CN102925458A - ABC transporter PgPDR2 gene and encoding protein and application of ABC transporter gene PgPDR2 - Google Patents

Abstract

The invention discloses a PgPDR2 (panax ginseng pleiotropic drug resistance) gene related to regulation of transport and accumulation of ginsenosides, as well as an encoding protein and an application of the PgPDR2 gene. A sequence of the ABC (ATP-binding cassette) PgPDR2 gene is shown as SEQ ID No. (sequence identification number) 1, and the sequence of the encoding protein of the ABC PgPDR2 gene is shown as SEQ ID No. 2. The research finds that PgPDR2 gene expression has tissue specificity and is related to the accumulation of the ginsenosides. The research preliminarily indicates that the PgPDR2 gene has functions of promoting synthesis, the transport and the accumulation of the ginsenosides. Panax ginseng or other plants can be screened or improved through the PgPDR2 gene or derivatives of the PgPDR2 gene to obtain excellent plant varieties with high ginsenoside content. Also, panax ginseng transgenic cells or plants can be adopted to produce ginsenoside monomers such as protopanaxadiol, Rb, Re, Rh2, Rg2 and Rg3.

Description

Ginseng ABC transporter gene PgPDR2 and proteins encoded thereof and application

Technical field

The invention belongs to technical field of biological genetic engineering, relate to the improvement plant trait, specifically genes involved and albumen and the application of short ginsenoside biosynthesis, transhipment and accumulation.The present invention has important using value aspect the content of ginsenoside in the plants such as raising ginseng.

Background technology

Ginseng (P.ginseng C.A.Meyer) is Araliaceae Panax per nnial herb, is rare traditional Chinese medicine.Contain multiple medicinal ingredients in the ginseng, wherein ginsenoside is the topmost activeconstituents of ginseng.Isolated at present more than 50 and plant ginsenoside from ginseng, modern medicine study proves that each saponin monomer has important pharmaceutical use, and has been widely used in clinical.The saponin content that but some of them have is antitumor, anti-ageing, inhibited apoptosis and strengthening immunity isoreactivity are strong is extremely low.In recent years, both at home and abroad carried out extensive research to utilizing the technology such as ginseng-cell cultivation to produce ginsenoside, but its content is still lower, demand that far can not satisfying the market.

In Secondary Metabolism of Plant regulation and control, can be by importing the multistep rate-limiting reaction in the gene regulating route of synthesis such as key enzyme, and promote the synthetic of secondary metabolite; Or by inducing transmembrane transporter to be accumulated outside the synthetic meta-bolites transporte to cells or in the organoid, utilize movement system that anabolism is changed to the accumulation direction, thereby improve its secondary metabolites content.By the transporting mechanism of ginsenoside, and then promote ginsenoside to synthesize and accumulation, be expected to become the New Policy of ginsenoside high yield regulation and control.

Plant ABC(ATP-binding cassette transporter) translocator is present known maximum, function is protein family the most widely, abc transport albumen belongs to the cross-film transport protein, energy transhipment organic acid, alkaloid, products of cellular metabolism and the medicine etc. that utilize hydrolysising ATP to discharge.Participate in bacterial drug resistance, secondary metabolite accumulation, coerce reaction and tumour resistance etc.The abc transport protein family comprises multidirectional resistance albumen (pleiotropic drug resistance, PDR), multidrug resistance albumen (multidrug resistance, MDR) etc.PDR is wherein maximum subtribe, is distinctive a kind of abc transport albumen in plant and the fungi.But very few to plant PDR gene-correlation research at present, in the in vitro cell culture, sclareol can be induced NpPDR1 gene up-regulated expression, in cell the accumulation of NpPDR1 albumen strengthened cell outside born of the same parents, transport sclareol with and the ability of analogue.The accumulation of sclareol also can be induced AtPDR12 gene up-regulated expression in Arabidopis thaliana (A.thaliana) cell, detect simultaneously the sclareol that AtPDR12 albumen is transported in the extracellular outside born of the same parents, infer that terpene substances may be the upstream regulation and control substance of AtPDR12 genetic expression.Therefore, PDR albumen may be by the antimycotic diterpenes material of transhipment endogenous involved in plant defensive raction.In addition, there is no the report of the more complicated terpene molecule of plant PDR albumen transhipment.

Summary of the invention

The present invention aim to provide a kind of can regulate and control ginsenoside transhipment and accumulate relevant gene, by albumen and the application thereof of this genes encoding, for the Future Development gene engineering product lays the foundation.

In order to achieve the above object, technical scheme provided by the invention is:

The invention provides a kind of ginseng ABC transporter gene PgPDR2, gene order is shown in SEQ IDNO.1.

The present invention also provides a kind of degenerated primer for amplification PgPDR2 gene pair, and described primer pair is shown in SEQ ID NO.5 and SEQ ID NO.6.

The present invention also provides and contained the PgPDR2 gene recombined vector, specifically comprises: plant expression vector pBI121 namely, in empty carrier pBI121, obtains recombinant vectors pBI121-PgPDR2 with the PgPDR2 gene clone; Prokaryotic expression carrier pET28a namely, in empty carrier pET28a, obtains recombinant vectors pET28a-PgPDR2 with the PgPDR2 gene clone.The PgPDR2 gene also can be cloned in other empty carriers, or is prepared into transgenic cell and recombinant bacterium.

The present invention also provides the application of PgPDR2 gene in the transhipment of regulation and control ginsenoside and accumulation; The application of PgPDR2 gene in the ginseng breeding.

The present invention also provides a kind of albumen by PgPDR2 genes encoding shown in the SEQ ID NO.1, and the sequence of this albumen is shown in SEQ ID NO.2.

The present invention also provides the application of albumen shown in the SEQ ID NO.2 in the transhipment of regulation and control ginsenoside and accumulation, and the application of this albumen in the ginseng breeding.

The present invention utilizes existing plant gene engineering technology, utilize plant PDR dna homolog design degenerated primer, adopt the methods such as degenerated primer round pcr and RACE to separate and identify the ginsenoside transhipment and accumulate relevant gene order, and change gene over to tobacco by agrobacterium tumefaciens-mediated transformation, through the transport function of heterogenous expression assay certificate PgPDR2 gene pairs ginsenoside.Ginseng ABC transporter gene PgPDR2 sequence of the present invention is shown in SEQ ID NO.1, and its Protein sequence is shown in SEQ ID NO.2.Research finds that PgPDR2 genetic expression has tissue specificity and relevant with the accumulation of ginsenoside.Tentatively show described PgPDR2 gene have promote that ginsenoside is synthetic, the function of transhipment and accumulation aspect.Can by this screening of gene or derivatives thereof or improvement ginseng even other plant, obtain the high good plant kind of content of ginsenoside.Can also adopt ginseng tissue to cultivate or the ginsenoside monomers such as transgenic plant production Rb, Re and Rg.

