CN103103194A - Gene promoter of ginseng PgPDR3 responded by methyl jasmonate and application thereof - Google Patents
Gene promoter of ginseng PgPDR3 responded by methyl jasmonate and application thereof Download PDFInfo
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Abstract
The invention discloses a gene promoter of ginseng PgPDR3 responded by methyl jasmonate and an application thereof. The sequences of the gene promoter of the ginseng PgPDR3 are shown in SEQ ID NO.1, or are the DNA sequences of which the homology with SEQ ID NO.1 is over 99%. According to the invention, a Genomewalker technology is adopted to clone, later the sequences of the gene promoter of the PDR transport protein is obtained, and then the expression vector Pcambia1301-ProPDR3::GUS of the promoter is built; if the promoter in the transgenic tobacco drives the GUS to express, and can be controlled by ginsenoside, salicylic acid, gibberellins and abscisic acid, the promoter participates in the transport and accumulation of the ginsenoside, which provides a basis for cloning the gene of key control recording factor in the ginsenoside transport signal pathway and controlling the transport and accumulation of the terpenoid, such as ginsenoside; and fine plants containing rich ginsenoside can be obtained by using a method for controlling or improving the ginseng and other plants via the promoter or the derivative thereof.
Description
Technical field
The invention belongs to the plant gene engineering technology field, be specifically related to a promotor that is subjected to the ginseng PDR transporter gene that methyl jasmonate replys.
Background technology
Ginseng (
P. ginsengC.A. being Meyer) Araliaceae Panax per nnial herb, is rare traditional Chinese medicine.Contain multiple medicinal ingredients in 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.That but some of them have is antitumor, anti-ageing, inhibited apoptosis and the strong saponin content of strengthening immunity isoreactivity extremely low.Utilize the relevant gene regulating technology of ginsenoside anabolism to promote the synthetic and accumulation of ginsenoside to have important theory value and application prospect.
Effective transcript and expression of gene is the essential condition of its gene function of performance, and gene success transcript and expression need to rely on promotor to its regulation and control, and promotor and RNA polymerase, transcriptional regulator etc. interact and bring into play the function of its regulation and control.TATA in eukaryote and CAAT box, they have determined initiation site and the efficient of transcribing, and are responsible for and the RNA polymerase combination, start the basic transcription process.Promoter component has many different types, comprises pathogeny evoked type functional element, organizing specific type functional element and chemical substance inducing action element etc.Wherein chemical substance induction type functional element is promoting secondary metabolite to play an important role aspect synthetic.In the synthetic expression process with accumulating genes involved of secondary metabolite, cis-acting elements is by bringing into play important regulating and controlling effect with transcription factor mutual: environmental stimulus is through after a series of signal transduction, transmitting, activate the mutual work of defence associated transcription factor and special cis-acting elements, thereby realize the expression of relevant defense function gene.Above-mentioned important regulating and controlling effect in view of cis-acting elements, use the resistant gene of inducible promoter mediation to have undoubtedly many-sided advantage, identify that therefore the synthetic inducible promoter relevant to accumulation of secondary metabolite become one of focus of present molecular biology of plants research.In the ginseng Secondary Metabolic Regulation of Callus, can be by the multistep rate-limiting reaction in the gene regulating route of synthesis such as importing key enzyme, the expression of perhaps synthesizing, transporting and accumulate genes involved at the transcriptional level control ginsenoside by cis-acting elements, thereby the content of raising ginsenoside.
Plant ABC transporter (ATP-binding cassette transporter) 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 fungi.But very few to the research of plant PDR gene-correlation at present, in vitro cell culture, sclareol can be induced
NpPDR1The gene up-regulated expression, in cell NpPDR1 albumen accumulation strengthened cell transport outside born of the same parents sclareol with and the ability of analogue.Arabidopis thaliana (
A. thaliana) in cell the accumulation of sclareol also can induce
AtPDR12The gene up-regulated expression detects the sclareol that AtPDR12 albumen is transported simultaneously outside born of the same parents in the extracellular, infer that terpene substances may be
AtPDR12The upstream regulation and control substance of genetic expression.In petunia, PhPDR1 albumen can be transported a kind of newfound plant hormone---solely angle gold lactone, and then promotion petunia branch.Thereby the PDR transporter gene has important potentiality aspect the accumulation of regulation and control secondary metabolite.Yet, before contriver's research, there is no the report of ginseng PDR protein gene and controlling element thereof, more there be not the report that affect secondary metabolite synthetic inducible promoter of with accumulation being correlated with relevant to this gene expression regulation.
