CN101481694A - Gentiana straminea beta-amyrin synthetase gene GsAS2 and use thereof - Google Patents
Gentiana straminea beta-amyrin synthetase gene GsAS2 and use thereof Download PDFInfo
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Abstract
The invention discloses a gentiana straminea beta-amyrin synthase gene GsAS2 and yeast and a plant expression vector containing the gene GsAS2. Meanwhile, the invention also discloses an application of the gentiana straminea beta-amyrin synthase gene GsAS2 and the plant expression vector containing the gene GsAS2 to preparing oleanolic acid. The inventon provides theoretical basis and practical foundation for increasing the content of target product oleanolic acid.
Description
Technical field
The invention belongs to technical field of biological genetic engineering, relate in particular to the Oleanolic Acid genes involved---β-amyrin synthase gene GsAS2 and functional verification.
Background technology
Secondary Metabolism of Plant is meant that some organism utilizes some nascent meta-bolites to be " raw material ", under the catalysis of a series of enzymes, form the process of some special chemical substances, these special chemical substances are secondary metabolite, as alkaloid, flavonoid, terpene, organic acid, xylogen etc., they are that a big class is not the necessary small molecules organic compound of growth in the plant, but play irreplaceable effect for plant from the survival and development in complex environment.
The Secondary Metabolism of Plant product has important economic value.The medicine of clinical application, having a lot all is the secondary metabolite of taking from plant.In the clinical medicine in west, about 25% is the secondary metabolite that extracts from plant directly or indirectly.In China, the applicating history of medicinal plant is more long.In addition, be used as also extremely people's the favor of Secondary Metabolism of Plant product of food-flavoring comps, daily perfume compound, agrochemical.
Constantly perfect along with genetic engineering technique modified and transformed vegetable cell metabolism network and accumulate a large amount of purpose products and become possibility.At present, the clone of secondary metabolite key gene and conversion are the research focuses of Secondary Metabolism of Plant engineering.The metabolic engineering of key application enzyme gene provides technology platform for the output that improves plant tissue, cell cultures and whole plant meta-bolites.
The Secondary Metabolism of Plant product is of a great variety, and biosynthetic pathway also varies, and only on the basis of understanding the specific secondary metabolism approach of target plant, could carry out the efficient gene operation selectively.Therefore, the research of Secondary Metabolism of Plant collection of illustrative plates is the work of Secondary Metabolism of Plant genetically engineered necessary precondition.
The Secondary Metabolism of Plant species is various, and chemical structure is totally different.Now, known nearly 10000 kinds of secondary metabolites, comprise phenols, flavonoid, tonka bean camphor, lignanoid, alkaloid, glucosides, terpene, steroid class, saponin(e, polyyne class, organic acid etc., can be divided into phenoloid, terpene compound, itrogenous organic substance three major types.Terpenoid (terpenoids) is maximum in a Secondary Metabolism of Plant product family, extensively distribute at occurring in nature, by isoprene (isoprene, C5) compound and the derivative thereof formed of unit can be divided into monoterpene (C10), sesquiterpene (C15), diterpene (C20), triterpene (C30) and polyterpene etc. by the number of carbon atom.Owing to have different carbocyclic ring numbers in most terpenoid molecules, therefore can be divided into chain terpene, monocyclic terpene, dicyclo terpene and tricyclene etc. again.The material formed by six isoprene units of triterpene compound wherein.Extensively be formed in the bodies of aminal and plant, to swim unbound state or to become the form of ester or glycosides to exist.Majority is a containing oxygen derivative, is one of main component of resin.For example the hay glycosides in the Radix Glycyrrhizae is called Potenlini, and because of the sweet glycyrrhizin that claims again of its flavor, the aglycon that hydrolysis obtains under acidic conditions is called glycyrrhetinic acid, dissolves in ethanol and the chloroform, is a pentacyclic triterpene compound.Squalene is a chain triterpene in the Oils,glyceridic,cod-liver that is present in shark, sweet oil, the rapeseed oil, it is that the mutual symmetry of fragment after being end-to-end by a pair of three isoprene units is formed by connecting, have effects such as blood fat reducing and vessel softening, be described as the blood vessel street cleaner.
Herbal medicine is the traditional medicinal plant of China, and its effective constituent overwhelming majority derives from secondary metabolite, and content is very little in plant, and it is very big to utilize cultivation step to improve the active constituent content difficulty significantly.By the genetically engineered of secondary metabolism, the research that improves the Chinese medicinal materials active constituent content is at the early-stage.The clone of effective constituent genes involved and the effective ways of Function Identification thereof are also less, and consuming time, and the genes involved in the secondary metabolism approach that is obtained is less.
High and cold Tibetan medicine fried dough twist Macrophylla is Gentianaceae (Gentianaceae), Gentiana (Gentiana), mainly be distributed in the extremely frigid zones of Tibet, Qinghai, Gansu height above sea level 2500-4700 rice, perennial herb, herb and root are used as medicine, owing to its minute spreading, vegetative period is long, and medicinal material is collected very difficulty, thereby has become endangered species.Cure mainly jaundice type hepatitis, rheumatic arthritis, tuberculosis hectic fever, asthma, tic, shock, anaphylaxis.Be rich in the pentacyclic triterpene secondary metabolite, β-amyrin (beta-amyrin) is the important triterpene compound of a class wherein.
