CN105002209A - Method for improving transgenosis efficiency of cotton - Google Patents
Method for improving transgenosis efficiency of cotton Download PDFInfo
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- CN105002209A CN105002209A CN201510519959.0A CN201510519959A CN105002209A CN 105002209 A CN105002209 A CN 105002209A CN 201510519959 A CN201510519959 A CN 201510519959A CN 105002209 A CN105002209 A CN 105002209A
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- plasmid dna
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Abstract
The invention discloses a method for improving the transgenosis efficiency of cotton. The method comprises the following steps: after mixing restriction enzyme digestion DNA with a plasmid DNA protective agent, introducing a target gene into cotton through the pollen tube pathway method by using lipidosome packaging to carry toxic protein genes on Ti plasmids. The method can effectively improve the transgenosis efficiency.
Description
Technical field
The present invention relates to transgenic field, a kind of concrete method improving cotton transgenic efficiency.
Background technology
Utilize transgenic technology foreign gene to be imported cotton cells and can be improvement cotton quality, obtain the important channel of new kind matter by genetic stability.Agrobacterium tumefaciens-mediated transformation and pollen tube (Pollen-Tube Pathway Method) introductory technique is mainly contained in cotton transgenic.After Umbeck in 1987 utilizes agrobacterium-mediated transformation that neomycin phosphotransferase gene nap ll and paraxin second phthalidyl transferase gene CAT is proceeded to cotton gene composition merit first, be widely used with agriculture bacillus mediated Cotton Transformation method.The genetic transformation mechanism of carrying Ti-plasmids about Agrobacterium after deliberation more clearly, and have obvious advantage, if be stably incorporated in host genome, copy number is lower, meets mendelian inheritance etc.But, this method needs host plant to occur and plant regeneration process through somatic embryo, great restriction is subject in the application of cotton transgenic technology, because cotton somatic embryos fetal hair is raw and plant regeneration is very large by variety and genetype restriction, most of hirsutum cultivar that production is applied are difficult to obtain conversion very soon by this method.Therefore, combine the conversion advantage of Ti-plasmids with other transgenic approach a kind of effective approach of can yet be regarded as.
Pollen tube passage method is not by the restriction of cotton variety, and cotton Floral organ structure is very suitable for the channel genes mode of pollen-tube pathway method, and required equipment condition is few, simple to operate, is subject to the favor of investigator and technician.But utilize the DNA molecular that how direct pollen tube pathway importing be exposed, maximum defect is that transformation mechanism is indefinite, transformation efficiency is low and unstable, to lose and the hereditary segregation ratio of most offspring changes greatly there is gene after the plantation of several generations, there is heredity and be separated the problem such as diversity.Therefore be how one of investigator's target of making great efforts in conjunction with Ti-plasmids configuration of superiorities and pollen tube passage method operational advantage.
At present, the Plastid transformation of existing research and utilization pollen tube passage method injection containing T-border, but efficiency is lower.Research show to utilize Agrobacterium carry T-DNA transform after integration efficiency will far away higher than the integration efficiency of Bombardment-Mediated Transformation method.Therefore, integration efficiency may be relevant with the Agrobacterium toxalbumin in T-mixture.Have a lot of Agrobacterium toxalbumin to participate in T-DNA integration process, wherein T-DNA pass through host cell core and be incorporated into that host DNA plays a crucial role albumen have VirD1, VirD2 and VirE2, VirD
1, VirD
2the tumor-necrosis factor glycoproteins of 25 bases in T-DNA border on Ti-plasmids is combined, and makes it loose, VirD
2cut at the tumor-necrosis factor glycoproteins place of border sequence, produce T-DNA strand and covalent attachment tight with right margin, and and VirE
2form complex body.Complex body is at VirD
2and VirE
2under nuclear localization signal (NLS) guides, with VirD
2be incorporated in host genome for guide's transhipment enters nucleus.Also have in addition and T-DNA integration mechanism is created " agrolistic " (AgrolisTic) in conjunction with particle bombardment, will the transient expression vector of virD1, virD2 be taken, utilize particle gun to squeeze in vegetable cell with the T-DNA carrier carrying goal gene.Substantially increase the efficiency of transgenosis.But particle bombardment is still subject to the puzzlement of plant regeneration problem in cotton gene importing.
