CN101532026A - Sutellaria viscidula phenyl alanine ammonialyase protein coded sequence - Google Patents
Sutellaria viscidula phenyl alanine ammonialyase protein coded sequence Download PDFInfo
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Abstract
The invention provides a sutellaria viscidula phenyl alanine ammonialyase protein coded sequence belonging to the genetic engineering field. Polypeptide nucleotide sequences having phenyl alanine ammonialyase activity are coded. The nucleotide sequences are composed of nucleotide sequences from positions 129 to 2261 in the SEQ ID NO.3 or simplified sequences. The sequences code polypeptide having amino acid sequences showed by SEQ ID NO.3. The invention obviously improves content of flavonoid compounds for instance scutelloside in sutellaria viscidula plants and has great application value.
Description
Technical field
What the present invention relates to is a kind of albumen coded sequence, and particularly a kind of Sutellaria viscidula phenyl alanine ammonialyase protein coded sequence belongs to the genetically engineered field.
Background technology
Sutellaria viscidula belongs to the perennial medicinal herb plant of Labiatae Scutellaria.The about kind more than 300 of this platymiscium, the world blazons, and China has more than 100 kinds, and can be used as the medicinal person of the root of large-flowered skullcap has about 7 kinds approximately, and Sutellaria viscidula is exactly wherein a kind of.The main medicinal ingredients of this plant is a baicalin, and modern pharmacology confirms that it has clearing heat and detoxicating, arresting bleeding and miscarriage prevention, antisepsis and anti-inflammation, hepatic cholagogic, step-down desensitization, sun-proof effect such as whiten.Along with in the world to the persistently overheating and understanding of research such as baicalin progressively deeply, think that baicalin all has effect in removing oxyradical, the ischemical reperfusion injury that alleviates tissue, adjusting immunity, promotion apoptosis and many-sides such as antitumor and HIV, has important pharmaceutical use and exploitation prospect.Yet the major technique that obtains Sutellaria viscidula at present is an artificial culture, but has long, drawback such as surviving rate is low, pesticide residue exceed standard and effective constituent is lower of production cycle, makes that this mode commercial promise is still uncertain.Thereby it is very necessary to seek new medicine source.Reaching its maturity of biological develop rapidly of Secondary Metabolism of Plant approach molecular biosciences and Plant Biotechnology makes and to utilize genetically engineered to obtain that important natural drug baicalin becomes possibility in the Sutellaria viscidula.
Genetic engineering technique is the important method that realizes genetic improvement and germplasm innovation, carry out the research of baicalin metabolic engineering, and then the novel medicine source of acquisition high yield and high quality, and prerequisite is to illustrate its biosynthetic molecule mechanism, the gene that promptly separates a plurality of enzymatic reaction steps on this approach, and investigate the effect of goal gene in the baicalin biosynthetic pathway, for the metabolic engineering of baicalin provides candidate gene and action target spot very necessary.(Phenylalanineammonia-lyase PAL) is first key enzyme of phenpropionate approach to phenylalanine ammonia lyase.PAL is present in plant and some fungi, bacterium and the algae, its catalysis L-phenylalanine nonoxidative deamination generates trans-cinnamic acid (cinnamic acid, CA), styracin is the general precursor of biosynthesizing of plant phenylpropyl alcohol alkanes secondary substance such as flavones, tonka bean camphor, xylogen and some phenols, so this enzyme has extremely important position in Secondary Metabolism of Plant.With upstream and downstream speed limit bottleneck in the metabolic engineering technological breakthrough PAL approach, be a practicable strategy of realizing a large amount expression of target product.Can infer, improve content of baicalin in the Sutellaria viscidula, PAL will be an effectively metabolic engineering target spot, yet regrettably, up to the present, yet there are no the report of cloning and identify the pal gene from Sutellaria viscidula.According to the nucleotide sequence of phenylalanine ammonia lyase among the GeneBank, we have cloned correlated series from Sutellaria viscidula in conjunction with round pcr, according to the Search Results of GeneBank, are the phenylalanine ammonia lyase gene with this unnamed gene.
In analysis to existing document, do not see the clone of the pal gene on the Sutellaria viscidula flavonoid compound biosynthetic pathway, do not find to have the report of close ties document so far as yet with theme of the present invention.
Summary of the invention
The objective of the invention is to overcome deficiency of the prior art, a kind of Sutellaria viscidula phenyl alanine ammonialyase protein coded sequence is provided.The invention discloses the aminoacid sequence of Sutellaria viscidula phenyl alanine ammonialyase and the nucleotide sequence of Sutellaria viscidula phenyl alanine ammonialyase gene, be the final rate-limiting reaction step of breaking on the flavonoid compound biosynthetic pathway that adopts, the metabolic engineering of realizing baicalin provides candidate gene and action target spot, and it is laid a good foundation in the application that utilizes transgenic technology to improve aspect the content of flavonoids such as baicalin.
