CN101168740B - Scopoloa acutangula tropinone reductase I gene and its coding protein and application - Google Patents
Scopoloa acutangula tropinone reductase I gene and its coding protein and application Download PDFInfo
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Abstract
The invention discloses anisodus tropinone reductase I gene and the protein and the use of the code thereof, and fills the blank of separating and cloning the tropinone reductase I gene from unique medicinal plants in Yunnan, China. The anisodus tropinone reductase I gene provided by the invention has nucleotide sequence shown in SEQ ID No.1, and the protein of the gene code has amino acid sequence shown in SEQ ID No.2. The anisodus tropinone reductase I gene provided by the invention has prominent effect for improving the content of tropane alkaloid in anisodus plants, etc. through an gene engineering technology, thereby being widely applied to the qauality improvement of resource plants producing tropane alkaloid.
Description
Technical field
The invention belongs to biological technical field, specifically, the acutangula tropinone reductase I gene that relates in Radix Anisodi Acutanguli, express and encoded protein matter and application.
Background technology
Tropane alkaloid such as tropine (hyoscyamine) and Scopolamine (scopolamine) etc., mainly from plant of Solanaceae such as belladonna, thorn apple, henbane and Radix Anisodi Acutanguli (Anisodus scutangulus) etc., extract, aspect medical is the anticholinergic drug that acts on parasympathetic nervous system, has anesthesia, spasmolysis, lenitive function.In addition, also have the effect of microcirculation improvement, can be used for treating diseases with microcirculatory disturbance clinically.Because the effort of Chinese scholar, the clinical application of tropane alkaloid spreads all over internal medicine, surgery, Obstetric and Gynecologic Department, neurology department, Dermatology Department, Otorhinolaryngologic Department etc., can treat more than 100 kind of disease, and the market requirement is very huge.
The medicinal plant Radix Anisodi Acutanguli is the Solanaceae per nnial herb, mainly is distributed in the Yunnan Province of China northwestward, in Yunnan among the people it use with a long history as the Chinese medicine of spasmolysis and analgesia.The Radix Anisodi Acutanguli root is rich in tropane alkaloid, it is reported, wild Radix Anisodi Acutanguli dry product total alkaloid content is up to 1.2%, and contained alkaloid is than all high many of plant of Solanaceae more commonly used such as belladonna, henbane thorn apple etc.According to record, living Radix Anisodi Acutanguli total alkaloid content can be up to 5% in 7 years.Experiment shows that the contained alkaloid overwhelming majority of Radix Anisodi Acutanguli is a tropane alkaloids.Along with the demand of market to tropane alkaloid constantly enlarges, the alternative method that searching can the mass production tropane alkaloid has become the focus of current research.The develop rapidly of genetic engineering technique and widespread use in recent years opened up a brand-new approach for utilizing modern biotechnology to improve the tropane alkaloid.Utilizing modern biotechnology that the key gene in the tropine biosynthetic pathway is imported in the resource plant, obtain transfer-gen plant, and cultivate on a large scale, is to realize one of optimal path that fundamentally improves tropane alkaloid content.
Acutangula tropinone reductase I (Tropinone reductase I, TRI) be the katalaze enzyme of a key in the whole tropine biosynthetic pathway, it and acutangula tropinone reductase II (Tropinone reductase II) distinguish two branches of an important branch point in the catalysis tropine building-up process.Wherein acutangula tropinone reductase I (TRI) thus the single-minded 3-ketone group with tropinone is reduced into 3 Alpha-hydroxies to be generated tropine (3 α-hydroxytropane) generates false tropine (3 β-hydroxytropane) thereby TRII then is reduced into the 3-ketone group of tropinone 3 beta-hydroxies.Tropine finally generates tropine and Scopolamine along TRI main road approach, and false tropine then finally generates Calystegines along another collateral branch's approach.The vigor ratio of these two enzymes will determine the metabolism stream flow direction at this important branch point because present in plant materials the change of tropine and false tropine also be not found.Therefore, the encoding gene of clone's acutangula tropinone reductase I also utilizes genetic engineering technique to improve acutangula tropinone reductase I (Anisodus acutangulusTropinone reductase I in medicinal plant such as the Radix Anisodi Acutanguli etc., AaTRI) activity or content, thereby the content of tropine and Scopolamine has crucial medical value in raising transgenic plant such as the Radix Anisodi Acutanguli.
In analysis to existing document, though " The Proceedings of the National Academyof Sciences of the United States of American (NAS's progress) 1993; 90; 9591-9595 " reported and cloned acutangula tropinone reductase I gene that from thorn apple any bibliographical information that clones and isolates acutangula tropinone reductase I from the distinctive medicinal plant Radix Anisodi Acutanguli of Yunnan Province of China is not arranged so far as yet.Because the enzyme of this genes encoding flows to tropine for metabolism stream and has direct decisive action, therefore, this step is to utilize genetic engineering technique to regulate and control tropine and the biosynthetic important point of penetration of Scopolamine.
Summary of the invention
Technical problem to be solved by this invention provides a kind of Scopoloa acutangula tropinone reductase I gene and encoded protein matter and purposes, to fill up the blank that clones and isolates acutangula tropinone reductase I gene from the distinctive medicinal plant Radix Anisodi Acutanguli of Yunnan Province of China.
Scopoloa acutangula tropinone reductase I gene provided by the present invention is homologous sequence or its allelotrope and the deutero-nucleotide sequence thereof that has the nucleotide sequence shown in the SEQ ID No.1 or add, replace, insert or delete one or more Nucleotide.
The protein of Scopoloa acutangula tropinone reductase I genes encoding provided by the present invention is to have the aminoacid sequence shown in the SEQ IDNo.2 or add, replace, insert or delete one or more amino acid whose homologous sequences.
