CN101199355A - Method of increasing resveratrol in peanut kernel - Google Patents
Method of increasing resveratrol in peanut kernel Download PDFInfo
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Abstract
The invention discloses a method to improve the resveratrol content in a peanut kernel. The method includes the following steps; (1) clone a full-long resveratrol synthase gene in a peanut;(2) get the specific promoter Arah1P which expresses specially in a peanut seminal leaf; (3) construct the plant binary expression vector pHT711; (4) translate the plants; (5)obtain a genetically modified peanut kernel;(6) measure the resveratrol content in the peanut kernel. The invention is simple in technical course, definite, easy to operate and low in cost with distinct gene expression, remarkably improving the resveratrol content in the peanut kernel.
Description
Technical field
The invention belongs to gene engineering technology field.Be specifically related to a kind of method that improves Resveratrol content in the shelled peanut; Be that tissue specificity expression promoter and resveratrol synzyme are transformed into peanut, thus Resveratrol content in the raising shelled peanut of safety, high yield, and the nutrition, the health care that improve peanut itself are worth.
Background technology
Resveratrol is a class polyphenolic plant compound, extensively is present in the seed plant, and is mainly synthetic by the phenylalanine metabolic pathway.It can not only make plant that some diseases is produced resistance, and human body is also had obvious health care effect, has antibiotic, anti-inflammatory, antiallergy, anti thrombotic action; Have the effect that suppresses platelet aggregation, protection ischemic organ, anti-oxidant, Green Tea Extract simultaneously, especially aspect cardiovascular disease and cancer prevention, have important effect [the resveratrol present Research with use prospect; Cheng Liying, Liu Shuxing, food research and development, 2005,26:25-27].
Under the natural conditions, the content of resveratrol in plant is very low, and in the highest Grape Skin of content, its content only is 10-100 μ g/g.After doctor Sanders studies have shown that " containing resveratrol in the edible peanut " since the U.S., cause in the world common concern to the peanut health care.China's peanut resource is abundant.Peanut also is rich in phytosterol, folic acid, vitamin and a large amount of trace elements simultaneously as important oil crops, and its nutrition and health care are worth all very high.Many scholars measure each position Resveratrol content of peanut, and the approaches and methods that improves Resveratrol content has been carried out many research and development.Method by high performance liquid chromatography (HPLC), Resveratrol content in the peanut different parts is measured, record the Resveratrol content the highest (94.3 μ g/g) in the peanut root, but do not find that in peanut leaf and shelled peanut [the HPLC method is measured Resveratrol content in the peanut different parts to resveratrol; Open Xuan, Hu Junping, Han Mei, Zhou Yong, Xinjiang Medicine University's journal, 2003,26:440-441].2004, the expert of United States Department of Agriculture confirmed that the content of resveratrol is about the developmental research of 25.7 a μ g/g[polyphenol compound-resveratrol in peanut red coat and the shelled peanut; Liu Dachuan, Liu Qiang, Chinese oil, 2004,29:9-11].
Along with the intensification to resveratrol biologically active and pharmacological function aspect understanding, its genetic engineering is subjected to researcher's extensive concern gradually in the application aspect plant nutrient, the health quality genetic improvement.The resveratrol synthase gene is imported in the target plant genome, be expected render transgenic expression of plants resveratrol synthase activity, start its secondary metabolism route of synthesis, thereby improve plant nutrient and health care proterties, satisfy people's health care needs.Resveratrol synzyme (resveratrolsynthase is called for short RS, EC 2.3.1.95) is last key enzyme that works in the resveratrol metabolic pathway of synthesizing, also is unique necessary synzyme in the route of synthesis.The expression mechanism that in plants such as grape, peanut, pine tree, all has induction type resveratrol synzyme, though but the substrate of enzyme is arranged in most of plant cell, but lack the resveratrol synthase gene, thereby lack the ability of synthetic such material under the incitant effect.In the genetic engineering of structural gene, promoter is to influence gene expression time, position, efficient and to different incitants influences, thereby influences a key factor of genetically modified plants using value.Shelled peanut, promptly peanut seed mainly is made up of kind of skin and embryo two parts; Embryo is divided into radicle, plumular axis, plumule and cotyledon four parts [Shandong peanut, Shandong Province Academy of Agricultural Sciences, Wang Zaixu, lid rear people chief editor, Shanghai science tech publishing house, 32 pages] again.Maturation along with the peanut genetic conversion system, can shelled peanut as bioreactor, utilize peanut cotyledon specific expression promoter, by engineered method, structure contains the plant expression vector of resveratrol synthase gene, import in the peanut plant and go, shelled peanut can be synthesized and the high yield resveratrol.
By the relevant resveratrol synthase gene of clone in grape, and then change tomato over to and attempt to improve its Resveratrol content [clone of stilbene synthase gene and the structure of plant expression vector; Tan Lin, Zheng Xueqin, Kang Youfa, Shi Jiang, tropical crops journal, 2003,24:42-45].But owing to the gene difference that relates between different plant species, so be difficult for obtaining Expected Results.
