CN101921735B - Encoding gene of Saussurea involucrate flavanonol-4-reductase and application thereof - Google Patents
Encoding gene of Saussurea involucrate flavanonol-4-reductase and application thereof Download PDFInfo
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- CN101921735B CN101921735B CN2010102263446A CN201010226344A CN101921735B CN 101921735 B CN101921735 B CN 101921735B CN 2010102263446 A CN2010102263446 A CN 2010102263446A CN 201010226344 A CN201010226344 A CN 201010226344A CN 101921735 B CN101921735 B CN 101921735B
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- flavanonol
- reductase
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Abstract
The invention discloses an encoding gene of Saussurea involucrate flavanonol-4-reductase and applications thereof. The invention provides a protein which is flavanonol-4-reductase and is named as SmDFR. The enzyme is the following protein 1) or following protein 2): 1) protein composed of the amino acid sequence shown in the sequence 2 of the sequence table; 2) protein which is the derivative of the protein 1) by replacing and/or losing and/or adding one or several amino acid residues to the amino acid sequence of the sequence 2 and has the same function. The experiments of the invention show that the cloned gene formed by constructing a Saussurea involucrate cDNA library, using the library and degenerate primer to screening out the encoding gene of Saussurea medusa SmDFR and performing yeast expression and in vitro enzymatic reaction, is the functional encoding gene of flavanonol-4-reductase. The related flavanonol synthetic protein of the invention and the encoding gene of the protein have important value for solving the shortage problem of the wild resources of Saussurea involucrate.
Description
Technical field
The present invention relates to a kind of saussurea involucrata flavanonol-4-reductase encoding sox and its application, relate in particular to a kind of saussurea involucrata flavanonol-4-reductase and encoding sox and application.
Background technology
Snow Lotus Herb has another name called saussurea involucrata, and the composite family hieracioides belongs to.Be one type of famous and precious medicinal plant commonly used among the people, belong to per nnial herb.Generally be distributed on the high mountain flowstone beach of height above sea level more than 4,000 meters.In " territory, northwest note " and " the little knowledge in garden mutually ", just relevant for the record of saussurea involucrata.Has dispelling cold and removing dampness; Functions such as promoting blood circulation to restore menstrual flow, strong muscle is supporing yang, anti-inflammatory, analgesia, contraction uterus; Among the peoplely be used to treat rheumatic arthritis, delay senility, arteriosclerosis, termination of pregnancy, diseases such as women's cold and pain in the lower abdomen, amenorrhoea, retention of placenta, measles without adequate eruption, lung cold cough, impotence.Snow Lotus Herb thallophyta kind is more, though all can be used as medicine on an equal basis according to the record of Chinese medicine document, its quality has the branch of quality.According to relevant, what Absatzvolumen was maximum in above several kinds of saussurea involucrata is Xinjiang Tianshan saussurea involucrata (snow lotus) (S.involucrata Kar.et Kir.) and Saussurea medusa (S.medusa Maxim).And Saussurea medusa (Saussurea medusa Maxim.) is the former plant of Tibetan medicine " saussurea involucrata " medicinal material, is distributed in ground such as China Qinghai, Gansu, Tibet.Saussurea medusa is the rare medicinal herbs in the Tibetan medicine, has effects such as dispelling cold and removing dampness, promoting blood circulation and removing obstruction in channels, anticancer, anti-inflammatory and antifatigue, can treat multiple diseases such as rheumatic arthritis, menoxenia, carbuncle pyogenic infections from tumour or sore and high mountain incompatibility.
Contain multiple compositions such as flavones, vegeto-alkali, lactone, sterol, volatile oil, polysaccharide in the saussurea involucrata, its effective constituent is mainly flavonoid compound.The pharmacological action of flavonoid compound comprises anticancer, antitumor, resisting cardiovascular disease, kobadrin, anti-inflammatory, analgesic activity, immunoregulation effect, the effect of female hormone appearance, antibiotic and antivirus action, anti-oxidant, anti-aging effects, radioprotective.As the promote blood circulation saussurea involucrata TONGMAI KOUFUYE of ball, Herba Saussureae Involueratae injection and treatment cerebral arteriosclerosis and ishemic stroke of the saussurea involucrata that is used for treating hemiplegia, all be quality standard with the flavonoid content.The saussurea involucrata flavones is the flavonols remarkable pharmacological action especially, like the Saussurea medusa flavone component cns is had restraining effect; The Herba Saussureae Involueratae injection treatment migraine effect that with the flavonoid compound is effective constituent is remarkable.
The biosynthesizing of flavonoid compound realizes through phenylpropyl alcohol alkanes biosynthetic pathway.Under chalcone synthase (CHS) catalysis, produce phenyl styryl ketone (chalkone) beginning by 1 molecule 4-coumaric acyl-CoA and 3 molecule malonyl--CoA, chalcone synthase is to guide phenylpropyl alcohol alkanes pathways metabolism into flavonoid synthetic first most critical enzyme.Flavanonol-4-reductase (DFR) is first key gene that leads to anthocyanin and proanthocyanidin in the flavonoid route of synthesis.Provide hydrogen DFR can 4 ketone group reduction of flavanonol be formed hydroxyl by NADPH and generate colourless proanthocyanidin, colourless proanthocyanidin instability is further generated anthocyanin and proanthocyanidin by cyanidin(e) aglycon synthetic enzyme (ANS) and colourless proanthocyanidin reductase enzyme enzyme catalysiss such as (LCR).Flavanonol-4-reductase (DFR) and flavonol synthetic enzyme (FLS) competition flavanonol substrate.
