CN1385530A - Method for raising taxol content by gene engineering technology - Google Patents
Method for raising taxol content by gene engineering technology Download PDFInfo
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- CN1385530A CN1385530A CN 02111944 CN02111944A CN1385530A CN 1385530 A CN1385530 A CN 1385530A CN 02111944 CN02111944 CN 02111944 CN 02111944 A CN02111944 A CN 02111944A CN 1385530 A CN1385530 A CN 1385530A
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Abstract
The present invention provides a cDNA of taxad diene synthetase gene which is one of key enzymes for rate-limiting step in biological synthesis of taxal and its application for raising taxol content by using gene engineering technique. Said invention relates to the method for obtaining cDNA to described gene and method for raising taxad alkal compound, for example taxal content by using gene engineering technique.
Description
Technical field
The present invention relates to fields such as molecular biology, zymetology, physiology, pharmacology and genetically engineered.Particularly, the present invention relates to a kind of taxol biosynthesizing key enzyme (Japanese yew diene synthetic enzyme) gene cDNA sequence, and utilize the efficient expression vector and the engineering strain that make up in host cell, to express the specific procedure of this gene, screening transformant.The invention still further relates to the Ramulus et folium taxi cuspidatae crown gall cell of the commentaries on classics Japanese yew diene synthase gene that utilizes the genetically engineered acquisition and filial generation, regeneration plant, plant tissue or the seed of hairly root and cultivation thereof.
Background technology
Taxol (taxol) is the natural product with unique antitumous effect that is extracted from yewtree (Taxus brevifolia) bark by Wani etc. the seventies in last century.Taxol is that it is the choice drug of treatment ovarian cancer through one of present best natural anti-cancer drugs of FDA authentication, and leukemia, lung cancer, the cancer of the brain and other some solid tumors etc. are all had better curative effect, and toxic side effect is very little.Now, unique difficulty of facing of taxol production is exactly the serious scarcity in medicine source.
Up to the present, find that taxol only is present in the Chinese yew genus plants of gymnosperm taxaceae.(Ramulus et folium taxi cuspidatae is the rare or endangered species of state guarantee owing to Ramulus et folium taxi cuspidatae quantity rareness; forbid to cut down); poor growth (20-30 just can be become a useful person); the content of taxol very low again (only be 100,000 of Ramulus et folium taxi cuspidatae dry weight/several to ten thousand/several; the age of tree can only provide a taxol that the cancer patients is required for the Ramulus et folium taxi cuspidatae in 100 years); can not satisfy people's needs growing (demand of whole world taxol is about 1000 kilograms/year, and output is less than 100 kilograms/year) far away so from natural Ramulus et folium taxi cuspidatae, extract the method for taxol to taxol.The magical curative effect of taxol and the critical shortage in medicine source make its price very expensive, up to 5000-6000 dollar/gram.
Research to searching and expansion taxol medicine source approach has in recent years obtained great progress, and main path roughly comprises: (1) screening high yield Ramulus et folium taxi cuspidatae (Taxus) Cultivar; (2) chemosynthesis; (3) microorganisms producing; (4) biotechnology.
In these research fields, the screening high yield Yew planting required cycle of kind is oversize, and efficient is very low, and also has many significant problems unclear in the biology of reproduction of Ramulus et folium taxi cuspidatae, and these have all limited the practical application of screening high yield Yew planting kind; Though the complete synthesis success of taxol chemistry, the synthetic step is too many, and other poisonous or useless byproducts are too many, taxol yield is too low, makes that the full chemosynthesis cost of taxol is too high and does not have commercial promise; The microorganism overwhelming majority who produces taxol is and the symbiotic fungi of Ramulus et folium taxi cuspidatae, and its content of taxol is atomic, and the large scale fermentation of these fungies cultivates very difficulty, and the decline of bacterial strain also is the difficult problem that can't overcome.
By contrast, utilize modern biotechnology, particularly genetic engineering technique is improved organism, produce taxol and just have very big advantage: (1) is in the biotechnology research field, the researchist has established good theory and practice basis to the long-term extensive and deep research of Ramulus et folium taxi cuspidatae isolated culture and the success repeatedly of Ramulus et folium taxi cuspidatae genetic transformation for utilizing biotechnology to produce taxol; (2) owing to illustrated taxol biosynthetic metabolism approach and key enzyme thereof at present substantially, on gene level, improve Ramulus et folium taxi cuspidatae and other life entities fast and efficiently so utilize genetic engineering technique, Ramulus et folium taxi cuspidatae (comprising seedling, hairly root, clone etc.) and other life entities of obtaining the high-content taxol of genetic modification become possibility, thereby to utilize genetic engineering technique be the optimal path in production taxol and medicine source thereof.
In the present invention, we are clone's Japanese yew diene synthase gene cDNA sequence from Taxus x media first, and member plant strongly expressed carrier, import Agrobacterium and genetic transformation Ramulus et folium taxi cuspidatae, Japanese yew diene synthase gene is expressed in Ramulus et folium taxi cuspidatae and improved the content of taxol in the Ramulus et folium taxi cuspidatae.
Before the present invention comes forth, any patent report that discloses or reported using gene engineering technique raising content of taxol mentioned in the present patent application is not arranged as yet.
Summary of the invention
The objective of the invention is at the deficiencies in the prior art, it is Japanese yew diene synthase gene that a kind of bearing taxanes and taxol biosynthetic pathway key gene are provided, and utilize engineered way, Japanese yew diene synthase gene is changed in the life entity, improve bearing taxanes and content of taxol in the life entity, make it to have more practical value and economic worth.
First purpose of the present invention just provides a kind of gene of the key enzyme effect of playing newly in the taxol biosynthesizing, this gene is a Japanese yew diene synthase gene.
Second purpose of the present invention provides the method for Japanese yew diene synthase gene in the life entity that shift;
The 3rd purpose of the present invention provides a kind of method that obtains to change the life entity of Japanese yew diene synthase gene;
The 4th purpose of the present invention provides a kind of method of expressing Japanese yew diene synthase gene in life entity, and this method helps improving the content of bearing taxanes and taxol.
In one aspect of the invention, a kind of isolated dna molecular is provided, this molecule comprises: the nucleotide sequence of the polypeptide of coding Japanese yew diene synthase activity, show at least 75% homology from the nucleotides sequence of Nucleotide 1-2589 position among described nucleotide sequence and the SEQ ID NO.3; Perhaps described nucleotide sequence can be under the rigorous condition of moderate with SEQ IDNO.3 in from the nucleotide sequence hybridization of Nucleotide 1-2589 position.Preferably, described sequence encoding has the polypeptide of the aminoacid sequence shown in the SEQ ID NO.4.More preferably, described sequence has among the SEQ ID NO.3 nucleotide sequence from Nucleotide 1-2589 position.
