CN106893737A - A kind of genetic transforming method of larch cells,primordial - Google Patents
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Abstract
The invention discloses a kind of genetic transforming method of larch cells,primordial.The invention provides a kind of genetic transforming method of larch cells,primordial, in turn include the following steps：(a1) larch cells,primordial is taken, recombinational agrobacterium is suspended in and is infected liquid and infected；(a2) after completing step (a1), larch cells,primordial is collected, carries out renewal cultivation；(a3) after completing step (a2), larch cells,primordial is taken, de- bacterium is carried out using the de- bacterium device of suction filtration.The method that the present invention is provided, can be used to carry out slewing genetic improvement to larch using genetic engineering means, and for Study of Exogenous gene, expression regulation and its functional verification and embryonic development research provide technology platform in larch body.
Description
Technical field
The present invention relates to a kind of genetic transforming method of larch cells,primordial.
Background technology
Larch (LarixLeptolepis) is the needle fast-growing afforestation material of alpine region in northern China and subtropical zone
Seeds, with widely distributed, strong adaptability, pest and disease damage is few, material is excellent the features such as, and early stage fast-growing character is all
Coniferous tree in rank among the best, be reproducting tree species that state forest farm and peasant pay much attention to, artificial afforestation area's mesh benefit
Expand.Therefore, the key areas that larch breeding research work is China's coniferous tree improvement are carried out.At present, I
State mainly by manually introducing a fine variety, fine-variety breeding, the traditional breeding method mode such as interspecific hybridization genetic improvement is carried out to larch.
Although having achieved the progress for attracting people's attention in this field, however from technological layer for, educated because these are traditional
Kind of mode exist human and material resources expend it is larger, by effect of natural conditions, breeding cycle is long, genetic manipulation difficulty is big etc.
Problem, larch breeding process can not meet the demand of modern Forest Tree Genetic Breeding.In recent years, genetic engineering breeding
Develop rapidly, be that woody energy opens a new way, using genetic engineering means, species boundary can be broken through,
The Studies of Transfer of Alien Genes Into Receptors of merit will be controlled, the genetic improvement of slewing is carried out to plant, so as to reach often
The target that rule breeding technique cannot be realized.Genetic engineering means have been successfully applied to many agriculture and forestry plant breeding techniques,
And had very much the new lines (kind) of application value.
Current coniferous tree genetic transformation mainly utilizes agrobacterium-mediated transformation and particle bombardment, at the same also attempted electric shocking method and
PEG revulsions.In many genetic transfoumations, Agrobacterium-mediated genetic transformation
(Agrobacterium-mediated transformation) occupies an leading position, wherein Agrobacterium tumefaciems
The genetic transformation of (Agrobacterium tumefaciens) mediation is that current most study, mechanism is most clear, technology
Most ripe gene transformation method.Agrobacterium-mediated transformation and other method for transformation (such as particle bombardment, electric shocking method, PEG
Mediated method) compared to have simple to operate, low cost, can import large fragment gene, gene integration copy number it is low, occur
The low advantage of gene silencing rate and used by more Plant Biotechnology laboratory, be also to be applied to coniferous tree genetic transformation
Widest method.Early in 1934, researchers just had learned that Agrobacterium tumefaciems can infect coniferous tree.Enter one later
Step research finds that coniferous tree is strictly the natural host of Agrobacterium, and this is found to be development with agriculture bacillus mediated coniferous tree
Study on Genetic Transformation simultaneously successful is laid a good foundation.Sederoff etc. is imported foreign gene using agrobacterium-mediated transformation
Torch pine, this is the report of successful conversion coniferous tree first, but does not obtain transfer-gen plant.The root of hair agriculture such as Huang
Bacillus is infected to European larch aseptic seedling, obtains the transformed plant of stable expression of exogenous gene, and this is to utilize
The first case transfer-gen plant that Agrobacterium-mediated Transformation coniferous tree obtains.It is existing in the world at present largely to utilize agriculture bacillus mediated pin
Leaf tree genetic transformation successfully obtains the report of stabilization express transgenic plant.
According to statistics, Pinaceae (Pinus, Abies, Picea, Pseudotsuga, Larch), Cupressaceae, Taxodiaceae in recent years
Transformant have successfully been obtained by Agrobacterium-mediated genetic transformation Deng tens kinds of coniferous trees.Wherein torch pine, move
Prestige dragon spruce, pine have good reconstituted receptors system, and its Study on Genetic Transformation makes fast progress, and transformation technology compares
It is ripe.Other coniferous species Study on Genetic Transformation work progresses are slow, and system less stable, conversion ratio is generally relatively low.
The report of comprehensive a large amount of coniferous tree Study on Genetic Transformation show that Agrobacterium infects conversion coniferous tree by multifactor impact, main
Including：Coniferous tree different tree species and genotype；Acceptor material and physiological status；The type of agrobacterium strains and carrier；
Infect and co-cultivation condition；Filtering system etc., the synthesis of various influence factors and optimization are favorably improved genetic transformation effect
Rate.Lack excellent regenerative system mostly due to coniferous species, Study on Genetic Transformation is made slow progress, lack systematicness.
The content of the invention
It is an object of the invention to provide a kind of genetic transforming method of larch cells,primordial.
The invention provides a kind of genetic transforming method of larch cells,primordial, in turn include the following steps：
(a1) larch cells,primordial is taken, recombinational agrobacterium is suspended in and is infected liquid and infected；
(a2) after completing step (a1), larch cells,primordial is collected, carries out renewal cultivation；
(a3) after completing step (a2), larch cells,primordial is taken, de- bacterium is carried out using the de- bacterium device of suction filtration；
The de- bacterium device of the suction filtration includes separator 1, fluid collection device 2 and air extractor 3；
The top of the separator is the sample-adding portion 1-1 of top end opening inner hollow, and bottom is in bottom end opening inside
Empty pars infundibularis 1-2, sample-adding portion with connect outside pars infundibularis, inside connect, the inside of sample-adding portion and pars infundibularis connects
Place is provided with the stainless (steel) wire 1-3 that aperture is 200-500 mesh；
The fluid collection device is closed hollow container, with upper shed and side opening；
The upper shed air tight communication of the bottom end opening of the pars infundibularis of the separator and the fluid collection device；
The side opening of the fluid collection device and air extractor air tight communication.
