CN104450779A - Fast and efficient agrobacterium-mediated rice stem tip genetic transformation method - Google Patents
Fast and efficient agrobacterium-mediated rice stem tip genetic transformation method Download PDFInfo
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Abstract
The invention discloses a fast and efficient agrobacterium-mediated rice stem tip genetic transformation method. The method comprises the following major steps: directly soaking mature rice seeds in tap water, accelerating germination, then transversely cutting the coleoptiles to expose stem tips as transformation receptors, performing genetic transformation by virtue of agrobacterium mediation without sterilization treatment, putting the transformed materials under an appropriate environment to allow plants to grow, and carrying out transgenic screening and identification to obtain the transgenic rice plants. According to the fast and efficient agrobacterium-mediated rice stem tip genetic transformation method, callus induction and a series of subsequent tissue culture processes are avoided, the defects of materials affected by bacteria, difficult regeneration, vitrification, browning, low survival rate of transplanted tissue cultured seedlings and the like in the tissue culture process are avoided, the rice genetic transformation cost is reduced, the restriction of the rice genotype to the transformation efficiency is effectively overcome, and a large quantity of transgenic rice plants can be obtained in short time; the fast and efficient agrobacterium-mediated rice stem tip genetic transformation method has a significant value in good-quality rice culture and rice genetic transformation research.
Description
Technical field
The invention belongs to agricultural biological technical field and plant genetic engineering field, specifically a kind of rapidly and efficiently agriculture bacillus mediated rice conversion method.
Background technology
Paddy rice is one of important in the world food crop, and the population exceeding half in the world take paddy rice as staple food, and its stable supplying directly affects the grain balance in the whole world.The paddy rice cultivating various high-quality is one of approach solving food problem and the environmental problem day by day highlighted.Conventional paddy rice cross breeding breeding, the cycle is long, takes effect slow, and there is the shortcoming of gene linkage and reproduction isolation.By means of the selection by mutation of some physico chemical factors, be difficult to the direction controlling mutagenesis, beneficial mutation rate is low.Obtain hybrid cell in the mode of cytogamy, cultivate and develop into the cell engineering breeding of plant, technical sophistication, efficiency is low.At present, adopting engineered technology to obtain new rice variety is one of the most efficient breeding mode.
Since the first transgenic paddy rice in 1988 succeeds, particle bombardment, electric shocking method, PEG method, pollen tube passage method and the multiple genetic transforming method such as agriculture bacillus mediated are comprised all in Transgenic Rice research successful Application.But due to PEG mediated method and electric shocking method, need by means of plant protoplast culture technique, difficulty is comparatively large, and plant protoplast may be subject to the impact of hormonal action or spontaneous fusion, produces cytometaplasia.Particle bombardment, although simple to operate, economic drain is large, and there is foreign gene and easily lose, easy gene silencing, and transgenic progeny such as easily to be suddenlyd change at the shortcoming.At present, infecting Rice Callus by Agrobacterium, to obtain transgenic paddy rice be the genetic transforming method that at present research is deep, the most ripe, and it has that transformation frequency is high, multiple copied frequency is low, low cost and other advantages and being widely adopted.But the method is subject to the impact of plant tissue culture technique, usually adopt mature seed or rataria evoked callus inductivity in tissue culture procedures, acquisition that low succeeding transfer culture efficiency all limits transfer-gen plant.Set up a rice transformation method not relying on plant tissue culture technique to be significant.
