CN102102096A - Mao bamboo phenyl alanine ammonialyase, encoding gene and in-vitro expression method thereof - Google Patents
Mao bamboo phenyl alanine ammonialyase, encoding gene and in-vitro expression method thereof Download PDFInfo
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Abstract
The invention provides mao bamboo phenyl alanine ammonialyase PePAL. The amino acid sequence of the mao bamboo phenyl alanine ammonialyase PePAL is shown as SEQ ID No.2, and the nucleotide sequence of the encoding gene of the mao bamboo phenyl alanine ammonialyase PePAL is shown as SEQ ID No.1. The mao bamboo phenyl alanine ammonialyase PePAL can catalyze L-phenylalanine to form trans-cinnamic acid, has high enzymatic activity, and provides a new selectable way for generating trans-cinnamic acid by catalyzing the L-phenylalanine. Moreover, recombinant PePAL is prepared by prokaryotic expression, so that the difficulty in separating egg white from bamboo leaves is overcome, and the cost is lowered.
Description
Technical field
The present invention relates to gene engineering technology field, be specifically related to a kind of phenylalanine ammonia lyase, its encoding gene and application, the invention still further relates to the vivoexpression method of this enzyme.
Background technology
Phenylpropyl alcohol alkanes pathways metabolism is very important approach in the plant metabolism, and all materials that contain phenylpropyl alcohol alkane skeleton all are directly or indirectly to be generated by this approach.The metabolism of phenylpropyl alcohol alkanes can generate multiple secondary metabolites such as flavonoid, xylogen, these secondary metabolites play an important role in growth and development of plant, disease-resistant, degeneration-resistant reaction, and phenylalanine ammonia lyase (phenylalanine ammonia-lyase, PAL) being the enzyme of connection elementary metabolism of plant and the metabolism of phenylpropyl alcohol alkanes, catalysis phenylpropyl alcohol alkanes metabolic regulation single step reaction, is the metabolic key enzyme of phenylpropyl alcohol alkanes.The deamination reaction of phenylalanine ammonia lyase catalysis phenylalanine makes NH
3Discharge, form trans-cinnamic acid, play an important role in secondary substance in plant materials (as the xylogen etc.) metabolism.
In recent years, increasing research concentrates on phenylalanine pathways metabolism key enzyme aspect, in the hope of expression activity by these enzymes of adjusting, realize regulating effectively the biosynthesizing of secondary metabolite correspondingly, cultivate the new variety of plant that is fit to the human being's production living needs, as be suitable for the low content of lignin seeds of papermaking, the soybean varieties of homoisoflavone content etc.
Bamboo is as the important forest reserves of China, because bamboo wood has characteristics such as intensity height, good toughness, hardness are big, is widely used in fields such as building, papermaking, finishing material.Mao bamboon (Phyllostachys edulis) is one of important economic bamboo kind of China, is the research that material carries out the PAL gene with the mao bamboon, has important practical significance for developing bamboo resource better.
Yet the research of bamboo phenylalanine pathways metabolism still belongs to blank.The report that bamboo phenylalanine ammonia lyase gene vivoexpression is not arranged as yet, the molecular basis of bamboo phenylalanine pathways metabolism is not clear.
Summary of the invention
The object of the present invention is to provide the gene and the application of a kind of mao bamboon phenylalanine ammonia lyase, this enzyme of encoding.
Another object of the present invention is to provide the method for the above-mentioned mao bamboon phenylalanine ammonia lyase of vivoexpression.
The present invention clones from mao bamboon (Phyllostachys edulis) and obtains a kind of new phenylalanine ammonia lyase gene (being PePAL with this unnamed gene now), its nucleotide sequence is shown in SEQ IDNo.1, this full length gene is 2 503bp, analysis revealed, this sequence comprises complete coding region 2 106bp, 5 ' non-translational region (UTR) 95bp, 3 ' non-translational region 274bp and polyA tail 28bp, GC content is 63.16%, the albumen (phenylalanine ammonia lyase) that 701 amino acid of encoding are formed.By the aminoacid sequence of the phenylalanine ammonia lyase of this genes encoding shown in SEQ ID No.2.This proteic molecular weight is 75.69KDa, and it is phenylalanine ammonia lyase-histidase (PAL-histidase) signal for the conservative territory of phenylalanine ammonia lyase at the 185th～201 that iso-electric point is 6.489, the 41～450, possesses the feature of phenylalanine ammonia lyase.It is to have higher enzyme work under 8.8 the condition at 30 ℃, pH value that experiment shows by the phenylalanine ammonia lyase of this genes encoding.For ease of statement, with this phenylalanine ammonia lyase called after PePAL.
Be to be understood that, those skilled in the art can be according to the aminoacid sequence (SEQ ID No.2) of phenylalanine ammonia lyase disclosed by the invention, do not influencing under its active prerequisite, replacing, lack and/or increase one or several amino acid, obtaining described proteic mutant nucleotide sequence.For example, the 675th L is replaced with I at nonactive section.Therefore, phenylalanine ammonia lyase of the present invention comprises that also aminoacid sequence shown in the SEQ ID No.2 is substituted, replaces and/or increases one or several amino acid, has the equal active protein of being derived and being obtained by PePAL with PePAL.Gene of the present invention comprises the nucleic acids encoding said proteins sequence.
In addition, should be understood that the degeneracy of considering codon and the preferences of different plant species codon, those skilled in the art can use as required and be fit to the codon that specific species are expressed.Thereby phenylalanine ammonia lyase gene of the present invention also comprises by nucleotide sequence shown in the SEQ ID No.1 and is substituted, lacks and/or increase one or several Nucleotide, the nucleotide sequence of the PePAL that obtains encoding.