Contain Scopoloa acutangula tropinone reductase I gene complete sequence of the present invention or the segmental plasmid of part and plant expression vector and all belong to protection scope of the present invention.
A kind of host cell, this cell contain the gene order of Scopoloa acutangula tropinone reductase I gene of the present invention or plasmid or plant expression vector.
Described host cell is Bacillus coli cells, agrobatcerium cell, yeast cell, tobacco cell or other vegetable cell.
Preferred Bacillus coli cells of described host cell or agrobatcerium cell or Radix Anisodi Acutanguli root of hair cell.
The application of Scopoloa acutangula tropinone reductase I gene of the present invention comprises with described plant expression vector transforming the Radix Anisodi Acutanguli cell or providing a kind of transgenosis Radix Anisodi Acutanguli with described Agrobacterium and Radix Anisodi Acutanguli co-culture of cells or with described Radix Anisodi Acutanguli root of hair cell culture male sterile plants or with described acutangula tropinone reductase I gene order.
The notion particular content that relates in the technical solution of the present invention is as follows:
The dna molecular of the said Scopoloa acutangula tropinone reductase I of the present invention gene comprises: coding has the nucleotide sequence of the active polypeptide of Scopoloa acutangula tropinone reductase I, and shows at least 70% homology from the nucleotides sequence of Nucleotide 70-891 position among described nucleotide sequence and the SEQ ID NO.1; Perhaps described nucleotide sequence can be under 40-55 ℃ of condition with SEQ ID NO.1 in from the nucleotide sequence hybridization of Nucleotide 70-891.Preferably, described sequence encoding has the polypeptide of the aminoacid sequence shown in the SEQ ID NO.2.More preferably, described sequence has among the SEQ ID NO.1 nucleotide sequence from Nucleotide 70-891 position.
The isolated Scopoloa acutangula tropinone reductase I of the present invention polypeptide comprises: polypeptide or its conservative property variation polypeptide or its active fragments or its reactive derivative with SEQ ID NO.2 aminoacid sequence.Preferably, this polypeptide is to have SEQ ID NO.2 polypeptide of sequence.
Dna molecular among the present invention comprises 8-100 continuous nucleotide in the described dna molecular.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.
Term among the present invention " Scopoloa acutangula tropinone reductase I (or polypeptide) gene " refers to: coding has the nucleotide sequence of the active polypeptide of Scopoloa acutangula tropinone reductase I, as 70-891 position nucleotide sequence and degenerate sequence thereof among the SEQ ID NO.1.This degenerate sequence is meant: be arranged in the encoder block 70-891 position Nucleotide of SEQ ID NO.1 sequence, 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.1 in 70-891 position nucleotide sequence homology be low to moderate about 70% the degenerate sequence described sequence of SEQ ID NO.2 of also encoding out.Also comprising can be under the rigorous condition of moderate, better under highly rigorous condition with SEQ ID NO.1 in from the nucleotide sequence of the nucleotide sequence hybridization of Nucleotide 70-891 position.Also comprise with SEQ ID NO.1 in from the homology of nucleotide sequence at least 70% of Nucleotide 70-891 position, preferably at least 80%, more preferably at least 90%, at least 95% nucleotide sequence best.Also comprising to encode has the variant form of open reading frame sequence among the proteic SEQ ID NO.1 with natural Scopoloa acutangula tropinone reductase I 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.
Term among the present invention " Scopoloa acutangula tropinone reductase I albumen or polypeptide " refers to: have the active SEQ ID of Scopoloa acutangula tropinone reductase I NO.2 polypeptide of sequence.This term also comprises the variant form that has with the SEQ ID NO.2 sequence of natural Scopoloa acutangula tropinone reductase I identical function.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 active fragments and the reactive derivative of Scopoloa acutangula tropinone reductase I, also comprises operationally being connected in the derivative that signal peptide, promotor or ribosome bind site sequence are formed.
The variant form of Scopoloa acutangula tropinone reductase I polypeptide of the present invention comprises: homologous sequence, conservative property varient, allelic variant, natural mutation, induced mutation body, under high or low rigorous condition can with the coded albumen of the DNA of Scopoloa acutangula tropinone reductase IDNA hybridization and the polypeptide or the albumen that utilize the serum of Scopoloa acutangula tropinone reductase I polypeptide to obtain.
Scopoloa acutangula tropinone reductase I conservative property variation polypeptide refers among the present invention: compare with the aminoacid sequence of SEQ ID NO.2, 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.
Replacement residue in the table 1. conservative property variation polypeptide
| 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 |
The present invention also comprises the analogue of Scopoloa acutangula tropinone reductase I or polypeptide, the difference of these analogues and natural acutangula tropinone reductase I 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 have 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.Described modification (not changing primary structure usually) form 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 Scopoloa acutangula tropinone reductase I polypeptide of the present invention, the nucleotide sequence of Scopoloa acutangula tropinone reductase I gene operationally can be connected in expression regulation sequence, thereby form Scopoloa acutangula tropinone reductase I expression vector.Described " operationally being connected in " refers 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.
Host cell is prokaryotic cell prokaryocyte or eukaryotic cell among the present invention.Prokaryotic host cell commonly used comprises intestinal bacteria; Eukaryotic host cell commonly used comprises yeast cell, tobacco cell and other vegetable cell.
Existence and the quantity of rna transcription thing in cell of Scopoloa acutangula tropinone reductase I is promptly analyzed in the expression of the also available Northern blotting of the present invention technical Analysis Scopoloa acutangula tropinone reductase I gene product.
In addition, the nucleic acid molecule that can be used as probe among the present invention has 8-100 continuous nucleotide of Scopoloa acutangula tropinone reductase I nucleotide coding sequence usually, preferably has 15-50 continuous nucleotide.This probe can be used for whether existing in the test sample nucleic acid molecule of coding Scopoloa acutangula tropinone reductase I.
