total serum IgE according to its specification.Step such as embodiment 1.

(3) the first chain of cDNA synthesizes

It using the RNA of extraction as template, is loaded according to 4 system of table, 42 DEG C of reaction 2min remove possible genomic DNA (gDNA), PrimeScript is used^TMRT reagent Kit with gDNA Eraser kit (TaKaRa, Dalian), by table 5 systems sample-adding, 37 DEG C of warm bath 30min, 85 DEG C of holding 5s terminate reaction and synthesize cDNA with reverse transcription, and save backup in -20 DEG C.

Table 4gDNA removes reaction system

Table 5cDNA synthetic system

(4) specific detection of qRT-PCR primer

The template reacted after 3 times of cDNA sample dilutions as qRT-PCR is compareed with 0h, using 50~65 DEG C of annealing temperature Gradient carries out qRT-PCR reaction to determine optimal annealing temperature, using 20 μ L reaction systems (table 6).Flavones anabolism way The primer sequence of key gene such as table 7 in diameter.

Table 6qRT-PCR reaction system

Table 7qRT-PCR primer sequence

Reaction tube uses the 0.2mL PCR plate after hundred happy silication.QRT-PCR is in iQ^TM 5thermal cycler(Bio- Rad USA) it carries out.Response procedures are as follows: 95 DEG C of initial denaturation 10s；Then by 95 DEG C of denaturation 10s, 50~65 DEG C of annealing 20s, 72 DEG C Extend 20s, and in this temperature collection fluorescence, the program of read plate (Plate read) carries out 46 circulations.Later since 50 DEG C, 95 DEG C are increased to 0.5 DEG C of temperature gradient of every step, every step temperature keeps 5s.

(5) qRT-PCR reacts

The template of qRT-PCR amplification will be used as after 3 times of cDNA sample dilutions of reverse transcription, after reaction tube uses silication The PCR plate of 0.2mL using 20 μ L reaction systems (table 6), and is set up with ddH₂O is the negative control of template.Single reverse transcription CDNA sample is set to be repeated three times.

QRT-PCR response procedures are as follows: then 95 DEG C of initial denaturation 10s press 95 DEG C of denaturation 10s, most suitable annealing temperature annealing 20s, 72 DEG C of extension 20s, and in this temperature collection fluorescence, the program of read plate (Plate read) carries out 46 circulations.Later most Afterwards since 50 DEG C, 95 DEG C are increased to 0.5 DEG C of speed of every step, each temperature keeps 5s, draws melting curve.

(6) qRT-PCR data are analyzed

This experiment is carried out using expression of double calibration curve methods to the key gene in Flavone metabolism route of synthesis Relative quantification.The relative expression quantity of gene is calculated using Δ Δ CT (Normalized Gene Expression) method.

Differential expression SPSS (the version between differential expression and each processing between 0h control and processing 10.0Inc.Chicago, IL) statistical analysis software analyzed.Set up two significance of difference water of P=0.05 and P=0.01 It is flat.(light pillar indicates the table of Anoectochilus roxburghii PAL gene to anoectochilus formosanus PAL gene different tissues expression pattern figure such as Fig. 4 Up to amount, dark pillar indicates that the expression quantity of anoectochilus formosanus, * indicate that significant difference, * * indicate that difference is extremely significant), Taiwan gold thread (light pillar indicates the table of Anoectochilus roxburghii PAL gene to the expression pattern figure such as Fig. 5 of lotus PAL gene at 4mg/LPhe in 12h Up to amount, dark pillar indicates that the expression quantity of anoectochilus formosanus, * indicate that significant difference, * * indicate that difference is extremely significant), 100mM (light pillar indicates the expression quantity of Anoectochilus roxburghii PAL gene, dark pillar table to expression pattern figure such as Fig. 6 under NaCl in 12h Show that the expression quantity of anoectochilus formosanus, * indicate that significant difference, * * indicate that difference is extremely significant), anoectochilus formosanus PAL gene exists (light pillar indicates the expression of Anoectochilus roxburghii PAL gene to expression pattern figure such as Fig. 7 under the ultraviolet stress of 253.7nm in 12h Amount, dark pillar indicate that the expression quantity of anoectochilus formosanus, * indicate that significant difference, * * indicate that difference is extremely significant), anoectochilus formosanus (light pillar indicates the expression of Anoectochilus roxburghii PAL gene to the expression pattern figure such as Fig. 8 of PAL gene under feux rouges stress in 12h Amount, dark pillar indicate that the expression quantity of anoectochilus formosanus, * indicate that significant difference, * * indicate that difference is extremely significant).Anoectochilus roxburghii Although PAL gene and anoectochilus formosanus PAL gene clip size amino acid number, contained structure function with it is identical, they Under phenylalanine, salt, ultraviolet and feux rouges stress, PAL gene expression amount is different.