Summary of the invention
The purpose of this invention is to provide flavanonol-4-reductase albumen and encoding sox thereof and application.
A kind of protein provided by the invention is flavanonol-4-reductase, derives from Saussurea medusa (Saussureamedusa Maxim), and called after SmDFR, this enzyme are following 1) or 2) protein:
1) protein of forming by the aminoacid sequence shown in the sequence in the sequence table 2;
2) with the aminoacid sequence of sequence 2 through the replacement of one or several amino-acid residue and/or disappearance and/or interpolation and have identical function by 1) deutero-protein.
Wherein, sequence 2 is made up of 342 amino-acid residues in the sequence table, the replacement of said one or several amino-acid residue and/or disappearance and/or be added to replacement and/or disappearance and/or the interpolation that is no more than 10 amino-acid residues.
In order to make 1) in SmDFR be convenient to purifying, label as shown in table 1 on proteinic N-terminal that can the aminoacid sequence shown in the sequence 2 is formed in by sequence table or C-terminal connect.
The sequence of table 1. label
| Label | Residue | Sequence |
| Poly-Arg | 5-6 (being generally 5) | RRRRR |
| Poly-His | 2-10 (being generally 6) | |
| FLAG | ||
| 8 | DYKDDDDK | |
| Strep-tag?II | 8 | WSHPQFEK |
| c- |
10 | EQKLISEEDL |
Above-mentioned 2) but in the SmDFR synthetic, also can synthesize its encoding sox earlier, carry out biology again and express and to obtain.Above-mentioned 2) encoding sox of the SmDFR in can be through lacking sequence in the sequence table 1 codon of one or several amino-acid residue in the dna sequence dna shown in the 5 ' terminal 61-1089; And/or carry out the missense mutation of one or several base pair, and/or obtain at the encoding sequence that its 5 ' end and/or 3 ' end connects the label shown in the table 1.
The proteic encoding sox of SmDFR also belongs to protection scope of the present invention.
Said encoding sox is following 1), 2), 3) or 4) or 5) shown in gene:
1) dna molecular shown in the sequence 1 in the sequence table;
2) in the sequence table sequence 1 from the dna molecular shown in 5 ' the terminal 61-1089 position Nucleotide;
3) in the sequence table sequence 1 from the dna molecular shown in 5 ' terminal the 56th-1129 Nucleotide;
4) under stringent condition with 1) or 2) or 3) dna molecule hybridize that limits and dna molecular with identical function;
5) with 1) or 2) or 3) dna sequence dna that limits has 90% homology at least and have the dna molecular of identical function.
Gene in the said step 5) is with 1) or 2) or 3) gene the homology more than 95% is preferably arranged.
Sequence 1 in the sequence table is the coding region by 1166 based compositions from 5 ' terminal 61-1089 position, the protein of sequence 2 in the code sequence tabulation.Above-mentioned stringent condition can be at 6 * SSC, in the solution of 0.5%SDS, 68 ℃ of hybridization down, uses 2 * SSC then, and 0.1%SDS and 1 * SSC, 0.1%SDS respectively wash film once.
The recombinant vectors, transgenic cell line, reorganization bacterium or the expression cassette that contain above-mentioned SmDFR encoding sox also belong to protection scope of the present invention.
Said recombinant vectors is for inserting the recombinant vectors that said encoding sox obtains between the Hind of carrier pYES2 III and EcoR I site.
Said reorganization bacterium is for importing the reorganization bacterium that the host bacterium obtains with described recombinant vectors.
Said host bacterium is yeast saccharomyces cerevisiae (Saccharomyces cerevisiae).
Available existing plant expression vector construction contains the recombinant expression vector of antisense SmDFR encoding sox.Said plant expression vector comprises double base agrobacterium vector and the carrier etc. that can be used for the plant micropellet bombardment, like pCAMBIA3301, pCAMBIA1300, pBI121, pBin19, pCAMBIA2301, pCAMBIA1301-UbiN or other plant expression vector of deriving.Conventional biological methods such as the plant expression vector that carries SmDFR encoding sox of the present invention can lead through Ti-plasmids, Ri plasmid, plant viral vector, directly DNA conversion, microinjection, electricity, agriculture bacillus mediated are transformed in vegetable cell or the tissue.
When using the SmDFR encoding sox to make up the recombinant plant expression vector; Before its transcription initiation Nucleotide, can add any enhancement type, composing type, organizing specific type or inducible promoter; Like cauliflower mosaic virus (CAMV) 35S promoter, general living plain gene Ubiquitin promotor (pUbi) etc., they can use separately or be used in combination with other plant promoter; In addition; When using SmDFR encoding sox of the present invention to make up plant expression vector; Also can use enhanser, comprise translational enhancer or transcriptional enhancer, these enhanser zones can be ATG initiator codon or neighboring region initiator codon etc.; But must be identical with the reading frame of encoding sequence, to guarantee the correct translation of whole sequence.The source of said translation wave and initiator codon is widely, can be natural, also can be synthetic.Translation initiation region can be from transcription initiation zone or structure gene.