The present invention is achieved by the following technical solutions:
At the isolated dna molecular of the present invention, this molecule comprises: coding has the Sutellaria viscidula phenyl alanine ammonialyase nucleotide sequence, and shows 70% homology from the nucleotides sequence of Nucleotide 129-2261 position among described nucleotide sequence and the SEQ ID NO.3; Perhaps described nucleotide sequence can be under 45-55 ℃ of conditions with SEQ ID NO.3 in from the nucleotide sequence hybridization of Nucleotide 129-2261 position.
The isolated Sutellaria viscidula phenyl alanine ammonialyase protein coded sequence polypeptide of the present invention, it comprises: have polypeptide or its conservative property variation polypeptide or its active fragments of SEQID NO.3 aminoacid sequence, or its reactive derivative.Preferably, this polypeptide is to have SEQ ID NO.3 polypeptide of sequence.
A kind of carrier provided by the present invention, it comprises above-mentioned dna molecular.A kind of nucleic acid molecule, it comprises 8-100 continuous nucleotide in the described dna molecular.
The present invention is an eukaryotic cell with above-mentioned carrier transformed host cells.
In the present invention, " isolating ", " purifying " DNA are meant, this DNA or fragment have been arranged in the sequence of its both sides and have separated under native state, refer to that also this DNA or fragment with under the native state follow the component of nucleic acid to separate, and separate with the protein of in cell, following it.
In the present invention, term " Sutellaria viscidula phenyl alanine ammonialyase protein coded sequence " refers to encode and has the nucleotide sequence of active polypeptide, as 129-2261 position nucleotide sequence and degenerate sequence thereof among the SEQ ID NO.3.This degenerate sequence is meant, is arranged in the encoder block 129-2261 position Nucleotide of SEQ ID NO.3 sequence, and having one or more codons to be encoded, the degenerate codon of same amino acid replaces the back and the sequence that produces.Because the degeneracy of codon, thus with SEQ ID NO.3 in 129-2261 position nucleotide sequence homology be low to moderate about 70% the degenerate sequence described sequence of SEQ ID NO.3 of also encoding out.This term also comprises can be under the moderate stringent condition, better under the height stringent condition with SEQ ID NO.3 in from the nucleotide sequence of the nucleotide sequence hybridization of Nucleotide 129-2261 position.This term also comprise with SEQ ID NO.3 in from the homology of nucleotide sequence at least 70% of Nucleotide 129-2261 position, preferably at least 80%, more preferably at least 90%, at least 95% nucleotide sequence best.
This term also comprises encoding to have the variant form of open reading frame sequence among the proteic SEQ IDNO.3 with the Sutellaria viscidula phenyl alanine ammonialyase identical function.These variant forms comprise (but being not limited to): several (are generally 1-90, preferably 1-60, more preferably 1-20,1-10 best) disappearance, insertion and/or the replacement of Nucleotide, and several (are generally in 60 to hold interpolation 5 ' and/or 3 ', preferably being in 30, more preferably is in 10, is in 5 best) Nucleotide.
In the present invention, term " Sutellaria viscidula phenyl alanine ammonialyase " refers to have the SEQ ID NO.3 polypeptide of sequence of protein-active.This term also comprises having variant form relevant identical function, SEQ ID NO.3 sequence.These variant forms comprise (but being not limited to): several (are generally 1-50, preferably 1-30, more preferably 1-20,1-10 best) amino acid whose disappearance, insertion and/or replacement, and add one or several at C-terminal and/or N-terminal and (be generally in 20, preferably being in 10, more preferably is in 5) amino acid.For example, in the art, when replacing, can not change proteinic function usually with the close or similar amino acid of performance.Again such as, add one or several amino acid at C-terminal and/or N-terminal and also can not change proteinic function usually.This term also comprises its active fragments and reactive derivative.
The variant form of Sutellaria viscidula phenyl alanine ammonialyase gene of the present invention comprises: homologous sequence, conservative property varient, allelic variant, natural mutation, induced mutation body, under high or low stringent condition can with the coded albumen of the DNA of DNA hybridization and the polypeptide or the albumen that utilize the serum of Sutellaria viscidula phenyl alanine ammonialyase protein polypeptide to obtain.
In the present invention, " Sutellaria viscidula phenyl alanine ammonialyase conservative property variation polypeptide " refers to compare with the aminoacid sequence of SEQ ID NO.3, have 10 at the most, preferably at the most 8, more preferably 5 amino acid similar performances or close amino acid are replaced and are formed polypeptide at the most.These conservative property variation polypeptide are preferably replaced according to table 1 and are produced.
