CN1958802A - Plant gene expression carrier, and application - Google Patents
Plant gene expression carrier, and application Download PDFInfo
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- CN1958802A CN1958802A CN 200610114564 CN200610114564A CN1958802A CN 1958802 A CN1958802 A CN 1958802A CN 200610114564 CN200610114564 CN 200610114564 CN 200610114564 A CN200610114564 A CN 200610114564A CN 1958802 A CN1958802 A CN 1958802A
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Abstract
This invention relates to a plant gene expression vector pBinAipt-bar and its application. The plant gene expression vector is constructed by inserting ipt gene, which can code the key enzyme for cytokinin synthesis, and herbicide-resistant bar gene into pBin19 cloning site. PBinAipt-bar can be used for breeding stress-resistant plants. When transforming rice mature embryo, the transgenic rice with cold resistance and herbicide resistance is obtained. The transgenic rice has improved characters, such as increased branches, short intersegments, dwarfed plant, retarded senescence, and increased unsaturated fatty acid content.
Description
Technical field
Technical field under the present invention is a biological technical field.Further, the present invention relates to structure, the application of a kind of plant gene expression vector pBinAipt-bar.
Background technology
In areas such as China, Korea S and Japan, once serious damaging to plants caused by sudden drop in temperature just takes place in average every 3--4, and China partial area almost all take place every year, cause damage every year to reach 30--50 hundred million kg.In central and west regions, the higher Guizhou of height above sea level, hybridisation rice Chang Yin in recent years is subjected to chilling injury, causes some areas to drop in production over a large area.According to Guizhou Province Department of Agriculture statistics, 2002 because Guizhou meets with autumn wind harm rarely seen in the history, and rate ratio calendar year 2001 reduces by 11.2 hundred million kg, and the underproduction reaches 24.4%.So the research of paddy cool injury is all paid much attention in countries in the world, in the hope of cultivating anti-cold rice varieties.
Prenyltransferase (ipt) is a key enzyme in the phytokinin synthesis step, it is activated under stress conditions and causes a large amount of synthetic of phytokinin (CTK), break the balance of CTK and growth hormone in the cell, thereby plant is produced coercing the resistance of the factor.Gan is connected anti-ageing promotor SAG12 with Amasino (1995) with the ipt gene, construct the PSAG12ipt of self regulating and control system of a delaying sanility, delays plant senescence by regulation and control plant leaf cell fission cellulose content.This cover system has all obtained checking in various plants such as tobacco, tomato, green vegetables, wheat, jielu grass, paddy rice.In addition, the plant tissue specific expressing promoter starts the ipt gene or itself and other gene constructed fusion gene genetic transformation plant is also had the report of success.
The bar gene source is the gene of anti-Glufosinate ammonium class weedicide in streptomyces hygroscopicus (Streptomyceshygroscopicus), both can be used as marker gene in transgenic research, can be used as functional gene again; The bar gene is imported paddy rice in seed removal of impurities guarantor is pure, have very big application potential.The report that the Bar gene is imported to paddy rice is more, and the commentaries on classics Bar trans-genetic hybrid rice of South China Botanical Garden has entered the production test stage at present.
Also do not find to change simultaneously the plant report that above-mentioned two kinds of genes are arranged on the prior art.
Summary of the invention
The present invention is directed to the blank in the above-mentioned field, use ipt gene and anti-herbicide gene (bar) gene fusion construct, change in the plant, obtain not only to resist the plant of cold but also antiweed.
A kind of plant gene expression vector is characterized in that: insert synthetic key enzyme ipt gene of phytokinin and antiweed bar gene at the pBin19 cloning site.
The application of said gene expression vector in transgenic plant.
Described application is above-mentioned expression vector to be transformed relevant plant make it to produce resistance.
Described method for transformation is agrobacterium-mediated transformation, pollen tube passage method, cotransformation method or particle bombardment.
Described plant is a monocotyledons.
Described monocotyledons is paddy rice, corn or turfgrass.
Described method for transformation is an agrobacterium-mediated transformation, and described plant is a paddy rice.
The method of described agriculture bacillus mediated transgenic paddy rice comprises the steps:
A, callus of induce: remove mature seed grain husk shell,, soak 15min with 0.1%HgCl again, use sterile water wash 5 times with 75% alcohol-pickled 90s.Suck dry moisture is connected to callus induction on the inducing culture, and per 15 days subcultures are once on subculture medium again.