Summary of the invention
The present invention is intended to isolate from ginseng and is subjected to methyl jasmonate to reply the gene induced type promotor of PgPDR3 of regulation and control, can regulate and control the ginsenoside transhipment and accumulate relevant genetic resources thereby provide a kind of, 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 regulate and control ginsenoside transhipment and accumulation
PgPDR3The promotor ProPDR3 of gene, its sequence is as shown in SEQ ID NO.1, or the DNA sequence dna that has 99% above homology with SEQ ID NO.1.
The preparation method of promotor ProPDR3 of the present invention is: extract the ginseng genome DNA, take the total DNA of ginseng as template, build four promoter libraries:
DraThe I library,
EcoR V library,
PvuThe II library and
StuThe I library; Respectively to build
DraThe I library,
EcoR V library,
PvuThe II library and
StuThe I library is template, according to ginseng
PgPDR5 ' terminal sequence design primer of 3 genes carries out pcr amplification; Glue reclaims the PCR product, and this product namely gets ProPDR3 promotor of the present invention.
Above-mentioned according to ginseng
PgPDRThe primer of 5 ' terminal sequence design of 3 genes comprises outside primer and nested primer, and outside primer Pro3-SP is as shown in SEQ ID NO.2, and nested primer Pro3-Nest-SP is as shown in SEQ ID NO.3.
The present invention also provides the recombinant vectors that contains the ProPDR3 promotor, specifically comprise: plant expression vector pCAMBIA1301, be about to the ProPDR3 promotor and be connected in empty carrier pCAMBIA1301, obtain the recombinant vectors pCAMBIA1301-ProPDR3::GUS of the gus reporter gene of ProPDR3 promoters driven.The ProPDR3 promotor also can be cloned in other empty carriers, or is prepared into transgenic cell and recombinant bacterium.
The present invention also provides application and the ProPDR3 promotor application among ginseng breedings of ProPDR3 promotor in the transhipment of regulation and control ginsenoside and accumulation.
The present invention utilizes existing plant gene engineering technology, 5 ' terminal sequence according to the ginseng PgPDR3 gene of having cloned, adopt the Genomewalker technical point from the ProPDR3 promoter sequence, build the recombinant expression vector of ProPDR3::GUS reporter gene, and changing gene over to tobacco by agrobacterium tumefaciens-mediated transformation, ProPDR3 transcribes gus reporter gene and expresses the driving effect that exists through the heterogenous expression assay certificate.Ginseng ABC transporter gene of the present invention
PgPDR3Promotor ProPDR3 sequence as shown in SEQ ID NO.1.The present invention studies and finds that gus gene that ProPDR3 drives expresses and have tissue specificity and be subjected to methyl jasmonate and the inducing of the chemical substance such as ginsenoside, and shows that described ProPDR3 promotor has the function that promotes the transhipment of regulation and control ginsenoside and accumulation aspect.Therefore, can by this regulation and control of promotor or derivatives thereof or improvement ginseng and other plant, obtain the high good plant kind of content of ginsenoside.For the regulative transcription factor gene of the key in follow-up clone's ginsenoside encoding transport signals approach, and regulate and control the transhipment of the terpenoids such as ginsenoside and accumulation etc. by transgenic method and lay a good foundation.
Description of drawings
Fig. 1 is the ginseng ProPDR3 promoter fragment electrophoresis result that the present invention increases; In figure, 1 expression pcr amplification product, M represents Marker;
Fig. 2 is the ginseng ProPDR3 promotor cis-acting elements analysis chart that the present invention increases;
Fig. 3 is the ginseng ProPDR3 promoter fragment electrophoresis result that increases in transgene tobacco; In figure, 1 ~ 10 expression pcr amplification product, M represents Marker;
Fig. 4 is that ProPDR3 promotor of the present invention starts the expression level figure of gus reporter gene in the transgene tobacco different tissues;
Fig. 5 is that ProPDR3 promotor startup gus reporter gene of the present invention is induced lower expression level view at 100 μ M MJ, 50 μ M SA, 20 μ M ABA and 20mg/LGS, and wherein MJ is that methyl jasmonate, SA are that Whitfield's ointment, ABA are that dormin, GS are ginsenoside.
