CN1995359A - Agrobacterium mediated large size tomato transformation method - Google Patents
Agrobacterium mediated large size tomato transformation method Download PDFInfo
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- CN1995359A CN1995359A CN 200610130675 CN200610130675A CN1995359A CN 1995359 A CN1995359 A CN 1995359A CN 200610130675 CN200610130675 CN 200610130675 CN 200610130675 A CN200610130675 A CN 200610130675A CN 1995359 A CN1995359 A CN 1995359A
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Abstract
The invention discloses a gene conversion method of large-scale tomato through agricillin, which is characterized by the following: adopting leaf as explant; improving germinating rate; increasing transition survival rate.
Description
[technical field]
The present invention relates in the bioengineering field plant be carried out the method for gene transformation, particularly relate to the method for tomato being carried out gene transformation, especially relate to the agriculture bacillus mediated method that large size tomato is carried out gene transformation of a kind of usefulness.
[background technology]
The first transgenic plant are cultivated successfully in the nineteen eighty-three world, Horch had created the Ye Panfa in the Agrobacterium-mediated Transformation in 1985, agrobacterium-mediated transformation, virus-mediated method, PEG mediated method, electric shock perforation method, microinjection, pollen tube passage method, supersonic method, particle bombardment etc. have henceforth been set up respectively, the species of transgenosis success constantly enlarge, more than 50 species that relate to 35 sections, totally 120 various plants.Through for many years the practice and the survival of the fittest, most method for transformation are progressively abandoned, have formed with agrobacterium-mediated transformation and particle bombardment prevailing two big plant transgene systems.Up to now, the transgenic plant of media agrobacterium co-cultivation acquisition account for transgenic plant sum about 85%.Dicotyledons is the natural host of Agrobacterium, utilizes Agrobacterium Ti-plasmids mediated method, has set up the gene transfer system of multiple dicotyledons, and the foreign gene that some are good changes dicotyledons over to, has bred transformed variety.
Tomato is a kind of typical dicotyledons, and its unique local flavor, abundant nutrition, bright colour and special medical value are attracting increasing human consumer.Therefore, utilize biotechnology that processing tomato is improved, in the ascendant with the research work that obtains the high-quality tomato variety.
[summary of the invention]
The purpose of this invention is to provide a kind of efficient, stable, easy agriculture bacillus mediated method of large size tomato being carried out gene transformation of utilizing.This method had overcome in the large-scale in the past plantation tomato conversion easy brownization of explant that occurs easily well, and the rate of sprouting is low, took root for being difficult to and moving shortcomings such as native surviving rate is low, had higher transformation efficiency.
Technical scheme of the present invention is a kind of method for transformation of agriculture bacillus mediated large size tomato, it is characterized in that it being that tomato explant and the Agrobacterium that contains the Ti-plasmids carrier are cultivated altogether, described Ti-plasmids carrier carries one or more foreign genes, foreign gene on the plasmid vector is transferred in the acceptor gene group, obtains transfer-gen plant through the plant transformation tissue culture.
The invention has the beneficial effects as follows: the present invention as explant, carries out tomato conversion by agrobacterium mediation method with cotyledon, is applicable to various large size tomatos, has higher transformation efficiency.This method has overcome easy brownization of explant that occurred easily in the large-scale in the past plantation tomato conversion well, the rate of sprouting is low, taking root is difficult to and moves shortcomings such as native surviving rate is low, for being the molecular biology research of material with the large size tomato and utilizing genetic engineering technique to carry out new quality variety the strong instrument that provides is provided.Utilize the method for above Agrobacterium-mediated Transformation tomato of setting up, the antisense RNA expression vector of LeCOP1LIKE gene has been imported among the large-scale processing tomato UC82B, detect by PCR and obtain the transgenic Fructus Lycopersici esculenti strain system that many strains are integrated into foreign gene.
[description of drawings]
Fig. 1 is plasmid pLC figure;
Fig. 2 is a transfer-gen plant;
Fig. 3 is the detection of transfer-gen plant.
[embodiment]
The inventive method generally comprises following content:
This method is that tomato explant and the Agrobacterium that contains the Ti-plasmids carrier are cultivated altogether, and Suo Shu Questions Ti-plasmids carrier carries one or more foreign genes, and the foreign gene on the plasmid vector is transferred in the acceptor gene group.
Described explant is from the cotyledon of sprouting 7-8 days large size tomato seedling.Described large size tomato is various processing tomatos.Described processing tomato can be UC82B.
Described Agrobacterium is LBA4404 bacterial strain or GV3101 bacterial strain.
In order to make changing effect higher, described Agrobacterium carries out resuspended with the MS substratum that contains 200 μ M Syringylethanones (AS) before conversion.
