CN101914571A - Method for cleaning DNA transformation plant without choosing marker gene - Google Patents
Method for cleaning DNA transformation plant without choosing marker gene Download PDFInfo
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Abstract
The invention discloses a method for cleaning a DNA transformation plant without choosing a marker gene. The method comprises the following steps of: 1), preparing a target gene expression frame: adopting an appropriate restriction enzyme to cut a constructed target gene expression frame DNA fragment from a plasmid vector according to the sequence character of the plasmid vector on which the target gene expression frame to be transferred is, and obtaining the target gene expression frame DNA by electrophoresing, separating, tapping and reclaiming a gel; 2), preparing a DNA particle vector suspension; 3), bombarding plant callus to transform the plant callus; 4), regenerating the plant; and 5), choosing transgenic plants. When the method is adopted to perform plant transgenosis, the influences of the selected marker gene and superfluous DNA sequences such as the plasmid vector frame DNA sequence on the transgenic plants and genetically modified foods can be overcome fundamentally, so that the transgenic plants without other superfluous DNAs except for the target gene expression frame are obtained.
Description
Technical field
The present invention relates to a kind of method of utilizing particle gun mediate foreign gene expression cassette (promotor-gene coding region-terminator) to transform plant, this method need not used any selectable marker gene, the transfer-gen plant that obtains does not contain any unnecessary rubbish DNA except that goal gene, be a kind of cleaning DNA transformation technology (Clean DNA transformation).Belong to field of biology, specially refer to genetic engineering technique.
Background technology
Since nineteen eighty-three first, routine transgenic plant were born, the plant gene leading-in technique became better and approaching perfection day by day, and has formed the plant gene conversion system based on particle gun and Agrobacterium two big transformation technologies.Tens of kinds of transgenic plant that comprise staple crops such as soybean, corn, rape, cotton, potato, paddy rice enter field experiment even commercialization plantation in a plurality of countries.Meanwhile, transgenic plant potential ecotope harm and by and the genetic modification food that comes to the mankind's edible safety, become the significant obstacle that transgenic crop is applied.The root that causes the transgenic plant safety problem has two: the resistance selectable marker gene in (1) transgenic plant.In the Plant Transformation process, because present gene leading-in technique can only import foreign gene in a few cell, transform and non-transformed cell so generally adopt selectable marker gene to distinguish, when importing goal gene, must introduce selectable marker gene and could realize effective conversion vegetable cell.Wherein giving the resistance selectable marker gene of transformant to weedicide or antibiotics resistance (resistant selected marker gene) and be and generally using in the Plant Transformation also is effective choice marker gene, after the vegetable cell conversion processing, screen and obtain transformant by in substratum, adding corresponding weedicide or microbiotic pair cell.Behind the transgenic plant regeneration, these resistance selectable marker genes no longer have using value usually, but herbicide resistance gene may pass to nearly edge species by genetic drift, produce malignant weed, and ecotope is worked the mischief; Antibiotics resistance gene may by genetic drift be transferred to soil microorganisms or by food chain transport to the human intestine microorganism, make the people produce resistance to corresponding microbiotic, influence human health.Therefore the resistance selectable marker gene is the major reason that causes the transgenic plant safety problem, has become the serious hindrance that the transgenic plant commercialization is used.(2) transform plasmid vector framework dna sequence dna.Because plant genetic generally uses complete plasmid when transforming, plasmid vector framework sequence is usually along with foreign gene is incorporated in the acceptor gene group together.Existing big quantity research evidence shows, at the direct integrate phenomenon ubiquity of carrier frame sequence in the transfer-gen plant of conversion system mediated transformation such as the transfer-gen plant of agrobacterium mediation converted and particle bombardment.Plasmid vector framework sequence contains inevitably and allows gene construct in intestinal bacteria or the antibiotics resistance selective marker and the replication orgin of cloning in the Agrobacterium, has potentially dangerous (the Fu Xiangdong that in external environment, escapes, et al.Linear transgene constructs lacking vector backbone sequences generate low-copy-number transgenic plants with simple integration patterns.Transgenic Research, 2000,9:11-19).