Description of drawings

Fig. 1 is that the present invention is by the ginseng PDR gene fragment diagram of degenerated primer amplification; Among the figure, 1 expression degenerated primer pcr amplification product, M represents Marker;

Fig. 2 is that the present invention is by ginseng PDR gene fragment speculating acid sequence alignment and the structural domain figure of degenerated primer amplification;

Fig. 3 is pcr amplification PgPDR2 gene diagram of the present invention; Among the figure, 1 expression pcr amplification PgPDR2 full length sequence product, M represents Marker;

Fig. 4 is PgPDR2 albumen membrane spaning domain prognostic chart of the present invention;

Fig. 5 be PgPDR2 gene coding amino acid sequence of the present invention and with other plant PDR dna homolog comparison diagram;

Fig. 6 is PgPDR2 gene coding amino acid sequence phylogenetic analysis figure of the present invention;

Fig. 7 is the expression level figure of PgPDR2 gene of the present invention in the ginseng different tissues;

Fig. 8 is expression level figure in the ginseng-cell of PgPDR2 gene of the present invention under 100 μ M MeJA induce.

Embodiment

The acquisition of embodiment 1PgPDR2 gene

1, ginseng suspension cell culture

(1) get the 4 years living ginsengs in Changbai mountain, Jilin ginseng producing region, tap water soaks the 45min post-flush 2 times, uses first 75% alcohol immersion 30 seconds (sec), aseptic water washing 2 times, with the 0.1%HgCl2 sterilization, constantly stir sterile distilled water flushing 4-5 time on the sterilisable chamber Bechtop.

(2) under aseptic condition, the explant of sterilizing is inoculated into contains 2,4-D(2.0mg/L) and KT(0.5mg/L) the MS substratum in.The complete dedifferentiation of explant behind 3 succeeding transfer culture, after being completed into callus, 6 explants of every 100ml triangular flask inoculation, culture condition such as aforementioned, cultivate after 45 days (d) with the capable succeeding transfer culture of callus, to replenish the required nutritive substance of culture and other requirement.Carry out the succeeding transfer culture first time behind the inoculation 30d, later every 3-4 week subculture once.

(3) after obtaining callus, it is transferred in the MS liquid nutrient medium.The callus lines diameter should be less than 3mm.If the large available aseptic scalper of tissue block is divided into fritter.Liquid nutrient medium is divided in the Erlenmeyer flask of 50ml, every 10ml liquid nutrient medium inoculation 1.5-2.0g callus, and concussion is cultivated after the inoculation, 120rpm, 24 ℃, the dark cultivation.Every the 4-7d subculture once.

2, cell is processed and the RNA extraction

(1) cell is processed

1) gets 460 μ L methyl jasmonates (MeJA) and be dissolved in 4540 μ L ethanol, add the 5ml distilled water to 10ml, be made into the MeJA mother liquor of 10ml 200mM, with the filter filtration sterilization of 0.22 μ m.

2) behind the 48h of suspension cell subculture, it is 200 μ M that the 10ml liquid nutrient medium adds 10 μ L mother liquor to final concentrations, 120rpm, and 24 ℃, vibration is secretly cultivated, and extracts total RNA of ginseng-cell behind the cultivation 24h.

(2) the total RNA of ginseng suspension cell extracts

1. the suspension cell collected of the centrifugal 5min of 3000 * g, in mortar with the abundant grind into powder of liquid nitrogen.The powder that every 40mg grinds places the 1.5ml centrifuge tube, adds 1ml TRIzol reagent, and 40 μ L beta-mercaptoethanols are with suction pipette head re-suspended cell precipitation, incubated at room 5-10min.

2. add the 0.2ml chloroform, jolting 15sec, incubated at room 10-15min.

3. 4 ℃, the centrifugal 15min of 12000 * g collect the colourless water in upper strata in the 1.5ml centrifuge tube, do not inhale moving middle layer, abandon precipitation.

4. add 0.4ml 3mol/L ammonium acetate (pH 5.2), 0.6ml Virahol, the mixing that softly vibrates, incubated at room 5-10min(RNA precipitation).4 ℃, the centrifugal 10min of 12000 * g abandon supernatant.

5. add 1ml 75% ethanol and vibration; 4 ℃, the centrifugal 5min of 7500 * g abandons supernatant, repeats this step.

6. air drying 10min adds 20 μ L DEPC water dissolution RNA.

7. the non-sex change agarose gel electrophoresis of RNA carries out in 0.5 * TBE, agarose concentration is the 10mg/ml EB that adds 2.5 μ L in the every 100ml gel of 1%(), 4 μ L applied sample amounts, 8v/cm constant voltage 40min, the then integrity of observation post's extraction RNA in the ultraviolet gel imaging system.

3, cDNA is synthetic

After the purified mRNA, take oligo (dT) as primer, the total mRNA of M-MuLV ThermoScript II reverse transcription.

(1) reaction system is as follows:

(2) soft stirring and evenly mixing; Room temperature moves to constant water bath box after placing 10min; 42 ℃, 1h; After the end, cooled on ice 2min.

4, degenerated primer pcr amplification

(1) the degenerated primer design is with synthetic

Utilize the 6 kind of plant PDR gene order tobacco (NpPDR1s of Bioedit software to having reported, AJ404328 and NtPDR1, AB109388.1), Arabidopis thaliana (AtPDR1, BK001001 and AtPDR12, BK0010011), (SpTUR2, Z70524) and wheat (TaPDR1, FJ185035) carry out sequence analysis, find out the zone of high conservative, adopt Primer 5.0 software design degenerated primers as follows:

P1：5’-CCAGGNGTDCTNACNGCNYTNATG-3’；（SEQID NO.3）

P2：5’-CATCCANGTYGCYGGRTTGTANCC-3’。（SEQID NO.4）

Primer is synthetic by the biological company limited of Shanghai English fine horse.

(2) pcr amplification

Take cDNA as template, carry out " touchdown " pcr amplification with degenerated primer P1, P2.

Reaction system is: 10 * PCR damping fluid, 5 μ L, MgCl ₂(25mmol/L) 3 μ L, dNTP(2.5mmol/L) 8 μ L, P1(20 μ mol/L) 2 μ L, P2(20 μ mol/L) 2 μ L, cDNA 2 μ L, dd H ₂O27.5 μ L and LATaq DNApolymerase(5U/ μ L) 0.5 μ L.