For the ease of transgenic plant cells or plant being identified and screening; Can process used plant expression vector, can in plant, express enzyme or the gene (gus gene, luciferase genes etc.) of luminophor, antibiotic marker thing (qingfengmeisu qiong affinity tag, kantlex affinity tag etc.) or the anti-chemical reagent marker gene (like anti-weedkiller gene) etc. that can produce colour-change with resistance as adding.
The application of above-mentioned protein s mDFR in external vat yellow ketone compounds, or the application of above-mentioned encoding sox SmDFR in external vat yellow ketone compounds also is the scope that the present invention protects; Said flavonoid compound is preferably the pure and mild 3-of flavanone and removes hydroxyl dihydro anthoxanthin alcohol; Said flavanonol is preferably Taxifoliol, and said 3-goes hydroxyl dihydro anthoxanthin alcohol to be preferably eriodictyol.
Another object of the present invention provides the method for the transgenic plant of cultivating flavonol and/or the raising of flavonoid content.
Method provided by the invention is with the afunction of the said proteic encoding sox in the purpose plant, obtains transgenic plant; Flavonol alcohol and/or flavonoid content are higher than said purpose plant in the said transgenic plant;
With the method for the afunction of the said proteic encoding sox in the purpose plant through realizing to wherein changing recombinant expression vector over to; Said recombinant expression vector is in the MCS of pCAMBIA1302 carrier, to insert dna fragmentation to obtain, and said dna fragmentation is connected in sequence by the reverse fragment and the Nos terminator of CaMV 35S promoter, said proteic encoding sox;
The reverse fragment of the said proteic encoding sox of said claim 1 is the reverse complemental fragment from dna fragmentation shown in 5 ' the terminal 56-1129 position Nucleotide of sequence 1 in the sequence table.
The sequence of said CaMV 35S promoter is a 1-364 position Nucleotide among the genbank V00140.1; Said Nos terminator promoter sequence, 1-318 position Nucleotide among the genbank AJ007623.1.
Said purpose plant is a dicotyledons, and said dicotyledons is preferably Herba Saussureae Involueratae;
Said flavonol is rutin, myricetin, the luxuriant and rich with fragrance alcohol of camphane and/or Quercetin, and said flavonoid is an apigenin.
Experiment of the present invention proves; Through making up saussurea involucrata cDNA library; And utilizing this library to screen the encoding sox of Saussurea medusa SmDFR through degenerated primer, the gene of being cloned into through yeast expression and external enzymatic reaction is the encoding sox that the flavanonol-4-reductase of function is arranged, and utilizes special primer to pass through the encoding sox that PCR has cloned SmDFR; And made up the recombinant expression vector that contains SmDFR, obtained antisense SmDFR Herba Saussureae Involueratae.The mensuration result of antisense SmDFR Herba Saussureae Involueratae flavonol shows that antisense SmDFR Herba Saussureae Involueratae is compared with the Herba Saussureae Involueratae that changes empty carrier, and flavonol content such as rutin and myricetin are significantly improved.Explanation imports antisense SmDFR in the saussurea involucrata, can obtain the saussurea involucrata that flavonol content raises.Flavonol synthesis associated protein of the present invention and encoding sox thereof have important value to solving saussurea involucrata wild resource problem of shortage.
Description of drawings
Fig. 1 is the external catalysis Taxifoliol of Saussurea medusa SmDFR
Fig. 2 is the external catalysis eriodictyol of Saussurea medusa SmDFR
Fig. 3 is the conversion and the transgenic plant regeneration of Herba Saussureae Involueratae embryo callus subculture
Fig. 4 is that antisense transgene saussurea involucrata PCR detects
Fig. 5 is the HPLC collection of illustrative plates of flavonoid in the Herba Saussureae Involueratae.
Embodiment
Employed experimental technique is ordinary method like no specified otherwise among the following embodiment.
Used material, reagent etc. like no specified otherwise, all can obtain from commercial sources among the following embodiment.
The clone and the vivoexpression of embodiment 1, saussurea involucrata flavanonol-4-reductase gene (SmDFR)
1. the structure in saussurea involucrata cDNA library
Build storehouse used Saussurea medusa 1-15 days callus red colour system, induce: with Saussurea medusa (Saussurea medusa Maxim through following method; Xie H H, Wang T, Matsuda H, et al.2005.Bioactiveconstituents from Chinese natural medicines XV
.1)Inhibitory effect on aldosereductase and structures of Saussureosides A and B from Saussurea medusa.ChemPharm Bull, 53 (11): 1416-1422; The public can obtain from Chinese Academy of Sciences's plant research.) stem and blade make explant; Containing 0.2mg/L benzyladenine, 2mg/L naphthylacetic acid, 30g/L sucrose; Induce callus on the MS substratum of pH value 5.8; And in the MS substratum that contains 2mg/L naphthylacetic acid, 0.5mg/L benzyladenine and 30g/L sucrose, 25 ± 1 ℃, cultivation, subculture expand numerous acquisition red colour system callus under 16h illumination/8h dark.