Particle bombardment can (only comprise promotor with the exogenous gene expression frame of removing plasmid vector framework sequence, the gene open reading frame, terminator) imports Plant Genome, fundamentally eliminate the disadvantageous effect of plasmid vector framework sequence, realize that cleaning DNA transforms (clean DNA transformation), existing a few studies person utilizes the cleaning DNA transformation technology to obtain paddy rice both at home and abroad at present, potato, grape vine, wheat, transfer-gen plants such as corn (Altpeter F et al.Particle bombardment and the genetic enhancement of crops:myths and realities.Mol.Breeding.2005,15:305-327), and research adopts the cotransformation method to obtain the transfer-gen plant (ZhaoY of marker-free, et al.Co-transformation of gene expression cassettes via particle bombardment to generate safe transgenic plant without any unwanted DNA.In Vitro Cellular andDevelopmental Biology-Plant, 2007,43 (4): 328-334; Shiva Prakash N et al.Marker-free transgenic corn plant production trough co-bombardment.Plant Cell Reports, 2009,28:1665-1668), but all used the resistance selectable marker gene in these cleaning DNA transformation systems, conversion process will could obtain the transfer-gen plant of non-resistant marker gene through the rejecting of the screening of transformant and resistant maker gene, program is very loaded down with trivial details, and the frequency of the transfer-gen plant of acquisition resistant maker gene is also very low.
Malnoy Michael etc. has reported method (Michael M etal.Genetic transformation of apple (Malus x domestica) the without use of a selectable marker gene.Tree Genetics ﹠amp that does not adopt selectable marker gene to transform apple tree recently; Genomics, 2010,6:423-433), be conversion method for agrobacterium but this system adopts, defective is to avoid the carrier frame sequence of complete plasmid of agrobacterium mediation converted in the genomic integration of transfer-gen plant.
The good grade of Amway has been invented a kind of plant transgenic method of marker-free, and (Amway is good etc., a kind of plant transgenic method of marker-free.Patent disclosure day: on July 3rd, 2002, patent publication No. CN 1356389A), but this method is examined the conserved sequence of DNA and the homology exchange reorganization realization gene transformation between the plant nuclear DNA by the plant that is arranged on conversion fragment two ends, the defective of this method is: must introduce extra DNA of plants conserved sequence for two sections in the transfering DNA fragment except goal gene, these extra DNA of plants conserved sequences are a kind of extra unnecessary rubbish DNA for the transformation receptor plant not only, and because the foundation of this promotion plant transformed DNA conserved sequence setting is the conserved sequence of the structure gene both sides of higher plant proteins encoded, when the external source goal gene when homologous recombination exchange is incorporated into Plant Genome, can destroy the original structure gene of recipient plant inevitably, cause the generation transgenation of transgene receptor plant and cause new security risk.
Summary of the invention
The technical problem to be solved in the present invention provides a kind of method that does not need the cleaning DNA transformation plant of any selectable marker gene, this method adopts particle bombardment directly the destination gene expression frame to be imported vegetable cell, implementation process does not need to adopt selectable marker gene, does not need to introduce the sequence that any extra promotion DNA transforms yet; Utilize the technology of the present invention to carry out plant transgene, can fundamentally solve of the influence of DNA redundant sequences such as selectable marker gene and plasmid vector framework dna sequence dna, obtain to remove the transfer-gen plant that the destination gene expression frame does not have other DNA redundants transgenic plant and genetically modified food.
In order to solve the problems of the technologies described above, the invention provides a kind of method of cleaning DNA transformation plant of not choosing marker gene, may further comprise the steps successively:
1), preparation destination gene expression frame:
Sequence signature according to the plasmid vector at destination gene expression frame to be imported place adopts suitable restriction enzyme with the destination gene expression frame that builds
Dna fragmentation cuts down from plasmid vector, separates rubber tapping by gel electrophoresis and reclaims, and obtains destination gene expression frame DNA, as the foreign DNA of particle gun bombardment vegetable cell;
2), the little missile-borne body of preparation DNA suspension:
Get bronze suspension 30～50 μ l of 600mg bronze (Φ 1.0 μ m)/ml, add the CaCl of 2.5mol/L
2Solution 45～55 μ L and 0.1mol/L spermidine solution 15～25 μ L behind the concussion mixing, add and contain the solution that the DNA total amount is the described destination gene expression frame DNA of 2 μ g, vibration, centrifugal, abandon supernatant, get the little bullet precipitation of DNA thereby make DNA be deposited to the bronze particle surface;
The little bullet precipitation of DNA is washed with dehydrated alcohol, vibration, centrifugal, abandon supernatant; Add dehydrated alcohol 60～80 μ l again, get the little missile-borne body of DNA suspension;
3), the plant callus bombardment transforms:
Utilize particle gun that the little missile-borne body of above-mentioned DNA suspension is bombarded in the plant embryo callus body, the callus after the bombardment leaves standstill dark cultivation, so that make the bombardment cellular-restoring;
4), plant regeneration:
With above-mentioned steps 3) the dark plant callus that recovered of cultivating is directly transferred to presorting on the pre-differentiation substratum after the bombardment of gained, then breaks up the acquisition regeneration plant;
5), the screening of transfer-gen plant.