Reaction conditions is: 94 ℃ of denaturation 3min; 94 ℃ of sex change 30s, 45 ℃ of annealing 2min, 72 ℃ of extension 2min, 2 circulations; 94 ℃ of sex change 30s, 42 ℃ of annealing 2min, 72 ℃ of extension 2min, 2 circulations; 94 ℃ of sex change 30s, 40 ℃ of annealing 2min, 72 ℃ of extension 2min, 2 circulations; 94 ℃ of sex change 30s, 50 ℃ of annealing 2min, 72 ℃ of extension 2min, 26 circulations; 72 ℃ are extended 10min.1.0% agarose gel electrophoresis is analyzed the PCR product.

(3) amplified fragments glue reclaims, connects, checks order and analyzes

To be connected on the pGEM-T Easy carrier after the recovery of PCR product glue, and transform bacillus coli DH 5 alpha.After the PCR preliminary evaluation is correct, recombinant plasmid pGEM-PDR is served the order-checking of Hai Yingjun company.Obtain the gene fragment that 2 sequence lengths are 693bp after the order-checking, sequencing result utilizes Blast instrument among the NCBI and dbEST database and the albumen database of GenBank to carry out sequence analysis, with other plant PDR gene height homology.

4, cDNA total length amplification

The 693bp PDR gene order design 5 ' end that amplification obtains according to degenerated primer and 3 ' end amplimer, 5 '-RACE primer (GSP5-2) and 3 '-RACE primer (GSP3-2) are respectively:

GSP5-2：5’-ATGCCGACCAACTGGACCCAC-3’；（SEQ IDNO.5）

GSP3-2：5’-GCTGCCGCAATCGTGATGAGA-3’。（SEQ IDNO.6）

1. RNA extracts and reverse transcription

RNA extracts and adopts aforesaid method to carry out, and ThermoScript II and primer among the SMART RACE cDNA AmplificationKit adopted in reverse transcription.

2. 5 ' hold amplification reaction system

3. 5 ' hold amplification reaction condition

94℃2min，1cycles:

94℃30sec，72℃4min 30sec，5cycles;

94℃30sec，70℃30sec，72℃4min 30sec，5cycles;

94℃30sec，68℃30sec，72℃4min 30sec，20cycles

4. 3 ' hold amplification system

5. 3 ' hold amplification reaction condition

94℃2min，1cycles:

94℃30sec，72℃2min，5cycles;

94℃30sec，70℃30sec，72℃2min，5cycles;

94℃30sec，68℃30sec，72℃2min，20cycles

6. electrophoresis and order-checking thereof

The PCR product is after 1.0% agarose gel electrophoresis detects, and glue reclaims and is connected with pGEM-T Easy carrier, transforms bacillus coli DH 5 alpha, extracts single bacterium colony plasmid.Plasmid carried out double digestion is identified and PCR detects, detect and serve Hai Yingjun company after errorless and check order.Obtaining 5 ' end cDNA length after the order-checking is 3376bp, and 3 ' end cDNA length is 1514bp, and the total length that obtains comprising complete encoder block after splicing is the sequence (PgPDR2) of 4725bp, is submitted to Genbank, and accession number is KC013237.

5, sequential analysis

Successfully having obtained length through checking order is the cDNA sequence of 4725bp, utilize on-line analysis tools BLAST and ORF Finder(http among the NCBI: //blast.ncbi.nlm.nih.gov/Blast.cgi and http://www.ncbi.nlm.nih.gov/gorf/orfig.cgi) compare to search with ORF and find that the CDS sequence length is 4338bp in this sequence, aminoacid sequence to its coding adopts CLUSTAL X software analysis relatively, with other plant PDR gene such as soybean (GmPDR1, XM003546171), tobacco (NpPDR1, AJ404328; NtPDR1, AB109388.1 and NpPDR2, BAD07484.1), paddy rice (OsPDR1, AJ535054; OsPDR2, AJ535053 and OsPDR3, AJ535052) higher homology arranged.Utilize TMHMM(http: //www.cbs.dtu.dk/services/TMHMM-2.0/) on-line analysis software analysis membrane spaning domain, the result shows that this albumen is transmembrane protein.And use MEGA4 software to set with adjacent method (Neighbor-Joining, NJ) constructing system.The confidence level of branch is estimated with bootstrap value (Bootstrap test) in the genealogical tree, and repeat 1000 times, the result shows that the PDR gene affinity of this gene and soybean is nearest, secondly is tobacco and paddy rice.

6, expression analysis

Get root, adventive root, bud, seed and the root of hair of fresh ginseng, extract respectively RNA; Induce respectively ginseng 0,1,3,6,12 and 24h with 100 μ MMeJA again, extract the total RNA of each time point ginseng.Take oligod(T) 18 be primer, the AMV ThermoScript II is synthesized cDNA, and take β-actin as confidential reference items, utilizes real-time pcr amplification PgPDR2 gene, and PgPDR2 and β-actin amplimer is respectively:

PgPDR2：F：5’-GTGGGGCTGGGAAGACCACTCT-3’；（SEQID NO.7）

R：5’-GCTCCCAGTATCCTGCTATGCGA-3’；（SEQID NO.8）

β-actin：F：5’-CGTGATCTTACAGATAGCTTCATGA-3’；（SEQID NO.9）

R：5’-AGAGAAGCTAAGATTGATCCTCC-3’。（SEQID NO.10）

Reaction system is:

Adopt two-step approach pcr amplification standard program: 95 ℃ of 30sec; 95 ℃ of 40sec, 60 ℃ of 30sec totally 40 circulations; 95 ℃ of 15sec, 60 ℃ of 1min, 95 ℃ of 15sec.Each sample repeats for three times.Confirm amplification curve and solubility curve after reaction finishes, adopt 2 ^{-Δ Δ Ct}Method is calculated the PgPDR2 gene is induced lower different time expression level at different tissues and MeJA difference.The result shows that it expresses the highlyest in seed, secondly be root of hair, adventive root and root, expresses minimum in the bud.MeJA can significantly induce the synthetic of ginsenoside, and PgPDR2 genetic expression increases constantly with induction time and increases, and is consistent with synthesizing with the cumulative change rule of ginsenoside.