The present invention relates to whether exist in the test sample method of Scopoloa acutangula tropinone reductase I nucleotide sequence, 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 Scopoloa acutangula tropinone reductase I nucleotide coding sequence, and can be positioned at the both sides or the centre of this encoding sequence.Primer length is generally 15-50 Nucleotide.
In addition, according to Scopoloa acutangula tropinone reductase I nucleotide sequence of the present invention and aminoacid sequence, can be on the homology basis of nucleic acid homology or marking protein, screening Scopoloa acutangula tropinone reductase I source gene or homologous protein.
In order to obtain the dot matrix with the Radix Anisodi Acutanguli cDNAs of Scopoloa acutangula tropinone reductase I gene-correlation, can screen Radix Anisodi Acutanguli cDNA library with dna probe, these probes are under low rigorous condition, with 32P Scopoloa acutangula tropinone reductase I gene all or part of cooked the radioactivity mark and.The cDNA library that most is suitable for screening is the library from Radix Anisodi Acutanguli.Structure is that biology field is well-known from the method in the cDNA library of interested cell or tissue.In addition, many such cDNA libraries also can buy, for example available from Clontech, and Stratagene, Palo Alto, Cal..This screening method can be discerned the nucleotide sequence with the gene family of Scopoloa acutangula tropinone reductase I.
Scopoloa acutangula tropinone reductase I 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 about nucleotide sequence, especially open reading frame sequence designs 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 relevant 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-PhasePeptide Synthesis, WH Freeman Co., San Francisco 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 Scopoloa acutangula tropinone reductase I of the present invention,, can filter out with Scopoloa acutangula tropinone reductase I interactional material takes place, perhaps acceptor, inhibitor or short of money dose etc. by various conventional screening methods.
Acutangula tropinone reductase I gene provided by the invention is to clone preparation first from Radix Anisodi Acutanguli, can be used for improving the content of tropane alkaloid in the plants such as Radix Anisodi Acutanguli by genetic engineering technique, transgene result shows, acutangula tropinone reductase I gene has remarkable effect for improving by the content of tropane alkaloid in the plants such as genetic engineering technique promotion Radix Anisodi Acutanguli, can be widely used in producing the quality-improving of the alkaloidal resource plant of tropane.
Embodiment
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used to the present invention is described and be not used in and limit the scope of the invention.Should be understood that in addition those skilled in the art can make various changes or modifications the present invention after the content of having read the present invention's instruction, these equivalent form of values fall within the application's appended claims institute restricted portion equally.
The experimental technique of unreceipted actual conditions in the following example, usually according to normal condition, for example the Sambrook equimolecular is cloned: laboratory manual (New York:Cold Spring Harbor LaboratoryPress, 1989) condition described in, or the condition of advising according to manufacturer.
Embodiment 1 (clone of Scopoloa acutangula tropinone reductase I gene)
1. separate tissue (isolation)
The Radix Anisodi Acutanguli plant derives from the Yunnan Lijing, takes the children to place the freezing preservation of liquid nitrogen immediately after tender.
2.RNA separation (RNA isolation)
Get portion of tissue and 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 TRI amino acid conserved sequence of henbane and other plant of Solanaceae, the design degenerated primer utilizes homologous genes clone principle, adopts Smart-RACE method (Clonetech test kit) to carry out the cDNA full-length clone, divides three phases to carry out:
(1)3′-RACE
PCR (UPM+F2) obtains AaTRIF2 ' (540bp), reclaims, and is connected on the T-Easy carrier,, adopt to stop thing fluorescent mark (Big-Dye, Perkin-Elmer as universal primer with SP6 or T7, USA) method, (Perkin-Elmer checks order on USA) at ABI 377 sequenators.Sequencing result GCG software package (Wisconsin group, USA) BLAST in and the existing database of FASTA software search (Genebank+EMBL), the homology of knowing its nucleotide sequence and proteins encoded and known acutangula tropinone reductase I gene (as henbane acutangula tropinone reductase I gene etc.) is very high, so think that tentatively it is an acutangula tropinone reductase I gene.
(2)5′-RACE
According to 3 ' RACE result, design reverse special primer R2, obtain AaTRIR2 ' (722bp) (process is with (1)) through PCR (UPM+R2).Reclaim, be connected on the T-Easy carrier, with SP6 or T7 as universal primer, adopt stop the thing fluorescent mark (Big-Dye, Perkin-Elmer, method USA), (Perkin-Elmer checks order on USA) at ABI 377 sequenators.Sequencing result and 3 ' RACE result than preface and splice, are obtained the full length fragment sequence.
(3) with 5 ' RACE sequencing result and 3 ' RACE sequencing result than preface and splice, obtain the full length fragment sequence information, and design a pair of special primer and carry out pcr amplification AaTRI coding region (AaTRIKF1+AaTRIKR1) and obtain AaTRI coding region (822bp) (the same step of process (1)).
The gene that result's proof of BLAST newly obtains from Radix Anisodi Acutanguli really is an acutangula tropinone reductase I gene.Because deriving from the acutangula tropinone reductase I gene of thorn apple, known homologous has the function (Ute Richter etc., 2005) that improves tropane alkaloid, so infer that this gene has identical functions.
By being used in combination above-mentioned 3 kinds of methods, obtained candidate's the proteic complete encoding sequence of Radix Anisodi Acutanguli AaTRI.Obtain on the total length basis of (comprising complete open reading frame at least) in splicing, further AaTRIF1:5 '-AAAGGAAAACCAATTGTCACATTTG-3 ' (SEQ ID NO.3) is a forward primer to the design primer, oligonucleotide AaTRIR1:5 '-ATGCTAACTATTTGCGACATTTTA-3 ' (SEQ ID NO.4) is a reverse primer, with total RNA is template, carry out the RT-PCR amplification, the PCR condition of F1/R2 be 94 ℃ 5 minutes, carried out 35 circulations in 2 minutes with 94 ℃ 1 minute, 60 ℃ 1 minute and 72 ℃ thereupon, extended 10 minutes with 72 ℃ at last.The electrophoresis detection pcr amplification product, the acquisition expanding fragment length is 1168bp.Clone, check order with pcr amplification product according to a conventional method then, obtain the sequence shown in the SEQ ID NO.1.