Agrobacterium is the Gram-negative bacteria extensively existed in a kind of soil.After plant sustains damage, some aldehydes matters can be produced, enter in agrobatcerium cell, cause the processing of T-DNA, enter vegetable cell and be incorporated on nuclear genome.If inserted by goal gene in the agrobatcerium T-DNA of suitable structure, foreign gene will insert in Plant Genome along with T-DNA, and along with the Differentiation of vegetable cell, produces transfer-gen plant.The acceptor material that agriculture bacillus mediated rice transformation institute adopts mainly contains: young fringe, rataria, mature embryo, anther callus, shoot apical meristem etc.The different developmental phases of above-mentioned materials originating species, has spatio-temporal difference.As desirable genetic transformation material, should have the following advantages: the first, material is relatively homogeneous, and repeatability is high with ubiquity.The second, material is relatively large, is convenient to obtain and genetic transformation.3rd, material is easily preserved, and reduces season to the impact of drawing materials, is convenient to test.4th, material differentiation degree is low, and cellular omnipotency is strong, is the success of material genetic transformation, develops into the basic guarantee of whole plant.Because Mature seed of rice individuality is comparatively large, material is homogeneous, reproducible, and easily obtains, and easily preserves, and cellular omnipotency is strong, so be the ideal material doing rice transformation research.Meanwhile, rice transformation often needs by plant tissue culture, and wherein the sterilization of explant is one of key factor, and disinfecting time is too short, the easy microbiological contamination of tissue culture procedures; Overlong time, disinfecting substance can kill and wound vegetable cell, affects group training effect.Due to genotypic difference, the genetic transformation efficiency of different rice varieties is different.Just report at present, the genetic transformation successful story report about japonica rice is a lot, but for long-grained nonglutinous rice, successful story is little.Affect by long-grained nonglutinous rice is genotypic, T-DNA insertion long-grained nonglutinous rice genomic efficiency ratio japonica rice is low.Although the many people comprising Hiei are proposed optimization long-grained nonglutinous rice being carried out to Agrobacterium-mediated genetic transformation, only having quite few rice variety to transform successfully, is largely by the genotypic restriction of material itself.So the rice transformation method setting up efficient transformation receptor genotype degree of dependence low is significant.
Summary of the invention
For the demand in above-mentioned field, the invention provides a kind of genetic transforming method, avoid technology in the plant tissue culture that traditional rice transformation must rely on loaded down with trivial details, strict sterilization and aseptic condition, culture cycle is long, economic input is many, and require high to the state of material and genotype, different materials often needs to grope different condition of tissue culture, even can not get a series of defects such as desirable effect, set up a kind of efficient paddy rice stem apex genetic transforming method agriculture bacillus mediated fast.
An agriculture bacillus mediated paddy rice stem apex genetic transforming method rapidly and efficiently, comprises following sequential steps:
(1) get Mature seed of rice to sprout, obtain its germination period individual;
(2) individual for material with the paddy rice of germination period, its plumule of crosscut, will comprise exposed shoot apical meristem as genetic transformation acceptor;
(3) take Agrobacterium as mediation, by vacuum infiltration process, Agrobacterium is imported shoot apical meristem cell, the T-DNA making it carry goal gene inserts Plant Genome;
(4) after rear plant to be transformed grows 3-4 sheet young leaves, Screening and Identification transfer-gen plant.
Described seed germination be rice paddy seed without the need to disinfecting, after directly showing money or valuables one carries unintentionally with tap water 42 DEG C of immersions, 37 DEG C of vernalization 24h.
Described shoot apical meristem be with complete radicle, endosperm and excision plumule after shoot apical meristem.
Described step (3) vacuum infiltration treatment process is the resuspended bacterium immersion bubble of paddy rice individuality AAM that will expose shoot apical meristem, vacuumizes process.
Described AAM re-suspension liquid resuspended bacterium liquid OD
600be add Syringylethanone and tensio-active agent Silwet L-77 in the resuspended bacterium liquid of 0.8, AAM, to promote transformation efficiency.
Described Agrobacterium is through artificial reconstructed, carries goal gene, for the agrobacterium tumefaciens (Agrobacterium tumefaciens) of Genetic Transformation in Higher Plants.
The kind of described paddy rice is japonica rice variety or rice variety.
