CN104293756A - Dammarenediol synthase gene of panax japonicus var and applications thereof - Google Patents

Abstract

The invention discloses a dammarenediol synthase gene of panax japonicus var and applications thereof. The dammarenediol synthase gene of panax japonicus var is prepared by the following steps: carrying out transcriptome sequencing and Denovo splicing on panax japonicus var rhizome by utilizing the second-generation Sole xaHiSeq2000 for the first time; analyzing and finding out a candidate gene for encoding the dammarenediol synthase (DS) in panax japonicus var and performing in vitro cloning, to finally obtain the dammarenediol synthase gene of panax japonicus var, wherein the sequence of the gene is SEQIDNO.1. The gene is genetically transformed into the panax japonicus var through agrobacterium tumefaciens mediated transformation, to obtain transgenic plant with high content of dammarane saponin, thus providing a technical support for the industrial production of the dammarane saponin.

Description

A kind of Rhizoma Panacis bipinnatifidi dammarenediol synthase gene and application thereof

Technical field

The invention belongs to biological technical field, relate generally to clone and the application of dammarenediol synthetic enzyme in Rhizoma Panacis bipinnatifidi (Dammarenediol synthase, DS) gene.

Background technology

Rhizoma Panacis bipinnatifidi (Panax japonicus.C.A.Mey.var.bipinnatifidus (Seem.) C.Y.Wu et K.M.Feng) belongs to Araliaceae Panax (Panax L.) plant, it is one of traditional rare medicinal herbs, there is higher pharmaceutical use, as clearing heat and detoxicating, pleasant stomach invigorating, the effects such as tired, strengthening by means of tonics of dispelling of invigorating blood circulation, its principle active component is saponins compound, wherein based on Triterpenoids sapogenins saponins, simultaneously containing a small amount of dammarane type four-ring triterpenoid saponins.

Rhizoma Panacis bipinnatifidi is that Chinese Pharmacopoeia records kind.Have broad application prospects and considerable economic worth, the wild resource of current Chinese Rhizoma Panacis bipinnatifidi is bordering in imminent danger, by the biosynthetic pathway of activeconstituents triterpene saponin componds and the molecular mechanism of regulation and control thereof in research Rhizoma Panacis bipinnatifidi, find out key enzyme wherein, realize the location of its gene, clone and high expression, on a molecular scale artificial regulatory is carried out to triterpenoid saponin biosynthesizing, realize the scale production of triterpene saponin componds further, to provide the demand of medical market.

Dammarane type four-ring triterpenoid saponins compound in Rhizoma Panacis bipinnatifidi comes from isoprene approach.The cyclisation of 2,3-oxidosqualene is the committed step in triterpenoid saponin synthesis downstream pathway, and this step produces protopanoxadiol by dammarenediol synthetic enzyme (DS) catalysis, based on this compound, finally synthesize various dammarane type saponin(e in downstream.Therefore, this reactions steps is the biosynthetic important regulating and controlling site of triterpenoid saponin.

Up to now, the biosynthesizing research about Rhizoma Panacis bipinnatifidi activeconstituents pathways metabolism still belongs to blank, and in Rhizoma Panacis bipinnatifidi, clone's research of dammarenediol synthetic enzyme (DS) gene also has no relevant report.This patent utilizes s-generation Solexa HiSeq2000 to carry out the order-checking of Rhizoma Panacis bipinnatifidi rhizome transcript profile first and De novo splices.Analysis is found in Rhizoma Panacis bipinnatifidi the candidate gene of dammarenediol synthetic enzyme (DS) of encoding and is carried out body outer clone, expression checking, and the dammarane type four-ring triterpenoid saponins compound in Rhizoma Panacis bipinnatifidi comes from isoprene approach.The cyclisation of 2,3-oxidosqualene is the committed step in triterpenoid saponin synthesis downstream pathway, and this step produces protopanoxadiol by dammarenediol synthetic enzyme (DS) catalysis, based on this compound, finally synthesize various dammarane type saponin(e in downstream.Therefore, this reactions steps is the biosynthetic important regulating and controlling site of triterpenoid saponin, has established Research foundation for understanding this enzyme further in Rhizoma Panacis bipinnatifidi plant gene expression in vivo, Function and operation mechanism.

Summary of the invention

The object of the present invention is to provide a kind of Rhizoma Panacis bipinnatifidi dammarenediol synthase (DS) gene, its sequence is for shown in SEQ ID NO.1.The dammarenediol synthase of this genes encoding plays key effect in dammarane type saponin compound route of synthesis.

The present invention's second object is to provide a kind of protein of Rhizoma Panacis bipinnatifidi dammarenediol synthase gene encodes, and its sequence is for shown in SEQ ID NO.2.