Structure and the conversion of embodiment 2 expression vectors

1, the structure of plant over-express vector and transfer-gen plant screening

(1) uses respectively BamHI and Sac I double digestion pBI121 and PgPDR2, reclaim pBI121 carrier and PgPDR2 gene fragment.Two fragments are connected and conversion DH5 α, filter out recon, recombinant plasmid called after pBI-PgPDR2.The upstream and downstream primer of PgPDR2 gene of wherein increasing is respectively:

PgPDR2：F1:5’-CG GGATCCCGCACCATGGATGGAAGTAATCTGTA-3’；（SEQID NO.11）

R1:5’-CGC GAGCTCGGCTCACCGCCTTTGGAAGTTGA-3’。（SEQID NO.12）

Wherein line part is BamH I and Sac I restriction enzyme site.

(2) preparation of agrobacterium tumefaciens competent cell

1. streak culture Agrobacterium LBA4404 on the YEB flat board that contains 100 μ g/ml rifomycins is secretly cultivated 2d for 28 ℃.

2. picking list colony inoculation is in the YEB substratum that contains 100 μ g/ml rifomycins, and 28 ℃ of shaking culture are spent the night.

3. the Agrobacterium of activation of will spending the night contains to 50ml by the dilution proportion of 1:50 in the YEB substratum of kanamycin of 100 μ g/ml, and 28 ℃ of shaking culture are about 0.4-0.6 to OD600, ice bath 30min.

4. in 4 ℃, the centrifugal 10min of 5000rpm abandons supernatant liquor, with 10ml 0.15mM NaCl suspension thalline.

5. in 4 ℃, the centrifugal 10min of 5000rpm abandons supernatant liquor, with 2ml 20mM CaCl2 suspension thalline.

6. every pipe 200 μ L packing, liquid nitrogen flash freezer ,-70 ℃ of preservations.

(3) conversion of agrobacterium tumefaciens competent cell

1. the agrobacterium tumefaciens competent cell is placed on ice, slowly thaw.

2. add 0.5 μ gpBI121-PgPDR2 plasmid DNA, mixing gently, ice bath 30min.

3. put freezing 2min in the liquid nitrogen, move into rapidly heat shock 5min in 37 ℃ of water-baths, rapid ice bath 2min adds 800 μ L YEB liquid nutrient mediums, afterwards in 28 ℃ of shaking culture 3-4h again.

4. the centrifugal 5min of 5000rpm precipitation thalline discards Eddy diffusion thalline behind the 800 μ L supernatants, evenly coats on the YEB substratum that contains 100 μ g/ml rifomycins and 50 μ g/ml kantlex, cultivates 2-3d for 28 ℃.

(4) agrobacterium tumefaciens plasmid extraction and evaluation

1. PCR identifies

The single colony inoculation of the Agrobacterium that picking transforms contains 37 ℃ of shaking culture in the LB liquid nutrient medium of kantlex in 1.5ml, uses the PgPDR2 gene-specific primer:

F：5’-GTGGGGCTGGGAAGACCACTCT-3’；（SEQ ID NO.7）

R：5’-GCTCCCAGTATCCTGCTATGCGA-3’；（SEQ ID NO.8）

Behind the pcr amplification, agarose gel electrophoresis detects whether contain the expection fragment.The PCR response procedures is as follows: 94 ℃ of 30sec, 55 ℃ of 45sec, 72 ℃ of 30sec, 35 circulations; 72 ℃ of 2min.

2. enzyme is cut evaluation

The single colony inoculation of picking Agrobacterium is in the YEB substratum of the kantlex of the rifomycin that contains 100 μ g/ml and 50 μ g/ml, 28 ℃ of overnight incubation are cultivated, extract recombinant plasmid pBI121-PgPDR2, adopt aforesaid method to cut the plasmid of extraction with BamH I and Sac I enzyme.

(5) agrobacterium tumefaciens that contains the pBI121-PgPDR2 plasmid is cultivated

The Agrobacterium that 28 ℃ streak culture contains the pBI121-PgPDR2 plasmid is 2d approximately.Picking list colony inoculation is in the YEB liquid nutrient medium that contains 100mg/L rifomycin and 50mg/L kantlex, and 28 ℃ of shaking culture are spent the night, and be about at 0.6 o'clock to OD600 and collect bacterium liquid, the centrifugal 10min of 4000rpm, precipitation is resuspended in the 1/2MS liquid nutrient medium.

(6) leaf dish method infects tobacco

The tobacco spire is placed tap water flushing 3-4 time, then soak 30-60sec in 70% ethanol, the 10-20min that sterilizes in 10% clorox fully washes with sterilized water again, then places sterile petri dish, and blade is cut into 0.5-1cm ²Small pieces, the blade that shears is put into respectively the Agrobacterium bacterium liquid that contains the pBI121-PgPDR2 plasmid infects 5min, the bacterium liquid on the blade is blotted with aseptic filter paper.

(7) cultivate altogether

Infected and blotted the leaf dish of surperficial bacterium liquid, pore faces up, and nestles up to be placed on (MS+6-BA 1.0mg/L+IAA 0.1mg/L) on the common substratum, and 2d is cultivated at dark place.

(8) screening and differentiation culture

Behind the dark cultivation 2d, be transformed on the screening division culture medium (MS+6-BA 1.0mg/L+IAA 0.1mg/L+Kan 100mg/L+Cef300mg/L), every 15d changes the primary screening division culture medium, goes in the root media when the tobacco that differentiates grows to 3-5cm.

(9) root culture

The tobacco that breaks up to 3-5cm is downcut respectively, be transferred to root media (1/2MS+IBA 2.0mg/L).

(10) transplant

When the root of regeneration plant grows to 5-8cm, open sealed membrane hardening 2-3d, with seedling replanting to flowerpot, the initial upper transparent plastics of 5-10d cover after transplanting, the humidity of maintenance 90%-100%, and beat a little apertures and be beneficial to gaseous interchange covering.The matrix of transplanting and flowerpot be in advance sterilization all.

(7) transfer-gen plant PCR identifies

Take the genomic dna of transfer-gen plant as template, adopt above-mentioned recombinant plasmid to identify that used PgPDR2 gene-specific primer carries out pcr amplification, PCR reaction system and reaction conditions are the same.Obtain the T of seedling differentiation through evaluation ₀For positive transfer-gen plant.

2, transport function is identified

Get the tobacco spire that turns the PgPDR2 gene, adopt above-mentioned same procedure to induce the generation suspension cell, utilize ginsenoside to feed and comparison Functional Plant Genomics technology, by passing through gas chromatography-mass spectrography (gaschromatography-mass spectrometry, GC/MS) change that the method direct quantitative is measured and the comparative analysis transgene tobacco is cultivated ginsenoside chemical composition stave type in the system determines that the PgPDR2 translocator is to the transport function of ginsenoside.