The present invention take Mature seed of rice as material, and through vernalization process, its shoot apical meristem of crosscut, as acceptor; Under non-sterile envrionment conditions, utilize agriculture bacillus mediated, by vacuum-assisted processing, by foreign gene Introduced into Rice genome, and then obtain transgenic paddy rice.
The vacuum infiltration treatment time is different according to the kind of material, determines during screening pressure Screening and Identification transfer-gen plant according to conversion carrier screening-gene.
The present invention is without the need to through tissue culture, low by genotype effect degree, and directly operates the seed sprouted, and has the following advantages.
The first, easy.Except spawn culture, do not need to operate under super-clean environment.A series of complicated processes such as removing the necessary substratum preparation of group training process, explant sterilization, callus induction, subculture, altogether training from, screen, regeneration bud is induced, take root.
The second, quick.Obtaining transfer-gen plant from soaking seed to, only needing paddy rice growth cycle, without the need to through callus induction, screening, break up again, the plant tissue culture cycle such as root induction.
Three, efficient.Genotype effect by converting material is very little, can be applicable to long-grained nonglutinous rice and japonica rice, and transformation efficiency is high simultaneously.
Four, economical.Directly infect rice transformation acceptor with bacterium liquid, adopt perlite or earth to cultivate paddy rice, a series of manpower and materials of removing from conversion process in material structure culturing process drop into, and can significantly reduce genetic transformation cost.
Term of the present invention and substratum:
In the present invention, Agrobacterium cultivates general employing YEP substratum (yeast extract 10g/L+ peptone 10g/L+NaCl 5g/L, pH 7.2, adds 15g/L agar in solid medium) cultivation.
Agrobacterium re-suspension liquid in the present invention is: AAM nutrient solution is with reference to Hiei, et al, and (2008) fill a prescription (KCl 2950.0mg/L, CaCl
22H
2o 150.0mg/L, MgSO
47H
2o 500.0mg/L, NaH
2pO
4h
2o 150.0mg/L, FeSO
47H
2o 278.0mg/L, EDTA 373.0mg/L, MnSO
44H
2o 13.2mg/L, ZnSO
47H
2o 2.0mg/L, CuSO
45H
2o 0.025mg/L, CoCl
26H
2o 0.025mg/L, H
3bO
33.0mg/L, KI 0.75mg/L, VB11.0mg/L, VB61.0mg/L, Nicotinic acid 1.0mg/L, Myo-Inositol 100.0mg/L, Glutamine 876.0mg/L, Asp 266.0mg/L, Arg 174.0mg/L, Gly 7.5mg/L, 100 μMs of AS, 68.5g sucrose, 36g glucose, 0.5g acid hydrolyzed casein, pH 5.2)
Accompanying drawing explanation
Fig. 1 pCambia-Ubi-CHI plant expression vector
Fig. 2 pCambia-35s-Pb plant expression vector
Red seed-grain of Fig. 3 Jianhe
The black glutinous seed in Fig. 4 Pingtang
Fig. 5 GUS dyes in the blue red paddy plant leaf of transgenosis Jianhe
The transgenosis Jianhe red paddy children fringe of Fig. 6 GUS stained positive
The black glutinous young fringe in transgenosis Pingtang of Fig. 7 GUS stained positive
Fig. 8 McCHIT1 gene PCR detected result
Fig. 9 P-10kDa sequence PCR detected result
Embodiment
Below in conjunction with embodiment, the present invention is described in further detail.
Embodiment 1: adopt present method to obtain the red paddy transfer-gen plant of the peculiar indica type red rice Jianhe of Guizhou Native
1. the preparation of rice transformation acceptor
Get the red paddy mature seed (as shown in Figure 3) of Jianhe, Seed soaking, 12h changes a water, break tripe to seed after 2d to show money or valuables one carries unintentionally, carry out vernalization at 37 DEG C and take root, treat that plumule grows into 1.5-2.0cm, cut plumule and coleoptile with scalpel from stem apex stem ring, expose its shoot apical meristem.