Last object of the present invention is to provide Rhizoma Panacis bipinnatifidi dammarenediol synthase gene improving the application in panax species dammarane type saponin compound content, is particularly improving the application in Rhizoma Panacis bipinnatifidi dammarane type saponin compound content.

In order to achieve the above object, the present invention takes following technical measures:

A kind of Rhizoma Panacis bipinnatifidi dammarenediol synthase gene (Dammarenediol synthase, hereinafter referred to as DS gene), its preparation method is as follows:

With the cDNA chain of Rhizoma Panacis bipinnatifidi for template, with forward primer P1:5'-ATGTGGAAGCTGAAGGTTGCTC AAG-3', reverse primer P2:5'-TTAAATTTTGAGCTGCTGGTGGT-3', carry out pcr amplification.Amplification system is as follows: 10 × buffer 2.5ul, dNTP 1 ul, each 1ul of primer P1 and P2, Taq enzyme 0.5 ul, masterplate 1ul, and all the other are supplied with water, and cumulative volume is 25ul altogether.Reaction conditions: 30 reaction cycle, 94 DEG C of sex change 1min, 42 DEG C of annealing 2min, 75 DEG C extend 3min, and last 75 DEG C extend 10min.

Finally obtain DS gene, its sequence is for shown in SEQ ID NO.1, and the protein of coding is for shown in SEQ ID NO.2.

A kind of Rhizoma Panacis bipinnatifidi dammarenediol synthetic enzyme (DS) gene is improving the application in Rhizoma Panacis bipinnatifidi dammarane type saponin compound content, and its application process is as follows:

DS gene (shown in preferred SE Q ID NO.1) corresponding for Rhizoma Panacis bipinnatifidi dammarenediol synthetase protein (SEQ ID NO.2 shown in) is proceeded to Rhizoma Panacis bipinnatifidi by Agrobacterium-mediated genetic transformation, can obtain high dammarane's saponin content transfer-gen plant.

Claimed content of the present invention also comprises:

Amino acid whose nucleotide sequence shown in coding SEQ ID NO.2; Nucleotide sequence shown in preferred SEQ ID NO.1

Recombinant vectors containing Rhizoma Panacis bipinnatifidi dammarenediol synthase (DS) gene complete sequence of the present invention or its ORF sequence, as protokaryon class carrier, eucaryon class expression vector and RNAi carrier all belong to protection scope of the present invention, and described protokaryon class carrier includes but not limited to pT7-Blue; Described eucaryon class expression vector includes but not limited to pYES2, pBI 121, pRS314.

Containing Rhizoma Panacis bipinnatifidi dammarenediol synthase (DS) gene complete sequence of the present invention or its ORF sequence host cell; as the host cell containing above-mentioned recombinant vectors also belongs to protection scope of the present invention, include but not limited to the living materials such as E.coli, tobacco, Arabidopis thaliana, yeast host bacterium GIL77, intestinal bacteria, Rhizoma Panacis bipinnatifidi.

The application of Rhizoma Panacis bipinnatifidi dammarenediol synthase (DS) gene of the present invention, comprises with described recombinant vectors, as plant expression vector transformed plant cells; Or with described hair root cells regeneration plant; Or with described Rhizoma Panacis bipinnatifidi dammarenediol synthase (DS) gene complete sequence or the Sequence Transformed acquisition transgenic organism of its ORF.

Utilize Rhizoma Panacis bipinnatifidi dammarenediol synthase (DS) of the present invention, by various conventional screening assays, can filter out, with Rhizoma Panacis bipinnatifidi dammarenediol synthase (DS), interactional material occur, or acceptor, inhibitor or antagonist etc.

Compared with prior art, the present invention has the following advantages:

Rhizoma Panacis bipinnatifidi dammarenediol synthase (DS) gene provided by the present invention is clone's preparation from Rhizoma Panacis bipinnatifidi first, utilize technology of the present invention can carry out genetic engineering modified to the medicinal plant that Rhizoma Panacis bipinnatifidi etc. contains similar compound, improved the content of the dammarane saponins compound in plant materials by transgenosis.Rhizoma Panacis bipinnatifidi dammarenediol synthetic enzyme (DS) gene can participate in the biosynthesizing of Rhizoma Panacis bipinnatifidi dammarane type saponin compound, and therefore the application provides theoretical foundation for the biosynthetic further research of Rhizoma Panacis bipinnatifidi dammarane type saponin compound and suitability for industrialized production.

Positively effect in its industry of further interpolation, the present invention utilizes transgenic technology to obtain the high yield strain of dammarane type saponin compound content increase, for the suitability for industrialized production of dammarane type saponin(e provides technical support.

Accompanying drawing explanation

Fig. 1 is Rhizoma Panacis bipinnatifidi RNA electrophorogram.

Fig. 2 is DS functional domain forecast analysis schematic diagram.

Fig. 3 is DS systematic evolution tree.

