CN100575494C - A kind of composition that is used for the conversion of plant direct gene - Google Patents
A kind of composition that is used for the conversion of plant direct gene Download PDFInfo
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- CN100575494C CN100575494C CN200710010192A CN200710010192A CN100575494C CN 100575494 C CN100575494 C CN 100575494C CN 200710010192 A CN200710010192 A CN 200710010192A CN 200710010192 A CN200710010192 A CN 200710010192A CN 100575494 C CN100575494 C CN 100575494C
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Abstract
The invention discloses a kind of composition, method for transformation and application thereof that the plant direct gene transforms that be used for, composition comprises the free nucleic acid component of (1) goal gene and expression regulation element thereof, and (2) transform damping fluid and (3) transform auxiliary component.Method for transformation is by cultivation altogether, soaks, and smear on the surface, and injection is instiled, and sprays, and libation at an ancient wedding ceremony flower method transforms; Also can transform by particle gun bombardment, ultrasonic wave, electric-shocking method.Composition can be applicable at the recipient plant cell, comprise the direct conversion of zygote, suspension cell, protoplastis, monolayer cell, mesophyll cell, at the recipient plant tissue, comprise immature embryo, embryo callus, merismatic direct conversion, be used at the direct conversion of seed and can be applicable to transform at the direct in-situ of recipient plant pollen, gynoecium, style, pollen tube, ovary.The present invention has great importance for plant genetics and breeding, breed improvement or gene function analysis.
Description
Technical field
The present invention relates to a kind of composition and application thereof that the plant direct gene transforms that be used for, belong to bioengineering field.
Background technology
Plant genetic engineering is deeply to develop on the basis that is gradually improved with recombinant DNA technology at molecular biology to grow up.Obtaining many transgenic plant aspect some composition of plant disease-resistant, pest-resistant, anti-green bristlegrass and change plant, the formation strain that has, be to improve crop yield, anti-adversity ability, improvement quality, carry out fast, the fine-variety breeding of high-quality, stable yields provides brand-new approach.In the higher plant gene engineering research, the transfer of foreign gene is a very important ring.Because plant and cell its own particularity thereof, thereby gene transfer method also there are some special requirements.Make a general survey of many foreign gene transfer methods, can be divided into two big classes: promptly infect plant and with the dna direct conversion plant that contains foreign gene with the agrobacterium tumefaciens that contains the foreign gene Ti-plasmids according to the form of carrier.The direct conversion that contains foreign gene DNA is to use physics, and chemistry and biological means import plant cell tissue or organ with exposed foreign DNA, and the gene that foreign DNA is comprised is expressed in vegetable cell.This method has fundamentally overcome Ti-plasmids can only infect the defective of dicotyledons, and the recipient plant scope is expanded greatly.
In the development of plant genetic engineering, studying the clearest and using the most successful is Agrobacterium tumefaciens mediated genetic transformation.1974, it is found that agrobacterium tumefaciens when infection plant, the section of DNA on the cyclic Ti-plasmids wherein can be inserted in the Plant Genome, cause the variation of plant genetic characteristic.From then on, people begin to attempt utilizing this natural carrier system, with exogenous gene transfered plant cell, and utilize the totipotency of vegetable cell, by means of cell cultures or tissue culture technique, make the complete transfer-gen plant of transformant regeneration, thereby realize the genetic transformation of foreign gene plant.Nineteen eighty-three has obtained the first routine transgenic plant.1985, people such as Horsh initiated leaf disc transformation method, made conversion process greatly simplify.After this, because of this method because of having the low copy of transgenosis, inheritance stability and can transform advantage such as large fragment DNA and be used widely.At present, in numerous transgenic plant 80% by agrobacterium mediation converted.Agrobacterium tumefaciens (Agrobacteriumtumefaciens), it contains Ti-plasmids, can induce the vegetable cell that is infected to form tumour, promptly brings out crown-gall nodule.Ti-plasmids has one section transfer DNA (claiming T-DNA again) on (comprising the Ri plasmid), when Agrobacterium was infected plant, this segment DNA can be inserted in the Plant Genome, and its gene that carries is expressed in plant.Because T-DNA can carry out high-frequency transfer, and can insert big foreign gene on Ti-plasmids and the Ri plasmid to 50kb.Therefore, Ti-plasmids and Ri plasmid just become the ideal carrier system that plant gene transforms.The transfer of T-DNA must could realize transforming by means of the toxalbumin virD2/E2virD2 of vir genes encoding generation on the Ti-plasmids and the effect of virE2 on the Ti-plasmids.But Agrobacterium-mediated Transformation generally need be passed through tissue culture, is subjected to the recipient plant type, and the restriction of regeneration system rapidly foundation.
