CN109486806B - Phenylalanine ammonia lyase of anoectochilus formosanus, and coding gene, recombinant vector, recombinant engineering bacterium and application thereof - Google Patents

Abstract

The invention provides phenylalanine ammonia lyase of anoectochilus formosanus, and a coding gene, a recombinant vector, recombinant engineering bacteria and application thereof, and belongs to the technical field of protein engineering. The sequence of the phenylalanine ammonia lyase of anoectochilus formosanus provided by the invention is a novel phenylalanine ammonia lyase gene sequence, is a key enzyme gene in the synthesis process of flavonoids which are main medicinal components of anoectochilus formosanus, and plays an important role in the anabolism pathway of flavonoids.

Description

Phenylalanine ammonia lyase of anoectochilus formosanus, and coding gene, recombinant vector, recombinant engineering bacterium and application thereof

Technical Field

The invention relates to the technical field of protein engineering, and particularly relates to phenylalanine ammonia lyase of anoectochilus formosanus, and a coding gene, a recombinant vector, recombinant engineering bacteria and application thereof.

Background

Anoectochilus formosanus (Anoectochilus formosanus) is a perennial herb, growing in Taiwan, and belongs to the Anoectochilus formosanus (Roxburgh) Roxburgh in the genus of Kalimerian in the family of Orchidaceae. The important pharmacological active substances in anoectochilus formosanus are mainly as follows: flavonoids, steroids, triterpenes, saccharides, alkaloids, cardiac glycosides, esters, taurine, various amino acids, trace elements, inorganic elements and the like. Folk agents are called "Yaowang", "gold grass", "Shencao", "bird ginseng" and the like. Among them, polysaccharides, flavonoids, steroids, etc. in anoectochilus formosanus are considered as important active substances, and the synthesis of these substances directly affects the medicinal value of anoectochilus formosanus.

Phenylalanine Ammonia Lyase (PAL) is a key enzyme gene in the anabolic pathway of flavonoids. In the anabolic pathway of flavonoids, the first catalytic enzyme is Phenylalanine Ammonia Lyase (PAL), which can catalyze L-Phenylalanine (L-Phenylalanine) to produce Trans-cinnamic acid (Trans-cinamic acid). The generated trans-Cinnamic acid can generate 4-hydroxycoumarin acid (4-hydroxycoumarin acid) under the catalysis of cinnamate 4-hydroxyzyme (C4H) under the condition that oxygen and Nicotinamide Adenine Dinucleotide Phosphate (NADPH) coexist. Then 4-hydroxycoumarin acid-coenzyme A ligase (4 CL) catalyzes the ligation to convert the 4-hydroxycoumarin acid generated in the previous step into thioesters such as 4-hydroxycoumarin-CoA, which requires ATP for energy supply. The produced 4-hydroxycoumarin-CoA can be reacted with malonyl-CoA under the action of Chalcone synthase to produce Chalcone (Chalcone). Then enters 5 branches of flavone anabolism (isoflavone branch, aurone branch, flavone branch, anthocyanin branch and flavonol branch). Therefore, phenylalanine ammonia lyase is an important rate-limiting enzyme for connecting primary metabolism and secondary metabolism of plants, plays an important role in the anabolic pathway of flavonoids, and has activity closely related to anabolism and accumulation of various flavonoids.

Disclosure of Invention

The first purpose of the invention is to provide phenylalanine ammonia lyase of anoectochilus formosanus.

The second purpose of the invention is to provide a coding gene of phenylalanine acidolysis enzyme of anoectochilus formosanus.

The third object of the present invention is to provide a recombinant vector containing a gene encoding phenylalanine lyase of anoectochilus formosanus.

The fourth purpose of the invention is to provide a recombinant engineering bacterium containing a coding gene of phenylalanine acidolysis enzyme of anoectochilus formosanus.

The fifth purpose of the invention is to provide the application of the coding gene of phenylalanine ammonia lyase of anoectochilus formosanus in expressing phenylalanine ammonia lyase.

In order to achieve the above purpose of the present invention, the following technical solutions are adopted:

a phenylalanine ammonia lyase of anoectochilus formosanus is disclosed, wherein the amino acid sequence of the phenylalanine ammonia lyase is shown as SEQ ID No. 1.

A coding gene of phenylalanine acidolysis enzyme of anoectochilus formosanus.

A recombinant vector containing the coding gene of phenylalanine acidolysis enzyme of anoectochilus formosanus.

A recombinant engineering bacterium containing the coding gene of phenylalanine acidolysis enzyme of anoectochilus formosanus.

The coding gene of phenylalanine ammonia lyase of anoectochilus formosanus is applied to expressing phenylalanine ammonia lyase.

Compared with the prior art, the invention has the beneficial effects that: the sequence of the phenylalanine ammonia lyase of anoectochilus formosanus provided by the invention is a novel phenylalanine ammonia lyase gene sequence, is a key enzyme gene in the synthesis process of flavonoids which are main medicinal components of anoectochilus formosanus, and plays an important role in the anabolism pathway of flavonoids.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the embodiments will be briefly described below, it should be understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and for those skilled in the art, other related drawings can be obtained according to the drawings without inventive efforts.

