CN103333902A - Method for increasing content of cyanidin in kochia scoparia - Google Patents
Method for increasing content of cyanidin in kochia scoparia Download PDFInfo
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- CN103333902A CN103333902A CN201310303320XA CN201310303320A CN103333902A CN 103333902 A CN103333902 A CN 103333902A CN 201310303320X A CN201310303320X A CN 201310303320XA CN 201310303320 A CN201310303320 A CN 201310303320A CN 103333902 A CN103333902 A CN 103333902A
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Abstract
The invention relates to a method for improving the anthocyanin content of hevea brasiliensis, which is characterized in that the full length of an arabidopsis anthocyanin synthesis regulatory factor 1 gene is obtained by adopting a gene cloning method, the arabidopsis anthocyanin synthesis regulatory factor 1 gene and a plant expression vector construct a recombinant expression vector, a positive plasmid of the recombinant expression vector is introduced into agrobacterium tumefaciens, the hevea brasiliensis is mediated and transformed by the agrobacterium tumefaciens to obtain transgenic hevea brasiliensis 5, and the anthocyanin content and the gum content of the obtained transgenic hevea brasiliensis are measured. The transgenic hevea brasiliensis has high anthocyanin content, and is beneficial to large-scale development and utilization and industrial development of the hevea brasiliensis.
Description
Technical field
The invention belongs to plant biotechnology field, specifically, relate to a kind of method that improves the russian dandelion anthocyanidin content.
Background technology
Russian dandelion has another name called Russian taraxacum (Taraxacum kok-saghyz Rodin), for composite family witloof family Dandelion plant, originates in ground (Chinese Plants will) such as Kazakhstan, Europe and Xinjiang of China.Russian dandelion can be grown in salinization grassy marshland, valley flat limit, has adaptability widely.Its root contains 6%～28% natural rubber, and the rubber relative molecular weight is bigger than paragutta, but structure similar with performance (An Feng etc., 2012).Along with rising steadily of oil price, the price of natural rubber also is sharply soaring, the natural rubber resource scarcity.Under this background, European Union and North America start " pearl plan " (EU-PEARLS) and " natural rubber substitute remarkable cooperative programme " (PENRA) project in succession, the development taraxacum rubber is as the second natural rubber resource, with reply paragutta shortage of resources (here girl, 2011).
Compare with Para rubber tree, the rubber separation and Extraction in the russian dandelion is loaded down with trivial details, and production cost is higher, and russian dandelion over-ground part gel content is low, is rejected usually, causes significant wastage, and these have all hindered large-scale developing and utilizing of russian dandelion.For reaching the profit purpose, all seeking the comprehensive development and utilization of russian dandelion both at home and abroad.Success at present relatively be process for processing rubber the time, obtain byproducts such as fermenting alcohol, synanthrin (can promote the propagation of bifidus bacillus and lactobacillus in the human body and suppress the growth of pathogenic bacteria such as intestinal bacteria, clostridium and Salmonellas).U.S. Delta has set up semi-automatic extraction source mill, produces rubber and obtains clean energy ethanol simultaneously with russian dandelion.In Europe, when utilizing russian dandelion to extract rubber, also synanthrin is extracted as natural food ingredients (here girl, 2011).In a word, increase the new approaches that the russian dandelion added value becomes the russian dandelion comprehensive development and utilization to greatest extent.
Anthocyanidin is a kind of functional natural pigment.Confirm in the 3rd the anthocyanidin international symposium that held in 2004 anthocyanidin have anti-oxidant, reduce liver injury, antimutagenic, hypotensive, the different physiological roles such as breeding of improving eyesight, antibiotic, anticancer, for diseases such as effective preventing cancer, diabetes, cardiovascular diseasess vital role (Yamakawa etc. are arranged, 2002Konczak etc., 2004; Wang Guanlin etc., 2005).In addition, the cyanin of acylations has advantages of higher stability, can replace the industrial synthetic colour that there is potential safety hazard in " Sudan red " etc., for the production of food and high value added products such as healthcare products and makeup such as beverage, wine, candies.Therefore, create high anthocyanidin transgenosis russian dandelion, in extracting root in the rubber, extract the anthocyanidin of russian dandelion over-ground part, with for the production of protective foods, not only can reduce industrial pollution, the russian dandelion added value can also be increased, the industrialized development of russian dandelion will be conducive to.