Particle bombardment is the most widely used a kind of method in the at present direct conversion method, this method claims particle bombardment (particlebombardment) again, high-velocity particles spraying technique (High-velocity particle microprojection) or particle gun bombardment technology (gene gun bombardment) is to equal nineteen eighty-three by the John.C.Santord of department of biochemistry of U.S. Comel university to study successfully.And in 1987, Vlein has reported that at first this technology of application is adsorbed onto the tungsten particle surface with TMV (tobacco mosaic virus (TMV)) RNA, the bombardment onion epidermis cell, find that after testing viral RNA can duplicate, and CAT (chloramphenicol acetyl transferasegene) gene is imported onion epidermis cell with same technology.Now, this technology success on various crop such as tobacco, paddy rice, wheat, rye grass, sugarcane, cotton, soybean, Kidney bean, onion, papaya, sweet orange, grape.This method has wide application, method is simple, transformation time is short, because the randomness of particle gun bombardment, foreign gene enters the integration site of host genome and relatively fixes, and copy number is often more, and sudden change appears in transgenic progeny easily like this, foreign gene is lost easily, cause the generation of phenomenons such as gene silencing easily, be unfavorable for the stably express of foreign gene at host plant.And particle gun costs an arm and a leg, the running expense height.
Pollen tube passage method is by the nineteen eighty-three foundation of period-luminosity space the earliest.Certain hour behind plant pollination injects the embryo position of cotton ovary with foreign DNA, makes DNA enter blastular along established pollen tube channel, thereby transforms zygote or the protoblast that does not still possess the normal cell wall.Because having plenty of total DNA of employing, this method imports, offspring's variation is at random, a lot of in addition tests lacks the molecule checking, and is difficult to get across for the mechanism of foreign DNA integration, so also exist dispute for being extensive use of of pollen tube channel method.In addition, because the restriction of principle of design and working specification itself, the conversion elements of external source is the DNA that exposes, enter blastular at it by pollen tube, in the cytolemma by zygote, the process that nuclear membrane is finished integration, be highly susceptible to being degraded, and foreign DNA enters blastular and mainly depends on gravity and osmosis, foreign DNA lacks the fundamental driving force that is integrated into the acceptor gene group, and transformation efficiency is low, the problems such as heredity that its offspring of the transfer-gen plant that is produced can not be stable.
Chemical method general using plant protoplast is not having under the situation of carrier, can absorb genetic material such as foreign DNA, plasmid by inducing of some chemical reagent, and might be incorporated into plant chromosome and get on.Common chemical reagent has PEG (polyoxyethylene glycol), PLD (poly-ornithine), and PVA (polyvinyl alcohol) etc., wherein that the most commonly used is PEG.Use this method, that carry out first in vegetable cell that direct gene dress moves is Dai Wei (Davey) and Krens etc.Chemical method has been broken the restriction that agrobacterium tumefaciens is infected scope, make it to be widely used in multiple monocotyledonous cereal plants, realized at present einkorn wheat, perennial rye, paddy rice, protoplastis and dicotyledons rapes such as high grain and wheat, the conversion of tobacco and petunia.But the applied chemistry method transforms difficult success separately, and transformation efficiency is low.The time of chemical method research is long, and method is ripe, and cost is low, good reproducibility, be easy to promote.But because the protoplast regeneration frequency is lower, the cultivation difficulty is big, and the cycle is long, thereby the universal of this method is restricted.
Simultaneously, the particle gun of widespread use at present and agriculture bacillus mediated methods for plant transformation, the potential safety issue that exists non-goal gene exogenous dna fragments such as selectable marker gene and carrier framework sequence to cause all the time.And be target to eliminate selectable marker gene and carrier framework sequence, domestic and international many laboratories have been adopted multiple strategy to carry out a large amount of research in recent years and have constantly been improved and obtained some progress.
Therefore inquire into to set up and a kind ofly new can eliminate the safe, practical, easy, efficient, stable of non-goal gene exogenous dna fragment fully, and need not pass through tissue culture, the direct conversion system that the acceptor scope is wide has great importance.