Take by weighing each 50mg of red colour system callus that cultivated 1-14 days respectively, amount to the 700mg biased sample in mortar, extract total RNA with TRIZOL Reagent (available from GIBCO company).Get the total RNA sample of 1-3 μ l, the synthetic first chain cDNA of the PolyT primer of the CDSIII restriction enzyme site that (CLONTECH) carries with SMART cDNALibrary Construction Kit (Catlog#:K1051-1).Adopt LD PCR (long distance PCR) to increase with cDNA first chain to obtain double-stranded cDNA.
Add protease K digesting and remove the Taq enzyme among the cDNA, cut cDNA with Sfi I enzyme again, (Catlog#:636076 Clontech) carries out size separation to cDNA to use CHROMASPIN-400 then.Collect the cDNA of size＞500bp; The cDNA that collects is connected with λ TripIEx2 Vector (Promega); After the DNA that connects is packed by packaging protein Packagene Lambda DNA Packaging System (available from Promega company); It is 0.01% that the adding gelatin makes final concentration, and the DMSO final concentration is 7%, can be constant in 1 year titre of-70 ℃ of following preservations.The library is divided into 20 Ya Wenku, avoids freezing repeatedly molten, each Ya Wenku comes from when original amplified library the regenerant of different petridish, and it is formed maybe be different, can preserve, and so just accomplished the saussurea involucrata phage library and made up.
2. from saussurea involucrata cDNA library screening saussurea involucrata flavanonol-4-reductase gene cDNA
Conservative region (69aa altogether) according to the gene nucleotide series of the DFR of the nearly source species (being mainly composite family) that landed on the Genbank, synthesize upstream and downstream part degenerated primer with software DNAMAN4.0 (U.S. Lynnon Biosoft company) design:
DFRgl：5’-GAG?AAT?GAA?GT(A/G)AT(A/C/T)AA(A/G)CC-3’，
DFRg2：5’-AAA?ATA?CAT?CCA?TCC(A/G/C/T)GT?CAT-3’。
It is template that each Ya Wenku gets 1 μ L, is primer with DFRg1, DFRg2, and 25 μ L reaction systems prior to 95 ℃ of heating 10min lytic phages, add the pcr amplification mixture, carry out pcr amplification.
10×PCR?Buffer 2.5μl
dNTPs 0.5μl
Taq (Beijing ancient cooking vessel state) 0.2 μ l
Primer?DFRg1 0.5μl
Primer?DFRg2 0.5μl
Model 3.0μl
ddH
2O 17.8μl
The PCR program:
95 ℃ of preparatory sex change 10min of template (phage of 1 * λ dilution buffer liquid wash-out);
94 ℃, 4min (heat start PCR)
72℃， 10min
Choose the strongest positive pond of signal, make 10 times of gradient dilutions to 10
6Doubly, it is template that each grade dilution is got 1 μ L, carries out PCR by same program and detects, and confirms the maximum dilution multiple N (can detect the Method of Limited Dilution multiple of positive signal) in positive pond at the corresponding levels when next stage screens.10 9cm LB flat boards of 1 μ L dilution ehec infection and shop system by its maximum dilution multiple N dilution, are got in the elementary positive pond of choosing, cultivate and reclaim eluate.Picking growth and separate good single plaque from the flat board places damping fluid, and the vortex concussion is centrifugal slightly, in 4 ℃ of refrigerator overnight.Getting 3 μ L eluates is template, and PCR program augmentation detection obtains positive monoclonal, and the positive colony note is made pTripIEX2-SmDFR.
3.PCR method clone saussurea involucrata flavanonol-4-reductase gene SmDFR full-length cDNA
Phage vector goal gene when building the storehouse inserts segmental two ends and designs following two primers:
VEC1：5’-AAG?CGC?GCC?ATT?GTG?TTG-3’，
VEC2：5’-AAG?TGA?GCT?CGA?ATT?GCG?G-3’，
The PCR reaction conditions:
10×PCR?Buffer 2.5μl
dNTPs 0.5μl
Taq (Beijing ancient cooking vessel state) 0.2 μ l
Primer?VEC1 0.5μl
Primer?VEC2 0.5μl
Model 3.0μl
ddH
2O 17.8μl
The PCR program:
95 ℃ of preparatory sex change 10min of template (phage of 1 * λ dilution buffer liquid wash-out);
94 ℃, 4min (heat start PCR);
72℃， 10min
PCR product electrophoresis detection, the clone obtains the fragment of size greater than 1000bp, and with this PCR product order-checking, this PCR product has sequence 1 in the sequence table (cDNA of gene) as a result.With the unnamed gene of this PCR product is SmDFR, and with the albumen called after SmDFR of this genes encoding, this proteic aminoacid sequence is the sequence 2 in the sequence table.The ORFs of SmDFR is the 61-1089 position Nucleotide of sequence 1.
To adopting the BLAST similarity analysis; Through relatively learning; Aminoacid sequence after SmDFR full length cDNA sequence and the cDNA translation is very high with the plant similarity that belongs to composite family together; Wherein the similarity of DFR gene in the spot Minor centaury (Centaurea maculosa) and aminoacid sequence is the highest, reaches 92%.Show that the SmDFR of from Saussurea medusa, being cloned into is the encoding sox of flavanonol-4-reductase.