Embodiment 2 (sequence information and the homology analysis of Scopoloa acutangula tropinone reductase I gene)
The length of the Scopoloa acutangula tropinone reductase I full-length cDNA that the present invention is new is 1168bp, and detailed sequence is seen SEQ ID NO.1, and wherein open reading frame is positioned at 70-891 position Nucleotide.Derive the aminoacid sequence of Scopoloa acutangula tropinone reductase I according to full-length cDNA, totally 273 amino-acid residues, molecular weight 29.463KD, pI are 6.45, detailed sequence is seen SEQ ID NO.2.
Full length cDNA sequence and the coded protein thereof of Scopoloa acutangula tropinone reductase I are carried out Nucleotide and protein homology retrieval with blast program in Non-redundant GenBank+EMBL+DDBJ+PDB and Non-redundant GenBank CDStranslations+PDB+SwissProt+Superdate+PIR database, found that it and henbane TRI gene (GenBank Accession No.D88156) have 94% homology (seeing Table 2); On amino acid levels, the 1-273 amino acids residue of it and henbane TRI (GenBank AccessionNo.BAA13547) has 90% homogeny and 96% similarity (seeing Table 3).Therefore, all there are higher homology in Scopoloa acutangula tropinone reductase I gene and henbane acutangula tropinone reductase I gene on nucleic acid still is protein level, so can think also have similar effect on the content of Scopoloa acutangula tropinone reductase I Scopolamine in improving resource plant.
The homology of the nucleotide sequence of table 2. Radix Anisodi Acutanguli AaTRI of the present invention and henbane (Hyoscyamus niger) HnTRI is (GAP) table relatively
Query 61
TAACCAGAAATG---GGAGAATCAAAAGTTTACATGAATGGCAACCATGGAGGAACTAGA?117
||||?||||||| |||||||||?||||||||||?|||||||||?|||||||||?||||
Sbjc?t 44
TAACAAGAAATGGCCGGAGAATCAGAAGTTTACATTAATGGCAACAATGGAGGAATTAGA 103
Query?118
TGGAGTCTCAAAGGCACCACTGCCCTTGTTACTGGTGGCTCTAAAGGCATTGGGTATGCA 177
|||||||||||||||||?|||||||||||||||||||||||||||||||||||||||||
Sbjct?104
TGGAGTCTCAAAGGCACAACTGCCCTTGTTACTGGTGGCTCTAAAGGCATTGGGTATGCA 163
Query?178
GTAGTGGAAGAACTGGCAGGTTTTGGAGCAACAGTATATACATGTTCACGTAATGAAAAG 237
||||||||||||||?||||||?||||?||||?||||||||||||||||||||||||||||
Sbjct?164
GTAGTGGAAGAACTAGCAGGTCTTGGTGCAAGAGTATATACATGTTCACGTAATGAAAAG 223
Query?238
GAACTCCAACAATGCCTTGAGATTTGGAGTAAA-GAAGGACTTAAAGTTGAAGGTTCTGT 296
||||||||||||||||||||?||||||||?|||?|||||||||?||||||||||||||||
Sbjct?224
GAACTCCAACAATGCCTTGATATTTGGAG-AAATGAAGGACTTCAAGTTGAAGGTTCTGT 282
Query?297
TTGTGATTTATTACTGCGCTCTGAACGTGAGAAGCTTATGCAGGCTGTTGGAGATTTATT 356
||||||||||||||||||||||||||||||?||?|||||||||?||||||?|||||||||
Sbjct?283
TTGTGATTTATTACTGCGCTCTGAACGTGACAAACTTATGCAGACTGTTGCAGATTTATT 342
Query?357
TAATGGAAAGCTCAATATTTTGGTAAATAATGCAGGGGTGGTGATACATAAAGAAGCTAA 416
||||||||||||||||||||||||||||||||||||?||||||||||||||||||||||
Sbjct?343
TAATGGAAAGCTCAATATTTTGGTAAATAATGCAGGTGTGGTGATACATAAAGAAGCTAA 402
Query?417
AGACTTCACAGAAGAAGATTATAACATCGTAATGGGCACTAATTTTGAAGCAGCTTATCA 476
|||?||||||?|||||||||| ||||||||?||||||||||||||||||||||||||||
Sbjct?403
AGATTTCACAAAAGAAGATTACGACATCGTATTGGGCACTAATTTTGAAGCAGCTTATCA 462
Query?477
CTTATCTCAACTTGCTTATCCCTTATTGAAGGCATCTGAAAATGGCAATGTTATTTTTCT 53
|||||?||||||||||||||||||?||||||||||||?||||||||||||||||||||||
Sbjct?463
CTTATGTCAACTTGCTTATCCCTTTTTGAAGGCATCTCAAAATGGCAATGTTATTTTTCT 522
Query?537
TTCTTCTATTGCTGGATTTTCAGCACTGCCTTCTGTTTCTCTTTATTCTGCTTCCAAAGC 596
|||||||||?||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct?523
TTCTTCTATAGCTGGATTTTCAGCACTGCCTTCTGTTTCTCTTTATTCTGCTTCCAAAGC 582
Query?597
TGCAATAAATCAAATGACGAAGAACTTGGCATGTGAATGGGCCAAGGAGAACATTCGGGT 656
|||||||||||||||?||||||||||||||||||||||||||||||||?|||||||||||
Sbjct?583
TGCAATAAATCAAATAACGAAGAACTTGGCATGTGAATGGGCCAAGGACAACATTCGGGT 642
Query?657
CAATTCAGTTGCTCCGGGAATCATTTTAACTCCACTCGTTGAAACTGCAATTAAGAAAAA 716