In example of the present invention, described gene is cloned acquisition by the following method: extract RNA from the mao bamboon blade, reverse transcription becomes cDNA, be cloned into earlier on the T-easy carrier by the method for RT-PCR, and, find that the conserved regions of inserting fragment and phenylalanine lyase gene family has higher similarity to positive colony that the filters out clonal analysis that checks order.According to obtaining the pulsating sequences Design primer of conserved regions, carry out 5 '-RACE and 3 '-RACE amplification, the PCR product is reclaimed, connects the back transform, in a large amount of clones that obtain, choose positive colony and check order.By the order-checking and with the conserved regions sequence assembly, remove lap, obtain full-length gene order 2 503bp.
Gene of the present invention can be operably connected with expression vector, obtain to express the proteic recombinant expression vector of the present invention, further this recombinant expression vector be imported in the appropriate host cell, obtain to express the genetic engineering bacterium of PePAL of the present invention.
The present invention also provides the vivoexpression method of PePAL, and it is by cultivation said gene engineering bacteria, and abduction delivering, obtains PePAL.
In example of the present invention, design comprises the primer of restriction enzyme site according to the PePAL coding region sequence, be cloned into earlier on the T-easy carrier by the method for RT-PCR, after order-checking is correct PePAL is cloned into the multiple clone site of prokaryotic expression carrier pGEX, transformed into escherichia coli BL21 (DE3), carry out abduction delivering, optimal conditions obtains to have active phenylalanine ammonia lyase.Experiment shows that PePAL can form trans-cinnamic acid by catalysis L-phenylalanine.
Preferably, grow to OD through fermentation culture bacterium liquid
600During for 0.6-0.8, add IPTG to final concentration 1mmol/L, cultivated 4 hours for 37 ℃, collect thalline, centrifuging and taking supernatant after the cracking obtains the purifying phenylalanine ammonia lyase with Histidine binding resin purifying.
In example of the present invention, treat that bacterium liquid grows to OD
600During for 0.6-0.8, add IPTG (final concentration 1mmolL
-1) carry out inducing culture.Get the bacterium liquid 250ml that induced, the centrifugal 10min of 5000rpm, collecting cell; Add the BugBuster solution of 9ml, the Benzonase nuclease of 9 μ l, the Lysozyme of 9 μ l, re-suspended cell is hatched 15min with enchylema under the room temperature on rocker; Vortex concussion gently, lysing cell; 4 ℃ of centrifugal 20min of following 1200g get supernatant liquor and prepare resin column (MERCK) according to IDA HisBind resin purification method; The extract for preparing carefully is added to the top of resin column; Use 1 * rinsing damping fluid of 1 * binding buffer liquid of 10 times of bed volumes, 6 times of bed volumes, 1 * elution buffer eluted protein of 6 times of bed volumes respectively, obtain the protein solution of purifying.
The invention provides a kind of new phenylalanine ammonia lyase, it can form trans-cinnamic acid by catalysis L-phenylalanine, has higher enzymic activity, provides a kind of new approach selected for catalysis L-phenylalanine generates trans-cinnamic acid.In addition, the present invention has overcome and directly separated this proteic difficulty from bamboo leaves by prokaryotic expression preparation reorganization PePAL, has reduced cost.
Description of drawings
Fig. 1 is conserved regions amplification and RACE product electrophorogram.
What Fig. 2 showed is the plasmid vector of prokaryotic expression carrier pGEX-PePAL.
That Fig. 3 shows is mono-clonal bacterium colony PCR electrophoresis result, wherein 1-4: the mono-clonal bacterium colony; 5: the recombinant expression vector plasmid; The 6:pGEX plasmid; 7:BL21 bacterium liquid; 8: water; The M:1kb molecular weight marker.
That Fig. 4 shows is different IP TG (final concentration 0.5mmolL
-1, 1mmolL
-1, 1.5mmolL
-1) electrophorogram of inductive PePAL recombinant protein, wherein 1,3,5: supernatant; 2,4,6: precipitation.
The BSA typical curve that is based on G-250 that Fig. 5 shows.
Embodiment
Following examples further specify content of the present invention, but should not be construed as limitation of the present invention.Without departing from the spirit and substance of the case in the present invention, modification or replacement to the inventive method, step or condition are done all belong to scope of the present invention.
If do not specialize the conventional means that used technique means is well known to those skilled in the art among the embodiment.
The acquisition of embodiment 1, mao bamboon chromobindins gene PePAL sequence
With the tender fresh blade of mao bamboon (Phyllostachys edulis) children is material, extract RNA, and reverse transcription becomes cDNA as template, according to the conserved sequence design primer of the grass chromobindins gene PAL family that announces, pcr amplification coding region sequence.
Upstream primer: 5 '-TGTCGACCAGCGTCAACGG-3 '
Downstream primer: 5 '-ACTCTTCGTCGTCGTCGCTGAC-3 '
Pcr amplification product is carried out agarose gel electrophoresis to be detected, downcutting purpose bar zone purification reclaims, the dna fragmentation that reclaims is connected on the pGEM-T easy carrier, transformed into escherichia coli (Esherichia coli) DH5 α competent cell, through blue hickie screening, extraction positive colony plasmid and restriction enzyme mapping check order mono-clonal after analyzing again, insert fragment bit 1 062bp, 340 amino acid of encoding.Use the online software of Blast, the sequence of order-checking and the gene of having reported are carried out homology relatively, find that the conserved regions of inserting fragment and phenylalanine lyase gene family has higher consistence, wherein the consistence with paddy rice reaches 95.9%.