In another aspect of this invention, provide a sharp Japanese yew diene synthetase protein polypeptide, it comprises: have polypeptide or its conservative property variation polypeptide or its active fragments of SEQ ID NO.4 aminoacid sequence, or its reactive derivative.Preferably, this polypeptide is to have SEQ ID NO.4 polypeptide of sequence.
In another aspect of this invention, also provide a kind of expression vector, it comprises above-mentioned dna molecular.
In another aspect of this invention, also provide a kind of usefulness above-mentioned carrier transformed host cells.This host cell is a Ramulus et folium taxi cuspidatae in example.Also provide a kind of genetic engineering technique that utilizes to transform the method for life entity with bearing taxanes and content of taxol in the raising life entity, its step is as follows:
(1) adopt any possible means (as methods such as gene clone, synthetic genes) to obtain bearing taxanes and taxol biosynthetic pathway key gene, as Japanese yew diene synthase gene.
(2) nucleotide sequence that coding is had a Japanese yew diene synthetase albumen active polypeptide operationally is connected in expression regulation sequence, form Japanese yew diene synthase activity protein expression vector, show at least 75% homology from the nucleotides sequence of Nucleotide 1-2589 position among described nucleotide sequence and the SEQID NO.3, perhaps described nucleotide sequence can be under the rigorous condition of moderate with SEQ ID NO.3 in from the nucleotide sequence hybridization of Nucleotide 1-2589 position.
(3) adopt any transgenic method (comprising the plasmid-mediated method of Agrobacterium rhizogenes Ri, agrobacterium tumefaciens Ti-plasmids mediated method etc.) transfer Japanese yew diene synthase gene or its carrier in any host cell that can produce bearing taxanes and taxol; In the plasmid-mediated method of Agrobacterium rhizogenes Ri, change the expression vector in the step (2) over to Agrobacterium rhizogenes, after the Agrobacterium that will contain expression vector is cultivated 8-24 hour (22-28 ℃) altogether with host cell, screening and evaluation transformant, the transformant of Japanese yew diene synthetase albumen gene is expressed in acquisition.
(4) bearing taxanes in the transformant and content of taxol are measured, cultivated the transformant that bearing taxanes and content of taxol improve, obtain genetically modified organism.
A kind of cell that is obtained by aforesaid method, it is the cell that can produce taxol.
The offspring of the transgenosis life entity that a kind of bearing taxanes that is obtained by aforesaid method and content of taxol improve.
In the present invention, term " Japanese yew diene synthetic enzyme " refers to have identical active SEQID NO.4 polypeptide of sequence with wild-type Japanese yew diene synthetic enzyme.This term also comprises having the variant form of expressing the SEQ IDNO.4 sequence of identical function with Japanese yew diene synthetic enzyme in plant.These variant forms comprise (but being not limited to): several (are generally 1-20, preferably 1-15, more preferably 1-10,1-5 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 15, preferably being in 10, more preferably is in 5) amino acid.
The variant form of Japanese yew diene synthase 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 DNA of Japanese yew diene synthase gene DNA hybridization.
In the present invention, " Japanese yew diene synthetic enzyme conservative property variation polypeptide " is meant with the aminoacid sequence of SEQ ID NO.4 and compares, have 10 at the most, preferably at the most 8, more preferably 5 amino acid is replaced by similar performance or close amino acid and is 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 |
(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 anti-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 Japanese yew diene synthase gene of in plant, expressing of the present invention, Japanese yew diene synthase gene encoding sequence operationally can be connected in expression regulation sequence, thereby form Japanese yew diene synthase gene expression vector.
As used herein, " 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.
In the present invention, term " life entity " refers to produce plant (as the various Ramulus et folium taxi cuspidatae of Taxus, fibert etc.), vegetable cell, plant organ, fungi, bacterium of bearing taxanes or taxol etc.
In the present invention, term " any possible means " specifically comprises homology clone (as RT-PCR, RACE etc.), library screening and synthetic Japanese yew diene synthase gene and varient thereof for obtaining the method for Japanese yew diene synthase gene.
In the present invention, term " any transgenic method " comprises that the conversion of agrobacterium tumefaciens Ti-plasmids mediated gene, the plasmid-mediated gene transformation of Agrobacterium rhizogenes Ri, plant viral vector mediated gene transform, transform as the conversion of PEG mediated gene, liposome-mediated gene transformation, the conversion of electric shocking method mediated gene, ultrasonic-mediated gene transformation, the conversion of microinjection mediated gene, the conversion of laser microbeam mediated gene, the conversion of particle bombardment mediated gene, the conversion of pollen tube channel mediated gene, sexual cell infusion method mediated gene.
In the present invention, term " screen under given conditions and identify transformant " is meant under the condition that is used in isolated culture and selects the transformant that contains goal gene with microbiotic (kantlex, Totomycin, G418 etc.) or other compound; Can use methods such as PCR, Southern hybridization, Northern hybridization and Western trace to identify transformant.
In the present invention, term " is cultivated the transformant that bearing taxanes and content of taxol improve; obtain genetically modified organism " and is meant the transformant isolated culture through identifying under the condition that is fit to, and detection bearing taxanes and content of taxol, screen high-load good transformant and cultivate, obtain genetically modified organism.
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.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 Laboratory Press, 1989) condition described in, or the condition of advising according to manufacturer.
Embodiment
Embodiment 1
Synthesizing of Japanese yew diene synthase gene
1. gene clone (gene synthesis)
Japanese yew diene synthase gene is cloned Japanese yew diene synthetic enzyme by the One Step RT-PCR test kit of this laboratory use TaKaRa company according to the explanation of service manual from total RNA extract of Taxus x media, be connected into the order-checking of pGEM-5zf carrier then.
2. enzyme is cut (restriction enzyme cutting)
The plasmid vector that will contain Japanese yew diene synthase gene downcuts Japanese yew diene synthase gene with restriction enzyme such as Bam HI and Sac I double digestion;
3. connect (ligation)
The gene that downcuts is connected in the big fragment of plasmid pCAMBIA2300 plant expression vector that cuts with BamHI and SacI, detects the intestinal bacteria positive colony that transforms with blue white screening.