5.SmDFR vivoexpression and enzymatic reaction product identify
1) structure of pYES2-SmDFR expression vector
Extract the RNA of Saussurea medusa, reverse transcription becomes cDNA, is template with cDNA, and with primer SmDFR-Yf and SmDFR-Yr amplification, the recognition site of Hind III and EcoRI is introduced at the two ends of primer SmDFR-Yf and SmDFR-Yr respectively, and the sequence of primer is following:
SmDFR-Yf：5’-TCC?AAG?CTT?ACAACATGGTACAAGATTCTC-3’
SmDFR-Yr：5’-GCG?GAA?TTC?AGTACAATGAGACATAAATGT-3’
The PCR product detects through agarose gel electrophoresis; Reclaim the also dna fragmentation of purifying 1092bp; Cut this fragment with Hind III and EcoRI enzyme; (Invitrogen Cat.no:v82520) connects fragment after agarose gel electrophoresis recovery enzyme is cut, obtains recombinant plasmid pYES2-SmDFR with the pYES2 carrier of the same double digestion of warp.The recombinant plasmid pYES2-SmDFR transformed into escherichia coli DH-5 α that obtains; Choose single bacterium colony with the 5ml liquid LB culture medium culturing extraction plasmid that spends the night through amicillin resistance screening; Identify with HindIII and EcoRI digested plasmid DNA; Obtain the positive pYES2-SmDFR of fragment of 1092bp; Positive pYES2-SmDFR is sent to order-checking, and sequencing result is: this positive plasmid contains the 56th-1129 Nucleotide (containing SmDFR) from 5 ' end of sequence 1 in the ordered list, proves positive pYES2-SmDFR.
2) pYES2-SmDFR transformed saccharomyces cerevisiae (Saccharomyces cerevisiae) INVSc1 and protein induce are expressed
(Invitrogen, Cat.no:C81000), bacterium INVSc1/pYES2-SmDFR obtains recombinating to adopt the LiAC method to change yeast saccharomyces cerevisiae (Saccharomycescerevisiae) INVSc1 over to the positive pYES2-SmDFR of above-mentioned acquisition.The plasmid that will from the reorganization bacterium, extract is a template; Obtain purpose fragment (1092bp with primer SmDFR-Yf and SmDFR-Yr amplification checking; Sequence 1 from 5 ' terminal the 56th-1129 Nucleotide), the bacterium called after positive of should the recombinate bacterium INVSc1/pYES2-SmDFR that recombinates.
Should carry out protein semi-lactosi abduction delivering by positive reorganization bacterium INVSc1/pYES2-SmDFR,, extract the yeast total protein behind the granulated glass sphere broken wall, obtain the SmDFR protein crude extract according to the pYES2 product description of Invitrogen company.
Adopt above-mentioned method that empty carrier pYES2 carrier is changed among yeast saccharomyces cerevisiae (Saccharomycescerevisiae) INVSc1, the INVSc1/pYES2 of acquisition, same abduction delivering obtains the protein crude extract of empty carrier.
3) external enzymatic reaction
With Taxifoliol (Dihydroquercetin, CAS24198-97-8, SIGMA-ALDRICH T4512-25MG) and eriodictyol (3-Deoxydihydroquercetin; CAS552-58-9; The inferior training in Shanghai bio tech ltd) be substrate, reaction solution TV 500 μ l, comprising:
100mM Tris-HCl damping fluid (pH=7.5) 370 μ l
SmDFR protein crude extract
(0.15 μ g μ l
-1, by above-mentioned acquisition.) 70μl
10mM NADPH (is dissolved in 100mM Tris-HCl, pH=7.5) 50 μ l
10 μ g μ l
-1Substrate (being dissolved in methyl alcohol) 10 μ l
1. place 30 ℃ of water-baths of 1.5ml centrifuge tube to be incubated 4h reaction solution.
2. reaction solution merges mutually with 400 μ l ethyl acetate extractions, twice, twice supernatant ETHYLE ACETATE and transfers in the new centrifuge tube uncovered being positioned in 50 ℃ of baking ovens the ETHYLE ACETATE evaporate to dryness.
3. residue is with 50 μ l propyl carbinols: (95 ℃ are boiled 5min to hydrochloric acid for 95:5, the v/v) dissolving again of enzymatic prepn, are used for HPLC-MS and identify.
4) HPLC-MS of external enzymatic reaction product identifies
Identify product with Agilent 1100 HPLC/MSD Trap VL.Chromatographic column: ZORBAX Eclipse XDB-C18,4.6 * 150mm, 5 μ m (Agilent Co, USA), 30 ℃ of column temperatures, sample size 10 μ l, flow velocity 1ml min
-1Moving phase: solution A acetonitrile, solution B pH 3.0 trifluoroacetic acids (TFA) water.Elution program: 0-20min:A 90%-10%, B 10%-90%.Detect wavelength 214nm.
Mass spectrum parameter: electron spray(ES) (ESI) ion trap, 325 ℃ of gasification temperatures, carrier gas (N
2) flow velocity 10.01min
-1, nebulizer gas pressure 40psi, ends of the earth radio frequency amplitude (octopole RF amplitude) 150vpp, skim 1 voltage34V, skim 2 voltage 6V, capillary exit 107V, cap exit offset 73V, MSn collision energy 40%.Analyze and adopt positive ion preference pattern (m/z M+H
+), full ion scan, ion range of choice m/z50-1000.