β-amyrin is one of modal triterpene compound in the plant.Its five carbocyclic ring skeletons are folded into chair conformation, are by 2, (Kushiro et al., 1998) that 3-oxidation squalene forms under β-amyrin synthetic enzyme catalysis through series reaction.And its synthetic enzyme is a very important rate-limiting enzyme in this approach, is controlling these medicinal ingredients precursor synthetic the first step reactions, and therefore the clone to this gene is extremely important.Oleanolic Acid is the final product in this reaction, and β in gentianaceae plant fried dough twist Macrophylla-amyrin synthase gene has very high promoter action for the accumulation of anti-hepatitis effective constituent Oleanolic Acid.
Till now, from many dicotyledonss, be separated to β-amyrin synthase gene, as the PNY1 in the dicotyledonous genseng (Kushiro et al., 1998), PNY2 (Kushiro et al., 1998); GgbAS1 in the hay (Hayashiet al., 2001); PSY in the pea (Morita et al., 2000); Also has the shape clover (Suzuki et al., 2002) of cutting and white birch (Zhang et al., 2003) etc.: the AsbAS1 in the monocotyledons oat (Haralampidis et al., 2001).And the checking to its function of article report arranged.For
Although in many species, be cloned into β-amyrin synthase gene at present, but this gene all clone, functional verification and in Plant Transformation, use and yet there are no report for gentianaceae plant fried dough twist Macrophylla, and also be not appear in the newspapers for the research of this gene and Oleanolic Acid relation.The present invention clones the gene GsAS2 that obtains and compares homology between 50-75% with the similar gene in other species in the fried dough twist Macrophylla, but all analyze discovery for its conserved regions: the aminoacid sequence of β in the fried dough twist Macrophylla-amyrin synthase gene GsAS2 contains 4 QW and 1 DCTAE primitive, β in known other species-amyrin synthase gene all contains this primitive, and this structural feature has certain effect for the performance tool of its function.
Summary of the invention
The purpose of this invention is to provide Oleanolic Acid genes involved in a kind of fried dough twist Macrophylla---β-amyrin synthase gene and the application in the preparation Oleanolic Acid thereof.
Technical scheme of the present invention is: separate obtaining β-amyrin synthase gene GsAS2 from the fried dough twist Macrophylla, authentication function in eukaryotic system forwards this gene to the transfer-gen plant that obtains high Oleanolic Acid content in the former plant fried dough twist Macrophylla then.
Fried dough twist Macrophylla β provided by the invention-amyrin synthase gene GsAS2 is characterized in that: described gene cDNA sequence is shown in SEQ ID No.1.
The present invention also provides a kind of Yeast expression carrier pPICZA that contains above-mentioned fried dough twist Macrophylla β-amyrin synthase gene GsAS2, it is characterized in that: described carrier cloning zone nucleotide sequence is shown in SEQ ID No.2.
The present invention also provides a kind of plant expression vector pROK II that contains above-mentioned fried dough twist Macrophylla β-amyrin synthase gene GsAS2, and it is characterized in that: described plant expression vector nucleotide sequence is shown in SEQID No.3.
This plant expression vector PROKII makes up mainly and finishes by following four steps: the primer amplification goal gene GsAS2 that 1) has SacI and XbalI restriction enzyme site; 2) goal gene GsAS2 is cloned into the pMD18-T carrier; 3) the pMD18-T carrier and the PROK II carrier that will contain the GsAS2 gene carries out double digestion with SacI and Xbal I respectively; 4) the GsAS2 gene that reclaims is connected with PROK II carrier.
The application of fried dough twist Macrophylla β of the present invention-amyrin synthase gene GsAS2 in the preparation Oleanolic Acid.
Experiment confirm: the GsAS1 gene transformation is advanced in the former plant fried dough twist Macrophylla, express making Oleanolic Acid content obtain big raising by crossing of this gene, wherein the content of Oleanolic Acid is measured by the HPLC method.
Beneficial effect of the present invention: utilize existing plant gene engineering technology, the present invention clones the β-amyrin synthase gene GsAS2 that has obtained the fried dough twist Macrophylla first and carry out functional verification in pichia spp, the method that transforms by particle gun changes this gene in the former plant fried dough twist Macrophylla over to, prove that through comparative analysis transfer-gen plant can improve 2-3 doubly than the Oleanolic Acid content of non-transgenic plant.So the research for this gene GsAS2 has important all meanings and application prospect.
Description of drawings
Fig. 1 is the cDNA electrophorogram of fried dough twist Macrophylla β-amyrin synthase gene GsAS2, and wherein: M is Marker.
Fig. 2 schemes for the positive callus screening of fried dough twist Macrophylla after changeing β-amyrin synthase gene GsAS2.