B, dip-dye and screening: Agrobacterium EHA105 28 ℃ of concussions in the YEP substratum that contains 50mg/L-kantlex and 20mg/L-Rifampin that will prepare expression vector pBinAipt-bar are cultured to OD600 and are about 0.7 o'clock collection bacterium, with the resuspended thalline of resuspended substratum.Select fresh and tender callus and in the resuspended liquid of bacterium liquid, soak 20min, blot bacterium liquid with aseptic filter paper and be connected on the common substratum, change on the screening culture medium after three days, keep selecting to press subculture 2 times.
C, break up, take root, transplant: will be connected on the division culture medium at the callus under the survival on the screening culture medium, pre-differentiation is about 10 days under dark culture condition, is transferred under the light.Move in the root media hardening, transplanting when seedling grows to the 10cm left and right sides during to the seedling 2cm left and right sides.
Inducing culture is: NB, 2.0mg/L 2,4 dichlorophenoxyacetic acid, pH5.8.
Subculture medium is: NB, 2.0mg/L 2,4 dichlorophenoxyacetic acid, 0.5mg/L 6-benzylaminopurine, pH5.8.
Screening culture medium is: NB, 2.0mg/L 2,4 dichlorophenoxyacetic acid, 4.0mg/L Glufosinate ammonium, pH5.8.
Resuspended substratum: NB (no agar), 100 μ mol/L Syringylethanones.
Substratum is altogether: NB, 2.0mg/L 2,4 dichlorophenoxyacetic acid, 100 μ mol/L Syringylethanones, pH5.2.
Division culture medium is: NB, 2.0mg/L 6-benzylaminopurine, 0.5mg/L kinetin, 2.0mg/L Glufosinate ammonium, pH5.8.
Root media is: 1/2N6,0.5mg/L naphthylacetic acid, 2.0mg/L Glufosinate ammonium, pH5.8.
Described NB is made up of following composition: a large amount of and micro-in the N6 substratum, the organic element in the B5 medium, 300mg/L caseinhydrolysate, 500mg/L proline(Pro), 500mg/L glutamine, 30g/L sucrose and 8g/L agar.
Described culture condition is: induce, subculture, screening be dark cultivation; Differentiation, bud propagation and take root and be that illumination cultivation, light intensity are 4000lux, 14h/d.
The present invention has made up bivalent expression carrier pBinAipt-bar, and it is transformed plant, makes it obtain the effect of not only anti-cold but also antiweed.Transfer-gen plant shows branch and increases in addition, and internode shortens, and plant is downgraded and delays feature such as vegetative organ aging.The present invention utilizes the paddy rice mature embryo to carry out genetic transformation and obtain transfer-gen plant for material.In detecting, the fatty acid content to transgenic paddy rice finds that its unsaturated fatty acids obviously increases.The present invention combines by transgenosis and traditional breeding method means, has reached the purpose of breed improvement.
The transgenic paddy rice saturated fatty acid content slightly changes compared with the control, but difference is not obvious.Unsaturated fatty acidss such as alpha-linolenic acid, oleic acid, linolic acid then obviously increase.Each transgenic line alpha-linolenic acid content all is higher than contrast, and wherein the R527ib1 strain is that content increases to 1.77% by 1.60%; Linoleic acid content also shows as increase, and the EY105ib00 strain is that linoleic relative content has increased by 2.2%.And the EY105ib00 strain to be oleic acid content reduce, all the other strains systems all increase.Illustrate to import the saturated fatty acid content that does not change paddy rice behind the ipt gene, and its unsaturated fatty acid content significantly increases.The film transformation temperature be to a great extent film fat, particularly membrane phospholipid lipid acid determined, the fatty acid content increase can reduce the film transformation temperature, guarantees the normal physiological metabolism function of vegetable cell.
Transform with rataria and to compare, mature embryo transforms difficulty of callus of induce, and the genetic transformation rate is also lower, but mature embryo can draw materials at all seasons, and rataria only in the middle of 1 year the specific time draw materials, limited the widespread use of rataria as the genetic transformation explant.It is that material carries out genetic transformation and obtains transfer-gen plant that the present invention adopts mature embryo.According to method provided by the invention the genetic transformation rate of paddy rice is reached 9.7%.
Description of drawings
Fig. 1: expression vector pBinAipt-bar structural representation of the present invention
Fig. 2: change the pBinAipt-bar rice plant
Fig. 3: transgenic paddy rice cold resistance qualification result
1a is a transgenic paddy rice before the deepfreeze, and 1b is a paddy rice adjoining tree before the deepfreeze, and 2a is a transgenic paddy rice after the deepfreeze, paddy rice adjoining tree after the 2b deepfreeze.
