CN102618574B - Method for direct genetic transformation of tobacco through vacuum infiltration - Google Patents
Method for direct genetic transformation of tobacco through vacuum infiltration Download PDFInfo
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- CN102618574B CN102618574B CN201210113686.6A CN201210113686A CN102618574B CN 102618574 B CN102618574 B CN 102618574B CN 201210113686 A CN201210113686 A CN 201210113686A CN 102618574 B CN102618574 B CN 102618574B
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Abstract
The invention discloses a method for direct genetic transformation of tobacco through vacuum infiltration. The method comprises the following steps of: immersing pre-cultured tobacco immature leaf explants in pAHC25 plasmid DNA (Deoxyribonucleic Acid) solution carrying target gene; performing vacuum infiltration treatment; then culturing the leaf explants on a screening culture medium containing phosphinothricin; inducing anti-phosphinothricin resistance calluses formed on the explants to differentiate adventitious buds; continuously growing the adventitious buds to form test-tube plantlets; and identifying a transgenic plant carrying the target gene by using a PCR (Polymerase Chain Reaction) amplification method. By using the technical scheme provided by the invention, the target gene is directly introduced into tobacco cells; compared with agrobacterium tumefaciens-mediated genetic transformation, the measure for suppressing the growth of the agrobacterium tumefaciens is saved; compared with the genetic transformation of chemical methods such as polyethylene glycol, steps of separation and culture from protoplast are saved; and compared with the genetic transformation of physical methods such as gene gun, expensive instrument equipment is not required and the operation technology is simple.
Description
Technical field
The present invention relates to the technology of plant transgene in a Plant Biotechnology, in particular to a kind of method of tobacco vacuum infiltration direct gene transfer.
Background technology
The genetic transformation of vegetable cell is a key link of genetically engineered plant.From (1986) such as Abel, tobacco mosaic virus (TMV) (TMV) capsid protein cDNA is imported to tobacco cell, obtain after the transgenic tobacco plant of anti-TMV virus, successively developed many plant genetic transformation methods and technology.These genetic transforming methods and technology can be divided into two large classes, and the one, the genetic transformation of bio-carrier mediation, another kind of is non-bio-carrier, the genetic transformation that DNA directly imports.
The genetic transformation of bio-carrier mediation is mainly Agrobacterium tumefaciens (Agrobacterium tumefaciens, be called for short Agrobacterium) mediation genetic transformation, De Cleene and De Ley (1976) report, there are 138 sections, 588 belong to, and 1193 kinds of dicotyledonss and gymnosperm are to Agrobacterium sensitivity.Although Zeng Youyong DNA virus is (as cauliflower mosaic virus, CaMV) and RNA viruses (as tobacco mosaic virus (TMV), TMV) be bio-carrier transformed plant cells, but because of the defect of its host's limitation and transformed plant generation illness, do not re-use virus for bio-carrier transformed plant cells.The advantage of agriculture bacillus mediated genetic transformation method is that economy and operative technique are simple, easily in common laboratory, implements; Shortcoming is insensitive to monocotyledons, and need in screening culture medium, add the microbiotic that suppresses Agrobacterium growth, and separation and the growth of these microbiotic on transgenic cell has impact.
In the genetic transformation directly importing at DNA, be divided into again the method for chemistry and physics.Chemical process has polyoxyethylene glycol (PEG) method (Golovkin etc., 1993), liposome method (Antonelli etc., 1990), calcium phosphate precipitation (Hain etc., 1985), polycation methyl-sulphoxide (The polycation DMSO) technology (Antonelli and Stadler, 1989) and diethyl aminoethyl dextran (diethyl amino ethyl dextran, DEAE) method.In theory, chemical process is not subject to floristic restriction, but the acceptor using due to chemical process is protoplastis, generally need to set up protoplastis and cultivate and regeneration system, and the protoplast regeneration system of many plants is not yet set up, the application of these methods is hindered.Although Naqvi etc. (2012) immerse leaf mustard (Brassica juncea) hypocotyl in the damping fluid of the calcium phosphate nano particle that contains parcel pCAMBIA 1300 plasmid DNA, after incubation, screening obtains transfer-gen plant, but prepares the technical sophistication of nanoparticle.Physical method has microprojectile bombardment methods or particle bombardment (Klein etc., 1987), micro-injection (Crossway etc., 1986), electric shock perforation method (Fromm etc., 1985), silicon carbide fiber mediated transformation method (Kaeppler etc., 1992), laser mediated method (Guo etc., 1995) and pollen tube passage method (period-luminosity space, 1978).The advantage of physical method genetic transformation is not to be subject to floristic restriction, and shortcoming is the apparatus expensive that most methods requires, and operative technique is not easy to grasp; Do not need expensive instrument, the simple method of technology although wherein also have, as electric shock perforation method and pollen tube passage method, it is acceptor that the perforation method that shocks by electricity generally needs protoplastis, and its application is also subject to setting up the restriction of protoplastis cultivation and regeneration system; Pollen tube passage method needs accurately to grasp fertilization process and the transformation time thereof of acceptor, and the method only succeeds in transformation of Gossypium hirsutum L so far.
