CN103045621A - P-coumaroyl ester 3'-hydroxylase gene LjC3'H in lonicera japonica thumb. and application thereof - Google Patents
P-coumaroyl ester 3'-hydroxylase gene LjC3'H in lonicera japonica thumb. and application thereof Download PDFInfo
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Abstract
The invention discloses a p-coumaroyl ester 3'-hydroxylase gene LjC3'H in lonicera japonica thumb. and a prokaryotic expression vector containing the gene LjC3'H, and also discloses application of the p-coumaroyl ester 3'-hydroxylase gene LjC3'H in lonicera japonica thumb. in preparation of chlorogenic acid. Experiments prove that coumaroyl quinic acid and coumaroyl shikimic acid can be respectively catalyzed into chlorogenic acid and caffeoyl shikimic acid by adding an LjC3'H protein purified in vitro into an enzymatic reaction system, so that a theoretical foundation and a practical basis for developing, producing and increasing the content of a target product namely chlorogenic acid are provided.
Description
Technical field
The invention belongs to technical field of biological genetic engineering, relate in particular to a kind of Japanese Honeysuckle p-coumaric acyl ester 3 '-'-hydroxylase gene LjC3 ' H and application thereof.
Background technology
Secondary Metabolism of Plant refers to 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, such as alkaloid, flavonoid, terpene, organic acid, xylogen etc., they are that a large 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 worth.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, a large amount of purpose products are modified and transformed to accumulate to the plant metabolism network has become possibility.At present, the clone of secondary metabolite key gene and conversion are the study hotspots of Plant secondary metabolic 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 selectively the efficient gene operation.Therefore, the research of Secondary Metabolism of Plant collection of illustrative plates is the prerequisite work of Genetic Engineering of Plant Secondary Metabolism necessity.
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 large to utilize cultivation step to improve significantly the active constituent content difficulty.By the genetically engineered of secondary metabolism, the research that improves active ingredients from traditional Chinese medicinal 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 obtains is less.
Shandong famous-region drug Japanese Honeysuckle (Lonicera japonica Thunb.) is Caprifoliaceae Lonicera (Lonicera) plant, has antibacterial, antiviral, analgesic, anti-inflammatory, protects the liver, the effect such as hemostasis, anti-oxidant, immunomodulatory.Contain multiple bioactive ingredients: volatile oil component class, phantol, palm fibre are put acid, flavonoid compound class (luteolin, former times honeysuckle etc.), chlorogenic acid (chlorogenic acid and isochlorogenic acid, coffic acid and palm fibre are put acid etc.), triterpene saponin (loniceroside A and loniceroside C), p-Sitosterol etc.It is generally acknowledged that the chlorogenic acid compound is the main effective constituent of Japanese Honeysuckle.Chlorogenic acid has critical function in the Enzymatic browning of removing free radical, fruits and vegetables and the aspect such as antimycotic and pest-resistant.Therefore, the raising chlorogenic acid content is significant to improving quality of Flos Lonicerae.
The biosynthesizing of chlorogenic acid belongs to the Phenylpropanoid Glycosides class pathways metabolism of plant.Plant has been synthesized a large amount of important secondary metabolites by this approach.Hydroxycinnamic acid is the synthetic intermediate product of xylogen, determines the synthetic and physicochemical property of cell walls.Simultaneously, the precursor that hydroxycinnamic acid or numerous volatilization class material are synthetic, these materials have critical function in Allelopathic Effect in Plants.Coffic acid and ferulic acid ester are important antioxidant in the plant materials.Other material that is generated by phenylpropyl alcohol alkane has also participated in biology and the abiotic stress of plant response environment, and for example toluylene and flavonoid all are important phytoalexins.