Fig. 3 is that transgenosis fried dough twist Macrophylla DNA identifies figure, and wherein: M is Marker, and 2-7,10,12,14-20,22-24 are the cDNA of fried dough twist Macrophylla transfer-gen plant.
Embodiment
The clone of embodiment 1, GsAS2
1.1 the extraction of the total RNA of fried dough twist Macrophylla
(1) fried dough twist Macrophylla material is put into the mortar of precooling, adds liquid nitrogen and grinds to form uniform powder rapidly.Note to guarantee that material is immersed in the liquid nitrogen in the process of lapping.
(2) treat after the liquid nitrogen volatilization material to be changed over to rapidly in the centrifuge tube of precooling, every 50-100mg organization material adds 1ml TRIZOL solution, behind the mixing, places 5min in room temperature, 12000r/min, and 2-8 ℃ of centrifugal 10min goes precipitation.
(3) add the 0.2ml chloroform in every 1ml TRIZOL solution, the abundant mixing of vibration 15sec is placed 5min, 12000r/min, 2-8 ℃ of centrifugal 15min in room temperature.
(4) get supernatant, every 1ml TRIZOL adds the Virahol of 0.5ml, mixing, and room temperature is placed 10～20min.
(5) the 2-8 ℃ of centrifugal 10min of 12000r/min abandons supernatant.
(6) add 1ml 75% washing with alcohol precipitation 2 times, 4 ℃ are no more than the centrifugal 5min of 7500r/min, abandon supernatant.
(7) after room temperature is placed and dried, add the distilled water dissolving RNA that 15 μ l DEPC handle.
1.2 reverse transcription synthesizes the first chain cDNA
(1) in the 0.2ml Eppendorf pipe, add following ingredients:
Above-mentioned total RNA (0.1 μ g/ μ l) 2.0 μ l with the DNaseI purifying
Olige(dT)anchored?primer(2μM) 2.0μl
(2) 0 ℃ of water-bath sex change 10 minutes place ice bath immediately.
(3) in 10 μ l reaction systems, add following ingredients: 2.0 μ l, 5 * MMLV RT buffer; 1.0 μ l 250 μ mol/L dNTP mix; 0.25 μ l Rnasin; 0.5 μ l (200u/ μ l) MMLV reversed transcriptive enzyme; The above-mentioned RNA sex change of 2 μ l liquid.42 ℃ are carried out reverse transcription reaction 1hr.The reverse transcription first chain cDNA is used for the reaction of back.
1.3?PCR
The synthetic first chain cDNA is a template with above-mentioned reverse transcription, PCR method amplification gene fragment.
(1) the PCR primer is synthetic by Shanghai Sangon Biological Engineering Technology And Service Co., Ltd
Primer P2:5 '-ATGTGGAGGCTAAAGATCG-3 '
Primer P3:5 '-AGAAGTTGATTATAAGTTAGCTGCA-3 '
(2) PCR reaction system
10×PCR?buffer 2μl
MgCl
2(25mmol/L) 1.5μl
dNTP(each?250μmol/L) 0.2μl
P2(10μmol/L) 1μl
P3(4μmol/L) 1μl
The reverse transcription first chain product 4 μ l
Ex?Taq 0.2μl
Sterilized water 10.1 μ l
(3) PCR response procedures
94 ℃ of 5min; 94 ℃ of 30sec, 50 ℃ of 30sec, 72 ℃ of 2min, 35 circulations; Last 72 ℃ are extended 7min.PCR product electrophoresis as shown in Figure 1.
1.4?5′-RACE
Operate according to step shown in TaKaRa 5 '-Full RACE Core Set specification sheets.Electrophoresis 5 '-the RACE product.
1.5 the segmental recovery of purpose
(1) downcuts the adhesive tape that contains the purpose band in the above-mentioned electrophoresis result with blade and place the 1.5mlEppendorf pipe.
(2) weigh, add the DR-I Buffer of 3-4 times of volume, 65 ℃ of heating 10min melt blob of viscose, guarantee once that every the 2min mixing blob of viscose can fully melt.
(3) add the DR-II Buffer of 1/2 volume of DR-I Buffer amount, uniform mixing.
(4) Spin Cloumn is placed on the Collection Tube, above-mentioned solution is transferred among the Spin Column, the centrifugal 1min of 5000rpm abandons filtrate.
(5) 500 μ l Rinse A are added among the Spin Column, the centrifugal 1min of 5000rpm abandons filtrate.
(6) 700 μ l Rinse B are added among the Spin Column, the centrifugal 1min of 5000rpm abandons filtrate.
(7) repeating step 6, then the centrifugal again 1min of 12000rpm.
(8) Spin Cloumn is placed on the centrifuge tube of new 1.5ml, adds water or the elution buffer of the 25 μ l that are preheating to 60 ℃ in the centre of Spin Cloumn film, room temperature leaves standstill 1min.
(9) the centrifugal 1min eluted dna of 12000rpm.