Summary of the invention
The objective of the invention is to improve the russian dandelion anthocyanidin content by creating high anthocyanidin transgenosis russian dandelion.
In order to realize purpose of the present invention, a kind of method that improves the russian dandelion anthocyanidin content of the present invention, adopt gene clone method to obtain synthetic regulatory factor 1 full length gene of Arabidopis thaliana anthocyanidin, with synthetic regulatory factor 1 gene of Arabidopis thaliana anthocyanidin and plant expression vector construction recombinant expression vector, the recombinant expression vector positive plasmid imports in the agrobacterium tumefaciens, adopt Agrobacterium tumefaciens mediated conversion russian dandelion, obtain high anthocyanidin transgenosis russian dandelion, and the transgenosis russian dandelion of gained is carried out anthocyanidin content and gel content mensuration.Method of the present invention comprises the steps:
1. the clone of Arabidopis thaliana
1) extraction of Arabidopis thaliana DNA: get Arabidopsis leaf in centrifuge tube, the centrifuge tube liquid nitrogen flash freezer, grinding rod pulverizes material rapidly, adds 65 ℃ of the CTAB extracting solutions insulation 30min of 600 μ l preheatings, during put upside down mixing frequently; Add the abundant mixing of 500 μ l chloroforms, room temperature, 12000rpm, 5min, centrifugal phase-splitting; Supernatant is forwarded in the new centrifuge tube, add the pre-cold isopropanol of 0.7 volume, mixing is placed 15min deposit D NA under the room temperature; Room temperature, 12000rpm, centrifugal 5min removes supernatant, 70% washing with alcohol precipitation; Room temperature, 12000rpm, centrifugal 3min removes ethanol, obtains Arabidopis thaliana DNA, and air drying 10min is dissolved in an amount of sterilized water.
2) polymerase chain reaction (PCR) amplification of synthetic regulatory factor 1 gene of Arabidopis thaliana anthocyanidin: design can amplify the primer of synthetic regulatory factor 1 full length gene of Arabidopis thaliana anthocyanidin, and introduce XbaI and SacI restriction enzyme site respectively at the upstream and downstream primer, upstream primer is 5'-AC
TCTAGAATGGAGGGTTCGTCCAAAGG-3', downstream primer are 5'-CC
GAGCTCThe CTAATCAAATTTCACAGTCTC-3'(underscore is denoted as Xba I and Sac I restriction enzyme site).Be template with Arabidopis thaliana DNA, (PCR) amplification through the polymerase chain reaction separates the amplified production that the obtains agarose gel electrophoresis in 1%, target fragment reclaim the back with
The Vector carrier connects, and changes bacillus coli DH 5 alpha over to, and sequence verification also obtains described gene order.
2. the structure of recombinant expression vector
DNA total length and the plant expression vector pBI121 of the gene that above-mentioned PCR method is obtained, the PCR that generates transcription factor 1 gene with XbaI and the described Arabidopis thaliana anthocyanidin of SacI double digestion reclaims product and pBI121 plant expression vector, reclaim to connect and transform DH5 α, the picking mono-clonal, the extraction plasmid is done the polymerase chain reaction detection and enzyme is cut checking, obtains containing the recombinant expression vector of described gene.The insertion exactness of this gene in the sequence verification recombinant expression vector.
3. contain the acquisition of recombinant expression vector agrobacterium tumefaciens
1) competence agrobacterium tumefaciens preparation: the single colony inoculation of EHA105 of picking is in containing 50mg/L Rif(Rifampin) the YEB substratum in, 200r/min overnight incubation under 28 ° of C; Get 400 μ l incubated overnight bacterium liquid and join 100ml and contain same antibiotic YEB liquid nutrient medium, 200r/min cultivates 3～4h under 28 ° of C, and OD600 is 0.6～0.8; Bacterium is transferred in the centrifuge tube of precooling then at ice-water bath cooling 10～15min, the centrifugal 20min of 5000r/min under 4 ° of C, the precipitation water dissolution of precooling; Repeated centrifugation 1 time is outwelled supernatant immediately, with remaining liquid re-suspended cell; Suspension is joined in the 50ml centrifuge tube of precooling, the centrifugal 10min of 5000r/min under 4 ° of C precipitates resuspendedly with isopyknic 10% glycerine, is loaded in the Eppendorf tube of precooling by the volume integral of per 200 μ l.