Adjacent method is adopted to build PpgDS phylogenetic tree.

Fig. 4 is that enzyme activity detects schematic diagram.

HPLC detects enzymatic preparation, and figure A enzyme product is detected by HPLC to be analyzed, and detection ultraviolet wavelength is 202nm; B figure is dammarenediol HPLC analysis chart.

Embodiment

Scheme of the present invention if not otherwise specified, is the conventional scheme of this area, agents useful for same if not otherwise specified, all purchased from biochemical shop.

Embodiment 1

The order-checking of Rhizoma Panacis bipinnatifidi rhizome transcript profile and data analysis

1, sample collecting

4 years raw Rhizoma Panacis bipinnatifidi (Panax japonicus.C.A.Mey.var.bipinnatifidus (Seem.) C.Y.Wu et K.M.Feng) pick up from enshi.Get rhizome, leaf, flower respectively, fruit is put in liquid nitrogen after quick-frozen, freezes save backup-80 DEG C of refrigerator and cooled.

2, the separation and detection of Rhizoma Panacis bipinnatifidi total serum IgE

P-80 DEG C of Different categories of samples preserved fully grind in liquid nitrogen, then the Trizol method of optimization is adopted sample to be carried out to the extraction of total serum IgE, whole process ensures to carry out under cryogenic, add certain density PVP solution (polyvinylpyrrolidone), and suitably strengthen the concentration of beta-mercaptoethanol, after centrifugal segregation PVP and beta-mercaptoethanol, adopt high density NaAc solution to separate out RNA, DNase and remove the purifying that residual DNA completes RNA.Detect the integrity (Fig. 1) of RNA by 1.0% agarose electrophoresis, measure A260, A280 ratio and concentration with Nanodrop2000 nucleic acid quantification instrument, it is for subsequent use that RNA sample is placed in-80 DEG C of refrigerators.

3, transcript profile order-checking (RNA-Seq)

With magnetic bead enrichment mRNA from total serum IgE of oligo (dT), connect and add fragmentation buffer mRNA is broken into short-movie section, take mRNA as template, Article 1 cDNA chain is synthesized with hexabasic base random primer (random hexamers), then damping fluid is added, dNTPs, RNase H and DNA polymerase I synthesizes Article 2 cDNA chain, end reparation is carried out after through QiaQuick PCR kit purifying and by EB buffer solution elution, add poly (A) and connect sequence measuring joints, agarose gel electrophoresis is separated and selects clip size, pcr amplification builds sequencing library, s-generation Solexa HiSeq2000 is utilized to carry out RNA order-checking, and De novo splices.

4, candidate gene tentatively sieves

Annotated by GO, the software analysis such as Blast compare of analysis and MEGA5.0 phylogenetic tree construction (Fig. 3) tentatively find in Rhizoma Panacis bipinnatifidi the candidate gene of dammarenediol synthase of encoding.

Embodiment 2:

The clone of Rhizoma Panacis bipinnatifidi dammarenediol synthase gene:

Utilize forward primer P1:5'-ATGTGGAAGCTGAAGGTTGC-3', reverse primer P2:5'-TTAAATTT TGAGCTGCTGGTGG-3', with Rhizoma Panacis bipinnatifidi rhizome cDNA library for template amplification candidate gene full length sequence enters performing PCR amplification.Amplification system is as follows: 10 × buffer 2.5ul, dNTP 1 ul, each 1ul of primer P1 and P2, Taq enzyme 0.5ul, masterplate 1ul, and all the other are supplied with water, and cumulative volume is 25ul altogether.Reaction conditions: 30 reaction cycle, 94 DEG C of sex change 1min, 42 DEG C of annealing 2min, 75 DEG C extend 3min.Last 75 DEG C extend 10min.

After clone's candidate gene full length sequence, being linked on cloning vector pT7-Blue and being transformed in competent escherichia coli cell E.coli DH5 α, step is as follows:

A) from-80 DEG C of Ultralow Temperature Freezers, get 100 μ L competent cell suspensions, thaw and be placed on ice;

B) add 5 μ L and connect product, blow and beat mixing gently with pipettor, place 30min on ice;

C) 42 DEG C of heat shock 90s, put rapidly 5min on ice;

D) in EP pipe, 1mL LB liquid nutrient medium (not containing microbiotic) is added, 37 DEG C of 200rpm 45min;

E) getting 100 μ L bacterium liquid after shaking bacterium coats containing on antibiotic flat board, and 37 DEG C of incubators spend the night;

F) picking list bacterium colony is in 4mL containing in antibiotic LB liquid nutrient medium, and 37 DEG C of 200rpm vibration training samples spend the night and choose the order-checking of positive colony sample presentation.

So far obtain Rhizoma Panacis bipinnatifidi dammarenediol synthase gene, its sequence is for shown in SEQ ID NO.1.