The biosynthesizing of Phenylpropanoid Glycosides class material needs two step hydroxylation reactions at least.At first be that No. 4 carbon atoms of phenyl ring are by styracin hydroxylase (C4H) catalysis hydroxylation.The C4H function is measured than being easier in plant, is one of P450 family member who identifies the earliest.The second step hydroxylation reaction occurs in No. 3 carbon atoms of phenyl ring, by p-coumaric acyl ester 3 '-hydroxylase (C3 ' H) catalysis.At present function and the enzyme characteristic alive of C3 ' H are also imperfectly understood.There are some researches show that C3 ' H is the oxydase that relies on xitix, NADPH or flavine.Other has the scholar to think that C3 ' H is positioned at plastid, utilizes plastoquinone or Triphosphopyridine nucleotide photoreductase to be electron donor.Also the someone think the hydroxylation reaction of No. 3 carbon atoms by P450 take coumaroyl guinic acid or coumaric acyl shikimic acid as substrate catalysis.Angiosperm C3 ' H belongs to Cytochrome P450 CYP98 family, and the hydroxylation reaction of its catalysis can not be take the coumaric acid monomer as substrate, can only the catalysis coumaric acyl and the mixture that forms of shikimic acid or quinic acid.C3 ' H afunction causes plant short and small in the Arabidopis thaliana, and highly sterile, shows that C3 ' H is essential in plant phenylalanine pathways metabolism.
Although in many species, be cloned at present p-coumaric acyl ester 3 '-'-hydroxylase gene, but this gene cloning, functional verification there is not yet report for the caprifoliaceae plant Japanese Honeysuckle, and also are to have no report for the research of this gene and chlorogenic acid relation.
Summary of the invention
The purpose of this invention is to provide a kind of Chlorogenic Acid of Flos Lonicerae genes involved---p-coumaric acyl ester 3 '-'-hydroxylase gene LjC3 ' H and the application in the preparation chlorogenic acid thereof.
Technical scheme of the present invention is: separate obtaining p-coumaric acyl ester 3 '-'-hydroxylase gene LjC3 ' H from Japanese Honeysuckle, authentication function in prokaryotic system, then itself and the biosynthetic relation of chlorogenic acid of research in former plant.
Japanese Honeysuckle p-coumaric acyl ester 3 '-'-hydroxylase gene LjC3 ' H of the present invention, it is characterized in that: described gene cDNA sequence is shown in SEQ ID No.1.
The present invention also provides a kind of prokaryotic expression carrier pET28a-LjC3 ' H that contains above-mentioned Japanese Honeysuckle p-coumaric acyl ester 3 '-'-hydroxylase gene, it is characterized in that: described carrier cloning zone nucleotide sequence is shown in SEQ ID No.2.
The structure of above-mentioned prokaryotic expression carrier pET28a-LjC3 ' H is mainly finished by following four steps: 1) with the primer amplification goal gene LjC3 ' H of EcoRI and XhoI restriction enzyme site; 2) goal gene LjC3 ' H is cloned into the pMD18-T carrier; PMD18-T carrier and the pET28a carrier that 3) will contain LjC3 ' H gene carry out double digestion with EcoRI and XhoI respectively; 4) LjC3 ' the H gene that reclaims is connected with the pET28a carrier.
The application of Japanese Honeysuckle p-coumaric acyl ester 3 '-'-hydroxylase gene LjC3 ' H of the present invention in the preparation chlorogenic acid.
The present invention clones the gene LjC3 ' H that obtains and compares homology between 46-77% with the similar gene in other species in Japanese Honeysuckle.Its conserved regions analysis is found that the aminoacid sequence of p-coumaric acyl ester 3 '-'-hydroxylase gene LjC3 ' H contains the conservative territory of 4 CYP families in the Japanese Honeysuckle: PPGP, I helix, PERF and PFGXGRRXCX.All contain this conservative territory in p-coumaric acyl ester 3 '-'-hydroxylase gene in known other species, this structural feature has certain effect for the performance tool of its function.
Experiment confirm: LjC3 ' the H albumen of purification is added in the enzymatic reaction system, coumaroyl guinic acid and the catalysis of coumaric acyl shikimic acid difference can be generated chlorogenic acid and caffeoyl shikimic acid, the content of wherein said chlorogenic acid and caffeoyl shikimic acid is measured by the HPLC method.