Utilize cell culture technology, the Grape Skin decorticationization tissue that Guo Bin etc. cross with agrobacterium tumefaciens infection by He-Ne laser irradiation carries out mutagenic treatment, can obtain resveratrol [the He-Ne mutation induced by laser breeding high-yield resveratrol cell of high level; Guo Bin, Wei Yahui, Cao Wei etc.; The photon journal, 2002,31:277-280].But operating parameter is loaded down with trivial details, and reappearance is relatively poor.
Publication number is that the Chinese patent of CN1382693 has been introduced separation resveratrol stilbene synthase gene from Boston ivy and kindred plant, and the further method that improves its synthetic quantity in the importing Boston ivy, but relate to separation, purification, the process of later stage synthetic product, technology cost height.
Summary of the invention
The object of the present invention is to provide a kind of simply, efficiently, easily, make the shelled peanut can synthesizing resveratrol and can improve the method for its content.This method nutrition, health care are laid equal stress on, and adapt to development and whole people's demand growing to health food of society.
The method of Resveratrol content in the raising shelled peanut of the present invention, step comprise total length resveratrol synthase gene RS1 in (1) clone peanut; (2) obtain the narrow spectrum specificity promoter Arah1P that in the peanut cotyledon, expresses; (3) make up plant binary expression vector pHT711; (4) plant transforms; (5) acquisition of transgenosis shelled peanut; (6) mensuration of Resveratrol content in the transgenosis shelled peanut; It is characterized in that: be to be template with the peanut genome among the total length resveratrol synthase gene clone in the described peanut, with RS1p1:5 '-TCC ATG GTG TCT GTG AGT GGA ATT C-3 '; RS1p2:5 '-GTATTA TAT GGC CAT GCT GCG GAG-3 ' carries out PCR for primer, and the method by reverse transcription PCR (RT-PCR) obtains the resveratrol synzyme that length is 1.5kb (being called for short RS1) gene complete sequence, and its coded product is the stilbene synthase.Wherein the PCR response procedures is: 94 ℃ of 10min, 94 ℃ of 40s, 55 ℃ of 30s, 72 ℃ of 2min, 30 circulations, 72 ℃ of 10min.The described narrow spectrum specificity promoter Arah1P that expresses in the peanut cotyledon is to be template with the peanut genome, with Arah1P1:5 '-TTA GGT TTT CCA TTT TTA CAC AGA G-3 '; Arah1P2:5 '-GCA GAA ACT GAAGCC AGG ACA AGG A-3 ' is a primer, by the method acquisition of PCR; Wherein the PCR response procedures is: 94 ℃ of 10min, 94 ℃ of 40s, 56 ℃ of 30s, 72 ℃ of 2min, 30 circulations, 72 ℃ of 10min.The method of described structure plant binary expression vector pHT711 is: pCAMBIA1301 transforms to carrier, by replace original CaMV35S constitutive promoter with Arah1P, be built into intermediate carrier pHT710, again the resveratrol synthase gene that obtains is built into carrier monoclonal site, obtains tissue specific expression carrier pHT711; Resistance marker is damp enzyme element in plant.It is to have the carrier pHT711 of specificity promoter and genes of interest by particle gun, electric shock, pollen tube channel or agrobacterium mediation method conversion peanut plant that described plant transforms; The preparation method of described transgenosis shelled peanut is that the first generation peanut seed that obtains is sowed, to the second generation seed sampling that obtains, respectively its DNA, RNA, protein are carried out multistage detection, screening obtains changing over to the genetically modified shelled peanut of resveratrol synzyme; The assay method of Resveratrol content is meant that with the resveratrol standard items be contrast in the described transgenosis shelled peanut, with high performance liquid chromatography (HPLC) method to sieve the transgenosis shelled peanut in Resveratrol content measure.The transgenosis shelled peanut that the genetic stability of choosing, Resveratrol content improve can be further used for producing.
Wherein: above-mentioned plant transforms the carrier pHT711 that preferably will have specificity promoter and genes of interest and transforms peanut plant by agrobacterium mediation method.
The inventive method has the following advantages:
(1) promoter and resveratrol synthase gene all derive from the peanut genome, with operating between species, help the stable of heredity, conversion system.
(2) the peanut cotyledon is a storage protein, is the strong promoter of expression vector with itself promoter, can better genes of interest be positioned at special position and express, and helps the expression of gene regulation and control.
(3) the peanut yield height, produce easily, itself is nutritious, after transforming in conjunction with technique for gene engineering, health care is worth and improves, also help enhance disease resistance, reduce the land for growing field crops produce in the agricultural chemicals use amount, improve its health quality.
(4) the whole technique route is simple, clear and definite, easy to operate, and cost is low, and income is big.
(5) content of resveratrol is significantly improved in the transgenosis shelled peanut of application this method preparation, is the 60%-80% without Resveratrol content in the genetic engineering modified shelled peanut.
Description of drawings
The structure schematic diagram of Fig. 1 plant binary expression vector pHT711.
The specific embodiment
The invention will be further described below in conjunction with embodiment, but be not limited thereto.