Improvement as the method for the cleaning DNA transformation plant of not choosing marker gene of the present invention, the plant callus bombardment of step 3) is converted into: the plant embryo callus of inducing culture oozes at height earlier before bombardment and places the dark 4～5h of cultivation on the substratum according to a conventional method, then carry out the particle gun bombardment, the callus of every culture dish bombards 2 rifles, and the step 2 of 1/6 volume is set in every rifle) the little missile-borne body of the DNA suspension of gained (BioRad PDS 1000/He particle gun bombardment parameter is by documents and materials); Callus after the bombardment leaves standstill dark cultivation 16～20h, so that make the bombardment cellular-restoring.
Further improvements in methods as the cleaning DNA transformation plant of not choosing marker gene of the present invention: the particle gun in the step 3) is a Biolistic PDS 1000/He particle gun.
Further improvements in methods as the cleaning DNA transformation plant of not choosing marker gene of the present invention, the screening of the transfer-gen plant of step 5) is: with the regeneration plant of step 4) gained directly on molecular level and the enterprising row filter of phenotypic level, thereby obtain transforming successful plant.
Further improvements in methods as the cleaning DNA transformation plant of not choosing marker gene of the present invention: plant is a paddy rice.
The present invention uses Biolistic PDS 1000/He particle gun device (BioRad) as the conversion instrument.Step 2) the little missile-borne preparation of DNA specifically can be: get bronze suspension 30～50 μ l of 600mg bronze (Φ 1.0 μ m)/ml, add the CaCl of 2.5mol/L
2Solution 50 μ L and 0.1mol/L spermidine solution 20 μ L, slight concussion mixing, the above-mentioned purpose gene expression frame dna solution (the DNA total amount is 2 μ g) that adds an amount of volume, vibration 3min mediates, the centrifugal 10min of 10000r/min, abandon supernatant, allow DNA be deposited to the bronze particle surface and get the little bullet precipitation of DNA.Add the little bullet of dehydrated alcohol 250 μ l washing DNA and precipitate, the centrifugal 3min of vibration concussion back 10000r/min that mediates abandons supernatant, repeats above-mentioned absolute ethanol washing once, adds dehydrated alcohol 80 μ l, gets the little missile-borne body of DNA suspension.During bombardment plant callus cell, every rifle needs the about 10 μ l of the little missile-borne body of DNA suspension, and the little missile-borne body of above-mentioned DNA suspension can bombard 6 rifles, because ethanol is volatile, so consider that the gram constant volume is the volume of 80 μ l.
The method that does not need the cleaning DNA transformation plant of any selectable marker gene of the present invention, owing to do not use any marker gene, do not need to carry out any screening behind the particle gun bombardment transformant yet, directly carry out pre-differentiation of cell and differentiation after the conversion processing and obtain regeneration plant, this can not only eliminate the inhibition and the interference of the differentiation of Screening Treatment pair cell fully, for example, differentiation produces a large amount of albefaction seedlings behind the use kantlex Screening of Rice cell.And shortened the transformation period greatly, and the pair cell screening needs two-wheeled after the general Plant Transformation, the every wheel about 15-35 of time days, and like this, the present invention makes the Plant Transformation cycle shorten about 30-70 days.