Summary of the invention
Technical problem to be solved by this invention is to overcome the deficiency that existing tobacco (Nicotiana tabacum) genetic transfoumation exists, in screening and culturing transgenic cell process, reduce the measure that suppresses Agrobacterium growth, thus the impact of the microbiotic of minimizing inhibition Agrobacterium growth on transgenic cell growth; Avoid utilizing the acceptor of protoplastis as genetic transformation; The expense that reduces institute's use instrument, easily carries out experimental implementation technology in common lab, and the method for a kind of tobacco vacuum infiltration direct gene transfer proposing.
Technical scheme provided by the present invention comprises the following steps:
(1) extract the restructuring pAHC25 plasmid that carries goal gene
The e. coli jm109 that contains restructuring pAHC25 plasmid is inoculated in the LB liquid nutrient medium containing 100mg/L penbritin to incubated overnight on the shaking table that is 190rpm at 37 DEG C, rotating speed; Alkali cracking method is extracted after restructuring pAHC25 plasmid DNA, and the concentration of plasmid DNA solution is adjusted into 0.5～1.5 μ g/ μ l.Enzyme is cut qualification restructuring pAHC25 plasmid.
(2) preculture spire explant
Clip spire from the potted plant seedling of tobacco of high 20cm left and right, carries out surface sterilization according to a conventional method, and the spire of surface sterilization is cut into 0.3～0.5cm
2leaf piece, be seeded in MS1 substratum and (in MS minimum medium, add 0.2mg/L 6-benzyl aminopurine, 0.02mg/L α-naphthaleneacetic acid, 3% sucrose and 0.65% agar; PH is 5.6～5.8) upper, be preculture 3～8 days under illumination in 16 hours and 8 hours dark conditions at temperature 26+1 DEG C, intensity of illumination 4000lx, photoperiod.
(3) prepare the leaf explant that genetic transformation uses
Getting the leaf piece after preculture, cut the leaf explant of long 3～5mm, the wide 1mm of leaf block edge left and right with scalper, is in the restructuring pAHC25 plasmid DNA solution of 0.5～1.5 μ g/ μ l by 20 leaf explants immersion 0.5ml, concentration.
(4) vacuum infiltration processing
Spire explant and restructuring pAHC25 plasmid DNA solution are placed in lens or glass moisture eliminator, it are connected with the vacuum suction mouth of vacuum pump with rubber hose; Switch on power, regulate negative pressure knob to make vacuum meter pointer move on to required negative pressure scale, under the condition that is 80kPa～20kPa at pressure, continue to process 6～15 minutes, then regulate negative pressure knob to make vacuum meter pointer be returned to zero-bit, be that air pressure in lens or glass moisture eliminator returns to atmospheric pressure state, under atmospheric pressure state, keep 30 seconds, regulate again negative pressure knob to make vacuum meter pointer move on to identical negative pressure scale, continue to process the identical time, then make the air pressure in lens or glass moisture eliminator return to atmospheric pressure state.Repeating identical vacuum infiltration processes 2～5 times.