Embodiment
The acquisition of embodiment 1 ProPDR3 promotor
1, the structure of ginseng DNA extraction and promoter library thereof
Get the 4 years living ginsengs in Changbai mountain, Jilin ginseng producing region, tap water soaks the 45min post-flush 2 times, first uses 75% alcohol immersion 30 seconds (sec), aseptic water washing 2 times.With reference to Omega Plant DNA Kit(Omega company) illustrate and extract genomic dna, take the total DNA of ginseng as template, build according to Genomewalker test kit (Takara company) specification sheets
DraI library (DL1),
EcoR V library (DL2),
PvuII library (DL3) and
StuI library (DL4).
2, design of primers and pcr amplification
(1) design of primers is with synthetic
According to the ginseng of having cloned
PgPDR5 ' end group of 3 genes (accession number is KC013238, and number of patent application is 201210462401.X) designs the outside and nested primer because of sequence, adopts Primer Premier 5.0 software design primers as follows:
Pro3-SP：5′-ATGCTCAAATCTGACTTCAATCGTGGG-3′；（SEQ?ID?NO.2）
Pro3-Nest-SP：5′-TCAACTTCCATACCATTAGTCCTCCAC-3′。（SEQ?ID?NO.3）
Primer is synthetic by Shanghai English fine horse biological company limited.
(2) pcr amplification
Respectively take four promoter libraries (DL1, DL2, DL3 and DL4) of building as template, utilize in Pro3-SP primer and test kit outside primer AP1 (5' – GTAATACGACTCACTATAGGGC – 3') carry out first round pcr amplification.
Reaction system is: 10 * PCR damping fluid, 5 μ L, MgCl
2(25mmol/L) 5 μ L, dNTP(2.5mmol/L) 8 μ L, Pro3-SP(10 μ mol/L) 2 μ L, AP1(10 μ mol/L) 2 μ L, library DNA 1 μ L, dd H
2O 26.5 μ L and LA Taq DNA polymerase(5U/ μ L) 0.5 μ L.
Reaction conditions is: 94 ℃ of sex change 25sec, and 72 ℃ are extended 3min, 7 circulations; 94 ℃ of sex change 25sec, 67 ℃ of annealing 3min, 32 circulations; 72 ℃ are extended 10min.
With 50 times of above-mentioned first round PCR product dilutions, then the nested primer AP2(5' – ACTATAGGGCACGCGTGGT – 3' to provide in test kit) and the Pro3-Nest-SP primer, with above-mentioned same system, 94 ℃ of sex change 25sec, 72 ℃ are extended 3min, 5 circulations; 94 ℃ of sex change 25sec, 67 ℃ of annealing 3min, 25 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
Be connected on pGEM-T Easy carrier after will PCR product glue reclaiming, and transform bacillus coli DH 5 alpha, recombinant plasmid pGEM-ProPDR3 will be served the order-checking of Hai Yingjun company.The sequence length that obtains after order-checking is the gene fragment of 917bp, to the gene order that obtains with
PgPDR3Gene 5' end overlap sequence is compared, through compare accurate after, sequence to the clone adopts PlantCARE (http://bioinformatiCS.psb.ugent.be/webtoo1s/plantCare/html/) on-line analysis instrument to carry out the cis element analysis to the promoter fragment of cloning, analytical results is seen Fig. 2, and result shows that clone's sequence contains the due primary element of promotor (TATA-Box and CAAT-Box) and multiple cis-acting elements.Its sequence is as shown in SEQ ID NO.1 in sequence table.
Structure and the conversion of embodiment 2 plant expression vectors
1, the screening of the structure of plant expression vector and transgene tobacco
(1) according to corresponding the comprising of promoter sequence design fragment
BamHI/
NcoThe Auele Specific Primer of I restriction enzyme site, the upstream and downstream primer is called after P1 and P2 respectively.
P1：5′-CGATGGATCCCACTTTGGTTGCTTCTCAATA-3′；（SEQ?ID?NO.4）
P2：5′-GGGCCATGGTCTTGCTTTCTATACATCACC-3′。（SEQ?ID?NO.5）
Use respectively
BamHI and
NcoI double digestion pCAMBIA1301 and ProPDR3 reclaim pCAMBIA1301 carrier and ProPDR3 promoter fragment.Two fragments are connected and transform DH5 α, by kantlex screening, PCR and
BamHI/
NcoThe I double digestion is identified and is obtained recon, recombinant plasmid called after pCAMBIA1301-ProPDR3::GUS.