2) electricity swashs conversion: the 200 μ l Agrobacteriums that will prepare are put on ice, add the recombinant expression vector that 5 μ l extract from contain the recombinant expression vector bacillus coli DH 5 alpha, mixing; Utilize electric method for transformation that positive plasmid is imported among the agrobacterium tumefaciens EHA105, in the sharp cup of the prior electricity in precooling on ice of the bacterium liquid immigration that adds plasmid DNA, jog makes liquid sink to the cuvette bottom, between the electricity of sharp glass of insertion conversion tank of electricity is swashed; Electric pulse parameter is set, and electric capacity is 25 μ F resistance, 200 Ω, and strength of electric field 20kv/cm carries out electricity and swashs; Swash adding 0.8ml YEB substratum in the cup at electricity immediately after electricity swashs, close with the rifle persorption and transfer in the 1.5ml centrifuge tube, 28 ° of C, 220r/min shaking culture 3h gives full expression to foreign gene in Agrobacterium; Draw above-mentioned culture 100 μ L, be laid on contain the Rif(Rifampin) 50mg/L, Kan(kantlex) among the 100mg/L, YEB flat board, 28 ° of C cultivate 36～48h, the screening transformant is selected single bacterium colony and is carried out polymerase chain reaction checking positive strain clone.
4. the acquisition of transgenosis russian dandelion plant
Picking contains the single bacterium colony of agrobacterium tumefaciens of recombinant expression vector, is inoculated in the YEB liquid nutrient medium of 10mL antibiotic-free, and 28 ° of C180r/min shaking culture 16 hours, the centrifugal 10min of 5000r/min collects thalline; Agrobacterium is diluted to OD560=0.5-0.8 with 50ml MS liquid nutrient medium.The russian dandelion blade is cut into 1～2cm
2, contaminated the russian dandelion blade 5～30 minutes, take out the blade callus, the bacterium liquid that it is surperficial blots with filter paper, transfers on the MS substratum.After cultivating 2～3 days altogether, explant is transferred on the division culture medium division culture medium of replacing in per 10～20 days from former substratum taking-up.When treating resistant buds length to the 0.5cm left and right sides, resistant buds is downcut, change the root media cultivation over to and take root, obtain the careless plant of regenerated rubber of resistance.
Described division culture medium is the additional BA1～2mg/L of MS substratum, NAA0.5～1mg/L, cephamycin 200mg/L, Kan(kantlex) 50～200mg/L.
Described root media is the additional NAA0.1～0.5mg/L of 1/2MS substratum, cephamycin 200mg/L, Kan(kantlex) 50～200mg/L.
5. the detection of transgenosis russian dandelion plant
With the vermiculite of the regenerated rubber of resistance grass plantlet of transplant to 1:1: turfy soil, clear water Routine Management.The CTAB method is extracted the genomic dna of above-mentioned plant, and the design gene specific detects primer, is respectively RT-F:5'-TTGCTGGAAGATTACCTGGT-3' and RT-R:5'-TCCAAAGTTGATCAACGTCA-3'; Amplifying target genes product 450bp.Be template with the total DNA of transgenic line blade, detect the positive transfer-gen plant of screening, filter out high anthocyanidin transgenosis russian dandelion.
6. anthocyanidin extracts and assay
1) anthocyanidin extracts: the anthocyanidin extracting method is in wild rubber grass (contrast) and the transgenosis russian dandelion plant: take by weighing 1g wild rubber grass (contrast) and transgenosis russian dandelion blade respectively and pulverize, each adds the methanol aqueous solution (70% methanol aqueous solution that contains 0.1% formic acid) of 3mL80%, behind 4 ℃ of following lixiviate 24h, centrifugal, get supernatant, cross 0.2 μ m aperture nylon leaching film, the filtered liquid that obtains to contain anthocyanidin is standby.