Eight, it is the subcellular localization of AfPAL albumen, is first the building of transient expression vector.According to anoectochilus formosanus PAL Gene ORF sequence, restriction enzyme site and protection alkali needed for its 5 ' and 3 ' end addition transient expression vector (Fig. 9) building Base does not contain the special primer [5 '-TC of terminator codon with 5.0 software design of PremierCCGGG(Sma I) ATGGACCATGCTAGGGAGAACG-3′/5′-CGCACTAGT(Spe I)GCAAATAGGGAGAGGAGCTTCA-3′]。

(1) it using the pDM19-T plasmid of above-mentioned insertion PAL gene ORF as template, is loaded by 4 reaction system of table, carries out PCR Amplification.The setting of PCR temperature cycles system program are as follows: 94 DEG C, 3min；98 DEG C of 10s, 62 DEG C of 30s, 72 DEG C of 60s, amplification cycles 38 It is secondary；Last 72 DEG C of extensions 5min.

(2) amplified production is separated with 1% non denatured agarose gel electrophoresis,ChemiDoc^TMXRS type gel at As system (Bio-Rad, the U.S.) imaging, with gel reclaims kit (Tiangeng, Beijing) recovery purifying target fragment.

(3) by 8 reaction system of table be loaded, using fast enzyme cutting under the conditions of 30 DEG C digestion 0.5h.Digestion products are with 1.2% Ago-Gel is separated by electrophoresis, with gel reclaims kit (Tiangeng, Beijing) recovery purifying.

8 target fragment Sma I/Spe I double enzyme digestion reaction system of table

(4) it is loaded according to 9 reaction system of table, with double enzyme products of T4DNA ligase connection recovery purifying, 16 DEG C of connections 8h。

9 coupled reaction system of table

(5) connection product is converted with heat-shock transformed method and PCR identification is carried out to recombination bacterium colony.

(6) Plasmid DNA in above-mentioned steps is extracted, after the fast enzyme cutting Sma I and Spe I double digestion identification of correspondence, is taken A small amount of plasmid send Sangon Biotech (Shanghai) Co., Ltd. to be sequenced, and checking carrier building is correct.Then, in onion bulb stem Transient expression detection in epidermal cell.

(7) the 5th layer of scale of fresh onion bulb stem is taken, 2cm × 2cm square is cut into, endepidermis is taken to be laid on 1/2MS plate, 25 DEG C culture 4h.

(8) it weighs 60mg bronze (60 μm of diameter) to be placed in 1.5mL centrifuge tube, add into the centrifuge tube equipped with bronze 70% ethyl alcohol of 1mL, acutely shakes 15min in vortex oscillation, with the revolving speed of 1300g, is centrifuged 1min at room temperature, and abandoning is taken to be centrifuged Supernatant fluid in pipe repeats the step three times.It is eventually adding sterile water, -20 DEG C is put into and saves backup.

(9) it takes 5 μ L bronze suspension in the import centrifuge tube of 1.5mL, 1 μ L transient expression vector DNA (1.0 μ g/ is added μ L), the autoclaved 2.5mol/L CaCl of 8 μ L₂, 4 μ L filter sterilizing 0.1mol/L spermidine, after mixing, in vortex oscillation 3min is acutely shaken on device, and stands 15min in ice bath.

(10) with the revolving speed of 1200g, under room temperature, moment is centrifuged 10s, takes abandoning supernatant fluid, and it is pure that 100 μ L analysis is added The dehydrated alcohol of concentration, bronze is resuspended in concussion on turbula shaker.

(11) with the revolving speed of 12000g, under room temperature, moment is centrifuged 10s, and except supernatant is abandoned, it is pure dense that 15 μ L analysis is added The dehydrated alcohol of degree shakes in turbula shaker and is resuspended, keeps liquid uniform.

(12) particle gun uses PDS-1000/He type particle gun (Bio-Rad, the U.S.), condition is arranged are as follows: sample vacuum chamber 26in Hg is spent, can split film pressure is 1100psi, at range 6cm.10 μ L suspension equalization points are taken to be coated in conversion slide glass Region is entreated, under the above conditions, the bronze that conversion slide glass middle section is wrapped in transient expression vector is uniformly squeezed into and is cultivated Onion bulb stem endepidermis cell.

(13) it is placed with the culture medium of the onion endepidermis after conversion with black plastic bag package, is cultivated at 25 DEG C.