Summary of the invention
The purpose of this invention is to provide a kind of be used for composition that the plant direct gene transforms and said composition safe, easy, efficiently direct gene method for transformation and said composition application.
The technical scheme that the present invention solves its technical problem employing is, a kind of composition that is used for the conversion of plant direct gene, said composition contains: the free nucleic acid component of (1) goal gene and expression regulation element thereof comprises plasmid DNA or does not contain the linear DNA fragment of carrier framework; (2) transform damping fluid, comprise the 1/2MS liquid nutrient medium, or TE (pH8.0) or 0.1 * SSC damping fluid; (3) transform the surfactant D OW CORNING Q2-5211 that auxiliary component comprises 0.005%-0.02%, 10mmol/L-50mmol/L Ca
2+, 1-10ppm 2,4-D, 0.5ppm 6-BA.Transform auxiliary component and also comprise 0.1% boric acid, agrobacterium tumefaciens excretory VirD2 albumen and VirE2 albumen.Agrobacterium tumefaciens is meant and contains Vir district, Con district and Ori district, but do not contain the Ti-plasmids of T-DNA transition range.VirD2 albumen and VirE2 albumen obtain by the Syringylethanone secretion inducing.Plasmid DNA is meant the cyclic DNA that possesses complete replication orgin, goal gene, expression regulation element and border sequence or possesses complete replication orgin, goal gene, expression regulation element, but do not contain the cyclic DNA of border sequence.The linear DNA component is meant the linear DNA fragment that comprises border sequence, goal gene and expression regulation sequence thereof or comprises goal gene and expression regulation sequence, but do not comprise the linear DNA fragment of border sequence.Plasmid DNA and linear DNA be free the existence in composition, do not need transduction to go in the agrobacterium tumefaciens.The linear DNA component comprises linear fragment of double-stranded DNA or the linear fragment of single stranded DNA.The double-stranded DNA linear fragment can obtain or cut acquisition by enzyme from the purpose plasmid from purpose plasmid amplification by PCR method.0 ℃ of cooling was rapidly obtained after the single stranded DNA fragment was passed through 99 ℃ of sex change by double-stranded DNA.
A kind of method for transformation that is used for the composition of plant direct gene conversion can soak by cultivating altogether, and smear on the surface, injects, and instils, and sprays, and dips in colored method and transform; Also can transform by particle gun bombardment, ultrasonic wave, electric-shocking method.
Said composition can be applicable at the recipient plant cell, comprise the direct conversion of zygote, suspension cell, protoplastis, monolayer cell, mesophyll cell, can be applicable at the recipient plant tissue, comprise immature embryo, embryo callus, merismatic direct conversion, also can be applicable at the direct conversion of seed and can be applicable to transform at the direct in-situ of recipient plant pollen, gynoecium, style, pollen tube, ovary.
Plant gene conversion composition provided by the invention and the application in directly transforming thereof; its core technology is to provide comprises free nucleic acid component, transform damping fluid and transform the conversion composition of auxiliary component, can solve a series of problems of the goal gene transformation power, transhipment, protection and the integration that are faced in the direct conversion.
Conversion auxiliary component provided by the present invention mainly comprises surfactant D ow Corning Q2-5211, Ca
2+, give birth to conditioning agent 2,4-D or 6-BA, boric acid, and through secreted VirD2 albumen and the VirE2 albumen outside born of the same parents of Syringylethanone inductive agrobacterium tumefaciens.Transform being used in combination of auxiliary component, can in conversion, play transformation power is provided, appraise and decide the position, and the effect of protection and promotion integration.
The super wetting agent of Dow Corning Q2-5211 is a kind of low-molecular-weight nonionic polyoxyalkylene modified silicone surfactants, when concentration is 0.01%, the super wetting agent of Dow Corning Q2-5211 can make surface tension be reduced to 23dynes/cm, this makes that the conversion damping fluid can be moistening rapidly, and be difficult to moistening explant such as wax shape leaf surfaces on rapidly diffusion and absorbed by explant, can significantly improve the wettability that transforms damping fluid, ductility and perviousness, thereby help to improve the efficient of conversion, and safety non-toxic, be ideal transformation power source.In the present invention, the concentration range of DOW CORNING Q2-5211 is 0.005%-0.02%.