2) anthocyanidin content is measured: adopt high-performance liquid chromatogram determination method to contrast the mensuration of anthocyanidin content in russian dandelion and the transgenosis russian dandelion plant, method is: adopt ODS-80TsQA C18column (4.6 * 250mm, i.d.5 μ m) chromatographic column, the automatic sampler sample introduction contains the filtered liquid sample size 4 μ l of anthocyanidin; Gradient eluent is: A is 0.2% formic acid water mutually, and B is acetonitrile mutually; Elution program: 0min, 5.0%B; 20min10.0%B; 35min20.0%B; 40min30.0%B; 50min40.0%B; 55min40.0%B; 60min5.0%B.30.0 ℃ of column temperatures; Flow velocity 0.4ml/min; Detect wavelength 515.0nm; Be with reference to product with Cyanidin-3 glucoside, according to its peak area by typical curve regression equation calculation anthocyanidin concentration.Adopt the high performance liquid chromatography mass spectrometric hyphenated technique to identify the anthocyanidin type, wherein anthocyanidin peak 1 retention time 22min molecular weight is 449.11, with above-mentioned consistent with reference to product (Cyanidin-3 glucoside); Anthocyanidin peak 2 and 3 is Cyanidin malonyl-glucoside, and molecular weight is 535.11, and retention time is respectively 32min and 34.5min(accompanying drawing 5)
7. rubber extracts and assay
1) rubber extracting method: the russian dandelion blade is carried out lyophilize, get 1g wild rubber grass (contrast) and transgenosis russian dandelion blade respectively and pulverize, each adds 20ml toluene, and the centrifugal 10min of 5000rpm collects the methylbenzene extraction thing behind 85 ℃ of lixiviate 20h; Again be dissolved in after the evaporated under reduced pressure in the 5ml toluene, adding the centrifugal 10min of 10ml methyl alcohol 5000rpm, be precipitated as the rubber of extraction;
2) rubber content measuring method: the rubber that extracts is dissolved in the 0.9ml toluene, the polystyrene liquid (as external standard) that adds 0.1ml 10mg/ml, the sample (about 20 μ l) that takes a morsel evenly is applied on the circular test zone of Potassium Bromide wafer, does examination of infrared spectrum then.Sweep limit 500～4000cm
-1, 32 scanning is averaged.Be with reference to product with polystyrene, according to the special absorption of 740nm(polystyrene) and the special absorption of 830nm(russian dandelion rubber) peak area ratio, the gel content of calculating russian dandelion.
A kind of method of russian dandelion anthocyanidin content that improves of the present invention is by making up plant expression vector, the synthetic regulatory factor of Arabidopis thaliana anthocyanidin is imported in the russian dandelion, do not influencing the russian dandelion plant strain growth and producing under the prerequisite of glue function, successfully improved the content of anthocyanidin in the russian dandelion, increased the utility value of russian dandelion greatly, for russian dandelion large-scale develops and utilizes, extract the anthocyanidin of russian dandelion, with for the production of protective foods and makeup and industrial synthetic colour, be conducive to the industrialized development of russian dandelion.
Description of drawings:
Fig. 1: recombinant expression vector collection of illustrative plates.Goal gene is the synthetic regulatory factor 1 of Arabidopis thaliana anthocyanidin, i.e. sequence table provides sequence.
Fig. 2: russian dandelion gene transformation process.
Among the figure: 1. screen red resistant buds; 2. resistant plant root culture; 3. complete red resistance plant.
Fig. 3: the comparison of wild rubber grass and transgenosis russian dandelion.
Fig. 4: the gene test of wild rubber grass and transgenosis russian dandelion.
Among the figure: M: standard molecular weight; 1: the wild rubber grass; 2: recombinant expression vector; 3～7: be respectively russian dandelion transgenic lines 1～5.
Fig. 5: the anthocyanidin color atlas of contrast russian dandelion and transgenosis russian dandelion compares and the anthocyanidin mass spectrum.