Table 1
| Initial residue | Representational replacement | The preferred replacement |
| Ala(A) | Val；Leu；Ile | Val |
| Arg(R) | Lys；Gln；Asn | Lys |
| Asn(N) | Gln；His；Lys；Arg | Gln |
| Asp(D) | Glu | Glu |
| Cys(C) | Ser | Ser |
| Gln(Q) | Asn | Asn |
| Glu(E) | Asp | Asp |
| Gly(G) | Pro；Ala | Ala |
| His(H) | Asn；Gln；Lys；Arg | Arg |
| Ile(I) | Leu；Val；Met；Ala；Phe | Leu |
| Leu(L) | Ile；Val；Met；Ala；Phe | Ile |
| Lys(K) | Arg；Gln；Asn | Arg |
| Met(M) | Leu；Phe；Ile | Leu |
| Phe(F) | Leu；Val；Ile；Ala；Tyr | Leu |
| Pro(P) | Ala | Ala |
| Ser(S) | Thr | Thr |
| Thr(T) | Ser | Ser |
| Trp(W) | Tyr；Phe | Tyr |
| Tyr(Y) | Trp；Phe；Thr；Ser | Phe |
| Val(V) | Ile；Leu；Met；Phe；Ala | Leu |
Table 2
90％ identity in 233aa overlap
Query 1 MAPAVEVSHRSNGFCVQLSDPLNWGAAAEALKGSHLDEVKRMVEEFRNPVVKIGGENLTI
MA H SNGFCV DPLNW AAAE LKGSHLDEVKRMVEEFRNPVVKIGGE LTI
Sb jct 1 MAAENGHEESNGFCVKQNDPLNWVAAAESLKGSHLDEVKRMVEEFRKPVVKLGGETLTI
Query 61 AQVAAIASRDNAVAVELAESARAGVKASSDWVMDSMNKGTDSYGVTTGFGATSHRRTKQGG
QVAAIA DNAVAVEL ES RAGVKASSDWVMDSM KGTDSYGVTTGFGATSHRRTKQGG
Sbjct 61 SQVAAIAAKDNAVAVELVESSRAGVKASSDWVMESMSKGTDSYGVTTGFGATSHRRTKQGG
Query 121 ALQKELIRFLNAGIFGKGTEGCHTLPHTATRAAMLVRINTLLQGYSGIRFEILEALTKFLN
ALQKELIRFLNAGIFGKGTE HTLPHTATRAAMLVRINTLLQGYSGIRFEILEALTKFLN
Sbjct 121 ALQKELIRFLNAGIFGNGTESNHTLPHTATRAAMLVRINTLLQGYSGIRFEILEAITKFLN
Query 181 HNITPCLPLRGTITASGDLVPLSYIAGLLTGRPNSKAVGPKGEELNAEEAFKLAGVSGGFF
NITPCLPLRGTITASGDLVPLSYIAGLLTGRPNSKAVGP GE LNAEEAFKLAGV GGFF
Sbjct 181 ENITPCLPLRGTITASGDLVPLSYIAGLLTGRPNSKAVGPNGEPLNAEEAFKLAGVKGGFF
Query 241 ELQPKEGLALVNGTAVGSGLASIALYEANVLALLAEVMSAIFAEVMNGKPEFTDHLTHKLK
ELQPKEGLALVNGTAVGSGLASIAL AN LA L EVMSA FAEVMNGKPEFTDHLTHKLK
Sbjct 241 ELQPKEGLALVNGTAVGSGLASIALFDANILAVLSEVMSAVFAEVMNGKPEFTDHLTHKLK
Query 241 HHPGQIEAAAIMEHILDGSAYVKAAQKMHEMDPLQKPKQDRYALRTSPQWLGPQIEVIRTA
HHPGQIEAAAIMEHILDGSAYVKAAQK HE DPLQKPKQDRYALRTSPQWLGPQIEVIRTA
Sbjct 241 HHPGQIEAAAIMEHILDGSGYVKAAQKLHEQDPLQKPKQDRYALRTSPQWLGPQIEVIRTA
Query 301 TKMIEREINSVNDNPLIDVSRNKAIHGGNFQGTPIGVSMDNTRLAIAAIGKLVFAQFSELV
TKMIEREINSVNDNPLIDVSRNKAIHGGNFQGTPIGVSMDN RLAIA IGKL FAQFSELV
Sbjct 301 TKMIEREINSVNDNPLIDVSRNKALHGGNFQGTPIGVSMDNARLAIASIGKLLFAQFSELV
Query 361 NDSYNNGLPSNLSGGRNPSLDYGFKGSEIAMASYCSELQFLANPVTNHVQSAEQHNQDVNS
ND YNNGLPSNLSGGRNPSLDYGFKGSEIAMASYCSELQFLANPVTNHVQSAEQHNQDVNS
Sbjct 361 NDLYNNGLPSNLSGGRNPSLDYGFKGSEIAMASYCSELQFLANPVTNHVQSAEQHNQDVNS
Query 421 LGLISSRKTVEALDILKLMSSTYLVALCQAVDLRHLEENLRLAVKNTVSQLAKRTLTMGAN
LGLISSRKTVEALDILKLMSSTYLVALCQAVDLRHLEENL AVKNTVSQ AKRTLTMG N
Sbjct 421 LGLISSRKTVEALDILKLMSSTYLVALCQAVDLRHLEENLKHAVKNTVSQVAKRTLTMGVN
Query 481 GELHPSRFCEKDLLRVVDREYVFAYIDDPCSANYPLMQKLRQVLVDHALKNGENEKNASTS
GELHPSRFCEKDLLRVVDREYVFAYIDDP SA YPLMQKLRQVLVDHALKNGEKNASTS
Sbjct 481 GELHPSRFCEKDLIRVVDREYVFAYIDDPSSATYPLMQKLRQVLVDHALKNGDLEKNASTS
Query 541 IFQKIEAFEEELKALLPKEVESARMALEAGNPAVPNRITECRSYPLYKFIREEAGTEFLTG
IFQKIEAFEEELKALLPKEV SARMALE P VPNRI ECRSYPLYKFIRE G FLTG
Sbjct 541 IFQKIEAFEEELKALLPKEVGSARMALESGSPTVANRIAECRSYPLYKFIREQLGAGFLTG
Query 601 EKVTSPGEECDKVFTALTNGFIIDSLLTCLEAWNGAPLPIC
EK SPGEECDKVFTAL NG IID LL CLWNG PLPIC
Sbjct 601 EKAVSPGEECEKVFTALSNGLIIDPLLECLQGWNGQPLPIC
Query: the aminoacid sequence of Sutellaria viscidula phenyl alanine ammonialyase
Sbjct: the aminoacid sequence of red sage root phenylalanine ammonia lyase (GenBank Accession No.ABR14606.1)
Table 2 is that the homology of the aminoacid sequence of Sutellaria viscidula phenyl alanine ammonialyase of the present invention and red sage root phenylalanine ammonia lyase compares (FASTA) table.Wherein, identical amino acid marks with the amino acid monocase between two sequences.