According to obtain the pulsating sequences Design 5 of conserved regions '-the RACE primer:
PAL5-1：5′-ATCTCGTTGACCTTCTTGGCGTGGCTC-3′；
3 '-the RACE primer:
PAL3-1：5′-GCTCCAGTTCCTTGCCAACCCGATCAC-3′
PAL3-2：5′-CGTGTTCAGCTACGCCGACGACCCG-3′
The product of primer PAL5-1 and UPM has a bright band about 1 100bp, and the product electrophoresis of primer PAL3-1 and UPM presents disperse state, does not have specific band.The PCR product of thing PAL3-1 and UPM is diluted 50 times as template, increase with primer PAL3-2 and NUP pairing, electrophoresis result is presented at a bright band (Fig. 1) about 800bp.The PCR product is reclaimed, connects the back transform, in a large amount of clones that obtain, choose positive colony and check order.By order-checking and in the conserved regions sequence assembly, remove lap, obtaining deoxyribonucleotide sequence total length is 2 503bp, comprises complete coding region 2 106bp, 5 ' non-translational region (UTR) 95bp, 3 ' non-translational region 274bp and polyA tail 28bp.
By the online comparative analysis of blast software, find that PePAL amino acid sequence coded and other monocotyledonous PAL have higher consistence, especially be both from paddy rice gramineous (Oryza sativa L.), corn (Zea mays L.), sugarcane (Saccharumofficinarum L.), little Bambusa ventricosa (Bambusa ventricosa McClure), the consistence of the PAL of green bamboo (Bambusa oldhamii Munro) and wheat (Triticum aestivum L.) is all more than 75%, wherein the consistence with the PAL (P14717) of paddy rice is the highest, reaching 94.4%, and is 76.7% and 98.1% from LLB1 (ABP96954) of mao bamboon and the consistence of PePAL1 (FJ195650).This shows that the PePAL gene that clones is a brand-new phenylalanine ammonia lyase gene.
With mao bamboon cDNA is template, according to sequence 1 design primer, the deoxyribonucleotide sequence of pcr amplification mao bamboon phenylalanine ammonia lyase gene coding region.The primer two ends are introduced EcoR I and Not I restriction enzyme site respectively, and primer sequence is as follows:
The upstream: 5 '-ACGAATTCATGGCGGGCAACGGGCTC-3 ', introduce EcoR I restriction enzyme site
The downstream: 5 '-ATTGCGGCCGCTTAGTTGATGGGCAGGGG-3 ', introduce Not I restriction enzyme site
Pcr amplification product is carried out agarose gel electrophoresis to be detected, downcutting purpose bar zone purification reclaims, the dna fragmentation that reclaims is connected on the pGEM-T easy carrier, transformed into escherichia coli (Esherichia coli) DH5 α competent cell, through blue hickie screening, after extracting positive colony plasmid and restriction enzyme mapping and analyzing,, obtain containing the plasmid pT-PePAL of the deoxyribonucleotide sequence of EcoR I and NotI restriction enzyme site and coding mao bamboon phenylalanine ammonia lyase again with the mono-clonal order-checking.
To use double digestion 6hr respectively under plasmid pT-PePAL and 37 ℃ of conditions of transformed plasmid pGEX (having added the His label before GST), enzyme is cut product and is carried out 1% agarose gel electrophoresis analysis respectively, reclaims, and uses T
44 ℃ of connections of dna ligase are spent the night.Connect product and transform DH5 α competent cell, the mono-clonal that grows on the picking Amp resistant panel extracts plasmid, carries out PCR evaluation and restriction enzyme mapping and identifies.With the recombinant expression vector called after pGEX-PePAL that obtains, recombinant expression vector pGEX-PePAL schemes as shown in Figure 2.
With recombinant expression vector pGEX-PePAL transformed into escherichia coli BL21 (DE3) competent cell of implementing to make up in 2, the single bacterium colony that grows on the picking Amp resistant panel carries out PCR to be identified.Mono-clonal bacterium colony with PePAL dna recombinant expression carrier is a template, carries out PCR with primer among the embodiment 2 and detects, and be contrast with recombinant plasmid, BL21 (DE3) and water simultaneously.The electrophoresis result (Fig. 3) of bacterium colony PCR product shows that the mono-clonal bacterium colony contains target gene fragment, can be used for the induction expression of protein experiment.
Embodiment 4PePAL Prokaryotic Expression
Contain penbritin (100mgL with implementing the correct clone's overnight incubation of evaluation in 3, again bacterium being diluted in for 100 times
-1) the LB substratum in cultivate, treat that bacterium liquid grows to OD
600During for 0.6-0.8, (final concentration is respectively 0.5mmolL to add IPTG
-1, 1mmolL
-1, 1.5mmolL
-1) carry out inducing culture.Get the bacterium liquid 250ml that induced, the centrifugal 10min of 5000rpm, collecting cell; Add the BugBuster solution of 9ml, the Benzonase nuclease of 9 μ l, the Lysozyme of 9 μ l, re-suspended cell is hatched 15min with enchylema under the room temperature on rocker; Vortex concussion gently, lysing cell; The centrifugal 20min of 1 200g under 4 ℃ gets supernatant to go in another new pipe; Precipitation is carried out the protein electrophoresis analysis after dissolving with sample-loading buffer.Protein electrophoresis result (Fig. 4) shows that the molecular weight of expressing protein is about 103KDa (comprising HisTag, GST and target protein).
The purifying of example 5PePAL gene prokaryotic product and active detection the thereof
Get the supernatant solution that obtains in the example 4, prepare resin column (MERCK) according to IDA HisBind resin purification method; The extract for preparing carefully is added to the top of resin column; Use 1 * rinsing damping fluid of 1 * binding buffer liquid of 10 times of bed volumes, 6 times of bed volumes, 1 * elution buffer eluted protein of 6 times of bed volumes respectively, obtain the protein solution of purifying.
Add each component by reaction system shown in the table 1,30 ℃ of incubations, oscillatory reaction 30min adds 200 μ l HCl (6molL-1) termination reactions then respectively.Measure the 278nm absorbance, the every variation 0.01 of OD value is enzyme unit alive (being equivalent to form in the 1ml reaction mixture 1 μ g styracin).Protein mass is measured with Xylene Brilliant Cyanine G (G-250) method.Xylene Brilliant Cyanine G G-250 is a kind of dyestuff, takes on a red color under unbound state, becomes cyan after it and protein bound.Protein content is in 0-1000 μ g scope, and the protein-absorbancy of pigment binding substances under 595nm is directly proportional with protein content, sets up the typical curve (Fig. 5) of standard protein BSA with colorimetry.