Embodiment 3:

The bioinformatic analysis of DS gene

Rhizoma Panacis bipinnatifidi dammarenediol synthase (DS) full length gene that the present invention obtains is 2310bp, and its sequence is for shown in SEQ ID NO.1, and wherein opening code-reading frame is positioned at 1 ~ 2310bp, and the protein sequence of coding is for shown in SEQ ID NO.2.In ncbi database, carry out Blast by splicing the dammarenediol synthase full-length sequence analyzed, this gene has typical ISOPREN_C2_like superfamily structural domain, as Fig. 2.

Embodiment 4:

The research of DS gene function

1, the structure of expression vector

With Rhizoma Panacis bipinnatifidi DS cDNA for masterplate, utilize forward primer P1:5'- gGTACCaTGTGGAAGCTGAAGG TTGC-3'; Reverse primer P2:5'- cTCGAGtTAAATTTTGAGCTGCTGGTGG-3' carries out PCR reaction, gets the gel electrophoresis of 5ul amplified production, takes a picture after half an hour, and observe glue figure, amplified fragments is 2322bp.Cut amplified production 2 hours with KpnI and XhoI enzyme, utilize and reclaim test kit (Takara company, China) purifying digestion products.Utilize Kp nI and XhoI enzyme enzyme at 37 DEG C to cut pRS314 carrier 2 hours simultaneously, then carry out agarose gel electrophoresis, observe glue figure, and utilize test kit recovery size to be about the fragment of 4761bp.

The two linked enzyme spends the night 16 DEG C of connections.Transformation of E. coli DH5a competent cell, the LB flat board containing ammonia benzyl mycin screens recon.Containing the pRS314 plasmid of DS clone through PCR and digestion with restriction enzyme electroresis appraisal and DNA sequence analysis, preserve and have the recombinant plasmid pRS314-DS of correct target for expressing conversion.This expression vector called after pRS314-DS.

2, the abduction delivering of albumen

With pRS314-DS Plastid transformation Partial digestion yeast host bacterium AB1380, cultivate and within 5 days, screen positive yeast afterwards in 2ml containing on the USM substratum of 2% glucose, after 4-5 days in picking mono-clonal 2ml USM liquid nutrient medium, 30 DEG C of concuss overnight incubation.5000rpm centrifugal cultivation 5min, abandons supernatant, dilutes 20 times with the USM liquid nutrient medium containing 2% glucose, and 30 DEG C of concuss cultivate 24h.Extract culturing cell total protein.Choose after high expression level transformant cultivates 20h with 50ml containing USM liquid nutrient medium 30 DEG C of concuss of 2% glucose, be inoculated into 1L and do not cultivate 40h containing USM liquid nutrient medium 30 DEG C of concuss of glucose, reclaim thalline, separating particles body, purifying protein.

3, enzymatic reaction qualification

External enzymatic reaction is with 2,3-oxygen MF59 (50 umolL ^-1) as reaction substrate, in system, add Triton X-100 (1 mg ml ^-1), 10 mmol L ^-1tris (pH6.0), 0.2 mmol L ^-1eDTA, 2ug purifying protein.Acutely rock 30min under 25 DEG C of conditions, after add 1ml 40%KOH stop enzymatic reaction.

HPLC analyzes its enzymatic preparation.Chromatographic column is silicagel column (4.6 mm × 250 mm); Moving phase is phosphate buffered saline buffer (50 mM, pH 4.5) and acetonitrile (90:10, v/v), flow velocity 1 mL/min; Determined wavelength is 220 nm; Column temperature 40 DEG C.

Enzymatic preparation and standard substance are contrasted, as shown in Figure 4, detect through HPLC, two kinds of material crests are completely the same, then provable, and enzymatic preparation is dammarenediol.

Embodiment 5:

DS carries out the mensuration of dammarane type saponin compound content in eukaryotic expression and transgenosis Rhizoma Panacis bipinnatifidi root of hair in Rhizoma Panacis bipinnatifidi

The acceptor material Rhizoma Panacis bipinnatifidi (Panax japonicus.C.A.Mey.var.bipinnatifidus (Seem.) C.Y.Wu et K.M.Feng) of transgenosis picks up from enshi city.

Adopt the ordinary skill in the art, according to the full length cDNA sequence (SEQ ID NO.1) of Rhizoma Panacis bipinnatifidi dammarenediol synthase gene, the both forward and reverse directions primer in amplification coding district is introduced restriction endonuclease sites (carrier depending on selecting), build plant expression vector, proceed to Rhizoma Panacis bipinnatifidi by Agrobacterium-mediated genetic transformation, screening transgenic plant detects the content of its dammarane type saponin(e.