4. the PCR of gene detects
Gene changed over to following primer contained gene is carried out PCR behind the expression vector and detect
Japanese yew diene synthase gene P1:5 '-cgggatccatggctcagctctcatttaatg-3 ' (SEQ ID NO.1)
Japanese yew diene synthase gene P2:5 '-cgagctcttacatgatatatcatacttg-3 ' (SEQ ID NO.2)
The PCR condition be 94 ℃ 3 minutes, carried out 35 circulations in 1 minute with 94 ℃ 1 minute, 54 ℃ 1 minute and 72 ℃ thereupon, extended 10 minutes with 72 ℃ at last.The electrophoresis detection pcr amplification product, the acquisition expanding fragment length is 2604bp.
Embodiment 2
The sequence information of gene and homology analysis
The total length of Japanese yew diene synthase gene of the present invention is 2589bp, and detailed sequence is seen SEQ ID NO.3, and wherein open reading frame is positioned at 1-2589 position Nucleotide.According to the aminoacid sequence of the total length Japanese yew diene synthase gene of deriving, totally 862 amino-acid residues, molecular weight is 98303.97, and pI is 5.56, and detailed sequence is seen SEQ ID NO.4.
Clone's Japanese yew diene synthase gene full length sequence and coded protein thereof are carried out Nucleotide and protein homology retrieval with blast program in Non-redundant+GenBank+EMBL+DDBJ+PDB and Non-redundant GenBank CDS translations+PDB+SwissProt+Superdate+PIR database, found that there are very high homology in it and Japanese yew diene synthetic enzyme that comes from Taxus brevifolia and Taxus chinensis and gene thereof.On nucleotide level, the whole coding sequence of the Japanese yew diene synthase gene nucleotide sequence (GenBank Accession No.U48796) of Japanese yew diene synthase gene Taxus chinensis has 98% homogeny (seeing Table 2) respectively, on amino acid levels, the amino-acid residue of the Japanese yew diene synthetic enzyme of it and Taxus chinensis has 97% similarity (GenPeptAccession No.AAG02257) (seeing Table 3) respectively.Therefore, still all there is higher homology in the Japanese yew diene synthase gene that we clone from Taxus x media with existing sequence at nucleic acid on protein level, can think that both are having the identical functions function, we further studies confirm that this point.
Embodiment 3 obtains the structure that the transgenosis Taxus x media contains goal gene (Japanese yew diene synthase gene) expression vector
The intermediate carrier PGEM-5zf that will contain Japanese yew diene synthase gene further is cloned in the plant binary expression vector (as pCAMBIA2300), guaranteeing to identify good expression vector under the correct prerequisite of reading frame, again it is changed in the Agrobacterium (as EHA105, LBA4404, A4, R1000, ATCC15834 etc.), utilize the tender stem section of children to transform Taxus x media.Utilize the genetic transformation 1. Agrobacterium rhizogenes A4 of the plasmid-mediated Taxus x media of Agrobacterium rhizogenes Ri.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 exists
Be inoculated in the YEB liquid nutrient medium 27 ℃ of overnight incubation before the use.2. through the grow aseptic shoot of the Taxus x media about 8 weeks of seed germination.3. the bacterium liquid through spending the night and cultivating (adds 100 μ mol/L Syringylethanones, the beet of 10mg/mL with conversion fluid in the B5 nutrient solution
Alkali) dilution is 100 bacterium/mL.Get plant different sites such as aseptic Ramulus et folium taxi cuspidatae terminal bud, lateral bud, leaf, stem, root, use
Aseptic scalper is drawn with "+" font wound, puts into above-mentioned conversion fluid, and 60rpm/min shaking culture 8h takes out, and uses
Aseptic water washing 3 times is put into the B5 that contains 250-500mg/L kantlex and different concns 6-BA (0.5mg/L-3mg/L)
In the substratum, transfer in per 2 weeks in the fresh culture 1 time, separate hairly root after waiting to grow hairly root, be transferred to and contain
The 250-500mg/L kantlex does not have in the B5 medium of hormone to be cultivated, and shifts 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 hairly root in solid medium, be inoculated in and 100mL is housed does not have hormone B5, the 500mL of substratum
In the triangular flask, culture condition such as culture temperature, illumination, rotating speed are identical with callus fluid suspension culture condition, cultivate
20 days, hairly root is put into freeze drier from the substratum taking-up carry out drying, weigh then, be stored in-70 ℃ and be equipped with
With.The method of Richard (1993), Saito (1992) is pressed in taxol extracting in the hairly root.The survey of content of taxol