The method of the cleaning DNA transformation plant of not choosing marker gene of the present invention, it adopts particle bombardment directly the destination gene expression frame to be imported vegetable cell, implementation process does not need to adopt selectable marker gene, does not need to introduce the sequence that any extra promotion DNA transforms yet; The transfer-gen plant that is obtained does not have other unnecessary rubbish DNA except that containing the destination gene expression frame.Utilize the technology of the present invention to carry out plant transgene, can fundamentally solve of the influence of DNA redundant sequences such as selectable marker gene and plasmid vector framework dna sequence dna, obtain to remove the transfer-gen plant that the destination gene expression frame does not have other DNA redundants transgenic plant and genetically modified food.The present invention does not have particular restriction and requirement to the foreign gene that is transformed, and is applicable to the transformable plant acceptor material of all particle bombardments.
In the invention process, the contriver is that example has been done following simultaneous test with the rice transformation:
Test 1: cleaning DNA transformation plant method (the marker-free cleanDNA transformation that does not need any selectable marker gene of the present invention, be called for short MFCT) and general conversion plant method (Selection transformation, changing effect contrast ST) of using the resistant maker gene screening.Adopting goal gene is 5-enol pyruvic acid shikimic acid-3-phosphate synthase (5-enolpyruvlyshikimate-3-phyosphate synthase is called for short EPSPS) gene, and its expression can be given the resistance of paddy rice to glyphosate herbicidal; The resistance selectable marker gene that adopts is that kalamycin resistance (Kanr) gene is a neomycin phosphotransferase II gene (nptII), be most widely used resistance selectable marker gene in plant gene transforms at present, its expression can be given the resistance of the rice cell of conversion to microbiotic G418.In EPSPS gene and the gene constructed plant expression plasmid p1501 of Kanr, particular content and step are as follows:
1), the preparation of destination gene expression frame:
MFCT group: adopt two kinds of restriction enzyme cuttings of HindIII and EcoRI plasmid p1501, reclaim through gel electrophoresis and obtain EPSPS destination gene expression frame (comprising CaMV35S promotor, EPSPS coding region and no terminator) dna fragmentation, as shown in Figure 1.The dna fragmentation that recovery obtains is dissolved in the sterile pure water, and concentration is adjusted into 200ng/ μ l.
ST group: adopt two kinds of restriction enzyme cuttings of HindIII and SacII plasmid p1501, reclaim the dna fragmentation that obtains EPSPS destination gene expression frame (comprising CaMV35S promotor, EPSPS coding region and no terminator) and kalamycin resistance (nptII) gene expression frame (comprising CaMV35S promotor, nptII coding region and CaMV35S terminator) through gel electrophoresis, as shown in Figure 2.Recovery obtains dna fragmentation and is dissolved in the sterile pure water, and concentration is adjusted into 200ng/ μ l.
2) preparation of the little missile-borne body of DNA suspension:
MFCT group: get the bronze suspension 40 μ l of 600mg bronze (Φ 1.0 μ m)/ml, add the CaCl of 2.5mol/L
2Solution 50 μ L and 0.1mol/L spermidine solution 20 μ L, slight concussion mixing, add 10 μ l above-mentioned steps 1) the EPSPS destination gene expression frame dna solution (200ng/ μ l) of gained, vibration 3min mediates, the centrifugal 10min of 10000r/min, abandon supernatant, allow DNA be deposited to the bronze particle surface and get the little bullet precipitation of DNA, add dehydrated alcohol 250 μ l washing precipitations, the centrifugal 3min of back 10000r/min is shaken in the vibration of mediating, and abandons supernatant, repeats above-mentioned absolute ethanol washing once, add dehydrated alcohol 80 μ l, get the little missile-borne body of DNA suspension.During bombardment rice callus histocyte, every rifle needs the about 10 μ l of the little missile-borne body of DNA suspension, the little missile-borne body of above-mentioned DNA suspension can bombard 6 rifles, because ethanol is volatile, so the above-mentioned steps constant volume is 80 μ l volumes when guaranteeing the bombardment of following step (thereby have an appointment 60 μ l).
The ST group: the little missile-borne body of DNA suspension preparation method is identical with the MFCT group, and just dna solution makes the dna fragmentation of the above-mentioned EPSPS of comprising destination gene expression frame and nptII gene expression frame into.