2. Agrobacterium is cultivated and activation
Agrobacterium tumefaciens bacterial strain LBA4404, carry balsam pear chitinase gene McCHIT1 and herbicide resistance gene PAT pCambia-Ubi-CHI carrier (this carrier be by Southwestern University Pei Yan teach laboratory build and be so kind as to give, I has preservation in school experiment room, can externally provide) as shown in Figure 1, be inoculated on the YEP solid medium containing 20mg/L Rifampin (Rif) and 50mg/L kantlex (Kan).After cultivating 2d in 28 DEG C of incubators, picking list bacterium colony is in 5ml YEP (containing 20mg/L Rif+50mg/L Kan) liquid nutrient medium, 180-200rpm, after 28 DEG C of shaking culture 24h, get 200 μ l bacterium liquid and join 50ml YEP (20mg/L Rifampin, 50mg/L kantlex) in substratum, 200rpm 28 DEG C of shaking culture, until OD
600value reaches 0.7.The Agrobacterium thalline of 5,000rpm collected by centrifugation, with AAM re-suspension liquid resuspended bacterium liquid OD
600to 0.8.
3. the genetic transformation of paddy rice
The paddy rice handled well is put into AAM re-suspension liquid, add the tensio-active agent Silwet L-77 (U.S. GE Company of 200 μ l/L, the allyloxypolyethyleneglycol methyl ether of the polyalkyleneoxide modified heptamethyltrisiloxane and 16% by 84% mixes), vacuum-treat 8min under 1.5Kpa, outwell re-suspension liquid, be placed on totally moistening perlite, 28 DEG C of light culture 3d.
4. the screening of transfer-gen plant, transplanting, management
Move into paddy seedbed by the plant after light culture, the 3-4 leaf phase carries out herbicide screening to the plant after conversion with 50mg/L basta (Bio Basic Inc Products).After 7d, transplant survival plant is in paddy field, transgenic plant Demonstration Base greenhouse, conventional fertilizer water management.
5. transfer-gen plant qualification
Resistant plant GUS histochemical stain detects: after transplanting two weeks, clip Rice Seedlings blade puts into PCR pipe, add X-Gluc (the chloro-3-indoles of the bromo-4-of the 5--β-D-Glucose glycosides of 15 μ l, Gold biotechnology Company) staining fluid, 37 DEG C are spent the night, outwell X-Gluc, 75% ethanol decolorization adding 200 μ l is completely to the greatest extent de-to chlorophyll.Result shows, and in blade dyeing process 224 strain paddy rice, 35 strain dye liquors are in blue, and positive plant accounts for 15.6%.And at rice ear sprouting period, clip children fringe, carries out GUS histochemical stain inspection further.See shown in Fig. 5, Fig. 6.
Transfer-gen plant PCR detects
Get the plant that GUS dye liquor detection blade is positive, adopt TIANGEN company DNAsecure Plant Kit test kit to extract rice leaf DNA.Concrete grammar is as follows:
(1) get the fresh blade 100mg of paddy rice that GUS detection in tillering phase is positive, shred in mortar, add liquid nitrogen, fully grind.
(2) by the material after grinding, add the 2ml centrifuge tube that 400 μ l damping fluid LP1 and 6 μ l RNase A (10mg/ml) are housed, vortex vibration 1min, room temperature places 10min.
(3) 130 μ l damping fluid LP2 are added, with liquid-transfering gun piping and druming mixing, vortex vibration 1min.
The centrifugal 5min of (4) 12,000rpm, moves to supernatant in new 2ml centrifuge tube.
(5) add the damping fluid LP3 of 1.5 times of volumes, fully vibration mixes 15sec immediately, until there is flocks.
(6) all add in an adsorption column CB3 (adsorption column puts into collection tube) by previous step gained solution and flocks, the centrifugal 30sec of 12,000rpm, outwells waste liquid, and adsorption column CB3 puts into collection tube.