Embodiment 1: the clone of resveratrol synthase gene RS1
1, the sequence (AF227963) according to the peanut of having delivered among the GenBank (Arachis hypogaea) resveratrol synthase 3 genes designs a pair of primer that comprises resveratrol synthase 3 initiation codons and termination codon, be respectively RS1p1 and RS1p2, the RS1 gene in the peanut is cloned out.Primer sequence is as follows: RS1p1:5 '-TCC ATG GTG TCT GTGAGT GGA ATT C-3 '; RS1p2:5 '-GTA TTA TAT GGC CAT GCT GCG GAG-3 '.
2, the extraction of genomic DNA
(1) takes by weighing the peanut leaf of 100mg, in mortar, fully be ground to fine powder, powder transfer to the 1.5ml centrifuge tube, is added 700 μ l " S " buffer solutions of preheating (65 ℃), mixing immediately, 65 ℃ of water-bath 1.5-2h; " S " buffer formulation: 100mM Tris-Cl pH8.5,50mM EDTA pH8.0,100mM NaCl, 2%SDS.
(2) add isopyknic phenol-chloroform (700 μ l), abundant mixing, the centrifugal 15min of 8000rpm;
(3) supernatant is transferred in another clean centrifuge tube, adds equal-volume chloroform (500 μ l), abundant mixing, the centrifugal 15min of 8000rpm;
(4) supernatant is transferred in another clean centrifuge tube, adds the isopropyl alcohol of 0.6-0.7 times of volume precooling (20 ℃), normal temperature leaves standstill 5min behind the mixing, the centrifugal 5min of 8000rpm;
(5) outwell supernatant, with 70% ethanol washing precipitation 2-3 time;
(6) super-clean bench dries up, and adds 200 μ l TE buffer solutions and 2 μ l RNaseA (10g/ml) dissolving;
(7) get 2 μ l, 1% agarose electrophoresis detects.
3, be primer with RS1p1 and RS1p2, peanut genome is that template is carried out PCR:
(1) PCR reaction system: Ex Taq enzyme buffer liquid (contains Mg
2+) 2.5 μ l, dNTP (2.5mM) 1 μ l, RS1p1, each 1 μ l of RS1p2 primer (10 μ M), peanut genome 5 μ l, Ex Taq enzyme (TaKaRa company) 0.3 μ l adds sterilization distilled water to 25 μ l.
(2) PCR response procedures: 94 ℃ of 10min, 94 ℃ of 40s, 55 ℃ of 30s, 72 ℃ of 2min, 30 circulations, 72 ℃ of 10min, 4 ℃ of preservations.Get 5 μ l and carry out the agarose gel electrophoresis detection, testing result has the band about one 1500 base-pair (bp).
4, the band about 1500bp is downcut from glue, reclaims kit with the quick glue of the Type B miniprep dna fragment of vast Imtech and reclaim as follows:
(1) gel is placed under the uviol lamp with clean blade band is downcut, in the centrifuge tube of the sterilization of packing into, take by weighing the weight of glue, the weight of glue can not surpass 300mg in each pipe.
(2) add the ratio of 700 μ l sol solutionses in 100mg glue, room temperature colloidal sol (or 55 ℃ of colloidal sols) shakes therebetween once in a while; The dress post, 9000rpm, centrifugal 30s; Remove waste liquid.
(3) 500 μ l rinsing liquid (wash buffer) rinsings, the centrifugal 30s of 12000rpm.Repeat rinsing once.Outwell after the waste liquid, again in the centrifugal 2min of 12000rpm;
(4) pillar is put into a new 1.5ml centrifuge tube, at the elution buffer (Eloution buffer uses 30 μ l-50 μ l usually) of pillar film central authorities adding suitable volumes, room temperature is placed 2-5min, the centrifugal 1min of 12000rpm, the liquid in the centrifuge tube is the dna fragmentation of recovery.
5, the PCR specific fragment behind the purifying is connected with the pMD18T-carrier:
Reactive component is as follows:
The dna fragmentation 7 μ l of a, recovery
B, pMD18T-carrier 1 μ l
C, T4 dna ligase 1 μ l
D, T4 dna ligase buffer 1 μ l
With above-mentioned a, b, c, d reactant mixing, 16 ℃ of reactions are spent the night, and are used for transformed into escherichia coli DH5 α.
6, the preparation of competent escherichia coli cell
(1) the single colony inoculation of picking E.coil DH5 α is to the LB fluid nutrient medium of about 5ml, and in 37 ℃, 250rpm shakes overnight incubation.
(2) next day this bacterium liquid is transferred in the fluid nutrient medium of 100ml with 1% inoculum concentration, continues to be cultured to absorbance (OD)
600About=0.4.
(3) with this bacterium liquid ice bath 10min, be transferred under aseptic condition in the ice-cold in advance 50ml centrifuge tube, 4 ℃, the centrifugal 10min of 5000rpm collects thalline.
(4) be resuspended in the CaCl of the 0.1mol/L of 10ml precooling
2In, ice bath 10min, 4 ℃, the centrifugal 10min of 5000rpm collects thalline.
(5) be resuspended in the CaCl of 0.1mol/L of the precooling of 2ml (containing 15% glycerine)
2In, being distributed into 100 μ l/ pipe ,-70 ℃ of preservations are standby.