In the step (a3), the process of the de- bacterium is：First with distillation water washing 3-5 times, each 2-5min,
3-5 times, each 5-10min are washed with 300-500mg/L antibiotic solutions again.
In the step (a3), the process of the de- bacterium is concretely：First with distillation water washing 4 times, each 3min,
4 times, each 8min are washed with 400mg/L antibiotic solutions again.
The antibiotic solution concretely cephalosporin aqueous solution.
In the step (a1), it is with infecting the liquid that liquid suspension recombinational agrobacterium is obtained that the recombinational agrobacterium infects liquid
Phase；The recombinational agrobacterium is the Agrobacterium containing target DNA.The liquid that infects is：Acetosyringone 2-40mg/L,
Balance of water；pH5.75-5.85.It is described to infect liquid concretely：Acetosyringone 20mg/L, balance of water；pH5.8.
In the step (a1), the condition that infects is：It is stored at room temperature 5-20 minutes.
In the step (a1), the condition for infecting is concretely：It is stored at room temperature 10 minutes.
In the step (a2), the renewal cultivation is in S1Carried out on culture medium；
The S1Culture medium is as follows：KNO31836-2836mg/L、NH4NO3243-360mg/L、KH2PO470-130mg/L、
CaCl2·2H2O300-450mg/L、MgSO4·7H2O130-250mg/L、H3BO32-11mg/L、ZnSO4·7H2O3-7mg/L、
MnSO4·H2O6.5-10.5mg/L、Na2MoO4·2H2O0.11-0.21mg/L、KI0.28-0.63mg/L、
CuSO4·5H2O0.01-0.035mg/L、CoCl20.01-0.035mg/L、FeSO4·7H2O13-21mg/L、
Na2·EDTA·H2O15-27mg/L, inositol 50-1500mg/L, vitamin B10.4-0.6mg/L, glutamine
400-700mg/L, sucrose 20000-55000mg/L, 2,4 dichlorophenoxyacetic acid 0.3-2.7mg/L, sour water solution junket egg
White 350-1050mg/L, 6-benzyl aminopurine 0.05-3.1mg/L, agar 2500-7000mg/L, acetosyringone
2-40mg/L, balance of water；pH5.75-5.85.
The S1Culture medium is specific as follows：KNO32803mg/L、NH4NO3331mg/L、KH2PO4103mg/L、
CaCl2·2H2O326mg/L、MgSO4·7H2O206mg/L、H3BO39.5mg/L、ZnSO4·7H2O5.2mg/L、
MnSO4·H2O10.2mg/L、Na2MoO4·2H2O0.15mg/L、KI0.51mg/L、CuSO4·5H2O0.0125mg/L、
CoCl20.0125mg/L、FeSO4·7H2O13.93mg/L、Na2·EDTA·H2O18.65mg/L, inositol 1000mg/L,
Vitamin B10.5mg/L, glutamine 450mg/L, sucrose 30000mg/L, 2,4 dichlorophenoxyacetic acid 0.4mg/L,
Acid hydrolyzed casein 500mg/L, 6-benzyl aminopurine 0.4mg/L, agar 3000mg/L, acetosyringone 20mg/L,
Balance of water；pH5.8.
In the step (a2), the condition of the renewal cultivation is：20-25 DEG C, stand 46-50 under dark condition
Hour.
In the step (a2), the condition of the renewal cultivation is concretely：23 DEG C, stand 48 under dark condition
Hour.
Methods described may also include the steps of (a4)：After completing step (a3), larch cells,primordial is taken, shifted
To S2On culture medium, 20-25 DEG C, quiescent culture 7-10 days under dark condition.
The S2Culture medium is as follows：KNO31836-2836mg/L、NH4NO3243-360mg/L、KH2PO470-130mg/L、
CaCl2·2H2O300-450mg/L、MgSO4·7H2O130-250mg/L、H3BO32-11mg/L、ZnSO4·7H2O3-7mg/L、
MnSO4·H2O6.5-10.5mg/L、Na2MoO4·2H2O0.11-0.21mg/L、KI0.28-0.63mg/L、
CuSO4·5H2O0.01-0.035mg/L、CoCl20.01-0.035mg/L、FeSO4·7H2O13-21mg/L、
Na2·EDTA·H2O15-27mg/L, inositol 50-1500mg/L, vitamin B10.4-0.6mg/L, glutamine
400-700mg/L, sucrose 20000-55000mg/L, 2,4 dichlorophenoxyacetic acid 0.3-2.7mg/L, sour water solution junket egg
White 350-1050mg/L, 6-benzyl aminopurine 0.05-3.1mg/L, agar 2500-7000mg/L, cephalosporin
200-700mg/L, balance of water；pH5.75-5.85.
The S2Culture medium is specific as follows：KNO32803mg/L、NH4NO3331mg/L、KH2PO4103mg/L、
CaCl2·2H2O326mg/L、MgSO4·7H2O206mg/L、H3BO39.5mg/L、ZnSO4·7H2O5.2mg/L、
MnSO4·H2O10.2mg/L、Na2MoO4·2H2O0.15mg/L、KI0.51mg/L、CuSO4·5H2O0.0125mg/L、
CoCl20.0125mg/L、FeSO4·7H2O13.93mg/L、Na2·EDTA·H2O18.65mg/L, inositol 1000mg/L,
Vitamin B10.5mg/L, glutamine 450mg/L, sucrose 30000mg/L, 2,4 dichlorophenoxyacetic acid 0.4mg/L,
Acid hydrolyzed casein 500mg/L, 6-benzyl aminopurine 0.4mg/L, agar 3000mg/L, cephalosporin 400mg/L,
Balance of water；pH5.8.