Table 1 PePAL is active to be detected
| Reagent | Blank pipe | The | Sample | 1 |
| Borate buffer (25mmolL -1，pH＝8.8) | 4 | 4.9 | 3.9 | |
| Purifying PePAL extracting |
0 | 0.1 | 0.1 | |
| L-Phe(50mmol·L -1) | 1 | 0 | 1 | |
| Cumulative volume | 5ml | 5ml | 5ml | |
| OD 278nm | -0.007 | 0.115 |
Content (y=103.52x-3.5021) according to typical curve calculating PePAL is about: 37 μ g/mL.Absorbance by 278nm calculates, and the every variation 0.01 of OD value is enzyme unit alive (being equivalent to form in the 1ml reaction mixture 1 μ g styracin), and 1 μ g PePAL can form 0.3 μ g trans-cinnamic acid by catalysis L-phenylalanine.
Sequence table
＜110〉International Center for Banboo and Rattan
＜120〉mao bamboon phenylalanine ammonia lyase, its encoding gene and vivoexpression method
<130>KHP09113879.8
<160>9
<170>PatentIn?version?3.5
<210>1
<211>2503
<212>DNA
<213>Phyllostachys?edulis
<220>
<221>5′UTR
<222>(1)..(95)
<220>
<221>CDS
<222>(96)..(2201)
<220>
<221>3′UTR
<222>(2202)..(2475)
<220>
<221>polyA_signal
<222>(2476)..(2503)
<400>1
acgcggggaa?gaagagctca?ccaccctctc?ctccggctct?tcttcgacct?cctcctcctc 60
ctcctaacct?acccaccacc?gccacccacc?gagca?atg?gcg?ggc?aac?ggg?ctc 113
Met?Ala?Gly?Asn?Gly?Leu
1 5
atc?atc?aag?aat?gac?ccg?ctc?aac?tgg?ggc?gcg?gcg?gcg?gcc?gag?ctc 161
Ile?Ile?Lys?Asn?Asp?Pro?Leu?Asn?Trp?Gly?Ala?Ala?Ala?Ala?Glu?Leu
10 15 20
acc?ggc?agc?cac?ctc?gac?gag?gtg?aag?cgg?atg?gtg?gcg?cag?ttc?cgc 209
Thr?Gly?Ser?His?Leu?Asp?Glu?Val?Lys?Arg?Met?Val?Ala?Gln?Phe?Arg
25 30 35
gag?ccc?gtg?gtc?aag?atc?gag?ggc?tcc?agc?ctc?cgc?gtc?ggc?cag?gtg 257
Glu?Pro?Val?Val?Lys?Ile?Glu?Gly?Ser?Ser?Leu?Arg?Val?Gly?Gln?Val
40 45 50
gcc?gcc?gtc?gcc?cag?gcc?aag?gac?gcc?gcc?ggc?gtc?gcc?gtc?gag?ctc 305
Ala?Ala?Val?Ala?Gln?Ala?Lys?Asp?Ala?Ala?Gly?Val?Ala?Val?Glu?Leu
55 60 65 70
gac?gag?gag?gcg?cgc?ccc?cgc?gtc?aag?gcc?agc?agc?gag?tgg?atc?ctc 353
Asp?Glu?Glu?Ala?Arg?Pro?Arg?Val?Lys?Ala?Ser?Ser?Glu?Trp?Ile?Leu
75 80 85
aac?tgc?ctc?gcc?cat?ggc?ggc?gac?atc?tac?ggc?gtc?acc?acc?ggc?ttc 401
Asn?Cys?Leu?Ala?His?Gly?Gly?Asp?Ile?Tyr?Gly?Val?Thr?Thr?Gly?Phe
90 95 100
ggc?ggc?acc?tcc?cac?cgc?cgc?acc?aag?gac?ggg?ccc?gcc?ctc?cag?gtc 449
Gly?Gly?Thr?Ser?His?Arg?Arg?Thr?Lys?Asp?Gly?Pro?Ala?Leu?Gln?Val
105 110 115
gag?ctc?ctc?agg?cat?ctc?aat?gcc?gga?atc?ttc?ggc?acc?ggc?acc?gac 497
Glu?Leu?Leu?Arg?His?Leu?Asn?Ala?Gly?Ile?Phe?Gly?Thr?Gly?Thr?Asp
120 125 130
ggg?cac?acg?ctg?ccg?tcg?gag?gtg?acg?cgt?gcg?gcc?atg?ctc?gtg?cgc 545
Gly?His?Thr?Leu?Pro?Ser?Glu?Val?Thr?Arg?Ala?Ala?Met?Leu?Val?Arg
135 140 145 150
atc?aac?acc?ctc?ctc?cag?ggc?tac?tcc?ggc?atc?cgc?ttc?gag?atc?ctt 593
Ile?Asn?Thr?Leu?Leu?Gln?Gly?Tyr?Ser?Gly?Ile?Arg?Phe?Glu?Ile?Leu
155 160 165
gag?gcc?atc?acc?aag?ctc?atc?aac?acc?ggc?gtc?agc?ccc?tgc?ctc?ccg 641
Glu?Ala?Ile?Thr?Lys?Leu?Ile?Asn?Thr?Gly?Val?Ser?Pro?Cys?Leu?Pro
170 175 180
ctc?agg?gga?acc?atc?acc?gcg?tcc?ggc?gac?ctg?gtc?ccg?ctg?tcc?tac 689
Leu?Arg?Gly?Thr?Ile?Thr?Ala?Ser?Gly?Asp?Leu?Val?Pro?Leu?Ser?Tyr
185 190 195
att?gcc?ggc?ctt?atc?act?ggc?cgc?ccc?aat?gcg?cag?gcc?gtc?gcc?ccc 737
Ile?Ala?Gly?Leu?Ile?Thr?Gly?Arg?Pro?Asn?Ala?Gln?Ala?Val?Ala?Pro
200 205 210
gac?ggc?agg?aag?gtg?gac?gcc?gcc?gag?gcg?ttc?aag?atc?gcc?ggc?atc 785
Asp?Gly?Arg?Lys?Val?Asp?Ala?Ala?Glu?Ala?Phe?Lys?Ile?Ala?Gly?Ile