Specific as follows:

The DS gene coding region (shown in SEQ ID NO.1) obtained in embodiment 2 is connected on the plasmid of pMD 18-T simple V ector, after carrying out pcr amplification, on forward and reverse primer, introduce restriction enzyme site KpnI and SalI respectively respectively, utilize forward primer P1:5'- gGTACCaTGTGGAAGCTGAAGGTTGC-3'; Reverse primer P2:5'- gTCGACtTAAATTTTGAGCTGCTGGTGG-3' carries out PCR reaction, and TA clones, and extracts plasmid.

With KpnI and SalI double digestion containing goal gene sequence PpgDS and prokaryotic expression carrier plasmid pBI121, digestion products reclaims corresponding fragment after agarose electrophoresis, pBI121 carrier and PpgDS goal gene fragment carry out ligation 16h through T4DNA ligase enzyme at 16 DEG C, connect product conversion competence e. coli bl21, screen with 50ug/mL kantlex, picking positive monoclonal bacterium colony enlarged culturing, extract plasmid and carry out enzyme and cut qualification, enzyme being cut the expression vector pBI-PpgDS identified proceeds in Agrobacterium, genetic transformation Rhizoma Panacis bipinnatifidi.

The genetic transformation of the Rhizoma Panacis bipinnatifidi utilizing Agrobacterium rhizogenes to mediate, required material and operation steps as follows:

1) Agrobacterium rhizogenesA4, take out in refrigerator before using, go down to posterity 2 times with YEB substratum, bacterial classification is inoculated in YEB liquid nutrient medium before use, 28 DEG C of overnight incubation.

2) the delicate blade of Rhizoma Panacis bipinnatifidi is got, clean to be placed in 70% alcohol and soak 1min, abandon alcohol, add 2% hypochlorite disinfectant 10min, period shake for several times, discard thimerosal, by rinsed with sterile water 4 ~ 5 times, be placed on aseptic filter paper and dry, by sterile razor blade, Rhizoma Panacis bipinnatifidi blade is cut into 5mm × 5mm small pieces, be placed on preculture solid medium, preculture 2d in (23 ± 1) DEG C camera bellows incubator.

3) after the Agrobacterium rhizogenesA4 bacterium liquid of incubated overnight is centrifugal, bacterial sediment 1/2MS is resuspended, takes out after being placed in 4 DEG C of 2h.Rhizoma Panacis bipinnatifidi blade preculture crossed is soaked in 5min in the resuspended bacterium liquid of 1/2MS, unnecessary bacterium liquid is sucked with aseptic filter paper, put into the 1/2MS solid medium containing 250-500mg/L kantlex, cultivate under (23 ± 1) DEG C dark condition, within every 2 weeks, to transfer in fresh culture 1 time, hairly root is separated after growing hairly root, be transferred to cultivating without in hormone 1/2MS solid medium containing 250-500mg/L kantlex, within every 2 weeks, to transfer in fresh culture to aseptic, and then be transferred to cultivating without in hormone 1/2MS substratum not containing kantlex.

4) the hairly root secondary culture on solid medium is inoculated in is equipped with in the 500ml triangular flask of 150ml without hormone 1/2MS liquid nutrient medium, the culture condition such as culture temperature, illumination, rotating speed are identical with callus fluid suspension culture condition, after cultivating 25d, hairly root is taken out from substratum and puts into freeze drier and carry out drying, then weigh, for subsequent use in storing-80 DEG C.

Positive strain utilizes conventional real-time PCR to carry out further screening verification, and method therefor of the present invention is specific as follows:

Extract and there is the total serum IgE of the conversion Rhizoma Panacis bipinnatifidi plant of kalamycin resistance, RNA reverse transcription is become cDNA, sxemiquantitative the primer be Rhizoma Panacis bipinnatifidi dammarenediol synthase gene special primer (forward primer 5 '- gGTACCaTGTGGAAACT GAAAGTGGC-3 '; Reverse primer 5 '- gTCGACaATTTTCAGCTGCTGATGTT-3 '), when response procedures is 94 DEG C 3 minutes, 94 DEG C of sex change 30 seconds, 61 DEG C of annealing 30 seconds, 72 DEG C extend 45 seconds, 25 circulations; After circulation completes, 72 DEG C extend 5 minutes.With the actin gene of Rhizoma Panacis bipinnatifidi as reference gene, forward primer 5'-GGAAAAGATTTGGCATC-3', reverse primer 5'-GGGCGTAACCCTCATA-3 '.The analysis of destination gene expression level is carried out under identical growth conditions is tied up to conversion to the wild-type of Rhizoma Panacis bipinnatifidi.Final selection is relative to WT lines, and the Fold-change value of gene expression amount carries out desired substance content detection higher than the transformed plant of more than 4 times (P<0.01) as positive strain.