Surely adopt HPLC (Catalatic, 1993) and taxol monoclonal antibody (Hawaii Biotechnology Group
Inc.) join enzyme immunoassay (Jiang Chenggan, 1984) indirectly.Culture content of taxol (taxol/cell freeze-drying heavy) callus 0.020% hairly root 0.29% utilizes the positive bacterium colony on the aseptic toothpick picking of genetic transformation 1. usefulness the YEB selection flat board of Taxus x media of agrobacterium tumefaciens Ti-plasmids mediation, be inoculated in 2ml YEB liquid (Sm+, Kan+), 28 degree, 200rpm shaking culture 24-36 hour; 2. under the room temperature 4, the centrifugal 10min of 000g; 3. abandon supernatant, thalline suspends with the 1/2MS liquid nutrient medium, is diluted to 5-20 times of original volume, makes the OD of bacterium liquid
600About=0.5-1.0; 4. the grow aseptic shoot of the Taxus x media about 8 weeks of the seed germination of learning from else's experience removes needle, and stem is cut into the 1cm segment, is put in respectively to contain 2.5mg/L2, in the B5 medium of 4-D, induces the formation callus under dark, 26 ℃ of conditions.5. the bacterium liquid through spending the night and cultivating is 100 bacterium/mL with conversion fluid (adding 100 μ mol/mL Syringylethanones, the trimethyl-glycine of 10mg/ mL in the B5 nutrient solution) dilution.Get aseptic Ramulus et folium taxi cuspidatae terminal bud, lateral bud, leaf, stem, plant different sites such as root, draw with "+" font wound with aseptic scalper, put into above-mentioned conversion fluid, take out behind the 60rpm/min shaking culture 8h, with aseptic water washing 3 times, put into the B5 medium that contains the 250-500mg/L kantlex, transfer in per 2 weeks in the fresh culture 1 time, wait to grow two-piece crown goiter tissue behind the crown gall tissue, be transferred in the B5 medium that contains the 250-500mg/L kantlex and do not have hormone and cultivate, shift 4～5 times till no bacterium, and then be transferred in the no hormone B5 medium that does not contain kantlex and cultivate.6. detect transformant with PCR and Southern blotting, detect the expression level of Japanese yew diene synthase gene with Northern blotting.7. the callus lines that induces put in the proliferated culture medium (B5+Phytagel 2.0g/L+ glucose 30g/L, pH5.8) in, cultivate down in 26-28 degree constant temperature, change a subculture every other month.8. the mensuration of content of taxol.Ramulus et folium taxi cuspidatae crown gall cell and callus solid culture be after 20 days, culture taken out from substratum put into freeze drier and carry out drying, weighs then, be stored in-70 ℃ standby.The method of Richard (1993), Saito (1992) is pressed in taxol extracting in the culture.The mensuration of content of taxol adopts HPLC (Catalatic, 1993) and taxol monoclonal antibody (Hawaii Biotechnology Group Inc.) to join enzyme immunoassay (Jiang Chenggan, 1984) indirectly.Culture content of taxol (taxol/cell freeze-drying is heavy) callus 0.019% transgenosis crown gall cell 0.18%
Sequence table＜110〉Fudan University＜120〉utilize technique for gene engineering to improve the method for content of taxol＜130〉11＜160〉2＜170〉PatentIn version 3.1＜210〉1＜211〉2589＜212〉nucleotides＜213〉Chinese yew＜220＜221〉coded sequence＜222〉(1) .. (2589)＜223〉＜400〉1atg gct cag ctc tca ttt aat gca gcg ctg aag atg aac gca ttg ggg 48Met Ala Gln Leu Ser Phe Asn Ala Ala Leu Lys Met Asn Ala Leu Gly1,5 10 15aac aag gca atc cac gat cca acg aat tgc aga gcc aaa tct gag cgc 96Asn Lys Ala Ile His Asp Pro Thr Asn Cys Arg Ala Lys Ser Glu Arg
20??????????????????25??????????????????30caa?atg?atg?tgg?gtt?tgc?tcc?aga?tca?ggg?cga?acc?aga?gta?aaa?atg??????144Gln?Met?Met?Trp?Val?Cys?Ser?Arg?Ser?Gly?Arg?Thr?Arg?Val?Lys?Met
35??????????????????40??????????????????45tcg?aga?gga?agt?ggt?ggt?cct?ggt?cct?gtc?gta?atg?atg?agc?agc?agc??????192Ser?Arg?Gly?Ser?Gly?Gly?Pro?Gly?Pro?Val?Val?Met?Met?Ser?Ser?Ser
50??????????????????55??????????????????60act?ggc?act?agc?aag?gtg?gtt?tcc?gag?act?tcc?agt?acc?att?gtg?gat??????240Thr?Gly?Thr?Ser?Lys?Val?Val?Ser?Glu?Thr?Ser?Ser?Thr?Ile?Val?Asp65??????????????????70??????????????????75??????????????????80gat?atc?cct?cga?ctc?tcc?gcc?aat?tat?cat?ggc?gat?ctg?tgg?cac?cac??????288Asp?Ile?Pro?Arg?Leu?Ser?Ala?Asn?Tyr?His?Gly?Asp?Leu?Trp?His?His
85??????????????????90??????????????????95aat?gtt?ata?caa?act?ctg?gag?aca?ccg?ttt?cgt?gag?agt?tct?act?tac??????336Asn?Val?Ile?Gln?Thr?Leu?Glu?Thr?Pro?Phe?Arg?Glu?Ser?Ser?Thr?Tyr
100?????????????????105?????????????????110caa?gaa?cgg?gca?gat?gag?ctg?gtt?gtg?aaa?att?aaa?gat?atg?ttc?aat??????384Gln?Glu?Arg?Ala?Asp?Glu?Leu?Val?Val?Lys?Ile?Lys?Asp?Met?Phe?Asn