FIG. 1 is a diagram showing the result of electrophoretic detection of CDS sequence of gene;

FIG. 2 is a three-level schematic of the predicted protein of PAL from Anoectochilus formosanus;

FIG. 3 shows the PAL protein generation relation tree analysis of Anoectochilus formosanus;

FIG. 4 is a diagram showing different tissue expression patterns of genes;

FIG. 5 is a graph showing the expression pattern of a gene at 4mg/L Phe over 12 hours;

FIG. 6 is a graph showing an expression pattern of a gene at 100mM NaCl over 12 hours;

FIG. 7 is a diagram of the expression pattern of the gene within 12h under 253.7nm ultraviolet stress;

FIG. 8 is a graph of the expression pattern of the gene within 12h under red light stress;

FIG. 9 shows the transient expression vector pC 2300-35S-PAL-eGFP;

FIG. 10 shows subcellular localization of the PAL protein of Anoectochilus formosanus;

FIG. 11 is a drawing showing the result of southern blotting of PAL gene of Anoectochilus formosanus;

FIG. 12 is a screening drawing of the PAL gene of Anoectochilus formosanus of Taiwan;

FIG. 13 is a PCR assay of transgenic Arabidopsis;

FIG. 14 shows the total flavone enrichment of transgenic Arabidopsis thaliana.

Detailed Description

Embodiments of the present invention will be described in detail below with reference to examples, but it will be understood by those skilled in the art that the following examples are only illustrative of the present invention and should not be construed as limiting the scope of the present invention. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.

The features and properties of the present invention are described in further detail below with reference to examples.

Examples

The method comprises the steps of extracting and purifying the total leaf RNA of anoectochilus formosanus, wherein the total RNA is extracted by using a Trizol extraction kit of Dalianbao biological wired company, and the method comprises the following steps.

(1) About 100mg of each ground powder was charged into a 1.5mL centrifuge tube, and then 1mL of RNAioso Plus was immediately added, and mixed by inversion.

(2) The well mixed homogenate was allowed to stand at room temperature for 5min, then 12000g, centrifuged at 4 ℃ for 5 min.

(3) Sucking 800ml of supernatant, transferring into a new centrifuge tube, adding 200ml of chloroform into the centrifuge tube, shaking vigorously, mixing until the homogenate is milky white, and standing at room temperature for 5 min.

(4) The mixture was centrifuged at 12000g at 4 ℃ for 15min, at which time the homogenate was divided into three layers. From top to bottom are respectively: supernatant (containing RNA), an intermediate white layer (mostly DNA) and a colored lower organic phase.

(5) Aspirate 400ml of supernatant and transfer to a new centrifuge tube without touching the middle layer. Then 400ml of isopropanol was added and mixed by inversion, and then left to stand at room temperature for 10min.

(6) The standing solution was centrifuged at 12000g at 4 ℃ for 10min, whereupon white flocculent RNA was visible. The supernatant was carefully discarded and 1mL of 75% ethanol was added, and the RNA was washed upside down and discarded.

(7) After the RNA was dried at room temperature for a few minutes by opening the centrifuge cap, 30. mu.l of RNase-free water was added to dissolve the RNA.

(8) Total RNA concentration was calculated by measuring the value of A260 using a ultramicrospectrophotometer (Bio-Rad, USA), and the value of OD260/OD280 was read to estimate total RNA purity and integrity. The RNA quality was rapidly checked by electrophoresis on a 1.2% agarose gel at 135V.

Secondly, sequencing transcriptome of anoectochilus formosanus by the following method:

extracting total RNA of the leaves of anoectochilus formosanus, detecting the RNA extraction quality, and meeting the requirement of library construction (RNA concentration)>250 ng/. mu.L, total amount>20μg，OD₂₆₀/OD₂₈₀Between 1.8 and 2.2, good integrity and RIN>6.5). Then, poly (A) mRNA is enriched by magnetic beads and is broken into segment fragments, the segment fragments are used as templates, the 1 st cDNA chain and the 2 nd cDNA chain are sequentially synthesized, and a sequencing joint is connected after purification, elution, end repair and poly (A) addition. Selecting 200 bp-700 bp fragments for PCR amplification, establishing a cDNA sequencing library, and sequencing by using IIIuma HiSeq 2000. The part of the experiment is finished by the Mei-Nei science and technology service cable company.

Using short reads assembly software Trinity (v2.4.0) to carry out De novo assembly to obtain a Contig assembly fragment without N, and using tgicl (v2.1) to carry out redundancy removal to remove sequences with low quality and uncertainty in the sequences. Sequences greater than 200bp were retained for subsequent analysis. And (3) performing gene structure prediction on the splicing result by using a transdecoder (v2.0.1), and performing subsequent analysis after predicting the structure.

Thirdly, the first strand synthesis of the Taiwan anoectochilus formosanus cDNA, the method is as follows:

the purified total RNA of Anoectochilus roxburghii leaves obtained in the above-mentioned step was used as a template, oligo (dT)18 was used as a Reverse transcription primer, and PrimeScript Reverse Transcriptase Transcriptase (Takara China) was used in accordance with SMART^TMThe PCR cDNA Synthesis Kit (Clontech USA) protocol indicates that the first strand of cDNA was synthesized. The total volume of the reaction system was 20. mu.L.

(1) Preparing a reverse transcription mixed solution 1 (table 1) in a 0.2mL PE tube;

(2) in another 0.2mL PE tube, the following reverse transcription mixture 2 (Table 2) was prepared;

(3) preserving the reverse transcription mixed solution prepared in the first step at 165 ℃ for 5min, rapidly cooling on ice for 2min, and centrifuging for several seconds to enable the mixed solution of template RNA, primers and the like to gather at the bottom of a PE tube;

(4) adding the reverse transcription mixed solution 2 prepared in the second step into the reaction solution prepared in the first step, gently mixing by using a pipette gun, and reacting for 90min at 42 ℃;

(5) keeping the temperature at 80 ℃ for 5min, and cooling on ice to obtain a cDNA solution which can be directly used for subsequent experiments.