The step (a4) is concretely：After completing step (a3), larch cells,primordial is taken, be transferred to S2Training
Support on base, 23 DEG C, quiescent culture 7 days under dark condition.
Methods described may also include the steps of (a5)：After completing step (a4), larch cells,primordial is taken, shifted
To S3On culture medium, 20-25 DEG C, quiescent culture 15-25 days under dark condition.
The S3Culture medium is as follows：KNO31836-2836mg/L、NH4NO3243-360mg/L、KH2PO470-130mg/L、
CaCl2·2H2O300-450mg/L、MgSO4·7H2O130-250mg/L、H3BO32-11mg/L、ZnSO4·7H2O3-7mg/L、
MnSO4·H2O6.5-10.5mg/L、Na2MoO4·2H2O0.11-0.21mg/L、KI0.28-0.63mg/L、
CuSO4·5H2O0.01-0.035mg/L、CoCl20.01-0.035mg/L、FeSO4·7H2O13-21mg/L、
Na2·EDTA·H2O15-27mg/L, inositol 50-1500mg/L, vitamin B10.4-0.6mg/L, glutamine
400-700mg/L, sucrose 20000-55000mg/L, 2,4 dichlorophenoxyacetic acid 0.3-2.7mg/L, sour water solution junket egg
White 350-1050mg/L, 6-benzyl aminopurine 0.05-3.1mg/L, agar 2500-7000mg/L, cephalosporin
200-700mg/L, the concentration of hygromycin are 3-20mg/L, balance of water；pH5.75-5.85.
The S3Culture medium is specific as follows：KNO32803mg/L、NH4NO3331mg/L、KH2PO4103mg/L、
CaCl2·2H2O326mg/L、MgSO4·7H2O206mg/L、H3BO39.5mg/L、ZnSO4·7H2O5.2mg/L、
MnSO4·H2O10.2mg/L、Na2MoO4·2H2O0.15mg/L、KI0.51mg/L、CuSO4·5H2O0.0125mg/L、
CoCl20.0125mg/L、FeSO4·7H2O13.93mg/L、Na2·EDTA·H2O18.65mg/L, inositol 1000mg/L,
Vitamin B10.5mg/L, glutamine 450mg/L, sucrose 30000mg/L, 2,4 dichlorophenoxyacetic acid 0.4mg/L,
Acid hydrolyzed casein 500mg/L, 6-benzyl aminopurine 0.4mg/L, agar 3000mg/L, cephalosporin 400mg/L,
The concentration of hygromycin is 5mg/L, balance of water；pH5.8.
The step (a5) is concretely：After completing step (a4), larch cells,primordial is taken, be transferred to S3Training
Support on base, 23 DEG C, quiescent culture 20 days under dark condition.
Methods described may also include the steps of (a6)：After completing step (a5), following operation more than twice is carried out：
The Embryogenic cell masses of survival are taken, the S is transferred to3On culture medium, 20-25 DEG C, quiescent culture 15-25 under dark condition
My god.
The step (a6) is concretely：After completing step (a5), following operation twice is carried out：Take the embryo of survival
Property cell mass, is transferred to the S3On culture medium, 25 DEG C, quiescent culture 20 days under dark condition.
In any of the above methods described, the step (a1) was carried out in the past, larch cells,primordial is first grasped as follows
Make：
(b1) larch cells,primordial is seeded to S0On culture medium, 20-25 DEG C, quiescent culture 15-20 under dark condition
My god；
(b2) after completing step (b1), picking larch cells,primordial adds Y0In culture medium, 20-25 DEG C,
Shaken cultivation under dark condition, acetosyringone is added during squamous subculture 5-7 days, addition per 7-10 days subcultures once
Larch cells,primordial is taken after acetosyringone 12-24 hours carries out the step (a1)；
The addition of acetosyringone is：Every 92 milliliters of Y0Culture medium adds 0.5-2mg acetosyringones.
The S0Culture medium is as follows：KNO31836-2836mg/L、NH4NO3243-360mg/L、KH2PO470-130mg/L、
CaCl2·2H2O300-450mg/L、MgSO4·7H2O130-250mg/L、H3BO32-11mg/L、ZnSO4·7H2O3-7mg/L、
MnSO4·H2O6.5-10.5mg/L、Na2MoO4·2H2O0.11-0.21mg/L、KI0.28-0.63mg/L、
CuSO4·5H2O0.01-0.035mg/L、CoCl20.01-0.035mg/L、FeSO4·7H2O13-21mg/L、
Na2·EDTA·H2O15-27mg/L, inositol 50-1500mg/L, vitamin B10.4-0.6mg/L, glutamine
400-700mg/L, sucrose 20000-55000mg/L, 2,4 dichlorophenoxyacetic acid 0.3-2.7mg/L, sour water solution junket egg
White 350-1050mg/L, 6-benzyl aminopurine 0.05-3.1mg/L, agar 2500-7000mg/L, balance of water；
pH5.4-6.2。
The S0Culture medium is specific as follows：KNO32803mg/L、NH4NO3331mg/L、KH2PO4103mg/L、
CaCl2·2H2O326mg/L、MgSO4·7H2O206mg/L、H3BO39.5mg/L、ZnSO4·7H2O5.2mg/L、
MnSO4·H2O10.2mg/L、Na2MoO4·2H2O0.15mg/L、KI0.51mg/L、CuSO4·5H2O0.0125mg/L、
CoCl20.0125mg/L、FeSO4·7H2O13.93mg/L、Na2·EDTA·H2O18.65mg/L, inositol 1000mg/L,
Vitamin B10.5mg/L, glutamine 450mg/L, sucrose 30000mg/L, 2,4 dichlorophenoxyacetic acid 0.4mg/L,
Acid hydrolyzed casein 500mg/L, 6-benzyl aminopurine 0.4mg/L, agar 3000mg/L, balance of water；pH5.8.