Invention also comprises the analogue of Sutellaria viscidula phenyl alanine ammonialyase protein or polypeptide.The difference of these analogues and Sutellaria viscidula phenyl alanine ammonialyase protein related polypeptide can be the difference on the aminoacid sequence, also can be the difference that does not influence on the modified forms of sequence, perhaps haves both at the same time.These polypeptide comprise natural or the inductive genetic variant.The induce variation body can obtain by various technology, as by radiation or be exposed to mutagenic compound and produce random mutagenesis, also can pass through site-directed mutagenesis method or the biological technology of other known moleculars.Analogue also comprises having the analogue that is different from the amino acid whose residue of natural L-(as D-amino acid), and has non-natural analogue that exist or synthetic amino acid (as β, gamma-amino acid).Should be understood that polypeptide of the present invention is not limited to the above-mentioned representational polypeptide that exemplifies.
(the not changing primary structure usually) form of modification comprises: the chemically derived form such as the acetylize or carboxylated of the polypeptide that body is interior or external.Modification also comprises glycosylation, carries out glycosylation modified and polypeptide that produce in the procedure of processing as those in the synthetic and processing of polypeptide or further.This modification can be carried out glycosylated enzyme (as mammiferous glycosylase or deglycosylating enzyme) and finishes by polypeptide is exposed to.Modified forms also comprises have the phosphorylated amino acid residue sequence of (as Tyrosine O-phosphate, phosphoserine, phosphothreonine).Thereby also comprise the polypeptide that has been improved its proteolysis performance or optimized solubility property by modifying.
In the present invention, can select various carrier known in the art for use, the carrier as commercially available comprises plasmid, clay etc.When producing Sutellaria viscidula phenyl alanine ammonialyase protein of the present invention, the encoding sequence of Sutellaria viscidula phenyl alanine ammonialyase gene operationally can be connected in expression regulation sequence, thereby form the Sutellaria viscidula phenyl alanine ammonialyase expression vector.
As used herein, " operationally being connected in " refer to a kind of like this situation, and promptly some part of linear DNA sequence can influence the activity of same other parts of linear DNA sequence.For example, if signal peptide DNA as precursor expression and participate in the secretion of polypeptide, signal peptide (secretion leader sequence) DNA operationally is connected in polypeptid DNA so; If transcribing of promotor control sequence, it is operationally to be connected in encoding sequence so; When if ribosome bind site is placed in the position that can make its translation, it is operationally to be connected in encoding sequence so.Generally, " operationally being connected in " means adjacent, then means in reading frame adjacent for the secretion leader sequence.
In the present invention, term " host cell " is an eukaryotic cell.Eukaryotic host cell commonly used comprises yeast cell, tobacco cell and other vegetable cell.
In addition, can be used as the nucleic acid molecule of probe among the present invention, this molecule has 8-100 continuous nucleotide of Sutellaria viscidula phenyl alanine ammonialyase gene coded sequence usually, preferably has 15-50 continuous nucleotide.This probe can be used for whether existing in the test sample nucleic acid molecule of the Sutellaria viscidula phenyl alanine ammonialyase of encoding.
The present invention relates to whether exist in the test sample method of Sutellaria viscidula phenyl alanine ammonialyase gene nucleotide series, it comprises with above-mentioned probe and sample and hybridizing whether detection probes combination has taken place then.Preferably, this sample is the product behind the pcr amplification, and wherein the pcr amplification primer is corresponding to Sutellaria viscidula phenyl alanine ammonialyase gene nucleotide encoding sequence, and can be positioned at the both sides or the centre of this encoding sequence.Primer length is generally 15-50 Nucleotide.