7, transform
(1) takes out competent cell from-70 ℃ of refrigerators and place dissolving on ice.
(2) the product 5 μ l that step 5 connected add in the competent cell, and fully mixing is placed 30min on ice.
(3) 42 ℃ of heat shock 90s do not rock centrifuge tube in this process.
(4) take out immediately after heat shock finishes and be put in 2min on ice.
(5) the LB fluid nutrient medium of adding 800 μ l, 37 ℃, 200rpm is hatched 1h.
(6) bacterium liquid is coated on ammonia benzyl (Amp) resistance and contain isopropyl-(IPTG) and the LB solid plate of 5-bromo-4-chloro-3-indoles-β-D-galactolipin (X-gal) on, cultivate 12h for 37 ℃.
(7) flat board is put into 4 ℃ of preservations.
8, the detection one bacterium liquid PCR of positive colony detects
(1) the picking white colony is inoculated into the LB fluid nutrient medium from the flat board that transforms, and 37 ℃, 3-4h is cultivated in the 250rpm concussion.
(2) PCR reaction system: Taq enzyme buffer liquid (contains Mg
2+) 2.5 μ l, dNTP (2.5mM) 1 μ l, RS1p1, each 1 μ l of RS1p2 primer (10 μ M), bacterium liquid 2 μ l, Taq enzyme (vast Tyke) 0.3 μ l adds sterilization distilled water to 25 μ l.
(3) PCR response procedures: 98 ℃ of 10min, 94 ℃ of 40s, 55 ℃ of 30s, 72 ℃ of 2min, 30 circulations, 72 ℃ of 10min, 4 ℃ of preservations.Get 10ul and carry out the agarose gel electrophoresis detection, testing result has a band about 1500bp.
9, the extraction of plasmid
(1) with the bacterium liquid of the bacterium liquid PCR positive in 37 ℃, 250rpm shakes overnight incubation.
(2) bacterium liquid is transferred in the centrifuge tube, the centrifugal 2min of room temperature 8000rpm collects thalline.
(3) get supernatant, centrifuge tube is tipped upside down on the blotting paper, remaining liquid is flowed out.
(4) solution I of adding 100 μ l precoolings, the resuspended thalline of concuss on the vortex oscillator, wherein said solution I prescription is: 50mmol/L glucose, 25mmol/L Tris-Cl (pH8.0), 10mmol/L EDTA (pH8.0).
(5) add the freshly prepared solution II of 200 μ l, put upside down mixing fast 10 times, ice bath 5min, wherein said solution II prescription is: 0.2mol/L Na0H, 1%SDS, face and use preceding preparation.
(6) solution III of adding 150 μ l precoolings is leniently put upside down 10 times, ice bath 5min, and 4 ℃ of centrifugal 10min of 12000rpm, wherein said solution III prescription is: 5mol/L potassium acetate 60ml, 3mol/L glacial acetic acid 11.5ml, ddH
20 28.5ml, pH5.2.
(7) supernatant is transferred in another clean centrifuge tube, added isopyknic phenol: chloroform: isoamyl alcohol (25: 24: 1), mixing repeatedly, 4 ℃ of centrifugal 10min of 12000rpm.
(8) supernatant is transferred in another clean centrifuge tube, added isopyknic chloroform: isoamyl alcohol (24: 1), mixing repeatedly, 4 ℃ of centrifugal 10min of 12000rpm.
(9) supernatant is transferred in another clean centrifuge tube, added the isopropyl alcohol of equal-volume precooling (20 ℃) ,-20 ℃ of precipitation 1h behind the mixing, 4 ℃ of centrifugal 10min of 12000rpm.
(10) outwell supernatant, add the ethanol washing precipitation twice of 500 μ l 70%, air drying.
(11) the TE solution and the 2 μ l RNaseA of adding proper volume, 37 ℃ of digestion 0.5h.
10, the enzyme of plasmid is cut detection
(1) the HindIII enzyme is cut system:
10×buffer?M 2μl
DNA 2 μ l
HindIII 1μl
Add water to 20 μ l.
(2) 37 ℃ of enzymes are cut 2h, get 10 μ l and carry out the agarose gel electrophoresis detection, and testing result has the band of a 1200bp and the band about 3000bp.
11, sequencing
The clone pMDRS1 of the above-mentioned detection positive is checked order by High Technology Center, academy of agricultural sciences, Shandong, and the result of mensuration is shown in SEQID NO.1; Shown in the nucleotide homology of result and the peanut resveratrol synthase gene (clone pGSG10) delivered be 95%, with resveratrol synzyme 3 genes (RS3) nucleotide homology in the peanut be 86%-84%.
12, the structure of plant binary expression vector.
Embodiment 2: the clone of peanut cotyledon specific expression promoter
1, according to document (Structure and organization of the genomic clone of a major peanutallergen gene, Ara h1.Viquez OM
*, Konan NK, Dodo HW, Mol Immunol 2003, the Arah1 promoter sequence of report designs a pair of primer 40:565-571), difference called after Arah1P1 and Arah1P2, and primer sequence is as follows: Arah1P1:5 '-TTA GGT TTT CCA TTT TTA CAC AGA G-3 '; Arah1P2:5 '-GCAGAA ACT GAA GCC AGG ACA AGG A-3 '.