(10) repeating step is 9,2 times, with all elutriant centralized collection in a pipe, mixing, the electrophoretic examinations organic efficiency that takes a morsel, all the other add the 3mol/L NaAc (pH5.3) of 1/10 volume and the dehydrated alcohol of 2 times of volumes ,-20 ℃ the precipitation 1hr.
(11) then 4 ℃, the centrifugal 15min of 12000rpm with 70% cold washing with alcohol precipitation, abandons most liquid, with 40 μ l sterilized water dissolution precipitations.The fragment that reclaims is used for the ligation of back.
1.6 connect
With above-mentioned recovery purpose fragment and pMD-18T carrier (available from precious biotechnology company limited, down with) in molar ratio the ratio of about 1:1 connect, linked system is as follows:
T-vector 1μl
10×T4?DNA?Ligase?Buffer 1μl
T4?DNA?Ligase 1μl
Mixing is also of short duration centrifugal, and 16 ℃ of connections are spent the night.Obtain recombinant plasmid pMD18T-AS1, be used for the reaction of back.
1.7 CaCl
2Legal system is equipped with competent cell
(1) the single colony inoculation of picking DH10B is in 5ml LB liquid nutrient medium, and 37 ℃ of shaking culture are spent the night.
(2) in the ratio of 1:100-1:50, to get 1ml bacterium liquid and be inoculated in the 100ml LB liquid nutrient medium, 37 ℃ of shaking culture are to bacterium liquid OD
600Be 0.3-0.6.
(3) with bacterium liquid ice bath 10min, 4 ℃ of centrifugal 10min of 4000rpm collect thalline.
(4) precipitation adds the 0.1M CaCl of 10ml precooling
2After the suspension, ice bath 30min.
(5) 4 ℃ of centrifugal 10min of 4000rpm.Pellet resuspended is in the 0.1M of 1ml precooling CaCl
2, preserve in mixing and the ice-water bath, standby or add 15% glycerine and place-70 ℃ of preservations.
1.8 thermal shock method Transformed E coli DH10B
(1) draw 100 μ l competent cells under aseptic condition, add in the aseptic Eppendorf pipe of 1.5ml precooling, add 10 μ l and connect product (recombinant plasmids in 1.6), mixing places 30min on ice immediately gently.
(2) heat shock 90sec (accurately clocking) in 42 ℃ of waters bath with thermostatic control.
(3) ice bath 3-5min.
(4) add 800 μ l and do not contain antibiotic LB liquid nutrient medium, mixing, 37 ℃ of shaking culture 45-60min.
(5) of short duration centrifugal collection thalline with the resuspended thalline of 150 μ l LB liquid nutrient mediums, goes on the LB solid plate that contains microbiotic Amp, X-gal, IPTG, with aseptic spreading rod coating.
(6) flat board is placed 15-30min in 37 ℃ of forwards and be absorbed to liquid, inversion is dull and stereotyped, in 37 ℃ of cultivation 12-16hr, observes flat board and has blue hickie.The plasmid that hickie is used for the back extracts.
1.9 alkaline lysis method of extracting escherichia coli plasmid
(1) the single bacterium colony of picking white, access contains in the 3ml LB liquid nutrient medium of microbiotic Amp (50mg/L), and the white single bacterium colony that also will preserve institute's picking simultaneously is in the solid LB flat board that contains microbiotic Amp (50mg/L), and 12-16h are cultivated in 37 ℃ of concussions;
(2) the centrifugal 30sec of 12000rpm collects the thalline in the culture, abandons most supernatant as far as possible;
(3) add the solution I that 100 μ l ice precooling, on vortice, make thalline fully resuspended;
(4) add 200 μ l solution II, immediately centrifuge tube is slowly put upside down for several times ice bath 5-10min;
(5) add the solution III that 150 μ l ice precooling, slowly put upside down centrifuge tube and fully form ice bath 5-10min until white precipitate for several times;
(6) the centrifugal 3min of 12000rpm gets supernatant and changes in another Eppendorf tube, adds 2 times of volume 95% ethanol, and behind the mixing, room temperature leaves standstill 3min; The centrifugal 3min of 12000rpm makes the plasmid DNA precipitation;
(7) abandon most supernatant, get 200 μ l TE solution dissolution precipitations;
(8) add the 5mol/L LiCl solution that equal-volume is iced precooling, ice bath 5min precipitates a large amount of RNA;
(9) 12000rpm, centrifugal 3min;
(10) shift supernatant in another centrifuge tube, add 95% ethanol of 2 times of volumes, leave standstill 3min in room temperature behind the mixing, the centrifugal 3min of 12000rpm makes the plasmid precipitation;
(11) abandon behind the supernatant with 1ml 70% washing with alcohol precipitation, abandon most liquid;
(12) after the drying at room temperature, get the TE solution dissolution precipitation that 20 μ l contain RNaseA (20 μ g/ml), 37 ℃ of water-bath 30～60min digestion RNA.