DNA can be deposited on the surface of cell by the form of calcium salt, and by causing endocytosis, realizes the transfer of DNA.CaCl with 10mmol/L-50mmol/L
2(or calcium phosphate) handles recipient cell, can cause cell expansion, increases the permeability of cell, makes recipient cell be easy to accept foreign DNA, improves transformation efficiency.Ca in the present invention
2+Concentration range be 10mmol/L-50mmol/L.
2,4-D and 6-BA are plant growth regulating substance under lower concentration, help cell to handle vigorous growth and splitting status, and the cell that is in vigorous division stage more helps to accept the DNA of external source, improves transformation efficiency.Among the present invention 2, the concentration range of 4-D is 1-10ppm; The concentration of 6-BA is 0.5ppm.
Boron is a kind of chemotropism material, can guide pollen tube by the transfer of column cap to the embryo strain, in conversion at floral organ, add 0.1% boric acid in the present invention and can help conversion elements to arrive the embryo strain through column cap or ovary surface, finish the directional transmissions and the integration of foreign gene.
In the conversion auxiliary component provided by the present invention, utilize the toxalbumin virD2 of Agrobacterium abduction delivering and virE2 to realize of the transfer of free nucleic acid especially, appraise and decide position, protection and integration to acceptor.Goal gene and regulating and controlling sequence thereof that toxalbumin virD2 can assist to have T-DNA left and right sides border sequence are finished transfer, are appraised and decided position and the integration in Plant Genome; VirE2 can be at cytolemma; and set up the specific passage of strand on the nuclear membrane; and play effect stable and protection with combining of single-chain nucleic acid; virE2 has nuclear localization signal; assist of location and the integration of virD2-single-chain nucleic acid mixture, solve various problems such as easily being degraded, transforming multiple copied, transformation efficiency are low, poor repeatability in the at present free direct conversion process of nucleic acid to plant nucleolus.
The present invention uses and only contains Vir district, Con district and Ori district, but does not contain the Agrobacterium Ti-plasmids of T-DNA transition range.VirD2 and virE2 induction expression of protein mainly are achieved through the following technical solutions: the Agrobacterium LBA4404 or the GV3101 that contain Ti-plasmids, be inoculated in the YEB substratum of Streptomycin sulphate of the Rifampin that contains 50mg/L and 25mg/L, wave and culture is to the logarithmic phase of growing (bacterium liquid absorbancy OD550 is 1.8-2.0) under 28 ℃ and 250rpm condition; With cultured bacterium liquid centrifugal I0min under the 5000rpm condition, thalline is suspended in the 1/2MS liquid nutrient medium again and makes bacterium liquid, and the bacterial concentration after the dilution is about 2 * 10
8Cfu/ml, adding Syringylethanone to final concentration then in bacterium liquid is 20mg/L, continuation under 28 ℃ and 250rpm condition wave and culture 3-4 hour, the centrifugal removal thalline of centrifugal 10min under the 3000rpm condition, supernatant liquor is the conversion damping fluid that contains virD2 and virE2.
The auxiliary direct conversion of Agrobacterium virD2/virE2 albumen comprises dual mode.A kind of is the cell at recipient plant, the conversion of tissue.The main technological route of this transformation system is: (1) sample pre-treatments: recipient cell or be organized in before the pre-inversion in 70% ethanol disinfection 1 minute, the flushing of MS substratum once, mercuric chloride sterilization 10 minutes, MS washes for several times, can select to place high osmotic buffer to handle then 10-20 minute, high osmotic buffer consist of MS liquid nutrient medium+47g/L N.F,USP MANNITOL+47g/L sorbyl alcohol.(2) transform: recipient cell after will handling or tissue and contain free conversion elements, conversion damping fluid and through Syringylethanone inductive Ti-plasmids bacterium liquid, or contain in the conversion composition of conversion assisted solution of virD2 and virE2 and cultivate 30min-1h.(3) transient expression analysis: explant got place the MS division culture medium dark under 20-24 ℃ of condition that contains 0.5ppm 6-BA to cultivate altogether 1-3 days,, select appropriate means to detect the transient expression of this gene according to the characteristic of encoding gene.As add in the above-mentioned conversion composition final concentration be 0.005%-0.02% DOW CORNINGQ2-5211, the CaCl of 10mmol/L-100mmol/L
2, 2 of 1-10ppm, the 6-BA of 4-D or 0.5ppm etc. transform auxiliary component, can further improve transformation efficiency.Second kind is the direct in-situ conversion at floral organ.Can inject by pollen tube passage method, ovary, or the ovary instillation, realize the plant converted in-situ.The main technological route of this method for transformation is: will contain free conversion elements, transform damping fluid and through Syringylethanone inductive Ti-plasmids bacterium liquid, or the conversion composition that contains the conversion assisted solution of virD2 and virE2 directly is added drop-wise to the column cap or the ovary surface of cutting, or be injected into the inside of column cap or ovary, realize of the conversion of free nucleic acid to zygote, promptly may include transforming gene in the seed of results, after the results seed directly becomes strain, analyze through screening, can obtain the stable transgenic progeny that continues.This method does not need through tissue culture procedures, and is easy, efficient, with low cost.Equally, adding final concentration in conversion composition is the Dow Corning Q2-5211 of 0.005%-0.02%, the CaCl of 10mmol/L-50mmol/L
20.1% boric acid; 2 of 1-10ppm, the 6-BA of 4-D or 0.5ppm can further improve transformation efficiency.