(14) 16, to after for 24 hours, take the onion epidermis film-making on culture medium, in BX63 type fluorescence microscope (Olimpas, day Originally it observes and takes pictures under).As a result such as Figure 10 (subcellular localization of A pC2300-35S-eGFP, B pC2300-35S-PAL-eGFP Subcellular localization), subcellular localization is in nucleus.

Nine, the copy number identification of PAL, the specific method is as follows:

According to anoectochilus formosanus PAL gene ORF sequence, with the special primer (5 '-of Premier 5.0 software design a pair AGCAAGATTACGCCTTGCCT-3′/5′-ATGAGGGGGTTGTCGTTGAC-3′).The PCR product of recycling uses digoxin mark Remember (random priming), reaction is illustrated to carry out by Southern blot kit, and steps are as follows:

(1) 1 μ g template DNA and autoclaved distilled water are added into 200 μ L PCR pipes, final volume is up to 16 μ L.

(2) then boiling water bath 10min is rapidly inserted into mixture of ice and water with denatured DNA.

(3) it is sufficiently mixed DIG-High Prime, and 4 μ L is taken to be added in denatured DNA, mixes and simply centrifugation is placed on 37 DEG C of overnight incubations in PCR instrument.

(4) 65 DEG C of heating 10min terminate reaction.

Then anoectochilus formosanus total DNA is extracted, setting reaction system is 50 μ L, chooses 3 kinds of restriction enzymes pair respectively Anoectochilus formosanus and anoectochilus formosanus total DNA are digested, and reaction overnight under optimum temperature, reaction system is shown in Table 10.

The digestion of 10 roxburgh anoectochilus terminal bud total DNA of table

It will take 2 μ L (0.8 μ g) in 0.8% Ago-Gel containing Goldenview from above-mentioned endonuclease reaction system 50V electrophoresis 6h, film recording electrophoresis result.Remaining sample point sample carries out electricity in 0.7% Ago-Gel of no DNA coloring agent Swimming.After electrophoresis, twice using distilled water detergent gel, removes gel redundance and cut left comer label front and back sides.Add Enter the hydrochloric acid depurination of 100ml 0.25mol/L, room temperature concussion 15min uses distilled water flushing until bromophenol blue turns yellow completely 2 times.It adds denaturing liquid room temperature concussion 40min: being completely recovered to original blue to bromophenol blue.In transfer mark slot, pour into 20 × SSC solution sets a solid support in slot, and be successively placed in from bottom to top on solid support: two gels are wide Filter paper is fallen in transfer mark slot (referred to as " bridge ") from solid support by filter paper is longitudinal, bottom surface in upper gel, nylon membrane (with Gel etc. is big), filter paper (big with gel etc., to be soaked in advance with 20 × SSC), blotting paper (is slightly less than filter paper, 5-8cm high), 400- 800g weight.Positively charged nylon membrane and gel are placed in and turned in blotter, surrounding is surrounded with PE film prevents short circuit, turns Film 18h.After transferring film, filter membrane is placed in 2 × SSC rinsing 5min, is blotted with filter paper.Nylon membrane is placed at 120 DEG C fixed 30min.Hybridization solution and hybridization instrument are now preheating to 37 DEG C, nylon membrane is placed in 10mL hybridization solution prehybridization 90min, and (film is placed in box In, can move freely).The probe for taking 5 μ L to mark is denaturalized 5min in boiling water bath, and is rapidly inserted into mixture of ice and water.It will The probe being denaturalized is added in 10mL hybridization solution, mixes well.Nylon membrane is placed in 42 DEG C of mild oscillation 20h in hybridization solution.[probe Hybridization temperature is calculated and is obtained according to G/C content and probe percentage similar to target segment, and formula is as follows: Tm=49.82+ 0.41 × 38 (%G+C)-(600/I), (length for the segment that I=can hybridize, calculated with base-pair)].Using 2 × SSC 0.1%SDS is washed 2 times in room temperature continuous oscillation, each 5min.Later, with the 0.5 × SSC 0.1% for being preheating to 65 DEG C SDS continuous oscillation is washed 2 times, every time 15 minutes.First nylon membrane 2min is eluted with washing buffer；By nylon membrane in 50mL 1 × It blockades to be stored at room temperature in liquid and blockades 35min；Antibody is centrifuged 5min in 12000r/min, takes 4 μ L of upper layer antibody-solutions, and 20 μ L are added It blockades in liquid and mixes, cover nylon membrane, be stored at room temperature 40min；Washing 2 times is mildly vibrated with enough washing buffers, every time 15min；Washing 2 times, each 15min are mildly vibrated with enough washing buffers；Nylon membrane is placed in 15mL developing solution, quiet It sets, is protected from light, closed colour developing 4-16h.It is taken pictures preservation using nylon membrane of the imager to closed colour developing, as a result as Figure 11 (uses BamH Two kinds of enzyme digestions of I and Sac I, southern botting are three copies) shown in.With two kinds of enzyme digestions of BamH I and Sac I, Southern botting is three copies.Illustrate that anoectochilus formosanus PAL gene is multi-copy gene.