TABLE 1 reverse transcription Mixed solution 1

TABLE 2 reverse transcription Mixed solution 2

And fourthly, extracting and purifying the total DNA of the anoectochilus formosanus leaves, wherein the total DNA is extracted by using a CTAB method, and the method comprises the following steps.

(1) About 100mg of tissue was taken and added with liquid nitrogen and milled thoroughly. 400 μ L of buffer FP1 and 6 μ L of RNase A (10mg/mL) were added, vortexed for 1min, and allowed to stand at room temperature for 10min.

(2) Add 130. mu.L of buffer FP2, mix well and vortex for 1 min.

(3) Centrifuge at 12000r/min for 5min and transfer the supernatant to a new centrifuge tube.

(4) And (3) repeating the step to remove precipitated impurities in the supernatant so as to ensure that the extracted genomic DNA has higher purity.

(5) 0.7 volume of pre-cooled isopropanol was added to the supernatant and mixed well, at which time flocculent genomic DNA appeared. Then, the mixture was centrifuged at 12000r/min for 2min, and the supernatant was discarded and the precipitate was retained.

(6) 1mL of 70% ethanol was added, vortexed for 10 seconds, centrifuged at 12000r/min for 2min, and the supernatant was discarded.

(7) And 6, repeating the step.

(8) And (3) opening the cover and inverting the cover, keeping the room temperature for 5-10 min, adding a proper amount of elution buffer TE to dissolve the DNA after the residual ethanol is completely volatilized, and inverting and uniformly mixing the solution for several times to finally obtain the DNA solution.

Fifthly, cloning of an open reading frame and a coding region of the PAL gene of anoectochilus formosanus by the following method.

On the basis of obtaining the CDS sequence of the PAL gene by sequencing and splicing the anoectochilus formosanus transcriptome, Primer Premier 5.0 software is adopted to design a Primer (PALF/PALR:5'-ATGGACCATGCTAGGGAGAACG-3'/5'-CTAGCAAATAGGGAGAGGAGCTTCA-3'), and the designed Primer has the specificity of an analyte Primer in Oligo6.0. The reaction procedure is as follows: pre-denaturation at 95 ℃ for 3 min; then, the mixture is denatured at 95 ℃ for 30s, annealed at 52 ℃ for 30s, and extended at 72 ℃ for 1min for amplification for 38 cycles. A50. mu.L system was used for PCR (Table 3).

TABLE 3 PCR reaction System

50ul of the amplified product was subjected to 180V electrophoresis on 1% nondenaturing agarose gel for 30min, and the amplified fragment was examined by UV after staining with GoLdenView, the results are shown in FIG. 1. Wherein, A is open reading course amplification, B is gene coding region (containing intron) amplification, M is marker, 1 is anoectochilus formosanus, and 2 is Fujian anoectochilus formosanus. The open reading frames and the fragments of the coding regions of the Anoectochilus formosanus and the Anoectochilus formosanus are similar in size.

And (3) recovering the target fragment:

the amplified specific bands were quickly and accurately removed from the agarose using a clean blade under an ultraviolet lamp, and placed in a 1.5ml centrifuge tube, and the DNA fragments in the Gel were recovered using a Gel recovery Kit (E.Z.N.A.TM. Gel Extraction Kit from OMEGA).

And (3) connection reaction:

the recovered DNA fragment was cloned into the Pmd19-T vector of TaKaRa. Ligation was carried out overnight at 16 ℃ in a total volume of 10ul using a ligation kit (TaKaRa).

Preparation of competent cells:

and (3) selecting a newly activated E.CoLiDH5a single colony from an LB plate, inoculating the colony in 3-5ml of an LB liquid culture medium, and carrying out shake culture at 37 ℃ and 225r/min for about 12h until the late logarithmic growth stage. The suspension was mixed with a suspension of 1: inoculating 10-1:50 of the extract into 100ml of LB liquid medium, and performing shake culture at 37 ℃ for 2-3h to OD₆₀₀About 0.35-0.5.

Transferring the culture solution into a centrifuge tube, standing on ice for 10min, and centrifuging at 4 deg.C at 3000r/min for 10min.

The supernatant was discarded and pre-cooled 0.05mol/L CaCl was used₂The solution 10ml gently suspends the cells, after placing on ice for 15-30min, centrifugates for 10min at 4 ℃ at 3000 r/min.

The supernatant was discarded and 4ml of pre-cooled 0.05mol/L CaCl containing 15% glycerol was added₂The solution, gently suspend the cells, and place on ice for several minutes to form a competent cell suspension.

The competent cells were divided into 100ul aliquots and stored at-70 ℃ for half a year.

Transformation of plasmid DNA:

a tube of E.coli competent cells DH5a was removed from the freezer at-70 ℃ and thawed on ice.

Adding 10ml of the ligation reaction solution under aseptic conditions, shaking gently and mixing uniformly, and standing on ice for 30 min.

The mixture is heated in a water bath at 42 ℃ for 90s without shaking, and then quickly placed in an ice bath for cooling for 2-3min.

Adding 890ul LB liquid culture medium without Amp, mixing, shaking at 37 deg.C and 150r/min, and incubating for 1 h.