3) bombardment of rice callus tissue transforms:
Induce according to a conventional method with the preceding height that inserts of the EMBRYO IN RICE callus bombardment of succeeding transfer culture and ooze substratum (NB minimum medium+2,4-D 2mg/L+ sorbyl alcohol 47g/L+ N.F,USP MANNITOL 47g/L), the quantity of callus and putting position particle gun routinely transform requirement, and 28 ℃ of dark cultivations are carried out the particle gun bombardment after 4 hours.The little missile-borne body of DNA suspension is transformed the mature embryo callus of japonica rice variety by the bombardment of BioRad PDS 1000/He particle gun, every ware callus bombards 2 rifles, callus after the bombardment leaves standstill dark cultivation 16h for 28 ℃, so that make the bombardment cellular-restoring, after this is divided into following two groups.
MFCT group: adopt MFCT method for transformation of the present invention, callus after the bombardment processing is transferred to last 28 ℃ of dark cultivations of pre-differentiation substratum (NB minimum medium+2mg/L 6-BA+1mg/L NAA+5mg/L ABA) to be broken up in 10 days in advance, and then be transformed into the last 28 ℃ of illumination cultivation of division culture medium (NB minimum medium+2mg/L 6-BA+1mg/L NAA) and carry out plant differentiation, 15-20 days, obtain regeneration plant.
ST group: screening culture medium (NB minimum medium+2 of the callus after the bombardment processing being transferred to the G418 that adds 300mg/L, the G418 of 4-D 2mg/L+300mg/L) carries out the two-wheeled screening on, the every wheel about 20 days, about totally 40 days, the resistant calli that the screening back obtains breaks up in advance and breaks up, method is organized with MFCT, thereby obtains regeneration plant.
4), adopt the PCR method that the reuse water rice plants is carried out molecular screening:
Two groups of transformation tissue cultures of rice plant organize to(for) above-mentioned MFCT group and ST all carries out PCR and screens.The pcr amplification primer designs at the no terminator sequence in the EPSPS destination gene expression frame, P1:GAA TCC TGT TGC CGG TCT TG (SEQID NO:1); P2:TTA TCC TAG TTT GCG CGC TA (SEQ ID NO:2).PCR reaction cycle parameter: 94 ℃ of pre-sex change 3min; Later 94 ℃ of sex change 40s of loop parameter, 59 ℃ of annealing 30s; 72 ℃ are extended 30s; Totally 35 circulations, last 72 ℃ are extended 3min.Agarose gel electrophoresis detects the successful plant of conversion and amplifies special 180kb band, and this 180kb specific band is document (Zhao Yan as has been publicly.The PCR of transgenic rice processed food detects investigative technique.China's grain and oil journal, 2006,21 (3): inform 198-201).Unconverted successful plant can not amplify special 180kb band; As shown in Figure 3.Simultaneously adopt blade to smear 0.3% glyphosate herbicidal in seedling stage and carry out the Herbicid resistant phenotypic evaluation, the PCR positive+glyphosate resistance plant is accredited as transfer-gen plant.
Concrete outcome sees Table 1:
Table 1, employing MFCT method of the present invention and conventional ST method compare the changing effect that the EPSPS gene imports paddy rice
Annotate: green seedling differentiation rate=(the green seedling number/bombardment callus number of regenerating) * 100%
White seedling differentiation rate=(the white seedling number/bombardment callus number of regenerating) * 100%
Transformation efficiency=(PCR positive plant number/bombardment callus number) * 100%
Testing data from above-mentioned table 1 is learnt and is proved: (1) MFCT method for transformation of the present invention, and the callus differentiation capability height after the bombardment, the green seedling differentiation rate of military fortune stalk and two kinds of P307 is respectively 85.7% and 89.8%, the albefaction seedling do not occur; And after ST contrast promptly passes through the screening two-wheeled of G418 of 300mg/L, the callus differentiation capability seriously reduces and differentiates the albefaction seedling, the green seedling differentiation rate of wherein military fortune stalk and two kinds of P307 is respectively 5.3% and 6.0%, and the white seedling differentiation rate of the two is respectively 0.8% and 1.2%.Confirmed that MFCT method for transformation of the present invention can eliminate in traditional method for transformation selective agent to the inhibition and the disadvantageous effect of callus differentiation capability.(2) MFCT method for transformation of the present invention is compared transformation efficiency with traditional ST method for transformation and is significantly improved, and in bombardment callus number, the EPSPS goal gene transformation efficiency of the military fortune stalk of MFCT group and two kinds of P307 has reached 40.5% and 37.0% respectively; And the EPSPS goal gene transformation efficiency of the military fortune stalk of ST group and two kinds of P307 only is respectively 4.5% and 6.0%.(3) MFCT method for transformation of the present invention is compared the transformation period shortening with traditional ST method for transformation.Because MFCT method for transformation of the present invention does not use any selection markers gene, do not need through screening, make from the bombardment callus to the time that obtains the rice regeneration plant and shortened about 40 days.