(13) for reducing the loss, can with TE cumulative volume be supplemented to 200 μ l earlier, add isopyknic phenol-chloroform-primary isoamyl alcohol, the 5-10min that fully vibrates, the centrifugal 5min of 12000rpm;
(14) get supernatant, add isopyknic chloroform-primary isoamyl alcohol, repeat above operation;
(15) get supernatant, add the 3mol/L sodium-acetate (pH5.3) of 1/10 volume and the dehydrated alcohol of 2 times of volumes, place 15min for-20 ℃ behind the mixing, the centrifugal 3min of 12000rpm makes the plasmid precipitation;
(16) abandon supernatant with 1ml70% washing with alcohol precipitation, abandon liquid, with 40 μ l TE or sterilized water dissolution precipitation.Plasmid is used for the reaction of back.
1.10 plasmid enzyme restriction checking
Cut above-mentioned plasmid with the EcoRI enzyme, the endonuclease reaction system is as shown in the table:
Recombinant plasmid pMD18T-AS2 16 μ l
10×Buffer 2μl
With above-mentioned reaction system mixing and the centrifugal 1min of 5000rpm, 37 ℃ of water-baths spend the night, and carry out 1% TAE agarose gel electrophoresis detection then.
1.11 dna sequencing
The positive single bacterium colony that picking contains recombinant plasmid shakes with the liquid LB that contains Amp (50mg/L) and spends the night, and serves the order-checking of Hai Boya Bioisystech Co., Ltd then, and obtain sequencing result: gene cDNA sequence is shown in sequence table SEQ ID No.1.
Analyze through NCBIblast, the PNY1 homology of above-mentioned cDNA sequence and genseng proves that testing what obtain is exactly the β-amyrin synthase gene of fried dough twist Macrophylla, called after fried dough twist Macrophylla β-amyrin synthase gene GsAS2.
The functional verification of embodiment 2, fried dough twist Macrophylla-amyrin synthase gene GsAS2
2.1 the structure of Yeast expression carrier
2.1.1 the acquisition of goal gene
(1) design of primers: design one couple of PCR amplified band EcoRI and XhoI restriction enzyme site be the GsAS2 gene all.
P01：5’-
GAATTC?ATGTGGAGGCTAAAGATCG-3’
EcoRI
P02：5’-
CTCGAG?AGAAGTTGATTATAAGTTAGCTGCA-3’
XhoI
(2) PCR reaction system:
10×PCR?buffer 2μl
MgCl
2(25mmol/L) 1.5μl
dNTP(each?250μmol/L) 0.2μl
P21(4μmol/L) 1μl
P32(4μmol/L) 1μl
Recombinant plasmid pMD18T-AS2 1 μ l
rTaq(TaKaRa) 0.2μl
Sterilized water 13.1 μ l
(3) the PCR reaction conditions is
94 ℃ of 5min; 94 ℃ of 30sec, 60 ℃ of 30sec, 72 ℃ of 2min, 35 circulations; Last 72 ℃ are extended 7min.After reclaiming, PCR product electrophoresis is used for the reaction of back.
2.1.2 the construction step of expression vector
Introduce Yeast expression carrier pPICZA (available from precious biotechnology company limited), EcoR and two restriction enzyme sites of XhoI are contained in institute in containing on this carrier.
To have the cDNA of EcoR and XhoI restriction enzyme site and the double digestion that plasmid carries out EcoR and XhoI respectively, the ratio according to 5:1 is connecting more than 16 hours under the catalysis of T4 ligase enzyme again.Reaction system and condition are as follows:
(1) cDNA endonuclease reaction system and condition:
cDNA/EcoR+XhoI 6μl
EcoR 1μl
XhoI 1μl
10×H?Buffer 2μl
37℃?16h；
(2) plasmid enzyme restriction reaction system and condition:
EcoR 1μl
XhoI 1μl
10×H?Buffer 2μl
37℃?16h；
Ligation is carried out with T4DNA Ligase, and system and condition are with the linked system and the condition of T carrier.With connecting the product transformed into escherichia coli, from the bacterium colony of kalamycin resistance, filter out recon, obtain Yeast expression carrier pPICZA-GS2.The plasmid that extracts positive colony pPICZA-GS1 carries out PCR evaluation and double digestion evaluation, and reaction system and condition are with identifying recon.
2.2 DNA imports yeast
(1) mono-clonal of picking yeast strain (pichia spp) is seeded to 10ml liquid YPD substratum, and 30 ℃, 200rpm cultivated 2-3 days;
(2) according to the ratio of 1:50 bacterium liquid is transferred in the 50ml YPD liquid nutrient medium 30 ℃, 200rpm cultivates 3～5h, the bacterium liquid OD 0.4～0.6 of this moment.