(5) screening and culturing transgenic cell, induction of resistance callus form and plant regeneration
Spire explant after treatment vacuum infiltration is inoculated on MS1 substratum, under dark condition, cultivate 3 days, then spire explant is transferred to MS2 screening culture medium and (in MS1 substratum, add 1.5mg/L grass fourth phosphine, pH is 5.6～5.8) on, be illumination in 16 hours and under dark condition, cultivate for 8 hours in 26 ± 1 DEG C of temperature, intensity of illumination 4000lx, photoperiod, in Initial stage of culture 2 weeks every 3～4 days subcultures once, once every two weeks subcultures later; In MS screening culture medium, cultivate after 3～5 weeks, the Calli Differentiation indefinite bud of the anti-careless fourth phosphine resistance forming on explant, indefinite bud continued growth forms test-tube plantlet.
(6) extract the total DNA of Ye and pcr amplification qualification transfer-gen plant
Extract the total DNA of Ye of regeneration plant by CTAB method, with goal gene sequences Design sense primer and antisense primer; Taking the total DNA of Ye as template, carry out pcr amplification with the primer of designed goal gene fragment, according to pcr amplification product qualification transfer-gen plant.
Compared with the prior art, tool of the present invention has the following advantages:
(1) be conducive to transgenic cell division and growth.The method of tobacco vacuum infiltration direct gene transfer belongs to the genetic transforming method of abiotic carrier, compared with agriculture bacillus mediated genetic transformation, need in screening culture medium, not add the microbiotic that suppresses Agrobacterium growth, can only in MS screening culture medium, add careless fourth phosphine, screening transgenic cell, is conducive to division and the growth of transgenic cell.
(2) easily obtain transgenic regenerated plant.The acceptor that the method for tobacco vacuum infiltration direct gene transfer is used is Tissues of Tobacco or the cell of isolated culture, instead of its protoplastis.Compared with cultivating with tobacco protoplast, tobacco leaf tissue or cell are more easily induced plant regeneration.Therefore,, compared with chemical process importing foreign DNA, present method easily obtains transgenic regenerated plant.
(3) required equipment is simple, and operative technique is easily grasped.The method of tobacco vacuum infiltration direct gene transfer can be included into again physical method class.Concerning other physical methods such as particle bombardment, silicon carbide fiber mediated transformation method and laser mediated method, need complicated and expensive plant and instrument, as particle gun, microscope, Laser emission instrument etc., and operative technique is not easy to grasp.The major equipment that present method needs is vacuum pump, and its price is low, easily operation, and do not need to prepare the complicated technical finesses such as foreign DNA particulate.
Brief description of the drawings
Fig. 1 is the product that Sac I and Xma I double digestion carry the restructuring pAHC25 plasmid of DFR gene;
M:DNA molecular weight marker; Two DNA fragmentations that produce after swimming lane 1:Sac I and Xma I double digestion restructuring pAHC25 plasmid, the fragment (7892bp) that molecular weight is larger is pAHC25 plasmid DNA fragment (swimming lane top), and the fragment (1054bp) of molecular weight is the DFR gene fragment (swimming lane below) that two ends have Sac I and Xma I part base sequence
Fig. 2 cultivates the callus and the regeneration plant (right side) thereof that on the explant of the contrast explant (left side) of 45 days and vacuum infiltration processing, form in MS2 screening culture medium
Fig. 3 is pcr amplification method qualification transfer-gen plant;
WT: non-transgenic plant; P25: the restructuring pAHC25 plasmid that carries DFR gene; H
2o: without DNA profiling, add equivalent redistilled water in reaction solution; 1～7: regeneration plant numbering, regeneration plant 1,2,5 and 7 has the 388bp DNA fragmentation identical with restructuring pAHC25 plasmid, and from non-transgenic contrast and regeneration plant 3,4 and 6, and without not occurring 388bp DNA fragmentation in the reaction solution of template DNA, show that regeneration plant 1,2,5 and 7 is transfer-gen plants, and regeneration plant 3,4 and 6 is false-positive non-transgenic plant; M:DNA molecular weight marker, unit: bp
Embodiment
Embodiment 1, extracts the restructuring pAHC25 plasmid that carries goal gene DFR
(1) cultivate e. coli jm109 cell
The goal gene that restructuring pAHC25 plasmid (8938bp) carries is flavanonol reductase gene (dihydroflavonol4-reductase, DFR) (1044bp; GenBank accession number: AY323494), be cloned in peace ancestral and spend the spathe cDNA of (Anthurium andreanumHort) flame kind, the base sequence of Sac I and Xma I restriction enzyme site is added at two ends; Glycerine after restructuring pAHC25 plasmid transformation escherichia coli JM109 cell is kept in-20 DEG C of refrigerators.