215 220 225 230
gag?ggc?ggg?ttc?ttc?aag?ctc?aac?ccc?aag?gag?ggt?ctc?gcc?atc?gtc 833
Glu?Gly?Gly?Phe?Phe?Lys?Leu?Asn?Pro?Lys?Glu?Gly?Leu?Ala?Ile?Val
235 240 245
aac?ggc?acg?tcc?gtg?ggc?tcc?gcc?ctc?gcg?gcc?acg?gtg?ctc?tat?gat 881
Asn?Gly?Thr?Ser?Val?Gly?Ser?Ala?Leu?Ala?Ala?Thr?Val?Leu?Tyr?Asp
250 255 260
tgc?aac?gtc?ctc?gcc?gtc?ctc?tcc?gag?gtc?ctg?tcc?gcc?gtg?ttc?tgc 929
Cys?Asn?Val?Leu?Ala?Val?Leu?Ser?Glu?Val?Leu?Ser?Ala?Val?Phe?Cys
265 270 275
gag?gtc?atg?aac?ggc?aag?ccg?gag?tac?acc?gac?cac?ctg?acc?cac?aag 977
Glu?Val?Met?Asn?Gly?Lys?Pro?Glu?Tyr?Thr?Asp?His?Leu?Thr?His?Lys
280 285 290
ctg?aag?cac?cac?ccg?ggc?tcg?atc?gag?gcc?gcg?gcc?atc?atg?gag?cac 1025
Leu?Lys?His?His?Pro?Gly?Ser?Ile?Glu?Ala?Ala?Ala?Ile?Met?Glu?His
295 300 305 310
atc?ctg?gcc?ggc?agc?tcg?ttc?atg?agc?cac?gcc?aag?aag?gtc?aac?gag 1073
Ile?Leu?Ala?Gly?Ser?Ser?Phe?Met?Ser?His?Ala?Lys?Lys?Val?Asn?Glu
315 320 325
atg?gac?ccg?ctg?ctc?aag?ccc?aag?cag?gac?agg?tac?gcg?ctc?cgc?aca 1121
Met?Asp?Pro?Leu?Leu?Lys?Pro?Lys?Gln?Asp?Arg?Tyr?Ala?Leu?Arg?Thr
330 335 340
tcg?ccg?cag?tgg?ctc?ggc?cca?cag?atc?gag?gtc?atc?cgg?gcg?gcc?acc 1169
Ser?Pro?Gln?Trp?Leu?Gly?Pro?Gln?Ile?Glu?Val?Ile?Arg?Ala?Ala?Thr
345 350 355
aag?tcc?atc?gag?cgc?gag?gtc?aac?tcg?gtc?aac?gac?aac?ccg?gtc?atc 1217
Lys?Ser?Ile?Glu?Arg?Glu?Val?Asn?Ser?Val?Asn?Asp?Asn?Pro?Val?Ile
360 365 370
gac?gtc?cac?cgc?ggc?aag?gca?ctc?cac?ggc?ggc?aac?ttc?cag?ggc?aca 1265
Asp?Val?His?Arg?Gly?Lys?Ala?Leu?His?Gly?Gly?Asn?Phe?Gln?Gly?Thr
375 380 385 390
ccc?atc?ggt?gtg?tcc?atg?gac?aac?acc?cgt?ctc?gcc?atc?gcc?aac?atc 1313
Pro?Ile?Gly?Val?Ser?Met?Asp?Asn?Thr?Arg?Leu?Ala?Ile?Ala?Asn?Ile
395 400 405
ggc?aag?ctc?atg?ttc?gcg?cag?ttc?tca?gag?ctc?gtg?aac?gag?ttc?tac 1361
Gly?Lys?Leu?Met?Phe?Ala?Gln?Phe?Ser?Glu?Leu?Val?Asn?Glu?Phe?Tyr
410 415 420
aac?aac?ggg?ctg?acg?tcc?aac?ctg?gcc?ggc?agc?cgc?aac?ccg?agc?ttg 1409
Asn?Asn?Gly?Leu?Thr?Ser?Asn?Leu?Ala?Gly?Ser?Arg?Asn?Pro?Ser?Leu
425 430 435
gac?tac?ggc?ttc?aag?ggc?acc?gag?atc?gcc?atg?gcc?tcc?tac?tgc?tct 1457
Asp?Tyr?Gly?Phe?Lys?Gly?Thr?Glu?Ile?Ala?Met?Ala?Ser?Tyr?Cys?Ser
440 445 450
gag?ctc?cag?tac?ctt?gcc?aac?ccg?atc?acc?aac?cat?gtc?cag?agc?gcg 1505
Glu?Leu?Gln?Tyr?Leu?Ala?Asn?Pro?Ile?Thr?Asn?His?Val?Gln?Ser?Ala
455 460 465 470
gag?cag?cac?aac?cag?gac?gtg?aac?tca?ctc?ggc?ctt?gtc?tca?gcc?agg 1553
Glu?Gln?His?Asn?Gln?Asp?Val?Asn?Ser?Leu?Gly?Leu?Val?Ser?Ala?Arg
475 480 485
aag?acc?gcc?gag?gcg?gtg?gac?atc?ctc?aag?ctc?atg?tcc?tcg?acg?tac 1601
Lys?Thr?Ala?Glu?Ala?Val?Asp?Ile?Leu?Lys?Leu?Met?Ser?Ser?Thr?Tyr
490 495 500
atg?gtc?gcg?ctg?tgc?cag?gcc?gtc?gac?ctc?cgc?cac?ctc?gag?gag?aac 1649
Met?Val?Ala?Leu?Cys?Gln?Ala?Val?Asp?Leu?Arg?His?Leu?Glu?Glu?Asn
505 510 515
atc?aag?agc?tcc?gtc?aag?aac?tgc?gtg?acg?cag?gta?gcc?aag?aag?gtg 1697
Ile?Lys?Ser?Ser?Val?Lys?Asn?Cys?Val?Thr?Gln?Val?Ala?Lys?Lys?Val
520 525 530
ctc?acc?atg?aac?ccc?acc?ggc?gac?ctc?tcc?agc?gcg?cgc?ttc?agc?gag 1745
Leu?Thr?Met?Asn?Pro?Thr?Gly?Asp?Leu?Ser?Ser?Ala?Arg?Phe?Ser?Glu
535 540 545 550
aag?aac?ctc?ctc?acc?gcc?atc?gac?cgc?gag?gtc?gtg?ttc?agc?tac?gcc 1793
Lys?Asn?Leu?Leu?Thr?Ala?Ile?Asp?Arg?Glu?Val?Val?Phe?Ser?Tyr?Ala
555 560 565
gat?gac?ccg?tgc?agc?gcc?aac?tac?ccg?ctg?atg?cag?aag?ctg?cgc?gcc 1841