(3) bacterium liquid branch is filled in the aseptic centrifuge tube of 10ml every pipe 5ml;
(4) the centrifugal 5min of 3000rpm, collecting cell.With the resuspended thalline of the aseptic ddH20 of 2.5ml;
(5) the centrifugal 5min of 3000rpm, collecting cell.With the resuspended thalline of 0.1ml100mM (1 *) LiAc/TE buffer;
(6) the centrifugal 1～2min sedimentation cell of 3000rpm (only need with thalline is centrifugal get final product at the bottom of centrifuge tube) carefully siphons away supernatant with micropipet;
(7) with the resuspended thalline of 50 μ l 100mM (1 *) LiAc/TE buffer, bacteria suspension is moved in the 1.5mlEppendorf pipe of sterilization;
(8) 1ml strand carrier DNA is boiled 5min, in frozen water, cool off fast; (annotate: all boil carrier DNA when need not to use, it is frozen to be distributed into aliquot at every turn; Should boil again after the freeze thawing 3～4 times, should remain on ice after the taking-up)
(9) sample that step 7 is prepared, the centrifugal 1～2min sedimentation cell of 3000rpm carefully siphons away supernatant with microscale sampler;
(10) add in the following order: 240 μ l PEG (50%w/v) (add the back and blow and beat up and down, thalline is suspended as far as possible) with the rifle head; 36 μ l 1M (10 *) LiAc; 25 μ l strand carrier DNA (2.0mg/ml), 50 μ l water and plasmid DNA (0.1～10 μ g) (generally speaking, plasmid adding 10～40 μ l get final product);
(11) vortex vibration Eppendorf pipe 1min, up to the complete mixing of cell, 30 ℃ of water-bath 30min;
(13) the centrifugal 1～2min of 5000rpm removes the conversion mixed solution with micropipet;
(14) draw 0.2～1.0ml sterilized water and be added in each reaction tubes, with pipettor gently extracting with the precipitation that suspends;
(15) get 50 μ l conversion fluids, be uniformly coated on the substratum that contains kalamycin resistance, cultivate 2～4d for 30 ℃.Do contrast with unconverted yeast.
2.3 the evaluation of yeast recon
2.3.1 bacterium colony PCR identifies primary dcreening operation
(1) bacterium colony on the flat board is long to naked eyes when visible.
(2) other PCR reaction solution components except template are ready to, and packing.
(3) with a sterilization toothpick picking colony, in the PCR pipe, survey, put into the 1.5ml centrifuge tube of a sterilization, PCR pipe and 1.5ml centrifuge tube numbering.
(4) pcr amplification, 0.8% agarose gel electrophoresis.To the clone that pcr amplification manifests specific band, place the half toothpick of 1.5ml centrifuge tube to throw away the 5mlYPD substratum, 30 ℃ of cultivations were extracted DNA, and were carried out PCR with DNA again as template and further determine in 24 ± 1 hours.
PCR reaction system and condition are as follows:
10×PCR?buffer 2μl
MgCl
2(25mmol/L) 1.5μl
dNTP(each?250μmol/L) 0.2μl
P2(4mol/L) 1μl
P3(4μmol/L) 1μl
rTaq(TaKaRa) 0.2μl
Sterilized water 13.1 μ l
The PCR reaction conditions is:
94 ℃ of 5min; 94 ℃ of 30sec, 60 ℃ of 30sec, 72 ℃ of 2min, 35 circulations; Last 72 ℃ are extended 7min.
2.3.2 being template, the total DNA of yeast carries out the secondary evaluation
The extraction of yeast genes group
(1) inoculation reorganization and empty plasmid transformant are in 5ml MD substratum, and bacterium compares in the YPD substratum, 30 ℃, cultivates 16-18 hour.
(2) under the room temperature, the centrifugal 5-10min of 1500g collects thalline.
(3) the resuspended back of 100uL TE (pH7.0) the centrifugal 5-10min of 1500g collects thalline.
(4) resuspended with the SCED (PH7.5) of 2ml.
(5) add 1～3mg helicase, 37 ℃, 50min.
(6) add 2ml 1% SDS, the jog mixing places 5min on ice then.
(7) add 1.5ml 5M Potassium ethanoate, mixing gently.
(8) the centrifugal 5-10min of 10000g abandons supernatant.
(9) add 2 times of volume dehydrated alcohols, room temperature is placed 15min.
(10) the centrifugal 20min of 10000g abandons supernatant.
(11) add 0.7ml TE damping fluid.
(12) add isopyknic phenol: chloroform (1:1V/V).
(13) the 7.5M Spirit of Mindererus of adding 1/2 volume.
(14) dehydrated alcohol of 2 times of volumes of adding is placed 1hr for-20 ℃.
(15) the centrifugal 20min of 10000g.
(16) add 1ml 75% ethanol rinsing precipitation once.
(17) after the drying, add TE or the H of 50ul
2The O dissolving ,-20 ℃ standby.
The composition of PCR reaction system and reaction conditions be with bacterium colony PCR, the 0.8% agarose gel electrophoresis analysis of PCR product.
The structure of embodiment 3, plant expression vector
3.1 the acquisition of goal gene
The system and the condition of design of primers and PCR reaction:
(1) the following one couple of PCR amplimer of design, two restriction enzyme site SacI that contained among the introduced plant expression vector pROK2 (available from precious biotechnology company limited) and XbalI.