Spreading 100ul of transformed bacterial liquid on a culture plate of LB Amp, standing for 30min with the front side facing upwards, sealing with a sealing film after the bacterial liquid is completely absorbed by the culture medium, then inverting the culture dish, and culturing at 37 ℃ in the dark for 12-16 h; subsequently, positive colonies were screened.

Identification and preservation of recombinant colonies

The colonies were generally white and round in appearance, and were identified by PCR.

Several white colonies were picked up on overnight-cultured plates with a sterilized small gun head, and seeded in 0.1g/L Amp LB liquid medium 1.5ml centrifuge tubes, respectively, and cultured at 37 ℃ for 4-6h with shaking at 150 r/min.

Taking 1ul of bacterial suspension as a template, carrying out PCR amplification by using a primer PALF/PALR, prolonging the cracking time in the PCR reaction condition to 5min, and detecting the amplification product by using 1% non-denaturing agarose gel electrophoresis. If the product is the target band, the colony is a positive clone, otherwise, the colony is a negative clone. Meanwhile, a negative control without adding bacterial suspension is set.

750ul of colony suspension of the identified positive clone is taken, 250ul of sterilized glycerol is added, the mixture is uniformly mixed, and then the mixture is quickly frozen by liquid nitrogen and stored in a refrigerator at the temperature of 70 ℃ below zero for later use.

And finally, sending the sequence to the Invitrogen biotechnology company for sequencing, and comparing and analyzing the obtained sequence in BLAstn of NCBI to verify that the cloned fragment is correct.

The product obtained by amplification is an ArPAL gene cDNA open reading frame sequence and a coding region sequence, wherein, the amino sequence is SEQ ID NO.1, the coding region nucleotide sequence is SEQ ID NO.2, and the open reading frame nucleotide sequence is SEQ ID NO. 3.

Sixthly, the method for analyzing the AfPAL protein bioinformatics is as follows:

the physical and chemical properties were analyzed using ProtParam (http:// web. expasy. org/ProtParam /). The total length of the PAL gene of anoectochilus formosanus is 2148bp, the sequence of the coding region is 2733bp, 1 intron with the length of 585bp is inserted between 408 bp and 993bp, and 715 amino acids are coded. PAL proteins have 2 PAL-typical conserved domains, a phenylalanine and histidine domain (GTITASGDLVPLSYIA) and an MIO region formed by the Ala-Ser-Gly active site. In addition, strictly conserved sites (Y109, L137, S202, N259, Q347, Y350, R353, F399 and Q487), deamination sites (L205, V206, L255 and A256), catalytic active sites (N259, G260, NDN 381-383 aa, H395 and HNQDV 485-488aa) and phosphorylation sites (VAKRVLTF 542-549aa) can be found at corresponding positions of the PAL protein of Anoectochilus formosanus. The physical and chemical properties of PAL gene coding protein are predicted by using an online tool ProtParam provided by ExPASy Proteomics Server, and the relative molecular mass of the protein is estimated to be 77.4kDa and the isoelectric point pI is 6.18.

GOR IV (http:// npsa-pbil. ibcp. fr/cgi-bin/npsa _ Automat. plpage ═ npsa _ GOR4.html) was used; the secondary structure of the PAL gene is predicted. The results of the on-line prediction of secondary structure showed that the Alpha-helix (Alpha helix) accounted for 48.53% of the total amino acids, the Extended strand (Extended strand) accounted for 10.07% of all amino acids, and the Random coil (Random coil) accounted for 41.40% of the total amino acids.

Prediction of the tertiary structure of the protein was carried out for the PAL gene of Anoectochilus formosanus by SWISS-MODEL (https:// swisssmall.expasy.org /), plotted by PyMOL Viewer and the three-dimensional MODEL is shown in FIG. 2.

Finally, the PAL amino acid sequence of the plant with higher homology is found, the comparison of the amino acid sequences is carried out by the ClustalW method, and the Neighbor-join phylogenetic tree is constructed by MEGA7.0 as shown in figure 3 (the position of anoectochilus formosanus in frame).

Seventhly, detecting the endogenous expression of the AfPAL by the following method:

(1) material treatment

Transplanting the tissue culture seedlings of the anoectochilus formosanus cultured for 4 months into a flowerpot filled with nutrient soil (nutrient soil: vermiculite: 3:1), and culturing at the temperature of 25 ℃ (day)/20 ℃ (night), with the relative humidity of 60-70% and the illumination of 200 mu mol/m²S (14 h)/dark (10 h). Adapting to 3-5 days, taking root, stem and leaf samples from untreated seedlings with uniform growth vigor, and quickly freezing the samples at-70 ℃ by liquid nitrogen for storage. The other seedlings with uniform growth are divided into two groups, one group is subjected to ultraviolet light irradiation treatment, and the other group is subjected to red light irradiation treatment. Ultraviolet irradiation treatment: placing potted anoectochilus formosanus in an ultraviolet and red light incubator, and respectively treating for 0h, 0.5h, 1h, 2h, 4h, 8h and 12h under the ultraviolet radiation wavelength of 253.7nm and the red light wavelength of 650 nm. Each treatment was repeated 3 times, and samples were immediately taken at each treatment time point and snap frozen in liquid nitrogen at-70 ℃ for storage.