The invention belongs to has breakthrough innovation on thinking because present transgenic technology think always transform in selectable marker gene be necessary, otherwise because the low and very difficult acquisition transfer-gen plant of transformation frequency; And the inventive method not need fully to find marker gene breakthroughly, has removed complicated screening step and but can obtain better changing effect.
In sum, the present invention does not need to use any other to promote the dna sequence dna element that transforms without any need for selectable marker gene yet, need not introduce any DNA redundant in the conversion process except that the destination gene expression frame, and schedule of operation is simple.The transfer-gen plant that obtains does not contain any unnecessary rubbish DNA except that containing the destination gene expression frame, realization transforms the cleaning DNA of vegetable cell, fundamentally eliminated antibiotic medicine, herbicide resistance gene and carrier backbone sequences disadvantageous effect, guaranteed the security of transfer-gen plant transgenic plant and genetically modified food.Transformation efficiency of the present invention is higher, is a kind of novel method of utilizing the particle gun mediated method to realize vegetable cell is transformed fast.The present invention does not have particular restriction and requirement to the foreign gene that is transformed, and is applicable to the transformable plant acceptor material of all particle bombardments.
Description of drawings
Below in conjunction with accompanying drawing the specific embodiment of the present invention is described in further detail.
Fig. 1 is an EPSPS destination gene expression mount structure synoptic diagram.
Fig. 2 is EPSPS destination gene expression frame and kalamycin resistance gene (nptII) expression cassette structural representation.
Fig. 3 is that agarose gel electrophoresis detects the no terminator pcr amplification result in the rice plant;
Among Fig. 3: M.DNA molecular weight marker; 1.nos positive control; 0.nos negative control; 2-21. regeneration plant, wherein 7, No. 14 plant are the non-transgenic plant, and 2,3,4,5,6,8,9,10,11,12,13,15,16,17,18,19,20, No. 21 plant are transfer-gen plant.
Fig. 4 is a gus gene expression cassette structural representation.
Fig. 5 forwards height to ooze treating on the substratum and bombard the rice callus tissue.
Fig. 6 is that the rice callus tissue differentiation went out regeneration plant after bombardment transformed.
Fig. 7 is the PCR product photo that obtains agarose gel electrophoresis detection gus gene in the paddy rice transfer-gen plant;
Among Fig. 7: M.DNA molecular weight marker; 0.GUS negative control; 1.GUS positive control; 2-23. regeneration plant, wherein 2,9,12,15,16,19,20, No. 21 plant are the non-transgenic plant, and 3,4,5,6,7,8,10,11,13,14,17,18,22,23 is transfer-gen plant.
Fig. 8 obtains to change gus gene rice plant blade GUS active coloring to show gus gene normal expression figure;
The left figure of Fig. 8 is the transgenic positive plant leaf, shows blue; Right figure is the non-transgenic negative control, does not show blue.
Embodiment
The present invention has realized that with aforesaid method (β-glucuronidase, the GUS) conversion of gene pairs paddy rice has successfully obtained the transgenic positive plant to beta-galactosidase enzymes.Employed culture plant cell method, gene gun conversion method and molecular biology method are known technology in following examples, and detailed explanation is all arranged in pertinent literature and reference book.
The preparation of embodiment 1, goal gene GUS expression cassette and cleaning DNA transform little missile-borne body suspension preparation:
Before bombardment, prepare, get bronze (Φ 1.0 μ m) the suspension 40 μ l of 600mg/ml, add the CaCl of 2.5mol/L
2Solution 50 μ L and 0.1mol/L spermidine solution 20 μ L, slight concussion mixing, the above-mentioned 200ng/ μ l goal gene GUS expression cassette dna solution that adds 10 μ l, making the DNA total amount is 2 μ g, vibration 3min mediates, the centrifugal 10min of 10000r/min, abandon supernatant, allow DNA be deposited to the bronze particle surface and get the little bullet precipitation of DNA, add dehydrated alcohol 250 μ l washing precipitations, the centrifugal 3min of vibration concussion back 10000r/min mediates, abandon supernatant, repeat above-mentioned absolute ethanol washing once, add dehydrated alcohol 80 μ l, this is the little missile-borne body of a DNA suspension.During bombardment plant callus cell, every rifle needs the little missile-borne body of DNA suspension 10 μ l, and the little missile-borne body of above-mentioned DNA suspension can bombard 6 rifles, because ethanol is volatile, so the above-mentioned steps constant volume is 80 μ l volumes.The little missile-borne body of the DNA for preparing suspension bombards immediately.