The free nucleic acid component that contains goal gene and expression regulation element thereof that provides among the present invention mainly comprises plasmid DNA component and linear DNA component.Plasmid DNA is meant to possess complete replication orgin, expression regulation element, comprise the cyclic DNA of (or not comprising) border sequence, and plasmid does not need transduction to go in the thalline in the present invention, exists so that the solution form is free in conversion composition.The linear DNA component is meant the linear DNA fragment that comprises (or not comprising) border sequence, goal gene and regulating and controlling sequence thereof, also exists so that the solution form is free in conversion composition.
Border sequence mainly refers to the conserved sequence on border, the T-DNA left and right sides in the Ti-plasmids, and the conserved sequence on goal gene and adding border, the T-DNA left and right sides, regulating and controlling sequence two ends thereof helps the identification of VirD2, and location and integration in the plant acceptor.In the present invention, sequence is the T-DNA right margin before the adding goal gene promotor, and its conserved sequence is: 5-gtttacccgccaatatatcctgtca; Joining goal gene terminator sequence afterwards is the T-DNA left margin, and its conserved sequence is: 5-tgtttacaccacaatatatcctgcca.Linear DNA provided by the invention can be that the DNA of strand also can double-stranded DNA.Double-stranded linear fragment can obtain from the amplification of purpose plasmid by PCR method, or cuts acquisition by enzyme from the purpose plasmid.The single stranded DNA fragment mainly obtains by 0 ℃ of cooling rapidly after 99 ℃ of sex change of double-stranded DNA.
The invention has the beneficial effects as follows that plant gene conversion composition provided by the invention and the application in directly transforming thereof can solve in the direct conversion process a series of problems of the transformation power that goal gene faced, location, protection and integration.Utilize the linear fragment of only forming, can realize the conversion process of marker-free gene, carrier free frame sequence, have biological safety by expression regulation sequence and goal gene; Direct in-situ at floral organ transforms, and can realize the conversion process that inorganization is cultivated, thereby has avoided the bottleneck of plant regeneration system foundation, has enlarged the scope of transformation receptor.Therefore, plant gene conversion composition provided by the invention has great importance for plant genetics and breeding, breed improvement or gene function analysis.
Embodiment
The present invention is further described below in conjunction with embodiment.
Rapidly and efficiently the moment expression of embodiment 1:GUS gene in onion epidermis cell
Get the onion lower epidermis and divide the tissue block of suitable size with blade into, ethanol disinfection in 70% 1 minute, the MS substratum washes once, mercuric chloride sterilization 5 minutes, MS washes for several times, places high osmotic buffer MS liquid nutrient medium+47g/L N.F,USP MANNITOL+47g/L sorbyl alcohol pre-treatment then 10-20 minute.