|||||||||||||||?|||?||||||||||?||||||?||||||||||||||||||||||
Sbjct?643
CAATTCAGTTGCTCCAGGAGTCATTTTAACCCCACTCATTGAAACTGCAATTAAGAAAAA 702
Query?717
TCCTCATCAAAAAGAAGAAATAGACAATTTTATTGTCAAGACTCCAATGGGCCGGGCCGG?776
|||||||||||||||||||||||||||||||||||||||||||||||||||||||||?||
Sbjct?703
TCCTCATCAAAAAGAAGAAATAGACAATTTTATTGTCAAGACTCCAATGGGCCGGGCTGG?762
Query?777
AAAACCCAAAGAAGTTTCTGCACTAATCTCTTTTCTTTGCTTCCCTGCTGCTTCTTATAT?836
|||?|||||?|||||?||||||||||| |?|||||||||||||||||||||||||||||
Sbjct?763
AAAGCCCAATGAAGTGTCTGCACTAATAGCCTTTCTTTGCTTCCCTGCTGCTTCTTATAT?822
Query?837
TACTGGCCAGATCATATGGGCTGATGGTGGATTCACAGCTAATGGTGGATTTTGAA?892
|||||||||?||?|||||||||||||||||||||||||||||||||||?|||||||
Sbjct?823
TACTGGCCAAATTATATGGGCTGATGGTGGATTCACAGCTAATGGTGGGTTTTGAA?878
Wherein: Query represents the nucleotide sequence of Radix Anisodi Acutanguli AaTRI; Sbjct represents the nucleotide sequence (GenBank Accession No.D88156) of henbane HnTRI.
The result: both have 94% similarity in the comparison of 832 Nucleotide.
The acutangula tropinone reductase I aminoacid sequence of the acutangula tropinone reductase I of table 3. Radix Anisodi Acutanguli of the present invention and henbane
Homology is (FASTA) table relatively
Query?2
GESKVYMNGNHGGTRWSLKGTTALVTGGSKGIGYAVVEELAGFGATVYTCSRNEKELQQC?61
GES+VY+NGN+GG?RWSLKGTTALVTGGSKGIGYAVVEELAG?GA?VYTCSRNEKELQQC
Sbjct?3
GESEVYINGNNGGIRWSLKGTTALVTGGSKGIGYAVVEELAGLGARVYTCSRNEKELQQC?62
Query?62
LEIWSKEGLKVEGSVCDLLLRSEREKLMQAVGDLFNGKLNILVNNAGVVIHKEAKDFTEE?121
L+IW?EGL+VEGSVCDLLLRSER+KLMQ?V?DLFNGKLNILVNNAGVVIHKEAKDFT+E
Sbjct?63
LDIWRNEGLQVEGSVCDLLLRSERDKLMQTVADLFNGKLNILVNNAGVVIHKEAKDFTKE?122
Query?122
DYNIVMGTNFEAAYHLSQLAYPLLKASENGNVIFLSSIAGFSALPSVSLYSASKAAINQM?181
DY+IV+GTNFEAAYHL?QLAYP?LKAS+NGNVIFLSSIAGFSALPSVSLYSASKAAINQ+
Sbjct 123
DYDIVLGTNFEAAYHLCQLAYPFLKASQNGNVIFLSSIAGFSALPSVSLYSASKAAINQI?182
Query?182
TKNLACEWAKENIRVNSVAPGIILTPLVETAIKKNPHQKEEIDNFIVKTPMGRAGKPKEV?241
TKNLACEWAK+NIRVNSVAPG+ILTPL+ETAIKKNPHQKEEIDNFIVKTPMGRAGKP?EV
Sbjct?183
TKNLACEWAKDNIRVNSVAPGVILTPLIETAIKKNPHQKEEIDNFIVKTPMGRAGKPNEV?242
Query?242?SALISFLCFPAASYITGQI?IWADGGFTANGGF?273
SALI+FLCFPAASYITGQIIWADGGFTANGGF
Sbjct?243?SALIAFLCFPAASYITGQI?IWADGGFTANGGF?274
Wherein: Query represents the aminoacid sequence of Radix Anisodi Acutanguli AaTRI; Subject represents the aminoacid sequence (GenBank Accession No.BAA13547) of henbane HnTRI; Identical amino acid marks with the amino acid monocase between two sequences.
The result: in 273 amino acid whose comparisons, both have 90% homogeny and 96% similarity respectively.Embodiment 3 (Scopoloa acutangula tropinone reductase I or polypeptide carry out prokaryotic expression and purification in intestinal bacteria)
In this embodiment, the Radix Anisodi Acutanguli AaTRI encoding sequence of total length or fragment are built into commercial protein merge among the expression vector, to express and purification of recombinant proteins.
1, construction of prokaryotic expression vector and transformed into escherichia coli
According to the nucleotide sequence of Radix Anisodi Acutanguli AaTRI, design amplifies the primer of protein-coding region, and introduces restriction endonuclease sites (this decides according to pET32a (+) carrier of selecting for use) on positive anti-primer respectively, so that construction of expression vector.Amplified production with acquisition among the embodiment 1 is a template, behind pcr amplification, Radix Anisodi Acutanguli AaTRI gene is being guaranteed to be cloned into pET32a (+) carrier (Novagen) under the correct prerequisite of reading frame.Identify that good expression vector utilizes CaCl
2Method changes e. coli bl21 over to, and Screening and Identification obtains containing engineering bacteria BL21-pET32a (+)-AaTRI of pET32a (+)-AaTRI expression vector.