3, prokaryotic expression analysis

(1) pcr amplification

Take the PgPDR2 gene that increased as template, the primer that adds respectively BamH I and Sac I restriction enzyme site sequence with upstream and downstream increases, primer following (the line part is respectively BamH I and Sac I restriction enzyme site) that its amplification is used:

5’-CGG GAATTCATGGATGGAAGTAATCTGTA-3’（SEQ ID NO.13）

5’-CGG GAATTCTCACCGCCTTTGGAAGTTGA-3’（SEQ ID NO.14）

Reaction system is the same, and the PCR response procedures is as follows: 94 ℃ of 30sec, 55 ℃ of 45sec, 72 ℃ of 4min 30sec, 35 circulations; 72 ℃ of 10min.

(2) enzyme is cut, is connected and transforms and screening

Use respectively BamH I and Sac I double digestion pET28a and PCR product, reclaim pET28a and PCR product fragment.Two fragments are connected and conversion BL21, cut by kantlex screening, pcr amplification and enzyme and identify recon, recombinant plasmid called after pET28a-PgPDR2.

(3) the protein induced expression of PgPDR2 and functional analysis thereof

Picking contains the bacterium colony BL21 (DE3) of restructuring pET28a-PgPDR2 plasmid, contains Kan 50 μ g/ml to 10ml LB() in 37 ℃ of incubated overnight.Get 1ml bacterium liquid and join and contain 100ml LB substratum (containing Kan 50 μ g/ml), 37 ℃ of concussions are cultured to OD600 and are about 0.4-0.6.Adding IPTG is that 1mM induces to final concentration, collects a bacterium liquid, the centrifugal 10min of 5000 * g every 1h, the results precipitation, the ratio that adds 4ml PBS damping fluid with the 1g thalline is resuspended, adds 5 * SDS-PAGE sample-loading buffer, mixing, boiling water bath 5min are got supernatant and are carried out the SDS-PAGE analysis.

Choose the high time point of PgPDR2 expressing quantity, add the substrates such as ginsenoside, analyze the transport function of PgPDR2 albumen, and carry out protein purification, its structure of external parsing in conjunction with His-tag.

SEQUENCE LISTING

＜110〉Central South University

＜120〉ginseng ABC transporter gene PgPDR2 and proteins encoded thereof and application