Transplanting the tissue culture seedlings of the anoectochilus formosanus cultured for 4 months onto a plastic foam plate with holes, culturing for 3-5 days by using Hoagland nutrient solution, and selecting the seedlings with uniform growth vigor for phenylalanine and salt stress treatment. Phenylalanine and salt stress treatment is to add Phe and NaCl into Hoagland nutrient solution to final concentration of 4mg/L and 100mmol/L, and treat for 0h, 0.5h, 1h, 2h, 4h, 8h and 12h respectively. Each treatment was repeated 3 times, and samples were immediately taken at each treatment time point and snap frozen in liquid nitrogen at-70 ℃ for storage.

(2) RNA extraction

Leaves of the 3 types of stress-treated and control seedlings were respectively subjected to liquid nitrogen quick-freezing and grinding, and total RNA was extracted using a total RNA extraction kit Trizol (TaKaRa, Dalian) according to the instructions thereof. The procedure is as in example 1.

(3) First Strand cDNA Synthesis

Using the extracted RNA as template, loading sample according to the system in Table 4, reacting at 42 deg.C for 2min, removing possible genomic DNA (gDNA), and using PrimeScript^TMRT reagent Kit with gDNAeraser Kit (TaKaRa, Large Link), adding sample according to the system in Table 5, warm bathing at 37 ℃ for 30min, keeping 5s at 85 ℃ to terminate the reaction for reverse transcription to synthesize cDNA, and storing at-20 ℃ for later use.

TABLE 4 gDNA removal reaction System

TABLE 5 cDNA Synthesis System

(4) Specific detection of qRT-PCR primers

The control cDNA sample (0 h) is diluted by 3 times and used as a template for qRT-PCR reaction, and qRT-PCR reaction is carried out at 50-65 ℃ to determine the optimal annealing temperature, and a 20 mu L reaction system is adopted (Table 6). The primer sequences of key enzyme genes in the flavone anabolic pathway are shown in Table 7.

TABLE 6 qRT-PCR reaction System

TABLE 7 qRT-PCR primer sequences

The reaction tube used 0.2mL PCR plate after siliconization. qRT-PCR at iQ^TM5thermal cycler (Bio-Rad USA). The reaction procedure is as follows: pre-denaturation at 95 ℃ for 10 s; then, the procedure of denaturation at 95 ℃ for 10s, annealing at 50-65 ℃ for 20s, and extension at 72 ℃ for 20s, and collecting fluorescence at this temperature, Plate reading (Plate read) was performed for 46 cycles. Thereafter, starting from 50 ℃ the temperature was increased to 95 ℃ with a temperature gradient of 0.5 ℃ per step, and the temperature was maintained for 5s per step.

(5) qRT-PCR reaction

The reverse transcribed cDNA sample was diluted 3-fold and used as a template for qRT-PCR amplification, and a siliconized 0.2mL PCR plate was used as a reaction tube, a 20. mu.L reaction system (Table 6) was used, and ddH was set up₂O is a negative control for the template. A single reverse transcribed cDNA sample was made in triplicate.

The qRT-PCR reaction program was: pre-denaturation at 95 ℃ for 10s, followed by annealing at 95 ℃ for 10s, annealing at the optimal annealing temperature for 20s, extension at 72 ℃ for 20s, and collection of fluorescence at this temperature, Plate reading (Plate read) was performed for 46 cycles. Then finally, starting at 50 ℃ and increasing the temperature to 95 ℃ at a rate of 0.5 ℃ per step, the melting curve is plotted for 5s per temperature.

(6) qRT-PCR data analysis

The experiment adopts a double-standard curve method to carry out relative quantification on the expression level of key enzyme genes in the pathway of the metabolism and synthesis of flavone. The relative expression level of the Gene was calculated by the Δ Δ CT (normalized Gene expression) method.

Differences in expression between the 0h control and treatment and between treatments were analyzed using SPSS (version 10.0inc. Two levels of differential significance were established, P0.05 and P0.01. The expression pattern of the taiwan anoectochilus formosanus PAL gene in different tissues is shown in fig. 4 (light color bar indicates the expression amount of fujian anoectochilus formosanus PAL gene, dark color bar indicates the expression amount of fujian anoectochilus formosanus, significant difference indicates the significant difference, extreme significant difference indicates the significant difference), the expression pattern of the taiwan anoectochilus formosanus PAL gene in 12h under 4mg/LPhe is shown in fig. 5 (light color bar indicates the expression amount of fujian anoectochilus PAL gene, dark color bar indicates the expression amount of taiwan anoectochilus formosanus, significant difference indicates the extreme significant difference), the expression pattern of the taiwan anoectochilus formosanus PAL gene in 12h under 100mM NaCl is shown in fig. 6 (light color bar indicates the expression amount of fujian anoectochilus formosanus PAL gene, dark color bar indicates the expression amount of taiwan anoectochilus formosanus, significant difference indicates the extreme significant difference), the expression pattern of the taiwan anoectochilus formosanus PAL gene in 12h under 253.7nm ultraviolet stress is shown in fig. 7 (light color bar indicates the expression amount of fujian anoectochilus formosanus, the dark color bar indicates the expression level of anoectochilus formosanus, the difference is significant, the difference is extremely significant), and the expression pattern of the anoectochilus formosanus PAL gene under red light stress within 12h is shown in FIG. 8 (the light color bar indicates the expression level of Fujian anoectochilus formosanus PAL gene, the dark color bar indicates the expression level of anoectochilus formosanus, the difference is significant, and the difference is extremely significant). Although the Fujian anoectochilus formosanus PAL gene and the Taiwan anoectochilus formosanus PAL gene have the same fragment size amino acid number and structure function, the PAL gene expression quantity is different under the stress of phenylalanine, salt, ultraviolet and red light.