Ten, the heterogenous expression for PAL gene in, the specific method is as follows:

(1) it takes appropriate wildtype Arabidopsis thaliana seed to be placed in 1.5mL centrifuge tube respectively, 75% alcohol is added and impregnates 30s, so After discard alcohol and be added 10%NaClO disinfection 10min.

(2) after sterilizing, with sterile water wash seed 3~4 times, after seed avales, the water on upper layer is abandoned.

(3) seed is seeded in 1/2MS culture medium to put down, is protected from light 4 DEG C of processing 48h.

(4) plate is placed in culturing room, 20~22 DEG C of temperature, humidity 60~70%, by seedling replanting to being equipped with after about 2 weeks In the culturing pot of Nutrition Soil (Nutrition Soil: frog stone=4:l), Nutrition Soil is inhaled permeable before transplanting seedlings.

(5) for the seedling short-day (10h illumination/14h is dark) after transplanting after culture 4 weeks, the long-day cultivates (16h illumination/8h It is dark), when plant bolting and while growing bud prepares dip dyeing.

(6) Agrobacterium for taking the recombinant plasmid containing pC2300-35S-PAL-eGFP, containing 50mg/L Kan and The flat lining out of the YEP of 50mg/L Rif, 28 DEG C of 2~3d of culture.

(7) picking Agrobacterium single colonie, which is seeded in 3mL YEP fluid nutrient medium, (contains Kan and Rif, concentration is 50mg/ L), 28 DEG C, 250r/min, shaken cultivation is overnight.

(8) (contain Kan and Rif, concentration is 50mg/L) in 100mL liquid YEP medium, inoculation 1mL is incubated overnight Starter Agrobacterium bacterium solution, 28 DEG C of shaken cultivations to bacterium solution OD600 value be 1.2~1.5.

(9) 4 DEG C, 5000r/min is centrifuged 10min and collects cell, with 5% sucrose solution suspension thalline and adjusts OD600 value extremely 0.8~1.0, surfactant silwetL-77 is added in 1/10000 to 2/10000 ratio.

(10) growth is had the culturing pot side of arabidopsis by the flower cutting established fruit pod before dip dyeing and having opened It puts, the bud of arabidopsis is made to be completely immersed in dip dyeing liquid for shell about 1~2min, the plant dark culturing after dip dyeing is stayed overnight.

(11) after dark culturing, the plant after dip dyeing is placed on culturing room and continues to cultivate, is collected after its maturation Seed carries out the screening of next step.

(12) seed after the dip dyeing of harvest is carried out disinfection by above-mentioned the surface of the seed sterilisation step.

(13) seed after surface sterilization is sowed to the 1/2MS culture medium containing 30mg/LHyg or the Kan of 50mg/L, is kept away After 4 DEG C of processing 2d of light, it is transferred to culturing room's culture.

(14) after cultivating about 2 weeks, positive transgenic plant is in green and grows normally, and negative Miao Ze is not grown or yellow is dead Die (Figure 12).Positive transgenic plantlet of transplant is cultivated into culturing pot, to its mature and single plant sowing.

(15) above-mentioned screening step is repeated, until the transgenic plant that harvest T3 generation is homozygous.

11, turn the PCR verifying of PAL trans-genetic hybrid rice, the specific method is as follows

(1) blade for the homozygous lines for taking T3 generation to be overexpressed and have complementary functions, extracts genomic DNA.

(2) special with Primerblast software (https: //blast.ncbi.nlm.nih.gov/Blast.cgi) design Different the primer (- CTAGCAAATAGGGAGAGGAGCTTCA-3 ' of 5 '-CATTTGGAGAGGACAGGGTACC-3 '/5 '), and expand across Promoter 2174bp segment.

(3) 1.2% non denatured agarose gel electrophoresis separation,ChemiDocTM XRS type gel imaging system (Bio-Rad, the U.S.) imaging, screens positive transgenic plant from regeneration plant.As a result as (F-1, F-2 and F-3 are to turn to Figure 13 The overexpression arabidopsis of anoectochilus formosanus PAL gene, R-1 and R-2 are the overexpression arabidopsis for turning Anoectochilus roxburghii PAL gene ,+ Indicate positive plasmid ,-indicate non-transgenic line).