(7) in adsorption column CB3, add 600 μ l rinsing liquid PW, the centrifugal 30sec of 12,000rpm, outwells waste liquid, and adsorption column CB3 is put into collection tube.
(8) previous action is repeated.
(9) put back in collection tube by adsorption column CB3, the centrifugal 2min of 12,000rpm, outwells waste liquid.Adsorption column CB3 is placed in room temperature and places several minutes, thoroughly dry rinsing liquid remaining in sorbing material.
(10) proceeded to by adsorption column CB3 in a clean centrifuge tube, the unsettled dropping in the middle part to adsorption film 50-200 μ l elution buffer TE, room temperature places the centrifugal 2min of 2-5min, 12,000rpm, by solution collection in centrifuge tube.
Agrobacterium plasmid extracts, and adopt the little extraction reagent kit of TIANGEN company plasmid to extract, concrete grammar is as follows:
(1) column equilibration step: the centrifugal 1min of the balance liquid BL adding 500 μ L to (adsorption column puts into collection tube) in adsorption column CP3,12,000rpm, outwell the waste liquid in collection tube, adsorption column is put back in collection tube.
(2) get the bacterium liquid 1-5ml of incubated overnight, add in centrifuge tube, the centrifugal 1min of 12,000rpm, draw supernatant.
(3) in the centrifuge tube leaving bacterial sediment, add 250 μ L solution P1, use pipettor or the thorough suspended bacterial precipitation of vortex oscillator.
(4) in centrifuge tube, add 250 μ L solution P2, leniently spin upside down and make the abundant cracking of thalline for 6-8 time.
(5) in centrifuge tube, add 350 μ L solution P3, leniently spin upside down 6-8 time immediately, fully mix, now will occur white flock precipitate.The centrifugal 10min of 12,000rpm, now forms precipitation bottom centrifuge tube.
(6) the supernatant liquor pipettor that previous step is collected is transferred to (adsorption column puts into collection tube) in adsorption column CP 3, note sucking-off precipitation of trying not.The centrifugal 30-60s of 12,000rpm, outwells the waste liquid in collection tube, and adsorption column CP 3 is put into collection tube.
(7) in adsorption column CP 3, add 600 μ L rinsing liquid PW, the centrifugal 30-60s of 12,000rpm, outwells the waste liquid in collection tube, and adsorption column CP 3 is put into collection tube.
(8) repeating step (7).
(9) adsorption column CP 3 is put into collection tube, the centrifugal 2min of 12,000rpm, remove rinsing liquid remaining in adsorption column.
(10) adsorption column CP 3 is placed in a clean centrifuge tube, the middle part to adsorption film drips 50-100 μ l elution buffer EB, and room temperature places the centrifugal 1min of 2min, 12,000rpm, is collected in centrifuge tube by plasmid solution.
Finally, PCR detection is carried out to pCambia-Ubi-CHI gene.Its PCR primer is synthesized by the handsome biological company limited in Shanghai.Primer sequence is Forward:5 '-ATCGGGAGACGTTGGCAG-3 '; Reverse primer:5 '-CTAGTGCTCTACCTGCTG-3 ', amplification CHI gene 401bp specific product.PCR response procedures is: 94 DEG C of 5min; 94 DEG C of 30s, 53 DEG C of 30s, 72 DEG C of 40s, 35 circulations; 72 DEG C extend 10min; 4 DEG C of preservations.PCR reaction is complete, gets 5 μ l amplified productions electrophoresis in the agarose gel electrophoresis of 1%, observes and take a picture under Bio-Rad gel imaging system instrument.See Fig. 8.