Sutellaria viscidula phenyl alanine ammonialyase gene nucleotide full length sequence of the present invention or its fragment can obtain with the method for pcr amplification method, recombination method or synthetic usually.For the pcr amplification method, can be disclosed according to the present invention relevant nucleotide sequence design primer, and with commercially available cDNA storehouse or by the prepared cDNA storehouse of ordinary method well known by persons skilled in the art as template, amplification and must be about sequence.When sequence is longer, usually needs to carry out twice or pcr amplification repeatedly, and then the fragment that each time amplifies is stitched together by proper order.
In case obtained relevant sequence, just can obtain relevant sequence in large quantity with recombination method.This normally is cloned into carrier with it, changes cell again over to, separates obtaining relevant sequence then from the host cell after the propagation by ordinary method.
In addition, also can will suddenly change and introduce in the protein sequence of the present invention by chemosynthesis.
Except producing with recombination method, the also available solid phase technique of the proteic fragment of the present invention is produced (people such as Stewart, (1969) Solid-Phase Peptide Synthesis, WH Freeman Co., SanFrancisco by direct peptide synthesis; Merrifield J. (1963) J.Am Chem.Soc 85:2149-2154).Can carry out by hand or automatically at external synthetic protein.For example, can (Foster City CA) synthesizes peptide automatically with the 431A type peptide synthesizer of Applied Biosystems.Can distinguish proteic each fragment of chemosynthesis the present invention, be connected to produce the molecule of total length with chemical process then.
Utilize Sutellaria viscidula phenyl alanine ammonialyase of the present invention,, can filter out the interactional material of relevant generation with Sutellaria viscidula phenyl alanine ammonialyase, perhaps acceptor, inhibitor or short of money dose etc. by various conventional screening methods.
The present invention has tangible effect on raising Sutellaria viscidula flavonoid content.Improve the output of this compound, to satisfy the great demand of the whole world to medicines such as baicalins.Therefore, the present invention has very big using value.
Embodiment
Below in conjunction with the concrete testing data in laboratory and in conjunction with specific embodiments, further set forth the present invention.These embodiment only are used to the present invention is described and are not used in and limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, usually according to normal condition, " molecular cloning: laboratory manual " (New York:ColdSpring Harbor Laboratory Press such as Sambrook for example, 1989) condition described in, or the condition of advising according to manufacturer.
Embodiment 1
The clone of Sutellaria viscidula phenyl alanine ammonialyase
1. separate tissue (isolation)
Sutellaria viscidula is bought from University Of Shanxi, and planting seed in the greenhouse, is treated that the Sutellaria viscidula seedling grows to 10cm when high, prepares DNA extraction or RNA.
2.RNA separation (RNA isolation)
Get portion of tissue, grind, add the 1.5mL EP pipe that fills lysate, fully after the vibration, move in the glass homogenizer again with mortar.Move to after the homogenate in the 1.5mL EP pipe, and extracted total RNA (TRIzol Reagents, GIBCO BRL, USA).Identify total RNA quality with the denaturing formaldehyde gel electrophoresis, on spectrophotometer, measure rna content then.
3. the full-length clone of gene (Cloning of Full-length cDNA)
According to the Nucleotide conserved sequence of the phenylalanine ammonia lyase gene of each kind of plant, utilize homologous genes clone principle, adopt RACE method (Clontech test kit) to carry out the cDNA full-length clone, divide three phases to carry out:
(1) the pulsating clone of core
PCR[BP001[5 '-AAGGC (G/T) AG (C/T) AGTGA (C/T) TGGGT (C/G) A-3 ']+BP002[5 '-AACCC (T/A/C) TTGAT (T/C) GA (T/C) GTTTC (C/A) AG-3 ']] the core segment of 594bp obtained, be connected to after the recovery on the pGEMT-Easy carrier, with SP6 or T7 as universal primer, adopt and stop thing fluorescent mark (Big-Dye, Perkin-Elmer, USA) method, (Perkin-Elmer checks order on USA) at the ABI377 sequenator.Sequencing result GCG software package (Wisconsin group, USA) BLAST in and the existing database of FASTA software search (Genebank+EMBL), the homology of PAL gene of knowing its nucleotide sequence and proteins encoded and the known root of large-flowered skullcap is very high, so think that tentatively it is a phenylalanine ammonia lyase gene.
(2)3’-RACE
First round PCR[AP+BP003 (5 '-AAGTTATGAATGGGAAGCCTGAG-3 ')] take turns PCR[AP+BP004 (5 '-CGAGAGGGAGATCAACTCGGTG-3 ') together with second] obtain 1172bp3 ' terminal sequence, reclaim, be connected on the pGEMT-Easy carrier, with SP6 or T7 as universal primer, adopt thing fluorescent mark (Big-Dye, Perkin-Elmer, method USA) of stopping, (Perkin-Elmer checks order on USA) at the ABI377 sequenator.Sequencing result GCG software package (Wisconsin group, USA) BLAST in and the existing database of FASTA software search (Genebank+EMBL), the homology of PAL gene of knowing its nucleotide sequence and proteins encoded and the root of large-flowered skullcap is very high, so think that tentatively it is a phenylalanine ammonia lyase gene.