Get that glycerine at-20 DEG C is preserved, containing the JM109 cell of restructuring pAHC25 plasmid, be coated on LB solid medium (containing 100mg/L penbritin, lower with) upper, 37 DEG C of cultivation (lower same).Picking list bacterium colony is in the LB of 2ml liquid nutrient medium, and incubated overnight on the shaking table that is 190rpm at rotating speed, then draws 0.5ml bacterium liquid in 50ml LB liquid nutrient medium, incubated overnight on the shaking table of same rotating speed.
(2) alkali cracking method extraction restructuring pAHC25 plasmid and enzyme are cut qualification
Adopt conventional alkali cracking method to extract restructuring pAHC25 plasmid, obtain after the restructuring pAHC25 plasmid of purifying, measure the concentration of plasmid DNA solution with NanoVue ultramicron ultraviolet spectrophotometer, the concentration of plasmid DNA solution is adjusted into 1.0 μ g/ μ l.
In 20 μ l endonuclease reaction systems, add 0.5 μ g restructuring pAHC25 plasmid, 15U Sac I, 15U Xma I and double digestion Tango
tMdamping fluid, reacts 6 hours in 37 DEG C of water-baths.Get 5 μ l enzymes and cut liquid, containing electrophoresis in 1% agarose gel plate of ethidium bromide, TAE electrophoretic buffer, voltage 4V/cm.Agarose gel plate is placed under gel imaging instrument, observes the DAN fragment (Fig. 1) that two sizes are 7892bp and 1054bp, show that the goal gene that in testing, the restructuring pAHC25 plasmid of use carries is DFR gene.
Embodiment 2, tobacco genetic transformation
(1) preculture spire explant
Taking tobacco K326 kind as material, clip spire from the potted plant seedling of high 20cm left and right, cleans and dries with tap water.On Bechtop, blade is put into aseptic beaker, with 70% alcohol-pickled blade 30 seconds, then receive solution soaking blade with 10% hypochlorous acid, surface sterilization 6 minutes, sterile water wash 5 times.Then, aseptic blade being cut into size is 0.3～0.5cm
2leaf piece, (MS minimum medium adds 0.2mg/L 6-benzyl aminopurine, 0.02mg/L α-naphthaleneacetic acid, 3% sucrose and 0.65% agar to be inoculated into MS1 substratum; PH is 5.6～5.8) upper, be under illumination in 16 hours and 8 hours dark conditions in 26 ± 1 DEG C of temperature, intensity of illumination 4000lx, photoperiod, cultivate 6 days.
(2) prepare the leaf explant that genetic transformation uses
Get the leaf piece after preculture, cut the leaf explant of long 3～5mm, the wide 1mm of leaf block edge left and right with scalper; In the flat finger-type Glass tubing (dactylethrae) that the restructuring pAHC25 plasmid DNA solution of drawing 0.5ml, concentration and be 1.0 μ g/ μ l is 1.2cm in internal diameter, 20 leaf explants are immersed to plasmid DNA solution, with sealed membrane (Shanghai Jia Feng garden supplies company limited) the sealing mouth of pipe that has breather hole, dactylethrae is placed in 100ml beaker.
(3) vacuum infiltration processing
The escape pipe of the pallet side of the vacuum suction pipe of AP-9925 oilless vacuum pump (Tianjin Ao Tesaiensi Instrument Ltd.) and lens below is connected with rubber hose, the beaker of placement dactylethrae is placed in to pallet central authorities, cover lens.Switch on power, (in lens, pressure is 30kPa) timing while regulating negative pressure knob to make move on to-70kPa of vacuum meter pointer, continue to process 10min, then regulate negative pressure knob to make vacuum meter pointer be returned to zero-bit, be that air pressure in lens returns to atmospheric pressure state, under atmospheric pressure state, keep 30 seconds; Timing while regulating again negative pressure knob to make move on to-70kPa of vacuum meter pointer, continues to process 10min, then regulates negative pressure knob to make vacuum meter pointer be returned to zero-bit, makes the pressure in lens return to atmospheric pressure state, powered-down.Vacuum infiltration is processed and is repeated 2 times.