The Y0Culture medium is as follows：KNO31836-2836mg/L、NH4NO3243-360mg/L、KH2PO470-130mg/L、
CaCl2·2H2O300-450mg/L、MgSO4·7H2O130-250mg/L、H3BO32-11mg/L、ZnSO4·7H2O3-7mg/L、
MnSO4·H2O6.5-10.5mg/L、Na2MoO4·2H2O0.11-0.21mg/L、KI0.28-0.63mg/L、
CuSO4·5H2O0.01-0.035mg/L、CoCl20.01-0.035mg/L、FeSO4·7H2O13-21mg/L、
Na2·EDTA·H2O15-27mg/L, inositol 50-1500mg/L, vitamin B10.4-0.6mg/L, glutamine
400-700mg/L, sucrose 20000-55000mg/L, 2,4 dichlorophenoxyacetic acid 0.3-2.7mg/L, sour water solution junket egg
White 350-1050mg/L, 6-benzyl aminopurine 0.05-3.1mg/L, balance of water；pH5.4-6.2；
The Y0Culture medium is specific as follows：KNO32803mg/L、NH4NO3331mg/L、KH2PO4103mg/L、
CaCl2·2H2O326mg/L、MgSO4·7H2O206mg/L、H3BO39.5mg/L、ZnSO4·7H2O5.2mg/L、
MnSO4·H2O10.2mg/L、Na2MoO4·2H2O0.15mg/L、KI0.51mg/L、CuSO4·5H2O0.0125mg/L、
CoCl20.0125mg/L、FeSO4·7H2O13.93mg/L、Na2·EDTA·H2O18.65mg/L, inositol 1000mg/L,
Vitamin B10.5mg/L, glutamine 450mg/L, sucrose 30000mg/L, 2,4 dichlorophenoxyacetic acid 0.4mg/L,
Acid hydrolyzed casein 500mg/L, 6-benzyl aminopurine 0.4mg/L, balance of water；pH5.8.
The step (b1) is concretely：Larch cells,primordial is seeded to the S0On culture medium, 23 DEG C, black
Quiescent culture 15 days under dark condition.
(b2) concretely：After completing step (b1), picking larch cells,primordial adds Y0Culture medium
In, 23 DEG C, shaken cultivation under dark condition, every 7 days subcultures once, acetosyringone are added during squamous subculture 7 days,
Adding acetosyringone that larch cells,primordial is taken after 12 hours carries out the step (a1)；
The addition of acetosyringone is：Every 92 milliliters of Y0Culture medium adds 1mg acetosyringones.
The recombinational agrobacterium infects the OD of liquid600nm=0.05-0.2, concretely 0.1.
In the de- bacterium device of suction filtration described in any of the above, the separator is made up of Buchner funnel and stainless (steel) wire 1-3；
The aperture of the stainless (steel) wire is 200-500 mesh, is placed on the bottom surface of the hollow cylindrical portion of the Buchner funnel, with
Its size shape is matched.
In the de- bacterium device of suction filtration described in any of the above, the top of the Buchner funnel is that only bottom surface does not have upper bottom surface
Hollow cylindrical portion, the bottom of the Buchner funnel is pars infundibularis, hollow cylindrical portion and pars infundibularis connect in outside, including
Portion is connected by some through holes on hollow cylindrical portion bottom surface.
In the de- bacterium device of suction filtration described in any of the above, the fluid collection device is Bu Shi flasks.
In the de- bacterium device of suction filtration described in any of the above, the air extractor is manual suction pump or minipump.
In the de- bacterium device of suction filtration described in any of the above, the air extractor be in working order under the speed of exhaust be
The air extractor of 20mL/s.
In the de- bacterium device of suction filtration described in any of the above, the air extractor is that power is the air extractor of below 6W.
The present invention also protects a kind of kit of the genetic transformation for larch cells,primordial, including described in any of the above
Infect S described in liquid, any of the above1S described in culture medium, any of the above2S described in culture medium and any of the above3Culture medium.
The kit may also include the de- bacterium device of suction filtration described in any of the above.
The kit may also include S described in any of the above0Y described in culture medium and any of the above0Culture medium.
The concretely larch-tree of larch described in any of the above.
The advantage of the genetic transforming method that the present invention is provided is as follows：
1. operate simple：All liquid used are culture medium in existing larch genetic conversion system, wherein de-
Bacterium washed once will use 2-3L or so, large usage quantity；In the method that the present invention is provided, culture medium is replaced with distilled water
Carry out that bacterium is resuspended and the washing of de- bacterium, eliminate culture medium configuration, packing, the tedious steps of sterilizing；Hair of the invention
A person of good sense has broken the conventional thinking of genetic transformation operation, is extremely improved and simplifies the complexity of genetic transformation operation,
Recovered culture finds, the recovery of later stage cells,primordial is bred without influence after simplifying operation；
2. take off that bacterium is thorough, contamination rate is low：During genetic transformation is carried out to embryo callus or cells,primordial,
Need to be suspended in embryo callus or cells,primordial and infect liquid and infected, how to infecting after embryo callus subculture group
Knit or cells,primordial carries out de- bacterium and turns into primary difficulty, to solve this problem, the present inventor devises suction filtration
De- bacterium device, washing is thorough, contamination rate is low and injury very little that is being caused to callus, is conducive to the extensive of cells,primordial
Multiple culture；
3. transformation efficiency high：Process acceptor material and recombinational agrobacterium respectively with acetosyringone before infecting, contribute to
The importing of foreign gene, the genetic transformation efficiency of existing larch genetic conversion system is 0.94/g, and the present invention is carried
The genetic transformation efficiency of the method for confession is improved to 2.5/g.
The method that the present invention is provided, can be used to carry out slewing genetic improvement to larch using genetic engineering means,
For Study of Exogenous gene, expression regulation and its functional verification and embryonic development research provide technology platform in larch body.
Brief description of the drawings
Fig. 1 is the flow chart of genetic transformation.