The result is as depicted in figs. 1 and 2, and A is the total ion figure behind the external catalysis Taxifoliol of Saussurea medusa SmDFR among Fig. 1, and the arrow indication is a substrate; B. the product Cyanidin aglycon (arrow indication) (m/z 287) that generates; C. the mass-spectrogram of Cyanidin aglycon.Total ion figure among Fig. 2 behind the external catalysis eriodictyol of A. Saussurea medusa SmDFR, the arrow indication is a substrate; B. the product (arrow indication) (m/z 271) that generates; C. the mass-spectrogram of product.Show that from the peak sequence of the passable product of Fig. 1 and mass spectrum the substrate Taxifoliol has been generated the Cyanidin aglycon by chemical catalysis external again after by the SmDFR reduction, Taxifoliol is a kind of of flavonol; Show that from the peak sequence and the mass spectrum of the product of Fig. 2 the substrate eriodictyol has been generated (-)-Leucofisetinidin by chemical catalysis external again after by the SmDFR reduction, eriodictyol is that 3-goes a kind of of hydroxyl dihydro anthoxanthin alcohol.Show that SmDFR can generate corresponding leucoanthocyanidin by external catalysis flavanonol substrate; SmDFR has the activity and the flavanone reductase enzyme (Flavanone4-reductase of flavanonol-4-reductase; It is FNR) active that (Taxifoliol is the most frequently used substrate of flavanonol-4-reductase; SmDFR can just show that it has the flavanonol-4-reductase function by the catalysis Taxifoliol, and SmDFR also possibly have flavanone reductase enzyme function in addition, removes hydroxyl dihydro anthoxanthin alcohol with 3-; Be that flavanone is a substrate, generate an other compounds.)。
Protein crude extract with the empty carrier of above-mentioned acquisition serves as that contrast detects, and the result is not for there being activity.
Embodiment 2 uses antisense Saussurea medusa flavanonol-4-reductase gene and improves Herba Saussureae Involueratae flavonol content
1.pCAMBIA1301-the structure of antisense SmDFR expression vector
The cDNA of the Saussurea medusa that obtains with embodiment is a template; The recognition site of BamHI and Sac I is introduced at the two ends of primer DFRa1 and DFRa2 respectively, and the sequence of primer DFRa1 and DFRa2 is following: DFRa1:5 '-TAC gag ctc ACAACA TGG TAC AAG ATT CTC-3 '; DFRa2:5 '-GCG gga tcc AGT ACA ATG AGA CAT AAATGT-3 ';
The PCR product detects through agarose gel electrophoresis; Reclaim and purifying DNA fragment; Cut this fragment with Sac I and BamHI enzyme; The fragment that agarose gel electrophoresis reclaims after enzyme is cut is connected with pBI121 (Clontech) carrier of the same double digestion of warp, and SmDFR cDNA oppositely is inserted between pBI121 support C aMV 35S promoter and the Nos terminator, obtains recombinant plasmid pBI121-antisense SmDFR.With recombinant plasmid pBI121-antisense SmDFR transformed into escherichia coli DH-5 α; Kantlex screening recon; Extract plasmid and identify that with Sac I and BamHI double digestion positive recombinant is sent to order-checking, and the result is for obtaining containing the reverse segmental positive recombinant of SmDFR cDNA.
PBI121-antisense SmDFR carrier is cut through EcoRI and PstI enzyme; Antisense SmDFR is downcut together with CaMV 35S promoter and Nos terminator; Product detects through agarose gel electrophoresis, reclaims and purifying fragment (2100bp), with this fragment and pCAMBIA1302 (the Cambia Institute that cuts through EcoRI and PstI enzyme; Austrilia) carrier connects, and obtains recombinant expression vector pCAMBIA1302-antisense SmDFR.
The sequence of said CaMV 35S promoter is a 1-364 position Nucleotide among the genbank V00140.1; Said Nos terminator promoter sequence, 1-318 position Nucleotide among the genbank AJ007623.1.
With recombinant plasmid transformed intestinal bacteria DH-5 α, kantlex and hygromycin selection recon extract plasmid and cut evaluation with EcoRI and PstI enzyme.With the frozen-thawed method pCAMBIA1302-antisense SmDFR is imported Agrobacterium EHA105 (Invitrogen); Obtain recon; With kantlex, Totomycin and Rifampin screening recon, be template with single bacterium colony, CaMV 35S primer (5 '-CCCACTATCCTTCG-3 ') is the primer PCR evaluation with DFRa1; Obtain the positive single bacterium colony of fragment of 1100bp, called after EHA105/pCAMBIA1302-antisense SmDFR.
2. the Herba Saussureae Involueratae embryo callus subculture induces
Herba Saussureae Involueratae (Saussurea involucrata Kar.et Kir.; Yi T; Chen H B; Zhao Z Z, etal.2009.Identification and determination of the major constituents in thetraditional uighur medicinal plant Saussurea involucrata by LC-DAD-MS.Chromatographia, 69:537-542; The public can obtain from Chinese Academy of Sciences's plant research.) aseptically process of seed: the Herba Saussureae Involueratae seed is degerming 20min in 0.3% chlorine bleach liquor; Aseptic water washing 5-6 time; The Herba Saussureae Involueratae seed of pouring out after the sterilized water degerming is carefully peelled off kind of a skin with tweezers and inoculating needle, places MS+5mg/L 2, evoked callus on the substratum of 4-D+0.5mg/L6-BA; Callus growth is good back in MS+3mg/L 2, succeeding transfer culture on the substratum of 4-D+0.5mg/L 6-BA.The Herba Saussureae Involueratae rataria was cultivated on inducing culture 40 days, and per 20 days subcultures once can grow the yellowish callus of color and luster.