(4) screening and culturing transgenic cell, induction of resistance callus form and plant regeneration
Spire explant after treatment vacuum infiltration is inoculated on MS1 substratum, under the dark condition of 26 ± 1 DEG C of temperature, cultivate 3 days, then (MS1 substratum adds 1.5mg/L grass fourth phosphine spire explant to be transferred to MS2 screening culture medium, pH is 5.6～5.8) on, be illumination in 16 hours and under dark condition, cultivate for 8 hours in 26 ± 1 DEG C of temperature, intensity of illumination 4000lx, photoperiod, in Initial stage of culture 2 weeks every 3～4 days subcultures once, once every two weeks subcultures later; On MS2 screening base, cultivate after 3～5 weeks, the Calli Differentiation indefinite bud of the anti-careless fourth phosphine resistance forming on explant, indefinite bud continued growth forms test-tube plantlet (Fig. 2).According to the explant number of inoculation explant number and generation resistant calli, under the condition that is 30kPa at pressure, the transformation efficiency of tobacco vacuum infiltration direct gene transfer is 13.75%.
Embodiment 3, pcr amplification qualification transfer-gen plant
(1) CTAB method is extracted the total DNA of blade
Get 0.05～0.1g regeneration plant blade, be placed in 1.5ml centrifuge tube, after immersion liquid nitrogen, pulverize fast, add the CTAB extracting solution of 0.5ml, 65 DEG C of preheatings, put upside down and mix, in 65 DEG C of water-baths, extract 30min; According to conventional CTAB method purify DNA, finally the DNA of purifying is dissolved in 20～30 μ l TE damping fluids again, gets 2 μ l DNA solution electrophoresis, estimate the concentration of DNA solution.
(2) pcr amplification
With DFR gene order design sense primer 5 '-AACCGCCTGACCCTCTGGAA-3 ' and antisense primer 5 '-CGATGTTGTTCTTCTCCGCG-3 ', amplified fragments size is 388bp.In the pcr amplification reaction system of 50 μ l, add the total DNA of 100ng Ye or 20ng restructuring pAHC25 plasmid DNA, 20 ρ mol/L justice and antisense primers, 200 μ mol/L dNTP, 2U archaeal dna polymerase and 5 μ l 10 × DNA polymerase reaction damping fluids.Pcr amplification condition is: 94 DEG C of denaturations 4 minutes, and then 94 DEG C of sex change 1 minute, 55 DEG C of annealing 1 minute, 72 DEG C are extended 2 minutes, and 35 circulations, finally 72 DEG C of downward-extensions 10 minutes.
Pcr amplification product is containing electrophoresis in 1.2% agarose gel plate of ethidium bromide, TAE electrophoretic buffer, voltage 4V/cm.Electrophoresis result shows (Fig. 3), and the PCR product of regeneration plant 1,2,5 and 7 plant has the 388bp DNA fragmentation the same with restructuring pAHC25 plasmid, shows that in the genome of DFR gene integration to 1,2,5 and 7 plant, they are transfer-gen plants; And the PCR product of regeneration plant 3,4 and 6 does not have 388bp DNA fragmentation, show that they are false-positive non-transgenic plant.
Claims (1)
1. a method for tobacco vacuum infiltration direct gene transfer, is characterized in that, clip spire from the potted plant seedling of high 20cm left and right, and after surface sterilization, being cut into size is 0.3～0.5cm
2leaf piece, be inoculated on MS substratum, be illumination in 16 hours and under dark condition, cultivate after 3～8 days for 8 hours in 26 ± 1 DEG C of temperature, intensity of illumination 4000lx, photoperiod, cut the leaf explant of long 3～5mm, the wide 1mm of leaf block edge left and right with scalper, it is in the pAHC25 plasmid DNA solution of 0.5～1.5 μ g/ μ l that this spire explant is immersed to concentration, under the condition that is 80kPa～20kPa at pressure, continue to keep 6～15 minutes, then under atmospheric pressure state, keep 30 seconds, repeat identical vacuum infiltration and process 2～5 times.
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| CN104789518A (en) * | 2015-04-03 | 2015-07-22 | 北京吉诺沃生物科技有限公司 | Efficient separation, transformation and regeneration system for tobacco protoplast |
| CN109385449A (en) * | 2017-08-10 | 2019-02-26 | 北京睿诚海汇健康科技有限公司 | Application of the plant as host in expression of insulin |
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