Fig. 2 is the structural representation of the de- bacterium device of suction filtration.
Fig. 3 is the photo in embodiment 2.
Fig. 4 is the agarose gel electrophoresis figure in embodiment 2.
Specific embodiment
Following embodiment facilitates a better understanding of the present invention, but does not limit the present invention.Experiment in following embodiments
Method, unless otherwise specified, is conventional method.Test material used in following embodiments, unless otherwise specified,
It is what is be commercially available from routine biochemistry reagent shop.Quantitative test in following examples, is respectively provided with three repetitions real
Test, results averaged.
Y0Culture medium (liquid) (pH5.8)：KNO32803mg/L、NH4NO3331mg/L、KH2PO4103mg/L、
CaCl2·2H2O326mg/L、MgSO4·7H2O206mg/L、H3BO39.5mg/L、ZnSO4·7H2O5.2mg/L、
MnSO4·H2O10.2mg/L、Na2MoO4·2H2O0.15mg/L、KI0.51mg/L、CuSO4·5H2O0.0125mg/L、
CoCl20.0125mg/L、FeSO4·7H2O13.93mg/L、Na2·EDTA·H2O18.65mg/L, inositol 1000mg/L,
Vitamin B10.5mg/L, glutamine 450mg/L, sucrose 30000mg/L, 2,4 dichlorophenoxyacetic acid 0.4mg/L,
Acid hydrolyzed casein 500mg/L, 6-benzyl aminopurine 0.4mg/L, balance of water.
S0Culture medium (solid) (pH5.8)：With Y0The difference of culture medium is only that and adds agar and make its concentration be
3000mg/L。
S1Culture medium (solid) (pH5.8)：With S0The difference of culture medium is only that and adds acetosyringone and make its dense
It is 20mg/L to spend.
S2Culture medium (solid) (pH5.8)：With S0The difference of culture medium is only that and adds cephalosporin and make its concentration
It is 400mg/L.
S3Culture medium (solid) (pH5.8)：With S0The difference of culture medium is only that and adds cephalosporin and hygromycin,
The concentration of cephalosporin is 400mg/L, and the concentration of hygromycin is 5mg/L.
Infect liquid (pH5.8)：Acetosyringone 20mg/L, balance of water.
Larch-tree cells,primordial：Zhu Caihong, Li Shuigen, Qi Liwang, Han Suying；Agriculture bacillus mediated Japan's fallen leaves
Loose cells,primordial Study on Genetic Transformation,《Chinese biological engineering magazine》05 phase in 2013.
Super1300 carriers：Shanghai Bei Nuo bio tech ltd.
Agrobacterium GV3101：Ocean bio tech ltd of Beijing CHMC.
The acceptor material fresh weight of resistance rate (individual/g)=resistant cell line quantity/genetic transformation.
The acceptor material fresh weight of genetic transformation rate (individual/g)=transgenic cell line quantity/genetic transformation.
The structural representation of the de- bacterium device of suction filtration is shown in Fig. 2, including separator 1, fluid collection device 2 and air extractor
3；Separator is made up of Buchner funnel and stainless (steel) wire.The top of Buchner funnel is that only bottom surface does not have upper bottom surface
Hollow cylindrical portion, the bottom of Buchner funnel is pars infundibularis, and hollow cylindrical portion and pars infundibularis connect in outside, internally lead to
The some through holes connection crossed on hollow cylindrical portion bottom surface.The aperture of stainless (steel) wire is 200-500 mesh, is placed in the cloth
On the bottom surface of the hollow cylindrical portion of family name's funnel, matched with its size shape.Fluid collection device is Bu Shi flasks.Take out
Device of air is manual suction pump (speed of exhaust under working condition is 20mL/s).
Embodiment 1, recombinant plasmid pSuper::The preparation of the structure and recombinational agrobacterium GV3101/MIR156 of MIR156
First, recombinant plasmid pSuper::The structure of MIR156
1st, primer pair is designed
The coded sequence of miR156 precursors is more than as shown in the sequence 1 of sequence table at the two ends of its loop-stem structure sequence
Primer pair (being made up of M156F and M156R) is designed at 90bp.
M156F：5’-CCCAAGCTTTTGTACTCAGCCGACAGAA-3’；
M156R：5’-GGACTAGTCCTCTAGCGGTAAATCTCAA-3’。
2nd, the genomic DNA of larch-tree is extracted as template, is constituted into performing PCR using M156F and M156R
Amplification, obtains pcr amplification product.
3rd, the pcr amplification product obtained with restriction enzyme Hind III and Spe I double digestion steps 2, reclaims
Digestion products.
4th, restriction enzyme Hind III and Spe I double digestion Super1300 carriers, reclaim the carrier of about 10kb
Skeleton.
5th, the carrier framework of the digestion products of step 3 and step 4 is connected, obtains recombinant plasmid pSuper::MIR156.
According to sequencing result, to recombinant plasmid pSuper::MIR156 carries out structure and is described as follows：In Super1300 carriers
The sequence 1 of sequence table from the nucleosides of 5 ' end the 84th to 469 is inserted between Hind III and Spe I restriction enzyme sites
Double chain DNA molecule shown in acid.Recombinant plasmid pSuper::In MIR156, external source is started by Super promoters and is inserted
The transcription of fragment.
2nd, the preparation of recombinational agrobacterium GV3101/MIR156
By recombinant plasmid pSuper::MIR156 imports Agrobacterium GV3101, obtains recombinational agrobacterium, is named as
GV3101/MIR156。
Embodiment 2, larchen genetic transformation
The flow chart of genetic transformation is shown in Fig. 1.
First, the preparation of acceptor material and preculture
1st, larch-tree cells,primordial is seeded to S0On culture medium, 23 DEG C, quiescent culture 15 days under dark condition.