2, express the isolation identification of the engineering bacteria of Trx-AaTRI recombinant protein
The BL21-pET32a (+) of picking list bacterium colony-AaTRI engineering bacteria contains jolting overnight incubation in the LB substratum of 100 μ g/mL penbritins in 3mL, draw nutrient solution by 1: 100 concentration and in new LB substratum (containing 100 μ g/mL penbritins), cultivated about 3 hours, to OD
600After reaching 0.5, adding IPTG continues at 37 ℃ to final concentration 1mmol/L and cultivated respectively 0,1,2,3 hour.It is centrifugal to get the different 1mL bacterium liquid of incubation time, in the bacterial precipitation thing, add lysate (2 * SDS sample-loading buffer, 50 μ L, distilled water 45 μ L, 3-mercaptoethanol 5 μ L), the suspendible bacterial precipitation, boiled in the boiling water bath 5 minutes, centrifugal 1 minute of 10000rpm, supernatant adds electrophoresis in the 12%SDS-PAGE glue.The bacterial strain that the protein content of dyeing back observation expection molecular weight size increases with the IPTG induction time is the engineering bacteria of expressing the Trx-AaTRI fusion rotein.
3, the extraction purifying of Trx-AaTRI fusion rotein
The proteic engineering bacteria BL21-pET32a of abduction delivering Trx-AaTRI amalgamation and expression (+)-AaTRI as stated above, collect thalline through centrifugation, and come the purifying inclusion body with BugBuster reagent and Benzonase nuclease according to the specification sheets of producer (Novagen).Inclusion body can with the dissolving damping fluid (50mM CAPS, pH11.0 0.3%N-lauroylsarcosine) dissolves, (200mM Tris-HCl pH8.5) dialyses to use dialysis buffer liquid again.Use Histidine to carry out affinity chromatography then, and collect the Trx-AaTRI fusion rotein through elution buffer (1M imidazole, 500mM NaCl, 20mM Tris-HCl pH7.9) wash-out in conjunction with (HisBind) resin.Fusion rotein is the expressing protein of the separable AaTRI that obtains after 20 ℃ of enzymes of enteropeptidase are cut 16 hours.
Embodiment 4 (Scopoloa acutangula tropinone reductase I or polypeptide carry out tropine and Scopolamine assay in eukaryotic cell expression and the transgenosis root of hair in Radix Anisodi Acutanguli)
The structure that contains the expression vector of goal gene (Scopoloa acutangula tropinone reductase I gene), full length sequence (SEQ ID NO.1) according to Scopoloa acutangula tropinone reductase I, 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 Scopoloa acutangula tropinone reductase I 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 over to genetic transformation resource plant Radix Anisodi Acutanguli.Utilize the genetic transformation of the plasmid-mediated Radix Anisodi Acutanguli of Agrobacterium rhizogenes Ri:
1) Agrobacterium rhizogenes A4.Take out from refrigerator before using, go down to posterity 2 times, going down to posterity with solid medium is the YEB substratum.Bacterial classification is inoculated in the YEB liquid nutrient medium before use, 28 ℃ of overnight incubation.
2) through the grow aseptic leaflet tablet of the Radix Anisodi Acutanguli about 8 weeks of seed germination.
3) the bacterium liquid through spending the night and cultivating is 100 bacterium/mL with the conversion fluid dilution.Get aseptic Radix Anisodi Acutanguli blade, draw with "+" font wound with aseptic scalper, put into above-mentioned conversion, 60rpm/min shaking culture 8h takes out, with aseptic water washing 3 times, put into the B5 medium that contains 250-500mg/L kantlex and different concns 6-BA (0.5mg/L-3mg/L), transfer in per 2 weeks in the fresh culture 1 time, separate hairly root after waiting to grow hairly root, be transferred in the B5 medium that contains the 250-500mg/L kantlex and do not have hormone and cultivate, shift 4-5 time till no bacterium, and then be transferred in the no hormone B5 medium that does not contain kantlex and cultivate.
4) the secondary culture of the hairly root in solid medium, be inoculated in and 100mL is housed does not have hormone B5, in the 500mL triangular flask of substratum, culture condition such as culture temperature, illumination, rotating speed are identical with callus fluid suspension culture condition, cultivated 20 days, hairly root taken out from substratum puts into freeze drier and carry out drying, weigh then, be stored in-70 ℃ standby.
5) contain the tropine and the Scopolamine assay of the transgenosis root of hair of Scopoloa acutangula tropinone reductase I gene
Press the method for Zhang etc. (PNAS, 2004) the transgenosis root of hair of expressing Scopoloa acutangula tropinone reductase I gene is carried out tropine and Scopolamine assay.The result shows: the tropane alkaloid significantly improves (P＜0.05) with comparing in the non-transgenic contrast in the transgenosis root of hair of expressing Scopoloa acutangula tropinone reductase I gene.Therefore transgene result proves: Scopoloa acutangula tropinone reductase I gene pairs promotes the raising of tropane alkaloid that obvious effect is arranged, and can be widely used in producing the quality-improving of the alkaloidal resource plant of tropane.