Among the figure: transgenosis: transgenic lines 1, contrast: wild rubber grass
Anthocyanidin peak 1 is Cyanidin-3 glucoside, and retention time 22min molecular weight is 449.11; Anthocyanidin peak 2 is Cyanidin malonyl-glucoside I, and retention time 32min molecular weight is 535.11; Anthocyanidin peak 3 is Cyanidin malonyl-glucoside II, and retention time 34.5min, molecular weight are 535.11.
Embodiment
Following examples are used for explanation the present invention, but are not used for limiting the scope of the invention.
Implement when of the present invention, the test method of unreceipted actual conditions among the embodiment is usually according to normal condition.The main agents of using: plasmid DNA extract test kit, dna gel reclaim test kit, Ex Taq archaeal dna polymerase,
Vector, endonuclease XbaI, Sac I and dNTPs are available from Takara company, TRIZOL Reagent, the T4 ligase enzyme, RevertAid First Strand cDNA Synthesis Kit is available from Thermo Fisher Scientific, the CTAB(cetyl trimethylammonium bromide), YEB, the Tris(Tutofusin tris), the EDTA(ethylenediamine tetraacetic acid (EDTA)), DH5 α, the Rif(Rifampin), the Kan(kantlex), biochemical reagents such as cephamycin are given birth to worker's biotechnology company limited available from Shanghai, Cyanidin-3 glucoside, polystyrene is available from Sigma-Aldrich Co.LLC..The enforcement concrete steps are as follows:
The clone of 1 Arabidopis thaliana
1) extraction of Arabidopis thaliana DNA: get the 100mg Arabidopsis leaf in the 1.5ml centrifuge tube, the centrifuge tube liquid nitrogen flash freezer, grinding rod pulverizes material rapidly, adds the CTAB extracting solution (2%CTAB of 600 μ l preheatings; 100mmol/L Tris-HCl pH=8.0; 20mmol/L EDTA pH8.0; 1.4mol/L NaCl), 65 ℃ the insulation 30min, during put upside down mixing frequently; Add the abundant mixing of 500 μ l chloroforms, room temperature, 12000rpm, 5min, centrifugal phase-splitting; Supernatant is forwarded in the new centrifuge tube, add the pre-cold isopropanol of 0.7 volume, mixing is placed 15min deposit D NA under the room temperature; Room temperature, 12000rpm, centrifugal 5min removes supernatant, 70% washing with alcohol precipitation; Room temperature, 12000rpm, centrifugal 3min removes ethanol, and air drying 10min is dissolved in an amount of sterilized water.
2) polymerase chain reaction (PCR) amplification of synthetic regulatory factor 1 gene of Arabidopis thaliana anthocyanidin: synthetic primer, upstream primer is 5'-ACTCTAGAATGGAGGGTTCGTCCAAAGG-3', and downstream primer is denoted as Xba I and Sac I restriction enzyme site for the 5'-CCGAGCTCCTAATCAAATTTCACAGTCTC-3'(underscore).Be template with Arabidopis thaliana DNA, through polymerase chain reaction (PCR) amplification, the amplified production that the obtains agarose gel electrophoresis in 1% separated, target fragment reclaim the back with
The Vector carrier connects, and changes bacillus coli DH 5 alpha over to, and random choose gained positive colony send invitrogen company to carry out sequence verification after the bacterium colony polymerase chain reaction is detected.
2. the structure of recombinant expression vector
(be the biomaterial of market sale with pBI121, clontech company produces) be plant expression vector, replace commentaries on classics beta-Glucuronidase gene (GUS) on it with synthetic regulatory factor 1 gene of Arabidopis thaliana anthocyanidin, namely use Xba I and Sac I (to be the reagent of market sale, originate from the scientific in Thermo) double digestion polymerase chain reaction (PCR) amplification product and pBI121 carrier, reclaim the back and connect conversion DH5 α, the picking mono-clonal, the extraction plasmid is done the polymerase chain reaction, enzyme is cut and sequence verification.