3. the acquisition of antisense SmDFR Herba Saussureae Involueratae
Single bacterium colony of picking EHA105/pCAMBIA1302-antisense SmDFR is inoculated into it in YEB liquid nutrient medium and (contains Kan (kantlex, Kanamycin) 100mg/L; Hyg (Totomycin, Hygromycin B) 50mg/L, Rif (Rifampin; Rifampicin) 50mg/L), 28 ℃ of shaking culture are spent the night.Agrobacterium (EHA105/pCAMBIA1302-antisense SmDFR) the bacterium liquid of drawing 200 μ l incubated overnight (contains Kan100mg/L in 20ml YEB liquid nutrient medium; Hyg50mg/L; Rif50mg/L), 28 ℃ of quick oscillation incubation growth to logarithmic phases (OD600=0.6), about 3-4h.The centrifugal 5min of room temperature 6000rpm collects thalline, uses MS
0Liquid nutrient medium is washed the thalline of once collecting, and guarantees not contain microbiotic in the substratum, then thalline is suspended in 10ml MS again
0For use in the liquid nutrient medium.
Select well-grown Herba Saussureae Involueratae callus, take out, place ready bacterium liquid with the tweezers after the calcination; Contaminate 30min; Pour out bacterium liquid, callus moves to MS+3mg/L 2, and dark the cultivation (measured too much for preventing bacterium in 48 hours on the 4-D+0.5mg/L 6-BA substratum; Disturb the later stage degerming, can put one deck filter paper) in media surface.At last material is moved to MS+3mg/L 2, screening and culturing on the 4-D+0.5mg/L 6-BA+Hyg40mg/L+Cef 400mg/L substratum.Can see after 20 days that green callus grows, the well-grown green callus that filters out is inoculated on the substratum of MS+1mg/L 6-BA+0.1mg/LNAA+Hyg40mg/L and induce 17 strain antisense SmDFR Herba Saussureae Involueratae seedlings.
Herba Saussureae Involueratae is seen shown in Figure 3 from embryo callus subculture to the process that differentiates seedling.
Adopt identical method that empty carrier pCAMBIA1302 is changed in the Herba Saussureae Involueratae, obtain to change the empty carrier Herba Saussureae Involueratae.
4. the detection of transgenic Herba Saussureae Involueratae
The antisense SmDFR Herba Saussureae Involueratae seedling of above-mentioned acquisition is extracted genomic dna with the CTAB method; With the genomic dna is template; With CaMV 35S primer and DFRa1 is whether primer PCR detection Saussurea medusa SmDFR cDNA has been incorporated in the Herba Saussureae Involueratae genome; Detected result is as shown in Figure 4, wherein C+: positive control is a template with pCAMBIA1302-antisense SmDFR; C-: negative control is a template with wild-type Herba Saussureae Involueratae genomic dna; M:2000PlusDNA Marker; A3, A5, A6, A8, A11, A12, A13, A16, A22, A23, A24, A26, A27, A30, A24, A36, A39 are T0 for antisense SmDFR Herba Saussureae Involueratae seedling, are template with T0 for antisense SmDFR Herba Saussureae Involueratae genomic dna.As can be seen from the figure, obtain the positive antisense SmDFR of 17 strains Herba Saussureae Involueratae seedling, show that SmDFRcDNA has been incorporated in the Herba Saussureae Involueratae genome.
5. the detection of transgenic Herba Saussureae Involueratae flavonol content
1) get and obtain antisense SmDFR Herba Saussureae Involueratae seedling system behind the antisense SmDFR Herba Saussureae Involueratae seedling subculture, the 10d sampling, each is four bottles; From every bottle, select the antisense SmDFR Herba Saussureae Involueratae seedling of robust growth, remove yellow leaf of cured leaf leaf and bottom callus, choose about tissue cultured seedling fresh weight 100mg for every bottle; Put into mortar at the liquid nitrogen grind into powder; Add 100 μ l, 80% methyl alcohol (v/v) according to every 10mg fresh weight blade, change in the 2ml centrifuge tube, fully mixing; Extract 24h under the room temperature, up and down stirred centrifuge tube once in every separated several hours therebetween.
2) with under the extracting solution room temperature 12, the centrifugal 10min of 000rpm carefully collects supernatant in the new centrifuge tube, and the deposition of gained is used again under half the 80% methyl alcohol (v/v) room temperature of former extracting liquid volume and extracted 24h.
3) under the room temperature, the centrifugal 10min of 12000rpm collects supernatant, and twice supernatant merged, and detects with being used for HPLC behind the 0.22 μ m membrane filtration.
HPLC detect parameters: chromatographic column: ZORBAX Eclipse XDB-C18,2.1 * 150mm, 5 μ m (Agilent Co, USA), 30 ℃ of column temperatures, sample size 5 μ l, flow velocity 0.25ml min
-1Moving phase: solution A 0.20% phosphate aqueous solution, solution B methyl alcohol.Elution program: 0-3min, 95%A, 5%B; 3-20min, A 95%-65%, B 5%-35%; 20-30min, A 65%-60%, B 35%-40%; 30-38min:A 60%, and B 40%; 38-45min:A 60%-80%, B 40%-20%; 45-49min:A 80%, and B 20%; 49-50min:A 80%-95%, B 20%-5%; 50-70min:A 95%, B5%.Detect wavelength 270nm.