2, be primer with Arah1P1 and Arah1P2, peanut genome is that template is carried out PCR:
(1) PCR reaction system: Ex Taq enzyme buffer liquid (contains Mg
2+) 5 μ l, dNTP (2.5 mM) 2 μ l, Arah1P1, each 1 μ l of Arah1P2 primer (10 μ M), peanut genome 5 μ l, Ex Taq enzyme (TaKaRa company) 0.5 μ l adds sterilization distilled water to 50 μ l.
(2) PCR response procedures: 94 ℃ of 10min, 94 ℃ of 40s, 56 ℃ of 30s, 72 ℃ of 2min, 30 circulations, 72 ℃ of 10min, 4 ℃ of preservations.Get 5 μ l and carry out the agarose gel electrophoresis detection, testing result has a band about 1900bp.
3, the band about 1900bp is downcut from glue, reclaim kit with the quick glue of the Type B miniprep dna fragment of vast Imtech and reclaim, recycling step is with embodiment 1.4.
4, the PCR specific fragment behind the purifying is connected with the pMD18T-carrier:
Reactive component is as follows:
The dna fragmentation 7 μ l of a, recovery
B, pMD18T-carrier 1 μ l
C, T4 dna ligase 1 μ l
D, T4 dna ligase buffer 1 μ l
With above-mentioned a, b, c, d reactant mixing, 16 ℃ of reactions are spent the night, and are used for transformed into escherichia coli DH5 α.
5, transform (step is with embodiment 1.7).
6, the detection of positive colony-bacterium liquid PCR detects
(1) the picking white colony is inoculated into the LB fluid nutrient medium from the flat board that transforms, and 37 ℃, 3-4h is cultivated in the 250rpm concussion.
(2) PCR reaction system: Taq enzyme buffer liquid (contains Mg
2+) 2.5 μ l, dNTP (2.5mM) 1 μ l, Arah1P1, each 1 μ l of Arah1P2 primer (10 μ M), bacterium liquid 2 μ l, Taq enzyme (vast Tyke) 0.3 μ l adds sterilization distilled water to 25 μ l.
(3) PCR response procedures: 98 ℃ of 10min, 94 ℃ of 40s, 55 ℃ of 30s, 72 ℃ of 2min, 30 circulations, 72 ℃ of 10min, 4 ℃ of preservations.Get 10 μ l and carry out the agarose gel electrophoresis detection, testing result has a band about 1900bp.
7, the extraction of plasmid (step is with embodiment 1.9).
8, the enzyme of plasmid is cut detection
(1) the HindIII enzyme is cut system:
10×buffer?M 2μl
DNA 2 μ l
HindIII 1μl
Add water to 20 μ l
(2) 37 ℃ of enzymes are cut 2h, get 10 μ l and carry out the agarose gel electrophoresis detection, and testing result has the band of a 1700bp and the band about 2800bp.
9, sequencing.
The clone pMDArah1P of the above-mentioned detection positive is checked order by High Technology Center, academy of agricultural sciences, Shandong, and the result of mensuration is shown in SEQ ID N0.2; Shown in result and the document reported sequence difference of minority base is only arranged.
10, the structure of plant binary expression vector.
Embodiment 3: the structure of plant binary expression vector pHT711
1, SmaI, BamHI enzyme are cut the pCAMBIA1301 plasmid, reclaim kit with the quick glue of the Type B miniprep dna fragment of vast Imtech behind the electrophoresis and reclaim, and recycling step is with embodiment 1.4.
2, EcoRV, BamHI enzyme are cut the pMDArah1P plasmid, and two bands are arranged behind the electrophoresis, cut the 1900bp fragment, reclaim kit with the quick glue of the Type B miniprep dna fragment of vast Imtech and reclaim, and recycling step is with embodiment 1.4.
3, reclaim fragment with two and connect, be built into intermediate carrier pHT710 with the T4 ligase.
4, with XbalI, a SalI enzyme carrier pHT710 plasmid that hits, behind the electrophoresis fragment is reclaimed, recycling step is with embodiment 1.4.
5, XbalI, SalI enzyme are cut the pMDRS1 plasmid, and two bands are arranged behind the electrophoresis, cut the 1500bp fragment, reclaim kit with the quick glue of the Type B miniprep dna fragment of vast Imtech and reclaim, and recycling step is with embodiment 1.4.
6, reclaim fragment with two and connect, be built into double base plant expression vector pHT711 with the T4 ligase.(see figure 1)
Embodiment 4: the agrobacterium mediation converted peanut plant
1, peanut seed sterilization: seed soaked in distilled water 10-15 minute, and add 75% alcohol and shook 1 minute, the distilled water flushing of usefulness sterilization 2 times, 1 ‰ mercury chloride soaked 10-15 minute, distilled water flushing 3 times, the peeling kind is gone in the MS culture medium.
2, the conversion of peanut:
(1) Agrobacterium that contains the purpose plasmid obtains: extract the positive colony plasmid, get trace and transform Agrobacterium LBA4404.