5) HPLC containing the dammarane type sapogenin content of the transgenosis root of hair of Rhizoma Panacis bipinnatifidi dammarenediol gene detects

In transgenosis Rhizoma Panacis bipinnatifidi root of hair, the detection of the content of dammarane type saponin(e can use the ordinary skill in the art, and the present invention specifically adopts following steps:

The pre-treatment of root of hair system sample, with liquid nitrogen by sample grind into powder, respectively gets 0.1g and puts into eggplant-shape bottle, add the methyl alcohol of 4ml, the eggplant-shape bottle that sample is housed is fixed on serpentine condenser, be placed on the water-bath of 80 DEG C, refluxing extraction 12h, eggplant-shape bottle is taken out, sucking-off methanol extract liquid, and by a small amount of methanol wash sample powder two to three times, proceed in the lump in volumetric flask, be settled to 5ml, after 0.45um organic system membrane filtration, namely can be used for HPLC sample detection.

Adopt the high performance liquid chromatograph of Thermo Fisher company LC-MS instrument of the U.S. (LCQ Fleet), detect the content of sapogenin protopanoxadiol (PPD) and Protopanaxatriol (PPT) in transgenosis root of hair, chromatographic column is Hypersil ODS2C18 post (250nm × 4.6mm, 5um), chromatographic condition is as follows: moving phase is methanol-water (90:10) flow velocity 1.0ml/min, column temperature 25 DEG C, determined wavelength 203nm.

With non-transgenic Rhizoma Panacis bipinnatifidi root of hair be contrast, control group and transgenosis group all detect 10 strains, compare the average content of its dammarane type saponin(e.

Detected result shows: compared with control group, in the transgenosis Rhizoma Panacis bipinnatifidi of process LAN Rhizoma Panacis bipinnatifidi dammarenediol synthase gene, the content of protopanoxadiol saponin(e (PPD) on average improves 2.3 times, and the content of Protopanaxatriol's saponin(e (PPT) on average improves 3.6 times.

Prove thus, Rhizoma Panacis bipinnatifidi dammarenediol synthase gene is to promoting that the raising of Rhizoma Panacis bipinnatifidi dammarane type saponin content has remarkable effect, in the research that the transgenic technology that can be used for Rhizoma Panacis bipinnatifidi dammarenediol synthase gene utilizing improves Rhizoma Panacis bipinnatifidi dammarane type saponin content and industrialization, there is certain application prospect.

SEQUENCE LISTING

The old sheet of the yellow fine jade fine jade of <110> little imperial Wang Qi Zhu Chen Yan of roc Zhao Zhang Xifeng that continues hears monarch 5 Chong king as peak Deng Chen

<120> Rhizoma Panacis bipinnatifidi dammarenediol synthase gene and application thereof

<130> Rhizoma Panacis bipinnatifidi dammarenediol synthase gene and application thereof