P41：5’-CGAGCTATGTGGAGGCTAAAGATCG-3′
SacI
P42：5’-GCTCTAGAAGAAGTTGATTATAAGTTAGCTGCA-3’
XbalI
(2) PCR reaction system:
10×PCR?buffer 2μl
MgCl
2(25mmol/L) 1.5μl
dNTP(each?250μmol/L) 0.2μl
P41(4μmol/L) 1μl
P42(4μmol/L) 1μl
Recombinant plasmid pMD18T-AS2 1 μ l
rTaq(TaKaRa) 0.2μl
Sterilized water 13.1 μ l
(3) the PCR reaction conditions is
94 ℃ of 5min; 94 ℃ of 30sec, 60 ℃ of 30sec, 72 ℃ of 2min, 35 circulations; Last 72 ℃ are extended 7min.After reclaiming, PCR product electrophoresis is used for the reaction of back.
3.2 the structure of expression vector
To have SacI, the cDNA of XbalI restriction enzyme site and plasmid carry out the double digestion of SacI and XbalI respectively, again according to the connection of spending the night under the catalysis of T4 ligase enzyme of the ratio of 5:1.Reaction system and condition are as follows:
(1) endonuclease reaction system and condition:
cDNA/SacI+XbalI 6μl
SacI 1μl
10?X?Buffer 2μl
ddH
2O 1μl
30 ℃ of 1h of last table system, 37 ℃ of 1h of following table system
XbalI 1μl
0.1％?BSA 2μl
0.1％TritonX-100 2μl
10X?Buffer 2μl
ddH
2O 3μl
(2) plasmid enzyme restriction reaction system and condition:
SacI 1μl
10?X?k?Buffer 2μl
ddH
2O 1μl
30 ℃ of 1h of last table system, 37 ℃ of 1h of following table system
XbalI 1μl
0.1％?BSA 2μl
0.1％TritonX-100 2μl
10?X?H?Buffer 2μl
ddH
2O 3μl
Ligation is carried out with T4DNA Ligase, and system and condition are with linked system and the condition of pMD18-T.With connecting the product transformed into escherichia coli, from the bacterium colony of kalamycin resistance, filter out recon, the plasmid that extracts positive colony pROK-AS2 carries out PCR evaluation and double digestion evaluation, and reaction system and condition are with identifying recon.
3.3 order-checking
The positive strain that will contain recombinant plasmid is served the order-checking of Hai Boya Bioisystech Co., Ltd, and the sequencing result that obtains is analyzed through NCBIblast, contains the cDNA sequence of fried dough twist Macrophylla β-amyrin synthase gene GsAS2 in the positive plasmid really.So far obtain recombinant expression vector, promptly contained the plant expression vector pROK-AS2 of gene GsAS2.
4.1 gene gun conversion method
Transform the fried dough twist Macrophylla embryo callus that uses two weeks of subculture.Carry out height and ooze processing going to before the micropellet bombardment on the MS subculture medium that contains permeate agent in dark, 26 ℃ of insulation 4h.Permeate agent is a N.F,USP MANNITOL, and concentration adopts 0.4mol/L.2mg goldc grains (diameter 1 μ m), each 2.5 μ l (1 μ g/ μ l) of plasmid DNA, 25 μ l spermidines (0.1mol/L) and 50 μ l CaCl2 (215mol/L) are mixed, it is even that vortex or ultrasonication repeatedly make it, remove supernatant behind the centrifugal 5s of 10000r/min, precipitation is with 250 μ l washing with alcohol and be suspended in 240 μ l ethanolic solns again.The goldc grains suspension that is coated with DNA is applied to little bullet carrying tablet, and every is coated with 3.5 μ l, is used for the particle gun bombardment after the placement several minutes drying; Before each masking liquid the processing of suspension vortex is made it even.The about 2912 μ g of little bullet amount of each bombardment.The particle gun model is PDS1000PHe (BioRad), and air pressure adopts 1100psi.
Select and regeneration system 4.2 cultivate
Bombardment back 16h oozes substratum with the embryo callus piece from height and moves to the enterprising row filter of IB subculture medium that contains 600mg/mlKan, and brown and black callus are removed, and finally obtains the green regenerating plant.
4.3 the CTAB method is extracted the transfer-gen plant genomic dna
(1) takes by weighing the bent grass leaf of 0.5G, shred the back liquid nitrogen grinding, rapidly with powder transfer to the 1.5ml centrifuge tube, add 700 μ l then and be preheated to 65 ℃ 2xCTAB extraction damping fluid and 0.1% thin basic ethanol, put upside down the centrifuge tube mixing gently, place 65 ℃ of water bath heat preservation 2h then, put upside down mixing frequently.
(2) mixture adds isopyknic phenol after being cooled to room temperature: atmosphere is imitative: primary isoamyl alcohol (25:24:1), 4 ℃ of centrifugal 10min of following 10000g.
(3) water is transferred in the clean centrifuge tube, adds isopyknic chloroform: primary isoamyl alcohol (24:1), 4 ℃ of centrifugal 10min of following 10000g.
(4) water is transferred in the clean centrifuge tube, adds the two volumes dehydrated alcohol, gentle mixing is placed 30min deposit D NA for-20 ℃.