The nucleotides sequence tabulation
＜110〉Shanghai Normal University
＜120〉Scopoloa acutangula tropinone reductase I gene and encoded protein matter and application
<160>4
<170>PatentIn?version?3.3
<210>1
<211>1188
<212>DNA
＜213〉Radix Anisodi Acutanguli (Anisodus acutangulus)
<221>CDS
<222>(70)..(891)
<400>1
aaaggaaaac?caattgtcac?atttgggcta?attccatatct?caaaatagt?tgcaagaaac 60
taaccagaa?atg?gga?gaa?tca?aaa?gtt?tac?atg?aat?ggc?aac?cat?gga?gga 111
Met?Gly?Glu?Ser?Lys?Val?Tyr?Met?Asn?Gly?Asn?His?Gly?Gly
1 5 10
act?aga?tgg?agt?ctc?aaa?ggc?acc?act?gcc?ctt?gtt?act?ggt?ggc?tct 159
Thr?Arg?Trp?Ser?Leu?Lys?Gly?Thr?Thr?Ala?Leu?Val?Thr?Gly?Gly?Ser
15 20 25 30
aaa?ggc?att?ggg?tat?gca?gta?gtg?gaa?gaa?ctg?gca?ggt?ttt?gga?gca 207
Lys?Gly?Ile?Gly?Tyr?Ala?Val?Val?Glu?Glu?Leu?Ala?Gly?Phe?Gly?Ala
35 40 45
aca?gta?tat?aca?tgt?tca?cgt?aat?gaa?aag?gaa?ctc?caa?caa?tgc?ctt 255
Thr?Val?Tyr?Thr?Cys?Ser?Arg?Asn?Glu?Lys?Glu?Leu?Gln?Gln?Cys?Leu
50 55 60
gag?att?tgg?agt?aaa?gaa?gga?ctt?aaa?gtt?gaa?ggt?tct?gtt?tgt?gat 303
Glu?Ile?Trp?Ser?Lys?Glu?Gly?Leu?Lys?Val?Glu?Gly?Ser?Val?Cys?Asp
65 70 75
tta?tta?ctg?cgc?tct?gaa?cgt?gag?aag?ctt?atg?cag?gct?gtt?gga?gat 351
Leu?Leu?Leu?Arg?Ser?Glu?Arg?Glu?Lys?Leu?Met?Gln?Ala?Val?Gly?Asp
80 85 90
tta?ttt?aat?gga?aag?ctc?aat?att?ttg?gta?aat?aat?gca?ggg?gtg?gtg 399
Leu?Phe?Asn?Gly?Lys?Leu?Asn?Ile?Leu?Val?Asn?Asn?Ala?Gly?Val?Val
95 100 105 110
ata?cat?aaa?gaa?gct?aaa?gac?ttc?aca?gaa?gaa?gat?tat?aac?atc?gta 447
Ile?His?Lys?Glu?Ala?Lys?Asp?Phe?Thr?Glu?Glu?Asp?Tyr?Asn?Ile?Val
115 120 125
atg?ggc?act?aat?ttt?gaa?gca?gct?tat?cac?tta?tct?caa?ctt?gct?tat 495
Met?Gly?Thr?Asn?Phe?Glu?Ala?Ala?Tyr?His?Leu?Ser?Gln?Leu?Ala?Tyr
130 135 140
ccc?tta?ttg?aag?gca?tct?gaa?aat?ggc?aat?gtt?att?ttt?ctt?tct?tct 543
Pro?Leu?Leu?Lys?Ala?Ser?Glu?Asn?Gly?Asn?Val?Ile?Phe?Leu?Ser?Ser
145 150 155
att?gct?gga?ttt?tca?gca?ctg?cct?tct?gtt?tct?ctt?tat?tct?gct?tcc 591
Ile?Ala?Gly?Phe?Ser?Ala?Leu?Pro?Ser?Val?Ser?Leu?Tyr?Ser?Ala?Ser
160 165 170
aaa?gct?gca?ata?aat?caa?atg?acg?aag?aac?ttg?gca?tgt?gaa?tgg?gcc 639
Lys?Ala?Ala?Ile?Asn?Gln?Met?Thr?Lys?Asn?Leu?Ala?Cys?Glu?Trp?Ala
175 180 185 190
aag?gag?aac?att?cgg?gtc?aat?tca?gtt?gct?ccg?gga?atc?att?tta?act 687
Lys?Glu?Asn?Ile?Arg?Val?Asn?Ser?Val?Ala?Pro?Gly?Ile?Ile?Leu?Thr
195 200 205
cca?ctc?gtt?gaa?act?gca?att?aag?aaa?aat?cct?cat?caa?aaa?gaa?gaa 735
Pro?Leu?Val?Glu?Thr?Ala?Ile?Lys?Lys?Asn?Pro?His?Gln?Lys?Glu?Glu
210 215 220
ata?gac?aat?ttt?att?gtc?aag?act?cca?atg?ggc?cgg?gcc?gga?aaa?ccc 783
Ile?Asp?Asn?Phe?Ile?Val?Lys?Thr?Pro?Met?Gly?Arg?Ala?Gly?Lys?Pro
225 230 235
aaa?gaa?gtt?tct?gca?cta?atc?tct?ttt?ctt?tgc?ttc?cct?gct?gct?tct 831
Lys?Glu?Val?Ser?Ala?Leu?Ile?Ser?Phe?Leu?Cys?Phe?Pro?Ala?Ala?Ser
240 245 250
tat?att?act?ggc?cag?atc?ata?tgg?gct?gat?ggt?gga?ttc?aca?gct?aat 879
Tyr?Ile?Thr?Gly?Gln?Ile?Ile?Trp?Ala?Asp?Gly?Gly?Phe?Thr?Ala?Asn
255 260 265 270
ggt?gga?ttt?tga?atcttacata?ttcaaaagaa?gtttcagctt?ttgaagcaat 931
Gly?Gly?Phe
tctgttttct?gtttgggctt?tgtaatcttt?tttaactttt?tgtggtttgt?accttgggcc 991
tataaaacgg?cccattatat?tttgaacaat?ttctgtccac?gggcttgtgt?tcttcggtct?1051
acatgcttcc?atattcatgg?gcttattatt?ttgggcctag?atgattcttg?tgtcagctag?1111
gcttctatct?gctttttgtt?gtcatcatca?caataaaatg?tcgcaaatag?ttagcataaa?1171
aaaaaaaaaaaaaaaaa 1188
<210>2
<211>273
<212>PRT
＜213〉Radix Anisodi Acutanguli (Anisodus acutangulus)
<400>2
Met?Gly?Glu?Ser?Lys?Val?Tyr?Met?Asn?Gly?Asn?His?Gly?Gly?Thr?Arg
1 5 10 15
Trp?Ser?Leu?Lys?Gly?Thr?Thr?Ala?Leu?Val Thr?Gly?Gly?Ser?Lys?Gly
20 25 30
Ile?Gly?Tyr?Ala?Val?Val?Glu?Glu?Leu?Ala?Gly?Phe?Gly?Ala?Thr?Val