Bombardment transforms to the rice callus tissue for embodiment 2, the little bullet of GUS expression cassette fragment cleaning DNA:
The inducing and cultivating of step 1, rice callus tissue: select the full fine mature embryo seed of japonica rice variety Japan in the sterilization Erlenmeyer flask, with the alcohol submergence of 70% (volumetric concentration); Surface cleaning 1 minute is outwelled filtrate.Use the chlorine bleach liquor of 30% (mass concentration) to embathe again 15 minutes, repeat once (promptly repeat alcohol embathe and repeat each sodium chlorate solution and embathe), use then sterile water wash 3-5 all over after, mature embryo be placed on the aseptic filter paper under super clean bench, dry up.Be seeded in NB inducing culture (NB minimum medium+2,4-D2mg/L) upward under 28 ℃ of dark cultivations, induce about 14 days, bud picking, callus is inserted fresh NB inducing culture succeeding transfer culture 10-12, treat that callus grows to diameter 4-5mm, callus is divided into 2-3 millimeter size, places new NB inducing culture to continue to cultivate 5 days as the bombardment callus.Insert height before the bombardment and ooze substratum (NB minimum medium+2,4-D 2mg/L+ sorbyl alcohol 47g/L+ N.F,USP MANNITOL 47g/L), the quantity of callus and putting position particle gun routinely transform requirement, and 28 ℃ of dark cultivations after 6 hours are bombarded; As shown in Figure 5.
The differentiation of embodiment 3, GUS expression cassette fragment cleaning DNA rice transformation callus and pre-differentiation:
Above-mentioned bombardment is transformed the dark callus of recovering of cultivating in back go on the pre-differentiation substratum (NB minimum medium+2mg/L6-BA+1mg/L NAA+5mg/L ABA) 28 ℃ of dark cultivations 12 days.The callus that will break up in advance goes to division culture medium (NB minimum medium+2mg/L 6-BA+1mg/L NAA) then, and 28 ℃ of 16 hours illumination cycles day break up, and breaks up to obtain regeneration plant in about 15 days; As shown in Figure 6.
The PCR molecular screening of embodiment 4, gus cleaning DNA rice transformation regeneration plant and the acquisition of transfer-gen plant
Press the rice regeneration plant that embodiment 3 obtains, get blade extraction DNA in seedling stage and carry out the PCR molecular screening, the PCR primer location is located at the gus gene coding region, and primer sequence is as follows: P1:CAACGAACTGAACTGGCAGA (SEQ ID NO:3); P2:TTTTTGTCACGCGCTATCA (SEQ ID NO:4).Pcr amplification condition: 94 ℃ of pre-sex change 5min; Later 94 ℃ of sex change 30s of loop parameter, 60 ℃ of annealing 30s; 72 ℃ are extended 30s; Totally 30 circulations, last 72 ℃ are extended 10min.Amplification back agarose gel electrophoresis detects, transforming successful plant is that the PCR positive (+) amplifies the 800kb specific band, the particular content reference of this 800kb specific band is disclosed document (Sivamani E, DeLong RK, Qu R.Protamine-mediated DNA coating remarkably improves bombardment transformation efficiency in plant cells.Plant Cell Rep.2009,28 (2): 213-21), unconverted successful then negative, there is not the 800kb specific band.The statistics conversion results is as shown in table 2.