(2) on the kanamycins culture medium, screen resistant strain, and the genes of interest plasmid bacterial strain that may contain that screens is carried out the PCR checking.
(3) breeding 50ml positive colony Agrobacterium bacterium liquid is used for peanut genetic transformation (genes of interest is changed over to the peanut plant genome).
(4) peanut of sprouting is downcut plumular axis, in thallus suspension liquid, soaked 10 minutes, bacterium liquid on the filter paper exhaustion plumular axis, insert in the blank inducing culture, behind the dark cultivation 48h, soaked 10 minutes with 1% cephalo, plumular axis inserts in the inducing culture (inducing culture+cephalo final concentration 250mg/l) with aqua sterilisa flushing 3 times again.
The bud of growing thickly that (5) will induce downcuts, and inserts by (antibiotic of elongation medium+cephalo final concentration 250mg/l+ screening) in the elongation medium.
(6) the grow thickly transplanting of blastogenesis root (root media+cephalo (final concentration 250mg/l)), hardening, transfer-gen plant.
Embodiment 5: the acquisition of transgenosis shelled peanut
1, results peanut seed, plantation and results second generation seed.
2, sprout a small amount of second generation seed, carry out methods such as PCR detection, Southern hybridization by GUS dyeing, extraction DNA, the checking transfer-gen plant obtains the transgenosis shelled peanut.
Embodiment 6: the mensuration of Resveratrol content in the transgenosis shelled peanut
1, using high performance liquid chromatography (HPLC) measures, (2.1mm * 250mm), chromatographic condition: chromatographic column LichrospherC18 flows phase: acetonitrile-water (25: 75) (if gradient elution, acetonitrile-water (1: 9)-(9: 1)), flow velocity 1mL/min, 30 ℃ of column temperatures, sample size 5 μ l, detect wavelength 303nm.
2, the preparation of calibration curve: precision takes by weighing trans Bai Lilu alcohol reference substance (sigma company) 5.4mg and add methyl alcohol to scale in the 10mL volumetric flask, shakes up.The accurate again 0.1ml that draws puts in the 25ml volumetric flask, adds methanol constant volume, and making every 1ml, to contain the reference substance test liquid of 2.16 μ g standby.Get this liquid auto injection 2,4,6,8,10,12 μ l respectively, measure each peak area by above-mentioned chromatographic condition.With sample size μ g peak area is mapped, obtain calibration curve.
3, the preparation of sample solution: get transgenosis shelled peanut grind into powder, 70 ℃ of oven dry 3h.Precision takes by weighing 0.2g, puts in the 10ml volumetric flask, and it is an amount of to add 90% methyl alcohol, and ultrasonic extraction 30min takes out, and puts and is chilled to room temperature, and 90% methanol constant volume is to scale.Leave standstill after shaking up, get supernatant with 0.45 μ m filtering with microporous membrane after, promptly.
4, sample size is measured: precision takes by weighing sample 0.2g, prepares with method by sample solution, and each sample introduction 10 μ l measures, and peak area is mapped the Resveratrol content of looking on calibration curve with sample size.
Experiment confirm repeatedly: the content of using resveratrol in the transgenosis shelled peanut of method for preparing is significantly improved, and is the 60-80% without Resveratrol content in the genetic engineering modified shelled peanut.
Sequence table
＜110〉High and New Technology Research Center, Shandong Prov. Academy of Agriculture S
＜120〉a kind of method that improves Resveratrol content in the shelled peanut
<141>2007-11-29
<160>2
<210>1
<211>1537
<212>DNA
＜213〉peanut (Arachis hypogaea L.)
<222>(1)…(1537)
<400>1
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tgtatgtcgt?tttctaaatg?tcatcaaggg?tattgatgga?tgtgaatttc?atattattat?540
ttcaggtgag?agaacacaga?tcaagaatag?acatatgtac?ttaacagaag?agatactgaa?600
agagaaccct?aacatgtgcg?catataaggc?accgtcgttg?gatgcaagag?aagacatgat?660
gatcagggag?gtaccaaggg?ttggaaaaga?ggctgcaacc?aaggccatca?aggaatgggg?720
ccggccaatg?tctaagatca?cacatttgat?cttctgcacc?accagcggcg?ttgcgttgcc?780
cggcgttgat?tatgaactca?tcgtactatt?agggctcgac?ccgtgtgtca?agaggtacat?840
gatgtaccac?caaggttgct?ttgctggtgg?cactgtcctt?cgtttggcta?aggacttggc?900