3. contain the acquisition of recombinant expression vector agrobacterium tumefaciens
1) competence agrobacterium tumefaciens preparation: the single colony inoculation of EHA105 of picking (for the product of market sale) is in containing 50mg/L Rif(Rifampin) the YEB substratum (be the biological reagent of market sale, Shanghai is given birth to worker's biotechnology company limited and is produced) in, 200r/min overnight incubation under 28 ° of C; Get 400 μ l incubated overnight bacterium liquid and join 100ml and contain same antibiotic YEB liquid nutrient medium, 200r/min cultivates 3～4h under 28 ° of C, and OD600 is 0.6-0.8; Bacterium is transferred in the centrifuge tube of precooling then at ice-water bath cooling 10～15min, the centrifugal 20min of 5000r/min under 4 ° of C, the precipitation water dissolution of precooling; Repeated centrifugation 1 time is outwelled supernatant immediately, with remaining liquid re-suspended cell; Suspension is joined in the 50ml centrifuge tube of precooling, the centrifugal 10min of 5000r/min under 4 ° of C precipitates resuspendedly with isopyknic 10% glycerine, is loaded in the Eppendorf tube of precooling by the volume integral of per 200 μ l.
2) electricity swashs conversion: the 200 μ l Agrobacteriums that will prepare are put on ice, add the recombinant expression vector that 5 μ l prepare, mixing; In the sharp cup of the prior electricity in precooling on ice of the bacterium liquid immigration that adds plasmid DNA, jog makes liquid sink to the cuvette bottom, between the electricity of sharp glass of insertion conversion tank of electricity is swashed; Electric pulse parameter is set, and electric capacity is 25 μ F, resistance 200 Ω, and strength of electric field 20kv/cm carries out electricity and swashs; Swash adding 0.8ml YEB substratum in the cup at electricity immediately after electricity swashs, close with the rifle persorption and transfer in the 1.5ml centrifuge tube, 28 ° of C, 220r/min shaking culture 3h gives full expression to foreign gene in Agrobacterium; Draw above-mentioned culture 100 μ L, be laid on contain the Rif(Rifampin) 50mg/L, Kan(kantlex) among the 100mg/L, YEB flat board, 28 ° of C cultivate 36～48h, the screening transformant is selected single bacterium colony and is carried out polymerase chain reaction checking positive strain clone.
4. the acquisition of transgenosis russian dandelion plant
The above-mentioned reorganization agrobacterium tumefaciens of picking mono-clonal from the YEB solid medium is inoculated into and contains 50mg/L Kan(kantlex) and 50mg/L Rif(Rifampin) the YEB liquid nutrient medium in, 28 ℃ are shaken bacterium and are cultured to logarithmic growth late period; Therefrom get again in the substratum that 1mL is forwarded to 50ml YEB, be cultured to OD600 ≈ 0.5 under the similarity condition; Behind the centrifugal 10min of 4000g, precipitation is diluted to OD560=0.5-0.8 as infecting bacterium liquid with 50ml MS liquid nutrient medium.
The russian dandelion blade is cut into the big leaflet dish of 1～2cm2, infects about 15min in the immersion bacterium liquid and will infect the taking-up of posterior lobe dish, transfer to after blotting with aseptic filter paper and select on the substratum.25 ℃ of dark cultivations after 3 days, after the leaf dish washs for several times with sterilized water, change division culture medium over to, change it over to root media cultivation after growing resistant buds, after several weeks, treat that plant grows 2～3 root systems, open the container closure film, hardening 2～3 days moves into seedling the nutrition soil of 1:1 then: cultivate on the vermiculite, obtain the transgenosis russian dandelion.
Described division culture medium adds 1～2mg/L BA, 0.5～1mg/L NAA and 200mg/L cephamycin and 50～200mg/L Kan(kantlex with the MS minimum medium);
Described root media adds 0.1～0.5mg/LNAA, 200mg/L cephamycin and 50～200mg/L Kan(kantlex with the 1/2MS minimum medium).