Table 2. adopts cleaning DNA method for transformation of the present invention that gus gene is transformed the fine result of japonica rice variety Japan
Annotate: green seedling differentiation rate=(the green seedling number/bombardment callus number of regenerating) * 100%;
Transformation efficiency=(PCR positive plant number/bombardment callus number) * 100%
The part paddy rice regeneration plant of embodiment 4 gained is got Fig. 7 to the check and analysis result that gus gene carries out the PCR molecular screening, Fig. 7 is that gus gene PCR product agarose gel electrophoresis detects photo, presentation of results: M.DNA molecular weight marker in the paddy rice transfer-gen plant; 0.GUS negative control; 1.GUS positive control; 2-23. regeneration plant, wherein 2,9,12,15,16,19,20, No. 21 plant are the non-transgenic plant, and 3,4,5,6,7,8,10,11,13,14,17,18,22,23 is transfer-gen plant.Fig. 7 presentation of results adopts the cleaning DNA method for transformation of any selectable marker gene that do not need of the present invention successfully the gus gene expression cassette successfully to be transformed in the paddy rice.
The part paddy rice regeneration plant blade of embodiment 4 gained is carried out the GUS active coloring obtain Fig. 8, the left side is that the transgenic positive plant leaf shows blue among Fig. 8, and it is blue that the right is that the non-transgenic negative control does not show.Fig. 8 presentation of results adopt the cleaning DNA method for transformation that does not need any selectable marker gene of the present invention import in the paddy rice can normal expression with the gus gene expression cassette.
At last, it is also to be noted that what more than enumerate only is some specific embodiments of the present invention.Obviously, the invention is not restricted to above embodiment, many distortion can also be arranged.All distortion that those of ordinary skill in the art can directly derive or associate from content disclosed by the invention all should be thought protection scope of the present invention.
Claims (5)
1. method of the cleaning DNA transformation plant of choosing marker gene not is characterized in that may further comprise the steps successively:
1), preparation destination gene expression frame:
Sequence signature according to the plasmid vector at destination gene expression frame to be imported place adopts suitable restriction enzyme with the destination gene expression frame that builds
Dna fragmentation cuts down from plasmid vector, separates rubber tapping by gel electrophoresis and reclaims, and obtains destination gene expression frame DNA, as the foreign DNA of particle gun bombardment vegetable cell;
2), the little missile-borne body of preparation DNA suspension:
Get bronze suspension 30～50 μ l of 600mg bronze/ml, add the CaCl of 2.5mol/L
2Solution 45～55 μ L and 0.1mol/L spermidine solution 15～25 μ L behind the concussion mixing, add and contain the solution that the DNA total amount is the described destination gene expression frame DNA of 2 μ g, vibration, centrifugal, abandon supernatant, get the little bullet precipitation of DNA thereby make DNA be deposited to the bronze particle surface;
The little bullet precipitation of DNA is washed with dehydrated alcohol, vibration, centrifugal, abandon supernatant; Add dehydrated alcohol 60～80 μ l again, get the little missile-borne body of DNA suspension;
3), the plant callus bombardment transforms:
Utilize particle gun that the little missile-borne body of above-mentioned DNA suspension is bombarded in the plant embryo callus body, the callus after the bombardment leaves standstill dark cultivation, so that make the bombardment cellular-restoring;
4), plant regeneration:
With above-mentioned steps 3) the dark plant callus that recovered of cultivating is directly transferred to presorting on the pre-differentiation substratum after the bombardment of gained, then breaks up the acquisition regeneration plant;
5), the screening of transfer-gen plant.
2. the method for the cleaning DNA transformation plant of not choosing marker gene according to claim 1, the plant callus bombardment that it is characterized in that described step 3) is converted into: the plant embryo callus of inducing culture oozes at height earlier before bombardment and places the dark 4～5h of cultivation on the substratum according to a conventional method, then carry out the particle gun bombardment, the callus of every culture dish bombards 2 rifles, and the step 2 of 1/6 volume is set in every rifle) the little missile-borne body of the DNA suspension of gained; Callus after the bombardment leaves standstill dark cultivation 16～20h, so that make the bombardment cellular-restoring.
3. the method for the cleaning DNA transformation plant of not choosing marker gene according to claim 2 is characterized in that the particle gun in the described step 3) is a Biolistic PDS 1000/He particle gun.
4. according to the method for the cleaning DNA transformation plant of claim 2 or 3 described not choosing marker genes, the screening that it is characterized in that the transfer-gen plant of described step 5) is: with the regeneration plant of step 4) gained directly on molecular level and the enterprising row filter of phenotypic level, thereby obtain transforming successful plant.
5. the method for the cleaning DNA transformation plant of not choosing marker gene according to claim 4 is characterized in that: described plant is a paddy rice.
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