12, transgenic arabidopsis general flavone content detects, the specific steps are as follows:

(1) rice leaf for turning anoectochilus formosanus PAL gene is weighed, drying grinding pulverized specimen 1.000g is in 20mL in cone In shape bottle, it is repeated 3 times.

(2) 95% ethyl alcohol 10mL is added, in KQ-100DV type ultrasonoscope (Kunshan Ultrasonic Instruments Co., Ltd.), 30 DEG C 30min is extracted, is filtered with 3 μm of filter paper, collect filtrate.

(3) residue adds same concentration ethanol, and by step (2), filtrate is collected in ultrasonic wave extraction, 3 μm of filter paper filterings again, with Step (2) filtrate merges.

(4) each 2mL of above-mentioned filtrate (ethyl alcohol of blank control addition 2mL 95%) is taken, 100g/L Al (NO is added₃)₃ 1mL, 9.8g/L potassium acetate 1mL, stand 30min after shaking up.

(5) 1cm cuvette is used, in UV-1800 type ultraviolet-visible spectrophotometer (Shimadzu, Japan), measures 415nm Light absorption value.As a result as (F-1, F-2 and F-3 are the overexpression arabidopsis for turning anoectochilus formosanus PAL gene to Figure 14, and R-1 and R-2 are Turn the overexpression arabidopsis of Anoectochilus roxburghii PAL gene, * indicates that significant difference, * * indicate that difference is extremely significant).

Embodiments described above is a part of the embodiment of the present invention, instead of all the embodiments.Reality of the invention The detailed description for applying example is not intended to limit the range of claimed invention, but is merely representative of selected implementation of the invention Example.Based on the embodiments of the present invention, obtained by those of ordinary skill in the art without making creative efforts Every other embodiment, shall fall within the protection scope of the present invention.

SEQUENCE LISTING

<110>Sanming College

<120>it a kind of phenylalanine lyase of anoectochilus formosanus and its encoding gene, recombinant vector, recombination engineering and answers With