(3)5’-RACE
First round PCR[UMP+BP005 (5 '-CAGAGTGTGGCAACCCTCCGTC-3 ')] take turns PCR[(NUP+BP006 (5 '-CACGGAGGGGCAAGCATGGAGT-3 ') together with second] obtain 446bp5 ' terminal sequence, reclaim, be connected on the pGEMT-Easy carrier, with SP6 or T7 as universal primer, adopt thing fluorescent mark (Big-Dye, Perkin-Elmer, method USA) of stopping, (Perkin-Elmer checks order on USA) at the ABI377 sequenator.Sequencing result GCG software package (Wisconsin group, USA) BLAST in and the existing database of FASTA software search (Genebank+EMBL), the homology of PAL gene of knowing its nucleotide sequence and proteins encoded and the red sage root is very high, so think that tentatively it is a phenylalanine ammonia lyase gene.
Overlap splicing with sequencing result, obtain the full length fragment sequence information, and design a pair of special primer and carry out pcr amplification PAL coding region (BP007F1 (5 '-ATGGCGCCCGCTGTCGAAGTCA-3 ')+BP007R1 (5 '-GCAGATGGGAAGAGGTGCACCA-3 ')) obtain the coding region (2133bp) of PAL.
The gene that result's proof of BLAST newly obtains from Sutellaria viscidula really is a phenylalanine ammonia lyase gene.Because known homology phenylalanine ammonia lyase gene has the function that the flavanonol reduction is generated leucoanthocyanidin, so infer that this gene has identical functions.
By being used in combination above-mentioned 3 kinds of methods, obtained the complete encoding sequence of candidate's Sutellaria viscidula phenyl alanine ammonialyase.Obtain on the total length basis of (comprising complete open reading frame) in splicing, further F1:5 '-ACGCGGGGAGGATCCCCTCTCT-3 ' (SEQ ID NO.1) is a forward primer to design primer BP008, BP009 R1:5 '-ACGCGGGGAGGATCCCCTCTCT-3 ' (SEQID NO.2) is a reverse primer, with total RNA is template, carry out the RT-PCR amplification, the PCR condition of F1/R2 is: 94 ℃ of pre-sex change 2min; 94 sex change 45s, 55 annealing 45s, 72 extend 2min then; 30 circulations; Last 72 extend 10min; Electrophoresis detection PCR product, obtaining the amplification fragment length is 1283bp.Clone with pcr amplification product according to a conventional method then, order-checking obtains the sequence shown in the SEQ IDNO.3.
Embodiment 2
Sutellaria viscidula phenyl alanine ammonialyase gene order information and homology analysis
The length of the phenylalanine ammonia lyase gene order full-length cDNA that the present invention is new is 2406bp, and detailed sequence is seen SEQID NO.3, and wherein open reading frame is positioned at 129-2261 position Nucleotide (2133 Nucleotide).Derive the Sutellaria viscidula phenyl alanine ammonialyase aminoacid sequence according to full-length cDNA, totally 711 amino-acid residues, molecular weight is 77.18kDa, iso-electric point (PI) is 5.97.Detailed sequence is seen SEQ ID NO.4.
The full length cDNA sequence and the coded protein thereof of Sutellaria viscidula phenyl alanine ammonialyase gene gene-correlation are carried out Nucleotide and protein homology retrieval with blast program in Non-redundant GenBank+EMBL+DDBJ+PDB and Non-redundant GenBankCDS translations+PDB+SwissProt+Superdate+PIR database, found that it and red sage root phenylalanine ammonia lyase gene gene order have 90% homogeny (subordinate list 2) on nucleotide level; On amino acid levels, it and red sage root phenylalanine ammonia lyase gene PAL have 81% homogeny, (subordinate list 3) on protein level.This shows, all there are higher homology in Sutellaria viscidula phenyl alanine ammonialyase gene and red sage root phenylalanine ammonia lyase gene on nucleic acid still is protein level, so can think that Sutellaria viscidula phenyl alanine ammonialyase also has similar effect on the flavonoid compound biosynthetic pathway.
Embodiment 3
Sutellaria viscidula phenyl alanine ammonialyase protein or polypeptide carry out the detection of content of baicalin in eukaryotic cell expression and the transfer-gen plant in Sutellaria viscidula.