5. the detection of transgenosis russian dandelion plant
The CTAB method is extracted the genomic dna of wild rubber grass (contrast) and above-mentioned transfer-gen plant, and the design gene specific detects primer, is respectively RT-F:5'-TTGCTGGAAGATTACCTGGT-3' and RT-R:5'-TCCAAAGTTGATCAACGTCA-3'; Be template with the total DNA of transgenic line blade, adopt PCR method to detect the positive transfer-gen plant of screening, amplifying target genes product size is 450bp.The PCR program is as follows:
PCR reaction system (25 μ l):
Taq polymerase(10u/mL) 0.2μl
10×buffer 2.5μl
dNTP(500mM) 1μl
Primer1(10mM) 1μl
Primer2(10mM) 1μl
H
2O 18.3μl
DNA 1μl
Total 25μl
PCR reaction system program: 94 ℃, 4min; 94 ℃ of 1min, 50 ℃～60 ℃ 1min, 72 ℃ of 1～2min, 32 circulations; 72 ℃ of 10min.
6. anthocyanidin extracts and assay
Take by weighing wild rubber grass (contrast) and each 1g of transgenosis russian dandelion blade respectively, each adds the methanol solution (methanol solution that contains 0.1% formic acid) of 3mL, and is centrifugal behind 4 ℃ of following lixiviate 24h, gets supernatant, and it is standby to cross 0.2 μ m aperture nylon leaching film.Adopt the anthocyanidin quantitative analysis of high performance liquid chromatography (HPLC) method to extracting.Be ODS-80TsQA C18column (4.6 * 250mm, i.d.5 μ m), (product of market sale originates from kromasil); The automatic sampler sample introduction, sample size 4 μ l; Gradient eluent is: A is that 0.2% formic acid water B is acetonitrile mutually mutually; Elution program: 0min, 5.0%B; 20min10.0%B; 35min20.0%B; 40min30.0%B; 50min40.0%B; 55min40.0%B; 60min5.0%B.30.0 ℃ of column temperatures; Flow velocity 0.4ml/min; Detect wavelength 515.0nm; Cyanidin-3 glucoside (for the product of market sale, originating from sigma) is for reference to product, according to its peak area by typical curve regression equation calculation anthocyanidin concentration.Adopt the high performance liquid chromatography mass spectrometric hyphenated technique to identify the anthocyanidin type, wherein anthocyanidin peak 1 is Cyanidin-3 glucoside; Retention time 22min molecular weight is 449.11, with above-mentioned consistent with reference to product; Anthocyanidin peak 2 and 3 is Cyanidin malonyl-glucoside, and molecular weight is 535.11, and retention time is respectively 32min and 34.5min, infers called after Cyanidin malonyl-glucoside I and Cyanidin malonyl-glucoside II(accompanying drawing 5 at this).The anthocyanidin content analysis in table 1 of contrast and transgenosis russian dandelion:
The anthocyanidin content analysis of table 1 wild rubber grass (contrast) and transgenosis russian dandelion
7. rubber extracts and assay
The russian dandelion blade is carried out lyophilize, get wild rubber grass (contrast) and each 1g of transgenosis russian dandelion blade respectively, pulverize, each adds 20ml toluene, and the centrifugal 10min of 5000rpm collects the methylbenzene extraction thing behind 85 ℃ of lixiviate 20h; Again be dissolved in after the evaporated under reduced pressure in the 5ml toluene, respectively add the centrifugal 10min of 10ml methyl alcohol 5000rpm again, be precipitated as the rubber of extraction.The rubber that extracts is dissolved in the 0.9ml toluene, adds the polystyrene liquid (as external standard) of 0.1ml10mg/ml, the sample that takes a morsel (about 20 μ l) evenly is applied on the circular test zone of Potassium Bromide wafer, does examination of infrared spectrum then.Sweep limit 500～4000cm-1,32 scanning is averaged.Be with reference to product with polystyrene, according to the special absorption of 740nm(polystyrene) and the special absorption of 830nm(russian dandelion rubber) peak area ratio, the gel content of calculating russian dandelion.The rubber content analysis in table 2 of contrast and transgenosis russian dandelion:
The rubber content analysis of table 2 wild rubber grass (contrast) and transgenosis russian dandelion
Though above the present invention is described in detail with a general description of the specific embodiments, on basis of the present invention, can make some modifications or improvements it, this will be apparent to those skilled in the art.Therefore, these modifications or improvements all belong to the scope of protection of present invention without departing from theon the basis of the spirit of the present invention.