The Agrobacterium that contains Ti-plasmids is inoculated in and contains in the antibiotic YEB substratum, and wave and culture is to the logarithmic phase of growing (bacterium liquid absorbancy OD under 28 ℃ and 250rpm condition
550Be 1.8-2.0); 3. with cultured bacterium liquid centrifugal 10min under the 5000rpm condition, thalline is suspended in the CaCl of the Dow corning Q2-5211+10mmol/L of 1/2MS liquid nutrient medium (additional Syringylethanone 20mg/L)+conversion elements+0.01% again then
2+ 0.1mmol/L spermidine, the bacterial concentration after the dilution about 2 * 10
8Cfu/ml; Conversion elements is the linear fragment that has RB+35S+GUS+NOS+LB; 4. will place bacterium liquid through pretreated onion lower epidermis, slowly shake 1.5h under the 25-26 ℃ of condition; 5. take out the onion lower epidermis and change on the MS substratum (Syringylethanone 200pmol/L), cultivated altogether 36-48 hour for 25-26 ℃; 6. the onion lower epidermis after will cultivating altogether with aseptic washing 3 times, with the aseptic washing that adds the 500mg/L cephamycin 1 time, is used the aseptic filter paper suck dry moisture again; 7. onion epidermis is placed 96 orifice plates, a slice onion epidermis is placed in every hole, and after paving, the surface drips the X-gluc20 microlitre, places the expression of observed and recorded gus gene under stereoscopic microscope then 37 ℃ of incubator 1-12 hours.The result shows, onion epidermis is under the mediation of Agrobacterium excretory toxalbumin virD2/E2, can help goal gene and regulating and controlling sequence thereof to enter into nucleus inside, goal gene can be realized the moment expression of goal gene under the effect of its regulating and controlling sequence, and the gus protein of expressing is evenly distributed in cell.Moment, the cell of expressing protein had 60-80%.
Embodiment 2: contain the efficient stable conversion of the linear fragment of gus gene by pollen tube passage method
Have the preparation of the damping fluid of Agrobacterium toxalbumin virD2/E2: the Agrobacterium LBA4404 that contains Ti-plasmids, be inoculated in the YEB substratum of Streptomycin sulphate of the Rifampin that contains 50mg/L and 25mg/L, wave and culture is to the logarithmic phase of growing (bacterium liquid absorbancy OD under 28 ℃ and 250rpm condition
550Be 1.8-2.0); With cultured bacterium liquid centrifugal 10min under the 5000rpm condition, thalline is suspended in the 1/2MS liquid nutrient medium again and makes bacterium liquid, and the bacterial concentration after the dilution is about 2 * 10
8Cfu/ml, adding Syringylethanone to final concentration then in bacterium liquid is 20mg/L, continuation under 28 ℃ and 250rpm condition wave and culture 3-4 hour, the centrifugal removal thalline of centrifugal 10min under the 3000rpm condition, supernatant liquor is the damping fluid that has virD2 and virE2.Be made into the conversion damping fluid with adding the 6-BA that final concentration is respectively CaCl2,0.1% boric acid and the 0.5ppm of 0.01% Dow Corning Q2-5211,10mmol/L in the damping fluid then.
The acquisition of conversion elements: with the pBI121-GUS plasmid is template, uses the primer PT1/PT2 that contains the T-DNA border sequence to carry out pcr amplification, makes up the gene transformation element.PT1:5 '-GTTTACCCGCCAATATATCCTGTCACTGCCTGCAGGTCCCCAGATTAGCCTT-3 ', PT2:5 '-TGGCAGGATATATTGTGGTGTAAACCGATCTAGTAACATAGATGACACCGC-3 ' uses high-fidelity LaTaq enzyme to carry out pcr amplification.Reaction conditions is as follows: 95 ℃, and behind the 5min, 94 ℃ of 30s, 55 ℃ of 1.5min, 72 ℃, 1.5min is totally 30 circulations, 72 ℃ of 7min; Pcr amplification product add the long-pending dehydrated alcohol of diploid, and the NaAc of the 3M of 1/10 volume precipitates after 1% agarose gel electrophoresis is identified, quantitatively, be dissolved in the conversion damping fluid that has toxalbumin virD2/E2, final concentration is 100ng μ l-1, is used for further conversion.
At 6-32 hour (corolla a little more than calyx 1mm about) complete excision column cap after the soybean self-pollination, 5ul is transformed the buffering drop in incision.If temperature is higher, mends and drip once.The flower that mark is handled, and wipe out the flower of operating.Ripe back results seed.Compare with the processing that only contains 1/2MS conversion damping fluid.Utilize PCR, Southern hybridization equimolecular means that the seed and the offspring thereof of results are detected, the result shows under the effect of the conversion damping fluid of toxalbumin virD2/E2 mediation, linear fragment with the form stable integration of single copy in the genome of soybean.