Under as above chromatographic condition, flavonoid standard substance (apigenin SIGMA-ALDRICH 460475; The luxuriant and rich with fragrance pure SIGMA-ALDRICH K1003 of camphane; Quercetin SIGMA-ALDRICH Q0125; Myricetin M6760; Rutin SIGMA-ALDRICHR5143) RT is shown in Fig. 5 A.The RT of various flavonol is respectively rutin 27.6min in the test sample, myricetin 29.2min, Quercetin 32.3min, the luxuriant and rich with fragrance pure 34.7min of camphane; The RT of the apigenin in the flavonoid (verivate of the luxuriant and rich with fragrance alcohol of camphane) is 35.1min in addition.
To change empty carrier Herba Saussureae Involueratae and wild-type (Herba Saussureae Involueratae) is contrast, experiment repetition four times, results averaged.
The result is as shown in Figure 5, wherein A: the flavonoid standard substance; B:SmDFR-A39 (being numbered the antisense SmDFR Herba Saussureae Involueratae seedling strain system of A39); C: wild-type contrast.The RT of SmDFR-A39 is shown in Fig. 5 B, and the RT of various flavonol is respectively rutin 27.5min in the test sample, myricetin 28.9min, Quercetin 32.8min, the luxuriant and rich with fragrance pure 34.4min of camphane; The RT of the apigenin in the flavonoid (verivate of the luxuriant and rich with fragrance alcohol of camphane) is 35.3min in addition.The RT of wild-type contrast is shown in Fig. 5 C, and the RT of various flavonol is respectively rutin 27.6min in the test sample, myricetin 29.1min, Quercetin 31.7min.
The content that shows flavonol such as rutin, myricetin among the SmDFR-A39 and/or flavonoid among the figure significantly raises than the wild-type contrast; Concrete calculating can be known; The rutin of wild-type contrast is that 25.16 ± 6.68 μ g/g fresh weights, myricetin are 22.43 ± 4.57 μ g/g fresh weights; The luxuriant and rich with fragrance alcohol of camphane is that 9.23 ± 0.49 μ g/g fresh weights, Quercetin are that 0.78 ± 1.35 μ g/g fresh weight, apigenin are 3.68 ± 1.19 μ g/g fresh weights, and the rutin of SmDFR-A39 is that 71.15 ± 17.64 μ g/g fresh weights, myricetin are that the luxuriant and rich with fragrance alcohol of 54.30 ± 9.81 μ g/g fresh weights, camphane is that 13.41 ± 4.07 μ g/g fresh weights, Quercetin are 10.76 ± 2.71 μ g/g fresh weights and apigenin 16.47 ± 12.07 μ g/g fresh weights.
The result who changes the contrast of empty carrier Herba Saussureae Involueratae and wild-type does not have marked difference.
Show that importing antisense Saussurea medusa SmDFR gene makes flavonol content increase in the Herba Saussureae Involueratae.Effective gene in the flavones pathways metabolism being imported in the Herba Saussureae Involueratae, and then generate high yield flavones transgenic seedling, carry out artificial culture, will be the effective way that solves the shortage of saussurea involucrata wild resource.
Claims (2)
1. a transgenic plant method of cultivating flavonol and/or the raising of flavonoid content is with the afunction of proteic encoding sox shown in the sequence 2 in the sequence table in the purpose plant, obtains transgenic plant; Flavonol and/or flavonoid content are higher than said purpose plant in the said transgenic plant;
Said purpose plant is a Herba Saussureae Involueratae, and said flavonol is rutin, myricetin, the luxuriant and rich with fragrance alcohol of camphane and/or Quercetin; Said flavonoid is an apigenin.
2. method according to claim 1 is characterized in that: with the method for the afunction of proteic encoding sox shown in the sequence 2 in the sequence table in the purpose plant through realizing to wherein changing recombinant expression vector over to; Said recombinant expression vector is in the MCS of pCAMBIA1302 carrier, to insert dna fragmentation to obtain, and said dna fragmentation is connected in sequence by the reverse fragment and the Nos terminator of proteic encoding sox shown in the sequence 2 in CaMV 35S promoter, the sequence table;
The reverse fragment of proteic encoding sox shown in the sequence 2 is the reverse complemental fragment from dna fragmentation shown in 5 ' the terminal 56-1129 position Nucleotide of sequence 1 in the sequence table in the said sequence table.
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Non-Patent Citations (4)
| Title |
|---|
| Lu,X..EF600682.<<GenBank>>.2007,1. * |
| 侯夫云等.植物花青素合成酶的研究进展.《中国农学通报》.2009,(第21期), * |
| 孙淑香.二氢黄酮醇的合成.《现代化工》.1998,(第12期), * |
| 颜华,李毅,宋云,邵莉,梁晓文,陈章良.二羟基黄酮醇还原酶基因的克隆、全序列分析及在大肠杆菌中的表达.《应用基础与工程科学学报》.1997,(第02期), * |
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