Embodiment 2: adopt present method to obtain the black glutinous transfer-gen plant in Guizhou Native peculiar round-grained rice type glutinous rice Pingtang
1. the preparation of rice transformation acceptor
To make even the black glutinous mature seed (as shown in Figure 4) in the pool, Seed soaking, 12h changes a water, break tripe to seed after 2d to show money or valuables one carries unintentionally, carry out vernalization at 37 DEG C and take root, treat that plumule grows into 1.5-2.0cm, cut plumule and coleoptile with scalpel from stem apex stem ring, expose its shoot apical meristem.
2. Agrobacterium is cultivated and activation
Agrobacterium tumefaciens bacterial strain LBA4404, (carrier is built by this laboratory to carry the pCambia-35s-Pb carrier of paddy rice pb gene and hygromycin gene HPTII, there is preservation in laboratory, can externally provide) as shown in Figure 2, be inoculated on the YEP solid medium containing 20mg/L Rifampin (Rif) and 40mg/L Totomycin (Hyg).After cultivating 2d in 28 DEG C of incubators, picking list bacterium colony is in 5ml YEP (containing 20mg/L Rif+35mg/L Hyg) liquid nutrient medium, 180-200rpm, after 28 DEG C of shaking culture 24h, getting 200 μ l bacterium liquid joins in 50ml YEP (containing 20mg/L Rif+35mg/L Hyg) substratum, 200rpm28 DEG C of shaking culture, until OD
600value reaches 0.7.The Agrobacterium thalline of 5,000rpm collected by centrifugation, with AAM re-suspension liquid resuspended bacterium liquid OD
600to 0.8.
3. the genetic transformation of paddy rice
The paddy rice handled well is put into AAM re-suspension liquid, and add the tensio-active agent Silwet L-77 of 200 μ l/L, under 1.5Kpa, vacuum-treat 5min, outwells re-suspension liquid, is placed on totally moistening perlite, 28 DEG C of light culture 3d.
5. the screening of transfer-gen plant, transplanting, management
Move into paddy seedbed by the plant after light culture, the 3-4 leaf phase screens with 40mg/L Totomycin (sigma Products) the plant after conversion.After 7d, transplant survival plant is in paddy field, transgenic plant Demonstration Base greenhouse, conventional fertilizer water management.
5. transfer-gen plant qualification
Resistant plant GUS histochemical stain detects: after transplanting two weeks, clip Rice Seedlings blade and heading stage children fringe put into PCR pipe, add the X-Gluc (5-Bromo-4-chloro-1H-indol-3-yl β-D-glucopyranosiduronic acid of 15 μ l, Gold biotechnology Company), 37 DEG C are spent the night, outwell X-Gluc, 75% ethanol decolorization adding 200 μ l is completely to the greatest extent de-to chlorophyll.Result shows, and in dyeing process 146 strain paddy rice, 45 strain dye liquors are in blue, and positive plant accounts for 30.8%.And at rice ear sprouting period, clip children fringe, carries out GUS histochemical stain inspection further.As shown in Figure 7.
Transfer-gen plant PCR detects
Get the plant that GUS dye liquor detection blade is positive, adopt TIANGEN company DNAsecure Plant Kit test kit to extract rice leaf DNA.Concrete grammar is as follows:
(1) get the fresh blade 100mg of paddy rice that GUS detection in tillering phase is positive, shred in mortar, add liquid nitrogen, fully grind.
(2) by the material after grinding, add the 2ml centrifuge tube that 400 μ l damping fluid LP1 and 6 μ l RNase A (10mg/ml) are housed, vortex vibration 1min, room temperature places 10min.
(3) 130 μ l damping fluid LP2 are added, with liquid-transfering gun piping and druming mixing, vortex vibration 1min.
The centrifugal 5min of (4) 12,000rpm, moves to supernatant in new 2ml centrifuge tube.
(5) add the damping fluid LP3 of 1.5 times of volumes, fully vibration mixes 15sec immediately, until there is flocks.
(6) all add in an adsorption column CB3 (adsorption column puts into collection tube) by previous step gained solution and flocks, the centrifugal 30sec of 12,000rpm, outwells waste liquid, and adsorption column CB3 puts into collection tube.