115?????????????????120?????????????????125gcg?ctc?gga?gac?gga?gat?atc?agt?ccg?tct?gca?tac?gac?act?gcg?tgg??????432Ala?Leu?Gly?Asp?Gly?Asp?Ile?Ser?Pro?Ser?Ala?Tyr?Asp?Thr?Ala?Trp
130?????????????????135?????????????????140gtg?gcg?agg?ctg?gcg?acc?att?tcc?tct?gat?gga?tct?gag?aag?cca?cgg??????480Val?Ala?Arg?Leu?Ala?Thr?Ile?Ser?Ser?Asp?Gly?Ser?Glu?Lys?Pro?Arg145?????????????????150?????????????????155?????????????????160ttt?cct?cag?gcc?ctc?aac?tgg?gtt?ttc?aac?aac?cag?ctc?cag?gat?gga??????528Phe?Pro?Gln?Ala?Leu?Asn?Trp?Val?Phe?Asn?Asn?Gln?Leu?Gln?Asp?Gly
165?????????????????170?????????????????175tcg?tgg?ggt?atc?gaa?tcg?cac?ttt?agt?tta?tgc?gat?cga?ttg?ctt?aac??????576Ser?Trp?Gly?Ile?Glu?Ser?His?Phe?Ser?Leu?Cys?Asp?Arg?Leu?Leu?Asn
180?????????????????185?????????????????190acg?acc?aat?tct?gtt?atc?gcc?ctc?tcg?gtt?tgg?aaa?aca?ggg?cac?agc??????624Thr?Thr?Asn?Ser?Val?Ile?Ala?Leu?Ser?Val?Trp?Lys?Thr?Gly?His?Ser
195?????????????????200?????????????????205caa?gta?caa?caa?ggt?gct?gag?ttt?att?gca?gag?aat?cta?aga?tta?ctc??????672Gln?Val?Gln?Gln?Gly?Ala?Glu?Phe?Ile?Ala?Glu?Asn?Leu?Arg?Leu?Leu
210?????????????????215?????????????????220aat?gag?gaa?gat?gag?ttg?tcc?ccg?gat?ttc?caa?ata?atc?ttt?cct?gct??????720Asn?Glu?Glu?Asp?Glu?Leu?Ser?Pro?Asp?Phe?Gln?Ile?Ile?Phe?Pro?Ala225?????????????????230?????????????????235?????????????????240ctg?ctg?caa?aag?gca?aaa?gcg?ttg?ggg?atc?aat?ctt?cct?tac?gat?ctt??????768Leu?Leu?Gln?Lys?Ala?Lys?Ala?Leu?Gly?Ile?Asn?Leu?Pro?Tyr?Asp?Leu
245?????????????????250?????????????????255cca?ttt?atc?aaa?tat?ttg?tcg?aca?aca?cgg?gaa?gcc?agg?ctt?aca?gat??????816Pro?Phe?Ile?Lys?Tyr?Leu?Ser?Thr?Thr?Arg?Glu?Ala?Arg?Leu?Thr?Asp
260?????????????????265?????????????????270gtt?tct?gcg?gca?gca?gac?aat?att?cca?gcc?aac?atg?ttg?aat?gcg?ttg??????864Val?Ser?Ala?Ala?Ala?Asp?Asn?Ile?Pro?Ala?Asn?Met?Leu?Asn?Ala?Leu
275?????????????????280?????????????????285gaa?ggt?ctc?gag?gaa?gtt?att?gac?tgg?aac?aag?att?atg?agg?ttt?caa??????912Glu?Gly?Leu?Glu?Glu?Val?Ile?Asp?Trp?Asn?Lys?Ile?Met?Arg?Phe?Gln
290?????????????????295?????????????????300agt?aaa?gat?gga?tct?ttc?ctg?agc?tcc?cct?gcc?tcc?act?gcc?tgt?gta?????960Ser?Lys?Asp?Gly?Ser?Phe?Leu?Ser?Ser?Pro?Ala?Ser?Thr?Ala?Cys?Val305?????????????????310?????????????????315?????????????????320ctg?atg?aat?aca?ggg?gac?gaa?aaa?tgt?ttc?act?ttt?ctc?aac?aat?ctg????1008Leu?Met?Asn?Thr?Gly?Asp?Glu?Lys?Cys?Phe?Thr?Phe?Leu?Asn?Asn?Leu
325?????????????????330?????????????????335ctc?gac?aaa?ttc?ggc?ggc?tgc?gtg?ccc?tgt?atg?tat?tcc?atc?gat?ctg????1056Leu?Asp?Lys?Phe?Gly?Gly?Cys?Val?Pro?Cys?Met?Tyr?Ser?Ile?Asp?Leu
340?????????????????345?????????????????350ctg?gaa?cgc?ctt?tcg?ctg?gtt?gat?aac?att?gag?cat?ctc?gga?atc?ggt????1104Leu?Glu?Arg?Leu?Ser?Leu?Val?Asp?Asn?Ile?Glu?His?Leu?Gly?Ile?Gly
355?????????????????360?????????????????365cgc?cat?ttc?aaa?caa?gaa?atc?aaa?gga?gct?ctt?gat?tat?gtc?tac?aga????1152Arg?His?Phe?Lys?Gln?Glu?Ile?Lys?Gly?Ala?Leu?Asp?Tyr?Val?Tyr?Arg
370?????????????????375?????????????????380cat?tgg?agt?gaa?agg?ggc?atc?ggt?tgg?ggc?aga?gac?agc?ctt?gtt?cca????1200His?Trp?Ser?Glu?Arg?Gly?Ile?Gly?Trp?Gly?Arg?Asp?Ser?Leu?Val?Pro385?????????????????390?????????????????395?????????????????400gat?ctc?aac?acc?aca?gcc?ctc?ggc?ctg?cga?act?ctt?cgc?atg?cac?gga????1248Asp?Leu?Asn?Thr?Thr?Ala?Leu?Gly?Leu?Arg?Thr?Leu?Arg?Met?His?Gly
405?????????????????410?????????????????415tac?aat?gtt?tct?tca?gac?gtt?ttg?aat?aat?ttc?aaa?gat?gaa?aac?ggg????1296Tyr?Asn?Val?Ser?Ser?Asp?Val?Leu?Asn?Asn?Phe?Lys?Asp?Glu?Asn?Gly
420?????????????????425?????????????????430cgg?ttc?ttc?tcc?tct?gcg?ggc?caa?acc?cat?gtc?gaa?ttg?aga?agc?gtg????1344Arg?Phe?Phe?Ser?Ser?Ala?Gly?Gln?Thr?His?Val?Glu?Leu?Arg?Ser?Val
435?????????????????440?????????????????445gtg?aat?ctt?ttc?aga?gct?tcc?gac?ctt?gca?ttt?cct?gac?gaa?aga?gct????1392Val?Asn?Leu?Phe?Arg?Ala?Ser?Asp?Leu?Ala?Phe?Pro?Asp?Glu?Arg?Ala
450?????????????????455?????????????????460atg?gac?gat?gct?aga?aaa?ttt?gca?gaa?cca?tat?ctt?aga?gag?gca?ctt????1440Met?Asp?Asp?Ala?Arg?Lys?Phe?Ala?Glu?Pro?Tyr?Leu?Arg?Glu?Ala?Leu465?????????????????470?????????????????475?????????????????480gca?acg?aaa?atc?tca?acc?aat?aca?aaa?cta?ttc?aaa?gag?att?gag?tac????1488Ala?Thr?Lys?Ile?Ser?Thr?Asn?Thr?Lys?Leu?Phe?Lys?Glu?Ile?Glu?Tyr
485?????????????????490?????????????????495gtg?gtg?gag?tac?cct?tgg?cac?atg?agt?atc?cca?cgc?tta?gaa?gcc?aga????1536Val?Val?Glu?Tyr?Pro?Trp?His?Met?Ser?Ile?Pro?Arg?Leu?Glu?Ala?Arg
500?????????????????505?????????????????510agt?tat?att?gat?tca?tat?gac?gac?aat?tat?gta?tgg?cag?agg?aag?act????1584Ser?Tyr?Ile?Asp?Ser?Tyr?Asp?Asp?Asn?Tyr?Val?Trp?Gln?Arg?Lys?Thr
515?????????????????520?????????????????525cta?tat?aga?atg?cca?tct?ttg?agt?aat?tca?aaa?tgt?tta?gaa?ttg?gca????1632Leu?Tyr?Arg?Met?Pro?Ser?Leu?Ser?Asn?Ser?Lys?Cys?Leu?Glu?Leu?Ala