Fig. 4: transgenic paddy rice and the contrast heading stage potted plant figure: a is a transfer-gen plant among the figure, and b is an adjoining tree.
Fig. 5: transgenic paddy rice and the experiment of contrast antiweed: a is a transfer-gen plant among the figure, and b is an adjoining tree.
Embodiment
The present invention is described in further detail below in conjunction with embodiment.
The structure of embodiment 1 bivalent expression carrier pBinAipt-bar:
The ipt gene source is in agrobacterium tumefaciens in this research, and the bar gene source is in streptomyces hygroscopicus (Streptomyceshygroscopicus), and one of used promotor is constitutive promoter pNOS, from the Ti-plasmids rouge alkali synthetase gene; Terminator is a NOS3 ' sequence, derives from the terminator sequence of Ti-plasmids rouge alkali synthetase gene.Another promotor act comes from paddy rice, and terminator also is NOS3 ' sequence.Expression element act-ipt-nos and Pnos-bar-nos are inserted pBin19.See Fig. 1.
The genetic transformation of embodiment 2 paddy rice mature embryos:
The Agrobacterium EHA105 28 ℃ of concussions in the YEP substratum that contains 50mg/L-kantlex and 20mg/L-Rifampin that prepare expression vector pBinAipt-bar are cultured to OD600 are about 0.7 o'clock collection bacterium, with the resuspended thalline of NBD2-As.Select fresh and tender callus and soak in the resuspended liquid of bacterium liquid (OD600 about 0.7), constantly shake therebetween, the 15min taking-up is blotted bacterium liquid with aseptic filter paper and is connected to the last cultivation of the solid medium NBD2-As that is covered with aseptic filter paper 3 days.Callus after cultivating is altogether transferred on the inducing culture recovery media, changes screening culture medium (PPT 4.0mg/L) after two weeks over to.Most of callus browning death after about 30 days, but the callus of brownization of part can grow the granular fresh callus of part.This part callus is transferred on new screening culture medium, change NBK division culture medium (PPT 2.0mg/L) after 15 days over to and go up cultivation.Begin to occur green point after 1 week, 3 weeks back formation budlet, budlet grew up to whole plant in about 1 month in cultivation on the root media.(seeing Fig. 2, Fig. 4)
The cold resistance of experimental example 1, transgenic rice plant is identified
Through the T that detects
0Carry out selfing for plant and obtain T
1, when being cultured to the 10cm left and right sides, transfer-gen plant and adjoining tree place 4 ℃ of growth cabinets to survey its cold resistance, and the result " sees Fig. 3 ".Wherein scheming a is the preceding transgenic paddy rice of deepfreeze and the growing state of contrast, and figure b is the growing state of material after the deepfreeze, and a left side is a transfer-gen plant among the figure, and the right side is an adjoining tree.
Experimental example 2, antiweed Performance Testing
Transgenic paddy rice and adjoining tree are cultured to the 1 core phase of 3 leaves, and with 0.25% Glufosinate ammonium spray solution plant, adjoining tree death after 5 days, and transfer-gen plant well-grown, the result " sees Fig. 5 ".
Experimental example 3, transgenic rice plant seed fatty acid compositional analysis
EY105, R527 are not genetically modified tissue cultured seedling, and EY105ib20, EY105ib00, R527ib1, R527ib2 are changes ipt-bar gene T3 for plant.
Measure the fatty acid content of four transgenic paddy rices and adjoining tree, measurement result is seen " table 1 ".The saturated fatty acid content no significant difference of transgenic paddy rice and contrast, unsaturated fatty acidss such as oleic acid, linolic acid, alpha linolenic acid then obviously increase, and wherein the EY105ib00 strain is that linoleic phase content has increased by 2.2%.
Table 1 transgenic paddy rice lipid acid is measured
| Lipid acid | Relative content (%) | |||||
| EY105 | EY105ib20 | EY105ib00 | R527 | R527ib1 | R527ib2 | |
| Myristic acid C 14:0 15 carbonic acid C 15:0 palmitic acid C 16:0 palmitoleic acid C 16:1 17 carbonic acid C 17:0 stearic acid C 18:0 oleic acid C 18:1 linoleic acid C 18:2 alpha linolenic acid C 18:3 arachidic acid C 20:0 20 carbon monoenoic acid C 20:1 behenic acid C 22:0 tetracosa carbon C 24:0 | 0.30 0.13 17.96 0.23 0.05 1.60 35.53 40.04 1.42 0.47 0.57 0.36 0.56 | 0.35 0.03 17.06 0.28 0.04 1.80 33.68 41.99 1.59 0.50 0.48 0.27 0.68 | 0.39 0.11 17.53 0.18 0.08 1.60 32.40 42.24 1.54 0.44 0.81 0.33 1.14 | 0.49 0.10 19.22 0.22 0.21 2.79 38.81 33.97 1.60 0.77 0.42 0.38 0.75 | 0.53 0.04 18.66 0.28 0.06 3.03 38.02 34.10 1.77 0.85 0.47 0.40 1.11 | 0.50 0.03 19.04 0.32 0.04 2.65 38.63 34.33 1.62 0.76 0.40 0.42 0.80 |
Claims (10)
1. a plant gene expression vector is characterized in that: insert synthetic key enzyme ipt gene of phytokinin and antiweed bar gene at the pBin19 cloning site.