The structure that contains the expression vector of goal gene, according to Sutellaria viscidula phenyl alanine ammonialyase gene coded sequence (SEQID NO.3), design amplifies the primer that complete coding is read frame, and on the upstream and downstream primer, introduce restriction endonuclease sites (this is decided by the carrier of selecting for use) respectively, so that construction of expression vector.Amplified production with acquisition among the embodiment 1 is a template, behind pcr amplification, with the Sutellaria viscidula phenyl alanine ammonialyase gene cDNA clone to intermediate carrier (as pBluescript), further be cloned into binary expression vector (as pBI121 and improved pCAMBIA1304), guaranteeing to identify good expression vector under the correct prerequisite of reading frame, again it is changed in the Agrobacterium, utilize leaf dish law technology to transform Sutellaria viscidula.
1. send out seedling: take the sophisticated seed of Sutellaria viscidula, with 0.1% (M/V) mercuric chloride (HgCl
2) solution soaking 5 minutes, with aseptic water washing 6 times; The Sutellaria viscidula seed is seeded in (substratum was contained in the triangular flask of 150ml, in 1210C sterilization 20 minutes) on the seed germination substratum, and this culture medium prescription is: the MS minimum medium, add 30gL
-1Sucrose, regulating the medium pH value is 5.8, adds 5% agar powder again.Cultivate the young shoot of Sutellaria viscidula in illumination box, culture condition is: 25 ℃, dark condition is cultivated down.After treating that seed is sprouted, the change culture condition is: 25 ℃, and illumination in 12 hours, intensity of illumination is 25 μ molm
-2S
-1By the time the plant length of a film can be used for genetic transformation when high to 3cm.
2. transform altogether and cultivate: the young leaflet tablet of Sutellaria viscidula aseptic seedling is whittled into about 1cm with small blade
2Putting into prior cultured bacterium liquid (OD value 0.4-0.6) infected 3-5 minute.Then take out and put into the dark cultivation of common substratum 2 days.
3. degerming is cultivated: after cultivating end altogether, explant is put into the degerming culture medium culturing.4 all subcultures once.After treating that end is cultivated in degerming, change the resistance screening substratum over to and carry out transgenosis hairly root preliminary screening.
Remove bacterium culture medium: MS+cb (250mgL
-1)
Screening culture medium: MS+hygr (30mgL
-1)
4. the isolated culture of hairly root: downcut when long when hairly root grows to about 4cm, do not containing on the MS substratum of any plant hormone succeeding transfer culture in the constant incubator of 25 ± 1.0 ℃ of unglazed photographs.Per three all subcultures once, through carrying out liquid fermentation and culture behind the succeeding transfer culture several times, further to carry out Molecular Detection.
5. the Molecular Detection of transgenosis Sutellaria viscidula
All (Sambrook is J.etal1993) according to the Ri plasmid gene sequential analysis design rolB of Furner etc., the Auele Specific Primer of rolC gene with reference to " molecular cloning experiment guide " for the extraction of hairly root DNA and purifying.The a pair of primer of rolB gene is: 5 '-GCTCTTGCAGTGCTAGATTT-3 ' and 5 '-GAAGGTGCAAGCTACCTCTC-3 '.The a pair of primer of rolC gene is 5 '-CTCCTGACATCAAACTCGTC-3 ' and 5 '-TGCTTCGAGTTATGGGTACA-3 '.The clip size of rolB, rolC gene is 423bp and 626bp respectively.Simultaneously gene being carried out PCR detects.
6. the northern blot of phenylalanine ammonia lyase in the Sutellaria viscidula hairly root analyzes
1) extraction of RNA: (GIBCO BRL USA) extracts and the preparation chapters and sections (Sambrook etc., 1989) of the relevant RNA of reference " molecular cloning experiment guide " extract each monoclonal RNA of Sutellaria viscidula transgenosis hairly root to utilize the TRIzol test kit.
2) RNA's is quantitative: with reference to " molecular cloning experiment guide " (Sambrook etc., 1989), and spectrophotometric instrumentation OD
260Rna content calculates: 1OD
260=40 μ gml
-1
3) total RNA agarose gel electrophoresis separates: 1. get 6ml 25 * (doubly) electrophoretic buffer, add the 117ml sterilized water, mixing.2. take by weighing the 1.5g agarose, join in the above-mentioned solution, heating and melting in microwave oven changes in 55 ℃ of water-baths.3. in stink cupboard, get 26.8ml formaldehyde, join in 55 ℃ the gelating soln mixing.4. pour into rapidly in the glue plate, room temperature water placing flat 30 minutes treats that gelling is solid.5. the RNA (20 μ g) that extracts is dissolved in the RNA denaturing soln, heated 10 minutes down, be placed on ice immediately then at 65 ℃.6. in sample, add 2ul 10 * sample-loading buffer, mixing.7. do not cover point sample under the condition of glue in electrophoresis liquid, 5V/cm voltage electrophoresis is about 5 hours.
4) shift on the RNA nylon membrane: before 1. shifting, nylon membrane is soaked with 10 * SSC.2. moistening film is covered exactly on film, two filter paper identical with film size are put in 2 * SSC solution moistening, cover on film, get rid of bubble.3. put one on the filter paper and fold and the identical thieving paper of film size, put a sheet glass and a weight on thieving paper, horizontal positioned shifted 12-20 hour.4. after shifting, film was toasted 2 hours in 80 ℃.