<160> 3

<170> PatentIn version 3.5

<210> 1

<211> 715

<212> PRT

<213> Anoectochilus formosanus

<400> 1

Met Asp His Ala Arg Glu Asn Gly His Val Met Glu Asn Gly His Val

1 5 10 15

Thr Glu Asn Gly Leu Cys Leu Lys Gly Lys Asp Pro Leu Gly Trp Ile

20 25 30

Ala Ala Ala Lys Ala Val Glu Gly Ser His Leu Glu Glu Val Lys Arg

35 40 45

Met Val Glu Asp Phe Arg Arg Pro Val Val Arg Leu Glu Gly Ala Glu

50 55 60

Leu Lys Ile Ser Gln Val Ala Ala Val Ala Ala Gly Val Val Ser Gln

65 70 75 80

Val Gln Leu Ala Glu Ser Ala Arg Ala Gly Val Asn Ala Ser Ser Asp

85 90 95

Trp Val Met Glu Ser Met Ser Ala Gly Gly Asp His Tyr Gly Val Thr

100 105 110

Thr Gly Phe Gly Ala Thr Ser His Arg Arg Thr Lys Gln Gly Gly Ala

115 120 125

Leu Gln Lys Glu Leu Ile Arg Phe Leu Asn Ala Gly Ile Phe Gly Ser

130 135 140

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

145 150 155 160

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

165 170 175

Ile Leu Glu Ala Ile Thr Ser Leu Leu Asn Ser Lys Ile Thr Pro Cys

180 185 190

Leu Pro Leu Arg Gly Thr Ile Thr Ala Ser Gly Asp Leu Val Pro Leu

195 200 205

Ser Tyr Ile Ala Gly Val Leu Thr Gly Arg Pro Asn Cys Lys Ala Ile

210 215 220

Thr Ala Asp Gly Val Thr Val Asn Ala Val Glu Ala Phe Arg Leu Ala

225 230 235 240

Gly Ile Ser Ser Gly Phe Phe Asp Leu Gln Pro Lys Glu Gly Leu Ala

245 250 255

Leu Val Asn Gly Thr Ala Val Gly Ser Gly Phe Ala Ser Ile Val Leu

260 265 270

Phe Glu Ala Asn Ile Leu Ala Leu Met Ala Glu Val Leu Ser Ala Leu

275 280 285

Phe Cys Glu Val Met Gln Gly Lys Pro Glu Phe Thr Asp His Leu Thr

290 295 300

His Lys Leu Lys His His Pro Gly Gln Ile Glu Ala Ala Ala Ile Met

305 310 315 320

Glu His Val Leu Glu Gly Ser Ser Tyr Met Lys Met Ala Lys Lys Leu

325 330 335

His Asp Leu Asp Pro Leu Gln Lys Pro Lys Gln Asp Arg Tyr Ala Leu

340 345 350

Arg Thr Ser Pro Gln Trp Leu Gly Pro Gln Ile Glu Val Ile Arg Ala

355 360 365

Ala Thr Lys Ser Ile Glu Arg Glu Ile Asn Ser Val Asn Asp Asn Pro

370 375 380

Leu Ile Asp Val Ser Arg Asn Lys Ala Ile His Gly Gly Asn Phe Gln

385 390 395 400

Gly Thr Pro Ile Gly Val Ser Met Asp Asn Thr Arg Leu Ala Ile Ala

405 410 415

Ala Ile Gly Lys Leu Met Phe Ala Gln Ile Ser Glu Leu Val Asn Asp

420 425 430

Phe Tyr Asn Asn Gly Leu Pro Ser Asn Leu Ser Gly Gly Arg Asn Pro

435 440 445

Ser Leu Asp Tyr Gly Phe Lys Gly Ala Glu Ile Ala Met Ala Ser Tyr

450 455 460

Cys Ser Glu Leu Gln Tyr Leu Ala Asn Pro Val Thr Asn His Val Gln

465 470 475 480

Ser Ala Glu Gln His Asn Gln Asp Val Asn Ser Leu Gly Leu Ile Ser

485 490 495

Ser Arg Lys Thr Gly Glu Ala Val Glu Ile Leu Lys Leu Met Thr Ser

500 505 510

Thr Phe Leu Val Ala Leu Cys Gln Ala Ile Asp Leu Arg His Leu Glu

515 520 525

Glu Asn Leu Lys Cys Ala Val Lys Asn Ala Val Ser Leu Ala Ala Lys

530 535 540

Arg Thr Leu Thr Phe Gly Ala Asn Gly Asp Leu His Pro Ser Arg Phe

545 550 555 560

Cys Glu Lys Asp Leu Ile Lys Val Val Asp Lys Glu Tyr Val Phe Ala

565 570 575

Tyr Ala Asp Asp Pro Cys Ser Ser Thr Tyr Pro Leu Met Gln Lys Leu

580 585 590

Arg Gln Val Leu Val Glu His Ala Leu Ser Asn Gly Asp Lys Glu Lys

595 600 605

Ala Arg Ser Thr Ser Ile Phe Gln Lys Ile Thr Asp Phe Glu Glu Asp

610 615 620

Ile Asn Ala Ala Leu Pro Lys Ala Val Glu Ala Ala Arg Ala Ala Phe

625 630 635 640

Glu Lys Gly Ser Ser Ala Ile Glu Asn Arg Ile Lys Glu Cys Arg Ser

645 650 655

Tyr Pro Leu Tyr Arg Leu Val Arg Glu Glu Leu Gly Ala Gly Phe Leu

660 665 670

Thr Gly Glu Lys Ala Met Ser Pro Gly Glu Glu Phe Asp Lys Val Phe

675 680 685

Asn Ala Ile Cys Glu Gly Arg Ala Ile Asp Pro Leu Leu Glu Cys Leu

690 695 700