(2) preparation of agrobacterium tumefaciens competent cell
1. streak culture Agrobacterium EHA105 on the YEB flat board that contains 100 μ g/mL rifomycins, secretly cultivate 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 spending the night contains to 50mL by the dilution proportion of l: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 20 mM CaCl
2The 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 thaws.
2. add 0.5 μ g pCAMBIA1301-ProPDR3::GUS plasmid DNA, mixing gently, ice bath 30min.
3. put freezing 2min in liquid nitrogen, move into rapidly heat shock 5min in 37 ℃ of water-baths, rapider ice bath 2min, add afterwards 800 μ L YEB liquid nutrient mediums, in 28 ℃ of shaking culture 3-4h.
4. the centrifugal 5min of 5000rpm precipitation thalline discards Eddy diffusion thalline after 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, differentiates with ProPDR3 promotor Auele Specific Primer, and primer is as follows:
P3：5′-CTCTGCTTCTCCAGCGAACT-3′；（SEQ?ID?N0.6）
P4：5′-TGTTCCATGCGTGTTGTGTG-3′；（SEQ?ID?N0.7）
After pcr amplification, agarose gel electrophoresis detects whether contain the expection fragment.The PCR response procedures is as follows: 94 ℃ of 30sec, 58 ℃ of 30sec, 72 ℃ of 30sec, 35 circulations; 72 ℃ of 2min.
2. enzyme is cut evaluation
Picking picking PCR is accredited as the single colony inoculation of positive Agrobacterium 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 pCAMBIA1301-ProPDR3::GUS, adopt aforesaid method with
BamHI/
NcoThe plasmid that the I double digestion extracts.After cutting and identify correctly, the process enzyme obtains to contain the Agrobacterium of recombinant plasmid pCAMBIA1301-ProPDR3::GUS.
2, the screening of gene transformation and transgene tobacco
(1) agrobacterium tumefaciens that contains the pCAMBIA1301-ProPDR3::GUS plasmid is cultivated
The Agrobacterium that 28 ℃ streak culture contains the pCAMBIA1301-ProPDR3::GUS 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.
(2) leaf dish method infects tobacco
With tobacco (
Nicotiana benthamiana) spire is placed in tap water and rinses 3-4 time, then soaks 30-60sec in 70% ethanol, the 10-20min that sterilizes in 10% clorox, more fully rinse with sterilized water, then be placed in sterile petri dish, blade is cut into 0.5-lcm
2Small pieces, the blade that shears is put into respectively the Agrobacterium bacterium liquid that contains the pCAMBIA1301-ProPDR3::GUS plasmid infects 5min, the bacterium liquid on blade is blotted with aseptic filter paper.
(3) 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 common substratum, and 2d is cultivated at dark place.
(4) screening and differentiation culture
After dark cultivation 2d, be transformed on the screening division culture medium (MS+6-BA 1.0mg/L+IAA 0.lmg/L+Kan 100mg/L+Cef 300mg/L), every 15d changes the primary screening division culture medium, goes in root media when the tobacco that differentiates grows to 3-5cm.
(5) root culture
The tobacco that breaks up to 3-5cm is downcut respectively, be transferred to root media (1/2MS+IBA 2.0mg/L).
(6) 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-l0d cover after transplanting, the humidity of maintenance 90%-100%, and beat a little apertures be beneficial to gaseous interchange on covering.The matrix of transplanting and flowerpot be sterilization in advance 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 ProPDR3 gene-specific primer P3 and P4 used carries out pcr amplification, PCR reaction system and reaction conditions are the same.Transgenic tobacco plant PCR detected result is seen Fig. 3, wherein 1 ~ 4,6 ~ 9 is respectively the T that transforms pCAMBIA1301-ProPDR3::GUS success and seedling differentiation
0For positive transfer-gen plant, No. 5 No. 10 for turning the also T of seedling differentiation of empty carrier pCAMBIA1301 success in order to transform failed plant
0For positive transfer-gen plant.
(8) ProPDR3 starts GUS expression and active detection the thereof
Utilize GUS can with substrate MUG(3,4-methyl umbellate form ketone-beta-glucuronidase) reaction produces fluorescent substance MU(4-methyl umbellate form ketone).The excitation wavelength of MU is 365nm, and emission wavelength is 456nm, and its content can be measured by spectrophotofluorometer.What of the fluorescent substance that produces within the unit time according to the plant total protein of unit mass come quantitative detection GUS content.