Eighthly, subcellular localization of AfPAL protein, firstly constructing transient expression vector. According to the PAL gene ORF sequence of anoectochilus formosanus at 5 'and 3'The restriction sites and the protective bases required for the construction of the transient expression vector (FIG. 9) were added to the ends, and the Premier 5.0 software was used to design specific primers [ 5' -TC ] that did not contain stop codonsCCGGG(Sma I)ATGGACCATGCTAGGGAGAACG-3′/5′-CGCACTAGT(Spe I)GCAAATAGGGAGAGGAGCTTCA-3′]。

(1) Using pDM19-T plasmid inserted with PAL gene ORF as a template, the reaction system in Table 4 was applied and PCR amplification was carried out. The PCR temperature cycle system program is set as follows: 94 ℃ for 3 min; amplification is circulated for 38 times at 98 ℃ for 10s, 62 ℃ for 30s and 72 ℃ for 60 s; finally, extension is carried out for 5min at 72 ℃.

(2) Separating the amplified product by using 1% non-denaturing agarose gel electrophoresis,

ChemiDoc^TMXRS type gel imaging system (Bio-Rad, USA) imaging, and gel recovery kit (Tiangen, Beijing) is used to recover and purify the target fragment.

(3) The reaction system was loaded as in Table 8 and digested with fast-cutting enzyme at 30 ℃ for 0.5 h. The digested product was separated by electrophoresis on 1.2% agarose gel, and recovered and purified using a gel recovery kit (Tiangen, Beijing).

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

(4) The purified double enzyme product was recovered by ligation with T4 DNA ligase following loading of the reaction system according to Table 9 and ligation at 16 ℃ for 8 h.

TABLE 9 ligation reaction System

(5) The ligation products were transformed by heat shock transformation and recombinant colonies were identified by PCR.

(6) Extracting the plasmid DNA in the steps, carrying out double enzyme digestion identification on corresponding fast-cutting enzymes Sma I and Spe I, taking a small amount of plasmid, sending the small amount of plasmid to a company Limited in bioengineering (Shanghai) for sequencing, and verifying that the vector is constructed correctly. Then, transient expression was detected in epidermal cells of onion bulbs.

(7) Cutting the 5th layer scale of fresh onion bulb into 2cm × 2cm squares, spreading the inner epidermis on 1/2MS plate, and culturing at 25 deg.C for 4 hr.

(8) 60mg of gold powder (diameter 60 μm) is weighed and placed in a 1.5mL centrifuge tube, 1mL 70% ethanol is added into the centrifuge tube filled with the gold powder, the centrifuge tube is vigorously shaken for 15min on vortex oscillation, the centrifuge tube is centrifuged for 1min at room temperature at the rotating speed of 1300g, the supernatant in the centrifuge tube is taken out, and the step is repeated for three times. Finally, sterile water is added, and the mixture is stored at the temperature of minus 20 ℃ for standby.

(9) mu.L of the gold powder suspension was placed in a 1.5mL inlet centrifuge tube and 1. mu.L of transient expression vector DNA (1.0. mu.g/. mu.L) and 8. mu.L of autoclaved 2.5mol/L CaCl were added₂4. mu.L of 0.1mol/L spermidine which has been sterilized by suction filtration, mixed, shaken vigorously on a vortex shaker for 3min, and left to stand in an ice bath for 15 min.

(10) Centrifuging for 10s at room temperature at 1200g, collecting supernatant, adding 100 μ L of anhydrous ethanol with analytically pure concentration, and shaking to resuspend gold powder on vortex shaker.

(11) Centrifuging at 12000g at room temperature for 10s, removing supernatant, adding 15 μ L of absolute ethanol with analytical pure concentration, and resuspending in a vortex oscillator to make the liquid uniform.

(12) Gene gun A PDS-1000/He type gene gun (Bio-Rad, USA) was used under the conditions: the sample chamber was at a vacuum of 26in Hg and a burst pressure of 1100psi at a target distance of 6 cm. And (3) taking 10 mu L of the suspension liquid to be evenly spotted on the central area of the transformation slide, and uniformly injecting gold powder which wraps the transient expression vector in the central area of the transformation slide into cultured onion bulb endothelial cells under the conditions.

(13) The medium containing the transformed onion inner epidermis was wrapped in a black plastic bag and cultured at 25 ℃.

(14) After 16 to 24h, the onion epidermis on the medium was sliced and photographed under a BX63 type fluorescence microscope (Olimpas, Japan). The results are shown in FIG. 10 (subcellular localization of ApC 2300-35S-eGFP, BpC 2300-35S-PAL-eGFP), with subcellular localization in the nucleus.

Ninthly, identifying the copy number of PAL by the following specific method:

a pair of specific primers (5'-AGCAAGATTACGCCTTGCCT-3'/5'-ATGAGGGGGTTGTCGTTGAC-3') was designed based on the ORF sequence of the PAL gene of Anoectochilus formosanus by using Premier 5.0 software. The recovered PCR product was labeled with digoxin (random primer method), and the reaction was performed as described in the Southern blot kit, and the procedure was as follows:

(1) to a 200. mu.L PCR tube, 1. mu.g template DNA and autoclaved double distilled water were added to a final volume of 16. mu.L.

(2) The DNA was denatured by boiling water bath for 10min and then quickly inserted into ice water mixture.

(3) DIG-High Prime was mixed well and 4. mu.L was added to the denatured DNA, mixed and centrifuged briefly and then placed in a PCR instrument to incubate overnight at 37 ℃.