Lys Glu Trp Asn Glu Ala Pro Leu Pro Ile Cys

705 710 715

<210> 2

<211> 2733

<212> DNA

<213> Anoectochilus formosanus

<400> 2

atggaccatg ctagggagaa cggtcacgtg atggagaacg ggcacgtgac ggagaacggg 60

ctatgcctaa aggggaagga cccgctggga tggatcgcgg cggcaaaggc ggtggagggg 120

agccaccttg aggaggtgaa gcggatggtg gaggatttcc ggcgtccggt ggtgaggctc 180

gaaggagcgg agctcaaaat atcgcaggta gccgcagtgg ctgccggcgt tgtttcccaa 240

gtacagctag cggagtctgc gcgcgctggg gtgaatgcca gcagcgactg ggtgatggag 300

agcatgagtg ctggtggcga ccactacggc gtcactaccg gcttcggcgc tacatctcac 360

cgccgcacca agcagggcgg cgccctgcag aaagaactca tcagattctg aaaatggcgg 420

agcatttttt gtgtttaact tggatggctt gatttgcttt gtcaagtttg cttctttttt 480

ccgaccggta aggctagggt tgggattttg gtggtaggtt ttggtttgtg tggtaggtgg 540

cgagaagaag atggagaact ctatcgtttg ttagctggat gtgttaaaga tctcagtttt 600

atggggattc ggcaggacaa aaaaaagggg aactttttat ttctcaatca tgaaagagaa 660

cattttgctt tgacaaatta acaaaatttt gttctatcag gctttttaaa actttggtgg 720

attgaattaa gcgaatattt tcatttagaa acttagctgg ttggtaggtt ttggttcttg 780

agcggtaggt actgaggatc gaattgggtg gtgccattaa tgaggggaag gtcctctgtt 840

ttctttagtc agctttattg gattcagctg aggaagaatc aaaggaggaa aaatgttgtt 900

ttttttatat aaaaaattct tttggtctct tcactttcca tttaaatggc agaacgaaag 960

cttttaaaaa gaattttttt attgttatcc agacttaatg cggggatctt cggatcaggg 1020

acaaacaaca cgctgccttc ggccgccagc agggctgcga tgcttgtgag gatcaacacc 1080

ctcctccaag gttactccgg catccgtttt gaaatcctgg aggccattac cagcctcctc 1140

aacagcaaga ttacgccttg cctgccgctg aggggaacca tcaccgcctc cggcgatctt 1200

gttccactat cttacattgc gggtgtctta accggccgtc ccaattgcaa ggctataacg 1260

gccgacggtg ttactgtcaa cgcagtagag gccttccgtc ttgcaggaat ctccagcggg 1320

ttcttcgatc ttcagcccaa ggaagggctc gcacttgtca atggaaccgc cgtcggctcc 1380

ggcttcgcct ccattgtcct gttcgaggca aacatcctcg cccttatggc agaggttctc 1440

tctgctctgt tctgcgaggt gatgcagggg aagccggagt tcaccgacca cctcacccac 1500

aagctgaaac accacccggg acaaatcgag gccgccgcca tcatggagca cgtgcttgaa 1560

ggaagctcct acatgaagat ggccaagaag ctccacgatt tggatcctct tcagaagcca 1620

aagcaggatc gctatgctct ccgcacctca ccccaatggc tcggccctca gatcgaagtg 1680

atccgagcag cgaccaagtc catcgagagg gagataaatt cagtcaacga caaccctctg 1740

attgatgtct cgaggaacaa ggccatccat ggaggcaact tccaagggac ccccattggc 1800

gtttccatgg acaacaccag gctcgccatt gctgccatcg ggaagctcat gttcgcccaa 1860

atatcagagc ttgtcaatga cttttataac aacggcttgc cttcaaatct atccggtggg 1920

agaaacccta gcttggatta tggcttcaaa ggcgcggaga tagccatggc ttcctactgc 1980

tccgagctcc agtacctcgc caatccggtc acaaaccatg tgcagagcgc cgagcagcac 2040

aaccaggacg tgaactccct gggactgata tcttcgagga agacggggga ggcggtggag 2100

atactaaagc tcatgacctc caccttcctg gttgcactct gccaagccat agacttgagg 2160

catctggagg agaacttgaa gtgtgccgtg aagaatgcgg tgagcctggc ggcaaagagg 2220

actctcactt tcggggccaa tggagatctt catccatcca ggttctgcga gaaggatttg 2280

atcaaggtgg tagataagga gtatgtgttc gcctacgccg acgatccctg cagctctacc 2340

taccctttga tgcagaagct caggcaggtg ctggttgagc atgccctcag caacggcgac 2400

aaggagaagg ccaggagcac ctccatcttc caaaagatca cagattttga ggaggatatc 2460

aatgccgcgc ttcccaaagc ggtcgaggcc gccagagcgg cgtttgagaa ggggtcgtcg 2520

gcgatagaga acagaatcaa agaatgcaga tcctacccac tgtacaggct tgtgagggaa 2580

gagctcgggg ccggctttct caccggagag aaggcgatgt cgccagggga ggaattcgac 2640

aaggtcttca atgccatttg cgaggggagg gcgatagatc ctctgctcga gtgcttgaag 2700

gagtggaatg aagctcctct ccctatttgc tag 2733

<210> 3

<211> 2148

<212> DNA

<213> Anoectochilus formosanus

<400> 3

atggaccatg ctagggagaa cggtcacgtg atggagaacg ggcacgtgac ggagaacggg 60