Reagent preparation: 1mol/L Na
2HPO
4Solution: 35.814g Na
2HPO
4Be dissolved in 100mL water.1mol/L NaH
2PO
4Solution: 15.601g NaH
2PO
4Be dissolved in 100mL water.0.1M phosphoric acid buffer (pH7.0): 1mol/L Na
2HPO
4Get 5.77mL, 1mol/L NaH
2PO
4Get 4.23mL, be settled to 100mL.10% SDS solution: 90mL water is heated a little, add 10g SDS, stirring and dissolving adds several concentrated hydrochloric acids to regulate pH to 7.2, then adds water and is settled to 100mL.0.5 M EDTA (pH8.0): add 18.61g Na in 80mL water
2EDTA2H
2O transfers pH to 8.0(approximately to need the solid NaOH of 2g left and right with NaOH), be settled to 100mL after dissolving.The GUS zyme extract: 0.1M phosphoric acid buffer (pH7.0) is got 50mL; 10% SDS gets 1mL; 0.5M EDTA (pH8.0) gets 2mL; Triton X-100 gets 100 μ L; Beta-mercaptoethanol 100 μ L; Water is settled to 100mL.MUG substrate: claim 8.8mg MUG, be dissolved in 10mL GUS zyme extract, be mixed with the working concentration of 2mmol/L.Reaction terminating liquid (0.2 mol/L Na
2CO
3): claim 2.12g Na
2CO
3, water is settled to 100mL.Coomassie brilliant blue G250 solution: Coomassie brilliant blue G250 10mg, 95% ethanol 5m1, H
3PO
410mL is settled to l00mL, filters rear 4 ℃ of preservations.1mg/mL BSA:20mg BSA is settled to 20mL with the GUS Extraction buffer.
1. the extraction of plant total protein
Get about the fresh plant tissue 100mg of transfer-gen plant, with the biomaterial IQF, then adopt the mode of liquid nitrogen grinding to grind tissue with liquid nitrogen in mortar.If do not grind immediately, the plant tissue that can first liquid nitrogen freezing be processed is stored in-80 ℃ of refrigerators.Forward in the Ep pipe grinding broken tissue, and add immediately the GUS Extraction buffer of 1mL, fully mixing.12,000rpm, 4 ℃ of centrifugal 5min go to another clean Ep pipe with supernatant, place stand-by on ice.
2. the mensuration of protein concentration
Get 7 Ep pipes, add respectively the BSA reference liquid of 0 μ L, 2 μ L, 4 μ L, 8 μ L, 12 μ L, 16 μ L, 20 μ L, water is mended to equal volume 20 μ L.The Coomassie brilliant blue G250 solution that adds 980 μ L, fully mixing, standing 5min on ice.Measure the absorption value at 595nm place with ultraviolet spectrophotometer.(mg/mL) does typical curve to absorption value A595 with protein concentration.Get testing protein sample 10 μ L, add 10 μ L water, add the Coomassie brilliant blue G250 solution of 980 μ L, abundant mixing, standing 5min, with the absorption value at ultraviolet spectrophotometer mensuration 595nm place, calculate the concentration of protein sample on ice.
3. the quantitative assay of GUS expression level
Get 100 μ L albumen supernatants, add in the GUS Extraction buffer of 37 ℃ of preheatings of 400 μ L, then add 500 μ L MUG substrates, 37 ℃ of temperature are bathed.Get respectively mixed reactant 200 μ L at 0min, 15min, 30min, 45min and 60min and join 800 μ L reaction terminating liquids, room temperature keeps in Dark Place.Under excitation wavelength 365nm, emission wavelength 455nm, measure the fluorescence intensity level of different time points with spectrophotofluorometer during slit 10nm.With fluorescence intensity level, the reaction times is made curve, the fluorescence intensity of obtaining the unit time changes.Fluorescence intensity with the unit time changes divided by the protein content of participating in reaction, and the fluorescence intensity of obtaining the albumen unit time of unit mass changes.