The concentration of described agroinfection liquid is with OD
600It is better when value is 0.6-0.8.
Described Agrobacterium is infected the time of tomato explant and was advisable with 10 minutes.
Culturing process is altogether, and described tomato explant and Agrobacterium were cultivated 24 hours at 28 ℃ of dark conditions, cultivates 24 hours under 24 ℃ of light afterwards.
Described culture medium altogether is to add: the MS substratum of 200 μ g/ml inositols, 200 μ M Syringylethanones (AS), 0.8 μ g/ml indolylacetic acid (IAA), 0.8 μ g/ml 6-benzyl aminopurine (6-BA).
The present invention is in conversion and regenerative process, and the selection substratum after the described end of cultivation altogether is for containing 200 μ g/ml inositols, 1.5 μ g/ml 6-benzyl aminopurines (6-BA), 0.1 μ g/ml indolylacetic acid, 500 μ g/ml Pyocianils, 3*10
-2μ g/mg VB
1The MS substratum; The regeneration bud of differentiation is taken root on the MS substratum that contains 200 μ g/ml inositols, 0.1 μ g/ml indolylacetic acid, 200 μ g/ml Pyocianils.
Below in conjunction with specific embodiment the inventive method is described further:
Embodiment
1. the acquisition of explant
Get the seed that strain is numbered the wild-type tomato of UC82B and carry out disinfection, 70% alcohol immersion 90 seconds, with aseptic water washing 3-4 time, with 10% antiformin immersion 12 minutes, usefulness aseptic water washing 7-8 time was transferred to seed on the 1/2MS substratum afterwards and is grown again.Treat that cotyledon launches to downcut the cotyledon of tomato seedling fully, place common culture medium (the MS substratum contains 200 μ g/ml inositols, 200 μ M Syringylethanones (AS), 0.8 μ g/ml indolylacetic acid (IAA), 0.8 μ g/ml 6-benzyl aminopurine (6-BA)), 24 degree illumination cultivation 24 hours.
2. the structure of expression vector
Utilize conventional gene engineering method from tomato, to clone the Partial cDNA fragment of LeCOP1LIKE gene, and utilize its non-conservative territory antisense rna construct expression vector-pLC.This expression vector is underlying carrier with pBI121, and LeCOP1LIKE gene antisense segment is driven by the CaMV 35S promoter.The NPTII gene (as Fig. 1) that also contains the NOS promoter regulation on this plasmid T-DNA.
3. the cultivation of Agrobacterium
The single bacterium colony of soil Agrobacterium LBA4404 that inoculation has changed the pLC plasmid over to contains in the liquid YEP substratum of kantlex (50mg/L) and Rifampin (20mg/L) in 10ml.28 ℃ of quick oscillation were cultivated 24 hours; Get wherein 1ml then and forward to and contain in the identical antibiotic 20ml liquid YEP substratum, 28 ℃ of quick oscillation were cultivated 24 hours.
4. cultivate altogether
Treat that Agrobacterium is long to OD
6000.6 between 0.8 the time,, and, make last OD with the resuspended Agrobacterium of liquid MS medium that contains 200 μ M Syringylethanones (AS) with centrifugal 5 minutes of Agrobacterium 4000rpm
600Value reaches about 0.7.
The cotyledon that scales off above-mentioned is placed on this dilutes in the good Agrobacterium bacterium liquid, infected 10 minutes.Explant is transferred on the previous common substratum, cultivated altogether two days, first day 28 ℃ of dark cultivation, second day 24 ℃ of light cultivations.
5. the selection of explant and plant regeneration
Forward explant to the selection substratum, promptly contain 200 μ g/ml inositols, 1.5 μ g/ml 6-benzyl aminopurines (6-BA), 0.1 μ g/ml indolylacetic acid, 3*10 in the MS substratum
-2μ g/mg VB
1And the microbiotic kantlex 50 μ g/ml that are used for transformation and selection.16 hours illumination cultivation of 24 degree.
When regeneration bud 3cm is high, they are gone to root media, promptly the MS substratum contains 200 μ g/ml inositols, 0.1 μ g/ml indolylacetic acid, 200 μ g/ml Pyocianils, 50 μ g/ml kantlex.
Will through after white silk seedling and cultivating seedling replanting to the greenhouse, obtain transfer-gen plant (Fig. 2).
In the above-described embodiments, the external source functional gene can be incorporated on the tomato karyomit(e).The present inventor utilizes ordinary method design at the primer that detects foreign gene, has increased among antisense segment (Fig. 3) figure of LeCOP1LIKE gene with the method for PCR: LC-A, and LC-C, LC-E are transfer-gen plant, WT, wild-type, P, plasmid is over against photograph, and N bears contrast.