<160> 2

<170> PatentIn version 3.1

<210> 1

<211> 2310

<212> DNA

<213> Rhizoma Panacis bipinnatifidi

<400> 1

atgtggaagc tgaaggttgc tcaaggaaat gatccatatt tgtatagcac taacaacttt 60

gttggcagac aatattggga gtttcagccc gatgctggta ctccagaaga gagggaagag 120

gttgaaaaag cacgcaagga ttatgtaaac aataagaagc tacatggaat tcatccatgc 180

agtgatatgc tgatgcgcag gcagcttatt aaagaaagtg gaatcgatct cctaagcata 240

ccgccggtga gattagatga aaacgaacaa gtgaactacg atgcagttac aaccgctgtg 300

aagaaagctc ttcgattgaa ccgggcaatt caagcacacg atggtcactg gccagctgaa 360

aatgcaggct ctttacttta tacacctccc cttatcattg ccctatatat cagcggaacg 420

attgacacta ttctgacaaa acaacacaag aaggaactga ttcgcttcgt ttacaaccat 480

caaaatgagg atggtggatg gggatcctat attgaggggc acagcacgat gattgggtca 540

gtacttagct acgtgatgtt acgtttgcta ggagaaggat tagctgaatc tgatggtgga 600

aatggtgcag ttgagagagg ccggaagtgg atacttgatc atggaggtgc agccagcata 660

ccctcttggg gaaagactta tctagcggtg cttggagtat atgagtggga agggtgcaac 720

ccgctgcccc cagaattctg gcttttccct tcaagttttc cttttcatcc agcaaaaatg 780

tggatctact gccggtgcac ttacatgcca atgtcgtatt tgtatgggaa gagatatcat 840

ggaccaataa ccgatcttgt tttatctttg agacaagaaa tttacaacat tccttatgag 900

cagataaagt ggaatcaaca gcgccataac tgttgcaagg aggatctcta ctaccctcat 960

acccttgtac aagacctggt ttgggatggt cttcactact ttagtgaacc attcctcaaa 1020

cgttggccct tcaacaaact gcgaaaaaga ggtctaaaaa gagtggttga actaatgcgc 1080

tatggtgcca ccgagaccag attcataacc acaggaaatg gggaaaaagc tttacaaata 1140

atgagttggt gggcagaaga tcccaatggt gatgagttta aacatcacct tgctagaatt 1200

cctgatttct tatggattgc tgaggatgga atgacagtac agagttttgg tagtcaacta 1260

tgggactgta ttcttgctac tcaagcaatt atcgccacca atatggttga agaatacgga 1320

gattctctta agaaggcgca tttcttcatc aaagaatcgc agataaaaga aaatccaaga 1380

ggagacttct taaaaatgtg tcgacagttt actaaaggtg cgtggacttt ctctgatcaa 1440

gatcatggtt gcgttgtctc ggactgcaca gctgaagcac taaagtgcct actgttactt 1500

tcacaaatgc cacaggatat tgtcggagaa aaacctgagg ttgagagatt atatgaggct 1560

gtgaatgttc ttctctattt gcagagtcgt gtaagtggtg gtttcgcagt ttgggagcct 1620

ccagttccaa aaccatattt ggagatgttg aatccttcag aaatttttgc agacattgtt 1680

gttgagagag agcacattga atgcactgca tctgtaatca aaggtctgat ggcatttaaa 1740

tgcttgcatc ctgggcatcg tcagaaagag atagaggatt ctgtggcgaa agccatccgt 1800

tatcttgaaa gaaaccaaat gcctgatggt tcatggtatg gcttttgggg aatttgtttc 1860

ctctatggga cattttttac cctatcaggg tttgcttctg ctgggaggac ttatgacaac 1920

agtgaagcag ttcgtaaggg tgttaaattt tacctttcaa cacaaaatga agaaggtggt 1980

tggggggaga gtcttgaatc atgcccaagc gaaaaattta caccactcaa gggaaacagg 2040

acaaatctag tacaaacatc atgggctatg ttaggtctta tgtttggtgg acaggccgag 2100

agagatccga cacctctgca tagagcagca aagttgttga tcaatgcgca aatggacaat 2160

ggagatttcc ctcaacagga aattactgga gtatactgta aaaatagtat gttacattat 2220

gcggagtaca gaaatatatt tcctctttgg gcacttggag aatatcggaa acgtgtttgg 2280

ttgcctaacc accagcagct caaaatttaa 2310

<210> 2

<211> 769

<212> PRT

<213> Rhizoma Panacis bipinnatifidi

<400> 2

Met Trp Lys Leu Lys Val Ala Gln Gly Asn Asp Pro Tyr Leu Tyr Ser

1 5 10 15

Thr Asn Asn Phe Val Gly Arg Gln Tyr Trp Glu Phe Gln Pro Asp Ala

20 25 30

Gly Thr Pro Glu Glu Arg Glu Glu Val Glu Lys Ala Arg Lys Asp Tyr

35 40 45

Val Asn Asn Lys Lys Leu His Gly Ile His Pro Cys Ser Asp Met Leu

50 55 60

Met Arg Arg Gln Leu Ile Lys Glu Ser Gly Ile Asp Leu Leu Ser Ile

65 70 75 80

Pro Pro Val Arg Leu Asp Glu Asn Glu Gln Val Asn Tyr Asp Ala Val

85 90 95

Thr Thr Ala Val Lys Lys Ala Leu Arg Leu Asn Arg Ala Ile Gln Ala

100 105 110

His Asp Gly His Trp Pro Ala Glu Asn Ala Gly Ser Leu Leu Tyr Thr

115 120 125

Pro Pro Leu Ile Ile Ala Leu Tyr Ile Ser Gly Thr Ile Asp Thr Ile

130 135 140

Leu Thr Lys Gln His Lys Lys Glu Leu Ile Arg Phe Val Tyr Asn His

145 150 155 160