530?????????????????535?????????????????540aaa?ttg?gac?ttc?aat?atc?gta?caa?tct?ttg?cat?caa?gag?gag?ttg?aag????1680Lys?Leu?Asp?Phe?Asn?Ile?Val?Gln?Ser?Leu?His?Gln?Glu?Glu?Leu?Lys545?????????????????550?????????????????555?????????????????560ctt?cta?aca?aga?tgg?tgg?aag?gaa?tcc?ggc?atg?gca?gat?ata?aat?ttc????1728Leu?Leu?Thr?Arg?Trp?Trp?Lys?Glu?Ser?Gly?Met?Ala?Asp?Ile?Asn?Phe
565?????????????????570?????????????????575act?cga?cac?cga?gtg?gcg?gag?gtt?tat?ttt?tca?tca?gct?aca?ttt?gaa????1776Thr?Arg?His?Arg?Val?Ala?Glu?Val?Tyr?Phe?Ser?Ser?Ala?Thr?Phe?Glu
580?????????????????585?????????????????590ccc?gaa?tat?tct?gcc?act?aga?att?gcc?ttc?aca?aaa?att?ggt?tgt?tta????1824Pro?Glu?Tyr?Ser?Ala?Thr?Arg?Ile?Ala?Phe?Thr?Lys?Ile?Gly?Cys?Leu
595?????????????????600?????????????????605caa?gtc?ctt?ttt?gat?gat?atg?gct?gac?atc?ttt?gca?aca?cta?gat?gaa????1872Gln?Val?Leu?Phe?Asp?Asp?Met?Ala?Asp?Ile?Phe?Ala?Thr?Leu?Asp?Glu
610?????????????????615?????????????????620ttg?aaa?agt?ttc?act?gag?gga?gta?aag?aga?tgg?gat?aca?tct?ttg?cta????1920Leu?Lys?Ser?Phe?Thr?Glu?Gly?Val??Lys?Arg?Trp?Asp?Thr?Ser?Leu?Leu625?????????????????630??????????????????635?????????????????640cat?gag?att?cca?gag?tgt?atg?caa?act?tgc?ttt?aaa?gtt?tgg?ttc?aaa????1968His?Glu?Ile?Pro?Glu?Cys?Met?Gln?Thr?Cys?Phe?Lys?Val?Trp?Phe?Lys
645?????????????????650?????????????????655tta?atg?gaa?gaa?gta?aat?aat?gat?gtg?gtt?aag?gta?caa?gga?cgt?gac????2016Leu?Met?Glu?Glu?Val?Asn?Asn?Asp?Val?Val?Lys?Val?Gln?Gly?Arg?Asp
660?????????????????665?????????????????670atg?ctc?gct?cac?ata?aga?aaa?ccc?tgg?gag?ttg?tac?ttc?aat?tgt?tat????2064Met?Leu?Ala?His?Ile?Arg?Lys?Pro?Trp?Glu?Leu?Tyr?Phe?Asn?Cys?Tyr
675?????????????????680?????????????????685gta?caa?gaa?agg?gag?tgg?ctt?gaa?gcc?ggg?tat?ata?cca?act?ttt?gaa????2112Val?Gln?Glu?Arg?Glu?Trp?Leu?Glu?Ala?Gly?Tyr?Ile?Pro?Thr?Phe?Glu
690?????????????????695?????????????????700gag?tac?tta?aag?act?tat?gct?ata?tca?gta?ggc?ctt?gga?ccg?tgt?acc????2160Glu?Tyr?Leu?Lys?Thr?Tyr?Ala?Ile?Ser?Val?Gly?Leu?Gly?Pro?Cys?Thr705?????????????????710?????????????????715?????????????????720cta?caa?cca?ata?cta?cta?atg?ggt?gag?ctt?gtg?aaa?gat?gat?gtt?gtt????2208Leu?Gln?Pro?Ile?Leu?Leu?Met?Gly?Glu?Leu?Val?Lys?Asp?Asp?Val?Val
725?????????????????730?????????????????735gag?aaa?gtg?cac?tat?ccc?tca?aat?atg?ttt?gag?ctt?gta?tcc?ttg?agc????2256Glu?Lys?Val?His?Tyr?Pro?Ser?Asn?Met?Phe?Glu?Leu?Val?Ser?Leu?Ser
740?????????????????745?????????????????750tgg?cga?cta?aca?aac?gac?acc?aaa?aca?tat?cag?gct?gaa?aag?gct?cga????2304Trp?Arg?Leu?Thr?Asn?Asp?Thr?Lys?Thr?Tyr?Gln?Ala?Glu?Lys?Ala?Arg
755?????????????????760?????????????????765gga?caa?caa?gcc?tca?ggc?ata?gca?tgc?tat?atg?aag?gat?aat?cca?gga????2352Gly?Gln?Gln?Ala?Ser?Gly?Ile?Ala?Cys?Tyr?Met?Lys?Asp?Asn?Pro?Gly
770?????????????????775?????????????????780gca?act?gag?gaa?gat?gcc?att?aag?cac?ata?tgt?cgt?gtt?gtt?gat?cgg????2400Ala?Thr?Glu?Glu?Asp?Ala?Ile?Lys?His?Ile?Cys?Arg?Val?Val?Asp?Arg785?????????????????790?????????????????795?????????????????800gcc?ttg?aaa?gaa?gca?agc?ttt?gaa?tat?ttc?aaa?cca?tcc?aat?gat?atc????2448Ala?Leu?Lys?Glu?Ala?Ser?Phe?Glu?Tyr?Phe?Lys?Pro?Ser?Asn?Asp?Ile
805?????????????????810?????????????????815cca?atg?ggt?tgc?aag?tcc?ttt?att?ttt?aac?ctt?aga?ttg?tgt?gtc?caa????2496Pro?Met?Gly?Cys?Lys?Ser?Phe?Ile?Phe?Asn?Leu?Arg?Leu?Cys?Val?Gln
820?????????????????825?????????????????830atc?ttt?tac?aag?ttt?ata?gat?ggg?tac?gga?atc?gcc?aat?gag?gag?att????2544Ile?Phe?Tyr?Lys?Phe?Ile?Asp?Gly?Tyr?Gly?Ile?Ala?Asn?Glu?Glu?Ile
835?????????????????840?????????????????845aag?gac?tat?ata?aga?aaa?gtt?tat?att?gat?cca?att?caa?gta?tga????????2589Lys?Asp?Tyr?Ile?Arg?Lys?Val?Tyr?Ile?Asp?Pro?Ile?Gln?Val
850 855 860＜210〉2＜211〉862＜212〉amino acid＜213〉Ramulus et folium taxi cuspidatae＜400〉2Met Ala Gln Leu Ser Phe Asn Ala Ala Leu Lys Met Asn Ala Leu Gly1,5 10 15Asn Lys Ala Ile His Asp Pro Thr Asn Cys Arg Ala Lys Ser Glu Arg
20??????????????????25??????????????????30Gln?Met?Met?Trp?Val?Cys?Ser?Arg?Ser?Gly?Arg?Thr?Arg?Val?Lys?Met
35??????????????????40??????????????????45Ser?Arg?Gly?Ser?Gly?Gly?Pro?Gly?Pro?Val?Val?Met?Met?Ser?Ser?Ser
50??????????????????55??????????????????60Thr?Gly?Thr?Ser?Lys?Val?Val?Ser?Glu?Thr?Ser?Ser?Thr?Ile?Val?Asp65??????????????????70??????????????????75??????????????????80Asp?Ile?Pro?Arg?Leu?Ser?Ala?Asn?Tyr?His?Gly?Asp?Leu?Trp?His?His
85??????????????????90??????????????????95Asn?Val?Ile?Gln?Thr?Leu?Glu?Thr?Pro?Phe?Arg?Glu?Ser?Ser?Thr?Tyr
100?????????????????105?????????????????110Gln?Glu?Arg?Ala?Asp?Glu?Leu?Val?Val?Lys?Ile?Lys?Asp?Met?Phe?Asn
115?????????????????120?????????????????125Ala?Leu?Gly?Asp?Gly?Asp?Ile?Ser?Pro?Ser?Ala?Tyr?Asp?Thr?Ala?Trp