<160> 14

<170> PatentIn version 3.3

<210> 1

<211> 4338

<212> DNA

＜213〉homo sapiens

<400> 1

atggatggaa gtaatctgta caacttaagc aacagtgtga ggaacagcag aagtataagg 60

gaaaggaaca gtttaagagc aagcagttct tcagtatgga ggagcaatgg aatggaggtc 120

ttctcaaagt cagcgcggga tgaagacgat gaagaagctc tcaagtgggc ttcccttgag 180

aaactcccga cctttgaccg actaaggaaa gggcttcttc ttggatcaca gggagcaggg 240

gcgagtgaag ttgatattga tgatcttggg attcaggaga ggaagaagtt actggagagg 300

ctagtcaacg ttgctgaaga agataacgag aggttcttgt tgaagctcag ggatcgagtc 360

gacagagttg gaattgattt gcccctaatc gaagtcagat tcgatcattt aactattgag 420

gcagaagctc atgtgggaag cagagctttg ccttctttct tgaacttcaa tattagtata 480

gtggaggggt tgttagacac cctccgctta cttccaaata gaaagcagcg cctaactatc 540

ctagaggatg ttagcggaat cctcaggcct tgcagaatga cattgctttt aggtcctcca 600

agttctggga agacaacatt attgctggct ttggctggga agctagatcc tacccttaag 660

ttttctggaa gggtgacgta caatggccat gaaatgactg agtttgtgcc ccagagaaca 720

gctgcctata ttagccaaca tgatctccat ataggagaaa tgactgtgag agaaaccttg 780

gcattctctg caagatgcca gggggttgga tcacgctatg aaatgttggc agagctgtca 840

agaagagaga aagacgcgaa tatcaaacct gatcctgatg ttgatatcta catgaaggct 900

gccgcaacag aaggtcaaga ggccaatgta gttacggatt atgttctaaa gattttggga 960

cttgatgttt gtgcagatac tatggttgga gaccagatga ttaggggtat atctggagga 1020

caaagaaagc gtgttacaac aggtgagatg ttggttggac catcaaaggc actctttatg 1080

gatgagatat ctactggatt ggacagttct acaacttatc aaattgtgaa agcgctcagg 1140

cacagtgtcc acattctcca aggaactgct ctcatctctc tcctgcagcc ggcacctgaa 1200

acttatgatt tatttgatga cattattctc ctatctgatg gccagatagt gtatcaaggt 1260

ccccgtgaaa atgtgctcca gttttttgag tctatgggtt tcaaatgtcc agaaaggaaa 1320

ggtgtggctg acttcttgca agaagtgaca tcaaagaaag atcagaaaca gtactggcta 1380

cgcaaagatg agccttacag atttattaca gccacagaat tctcagaggc tttccagtcg 1440

ttccatgttg gactcaggct gggaggtgat cttgcaacac cctatgacaa gggcagaagc 1500

cacccagctg ctttgacaac tgataagtac ggagttagca agacagagct cttgaaagct 1560

gtgactgcaa gagaaatttt gcttatgaaa agaaactcgt ttgtatacat attcaaattg 1620

tttcaactta ccgtaatgtc cattgttacc atggcactgt tcctgagaac tgagttgaac 1680

cacaatacaa caactgatgg agggatatac atgggtgctc tattttttgc cgtggttatg 1740

cttatgttta atggattggc agagcttgcc atgacaatag caaagcttcc cattttttac 1800

aagcaaaggg accttctctt ttatccaaca tggtcatatg ctcttccatc gtggatcatc 1860

aagataccta ttaccttttt agaagttgct gtttgggtag tcctcacgta ttatgttatt 1920

ggatttgatc caaatgttgg aagatttgtt aaacagtaca ttgtactctt actcatcaac 1980

cagatggctt cagcattgtt ccgacttatg gcagcactgg gtaggaacat gattcttgca 2040

ttcacatttg ggggctttgc actgcttgtg ctttttgcat taggtggctt tgtcctagca 2100

cgagacgatg tagcgaaatg gtggctatgg ggttactatt catcacccat gatgtatggg 2160

atgaatgcaa ttgcagtgaa tgaatttctt ggtcatcaat ggagcaaatt aactgcgaat 2220

ggaaacgaga caataggagt tgcaattttg aggtctcgag gtttcttccc atatgcatac 2280

tggtattgga taggagtagg ggcattggtt ggattcgttt tgatactcaa cttcgctgtt 2340

acagtggcac ttaattatct tgaccctctg gggaaacctc aagctgttat accagaaaaa 2400

ggtgacaccg cagaacccat tgagttatca tcccgaggaa gtagctccaa agggaaagct 2460

ggatctcaag aaatcaatgt tgaggccaat ccgaacaaga aaagaggaat gattcttcca 2520

tttgaaccac attccatcac ttttgatgaa gttaaatatt cagttgacat gccacaggaa 2580

atgaaagatc aaagtgttgt tgaagataag ctgttgcttc ttaagggtgt gagtggagca 2640

ttcagaccag gtgttctcac tgctctaatg ggtgtcagcg gtgctggtaa aacaactttg 2700

atggatgtgc ttgctggtcg gaaaactggt ggatatattg agggtaacat tacaatttcc 2760

gggtttccaa agaagcaaga aacctttgct cgaatatctg gatactgtga gcagaatgac 2820

atccactcgc ctcatgttac tgtttatgag tccttgatct actcagcatg gctgcggttg 2880

ccttcagaag ttgattctgt gaccagaaag atgtttgtcg acgaggtcct agaactagtt 2940

gaactaaaca cactgaagga tggactagtt gggttgccgg gtgtaaatgg tctctcaact 3000

gagcagcgca agaggctaac cattgcagtt gagctggtgg caaacccctc cataatattc 3060

atggatgagc caacttcagg gctagatgca agagctgccg caatcgtgat gagaacagta 3120

aggaatactg ttgatacagg aagaacagtt gtttgcacca tccatcaacc tagcatcgac 3180

atatttgaag cttttgatga gcttttcctc atgaaacgag gaggacaaga gctatatgtg 3240

ggtccagttg gtcggcattc tagccagtta atcaattact ttgaggatat tgacggaata 3300

agtaaaatca aagatggata taatccagcc acctggatgt tggaagttac tacttctgca 3360

caagaaatgg ttctaggtgt tgatttcact gaggtttaca gaaattcaga tctgtacagg 3420

agaaacaaag ccctgattag agaattaagt acacctcgtc ctggtacaaa ggatatctat 3480

ttctcaagtc aatattcaca atcttttctc acccaatgct tggcttgcct atggaaacaa 3540

cgatgctcat actggcgaaa cacatcatac actgctgtga ggtttctatt tgctaccgcc 3600

atagcattga tgcttggatc aatgttctgg gaccttggtt ccaaaatgcg aactcaacaa 3660

gatctcttca atgctacggg ttctatgtat gccgcctgcc tcttcttagg ggtccaaaat 3720

gcctcatcag tgcagccagt ggtggacgtt gaacgaacag tcttttacag agaaagagct 3780

gcaggaatgt attcagcctt gccatatgcg tttgcgcagg ttttggtaga agtaccatat 3840

atttttacac aagctgcagt atacggtgtt attgtgtatg caatgatcgg gtttgattgg 3900

actgtcgtga aattcatttg gtatatattc ttcacgttct tcacattgtt atacttcaca 3960

ctcttcggca tgatgactgt ggccgtgact cccaatgcgg acattgctgc tattattgcg 4020

gctgcatttt ttgcattatg gaatgtgttt tcaggattta taatcccaag accaaaaatg 4080

cctgtatggt ggagatggta ttcttgggta tgtccagttg cttggacttt gtacggtatg 4140

attgcatcac agtttggaga cattgacgac aagacccttg tggatgctaa ggtaaacgtg 4200