Summary of the invention
For solving the problem, the invention provides a kind of method improving cotton transgenic efficiency, it effectively can improve the bolling rate of the bright and transgene cotton of transgene efficiency.
For achieving the above object, the technical scheme that the present invention takes is:
Improve a method for cotton transgenic efficiency, comprise the steps;
S1, to operate on ice, Agrobacterium toxalbumin VirD1, VirD2, VirE2 concentration after expression and purification is adjusted to 100 μ g/ μ l respectively;
S2, build exogenous plasmid dna with being connected to after digestion with restriction enzyme DNA on pBin438 plasmid;
S3, be resuspended in plasmid DNA protective material by gained exogenous plasmid dna, the concentration obtaining exogenous plasmid dna is the mixed solution of 1 μ g/ μ L;
S4, one or more the totally 2 μ l got in step S1 gained solution, add the mixed solution of the step S3 gained containing 8 μ l exogenous plasmid dnas successively, after 6 μ l liposome mixing, add in the damping fluid of 30 μ l, place after 2 hours for 4 DEG C, take out, by pollen tube passage method, goal gene is imported in cotton;
Wherein, described damping fluid comprises 5-15mmol/L Tutofusin tris-hydrochloric acid, 0.4-0.6mmol/L diaminoethanes tetraacethyl, 5-15mmol/L sodium-chlor, 0.015 μ g μ l
-1-0.055 μ g μ l
-1ethylmethane sulfonate.
Wherein, plasmid DNA protective material is by aluminum hydroxide sol protective material, calcium phosphate protective material and Tween20 protective material 1: 2: 1 mixing gained by volume.
Wherein, restriction enzyme is restriction enzyme Hind III.+EcoR I enzyme.
The present invention has following beneficial effect:
Entering with the foreign DNA of liposome can from the degraded of intracellular nucleic acid enzyme in the process of cell, simultaneously due to sperm enter ovum time, now cell is similar to plasmic state, and the foreign gene after liposome packaging is more easily integrated into ovum inside.Liposome packaging has very large effect to the stability of Agrobacterium toxalbumin, avoids some composition in plant tissue to the degraded of allogenic material.Contrast adds the transformation efficiency between the combination of liposome, the combination finding to add VirD1, VirD2, VirE2 tri-kinds of Agrobacterium toxalbumin significantly improves than the successful ratio of the combination transgenosis only adding VirD1, VirD2, bring up to 9.7% from 6.8%, the combination only adding VirD1, VirD2 two kinds of Agrobacterium toxalbumin is also high than the combined efficiency not adding any Agrobacterium toxalbumin; After importing cotton by digestion with restriction enzyme DNA, the aberration rate of offspring imports than global DNA and significantly improves, and steady speed is fast; Adopt aluminum hydroxide sol, calcium phosphate and Tween20 as exogenous plasmid dna protective material respectively, improve transformation efficiency; 0.015 μ g μ l is with the addition of in damping fluid
-1-0.055 μ g μ l
-1ethylmethane sulfonate, after testing, average conversion improves twice than the average conversion of traditional damping fluid.
Embodiment
In order to make objects and advantages of the present invention clearly understand, below in conjunction with embodiment, the present invention is further elaborated.Should be appreciated that specific embodiment described herein only in order to explain the present invention, be not intended to limit the present invention.