130?????????????????135?????????????????140Val?Ala?Arg?Leu?Ala?Thr?Ile?Ser?Ser?Asp?Gly?Ser?Glu?Lys?Pro?Arg145?????????????????150?????????????????155?????????????????160Phe?Pro?Gln?Ala?Leu?Asn?Trp?Val?Phe?Asn?Asn?Gln?Leu?Gln?Asp?Gly
165?????????????????170?????????????????175Ser?Trp?Gly?Ile?Glu?Ser?His?Phe?Ser?Leu?Cys?Asp?Arg?Leu?Leu?Asn
180?????????????????185?????????????????190Thr?Thr?Asn?Ser?Val??Ile?Ala?Leu?Ser?Val?Trp?Lys?Thr?Gly?His?Ser
195??????????????????200?????????????????205Gln?Val?Gln?Gln?Gly?Ala?Glu?Phe?Ile?Ala?Glu?Asn?Leu?Arg?Leu?Leu
210?????????????????215?????????????????220Asn?Glu?Glu?Asp?Glu?Leu?Ser?Pro?Asp?Phe?Gln?Ile?Ile?Phe?Pro?Ala225?????????????????230?????????????????235?????????????????240Leu?Leu?Gln?Lys?Ala?Lys?Ala?Leu?Gly?Ile?Asn?Leu?Pro?Tyr?Asp?Leu
245?????????????????250?????????????????255Pro?Phe?Ile?Lys?Tyr?Leu?Ser?Thr?Thr?Arg?Glu?Ala?Arg?Leu?Thr?Asp
260?????????????????265?????????????????270Val?Ser?Ala?Ala?Ala?Asp?Asn?Ile?Pro?Ala?Asn?Met?Leu?Asn?Ala?Leu
275?????????????????280?????????????????285Glu?Gly?Leu?Glu?Glu?Val?Ile?Asp?Trp?Asn?Lys?Ile?Met?Arg?Phe?Gln
290?????????????????295?????????????????300Ser?Lys?Asp?Gly?Ser?Phe?Leu?Ser?Ser?Pro?Ala?Ser?Thr?Ala?Cys?Val305?????????????????310?????????????????315?????????????????320Leu?Met?Asn?Thr?Gly?Asp?Glu?Lys?Cys?Phe?Thr?Phe?Leu?Asn?Asn?Leu
325?????????????????330?????????????????335Leu?Asp?Lys?Phe?Gly?Gly?Cys?Val?Pro?Cys?Met?Tyr?Ser?Ile?Asp?Leu
340?????????????????345?????????????????350Leu?Glu?Arg?Leu?Ser?Leu?Val?Asp?Asn?Ile?Glu?His?Leu?Gly?Ile?Gly
355?????????????????360?????????????????365Arg?His?Phe?Lys?Gln?Glu?Ile?Lys?Gly?Ala?Leu?Asp?Tyr?Val?Tyr?Arg
370?????????????????375?????????????????380His?Trp?Ser?Glu?Arg?Gly?Ile?Gly?Trp?Gly?Arg?Asp?Ser?Leu?Val?Pro385?????????????????390?????????????????395?????????????????400Asp?Leu?Asn?Thr?Thr?Ala?Leu?Gly?Leu?Arg?Thr?Leu?Arg?Met?His?Gly
405?????????????????410?????????????????415Tyr?Asn?Val?Ser?Ser?Asp?Val?Leu?Asn?Asn?Phe?Lys?Asp?Glu?Asn?Gly
420?????????????????425?????????????????430Arg?Phe?Phe?Ser?Ser?Ala?Gly?Gln?Thr?His?Val?Glu?Leu?Arg?Ser?Val
435?????????????????440?????????????????445Val?Asn?Leu?Phe?Arg?Ala?Ser?Asp?Leu?Ala?Phe?Pro?Asp?Glu?Arg?Ala
450?????????????????455?????????????????460Met?Asp?Asp?Ala?Arg?Lys?Phe?Ala?Glu?Pro?Tyr?Leu?Arg?Glu?Ala?Leu465?????????????????470?????????????????475?????????????????480Ala?Thr?Lys?Ile?Ser?Thr?Asn?Thr?Lys?Leu?Phe?Lys?Glu?Ile?Glu?Tyr
485?????????????????490?????????????????495Val?Val?Glu?Tyr?Pro?Trp?His?Met?Ser?Ile?Pro?Arg?Leu?Glu?Ala?Arg
500?????????????????505?????????????????510Ser?Tyr?Ile?Asp?Ser?Tyr?Asp?Asp?Asn?Tyr?Val?Trp?Gln?Arg?Lys?Thr
515?????????????????520?????????????????525Leu?Tyr?Arg?Met?Pro?Ser?Leu?Ser?Asn?Ser?Lys?Cys?Leu?Glu?Leu?Ala
530?????????????????535?????????????????540Lys?Leu?Asp?Phe?Asn?Ile?Val?Gln?Ser?Leu?His?Gln?Glu?Glu?Leu?Lys545?????????????????550?????????????????555?????????????????560Leu?Leu?Thr?Arg?Trp?Trp?Lys?Glu?Ser?Gly?Met?Ala?Asp?Ile?Asn?Phe
565?????????????????570?????????????????575Thr?Arg?His?Arg?Val?Ala?Glu?Val?Tyr?Phe?Ser?Ser?Ala?Thr?Phe?Glu
580?????????????????585?????????????????590Pro?Glu?Tyr?Ser?Ala?Thr?Arg?Ile?Ala?Phe?Thr?Lys?Ile?Gly?Cys?Leu
595?????????????????600?????????????????605Gln?Val?Leu?Phe?Asp?Asp?Met?Ala?Asp?Ile?Phe?Ala?Thr?Leu?Asp?Glu
610?????????????????615?????????????????620Leu?Lys?Ser?Phe?Thr?Glu?Gly?Val?Lys?Arg?Trp?Asp?Thr?Ser?Leu?Leu625?????????????????630?????????????????635?????????????????640His?Glu?Ile?Pro?Glu?Cys?Met?Gln?Thr?Cys?Phe?Lys?Val?Trp?Phe?Lys
645?????????????????650?????????????????655Leu?Met?Glu?Glu?Val?Asn?Asn?Asp?Val?Val?Lys?Val?Gln?Gly?Arg?Asp
660?????????????????665?????????????????670Met?Leu?Ala?His?Ile?Arg?Lys?Pro?Trp?Glu?Leu?Tyr?Phe?Asn?Cys?Tyr
675?????????????????680?????????????????685Val?Gln?Glu?Arg?Glu?Trp?Leu?Glu?Ala?Gly?Tyr?Ile?Pro?Thr?Phe?Glu
690?????????????????695?????????????????700Glu?Tyr?Leu?Lys?Thr?Tyr?Ala?Ile?Ser?Val?Gly?Leu?Gly?Pro?Cys?Thr705?????????????????710?????????????????715?????????????????720Leu?Gln?Pro?Ile?Leu?Leu?Met?Gly?Glu?Leu?Val?Lys?Asp?Asp?Val?Val
725?????????????????730?????????????????735Glu?Lys?Val?His?Tyr?Pro?Ser?Asn?Met?Phe?Glu?Leu?Val?Ser?Leu?Ser
740?????????????????745?????????????????750Trp?Arg?Leu?Thr?Asn?Asp?Thr?Lys?Thr?Tyr?Gln?Ala?Glu?Lys?Ala?Arg
755?????????????????760?????????????????765Gly?Gln?Gln?Ala?Ser?Gly?Ile?Ala?Cys?Tyr?Met?Lys?Asp?Asn?Pro?Gly
770?????????????????775?????????????????780Ala?Thr?Glu?Glu?Asp?Ala?Ile?Lys?His?Ile?Cys?Arg?Val?Val?Asp?Arg785?????????????????790?????????????????795?????????????????800Ala?Leu?Lys?Glu?Ala?Ser?Phe?Glu?Tyr?Phe?Lys?Pro?Ser?Asn?Asp?Ile
805?????????????????810?????????????????815Pro?Met?Gly?Cys?Lys?Ser?Phe?Ile?Phe?Asn?Leu?Arg?Leu?Cys?Val?Gln