2nd, after completing step 1, picking superficial growth vigorous cells,primordial about 1g is added and 92mlY is housed0Culture medium
250ml triangular flasks in, 23 DEG C, 100rpm shaken cultivations under dark condition.Every 7 days subcultures once (each subculture
Method it is as follows：Cells,primordial about 1g is taken, is added and 92ml Y is housed0In the 250ml triangular flasks of culture medium, 23 DEG C,
100rpm shaken cultivations under dark condition).During squamous subculture 7 days, 1mg acetosyringones are added in each triangular flask,
Since the timing adding acetosyringone, take cells,primordial, as the acceptor material of genetic transformation after 12 hours.
2nd, Agrobacterium infects liquid preparation
With liquid suspension recombinational agrobacterium GV3101/MIR156 is infected, OD is obtained600nm=0.1 bacteria suspension, as Agrobacterium
Infect liquid.
3rd, infect, co-culture and resistance screening
1st, 6g steps one are taken and obtains cells,primordial (acceptor material of genetic transformation), be suspended in the agriculture bar of step 2 preparation
Bacterium is infected in liquid, is stored at room temperature 10 minutes after fully mixing.
2nd, after completing step 1, cells,primordial is collected, is transferred to S1On culture medium, 23 DEG C, stand under dark condition
Culture 48 hours.
3rd, after completing step 2, cells,primordial is taken, de- bacterium is carried out using the de- bacterium device of suction filtration, comprised the following steps that：Will
Cells,primordial pours separator into, opens manual suction pump, first with distillation water washing 4 times (each 3min), then uses
The 400mg/L cephalosporins aqueous solution washs 4 times (each 8min).
4th, after completing step 3, cells,primordial is taken, is transferred to the S for being equipped with aseptic filter paper2On culture medium, 23 DEG C,
Quiescent culture 7 days under dark condition.
5th, after completing step 4, cells,primordial is taken, is transferred to S3On culture medium, 23 DEG C, training is stood under dark condition
Support 20 days, (photo is shown in Fig. 3 to 22 Embryogenic cell masses of survival, and 1 S is transferred to per 3g cells,primordials in Fig. 33
Culture medium, displaced 6g cells,primordials altogether, each S311 Embryogenic cell masses of survival are obtained on culture medium, altogether
Obtain 22 Embryogenic cell masses of survival).
6th, after completing step 5, the Embryogenic cell masses of survival are taken, is transferred to new S3On culture medium, 25 DEG C, dark
Under the conditions of quiescent culture 20 days.
7th, after completing step 6, the Embryogenic cell masses of survival are taken, is transferred to new S3On culture medium, 25 DEG C, dark
Under the conditions of quiescent culture 20 days, (each Embryogenic cell masses is defined as a resisting cell to 15 Embryogenic cell masses of survival
System).Resistance rate is 2.5/g acceptor materials.
In above-mentioned steps 4 to step 7, without any microbiological contamination phenomenon of generation.
4th, PCR identifications
15 resistant cell lines that step 3 is obtained are identified as follows respectively：
The genomic DNA of resistant cell line is extracted, the primer pair constituted using F1 and R1 is entered performing PCR and expanded, then
Carry out 1.0% agarose gel electrophoresis.Using the genomic DNA of larch-tree cells,primordial as negative control.With weight
Group plasmid pSuper::MIR156 is used as positive control.
F1：5’-ACAGCAAGAACGGAATGC-3’；
R1：5’-GATAATCATCGCAAGACCG-3’.
Nucleotide sequence on F1 Super1300 carriers corresponding with R1, across external source Insert Fragment, if obtaining 657bp
Amplified production, PCR qualification results for the positive, the resistant cell line be the successful transgenic cell line of genetic transformation.
Agarose gel electrophoresis figure is shown in Fig. 4.In Fig. 4, Marker be followed successively by from bottom to top 300bp, 500bp, 800bp,
1000bp, 1500bp, 2000bp, 3000bp, 5000bp, plasmid represent positive control, and non-transgenic represents feminine gender
Control, No. 1 to No. 15 represents 15 resistant cell lines.
Genetic transformation rate is 2.5/g acceptor materials.
Claims (10)
1. a kind of genetic transforming method of larch cells,primordial, in turn includes the following steps：
(a1) larch cells,primordial is taken, recombinational agrobacterium is suspended in and is infected liquid and infected；
(a2) after completing step (a1), larch cells,primordial is collected, carries out renewal cultivation；
(a3) after completing step (a2), larch cells,primordial is taken, de- bacterium is carried out using the de- bacterium device of suction filtration；
The de- bacterium device of the suction filtration includes separator (1), fluid collection device (2) and air extractor (3)；
The top of the separator is the sample-adding portion (1-1) of top end opening inner hollow, and bottom is inside bottom end opening
Hollow pars infundibularis (1-2), sample-adding portion with connect outside pars infundibularis, inside connect, the inside of sample-adding portion and pars infundibularis
Junction is provided with the stainless (steel) wire (1-3) that aperture is 200-500 mesh；
The fluid collection device is closed hollow container, with upper shed and side opening；
The upper shed air tight communication of the bottom end opening of the pars infundibularis of the separator and the fluid collection device；
The side opening of the fluid collection device and air extractor air tight communication.
2. the method for claim 1, it is characterised in that：In the step (a3), the process of the de- bacterium
For：First with distillation water washing 3-5 times, each 2-5min, then washed with 300-500mg/L antibiotic solutions 3-5 times,
Each 5-10min.
3. method as claimed in claim 2, it is characterised in that：
In the step (a1), it is with infecting the liquid that liquid suspension recombinational agrobacterium is obtained that the recombinational agrobacterium infects liquid
Phase；The recombinational agrobacterium is the Agrobacterium containing target DNA；
The liquid that infects is：Acetosyringone 2-40mg/L, balance of water；pH5.75-5.85；
In the step (a1), the condition that infects is：It is stored at room temperature 5-20 minutes.