Fig. 4 is the active relative value (multiple) with contrasting GUS expression level quantitative assay in tobacco (turning the pCAMBIA1301 empty carrier) different tissues of GUS in the transgene tobacco different tissues.Fig. 4 shows that the ProPDR3 promoter expression has obvious tissue specificity, and its activity in root is the highest.Fig. 5 is the active relative value (multiple) with the quantitative assay of contrast tobacco (turning the pCAMBIA1301 empty carrier) GUS expression level of GUS in transgene tobacco under different inductor effects.Fig. 5 result shows that the ProPDR3 promotor is subjected to methyl jasmonate (MJ) and ginsenoside (ginsenosides, inducing GS), and ProPDR1 comprises the cis-acting elements (CGTCA-motif, TGACG-motif and T/G-box) that methyl jasmonate is induced in starting, and infers that the responsing reaction of ProPDR3 promotor is relevant with these cis-acting elements.This promotor is subjected to inducing of ginsenoside, shows that it may participate in ginsenoside transhipment and accumulation.
Claims (9)
1. the methyl jasmonate ginseng PgPDR3 gene promoter of replying is characterized in that: the DNA sequence dna of this promotor is as shown in SEQ ID NO.1, or the DNA sequence dna that has 99% above homology with SEQ ID NO.1.
2. the preparation method of promotor as claimed in claim 1 is characterized in that being followed successively by following steps: extract the ginseng genome DNA, take the total DNA of ginseng as template, build four promoter libraries:
DraThe I library,
EcoR V library,
PvuThe II library and
StuThe I library; Respectively to build
DraThe I library,
EcoR V library,
PvuThe II library and
StuThe I library is template, according to ginseng
PgPDR5 ' terminal sequence design primer of 3 genes carries out pcr amplification; Glue reclaims the PCR product, and this product namely gets promotor ProPDR3 of the present invention.
3. preparation method according to claim 2, is characterized in that: described according to ginseng
PgPDRThe primer of 5 ' terminal sequence design of 3 genes comprises outside primer Pro3-SP and nested primer Pro3-Nest-SP, and outside primer Pro3-SP is as shown in SEQ ID NO.2, and nested primer Pro3-Nest-SP is as shown in SEQ ID NO.3.
4. contain the recombinant vectors of the described promotor of claim 1, it is characterized in that: described recombinant vectors is pCAMBIA1301-ProPDR3::GUS, and described empty carrier is the pCAMBIA1301 carrier, connects gus reporter gene in the downstream of ProPDR3 promotor.
5. the construction process of recombinant vectors as claimed in claim 4 is characterized in that construction step is: (1) is according to corresponding the comprising of promoter sequence design fragment
BamHI/
NcoThe special primer of I restriction enzyme site; (2) use respectively
BamHI and
NcoI double digestion pCAMBIA1301 and promotor ProPDR3 reclaim pCAMBIA1301 carrier and ProPDR3 promoter fragment; (3) two fragments connected and transform DH5 α, by kantlex screen, PCR identifies and
BamHI/
NcoThe I double digestion is identified, is namely obtained described recombinant vectors pCAMBIA1301-ProPDR3::GUS.
6. method according to claim 5, is characterized in that: design corresponding the comprising of fragment according to promoter sequence in described step (1)
BamHI/
NcoThe special primer of I restriction enzyme site is as shown in SEQ ID NO.4 and SEQ ID NO.5.
7. according to claim 5 or 6 described methods is characterized in that: in described step (3), PCR identifies that designed primer pair is as shown in SEQ ID NO.6 and SEQ ID NO.7.
8. the application of promotor ProPDR3 in regulation and control ginsenoside transhipment and accumulation or ginseng breeding according to claim 1.
9. the application of recombinant vectors pCAMBIA1301-ProPDR3::GUS according to claim 4 in regulation and control ginsenoside transhipment and accumulation or ginseng breeding.
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| CN105087599A (en) * | 2015-09-07 | 2015-11-25 | 昆明理工大学 | Application of panax japonicus transcription factor gene PjERF1 |
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| CN111575333A (en) * | 2020-04-03 | 2020-08-25 | 东北林业大学 | Biological preparation method of ginsenoside |
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| CN114891803A (en) * | 2022-05-30 | 2022-08-12 | 湖南工程学院 | Ginseng PgWRKY40 gene induced by methyl jasmonate and application thereof |
| CN117625628A (en) * | 2024-01-26 | 2024-03-01 | 湖南工程学院 | ProPgJOX2 promoter for enhancing stress resistance of ginseng and application thereof |
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