ctatgcctaa aggggaagga cccgctggga tggatcgcgg cggcaaaggc ggtggagggg 120

agccaccttg aggaggtgaa gcggatggtg gaggatttcc ggcgtccggt ggtgaggctc 180

gaaggagcgg agctcaaaat atcgcaggta gccgcagtgg ctgccggcgt tgtttcccaa 240

gtacagctag cggagtctgc gcgcgctggg gtgaatgcca gcagcgactg ggtgatggag 300

agcatgagtg ctggtggcga ccactacggc gtcactaccg gcttcggcgc tacatctcac 360

cgccgcacca agcagggcgg cgccctgcag aaagaactca tcagattcct taatgcgggg 420

atcttcggat cagggacaaa caacacgctg ccttcggccg ccagcagggc tgcgatgctt 480

gtgaggatca acaccctcct ccaaggttac tccggcatcc gttttgaaat cctggaggcc 540

attaccagcc tcctcaacag caagattacg ccttgcctgc cgctgagggg aaccatcacc 600

gcctccggcg atcttgttcc actatcttac attgcgggtg tcttaaccgg ccgtcccaat 660

tgcaaggcta taacggccga cggtgttact gtcaacgcag tagaggcctt ccgtcttgca 720

ggaatctcca gcgggttctt cgatcttcag cccaaggaag ggctcgcact tgtcaatgga 780

accgccgtcg gctccggctt cgcctccatt gtcctgttcg aggcaaacat cctcgccctt 840

atggcagagg ttctctctgc tctgttctgc gaggtgatgc aggggaagcc ggagttcacc 900

gaccacctca cccacaagct gaaacaccac ccgggacaaa tcgaggccgc cgccatcatg 960

gagcacgtgc ttgaaggaag ctcctacatg aagatggcca agaagctcca cgatttggat 1020

cctcttcaga agccaaagca ggatcgctat gctctccgca cctcacccca atggctcggc 1080

cctcagatcg aagtgatccg agcagcgacc aagtccatcg agagggagat aaattcagtc 1140

aacgacaacc ctctgattga tgtctcgagg aacaaggcca tccatggagg caacttccaa 1200

gggaccccca ttggcgtttc catggacaac accaggctcg ccattgctgc catcgggaag 1260

ctcatgttcg cccaaatatc agagcttgtc aatgactttt ataacaacgg cttgccttca 1320

aatctatccg gtgggagaaa ccctagcttg gattatggct tcaaaggcgc ggagatagcc 1380

atggcttcct actgctccga gctccagtac ctcgccaatc cggtcacaaa ccatgtgcag 1440

agcgccgagc agcacaacca ggacgtgaac tccctgggac tgatatcttc gaggaagacg 1500

ggggaggcgg tggagatact aaagctcatg acctccacct tcctggttgc actctgccaa 1560

gccatagact tgaggcatct ggaggagaac ttgaagtgtg ccgtgaagaa tgcggtgagc 1620

ctggcggcaa agaggactct cactttcggg gccaatggag atcttcatcc atccaggttc 1680

tgcgagaagg atttgatcaa ggtggtagat aaggagtatg tgttcgccta cgccgacgat 1740

ccctgcagct ctacctaccc tttgatgcag aagctcaggc aggtgctggt tgagcatgcc 1800

ctcagcaacg gcgacaagga gaaggccagg agcacctcca tcttccaaaa gatcacagat 1860

tttgaggagg atatcaatgc cgcgcttccc aaagcggtcg aggccgccag agcggcgttt 1920

gagaaggggt cgtcggcgat agagaacaga atcaaagaat gcagatccta cccactgtac 1980

aggcttgtga gggaagagct cggggccggc tttctcaccg gagagaaggc gatgtcgcca 2040

ggggaggaat tcgacaaggt cttcaatgcc atttgcgagg ggagggcgat agatcctctg 2100

ctcgagtgct tgaaggagtg gaatgaagct cctctcccta tttgctag 2148

Claims (6)

1. a kind of phenylalanine lyase of anoectochilus formosanus, which is characterized in that the phenylalanine solution ammonia of the anoectochilus formosanus The amino acid sequence of enzyme is as shown in SEQ ID NO.1.

2. encoding the encoding gene of the phenylalanine solution enzyme of anoectochilus formosanus as described in claim 1.

3. the encoding gene of the phenylalanine solution enzyme of anoectochilus formosanus according to claim 2, which is characterized in that the base Because nucleotide sequence is as shown in SEQ ID NO.2.

4. the recombinant vector of the encoding gene of the phenylalanine solution enzyme containing anoectochilus formosanus as claimed in claim 2 or claim 3.

5. the recombination engineering of the encoding gene of the phenylalanine solution enzyme containing anoectochilus formosanus as claimed in claim 2 or claim 3.

6. the encoding gene of the phenylalanine solution enzyme of anoectochilus formosanus as claimed in claim 2 or claim 3 is in expression phenylalanine solution Application in adnosine deaminase.