4. the method as described in any in claims 1 to 3, it is characterised in that：
In the step (a2), the renewal cultivation is in S1Carried out on culture medium；
The S1Culture medium is as follows：KNO31836-2836mg/L、NH4NO3243-360mg/L、KH2PO470-130mg/L、
CaCl2·2H2O300-450mg/L、MgSO4·7H2O130-250mg/L、H3BO32-11mg/L、ZnSO4·7H2O3-7mg/L、
MnSO4·H2O6.5-10.5mg/L、Na2MoO4·2H2O0.11-0.21mg/L、KI0.28-0.63mg/L、
CuSO4·5H2O0.01-0.035mg/L、CoCl20.01-0.035mg/L、FeSO4·7H2O13-21mg/L、
Na2·EDTA·H2O15-27mg/L, inositol 50-1500mg/L, vitamin B10.4-0.6mg/L, glutamine
400-700mg/L, sucrose 20000-55000mg/L, 2,4 dichlorophenoxyacetic acid 0.3-2.7mg/L, sour water solution junket egg
White 350-1050mg/L, 6-benzyl aminopurine 0.05-3.1mg/L, agar 2500-7000mg/L, acetosyringone
2-40mg/L, balance of water；pH5.75-5.85；
In the step (a2), the condition of the renewal cultivation is：20-25 DEG C, stand 46-50 under dark condition
Hour.
5. the method as described in any in Claims 1-4, it is characterised in that：Methods described also comprises the following steps
(a4)：After completing step (a3), larch cells,primordial is taken, be transferred to S2On culture medium, 20-25 DEG C, dark
Under the conditions of quiescent culture 7-10 days；
The S2Culture medium is as follows：KNO31836-2836mg/L、NH4NO3243-360mg/L、KH2PO470-130mg/L、
CaCl2·2H2O300-450mg/L、MgSO4·7H2O130-250mg/L、H3BO32-11mg/L、ZnSO4·7H2O3-7mg/L、
MnSO4·H2O6.5-10.5mg/L、Na2MoO4·2H2O0.11-0.21mg/L、KI0.28-0.63mg/L、
CuSO4·5H2O0.01-0.035mg/L、CoCl20.01-0.035mg/L、FeSO4·7H2O13-21mg/L、
Na2·EDTA·H2O15-27mg/L, inositol 50-1500mg/L, vitamin B10.4-0.6mg/L, glutamine
400-700mg/L, sucrose 20000-55000mg/L, 2,4 dichlorophenoxyacetic acid 0.3-2.7mg/L, sour water solution junket egg
White 350-1050mg/L, 6-benzyl aminopurine 0.05-3.1mg/L, agar 2500-7000mg/L, cephalosporin
200-700mg/L, balance of water；pH5.75-5.85.
6. method as claimed in claim 5, it is characterised in that：Methods described also comprises the following steps (a5)：It is complete
Into after step (a4), larch cells,primordial is taken, be transferred to S3On culture medium, 20-25 DEG C, quiet under dark condition
Put culture 15-25 days；
The S3Culture medium is as follows：KNO31836-2836mg/L、NH4NO3243-360mg/L、KH2PO470-130mg/L、
CaCl2·2H2O300-450mg/L、MgSO4·7H2O130-250mg/L、H3BO32-11mg/L、ZnSO4·7H2O3-7mg/L、
MnSO4·H2O6.5-10.5mg/L、Na2MoO4·2H2O0.11-0.21mg/L、KI0.28-0.63mg/L、
CuSO4·5H2O0.01-0.035mg/L、CoCl20.01-0.035mg/L、FeSO4·7H2O13-21mg/L、
Na2·EDTA·H2O15-27mg/L, inositol 50-1500mg/L, vitamin B10.4-0.6mg/L, glutamine
400-700mg/L, sucrose 20000-55000mg/L, 2,4 dichlorophenoxyacetic acid 0.3-2.7mg/L, sour water solution junket egg
White 350-1050mg/L, 6-benzyl aminopurine 0.05-3.1mg/L, agar 2500-7000mg/L, cephalosporin
200-700mg/L, the concentration of hygromycin are 3-20mg/L, balance of water；pH5.75-5.85.
7. method as claimed in claim 6, it is characterised in that：Methods described also comprises the following steps (a6)：It is complete
Into after step (a5), following operation more than twice is carried out：The Embryogenic cell masses of survival are taken, the S is transferred to3Culture
On base, 20-25 DEG C, quiescent culture 15-25 days under dark condition.
8. the method as described in any in claim 1 to 7, it is characterised in that：Carried out the step (a1) in the past,
Larch cells,primordial is first proceeded as follows：
(b1) larch cells,primordial is seeded to S0On culture medium, 20-25 DEG C, quiescent culture 15-20 under dark condition
My god；
The S0Culture medium is as follows：KNO31836-2836mg/L、NH4NO3243-360mg/L、KH2PO470-130mg/L、
CaCl2·2H2O300-450mg/L、MgSO4·7H2O130-250mg/L、H3BO32-11mg/L、ZnSO4·7H2O3-7mg/L、
MnSO4·H2O6.5-10.5mg/L、Na2MoO4·2H2O0.11-0.21mg/L、KI0.28-0.63mg/L、
CuSO4·5H2O0.01-0.035mg/L、CoCl20.01-0.035mg/L、FeSO4·7H2O13-21mg/L、
Na2·EDTA·H2O15-27mg/L, inositol 50-1500mg/L, vitamin B10.4-0.6mg/L, glutamine
400-700mg/L, sucrose 20000-55000mg/L, 2,4 dichlorophenoxyacetic acid 0.3-2.7mg/L, sour water solution junket egg
White 350-1050mg/L, 6-benzyl aminopurine 0.05-3.1mg/L, agar 2500-7000mg/L, balance of water；
pH5.4-6.2；
(b2) after completing step (b1), picking larch cells,primordial adds Y0In culture medium, 20-25 DEG C,
Shaken cultivation under dark condition, acetosyringone is added during squamous subculture 5-7 days, addition per 7-10 days subcultures once
Larch cells,primordial is taken after acetosyringone 12-24 hours carries out the step (a1)；
The Y0Culture medium is as follows：KNO31836-2836mg/L、NH4NO3243-360mg/L、KH2PO470-130mg/L、
CaCl2·2H2O300-450mg/L、MgSO4·7H2O130-250mg/L、H3BO32-11mg/L、ZnSO4·7H2O3-7mg/L、
MnSO4·H2O6.5-10.5mg/L、Na2MoO4·2H2O0.11-0.21mg/L、KI0.28-0.63mg/L、
CuSO4·5H2O0.01-0.035mg/L、CoCl20.01-0.035mg/L、FeSO4·7H2O13-21mg/L、
Na2·EDTA·H2O15-27mg/L, inositol 50-1500mg/L, vitamin B10.4-0.6mg/L, glutamine
400-700mg/L, sucrose 20000-55000mg/L, 2,4 dichlorophenoxyacetic acid 0.3-2.7mg/L, sour water solution junket egg
White 350-1050mg/L, 6-benzyl aminopurine 0.05-3.1mg/L, balance of water；pH5.4-6.2；
The addition of acetosyringone is：Every 92 milliliters of Y0Culture medium adds 0.5-2mg acetosyringones.