aagaaatttg ttgaagatta ctttggattt gagcacggta atgtgtgggt agccggaact 4260

gccgtagccg gcttcactgt tgtttttgca ttcacctttg ccttctccat caagtcattc 4320

aacttccaaa ggcggtga 4338

<210> 2

<211> 1445

<212> PRT

＜213〉homo sapiens

<400> 2

Met Asp Gly Ser Asn Leu Tyr Asn Leu Ser Asn Ser Val Arg Asn Ser

1 5 10 15

Arg Ser Ile Arg Glu Arg Asn Ser Leu Arg Ala Ser Ser Ser Ser Val

20 25 30

Trp Arg Ser Asn Gly Met Glu Val Phe Ser Lys Ser Ala Arg Asp Glu

35 40 45

Asp Asp Glu Glu Ala Leu Lys Trp Ala Ser Leu Glu Lys Leu Pro Thr

50 55 60

Phe Asp Arg Leu Arg Lys Gly Leu Leu Leu Gly Ser Gln Gly Ala Gly

65 70 75 80

Ala Ser Glu Val Asp Ile Asp Asp Leu Gly Ile Gln Glu Arg Lys Lys

85 90 95

Leu Leu Glu Arg Leu Val Asn Val Ala Glu Glu Asp Asn Glu Arg Phe

100 105 110

Leu Leu Lys Leu Arg Asp Arg Val Asp Arg Val Gly Ile Asp Leu Pro

115 120 125

Leu Ile Glu Val Arg Phe Asp His Leu Thr Ile Glu Ala Glu Ala His

130 135 140

Val Gly Ser Arg Ala Leu Pro Ser Phe Leu Asn Phe Asn Ile Ser Ile

145 150 155 160

Val Glu Gly Leu Leu Asp Thr Leu Arg Leu Leu Pro Asn Arg Lys Gln

165 170 175

Arg Leu Thr Ile Leu Glu Asp Val Ser Gly Ile Leu Arg Pro Cys Arg

180 185 190

Met Thr Leu Leu Leu Gly Pro Pro Ser Ser Gly Lys Thr Thr Leu Leu

195 200 205

Leu Ala Leu Ala Gly Lys Leu Asp Pro Thr Leu Lys Phe Ser Gly Arg

210 215 220

Val Thr Tyr Asn Gly His Glu Met Thr Glu Phe Val Pro Gln Arg Thr

225 230 235 240

Ala Ala Tyr Ile Ser Gln His Asp Leu His Ile Gly Glu Met Thr Val

245 250 255

Arg Glu Thr Leu Ala Phe Ser Ala Arg Cys Gln Gly Val Gly Ser Arg

260 265 270

Tyr Glu Met Leu Ala Glu Leu Ser Arg Arg Glu Lys Asp Ala Asn Ile

275 280 285

Lys Pro Asp Pro Asp Val Asp Ile Tyr Met Lys Ala Ala Ala Thr Glu

290 295 300

Gly Gln Glu Ala Asn Val Val Thr Asp Tyr Val Leu Lys Ile Leu Gly

305 310 315 320

Leu Asp Val Cys Ala Asp Thr Met Val Gly Asp Gln Met Ile Arg Gly

325 330 335

Ile Ser Gly Gly Gln Arg Lys Arg Val Thr Thr Gly Glu Met Leu Val

340 345 350

Gly Pro Ser Lys Ala Leu Phe Met Asp Glu Ile Ser Thr Gly Leu Asp

355 360 365

Ser Ser Thr Thr Tyr Gln Ile Val Lys Ala Leu Arg His Ser Val His

370 375 380

Ile Leu Gln Gly Thr Ala Leu Ile Ser Leu Leu Gln Pro Ala Pro Glu

385 390 395 400

Thr Tyr Asp Leu Phe Asp Asp Ile Ile Leu Leu Ser Asp Gly Gln Ile

405 410 415

Val Tyr Gln Gly Pro Arg Glu Asn Val Leu Gln Phe Phe Glu Ser Met

420 425 430

Gly Phe Lys Cys Pro Glu Arg Lys Gly Val Ala Asp Phe Leu Gln Glu

435 440 445

Val Thr Ser Lys Lys Asp Gln Lys Gln Tyr Trp Leu Arg Lys Asp Glu

450 455 460

Pro Tyr Arg Phe Ile Thr Ala Thr Glu Phe Ser Glu Ala Phe Gln Ser

465 470 475 480

Phe His Val Gly Leu Arg Leu Gly Gly Asp Leu Ala Thr Pro Tyr Asp

485 490 495

Lys Gly Arg Ser His Pro Ala Ala Leu Thr Thr Asp Lys Tyr Gly Val

500 505 510

Ser Lys Thr Glu Leu Leu Lys Ala Val Thr Ala Arg Glu Ile Leu Leu

515 520 525

Met Lys Arg Asn Ser Phe Val Tyr Ile Phe Lys Leu Phe Gln Leu Thr

530 535 540

Val Met Ser Ile Val Thr Met Ala Leu Phe Leu Arg Thr Glu Leu Asn

545 550 555 560

His Asn Thr Thr Thr Asp Gly Gly Ile Tyr Met Gly Ala Leu Phe Phe

565 570 575

Ala Val Val Met Leu Met Phe Asn Gly Leu Ala Glu Leu Ala Met Thr

580 585 590

Ile Ala Lys Leu Pro Ile Phe Tyr Lys Gln Arg Asp Leu Leu Phe Tyr

595 600 605

Pro Thr Trp Ser Tyr Ala Leu Pro Ser Trp Ile Ile Lys Ile Pro Ile

610 615 620

Thr Phe Leu Glu Val Ala Val Trp Val Val Leu Thr Tyr Tyr Val Ile

625 630 635 640

Gly Phe Asp Pro Asn Val Gly Arg Phe Val Lys Gln Tyr Ile Val Leu

645 650 655

Leu Leu Ile Asn Gln Met Ala Ser Ala Leu Phe Arg Leu Met Ala Ala

660 665 670

Leu Gly Arg Asn Met Ile Leu Ala Phe Thr Phe Gly Gly Phe Ala Leu

675 680 685

Leu Val Leu Phe Ala Leu Gly Gly Phe Val Leu Ala Arg Asp Asp Val

690 695 700

Ala Lys Trp Trp Leu Trp Gly Tyr Tyr Ser Ser Pro Met Met Tyr Gly

705 710 715 720

Met Asn Ala Ile Ala Val Asn Glu Phe Leu Gly His Gln Trp Ser Lys

725 730 735

Leu Thr Ala Asn Gly Asn Glu Thr Ile Gly Val Ala Ile Leu Arg Ser

740 745 750

Arg Gly Phe Phe Pro Tyr Ala Tyr Trp Tyr Trp Ile Gly Val Gly Ala

755 760 765

Leu Val Gly Phe Val Leu Ile Leu Asn Phe Ala Val Thr Val Ala Leu

770 775 780

Asn Tyr Leu Asp Pro Leu Gly Lys Pro Gln Ala Val Ile Pro Glu Lys

785 790 795 800

Gly Asp Thr Ala Glu Pro Ile Glu Leu Ser Ser Arg Gly Ser Ser Ser

805 810 815

Lys Gly Lys Ala Gly Ser Gln Glu Ile Asn Val Glu Ala Asn Pro Asn

820 825 830

Lys Lys Arg Gly Met Ile Leu Pro Phe Glu Pro His Ser Ile Thr Phe

835 840 845

Asp Glu Val Lys Tyr Ser Val Asp Met Pro Gln Glu Met Lys Asp Gln

850 855 860

Ser Val Val Glu Asp Lys Leu Leu Leu Leu Lys Gly Val Ser Gly Ala

865 870 875 880

Phe Arg Pro Gly Val Leu Thr Ala Leu Met Gly Val Ser Gly Ala Gly

885 890 895

Lys Thr Thr Leu Met Asp Val Leu Ala Gly Arg Lys Thr Gly Gly Tyr

900 905 910

Ile Glu Gly Asn Ile Thr Ile Ser Gly Phe Pro Lys Lys Gln Glu Thr

915 920 925

Phe Ala Arg Ile Ser Gly Tyr Cys Glu Gln Asn Asp Ile His Ser Pro

930 935 940

His Val Thr Val Tyr Glu Ser Leu Ile Tyr Ser Ala Trp Leu Arg Leu

945 950 955 960

Pro Ser Glu Val Asp Ser Val Thr Arg Lys Met Phe Val Asp Glu Val

965 970 975

Leu Glu Leu Val Glu Leu Asn Thr Leu Lys Asp Gly Leu Val Gly Leu

980 985 990

Pro Gly Val Asn Gly Leu Ser Thr Glu Gln Arg Lys Arg Leu Thr Ile

995 1000 1005