(5) 4 ℃ of centrifugal 10min of following 10000g discard liquid, and with 70% washing with alcohol twice, behind the drying at room temperature DNA, add the 100 TE liquid dissolvings of dodging the RNA enzyme that contains no DNA enzyme, 37 ℃ of water-bath 30min
(6) add 200 μ l dehydrated alcohols, gentle mixing is placed NA.4 ℃ of centrifugal 10min of following 10000g of 30min deposit D for-20 ℃, discards liquid, and with 70% washing with alcohol twice, behind the drying at room temperature DNA, adds 40 μ l sterilized water dissolving DNAs, and 4 ℃ of preservations are stand-by.
4.4 PCR detects transfer-gen plant
The DNA that extracts with the CTAB method is a template, and the PCR method increases with the 35S promoter fragment.
35S fragment promoter primer sequence:
35SS：5’-GCAGAGGCATCTTCAACG-3’
35SA：5’-GACGATCTACCCGAGCAA-3’
(1) PCR reaction system
10×PCR?buffer 2μl
MgCl
2(25mmol/L) 1.5μl
dNTP(each?250μmol/L) 0.2μl
35SS(4mol/L) 1μl
35SA(4mol/L) 1μl
rTaq(TaKaRa) 0.2μl
Sterilized water 13.1 μ l
(2) PCR response procedures
94 ℃ of 5min; 94 ℃ of 30sec, 53 ℃ of 30sec, 72 ℃ of 2min, 35 circulations; Last 72 ℃ are extended 7min.
After reaction finished, reaction solution detected in 0.8% TAE agarose gel electrophoresis.
The positive plant that screening obtains after transforming adds the 10ml chloroform, the centrifugal supernatant that goes behind the ultrasonic 40min after liquid nitrogen grinding.At last supernatant liquor is carried out HPLC and measure Oleanolic Acid content.
The result confirms: transfer-gen plant of the present invention improves more than 1 times than the Oleanolic Acid content of non-transgenic plant.
Annotate: the reagent of using in the above-mentioned experiment etc. are available from companies such as TAKARA, Promega, Sigma, Invitrogen.The LB culture medium prescription of using in the experiment (being weight ratio): 0.5% yeast powder, 1% NaCl, 1% peptone.Other agent prescription: see " molecular cloning " third edition.
Sequence table
＜110〉Shandong University
＜120〉fried dough twist Macrophylla β-amyrin synthase gene GsAS2 and application thereof
<141>2009-1-18
<160>3
<210>1
<211>2335
<212>cDNA
＜213〉fried dough twist Macrophylla
＜221〉fried dough twist Macrophylla β-amyrin synthase gene GsAS2
<222>(1)…(2335)
<400>1
<210>2
<211>8621
<212>DNA
＜213〉artificial sequence
＜221〉Yeast expression carrier pPICZA-GS2
<222>(1)…(8621)
<400>2
<210>3
<211>15175
<212>DNA
＜213〉artificial sequence
＜221〉plant expression vector pROK-AS2
<222>(1)…(15175)
<400>3
Claims (5)
1, a kind of fried dough twist Macrophylla β-amyrin synthase gene GsAS2, it is characterized in that: described gene cDNA sequence is shown in SEQID No.1.
2, a kind of Yeast expression carrier pPICZA-GS2 that contains the described gene GsAS2 of claim 1 is characterized in that: described carrier cloning zone nucleotide sequence is shown in SEQ ID No.2.
3, a kind of plant expression vector pROK-AS2 that contains the described gene GsAS2 of claim 1, it is characterized in that: described plant expression vector nucleotide sequence is shown in SEQ ID No.3.
4, the application of the described fried dough twist Macrophylla β of claim 1-amyrin synthase gene GsAS2 in the preparation Oleanolic Acid.
5, the application of the described plant expression vector pROK of claim 3 II in the preparation Oleanolic Acid.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103484389A (en) * | 2013-09-05 | 2014-01-01 | 中国科学院天津工业生物技术研究所 | Recombinant saccharymyces cerevisiae for producing ginsengenins as well as construction method and application of same |
| CN104293758A (en) * | 2014-09-17 | 2015-01-21 | 陈平 | Rhizoma panacis majoris beta-amyrin synthase gene and application thereof |
| CN113755478A (en) * | 2020-06-02 | 2021-12-07 | 东北林业大学 | Method for changing activity or function of 2,3-oxidosqualene cyclase |
-
2009
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103484389A (en) * | 2013-09-05 | 2014-01-01 | 中国科学院天津工业生物技术研究所 | Recombinant saccharymyces cerevisiae for producing ginsengenins as well as construction method and application of same |
| CN104293758A (en) * | 2014-09-17 | 2015-01-21 | 陈平 | Rhizoma panacis majoris beta-amyrin synthase gene and application thereof |
| CN113755478A (en) * | 2020-06-02 | 2021-12-07 | 东北林业大学 | Method for changing activity or function of 2,3-oxidosqualene cyclase |
| CN113755478B (en) * | 2020-06-02 | 2023-05-26 | 东北林业大学 | Method for changing activity or function of 2, 3-oxidation squalene cyclase |
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