5) the detecting of hybridization signal on the film: 1. film is immersed in 5 * Dendart ' s, 0.1% SDS, 0.1mgml
-1Salmon sperm dna, 65 ℃ of following prehybridizations 2 hours.2. will use Gene Images
TMContents CDP-Star
TMThe sex change 5 minutes in boiling water of the probe of labelling module mark directly adds in the hybridization solution 1., in 65 ℃ of hybridization 16-24 hour.3. take out film, place among the film washing liquid I (1 * SSC, 1% SDS), in 65 ℃ of rinsings 3 times, each 15 minutes.Change among the film washing liquid II (0.1 * SSC, 1% SDS) in 65 ℃ of rinsings 3 times each 15 minutes over to.4. use X-ray sheet compressing tablet 60-90 minute, then development, photographic fixing (method is with reference to Roche DIG labeled test kit specification sheets).
7. the HPLC that changes the Sutellaria viscidula hairly root baicalin of phenylalanine ammonia lyase analyzes
The extraction of baicalin: the mono-clonal hairly root that will cultivate 30d is cleaned, and uses the thieving paper suck dry moisture, claims fresh weight postlyophilization 30h to constant weight, pulverize, take by weighing 100mg dry powder and add 10mL methyl alcohol ultrasonication 20min, filter, extract once again, merging filtrate, methanol extract liquid is concentrated evaporate to dryness, with methanol constant volume to 10ml ,-20 ℃ of preservations, as for test agent solution, can be used for HPLC and analyze.
The preparation of high-pressure liquid phase examination criteria product: precision takes by weighing 2mg baicalin standard reference material, filters with the chromatographically pure dissolve with methanol, and being made into concentration is 1000 μ gmL
-1, gradient dilution becomes 280,140,70,35,17.5 μ gmL respectively
-1Sample introduction under corresponding chromatographic condition, and each concentration sample introduction three times, record collection of illustrative plates and chromatographic parameter, respectively with peak area to the sample size regression analysis, the equation of linear regression and the typical curve of reference substance.
The high-pressure liquid phase testing conditions: chromatographic column is Symmetry-C
18Post (4.6mm * 150mm, 5 μ m), mobile phase methanol: 0.2molL
-1Sodium dihydrogen phosphate (45: 55), flow velocity 0.8mLmin
-1, 25 ℃ of column temperatures.Detect wavelength: 280nm.Baicalin goes out the peak about the 7.7min place greatly.
Sequence table
SEQ ID NO.1
＜110〉Southwestern University
＜120〉Sutellaria viscidula phenyl alanine ammonialyase protein coded sequence
<140>
<141>
<160>4
<170>PatentIn version 3.1
<210>1
<211>22bp
<212>DNA
＜213〉Sutellaria viscidula (Scutellaria viscidula Bunge)
<400>1
SEQ ID NO.2
<210>2
<211>22bp
<212>DNA
＜213〉Sutellaria viscidula (Scutellaria viscidula Bunge)
<400>2
SEQ ID NO.3
<210>3
<211>2406
<212>DNA
＜213〉Sutellaria viscidula (Scutellaria viscidula Bunge)
<400>3
SEQ ID NO.4
<210>4
<211>711
<212>PRT
＜213〉Sutellaria viscidula (Scutellaria viscidula Bunge)
<400>4
Claims (4)
1, a kind of Sutellaria viscidula phenyl alanine ammonialyase protein coded sequence, coding has the nucleotide sequence of the active polypeptide of phenylalanine ammonia lyase, it is characterized in that described nucleotide sequence is made of nucleotide sequence or its degenerate sequence from Nucleotide 129-2261 position among the SEQ ID NO.3.
2, Sutellaria viscidula phenyl alanine ammonialyase protein coded sequence according to claim 1, it is characterized in that, described degenerate sequence is meant the encoder block 129-2261 position Nucleotide that is arranged in SEQ ID NO.3 sequence, and having one or more codons to be encoded, the degenerate codon of same amino acid replaces the back and the sequence that produces.
3, Sutellaria viscidula phenyl alanine ammonialyase protein coded sequence according to claim 1 and 2 is characterized in that, described sequence encoding has the polypeptide of the aminoacid sequence shown in the SEQ ID NO.4.
4, Sutellaria viscidula phenyl alanine ammonialyase protein coded sequence transformed host cells according to claim 3 is characterized in that, described host cell is an eukaryotic cell.
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Cited By (3)
| 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 |
| CN104131017A (en) * | 2014-07-28 | 2014-11-05 | 江南大学 | Rhodotorula glutinis phenylalanine deaminase gene and application thereof |
-
2009
- 2009-04-17 CN CN200910103635A patent/CN101532026A/en active Pending
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 |
| 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 |
| CN104131017A (en) * | 2014-07-28 | 2014-11-05 | 江南大学 | Rhodotorula glutinis phenylalanine deaminase gene and application thereof |
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