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

1010 1015 1020

Pro Thr Ser Gly Leu Asp Ala Arg Ala Ala Ala Ile Val Met Arg

1025 1030 1035

Thr Val Arg Asn Thr Val Asp Thr Gly Arg Thr Val Val Cys Thr

1040 1045 1050

Ile His Gln Pro Ser Ile Asp Ile Phe Glu Ala Phe Asp Glu Leu

1055 1060 1065

Phe Leu Met Lys Arg Gly Gly Gln Glu Leu Tyr Val Gly Pro Val

1070 1075 1080

Gly Arg His Ser Ser Gln Leu Ile Asn Tyr Phe Glu Asp Ile Asp

1085 1090 1095

Gly Ile Ser Lys Ile Lys Asp Gly Tyr Asn Pro Ala Thr Trp Met

1100 1105 1110

Leu Glu Val Thr Thr Ser Ala Gln Glu Met Val Leu Gly Val Asp

1115 1120 1125

Phe Thr Glu Val Tyr Arg Asn Ser Asp Leu Tyr Arg Arg Asn Lys

1130 1135 1140

Ala Leu Ile Arg Glu Leu Ser Thr Pro Arg Pro Gly Thr Lys Asp

1145 1150 1155

Ile Tyr Phe Ser Ser Gln Tyr Ser Gln Ser Phe Leu Thr Gln Cys

1160 1165 1170

Leu Ala Cys Leu Trp Lys Gln Arg Cys Ser Tyr Trp Arg Asn Thr

1175 1180 1185

Ser Tyr Thr Ala Val Arg Phe Leu Phe Ala Thr Ala Ile Ala Leu

1190 1195 1200

Met Leu Gly Ser Met Phe Trp Asp Leu Gly Ser Lys Met Arg Thr

1205 1210 1215

Gln Gln Asp Leu Phe Asn Ala Thr Gly Ser Met Tyr Ala Ala Cys

1220 1225 1230

Leu Phe Leu Gly Val Gln Asn Ala Ser Ser Val Gln Pro Val Val

1235 1240 1245

Asp Val Glu Arg Thr Val Phe Tyr Arg Glu Arg Ala Ala Gly Met

1250 1255 1260

Tyr Ser Ala Leu Pro Tyr Ala Phe Ala Gln Val Leu Val Glu Val

1265 1270 1275

Pro Tyr Ile Phe Thr Gln Ala Ala Val Tyr Gly Val Ile Val Tyr

1280 1285 1290

Ala Met Ile Gly Phe Asp Trp Thr Val Val Lys Phe Ile Trp Tyr

1295 1300 1305

Ile Phe Phe Thr Phe Phe Thr Leu Leu Tyr Phe Thr Leu Phe Gly

1310 1315 1320

Met Met Thr Val Ala Val Thr Pro Asn Ala Asp Ile Ala Ala Ile

1325 1330 1335

Ile Ala Ala Ala Phe Phe Ala Leu Trp Asn Val Phe Ser Gly Phe

1340 1345 1350

Ile Ile Pro Arg Pro Lys Met Pro Val Trp Trp Arg Trp Tyr Ser

1355 1360 1365

Trp Val Cys Pro Val Ala Trp Thr Leu Tyr Gly Met Ile Ala Ser

1370 1375 1380

Gln Phe Gly Asp Ile Asp Asp Lys Thr Leu Val Asp Ala Lys Val

1385 1390 1395

Asn Val Lys Lys Phe Val Glu Asp Tyr Phe Gly Phe Glu His Gly

1400 1405 1410

Asn Val Trp Val Ala Gly Thr Ala Val Ala Gly Phe Thr Val Val

1415 1420 1425

Phe Ala Phe Thr Phe Ala Phe Ser Ile Lys Ser Phe Asn Phe Gln

1430 1435 1440

Arg Arg

1445

<210> 3

<211> 24

<212> DNA

＜213〉homo sapiens

<220>

<221> misc_feature

<222> (6)..(6)

<223> n is a, c, g, or t

<220>

<221> misc_feature

<222> (12)..(12)

<223> n is a, c, g, or t

<220>

<221> misc_feature

<222> (15)..(15)

<223> n is a, c, g, or t

<220>

<221> misc_feature

<222> (18)..(18)

<223> n is a, c, g, or t

<220>

<221> misc_feature

<222> (21)..(21)

<223> n is a, c, g, or t

<400> 3

ccaggngtdc tnacngcnyt natg 24

<210> 4

<211> 24

<212> DNA

＜213〉homo sapiens

<220>

<221> misc_feature

<222> (7)..(7)

<223> n is a, c, g, or t

<220>

<221> misc_feature

<222> (22)..(22)

<223> n is a, c, g, or t

<400> 4

catccangty gcyggrttgt ancc 24

<210> 5

<211> 21

<212> DNA

＜213〉homo sapiens

<400> 5

atgccgacca actggaccca c 21

<210> 6

<211> 21

<212> DNA

＜213〉homo sapiens

<400> 6

gctgccgcaa tcgtgatgag a 21

<210> 7

<211> 22

<212> DNA

＜213〉homo sapiens

<400> 7

gtggggctgg gaagaccact ct 22

<210> 8

<211> 23

<212> DNA

＜213〉homo sapiens

<400> 8

gctcccagta tcctgctatg cga 23

<210> 9

<211> 25

<212> DNA

＜213〉homo sapiens

<400> 9

cgtgatctta cagatagctt catga 25

<210> 10

<211> 23

<212> DNA

＜213〉homo sapiens

<400> 10

agagaagcta agattgatcc tcc 23

<210> 11

<211> 34

<212> DNA

＜213〉homo sapiens

<400> 11

cgggatcccg caccatggat ggaagtaatc tgta 34

<210> 12

<211> 32

<212> DNA

＜213〉homo sapiens

<400> 12

cgcgagctcg gctcaccgcc tttggaagtt ga 32

<210> 13

<211> 29

<212> DNA

＜213〉homo sapiens

<400> 13

cgggaattca tggatggaag taatctgta 29

<210> 14

<211> 29

<212> DNA

＜213〉homo sapiens

<400> 14

cgggaattct caccgccttt ggaagttga 29

Claims (9)

1. a ginseng ABC transporter gene PgPDR2 is characterized in that, described PgPDR2 gene order is shown in SEQ ID N0.1.

2. a primer pair that is used for the described PgPDR2 gene of amplification claim 1 is characterized in that, described primer pair is shown in SEQ ID N0.5 and SEQ ID N0.6.

3. a recombinant vectors is comprised of empty carrier and the goal gene that inserts this empty carrier, it is characterized in that, described goal gene is PgPDR2 gene claimed in claim 1.

4. recombinant vectors according to claim 2 is characterized in that, described empty carrier is pET28a or pBI121.

5. the according to claim 1 application of described PgPDR2 gene in the transhipment of regulation and control ginsenoside and accumulation.

6. the according to claim 1 application of described PgPDR2 gene in the ginseng breeding.

7. a ginseng abc transport albumen is characterized in that, described albumen is by the genes encoding shown in the SEQ ID N0.1, and its aminoacid sequence is shown in SEQ ID N0.2.

8. the according to claim 6 application of described albumen in the transhipment of regulation and control ginsenoside and accumulation.

9. the according to claim 6 application of described albumen in the ginseng breeding.

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