(7) in adsorption column CB3, add 600 μ l rinsing liquid PW, the centrifugal 30sec of 12,000rpm, outwells waste liquid, and adsorption column CB3 is put into collection tube.
(8) previous action is repeated.
(9) put back in collection tube by adsorption column CB3, the centrifugal 2min of 12,000rpm, outwells waste liquid.Adsorption column CB3 is placed in room temperature and places several minutes, thoroughly dry rinsing liquid remaining in sorbing material.
(10) proceeded to by adsorption column CB3 in a clean centrifuge tube, the unsettled dropping in the middle part to adsorption film 50-200 μ l elution buffer TE, room temperature places the centrifugal 2min of 2-5min, 12,000rpm, by solution collection in centrifuge tube.
Agrobacterium plasmid extracts, and adopt the little extraction reagent kit of TIANGEN company plasmid to extract, concrete grammar is as follows:
(1) column equilibration step: the centrifugal 1min of the balance liquid BL adding 500 μ L to (adsorption column puts into collection tube) in adsorption column CP3,12,000rpm, outwell the waste liquid in collection tube, adsorption column is put back in collection tube.
(2) get the bacterium liquid 1-5ml of incubated overnight, add in centrifuge tube, the centrifugal 1min of 12,000rpm, draw supernatant.
(3) in the centrifuge tube leaving bacterial sediment, add 250 μ L solution P1, use pipettor or the thorough suspended bacterial precipitation of vortex oscillator.
(4) in centrifuge tube, add 250 μ L solution P2, leniently spin upside down and make the abundant cracking of thalline for 6-8 time.
(5) in centrifuge tube, add 350 μ L solution P3, leniently spin upside down 6-8 time immediately, fully mix, now will occur white flock precipitate.The centrifugal 10min of 12,000rpm, now forms precipitation bottom centrifuge tube.
(6) the supernatant liquor pipettor that previous step is collected is transferred to (adsorption column puts into collection tube) in adsorption column CP 3, note sucking-off precipitation of trying not.The centrifugal 30-60s of 12,000rpm, outwells the waste liquid in collection tube, and adsorption column CP 3 is put into collection tube.
(7) in adsorption column CP 3, add 600 μ L rinsing liquid PW, the centrifugal 30-60s of 12,000rpm, outwells the waste liquid in collection tube, and adsorption column CP 3 is put into collection tube.
(8) repeating step (7).
(9) adsorption column CP 3 is put into collection tube, the centrifugal 2min of 12,000rpm, remove rinsing liquid remaining in adsorption column.
(10) adsorption column CP 3 is placed in a clean centrifuge tube, the middle part to adsorption film drips 50-100 μ l elution buffer EB, and room temperature places the centrifugal 1min of 2min, 12,000rpm, is collected in centrifuge tube by plasmid solution.
Finally, PCR detection is carried out to pCambia-35s-Pb gene.Its PCR primer is synthesized by the handsome biological company limited in Shanghai, and product is Pb Gene Partial sequence and 10kDa prolamine terminator partial sequence P-10kDa (this sequence does not exist in normal rice genome).Primer sequence is Forward:5 '-CAAGAAGCAGGCGAAAG-3 '; Reverse primer:5 '-ACAGAAATCCAAATACCCAAT-3 ', amplification 500bp specific product.PCR response procedures is: 94 DEG C of 5min; 94 DEG C of 30s, 53 DEG C of 30s, 72 DEG C of 40s, 35 circulations; 72 DEG C extend 10min; 4 DEG C of preservations.PCR reaction is complete, gets 5 μ l amplified productions electrophoresis in the agarose gel electrophoresis of 1%, observes and take a picture under Bio-Rad gel imaging system instrument.See Fig. 9.