(4) The reaction was terminated by heating at 65 ℃ for 10min.

Then, total DNA of Anoectochilus formosanus was extracted, a reaction system of 50. mu.L was set, 3 restriction enzymes were selected to digest the total DNA of Anoectochilus formosanus and Anoectochilus formosanus, respectively, and the reaction system was shown in Table 10.

TABLE 10 restriction of Total DNA of Anoectochilus roxburghii

mu.L (0.8. mu.g) of the resulting digested product was electrophoresed in a 0.8% agarose gel containing Goldenvew at 50V for 6 hours, and the electrophoresis results were recorded by photography. The remaining samples were spotted on a 0.7% agarose gel without DNA stain for electrophoresis. After electrophoresis, the gel was washed twice with double distilled water, the excess portion of the gel was removed and the left corner was cut off to mark the front and back sides. Adding 100ml 0.25mol/L hydrochloric acid to depurinate, shaking at room temperature for 15min until bromophenol blue becomes yellow completely, and washing with distilled water for 2 times. Adding a denatured liquid, and carrying out warm shaking for 40 min: until the bromophenol blue is completely restored to the original blue color. Pouring 20 XSSC solution into a transfer printing groove, placing a solid phase support in the groove, and placing the following components on the solid phase support from bottom to top in sequence: two pieces of filter paper with equal width of gel, vertically hanging the filter paper from the solid phase support in the transfer track groove (abbreviated as "bridge"), gel with the bottom surface on top, nylon membrane (equal to gel), filter paper (equal to gel, soaked with 20 XSSC in advance), absorbent paper (slightly smaller than filter paper, 5-8cm high), 400-. Placing the nylon membrane with positive charges and the gel in a transfer printing device, surrounding the periphery by a PE film to prevent short circuit, and transferring the membrane for 18 h. After the membrane transfer is finished, the filter membrane is rinsed for 5min in 2 XSSC and then is dried by suction with filter paper. The nylon membrane is fixed for 30min at 120 ℃. The hybridization solution and the hybridization apparatus were preheated to 37 ℃ and the nylon membrane was placed in 10mL of the hybridization solution for prehybridization for 90min (the membrane was placed in a box and was free to move). mu.L of the labeled probe was denatured in a boiling water bath for 5min and quickly inserted into an ice-water mixture. The denatured probe was added to 10mL of the hybridization solution and mixed well. The nylon membrane is placed in the hybridization solution and gently shaken for 20h at 42 ℃. [ Probe hybridization temperature is calculated from GC content and the percent similarity of the probe to the target fragment, and the formula is as follows: tm 49.82+0.41 × 38 (% G + C) - (600/I), (I) the length of the fragment capable of hybridizing, calculated in base pairs) ]. Washing was performed 2 times with 2 × SSC 0.1% SDS at room temperature for 5min with continuous shaking. Thereafter, the column was washed with 0.5 XSSC 0.1% SDS pre-heated to 65 ℃ for 15 minutes each time with continuous shaking 2 times. Firstly, washing the nylon membrane for 2min by using a washing buffer solution; standing and blocking the nylon membrane in 50mL of 1 × blocking solution at room temperature for 35 min; centrifuging the antibody at 12000r/min for 5min, collecting upper layer antibody solution 4 μ L, adding into blocking solution 20 μ L, mixing, covering with nylon membrane, and standing at room temperature for 40 min; washing with sufficient washing buffer solution for 15min for 2 times; washing with sufficient washing buffer solution for 15min for 2 times; and (3) placing the nylon membrane in 15mL of developing solution, standing, keeping out of the sun, and carrying out closed development for 4-16 h. The nylon membrane with the closed color development was photographed and stored by using an imager, and the result is shown in FIG. 11 (three copies of southern mounting was obtained by digesting with both BamH I and Sac I enzymes). The enzyme digestion is carried out by two enzymes of BamH I and Sac I, and the southern blotting is carried out by three copies. The PAL gene of anoectochilus formosanus is indicated to be a multi-copy gene.

Tenth, for the heterologous expression of PAL gene in the medium, the specific method is as follows:

(1) respectively putting appropriate amount of wild type Arabidopsis seeds into a 1.5mL centrifuge tube, adding 75% alcohol, soaking for 30s, then removing alcohol, and adding 10% NaClO for disinfection for 10min.

(2) And after the disinfection is finished, cleaning the seeds for 3-4 times by using sterile water, and after the seeds are settled, discarding the water on the upper layer.

(3) Seeds were sown on a level of 1/2MS medium and protected from light for 48h at 4 ℃.

(4) And (3) placing the flat plate in a culture chamber, wherein the temperature is 20-22 ℃, the humidity is 60-70%, transplanting the seedlings into a culture pot filled with nutrient soil (the nutrient soil: the frogsite is 4: l) after about 2 weeks, and the nutrient soil absorbs water before transplanting.

(5) After the transplanted seedlings are cultured for 4 weeks in short day (10h light/14 h dark), the seedlings are cultured in long day (16h light/8 h dark), and the seedlings are ready to be impregnated when bolting and buds grow.

(6) Taking agrobacterium containing the recombinant plasmid of pC2300-35S-PAL-eGFP, streaking on a YEP plate containing 50mg/L Kan and 50mg/L Rif, and culturing at 28 ℃ for 2-3 d.

(7) A single colony of Agrobacterium was picked and inoculated in 3mL of YEP liquid medium (containing Kan and Rif at a concentration of 50mg/L) at 28 ℃ at 250r/min, and cultured overnight with shaking.