Asp?Asp?Pro?Cys?Ser?Ala?Asn?Tyr?Pro?Leu?Met?Gln?Lys?Leu?Arg?Ala
570 575 580
gtg?ctc?gtc?gac?cac?gcc?ctc?acc?agc?ggc?gac?gcc?gag?agg?gag?ccc 1889
Val?Leu?Val?Asp?His?Ala?Leu?Thr?Ser?Gly?Asp?Ala?Glu?Arg?Glu?Pro
585 590 595
tcc?gtg?ttc?tcc?aag?atc?acc?aag?ttc?gag?gag?gag?ctg?cgc?tcg?gcg 1937
Ser?Val?Phe?Ser?Lys?Ile?Thr?Lys?Phe?Glu?Glu?Glu?Leu?Arg?Ser?Ala
600 605 610
ctg?ccc?cgg?gag?gtc?gag?gcc?gcc?cgc?gtg?gcc?gtg?gag?aac?ggc?tcc 1985
Leu?Pro?Arg?Glu?Val?Glu?Ala?Ala?Arg?Val?Ala?Val?Glu?Asn?Gly?Ser
615 620 625 630
gcg?ccg?atc?gcc?aac?cgg?atc?aag?gag?agc?cgg?tcg?ttc?ccc?gtg?tac 2033
Ala?Pro?Ile?Ala?Asn?Arg?Ile?Lys?Glu?Ser?Arg?Ser?Phe?Pro?Val?Tyr
635 640 645
cgc?ttc?gtc?cgc?gag?gag?ctg?ggc?tgc?gtg?tac?ctc?acc?ggc?gag?aag 2081
Arg?Phe?Val?Arg?Glu?Glu?Leu?Gly?Cys?Val?Tyr?Leu?Thr?Gly?Glu?Lys
650 655 660
ctc?aag?tcg?ccc?ggc?gag?gag?tgc?aac?aag?gtg?ttc?ctc?ggc?atc?agc 2129
Leu?Lys?Ser?Pro?Gly?Glu?Glu?Cys?Asn?Lys?Val?Phe?Leu?Gly?Ile?Ser
665 670 675
cag?ggc?aag?ctc?atc?gac?ccc?atg?ctc?gaa?tgc?ctc?aag?gag?tgg?aac 2177
Gln?Gly?Lys?Leu?Ile?Asp?Pro?Met?Leu?Glu?Cys?Leu?Lys?Glu?Trp?Asn
680 685 690
ggc?gag?ccc?ctg?ccc?atc?aac?taa?gcatccgtca?tccacccgtg?agatcgtgga 2231
Gly?Glu?Pro?Leu?Pro?Ile?Asn
695 700
ggagaggacg?ctacagaata?catcaaagaa?aataaaccgc?gtcgtgtatg?ttcggatcgt 2291
gtcgtcgctt?ttgccttttt?tcgttcgttg?gtggcgttgt?tctttgtgta?gctgttctgc 2351
catcgcctgt?aatgcgcatg?ccctggcggg?cggctttttt?taagatatgt?ttgttgtatt 2411
ttgggctgaa?gtatgttacc?ggattcccac?tttttaattg?tcctaataga?atgttttgct 2471
cctcaaaaaa?aaaaaaaaaa?aaaaaaaaaa?aa 2503
<210>2
<211>701
<212>PRT
<213>Phyllostachys?edulis
<400>2
Met?Ala?Gly?Asn?Gly?Leu?Ile?Ile?Lys?Asn?Asp?Pro?Leu?Asn?Trp?Gly
1 5 10 15
Ala?Ala?Ala?Ala?Glu?Leu?Thr?Gly?Ser?His?Leu?Asp?Glu?Val?Lys?Arg
20 25 30
Met?Val?Ala?Gln?Phe?Arg?Glu?Pro?Val?Val?Lys?Ile?Glu?Gly?Ser?Ser
35 40 45
Leu?Arg?Val?Gly?Gln?Val?Ala?Ala?Val?Ala?Gln?Ala?Lys?Asp?Ala?Ala
50 55 60
Gly?Val?Ala?Val?Glu?Leu?Asp?Glu?Glu?Ala?Arg?Pro?Arg?Val?Lys?Ala
65 70 75 80
Ser?Ser?Glu?Trp?Ile?Leu?Asn?Cys?Leu?Ala?His?Gly?Gly?Asp?Ile?Tyr
85 90 95
Gly?Val?Thr?Thr?Gly?Phe?Gly?Gly?Thr?Ser?His?Arg?Arg?Thr?Lys?Asp
100 105 110
Gly?Pro?Ala?Leu?Gln?Val?Glu?Leu?Leu?Arg?His?Leu?Asn?Ala?Gly?Ile
115 120 125
Phe?Gly?Thr?Gly?Thr?Asp?Gly?His?Thr?Leu?Pro?Ser?Glu?Val?Thr?Arg
130 135 140
Ala?Ala?Met?Leu?Val?Arg?Ile?Asn?Thr?Leu?Leu?Gln?Gly?Tyr?Ser?Gly
145 150 155 160
Ile?Arg?Phe?Glu?Ile?Leu?Glu?Ala?Ile?Thr?Lys?Leu?Ile?Asn?Thr?Gly
165 170 175
Val?Ser?Pro?Cys?Leu?Pro?Leu?Arg?Gly?Thr?Ile?Thr?Ala?Ser?Gly?Asp
180 185 190
Leu?Val?Pro?Leu?Ser?Tyr?Ile?Ala?Gly?Leu?Ile?Thr?Gly?Arg?Pro?Asn
195 200 205
Ala?Gln?Ala?Val?Ala?Pro?Asp?Gly?Arg?Lys?Val?Asp?Ala?Ala?Glu?Ala
210 215 220
Phe?Lys?Ile?Ala?Gly?Ile?Glu?Gly?Gly?Phe?Phe?Lys?Leu?Asn?Pro?Lys
225 230 235 240
Glu?Gly?Leu?Ala?Ile?Val?Asn?Gly?Thr?Ser?Val?Gly?Ser?Ala?Leu?Ala
245 250 255
Ala?Thr?Val?Leu?Tyr?Asp?Cys?Asn?Val?Leu?Ala?Val?Leu?Ser?Glu?Val
260 265 270
Leu?Ser?Ala?Val?Phe?Cys?Glu?Val?Met?Asn?Gly?Lys?Pro?Glu?Tyr?Thr
275 280 285
Asp?His?Leu?Thr?His?Lys?Leu?Lys?His?His?Pro?Gly?Ser?Ile?Glu?Ala
290 295 300
Ala?Ala?Ile?Met?Glu?His?Ile?Leu?Ala?Gly?Ser?Ser?Phe?Met?Ser?His
305 310 315 320
Ala?Lys?Lys?Val?Asn?Glu?Met?Asp?Pro?Leu?Leu?Lys?Pro?Lys?Gln?Asp
325 330 335