Claims (5)
1. method that improves the russian dandelion anthocyanidin content, it is characterized in that adopting gene clone method to obtain synthetic regulatory factor 1 full length gene of Arabidopis thaliana anthocyanidin, with synthetic regulatory factor 1 gene of Arabidopis thaliana anthocyanidin and plant expression vector construction recombinant expression vector, the recombinant expression vector positive plasmid imports in the agrobacterium tumefaciens, adopt Agrobacterium tumefaciens mediated conversion russian dandelion, obtain high anthocyanidin transgenosis russian dandelion, and transgenosis russian dandelion plant is detected.
2. according to the described a kind of method that improves the russian dandelion anthocyanidin content of claim 1, it is characterized in that adopting gene clone method to obtain Arabidopis thaliana anthocyanidin and generate transcription factor 1 full length gene, be masterplate with the arabidopsis thaliana genomic dna, employing contains XbaI upstream primer 5'-ACTCTAGAATGGAGGGTTCGTCCAAAGG-3', and the downstream primer 5' – CCGAGCTCCTAATCAAATTTCACAGTCTC-3' that contains SacI, (PCR) amplification through the polymerase chain reaction, the amplified production that obtains separates in 1% agarose gel electrophoresis, target fragment reclaim the back with
The Vector carrier connects, and changes bacillus coli DH 5 alpha over to, and sequence verification also obtains the encoding sequence of described gene.
3. according to the described a kind of method that improves the russian dandelion anthocyanidin content of claim 1, it is characterized in that the method for synthetic regulatory factor 1 gene of Arabidopis thaliana anthocyanidin and plant expression vector construction recombinant expression vector being, the PCR that generates transcription factor 1 gene with XbaI and the described Arabidopis thaliana anthocyanidin of SacI double digestion reclaims product and pBI121 plant expression vector, reclaim to connect and transform DH5 α, the picking mono-clonal, the extraction plasmid is done the polymerase chain reaction detection and enzyme is cut checking, obtains containing the recombinant expression vector of described gene.
4. according to the described a kind of method that improves the russian dandelion anthocyanidin content of claim 1, it is characterized in that adopting the method for Agrobacterium tumefaciens mediated conversion russian dandelion to be, picking contains the single bacterium colony of agrobacterium tumefaciens of recombinant expression vector, be inoculated in the YEB liquid nutrient medium of 10mL antibiotic-free, 28 ° of C180r/min shaking culture 16 hours, the centrifugal 10min of 5000r/min collects thalline; Agrobacterium is diluted to OD560=0.5-0.8 with 50ml MS liquid nutrient medium.The russian dandelion blade is cut into 1～2cm
2, contaminated the russian dandelion blade 5～30 minutes, take out the blade callus, the bacterium liquid that it is surperficial blots with filter paper, transfers on the MS substratum.After cultivating 2～3 days altogether, explant is taken out from former substratum, be transferred on the division culture medium, division culture medium of replacing in per 10～20 days.When treating resistant buds length to the 0.5cm left and right sides, resistant buds is downcut, change the root media cultivation over to and take root, obtain the careless plant of regenerated rubber of resistance.
5. according to the described a kind of method that improves the russian dandelion anthocyanidin content of claim 1, it is characterized in that it is with the vermiculite of the careless plantlet of transplant of the regenerated rubber of resistance to 1:1 that transgenosis russian dandelion plant detects: turfy soil, clear water Routine Management.The CTAB method is extracted the genomic dna of above-mentioned plant, and the design gene specific detects primer, is respectively RT-F:5'-TTGCTGGAAGATTACCTGGT-3' and RT-R:5'-TCCAAAGTTGATCAACGTCA-3'; Amplifying target genes product 450bp.Be template with the total DNA of transgenic line blade, detect the positive transfer-gen plant of screening, filter out high anthocyanidin transgenosis russian dandelion.
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| CN107459504A (en) * | 2017-07-18 | 2017-12-12 | 中国科学院南京土壤研究所 | A kind of method that anthocyanidin is extracted in the arabidopsis from water planting |
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