Claims (7)
1. an agriculture bacillus mediated paddy rice stem apex genetic transforming method rapidly and efficiently, comprises following sequential steps:
(1) get Mature seed of rice to sprout, obtain its germination period individual;
(2) individual for material with the paddy rice of germination period, its plumule of crosscut, will comprise exposed shoot apical meristem as genetic transformation acceptor;
(3) take Agrobacterium as mediation, by vacuum infiltration process, Agrobacterium is imported shoot apical meristem cell, the T-DNA making it carry goal gene inserts Plant Genome;
(4) after rear plant to be transformed grows 3-4 sheet young leaves, Screening and Identification transfer-gen plant.
2. method for transformation according to claim 1, described seed germination be rice paddy seed without the need to disinfecting, after directly showing money or valuables one carries unintentionally with tap water 42 DEG C of immersions, 37 DEG C of vernalization 24h.
3. method for transformation according to claim 1, described shoot apical meristem be with complete radicle, endosperm and excision plumule after shoot apical meristem.
4. method for transformation according to claim 1, described step (3) vacuum infiltration treatment process is the resuspended bacterium immersion bubble of paddy rice individuality AAM that will expose shoot apical meristem, vacuumizes process.
5. method for transformation according to claim 4, described AAM re-suspension liquid resuspended bacterium liquid OD
600be add Syringylethanone and tensio-active agent Silwet L-77 in the resuspended bacterium liquid of 0.8, AAM, to promote transformation efficiency.
6. method for transformation according to claim 1, described Agrobacterium is through artificial reconstructed, carries goal gene, for the agrobacterium tumefaciens (Agrobacterium tumefaciens) of Genetic Transformation in Higher Plants.
7. method for transformation according to claim 1, the kind of described paddy rice is japonica rice variety or rice variety.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103233039A (en) * | 2013-05-06 | 2013-08-07 | 福建农林大学 | Agrobacterium tumefaciens-mediated rice seedling gene transformation method |
| CN103451230A (en) * | 2013-09-23 | 2013-12-18 | 江苏省农业科学院 | Rice transgenosis method utilizing agrobacterium tumefaciens to mediate |
-
2014
- 2014-12-15 CN CN201410770462.1A patent/CN104450779A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103233039A (en) * | 2013-05-06 | 2013-08-07 | 福建农林大学 | Agrobacterium tumefaciens-mediated rice seedling gene transformation method |
| CN103451230A (en) * | 2013-09-23 | 2013-12-18 | 江苏省农业科学院 | Rice transgenosis method utilizing agrobacterium tumefaciens to mediate |
Non-Patent Citations (1)
| Title |
|---|
| 曹孟良: "农杆菌介导的水稻高效遗传转化体系的建立", 《湖南农业大学学报：自然科学版》 * |
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| CN106148394A (en) * | 2015-05-18 | 2016-11-23 | 东北林业大学 | A kind of nonobstructive azoospermia method utilizing open country chrysanthemum foot bud to be outer implant |
| CN106244625A (en) * | 2016-10-09 | 2016-12-21 | 贵州大学 | A kind of agriculture bacillus mediated tobacco seed genetic transforming method rapidly and efficiently |
| CN106244625B (en) * | 2016-10-09 | 2019-07-12 | 贵州大学 | A kind of tobacco seed genetic transforming method of mediated by agriculture bacillus rapidly and efficiently |
| CN108998469A (en) * | 2018-08-02 | 2018-12-14 | 天津大学 | A kind of quick Mature Embryos of Rice original position transgenic method |
| CN110656129A (en) * | 2019-11-05 | 2020-01-07 | 贵州大学 | Genetic transformation method of agrobacterium-mediated coix lacryma-jobi |
| CN115029379A (en) * | 2022-07-04 | 2022-09-09 | 贵州大学 | Rapid and efficient agrobacterium-mediated pepper root tip genetic transformation method |
| CN117925706A (en) * | 2024-03-19 | 2024-04-26 | 云南省农业科学院花卉研究所 | Genetic transformation method of pigment marigold |
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