Claims (17)
1, a kind of composition that is used for the conversion of plant direct gene, contain: the free nucleic acid component of (1) goal gene and expression regulation element thereof, it is a plasmid DNA, or does not contain the linear DNA fragment of carrier framework; (2) transform damping fluid, it is the 1/2MS liquid nutrient medium, or TE, or 0.1 * SSC damping fluid; (3) transform auxiliary component, it is the surfactant D OW CORNING Q2-5211 of 0.005%-0.02%, 10mmol/L-50mmol/L Ca
2+And 1-10ppm 2,4-D or 0.5ppm6-BA.
2, the composition that is used for the conversion of plant direct gene according to claim 1, conversion auxiliary component wherein also comprises 0.1% boric acid.
3, the composition that is used for the conversion of plant direct gene according to claim 1, conversion auxiliary component wherein also comprises agrobacterium tumefaciens excretory VirD2 albumen and VirE2 albumen.
4, the composition that is used for the conversion of plant direct gene according to claim 3, agrobacterium tumefaciens excretory VirD2 albumen wherein and VirE2 albumen obtain by the Syringylethanone secretion inducing.
5, the composition of plant direct gene conversion according to claim 1, wherein plasmid DNA is meant the cyclic DNA that possesses complete replication orgin, goal gene, expression regulation element and border sequence; Or possess complete replication orgin, goal gene, expression regulation element, but do not contain the cyclic DNA of border sequence.
6, the composition of plant direct gene conversion according to claim 1, wherein the linear DNA component is meant the linear DNA fragment that comprises border sequence, goal gene and expression regulation sequence thereof; Or comprise goal gene and expression regulation sequence thereof, but do not comprise the linear DNA fragment of border sequence.
7, the composition of plant direct gene conversion according to claim 1, plasmid DNA wherein and linear DNA be free the existence in composition, do not need transduction to go in the agrobacterium tumefaciens.
8, the composition of plant direct gene conversion according to claim 6, wherein the linear DNA component comprises the double-stranded DNA linear fragment; Or single stranded DNA linear fragment.
9, the composition of plant direct gene conversion according to claim 8, wherein the double-stranded DNA linear fragment can obtain from the amplification of purpose plasmid by PCR method; Or from the purpose plasmid, cut acquisition by enzyme.
10, the composition of plant direct gene conversion according to claim 8,0 ℃ of cooling was rapidly obtained after wherein the single stranded DNA fragment was passed through 99 ℃ of sex change by double-stranded DNA.
11, the method that transforms of the composition that utilizes the described plant direct gene of claim 1 to transform by cultivating altogether, is soaked, and smear on the surface, and injection is instiled, and sprays, and dips in colored method and transforms; Or by particle gun bombardment, ultrasonic wave, electric-shocking method conversion.
12, the purposes of the composition of plant direct gene conversion according to claim 1 is applied to the direct conversion at the recipient plant cell.
13, the purposes of the composition of plant direct gene conversion according to claim 12, recipient plant cell wherein is zygote, suspension cell, protoplastis, monolayer cell or mesophyll cell.
14, the purposes of the composition of plant direct gene conversion according to claim 1 is applied to the direct conversion at the recipient plant tissue.
15, the purposes of the composition of plant direct gene conversion according to claim 14, recipient plant wherein is organized as immature embryo, embryo callus or meristematic tissue.
16, the purposes of the composition of plant direct gene conversion according to claim 1 is applied to the direct conversion at seed.
17, the purposes of the composition of plant direct gene conversion according to claim 1 is applied to the direct conversion at recipient plant pollen, gynoecium, style, pollen tube, ovary.
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| CN103070067A (en) * | 2013-02-05 | 2013-05-01 | 中国农业科学院棉花研究所 | Gene gun living quick transformation method of cotton |
| CN108070616B (en) * | 2016-11-07 | 2022-02-11 | 沈阳农业大学 | Low-pressure gene gun-mediated plant in-situ transformation method |
| CN108374020A (en) * | 2017-12-28 | 2018-08-07 | 南京农业大学 | A kind of agriculture bacillus mediated soybean transformation in planta method of improvement |
| EP3959967A4 (en) * | 2019-03-21 | 2023-06-28 | Masashi Mori | Method for producing transformed plant and transformation agent |
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