(8) Inoculating 1mL of overnight-cultured initial agrobacterium liquid into 100mL of liquid YEP culture medium (containing Kan and Rif, the concentration of which is 50mg/L), and performing shaking culture at 28 ℃ until the OD600 value of the liquid is 1.2-1.5.

(9) Centrifuging at 4 ℃ and 5000r/min for 10min to collect cells, suspending thalli by using 5% of sucrose solution, adjusting the OD600 value to 0.8-1.0, and adding a surfactant silwet L-77 according to the proportion of 1/10000-2/10000.

(10) Cutting off formed fruit pods and opened flowers before dip dyeing, placing the culture pot with the arabidopsis thaliana on the side, completely immersing the flower buds of the arabidopsis thaliana in the dip dyeing solution for about 1-2 min, and culturing the dipped plants in the dark overnight.

(11) And after the dark culture is finished, placing the impregnated plant in a culture room for continuous culture, collecting seeds after the plant is mature, and carrying out the next screening.

(12) And disinfecting the harvested dip-dyed seeds according to the step of disinfecting the surfaces of the seeds.

(13) The seeds with the sterilized surfaces are sown to 1/2MS culture medium containing 30mg/LHyg or 50mg/L Kan, are treated for 2 days at 4 ℃ in the absence of light, and are transferred to a culture room for culture.

(14) After about 2 weeks of culture, positive transgenic plants were green and grew normally, while negative seedlings did not grow or yellow to die (FIG. 12). And transplanting the positive transgenic plant into a culture pot for culture, and harvesting the single plant after the positive transgenic plant is mature.

(15) The screening steps are repeated until T3 homozygous transgenic plants are harvested.

PCR verification of eleven-PAL gene rice is carried out by the following specific method

(1) Taking the leaves of a T3 generation overexpression and function complementation homozygous strain, and extracting genome DNA.

(2) Specific primers (5'-CATTTGGAGAGGACAGGGTACC-3'/5'-CTAGCAAATAGGGAGAGGAGCTTCA-3') were designed using Primerlest software (https:// blast. ncbi. nlm. nih. gov/blast. cgi) and a 2174bp fragment spanning the promoter was amplified.

(3) 1.2% native agarose gel electrophoresis separation,

positive transgenic plants were screened from regenerated plants by imaging with a ChemiDocTM XRS type gel imaging system (Bio-Rad, USA). The results are shown in FIG. 13(F-1, F-2 and F-3 are the over-expressed Arabidopsis thaliana of the PAL gene of Anoectochilus formosanus, R-1 and R-2 are the over-expressed Arabidopsis thaliana of the PAL gene of Anoectochilus formosanus, + positive plasmid, and-non-transgenic line).

Twelve steps of detecting the content of total flavonoids in transgenic arabidopsis thaliana are as follows:

(1) weighing the rice leaves of PAL gene of anoectochilus formosanus, drying and grinding 1.000g of the powdery sample into 20mL of conical flask, and repeating for 3 times.

(2) Adding 10mL of 95% ethanol, extracting at 30 deg.C for 30min in KQ-100DV type ultrasonic instrument (ultrasonic instruments, Inc. of Kunshan), filtering with 3 μm filter paper, and collecting filtrate.

(3) Adding ethanol with the same concentration into the residue, performing ultrasonic extraction again according to the step (2), filtering with 3 μm filter paper, collecting filtrate, and combining the filtrate with the filtrate in the step (2).

(4) 2mL of each of the above filtrates were taken (2 mL of 95% ethanol was added to the blank control), and 100g/L of Al (NO) was added₃)₃1mL and 9.8g/L potassium acetate 1mL, shaking up and standing for 30 min.

(5) The absorbance at 415nm was determined on a UV-1800 UV-visible spectrophotometer (Shimadzu, Japan) using a 1cm cuvette. The results are shown in FIG. 14(F-1, F-2 and F-3 are overexpressed Arabidopsis thaliana of the PAL gene of Anoectochilus formosanus, R-1 and R-2 are overexpressed Arabidopsis thaliana of the PAL gene of Anoectochilus formosanus, indicating significant difference, indicating very significant difference).

The embodiments described above are some, but not all embodiments of the invention. The detailed description of the embodiments of the present invention is not intended to limit the scope of the invention as claimed, but is merely representative of selected embodiments of the invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

SEQUENCE LISTING

<110> Sanming academy of academic

<120> phenylalanine ammonia lyase of anoectochilus formosanus, and coding gene, recombinant vector, recombinant engineering bacterium and application thereof

<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. The phenylalanine ammonia lyase of anoectochilus formosanus is characterized in that the amino acid sequence of the phenylalanine ammonia lyase of the anoectochilus formosanus is shown as SEQ ID No. 1.

2. The gene encoding phenylalanine acidolysis enzyme of anoectochilus formosanus according to claim 1.

3. The gene encoding phenylalanine ammonia lyase of anoectochilus formosanus according to claim 2, wherein the nucleotide sequence of the gene is represented by SEQ ID No. 2.

4. A recombinant vector containing the gene encoding phenylalanine lyase of Anoectochilus formosanus as defined in claim 2 or 3.

5. A recombinant engineered bacterium containing the gene encoding phenylalanine acidolysis enzyme of Anoectochilus formosanus as defined in claim 2 or 3.

6. The use of the gene encoding phenylalanine ammonia lyase of anoectochilus formosanus according to claim 2 or 3 for expressing phenylalanine ammonia lyase.

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