35 40 45
Tyr?Thr?Cys Ser?Arg?Asn?Glu?Lys?Glu?Leu?Gln?Gln?Cys?Leu?Glu?Ile
50 55 60
Trp?Ser?Lys?Glu?Gly?Leu?Lys?Val?Glu?Gly?Ser?Val?Cys?Asp?Leu?Leu
65 70 75 80
Leu?Arg?Ser?Glu?Arg?Glu?Lys?Leu?Met?Gln?Ala?Val?Gly?Asp?Leu?Phe
85 90 95
Asn?Gly?Lys?Leu?Asn?Ile?Leu?Val?Asn?Asn?Ala?Gly?Val?Val?Ile?His
100 105 110
Lys?Glu?Ala?Lys?Asp?Phe?Thr?Glu?Glu?Asp?Tyr?Asn?Ile?Val?Met?Gly
115 120 125
Thr?Asn?Phe?Glu?Ala?Ala?Tyr?His?Leu?Ser?Gln?Leu?Ala?Tyr?Pro?Leu
130 135 140
Leu?Lys?Ala?Ser?Glu?Asn?Gly?Asn?Val?Ile?Phe?Leu?Ser?Ser?Ile?Ala
145 150 155 160
Gly?Phe?Ser?Ala?Leu?Pro?Ser?Val Ser?Leu?Tyr?Ser?Ala?Ser?Lys?Ala
165 170 175
Ala?Ile?Asn?Gln?Met?Thr?Lys?Asn?Leu?Ala?Cys?Glu?Trp?Ala?Lys?Glu
l80 185 190
Asn?Ile?Arg?Val?Asn?Ser?Val?Ala?Pro?Gly?Ile?Ile?Leu?Thr?Pro?Leu
195 200 205
Val?Glu?Thr?Ala?Ile?Lys?Lys?Asn?Pro?His?Gln?Lys?Glu?Glu?Ile?Asp
210 215 220
Asn?Phe?Ile?Val?Lys?Thr?Pro?Met?Gly?Arg?Ala?Gly?Lys?Pro?Lys?Glu
225 230 235 240
Val?Ser?Ala?Leu?Ile?Ser?Phe?Leu?Cys?Phe?Pro?Ala?Ala?Ser?Tyr?Ile
245 250 255
Thr?Gly?Gln?Ile?Ile?Trp?Ala?Asp?Gly?Gly?Phe?Thr?Ala?Asn?Gly?Gly
260 265 270
Phe
<210>3
<211>25
<212>DNA
＜213〉Radix Anisodi Acutanguli (Anisodus acutangulus)
<400>3
aaaggaaaac?caattgtcac?atttg
<210>4
<211>24
<212>DNA
＜213〉Radix Anisodi Acutanguli (Anisodus acutangulus)
<400>4
atgctaacta?tttgcgacat?ttta
Claims (8)
1. a Scopoloa acutangula tropinone reductase I gene is characterized in that, its nucleotide sequence is as described in the SEQ IDNo.1.
2. the protein of the described Scopoloa acutangula tropinone reductase I of claim 1 genes encoding is characterized in that, its aminoacid sequence is as described in the SEQ ID No.2.
3. a plasmid is characterized in that, it contains the described gene complete sequence of claim 1.
4. a plant expression vector is characterized in that, it contains the described gene complete sequence of claim 1.
5. a host cell is characterized in that, it contains the described gene order of claim 1.
6. host cell according to claim 5 is characterized in that, described host cell is Bacillus coli cells, agrobatcerium cell, yeast cell, tobacco cell or other vegetable cell.
7. host cell according to claim 6 is characterized in that, described host cell is Bacillus coli cells or agrobatcerium cell or Radix Anisodi Acutanguli root of hair cell.
8. the application of the described Scopoloa acutangula tropinone reductase I of claim 1 gene is characterized in that, provides a kind of transgenosis Radix Anisodi Acutanguli with the described acutangula tropinone reductase I of claim 1 gene order.
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| CN102212548A (en) * | 2011-04-02 | 2011-10-12 | 上海师范大学 | Method for carrying out cotransformation on double genes of AaPMT and AaTRI to improve content of tropine alkaloid in anisodus acutangulus hairy roots |
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Non-Patent Citations (4)
| Title |
|---|
| KEUI NAKAJIMA等.Two tropinone reductases with different stereospecificitiesareshort-chain dehydrogenases evolved from acommonancestor..PNAS90 20.1993,90(20),附图2和3A，正文9592页左栏第2段-右栏第3段. |
| KEUI NAKAJIMA等.Two tropinone reductases with different stereospecificitiesareshort-chain dehydrogenases evolved from acommonancestor..PNAS90 20.1993,90(20),附图2和3A,正文9592页左栏第2段-右栏第3段. * |
| 刘涛.唐古特山莨菪毛状根托品烷类生物碱生物合成相关基因的研究..中国协和医科大学药物研究所,2006,第四章 唐古特山莨菪毛状根托品酮还原酶的克隆. * |
| 刘涛.唐古特山莨菪毛状根托品烷类生物碱生物合成相关基因的研究。.中国协和医科大学药物研究所,2006,第四章 唐古特山莨菪毛状根托品酮还原酶的克隆. |
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