Gln Asn Glu Asp Gly Gly Trp Gly Ser Tyr Ile Glu Gly His Ser Thr

165 170 175

Met Ile Gly Ser Val Leu Ser Tyr Val Met Leu Arg Leu Leu Gly Glu

180 185 190

Gly Leu Ala Glu Ser Asp Gly Gly Asn Gly Ala Val Glu Arg Gly Arg

195 200 205

Lys Trp Ile Leu Asp His Gly Gly Ala Ala Ser Ile Pro Ser Trp Gly

210 215 220

Lys Thr Tyr Leu Ala Val Leu Gly Val Tyr Glu Trp Glu Gly Cys Asn

225 230 235 240

Pro Leu Pro Pro Glu Phe Trp Leu Phe Pro Ser Ser Phe Pro Phe His

245 250 255

Pro Ala Lys Met Trp Ile Tyr Cys Arg Cys Thr Tyr Met Pro Met Ser

260 265 270

Tyr Leu Tyr Gly Lys Arg Tyr His Gly Pro Ile Thr Asp Leu Val Leu

275 280 285

Ser Leu Arg Gln Glu Ile Tyr Asn Ile Pro Tyr Glu Gln Ile Lys Trp

290 295 300

Asn Gln Gln Arg His Asn Cys Cys Lys Glu Asp Leu Tyr Tyr Pro His

305 310 315 320

Thr Leu Val Gln Asp Leu Val Trp Asp Gly Leu His Tyr Phe Ser Glu

325 330 335

Pro Phe Leu Lys Arg Trp Pro Phe Asn Lys Leu Arg Lys Arg Gly Leu

340 345 350

Lys Arg Val Val Glu Leu Met Arg Tyr Gly Ala Thr Glu Thr Arg Phe

355 360 365

Ile Thr Thr Gly Asn Gly Glu Lys Ala Leu Gln Ile Met Ser Trp Trp

370 375 380

Ala Glu Asp Pro Asn Gly Asp Glu Phe Lys His His Leu Ala Arg Ile

385 390 395 400

Pro Asp Phe Leu Trp Ile Ala Glu Asp Gly Met Thr Val Gln Ser Phe

405 410 415

Gly Ser Gln Leu Trp Asp Cys Ile Leu Ala Thr Gln Ala Ile Ile Ala

420 425 430

Thr Asn Met Val Glu Glu Tyr Gly Asp Ser Leu Lys Lys Ala His Phe

435 440 445

Phe Ile Lys Glu Ser Gln Ile Lys Glu Asn Pro Arg Gly Asp Phe Leu

450 455 460

Lys Met Cys Arg Gln Phe Thr Lys Gly Ala Trp Thr Phe Ser Asp Gln

465 470 475 480

Asp His Gly Cys Val Val Ser Asp Cys Thr Ala Glu Ala Leu Lys Cys

485 490 495

Leu Leu Leu Leu Ser Gln Met Pro Gln Asp Ile Val Gly Glu Lys Pro

500 505 510

Glu Val Glu Arg Leu Tyr Glu Ala Val Asn Val Leu Leu Tyr Leu Gln

515 520 525

Ser Arg Val Ser Gly Gly Phe Ala Val Trp Glu Pro Pro Val Pro Lys

530 535 540

Pro Tyr Leu Glu Met Leu Asn Pro Ser Glu Ile Phe Ala Asp Ile Val

545 550 555 560

Val Glu Arg Glu His Ile Glu Cys Thr Ala Ser Val Ile Lys Gly Leu

565 570 575

Met Ala Phe Lys Cys Leu His Pro Gly His Arg Gln Lys Glu Ile Glu

580 585 590

Asp Ser Val Ala Lys Ala Ile Arg Tyr Leu Glu Arg Asn Gln Met Pro

595 600 605

Asp Gly Ser Trp Tyr Gly Phe Trp Gly Ile Cys Phe Leu Tyr Gly Thr

610 615 620

Phe Phe Thr Leu Ser Gly Phe Ala Ser Ala Gly Arg Thr Tyr Asp Asn

625 630 635 640

Ser Glu Ala Val Arg Lys Gly Val Lys Phe Tyr Leu Ser Thr Gln Asn

645 650 655

Glu Glu Gly Gly Trp Gly Glu Ser Leu Glu Ser Cys Pro Ser Glu Lys

660 665 670

Phe Thr Pro Leu Lys Gly Asn Arg Thr Asn Leu Val Gln Thr Ser Trp

675 680 685

Ala Met Leu Gly Leu Met Phe Gly Gly Gln Ala Glu Arg Asp Pro Thr

690 695 700

Pro Leu His Arg Ala Ala Lys Leu Leu Ile Asn Ala Gln Met Asp Asn

705 710 715 720

Gly Asp Phe Pro Gln Gln Glu Ile Thr Gly Val Tyr Cys Lys Asn Ser

725 730 735

Met Leu His Tyr Ala Glu Tyr Arg Asn Ile Phe Pro Leu Trp Ala Leu

740 745 750

Gly Glu Tyr Arg Lys Arg Val Trp Leu Pro Asn His Gln Gln Leu Lys

755 760 765

Ile

Claims (7)

1. the protein be separated, its aminoacid sequence is for shown in SEQ ID NO.2.

2. the nucleotide sequence of protein described in coding claim 1.

3. nucleotide sequence according to claim 2, its sequence is for shown in SEQ ID NO.1.

4. the plant expression vector containing nucleotide sequence described in claim 2.

5. the transfer-gen plant containing plant expression vector described in claim 4.

6. protein described in claim 1 or gene according to claim 2 are improving the application in panax species dammarane type saponin content.

7. application according to claim 6, is characterized in that: protein described in claim 1 or gene according to claim 2 are improving the application in Rhizoma Panacis bipinnatifidi dammarane type saponin content.