Beneficial effect of the present invention: utilize existing plant gene engineering technology, the present invention clones first and has obtained p-coumaric acyl the ester 3 '-'-hydroxylase gene LjC3 ' H in the Japanese Honeysuckle and carried out functional verification.Process the Japanese Honeysuckle plant by different elicitors, prove that through comparative analysis LjC3 ' H gene and chlorogenic acid biosynthesizing are closely related.So the research for this gene LjC3 ' H has important all meanings and application prospect.
Description of drawings
Fig. 1 is the reading frame electrophorogram of Japanese Honeysuckle p-coumaric acyl ester 3 '-'-hydroxylase gene LjC3 ' H
Wherein: 1 is LjC3 ' H gene, and M is Marker.
Fig. 2 is Japanese Honeysuckle p-coumaric acyl ester 3 '-'-hydroxylase gene LjC3'H protein purification figure
Wherein: M, protein molecular standard; 1, induce total protein without IPTG; 2, the soluble proteins of inducing without IPTG; 3, induce total protein through IPTG; 4, the soluble proteins of inducing through IPTG; 5, LjC3 ' the H albumen of recovery.
LjC3 ' the H albumen catalysis coumaroyl guinic acid of Fig. 3 purifying and the HPLC of coumaric acyl shikimic acid detect.
Under the different elicitor treatment condition of Fig. 4, the expression analysis of LjC3 ' H gene in the Japanese Honeysuckle plant.
Embodiment
The clone of embodiment 1, LjC3 ' H
1.1 the extraction of the total RNA of Japanese Honeysuckle
(1) the Japanese Honeysuckle material is put into the mortar of precooling, adds liquid nitrogen and grinds to form rapidly uniform powder.Note to guarantee that material is immersed in the liquid nitrogen in the process of lapping.
(2) after the liquid nitrogen volatilization material is changed over to rapidly in the centrifuge tube of precooling, every 50-100mg organization material adds 1mlTRIZOL 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 1ml75% 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 process.
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 with the DNaseI purifying) 2.0 μ l
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.Reverse transcription the first chain cDNA is used for the reaction of back.
1.3 PCR
Take synthetic the first chain cDNA of above-mentioned reverse transcription as template, PCR method amplification gene fragment.
(1) the PCR primer is synthetic by Shanghai Sangon Biological Engineering Technology And Service Co., Ltd
Primer P0:5'-GTYGGHAACCTCTACGAC-3'
Primer P1:5'-RTCWCKVGCNACHGCCCA-3'
W=T or A wherein; R=A or G; Y=C or T; N=C, T, A or G;
H=A,T or C;K=G or T;V=G， A or C
(2) PCR reaction system
(3) PCR response procedures
94 ℃ of 5min; 94 ℃ of 30sec, 50 ℃ of 30sec, 72 ℃ of 2min, 35 circulations; Last 72 ℃ are extended 7min.
1.4 3 ＇ and 5 ＇-RACE
Operate according to step shown in the TaKaRa Full RACE Core Set specification sheets.Electrophoresis 3 ＇ and 5 ＇-RACE product.
1.5 the recovery of purpose fragment
(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) the DR-II Buffer of 1/2 volume of adding DR-I Buffer amount evenly mixes.
(4) Spin Cloumn is placed on the Collection Tube, 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 12000rpm centrifugal 1min again.
(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, and 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 with all elutriant centralized collection) and the dehydrated alcohol of 2 times of volumes ,-20 ℃ of 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, lower with) in molar ratio the ratio of about 1:1 connect, linked system is as follows:
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
2The standby competent cell of legal system
(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, for subsequent use or add 15% glycerine and place-70 ℃ of preservations.
1.8 thermal shock method Transformed E coli DH5 α
(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 clocks 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.Hickie is used for the plasmid extraction of back.
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), the white single bacterium colony that simultaneously also will preserve institute's picking is in the solid LB flat board that contains microbiotic Amp (50mg/L), and 12-16h is 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, make thalline fully resuspended at vortice;
(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 for several times until white precipitate fully forms ice bath 5-10min;
(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 1ml70% 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 first, 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 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 then carry out 1% TAE agarose gel electrophoresis detection.