9. the kit of a kind of genetic transformation for larch cells,primordial, including infect liquid, S1Culture medium, S2Training
Support base and S3Culture medium；
The liquid that infects is：Acetosyringone 2-40mg/L, balance of water；pH5.75-5.85；
The S1Culture medium is as follows：KNO31836-2836mg/L、NH4NO3243-360mg/L、KH2PO470-130mg/L、
CaCl2·2H2O300-450mg/L、MgSO4·7H2O130-250mg/L、H3BO32-11mg/L、ZnSO4·7H2O3-7mg/L、
MnSO4·H2O6.5-10.5mg/L、Na2MoO4·2H2O0.11-0.21mg/L、KI0.28-0.63mg/L、
CuSO4·5H2O0.01-0.035mg/L、CoCl20.01-0.035mg/L、FeSO4·7H2O13-21mg/L、
Na2·EDTA·H2O15-27mg/L, inositol 50-1500mg/L, vitamin B10.4-0.6mg/L, glutamine
400-700mg/L, sucrose 20000-55000mg/L, 2,4 dichlorophenoxyacetic acid 0.3-2.7mg/L, sour water solution junket egg
White 350-1050mg/L, 6-benzyl aminopurine 0.05-3.1mg/L, agar 2500-7000mg/L, acetosyringone
2-40mg/L, balance of water；pH5.75-5.85；
The S2Culture medium is as follows：KNO31836-2836mg/L、NH4NO3243-360mg/L、KH2PO470-130mg/L、
CaCl2·2H2O300-450mg/L、MgSO4·7H2O130-250mg/L、H3BO32-11mg/L、ZnSO4·7H2O3-7mg/L、
MnSO4·H2O6.5-10.5mg/L、Na2MoO4·2H2O0.11-0.21mg/L、KI0.28-0.63mg/L、
CuSO4·5H2O0.01-0.035mg/L、CoCl20.01-0.035mg/L、FeSO4·7H2O13-21mg/L、
Na2·EDTA·H2O15-27mg/L, inositol 50-1500mg/L, vitamin B10.4-0.6mg/L, glutamine
400-700mg/L, sucrose 20000-55000mg/L, 2,4 dichlorophenoxyacetic acid 0.3-2.7mg/L, sour water solution junket egg
White 350-1050mg/L, 6-benzyl aminopurine 0.05-3.1mg/L, agar 2500-7000mg/L, cephalosporin
200-700mg/L, balance of water；pH5.75-5.85.
The S3Culture medium is as follows：KNO31836-2836mg/L、NH4NO3243-360mg/L、KH2PO470-130mg/L、
CaCl2·2H2O300-450mg/L、MgSO4·7H2O130-250mg/L、H3BO32-11mg/L、ZnSO4·7H2O3-7mg/L、
MnSO4·H2O6.5-10.5mg/L、Na2MoO4·2H2O0.11-0.21mg/L、KI0.28-0.63mg/L、
CuSO4·5H2O0.01-0.035mg/L、CoCl20.01-0.035mg/L、FeSO4·7H2O13-21mg/L、
Na2·EDTA·H2O15-27mg/L, inositol 50-1500mg/L, vitamin B10.4-0.6mg/L, glutamine
400-700mg/L, sucrose 20000-55000mg/L, 2,4 dichlorophenoxyacetic acid 0.3-2.7mg/L, sour water solution junket egg
White 350-1050mg/L, 6-benzyl aminopurine 0.05-3.1mg/L, agar 2500-7000mg/L, cephalosporin
200-700mg/L, the concentration of hygromycin are 3-20mg/L, balance of water；pH5.75-5.85.
10. kit as claimed in claim 9, it is characterised in that：The kit also includes the de- bacterium device of suction filtration；
The de- bacterium device of the suction filtration includes separator (1), fluid collection device (2) and air extractor (3)；
The top of the separator is the sample-adding portion (1-1) of top end opening inner hollow, and bottom is inside bottom end opening
Hollow pars infundibularis (1-2), sample-adding portion with connect outside pars infundibularis, inside connect, the inside of sample-adding portion and pars infundibularis
Junction is provided with the stainless (steel) wire (1-3) that aperture is 200-500 mesh；
The fluid collection device is closed hollow container, with upper shed and side opening；
The upper shed air tight communication of the bottom end opening of the pars infundibularis of the separator and the fluid collection device；
The side opening of the fluid collection device and air extractor air tight communication.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110004176A (en) * | 2019-04-12 | 2019-07-12 | 东北林业大学 | The construction method of hybrid larch genetic conversion system |
| CN110004176B (en) * | 2019-04-12 | 2023-03-10 | 东北林业大学 | Construction method of hybrid larch genetic transformation system |
| CN111280065A (en) * | 2020-04-07 | 2020-06-16 | 北京林业大学 | Method for regenerating larch somatic embryos |
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