Claims (9)
1, a kind of method for transformation of agriculture bacillus mediated large size tomato, it is characterized in that it being that large size tomato explant and the Agrobacterium that contains the Ti-plasmids carrier are cultivated altogether, described Ti-plasmids carrier carries one or more foreign genes, foreign gene on the plasmid vector is transferred in the acceptor gene group, obtains transfer-gen plant through the plant transformation tissue culture.
2, the method for transformation of agriculture bacillus mediated large size tomato according to claim 1 is characterized in that said explant is from the cotyledon of sprouting 7-8 days large size tomato seedling; Said large size tomato refers to processing tomato.
3, the method for transformation of agriculture bacillus mediated large size tomato according to claim 1 and 2 is characterized in that said Agrobacterium is LBA4404 bacterial strain or GV3101 bacterial strain.
4, the method for transformation of agriculture bacillus mediated large size tomato according to claim 3 is characterized in that said Agrobacterium carries out resuspended with the MS substratum that contains 200 μ M Syringylethanones (AS) before conversion.
5,, it is characterized in that the concentration OD of said agroinfection liquid according to the method for transformation of claim 1 or 4 described agriculture bacillus mediated large size tomatos
600Value is 0.6-0.8; The time that Agrobacterium is infected the tomato explant is 10 minutes.
6, the method for transformation of agriculture bacillus mediated large size tomato according to claim 1 is characterized in that said culturing process altogether is that the tomato explant was cultivated 24 hours at 28 ℃ of dark conditions with Agrobacterium, cultivation 24 hours under 24 ℃ of light afterwards.
7,, it is characterized in that said culture medium altogether is the MS substratum that adds following composition: 200 μ g/ml inositols, 200 μ M Syringylethanones (AS), 0.8 μ g/ml indolylacetic acid (IAA), 0.8 μ g/ml 6-benzyl aminopurine (6-BA) according to the method for transformation of claim 1 or 6 described agriculture bacillus mediated large size tomatos.
8, the method for transformation of agriculture bacillus mediated large size tomato according to claim 1, it is characterized in that saidly in conversion process, describedly cultivate altogether selection substratum after finishing for adding 200 μ g/ml inositols, 1.5 μ g/ml 6-benzyl aminopurines (6-BA), 0.1 μ g/ml indolylacetic acid, 500 μ g/ml Pyocianils, 3*10
-2The MS substratum of μ g/mg VB1.
9, according to the method for transformation of claim 1 or 8 described agriculture bacillus mediated large size tomatos, it is characterized in that in the said regenerative process that the regeneration bud of differentiation is taken root on the MS substratum that contains 200 μ g/ml inositols, 0.1 μ g/ml indolylacetic acid, 200 μ g/ml Pyocianils.
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| CN 200610130675 CN1995359A (en) | 2006-12-29 | 2006-12-29 | Agrobacterium mediated large size tomato transformation method |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103026966A (en) * | 2012-12-17 | 2013-04-10 | 天津市农业生物技术研究中心 | Method for identifying tomato yellow leaf curl virus resistance by utilizing detached leaf |
| CN103074364A (en) * | 2012-04-17 | 2013-05-01 | 吉林师范大学 | Tomato genetic transformation method |
| CN112889667A (en) * | 2021-01-18 | 2021-06-04 | 许昌仓羽生物科技有限公司 | Convenient and efficient tomato transgenic method |
| CN117701625A (en) * | 2023-12-12 | 2024-03-15 | 合肥润初生物科技有限公司 | Her-2 antibody production method based on transgenic plants |
-
2006
- 2006-12-29 CN CN 200610130675 patent/CN1995359A/en active Pending
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103074364A (en) * | 2012-04-17 | 2013-05-01 | 吉林师范大学 | Tomato genetic transformation method |
| CN103074364B (en) * | 2012-04-17 | 2014-11-12 | 吉林师范大学 | Tomato genetic transformation method |
| CN103026966A (en) * | 2012-12-17 | 2013-04-10 | 天津市农业生物技术研究中心 | Method for identifying tomato yellow leaf curl virus resistance by utilizing detached leaf |
| CN103026966B (en) * | 2012-12-17 | 2013-12-11 | 天津市农业生物技术研究中心 | Method for identifying tomato yellow leaf curl virus resistance by utilizing detached leaf |
| CN112889667A (en) * | 2021-01-18 | 2021-06-04 | 许昌仓羽生物科技有限公司 | Convenient and efficient tomato transgenic method |
| CN117701625A (en) * | 2023-12-12 | 2024-03-15 | 合肥润初生物科技有限公司 | Her-2 antibody production method based on transgenic plants |
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