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 then serves the order-checking of Hai Boya Bioisystech Co., Ltd, and obtain sequencing result: full-length gene cDNA sequence is shown in sequence table SEQ ID No.1.
Analyze through NCBIblast, prove that testing what obtain is exactly p-coumaric acyl ester 3 '-'-hydroxylase gene of Japanese Honeysuckle, called after p-coumaric acyl ester 3 '-'-hydroxylase gene LjC3 ' H.
The functional verification of embodiment 2, Japanese Honeysuckle p-coumaric acyl ester 3 '-'-hydroxylase gene LjC3 ' H
2.1 the structure of prokaryotic expression carrier
2.1.1 the acquisition of goal gene
(1) design of primers: LjC3 ' the H gene of design one couple of PCR amplified band EcoRI and XhoI restriction enzyme site.
P2:5'-G
AATTCATGGCATTCGCTCTCCTC-3'
EcoRI
P3:5'-C
TCGAGCTAGTAATCCACTGGGACACG-3'
XhoI
(2) PCR reaction system:
(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.PCR product electrophoresis as shown in Figure 1.
2.1.2 the construction step of prokaryotic expression carrier
Introduce prokaryotic expression carrier pET28a(available from precious biotechnology company limited), contained EcoRI and two restriction enzyme sites of XhoI in containing on this carrier.
The double digestion that will carry out respectively EcoRI and XhoI with cDNA and the plasmid of EcoRI and XhoI restriction enzyme site, 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:
37℃l6h；
(2) plasmid enzyme restriction reaction system and condition:
37℃l6h；
Ligation is carried out with T4 DNA Ligase, and system and condition are with linked system and the condition of T carrier.Transform intestinal bacteria with connecting product, from the bacterium colony of kalamycin resistance, filter out recon, obtain prokaryotic expression carrier pET28a-LjC3 ' H.The plasmid that extracts positive colony pET28a-LjC3 ' H carries out PCR evaluation and double digestion evaluation, and reaction system and condition are with identifying recon.
2.2 DNA imports e. coli bl21-Rosetta
(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 2.1), mixing places 30min on ice immediately gently.
(2) heat shock 40sec(accurately clocks 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 Ka Na, 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, cultivates 12-16hr in 37 ℃.
2.3 the evaluation of recon
2.3.1 bacterium colony PCR identifies primary dcreening operation
When (1) length of the bacterium colony on the flat board is visible to naked eyes.
(2) other PCR reaction solution components except template are ready to, and packing.
(3) with a sterilizing toothpick picking colony, in the PCR pipe, survey, put into the 1.5m1 centrifuge tube of a sterilization, to PCR pipe and 1.5m1 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 1.5m1 centrifuge tube to throw away the 5m1LB substratum, 37 ℃ of concussions are cultivated, and extract DNA, and carry out PCR with DNA as template again and further determine in 3 ± 1 hours.
PCR reaction system and condition are as follows:
The PCR reaction conditions is:
94 ℃ of 5min; 94 ℃ of 30sec, 60 ℃ of 30sec, 72 ℃ of 2min, 35 circulations; Last 72 ℃ are extended 7min.
3.1 the abduction delivering of target protein
The e. coli bl21 mono-clonal that contains recombinant expression plasmid pETLjC3 ' H and pET28a accesses respectively the LB substratum that 5mL contains 50 μ g/mL kantlex, and 37 ℃ of overnight incubation are diluted 100 times next day and continued to be cultured to OD
600Be that 0.6 o'clock adding final concentration is the IPTG (Isopropyl-beta-D-thio galactoside) of 0.5mmol/L, 28 ℃ of abduction delivering 4h.Centrifugal results bacterial cultures, (50mmol/L PBS, pH 7.2) is resuspended for phosphate buffered saline buffer, and ultrasonic wave is broken on ice, and SDS-PAGE detects total protein and solubilized albumen.