agagaacaac?aaggatgctc?gtgtgcttat?tgtttgttct?gggaatactg?cagtcacctt?960
ccgtggtcct?agtgagacag?atatggatag?tcttgtaggg?caagcattgt?ttgccgatgg?1020
agctgctgcg?attatcattg?gttctgatcc?tataccagag?gttgagaatc?ctatctttgg?1080
gatcgtttca?accgatcaaa?aacttgtccc?tggcagccac?ggagccatcg?gtggtctcct?1140
tcgtgaagtt?gggcttacgt?tctatcttaa?caagagtgtt?cctgatatta?tttcacaaaa?1200
catcaacgac?gcactcagta?aagcttttga?tccattgggt?atatctgatt?ataactcaat?1260
attttggatt?gcacaccctg?gtggacgtgc?aattctggac?caggttgaac?aaaaggtaaa?1320
cttgaagcca?gagaagatga?aagccaccag?agatgtgctt?agcaattatg?gaaacatgtc?1380
aagtgcatgt?gtgtttttca?tcatggattt?gatgaggaag?agatctcttg?aaggaaaact?1440
taaaactact?ggagaaggat?ttgattgggg?tgtgcttttt?ggctttggtc?ctggtctcac?1500
tattgaaact?gttgtcctcc?gcagcatggc?catataa 1537
<210>2
<211>1931
<212>DNA
＜213〉artificial sequence
<222>(1)…(1931)
<400>2
agagtagcat?tttgaactgg?cagcttctca?ttttttcacc?gttggatcga?cgtgaaatttg 60
gacagtaaat?tcttcatatc?ttgttcttca?tagttgctgc?cacattgtgt?tgtgagtgct 120
tccatactat?tgttgtattg?gatattgtgt?tatttttgct?gatagactc?tttcaataat 180
gtctaggaaa?atagtagata?aacaaaagct?taattatgaa?gaaaaagaga?aacttaaacg 240
aaaagataaa?tgttggttaa?aactgttgat?tattccaatc?ttttcccaat?agaataaatt 300
atgccccaat?tattttatca?atatgttaat?taggaatgaa?cattttggag?attgtatcat 360
aagttttaat?taagaatgtg?attactttgt?ttccatagtt?ggattatctt?ttgtagaata 420
acgagggtat?aatctctaat?atacatttgt?cggccttagg?ttgtgttatt?aatgcttctc 480
atacggtacc?acaactctgt?agctagagct?tgcgattata?ttgaacagaa?tagagatgat 540
atcaaaaaga?aagaaggaac?aaattaaacg?tatgtgatgt?ttaaatggat?atgaaatttt 600
ggtcaatgaa?gacatgtatg?atttcgcttg?tgatacgcag?atacgctaaa?aggcaagcca 660
ataccgtaag?gtagggggtg?catatgtgcc?gggtgaagtc?gggtttgatg?tgaccccaga 720
cccgatccga?aatatacacc?gggtttattt?attagacccg?aacccgaccc?tagacccgat 780
gaaaccaata?cactttcggg?ccacaattat?accgggtaaa?aaccgggtga?aaaccgggcc 840
attaacatta?cattacgttg?ataccttctt?gtaagctagc?atgtaaaaat?atccaaattt 900
tcaagactcc?aaccattatt?taacatggta?aaattcactt?agaaaaatat?aacaaaaacc 960
aacccttctt?taaaattaaa?gcataaccac?aatccatact?aaagcaaaac?cacaatcaat 1020
actaatattg?tctaataata?ccaaatattt?aaatcaatac?aaataacaca?atattatgca 1080
ttagtctaaa?gtcttatgca?ttctaaacat?aaaatattaa?cttatagtct?tataatgacg 1140
taataaaaca?aaatattaaa?gtttacaata?cttaaattcc?acataagaat?agtcatgatc 1200
catcactaat?aacacaaaat?attaatgcca?ccaggccgac?ttcgggtgac?ccgagctatg 1260
gcccggacgc?gacccgaaat?aatgaccggg?tctatttttg?agacccttac?ccgaccctaa 1320
accccgatga?aatcatacca?aattagcccc?taaaatgttc?gggaccgggc?cgggccttcg 1380
ggccgggccg?ggtctgtgca?cccctaccgt?aaggatagct?gaatgggtga?tgatatttta 1440
tcaaaagaca?aaaggaatcc?atcctgtatg?tgcagtggaa?ccttttccca?gacaatggag 1500
tgatgaacat?tatatatatc?gccgcgtcca?tcaccataca?tatccaaagc?aaaaaattta 1560
acaaatactg?aaccccaatt?gccatgcata?ttaacgcgta?cactctctat?gtcaatgtgt 1620
ccaccatgca?ccacaatcca?tccatctctt?gtttatctct?aagccaagac?ctagtgttgt?1680
tgccacctca?catttctcaa?tttcaacact?cgtcaacctg?catgccactc?caatgcccaa?1740
gtgtacatcc?atatcatcct?aacgcaacta?tataaatacc?actcacctcc?ctccatcatt?1800
tcaccatcca?cactcataat?aatcatatat?attcatcaat?catctatata?agtagtagca?1860
ggagcaatga?gagggagggt?ttctccactg?atgctgttgc?tagggatcct?tgtcctggct?1920
tcagtttctg?c 1931
Claims (2)
1. method that improves in the shelled peanut from veratryl alcohol content, step comprises total length resveratrol synthase gene in (1) clone peanut; (2) obtain the narrow spectrum specificity promoter Arah1P that in the peanut cotyledon, expresses; (3) make up plant binary expression vector pHT711; (4) plant transforms; (5) acquisition of transgenosis shelled peanut; (6) mensuration of Resveratrol content in the transgenosis shelled peanut; It is characterized in that: be to be template with the peanut genome among the total length resveratrol synthase gene clone in the described peanut, with RS1p1:5 '-TCC ATG GTG TCT GTG AGT GGA ATT C-3 '; RS1p2:5 '-GTA TTA TAT GGC CAT GCT GCG GAG-3 ' carries out PCR for primer, and wherein the PCR response procedures is: 94 ℃ of 10min, 94 ℃ of 40s, 55 ℃ of 30s, 72 ℃ of 2min, 30 circulations, 72 ℃ of 10min; The described narrow spectrum specificity promoter Arah1P that expresses in the peanut cotyledon is to be template with the peanut genome, with Arah1P1:5 '-TTA GGT TTT CCA TTT TTA CAC AGA G-3 '; Arah1P2:5 '-GCA GAA ACT GAAGCC AGG ACA AGG A-3 ' is a primer, by the method acquisition of PCR; Wherein the PCR response procedures is: 94 ℃ of 10min, 94 ℃ of 40s, 56 ℃ of 30s, 72 ℃ of 2min, 30 circulations, 72 ℃ of 10min; The method of described structure plant binary expression vector pHT711 is: pCAMBIA1301 transforms to carrier, by replace original CaMV35S constitutive promoter with Arah1P, be built into intermediate carrier pHT710, again the resveratrol synthase gene that obtains is built into carrier monoclonal site, obtains tissue specific expression carrier pHT711; It is to have the carrier pHT711 of specificity promoter and genes of interest by particle gun, electric shock, pollen tube channel or agrobacterium mediation method conversion peanut plant that described plant transforms; The preparation method of described transgenosis shelled peanut is that the first generation peanut seed that obtains is sowed, to the second generation seed sampling that obtains, respectively its DNA, RNA, protein are carried out multistage detection, screening obtains changing over to the genetically modified shelled peanut of resveratrol synzyme; The assay method of Resveratrol content is meant that with the resveratrol standard items be contrast in the described transgenosis shelled peanut, with high performance liquid chromatography to sieve the transgenosis shelled peanut in Resveratrol content measure.
2. improve the method for Resveratrol content in the shelled peanut according to claim 1, it is characterized in that: it is that the carrier pHT711 that will have specificity promoter and genes of interest transforms peanut plant by agrobacterium mediation method that described plant transforms.
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|---|---|---|---|
| CNA2007101148364A CN101199355A (en) | 2007-11-29 | 2007-11-29 | Method of increasing resveratrol in peanut kernel |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA2007101148364A CN101199355A (en) | 2007-11-29 | 2007-11-29 | Method of increasing resveratrol in peanut kernel |
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| CN101199355A true CN101199355A (en) | 2008-06-18 |
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| CNA2007101148364A Pending CN101199355A (en) | 2007-11-29 | 2007-11-29 | Method of increasing resveratrol in peanut kernel |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102911941A (en) * | 2012-11-02 | 2013-02-06 | 中国农业科学院植物保护研究所 | Root-specific promoter and application thereof |
| CN103898130A (en) * | 2014-03-04 | 2014-07-02 | 天津大学 | Cloning of mulberry resveratrol synthase gene and construction of plant expression vector |
| CN111944818A (en) * | 2020-08-27 | 2020-11-17 | 山东省花生研究所 | Promoter Arah1-P of peanut allergen gene Arah1 as well as cloning and application thereof |
| CN114073273A (en) * | 2020-08-17 | 2022-02-22 | 丰益(上海)生物技术研发中心有限公司 | Peanut-flavor grease with light coloring and low residual oil and preparation method thereof |
| CN115141838A (en) * | 2022-06-07 | 2022-10-04 | 珠海科技学院 | Construction of resveratrol synthase gene transformation peanut system |
-
2007
- 2007-11-29 CN CNA2007101148364A patent/CN101199355A/en active Pending
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102911941A (en) * | 2012-11-02 | 2013-02-06 | 中国农业科学院植物保护研究所 | Root-specific promoter and application thereof |
| CN102911941B (en) * | 2012-11-02 | 2014-03-19 | 中国农业科学院植物保护研究所 | Root-specific promoter and application thereof |
| CN103898130A (en) * | 2014-03-04 | 2014-07-02 | 天津大学 | Cloning of mulberry resveratrol synthase gene and construction of plant expression vector |
| CN103898130B (en) * | 2014-03-04 | 2016-04-20 | 天津大学 | The clone of mulberry fruit resveratrol synthase gene and the structure of plant expression vector |
| CN114073273A (en) * | 2020-08-17 | 2022-02-22 | 丰益(上海)生物技术研发中心有限公司 | Peanut-flavor grease with light coloring and low residual oil and preparation method thereof |
| CN111944818A (en) * | 2020-08-27 | 2020-11-17 | 山东省花生研究所 | Promoter Arah1-P of peanut allergen gene Arah1 as well as cloning and application thereof |
| CN111944818B (en) * | 2020-08-27 | 2021-12-07 | 山东省花生研究所 | Promoter Arah1-P of peanut allergen gene Arah1 as well as cloning and application thereof |
| CN115141838A (en) * | 2022-06-07 | 2022-10-04 | 珠海科技学院 | Construction of resveratrol synthase gene transformation peanut system |
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