Embodiment 1
S1, to operate on ice, Agrobacterium toxalbumin VirD1, VirD2, VirE2 concentration after expression and purification is adjusted to 100 μ g/ μ l respectively;
S2, cut DNA with restriction enzyme Hind III.+EcoR I enzyme enzyme after be connected on pBin438 plasmid and build exogenous plasmid dna;
S3, be resuspended in plasmid DNA protective material by gained exogenous plasmid dna, the concentration obtaining exogenous plasmid dna is the mixed solution of 1 μ g/ μ L; Plasmid DNA protective material is by aluminum hydroxide sol protective material, calcium phosphate protective material and Tween20 protective material 1: 2: 1 mixing gained by volume;
S4, one or more the totally 2 μ l got in step S1 gained solution, add the mixed solution of the step S3 gained containing 8 μ l exogenous plasmid dnas successively, after 6 μ l liposome mixing, add in the damping fluid of 30 μ l, place after 2 hours for 4 DEG C, take out, by pollen tube passage method, goal gene is imported in cotton; Described damping fluid comprises 5mmol/L Tutofusin tris-hydrochloric acid, 0.4mmol/L diaminoethanes tetraacethyl, 5mmol/L sodium-chlor, 0.015 μ g μ l
-1ethylmethane sulfonate.
Embodiment 2
S1, to operate on ice, Agrobacterium toxalbumin VirD1, VirD2, VirE2 concentration after expression and purification is adjusted to 100 μ g/ μ l respectively;
S2, cut DNA with restriction enzyme Hind III.+EcoR I enzyme enzyme after be connected on pBin438 plasmid and build exogenous plasmid dna;
S3, be resuspended in plasmid DNA protective material by gained exogenous plasmid dna, the concentration obtaining exogenous plasmid dna is the mixed solution of 1 μ g/ μ L; Plasmid DNA protective material is by aluminum hydroxide sol protective material, calcium phosphate protective material and Tween20 protective material 1: 2: 1 mixing gained by volume;
S4, one or more the totally 2 μ l got in step S1 gained solution, add the mixed solution of the step S3 gained containing 8 μ l exogenous plasmid dnas successively, after 6 μ l liposome mixing, add in the damping fluid of 30 μ l, place after 2 hours for 4 DEG C, take out, by pollen tube passage method, goal gene is imported in cotton; Described damping fluid comprises 15mmol/L Tutofusin tris-hydrochloric acid, 0.6mmol/L diaminoethanes tetraacethyl, 15mmol/L sodium-chlor, 0.055 μ g μ l
-1ethylmethane sulfonate.
Embodiment 3
S1, to operate on ice, Agrobacterium toxalbumin VirD1, VirD2, VirE2 concentration after expression and purification is adjusted to 100 μ g/ μ l respectively;
S2, cut DNA with restriction enzyme Hind III.+EcoR I enzyme enzyme after be connected on pBin438 plasmid and build exogenous plasmid dna;
S3, be resuspended in plasmid DNA protective material by gained exogenous plasmid dna, the concentration obtaining exogenous plasmid dna is the mixed solution of 1 μ g/ μ L; Plasmid DNA protective material is by aluminum hydroxide sol protective material, calcium phosphate protective material and Tween20 protective material 1: 2: 1 mixing gained by volume;
S4, one or more the totally 2 μ l got in step S1 gained solution, add the mixed solution of the step S3 gained containing 8 μ l exogenous plasmid dnas successively, after 6 μ l liposome mixing, add in the damping fluid of 30 μ l, place after 2 hours for 4 DEG C, take out, by pollen tube passage method, goal gene is imported in cotton; Described damping fluid comprises 10mmol/L Tutofusin tris-hydrochloric acid, 0.5mmol/L diaminoethanes tetraacethyl, 10mmol/L sodium-chlor, 0.035 μ g μ l
-1ethylmethane sulfonate.