3.2 the purifying of target protein
Supernatant is crossed chelating Ni
2+Nickel sepharose post (5ml), then reduce the concentration (making the urea concentration on the post be followed successively by 8M/L, 6M/L, 4M/L, 2M/L) of urea by gradient, use again the lavation buffer solution (20mmol/LTris-HCl of 50ml, pH8.0,0.5mol/L NaCl, the 30mmol/L imidazoles, 0.1%Triton x-100) flushing, use at last elution buffer (20mmol/L Tri s-HCl, pH8.0,0.5mol/L NaCl, 0.5mol/L imidazoles, 0.1%Triton x-100) wash-out is got an amount of elutriant SDS-PAGE and is detected.Detected result as shown in Figure 2.
The outer enzymatic reaction system of 100 μ l standard bodies contains the recombinant protein of NADPH, 100 μ M coumaric acyl shikimic acids (or coumaroyl guinic acid), 0.1M potassium phosphate buffer (pH7.5) and the 2.0 μ g of 600 μ M.Above-mentioned reaction system places 30 ° of C after lower 30 minutes, adds final concentration and be 5% acetic acid termination reaction, uses afterwards the ethyl acetate extracting of 250 μ l and under 10,000g centrifugal 10 minutes.After getting supernatant vacuum-drying, add the methanol aqueous solution of 50 μ l50% (v/v).
The analysis of enzymatic preparation is used and is equipped with Kromosil C18 reversed-phase column (5 μ m, 250mm * 4.6mm; Macherey Nagel) Waters Alliance 2695 highly effective liquid phase chromatographic systems (HPLC) are finished.Moving phase is water (A) and methyl alcohol (B), and flow velocity is 0.6ml min
-1, use following gradient condition: 30%B 3 minutes, 30 – 70%B 27 minutes, 70 – 80%B 2 minutes, 80 – 95%B 3 minutes and 95%B 5 minutes.Detect the long 320nm of wavelength.With corresponding standard substance various compounds are carried out quantitatively.The HPLC detected result as shown in Figure 3.
The mensuration of this research recombinant protein kinetic constant is in the saturated situation of concentration of substrate, with another substrate between its K
m0.2 – 6.0 scopes in 5 concentration values calculate acquisition.250 μ l0.1M potassium phosphate buffer reaction systems of above-mentioned standard are used in experiment, and each experiment repeats 3 times.Finish under the optimal reactive temperature that is determined at its principal product of constant parameter and the optimal pH condition.K
mAnd K
CatValue calculation result such as following table:
The analysis of LjC'H genetic expression and chlorogenic acid content in embodiment 5, the rear Japanese Honeysuckle plant of different elicitor processing
5.1 the cultivation of Japanese Honeysuckle
Clip aseptic seedling side shoot, at MS substratum (3% sucrose, 0.7% agar) about upper root culture 20d, open sealed membrane hardening 2-3d, then be transplanted to peat soil mid-greenhouse growth, culture condition is: 27/18 ° of C of temperature, periodicity of illumination 16/8h (light/dark) cultivated after 50 days, choose the consistent material of growing way, random packet repeats for 3 times totally.Spray 0.1mM ABA, 0.1mM MeJA, 1.0mM salicylic acid (SA) with atomizer, contrast sprays distilled water.UV processes 2W m
-2UV-B shines 5h.For bulk testing repeats once, guarantee the reliable of testing data.
5.2 LjC ' H gene expression analysis
Get and process rear Japanese Honeysuckle blade, extract total RNA, method is with 1.1; Reverse transcription synthesizes the first chain cDNA, and method is with 1.2; Pcr amplification LjC ' H gene, the Actin gene is as contrast in the Japanese Honeysuckle.The PCR condition is seen as shown in Figure 4 with the 2.1.1. detected result.