Arg?Tyr?Ala?Leu?Arg?Thr?Ser?Pro?Gln?Trp?Leu?Gly?Pro?Gln?Ile?Glu
340 345 350
Val?Ile?Arg?Ala?Ala?Thr?Lys?Ser?Ile?Glu?Arg?Glu?Val?Asn?Ser?Val
355 360 365
Asn?Asp?Asn?Pro?Val?Ile?Asp?Val?His?Arg?Gly?Lys?Ala?Leu?His?Gly
370 375 380
Gly?Asn?Phe?Gln?Gly?Thr?Pro?Ile?Gly?Val?Ser?Met?Asp?Asn?Thr?Arg
385 390 395 400
Leu?Ala?Ile?Ala?Asn?Ile?Gly?Lys?Leu?Met?Phe?Ala?Gln?Phe?Ser?Glu
405 410 415
Leu?Val?Asn?Glu?Phe?Tyr?Asn?Asn?Gly?Leu?Thr?Ser?Asn?Leu?Ala?Gly
420 425 430
Ser?Arg?Asn?Pro?Ser?Leu?Asp?Tyr?Gly?Phe?Lys?Gly?Thr?Glu?Ile?Ala
435 440 445
Met?Ala?Ser?Tyr?Cys?Ser?Glu?Leu?Gln?Tyr?Leu?Ala?Asn?Pro?Ile?Thr
450 455 460
Asn?His?Val?Gln?Ser?Ala?Glu?Gln?His?Asn?Gln?Asp?Val?Asn?Ser?Leu
465 470 475 480
Gly?Leu?Val?Ser?Ala?Arg?Lys?Thr?Ala?Glu?Ala?Val?Asp?Ile?Leu?Lys
485 490 495
Leu?Met?Ser?Ser?Thr?Tyr?Met?Val?Ala?Leu?Cys?Gln?Ala?Val?Asp?Leu
500 505 510
Arg?His?Leu?Glu?Glu?Asn?Ile?Lys?Ser?Ser?Val?Lys?Asn?Cys?Val?Thr
515 520 525
Gln?Val?Ala?Lys?Lys?Val?Leu?Thr?Met?Asn?Pro?Thr?Gly?Asp?Leu?Ser
530 535 540
Ser?Ala?Arg?Phe?Ser?Glu?Lys?Asn?Leu?Leu?Thr?Ala?Ile?Asp?Arg?Glu
545 550 555 560
Val?Val?Phe?Ser?Tyr?Ala?Asp?Asp?Pro?Cys?Ser?Ala?Asn?Tyr?Pro?Leu
565 570 575
Met?Gln?Lys?Leu?Arg?Ala?Val?Leu?Val?Asp?His?Ala?Leu?Thr?Ser?Gly
580 585 590
Asp?Ala?Glu?Arg?Glu?Pro?Ser?Val?Phe?Ser?Lys?Ile?Thr?Lys?Phe?Glu
595 600 605
Glu?Glu?Leu?Arg?Ser?Ala?Leu?Pro?Arg?Glu?Val?Glu?Ala?Ala?Arg?Val
610 615 620
Ala?Val?Glu?Asn?Gly?Ser?Ala?Pro?Ile?Ala?Asn?Arg?Ile?Lys?Glu?Ser
625 630 635 640
Arg?Ser?Phe?Pro?Val?Tyr?Arg?Phe?Val?Arg?Glu?Glu?Leu?Gly?Cys?Val
645 650 655
Tyr?Leu?Thr?Gly?Glu?Lys?Leu?Lys?Ser?Pro?Gly?Glu?Glu?Cys?Asn?Lys
660 665 670
Val?Phe?Leu?Gly?Ile?Ser?Gln?Gly?Lys?Leu?Ile?Asp?Pro?Met?Leu?Glu
675 680 685
Cys?Leu?Lys?Glu?Trp?Asn?Gly?Glu?Pro?Leu?Pro?Ile?Asn
690 695 700
<210>3
<211>19
<212>DNA
＜213〉artificial sequence
<400>3
tgtcgaccag?cgtcaacgg 19
<210>4
<211>22
<212>DNA
＜213〉artificial sequence
<400>4
actcttcgtc?gtcgtcgctg?ac 22
<210>5
<211>27
<212>DNA
＜213〉artificial sequence
<400>5
atctcgttga?ccttcttggc?gtggctc 27
<210>6
<211>27
<212>DNA
＜213〉artificial sequence
<400>6
gctccagttc?cttgccaacc?cgatcac 27
<210>7
<211>25
<212>DNA
＜213〉artificial sequence
<400>7
cgtgttcagc?tacgccgacg?acccg 25
<210>8
<211>26
<212>DNA
＜213〉artificial sequence
<400>8
acgaattcat?ggcgggcaac?gggctc 26
<210>9
<211>29
<212>DNA
＜213〉artificial sequence
<400>9
attgcggccg?cttagttgat?gggcagggg 29
Claims (10)
1. mao bamboon phenylalanine ammonia lyase PePAL, its aminoacid sequence is:
A) aminoacid sequence shown in the SEQ ID No.2;
B) aminoacid sequence shown in the SEQ ID No.2 is through replacing, lack or add the aminoacid sequence with same function of one or several amino-acid residue formation.
2. the gene of coding claim 1 described phenylalanine ammonia lyase PePAL.
3. gene as claimed in claim 2, its nucleotide sequence is shown in SEQ ID No.1.
4. the recombinant vectors that contains claim 2 or 3 described genes.
5. recombinant vectors as claimed in claim 4, it is pGEX-PePAL.
6. claim 4 or 5 described recombinant vectors transformed host cells.
7. host cell as claimed in claim 6, it is e. coli bl21 (DE3).
8. the method for a vivoexpression mao bamboon phenylalanine ammonia lyase PePAL, it induces PePAL to express by cultivating claim 6 or 7 described host cells.