820?????????????????825?????????????????830Ile?Phe?Tyr?Lys?Phe?Ile?Asp?Gly?Tyr?Gly?Ile?Ala?Asn?Glu?Glu?Ile
835?????????????????840?????????????????845Lys?Asp?Tyr?Ile?Arg?Lys?Val?Tyr?Ile?Asp?Pro?Ile?Gln?Val
850?????????????????855?????????????????860
Claims (7)
1. isolated dna molecular, it is characterized in that, it comprises: have the nucleotide sequence of coding Japanese yew diene synthetic enzyme, and show at least 75% homology from the nucleotides sequence of Nucleotide 1-2589 position among described nucleotide sequence and the SEQ ID NO.3; Perhaps described nucleotide sequence can be under the rigorous condition of moderate with SEQ ID NO.3 in from the nucleotide sequence hybridization of Nucleotide 1-2589 position.
2. dna molecular as claimed in claim 1 is characterized in that, this sequence has among the SEQ ID NO.3 nucleotide sequence from Nucleotide 1-2589 position.
3. the enzyme by the described dna encoding of claim 1 is characterized in that: have SEQ ID NO.4 aminoacid sequence or its conservative property variation polypeptide or its active fragments or its reactive derivative.
4. method of utilizing transgenic technology to improve bearing taxanes and content of taxol in the life entity is characterised in that its step is as follows:
(1) adopt gene clone or synthetic gene method to obtain bearing taxanes and taxol biosynthetic pathway key gene Japanese yew diene synthase gene;
(2) key gene operationally is connected in expression regulation sequence, forms the expression vector that contains key gene;
(3) adopt any transgenic method transfer key gene or its expression vector in any host cell that can produce bearing taxanes and taxol; Screening and evaluation transformant, the transformant of Japanese yew diene synthetase albumen gene is expressed in acquisition;
(4) bearing taxanes in the transformant and content of taxol are measured, cultivated the transformant that bearing taxanes and content of taxol improve, obtain genetically modified organism.
5. a carrier is characterized in that, it comprises the described DNA of claim 1.
6. a cell that is obtained by the described method of claim 5 is characterized in that it is the cell that can produce taxol.
7. offspring who obtains the transgenosis life entity that bearing taxanes and content of taxol improve by the described method of claim 5.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 02111944 CN1385530A (en) | 2002-06-04 | 2002-06-04 | Method for raising taxol content by gene engineering technology |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 02111944 CN1385530A (en) | 2002-06-04 | 2002-06-04 | Method for raising taxol content by gene engineering technology |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN1385530A true CN1385530A (en) | 2002-12-18 |
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN 02111944 Pending CN1385530A (en) | 2002-06-04 | 2002-06-04 | Method for raising taxol content by gene engineering technology |
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| CN (1) | CN1385530A (en) |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100393879C (en) * | 2005-12-26 | 2008-06-11 | 合肥宏源生物技术开发有限公司 | Process of inducing root growing of Chinese yew by electric punching transforming Ri plasmid |
| CN101074443B (en) * | 2006-11-14 | 2011-08-03 | 西南大学 | Istp-ads carrying plastid transporting peptide and its use |
| CN103146728A (en) * | 2013-02-28 | 2013-06-12 | 天津大学 | Microzyme for producing taxadiene and construction method thereof |
| CN112574968A (en) * | 2019-09-30 | 2021-03-30 | 中国科学院分子植物科学卓越创新中心 | Fusion protein for producing 5 alpha-hydroxy taxadiene and application thereof |
-
2002
- 2002-06-04 CN CN 02111944 patent/CN1385530A/en active Pending
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100393879C (en) * | 2005-12-26 | 2008-06-11 | 合肥宏源生物技术开发有限公司 | Process of inducing root growing of Chinese yew by electric punching transforming Ri plasmid |
| CN101074443B (en) * | 2006-11-14 | 2011-08-03 | 西南大学 | Istp-ads carrying plastid transporting peptide and its use |
| CN103146728A (en) * | 2013-02-28 | 2013-06-12 | 天津大学 | Microzyme for producing taxadiene and construction method thereof |
| CN103146728B (en) * | 2013-02-28 | 2015-05-20 | 天津大学 | Microzyme for producing taxadiene and construction method thereof |
| CN112574968A (en) * | 2019-09-30 | 2021-03-30 | 中国科学院分子植物科学卓越创新中心 | Fusion protein for producing 5 alpha-hydroxy taxadiene and application thereof |
| CN112574968B (en) * | 2019-09-30 | 2023-03-17 | 中国科学院分子植物科学卓越创新中心 | Fusion protein for producing 5 alpha-hydroxy taxadiene and application thereof |
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