5.3 determination of chlorogenic acid
Get 25mg Japanese Honeysuckle blade dry powder, the extraction of spending the night of 20ml sherwood oil, evaporate to dryness after filtering, the 5ml dissolve with methanol filters behind the mixing, gets 20 μ l HPLC and detects.Testing conditions is: moving phase is water (A) and methyl alcohol (B), and flow velocity is 0.6ml min
-1, use following gradient condition: 30%B 3 minutes, 30 – 70%B 27 minutes, 70 – 80%B 2 minutes, 80 – 95%B 3 minutes and 95%B 5 minutes.Detect the long 320nm of wavelength.Detected result is seen such as following table:
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 (being weight ratio) of using in the experiment: 0.5% yeast powder, 1%NaCl, 1% peptone.
Other agent prescription: see " molecular cloning " third edition.
Claims (3)
1. Japanese Honeysuckle p-coumaric acyl ester 3 '-'-hydroxylase gene LjC3 ' H, it is characterized in that: described gene cDNA sequence is shown in SEQ ID No.1.
2. prokaryotic expression carrier pET28a-LjC3 ' H who contains the described gene LjC3 ' of claim 1 H is characterized in that: described carrier cloning zone nucleotide sequence is shown in SEQ ID No.2.
3. the application of the described Japanese Honeysuckle p-of claim 1 coumaric acyl ester 3 '-'-hydroxylase gene LjC3 ' H in the preparation chlorogenic acid.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103320469A (en) * | 2013-07-08 | 2013-09-25 | 山东大学 | Method for acquiring transgenic seedlings by converting adventitious buds of honeysuckle |
| CN107099543A (en) * | 2017-06-21 | 2017-08-29 | 山东中医药大学 | Honeysuckle clone LjHQT genes, cloning process, plant expression vector and purposes |
| CN112391397A (en) * | 2020-11-25 | 2021-02-23 | 云南中烟工业有限责任公司 | Tobacco flavone monooxygenase gene NtCYP75B2 and application thereof |
| CN115531365A (en) * | 2022-02-07 | 2022-12-30 | 四川九章生物科技有限公司 | Application of chlorogenic acid composition in preparing medicine for preventing or treating pain |
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| CN102465136A (en) * | 2010-11-05 | 2012-05-23 | 中国中医科学院中药研究所 | Lonicera japonica chalcone isomerase (LjCHI) gene, LjCHI gene coded protein and their application |
| CN102465133A (en) * | 2010-11-05 | 2012-05-23 | 中国中医科学院中药研究所 | Lonicera japonica chalcone synthase (LjCHS) gene, protein coded by LjCHS gene and application of LjCHS gene |
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| CN102465136A (en) * | 2010-11-05 | 2012-05-23 | 中国中医科学院中药研究所 | Lonicera japonica chalcone isomerase (LjCHI) gene, LjCHI gene coded protein and their application |
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103320469A (en) * | 2013-07-08 | 2013-09-25 | 山东大学 | Method for acquiring transgenic seedlings by converting adventitious buds of honeysuckle |
| CN103320469B (en) * | 2013-07-08 | 2014-11-05 | 山东大学 | Method for acquiring transgenic seedlings by converting adventitious buds of honeysuckle |
| CN107099543A (en) * | 2017-06-21 | 2017-08-29 | 山东中医药大学 | Honeysuckle clone LjHQT genes, cloning process, plant expression vector and purposes |
| CN112391397A (en) * | 2020-11-25 | 2021-02-23 | 云南中烟工业有限责任公司 | Tobacco flavone monooxygenase gene NtCYP75B2 and application thereof |
| CN112391397B (en) * | 2020-11-25 | 2023-01-31 | 云南中烟工业有限责任公司 | Tobacco flavone monooxygenase gene NtCYP75B2 and application thereof |
| CN115531365A (en) * | 2022-02-07 | 2022-12-30 | 四川九章生物科技有限公司 | Application of chlorogenic acid composition in preparing medicine for preventing or treating pain |
| CN115531365B (en) * | 2022-02-07 | 2023-10-13 | 四川九章生物科技有限公司 | Application of chlorogenic acid composition in preparation of medicines for preventing or treating pain |
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| CN103045621B (en) | 2014-05-21 |
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