2. the application of claim 1 expression vector in transgenic plant.
3. application according to claim 2 is characterized in that the described plant gene expression vector of claim 1 is transformed relevant plant makes it to produce resistance.
4. application according to claim 3, described method for transformation are agrobacterium-mediated transformation, pollen tube passage method, cotransformation method or particle bombardment.
5. application according to claim 3, described plant are monocotyledons.
6. application according to claim 5, described monocotyledons are paddy rice, corn or turfgrass.
7. application according to claim 6, described method for transformation are agrobacterium-mediated transformation, and described plant is a paddy rice.
8. application according to claim 7, the method for described agriculture bacillus mediated transgenic paddy rice comprises the steps:
A, callus of induce: remove mature seed grain husk shell,, soak 15min with 0.1%HgCl again with 75% alcohol-pickled 90s, usefulness sterile water wash 5 times, suck dry moisture is connected to callus induction on the inducing culture, and per 15 days subcultures are once on subculture medium again.
B, dip-dye and screening: Agrobacterium EHA105 28 ℃ of concussions in the YEP substratum that contains 50mg/L-kantlex and 20mg/L-Rifampin that will prepare expression vector pBinAipt-bar are cultured to OD600 and are about 0.7 o'clock collection bacterium, with the resuspended thalline of resuspended substratum, select fresh and tender callus and in the resuspended liquid of bacterium liquid, soak 20min, blotting bacterium liquid with aseptic filter paper is connected on the common substratum, change on the screening culture medium after three days, keep selecting to press subculture 2 times.
C, break up, take root, transplant: will be connected on the division culture medium at the callus under the survival on the screening culture medium, pre-differentiation is about 10 days under dark culture condition, be transferred under the light, move in the root media hardening, transplanting when seedling grows to the 10cm left and right sides during to the seedling 2cm left and right sides.
9. application according to claim 8, described inducing culture is: NB, 2.0mg/L 2,4 dichlorophenoxyacetic acid, pH5.8; Subculture medium is: NB, 2.0mg/L 2,4 dichlorophenoxyacetic acid, 0.5mg/L 6-benzylaminopurine, pH5.8; Screening culture medium is: NB, 2.0mg/L 2,4 dichlorophenoxyacetic acid, 4.0mg/L Glufosinate ammonium, pH5.8; Resuspended substratum: NB (no agar), 100 μ mol/L Syringylethanones; Substratum is altogether: NB, 2.0mg/L 2,4 dichlorophenoxyacetic acid, 100 μ mol/L Syringylethanones, pH5.2; Division culture medium is: NB, 2.0mg/L 6-benzylaminopurine, 0.5mg/L kinetin, 2.0mg/L Glufosinate ammonium, pH5.8; Root media is: 1/2N6,0.5mg/L naphthylacetic acid, 2.0mg/L Glufosinate ammonium, pH5.8; Described NB is made up of following composition: a large amount of and micro-in the N6 substratum, the organic element in the B5 medium, 300mg/L caseinhydrolysate, 500mg/L proline(Pro), 500mg/L glutamine, 30g/L sucrose and 8g/L agar.
10. application according to claim 8, described culture condition is: induce, subculture, screening be dark cultivation; Differentiation, bud propagation and take root and be that illumination cultivation, light intensity are 4000lux, 14h/d.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105039389A (en) * | 2015-06-29 | 2015-11-11 | 广东省农业科学院作物研究所 | Sugarcane carrier-free frame transgenic method |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN105039389A (en) * | 2015-06-29 | 2015-11-11 | 广东省农业科学院作物研究所 | Sugarcane carrier-free frame transgenic method |
| CN105039389B (en) * | 2015-06-29 | 2020-07-28 | 广东省农业科学院作物研究所 | Sugarcane carrier-free frame transgenic method |
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