Compare between the combination of liposome packaging, the transgene efficiency adding Agrobacterium toxalbumin significantly improves a lot, the two kinds of combinations adding toxicity albumen improve 5.4% and 8.3% respectively than the combination not adding toxalbumin, entering with the foreign DNA of liposome can from the degraded of intracellular nucleic acid enzyme in the process of cell, simultaneously due to sperm enter ovum time, now cell is similar to plasmic state, and the foreign gene after liposome packaging is more easily integrated into ovum inside.Liposome packaging has very large effect to the stability of Agrobacterium toxalbumin, avoids some composition in plant tissue to the degraded of allogenic material.Contrast adds the transformation efficiency between the combination of liposome, the combination finding to add VirD1, VirD2, VirE2 tri-kinds of Agrobacterium toxalbumin significantly improves than the successful ratio of the combination transgenosis only adding VirD1, VirD2, bring up to 9.7% from 6.8%, the combination only adding VirD1, VirD2 two kinds of Agrobacterium toxalbumin is also high than the combined efficiency not adding any Agrobacterium toxalbumin; Adopt aluminum hydroxide sol, calcium phosphate and Tween20 as exogenous plasmid dna protective material respectively, improve transformation efficiency; 0.015 μ g μ l is with the addition of in damping fluid
-1-0.055 μ g μ l
-1ethylmethane sulfonate, after testing, average conversion improves twice than the average conversion of traditional damping fluid.
The above is only the preferred embodiment of the present invention; it should be pointed out that for those skilled in the art, under the premise without departing from the principles of the invention; can also make some improvements and modifications, these improvements and modifications also should be considered as protection scope of the present invention.
Claims (4)
1. improve a method for cotton transgenic efficiency, it is characterized in that, comprise the steps;
S1, to operate on ice, Agrobacterium toxalbumin VirD1, VirD2, VirE2 concentration after expression and purification is adjusted to 100 μ g/ μ l respectively;
S2, build exogenous plasmid dna with being connected to after digestion with restriction enzyme DNA on pBin438 plasmid;
S3, be resuspended in plasmid DNA protective material by gained exogenous plasmid dna, the concentration obtaining exogenous plasmid dna is the mixed solution of 1 μ g/ μ L;
S4, one or more the totally 2 μ l got in step S1 gained solution, add the mixed solution of the step S3 gained containing 8 μ l exogenous plasmid dnas successively, after 6 μ l liposome mixing, add in the damping fluid of 30 μ l, place after 2 hours for 4 DEG C, take out, by pollen tube passage method, goal gene is imported in cotton.
2. a kind of method improving cotton transgenic efficiency according to claim 1, it is characterized in that, described damping fluid comprises 5-15mmol/L Tutofusin tris-hydrochloric acid, 0.4-0.6mmol/L diaminoethanes tetraacethyl, 5-15mmol/L sodium-chlor, 0.015 μ g μ l
-1-0.055 μ g μ l
-1ethylmethane sulfonate.
3. a kind of method improving cotton transgenic efficiency according to claim 1, is characterized in that, plasmid DNA protective material is by aluminum hydroxide sol protective material, calcium phosphate protective material and Tween20 protective material 1: 2: 1 mixing gained by volume.
4. a kind of method improving cotton transgenic efficiency according to claim 1, is characterized in that, restriction enzyme is restriction enzyme Hind III.+EcoR I enzyme.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105543173A (en) * | 2016-01-30 | 2016-05-04 | 新乡医学院第一附属医院 | Solid tumor tissue digestive juice |
| CN105969793A (en) * | 2016-05-10 | 2016-09-28 | 广西兆和种业有限公司 | Method for breeding rice |
| CN112458114A (en) * | 2020-12-25 | 2021-03-09 | 苏州农业职业技术学院 | Method for rapidly identifying functions of genes related to fruit color development of pepper fruits |
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Patent Citations (7)
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| CN1197482A (en) * | 1995-09-25 | 1998-10-28 | 诺瓦提斯公司 | Improved integration of exogenous DNA delivered to eukaryoltic cells |
| US6051409A (en) * | 1995-09-25 | 2000-04-18 | Novartis Finance Corporation | Method for achieving integration of exogenous DNA delivered by non-biological means to plant cells |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105543173A (en) * | 2016-01-30 | 2016-05-04 | 新乡医学院第一附属医院 | Solid tumor tissue digestive juice |
| CN105969793A (en) * | 2016-05-10 | 2016-09-28 | 广西兆和种业有限公司 | Method for breeding rice |
| CN112458114A (en) * | 2020-12-25 | 2021-03-09 | 苏州农业职业技术学院 | Method for rapidly identifying functions of genes related to fruit color development of pepper fruits |
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Application publication date: 20151028 |