9. method as claimed in claim 8 is characterized in that, described host cell grows to OD for transforming the e. coli bl21 (DE3) of pGEX-PePAL recombinant vectors through fermentation culture bacterium liquid
600During for 0.6-0.8, add IPTG to final concentration 1mmol/L, cultivated 4 hours for 37 ℃, collect thalline, centrifuging and taking supernatant after the cracking obtains the purifying phenylalanine ammonia lyase with Histidine binding resin purifying.
10. the application of the described mao bamboon phenylalanine ammonia lyase of claim 1 PePAL in catalysis L-phenylalanine generation trans-cinnamic acid.
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|---|---|---|---|
| CN2009102612054A CN102102096B (en) | 2009-12-17 | 2009-12-17 | Mao bamboo phenyl alanine ammonialyase, encoding gene and in-vitro expression method thereof |
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| CN2009102612054A CN102102096B (en) | 2009-12-17 | 2009-12-17 | Mao bamboo phenyl alanine ammonialyase, encoding gene and in-vitro expression method thereof |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102358890A (en) * | 2011-11-01 | 2012-02-22 | 北京三元基因工程有限公司 | Preparation method of lactococcus lactis product for expressing phenylalanine ammonialyase |
| CN102358889A (en) * | 2011-11-01 | 2012-02-22 | 北京三元基因工程有限公司 | Preparation method of lactococcus lactis product expressing phenylalanine ammonia lyase |
| CN103468665A (en) * | 2013-10-08 | 2013-12-25 | 南京林业大学 | Corn phenylalanine ammonia enzyme and application thereof |
| CN112662696A (en) * | 2020-12-30 | 2021-04-16 | 上海予泉生物科技有限公司 | Engineering cyanobacteria for biologically synthesizing p-coumaric acid and preparation method thereof |
-
2009
- 2009-12-17 CN CN2009102612054A patent/CN102102096B/en not_active Expired - Fee Related
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102358890A (en) * | 2011-11-01 | 2012-02-22 | 北京三元基因工程有限公司 | Preparation method of lactococcus lactis product for expressing phenylalanine ammonialyase |
| CN102358889A (en) * | 2011-11-01 | 2012-02-22 | 北京三元基因工程有限公司 | Preparation method of lactococcus lactis product expressing phenylalanine ammonia lyase |
| CN102358890B (en) * | 2011-11-01 | 2013-07-17 | 北京三元基因工程有限公司 | Preparation method of lactococcus lactis product for expressing phenylalanine ammonialyase |
| CN103468665A (en) * | 2013-10-08 | 2013-12-25 | 南京林业大学 | Corn phenylalanine ammonia enzyme and application thereof |
| CN103468665B (en) * | 2013-10-08 | 2015-12-23 | 南京林业大学 | A kind of Corn phenylalanine ammonia enzyme and application thereof |
| CN112662696A (en) * | 2020-12-30 | 2021-04-16 | 上海予泉生物科技有限公司 | Engineering cyanobacteria for biologically synthesizing p-coumaric acid and preparation method thereof |
| CN112662696B (en) * | 2020-12-30 | 2023-07-07 | 江苏甬泽生物科技有限公司 | Engineering cyanobacteria for biosynthesizing p-soyabean aromatic acid and preparation method thereof |
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| Publication number | Publication date |
|---|---|
| CN102102096B (en) | 2012-11-07 |
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