CN109652445A - A kind of construction method of high frequency zone transgenic plant carrier - Google Patents
A kind of construction method of high frequency zone transgenic plant carrier Download PDFInfo
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- 230000009466 transformation Effects 0.000 claims abstract description 9
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- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
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Abstract
This application discloses a kind of construction methods of high frequency zone transgenic plant carrier, comprising the following steps: the acquisition of A, DsRed gene；B, DsRed gene is connected on pCAMBIA1300 plant binary expression vector.The present invention provides a kind of based on DsRED fluorescence protein gene be reporter gene and hygromycin be antibiotic-screening engineered strain transformation.Whether successfully the present invention also provides one kind can be observed that a kind of transgenosis efficiently reporter gene detection method at plant transgene initial stage.Foundation is provided for subsequent efficient positive plant screening.Invention further provides one kind be reporter gene based on DsRED fluorescence protein gene and hygromycin gene is the simultaneous engineered strain of screening-gene.The present invention provides one kind again can be with a kind of whether successful efficiently detection method of Double Selection transgenosis.
Description
Technical field
The present invention relates to genetic engineering genetic thremmatology and biological heredity improving technology field, in particular to a kind of high efficiency
The method quickly screened from Transgenic rice cells.
Background technique
Rice is one of most important cereal crops at past half as the staple food for being in the world more than half population
In more centuries, the specific yield that rice breeding achieves huge success rice realizes multiplication, and some areas are even improved extremely
3 times, this has made safely huge contribution for guarantee world food.
But the yield stagnation of rice in recent ten years, this aspect is due to not having new breakthrough on breeding technique
And genetic diversity gradually narrowing in cultivar, on the other hand also in that the pest and disease damage and drought frequently occurred
Etc. natural calamities make Rice Production suffer heavy losses however, the sustainable growth of world population and the fast development of social economy
Cause to be continuously increased the demand of grain.
For these problems, Chinese scholar proposes the imagination for cultivating green super hybridization rice, around Rice Resistance disease pest, drought resisting,
The five big important characters such as nutrient efficient utilization, high-quality, high yield carry out improvement comprehensively to rice varieties to realize the sustainable of agricultural
Development.And transgenic technology will play a significant role on realizing green super hybridization rice target as a kind of emerging breeding technique.
The transgenic research of rice starts from phase late 1980s, has a large amount of transgenic paddy rice research so far and is reported
Road.
Transgenic Rice technology starts from Protoplast cuhnre, and 1985 successfully complete from rice protoplast regeneration for the first time
Plant；First transgenic rice plant is obtained in japonica rice variety within 1988；Nineteen ninety is from rice variety Chinsurah
First case transgenic indica type rice plant is obtained in Boro II；Nineteen ninety Li Baojian etc. infects rice tissue with Agrobacterium and is converted
Callus；Transgenic plant is successfully obtained with particle bombardment using Rice Young Embryo as acceptor material, and is turned within 1991
Change efficiency to significantly improve.
From this, rataria is widely studied application as transgenic acceptor.1993 with agrobacterium co-cultivation in japonica rice product
Transgenic plant is obtained on kind, hereafter, particle bombardment and agrobacterium-mediated transformation are wide as the path for transformation of most worthy
It is general to be studied for Transgenic Rice.
Using transgenic method obtain transgenic plant and its generation offspring, how to prove foreign gene be transferred to by
Body, or stablize heredity? following level is mainly divided to identify:
1) identification of exogenous origin gene integrator mainly has Southern hybridization and the detection of PCR method positive.
2) identification of transcription of foreign genes level has Northern hybridization and RT-PCR (reverse transcribed
PCR) detection method.
3) detection of exogenous gene expression protein, there are mainly three types of: 1. biochemical reaction detection method: mainly pass through enzyme reaction
To detect；2. immunological detection: being detected by destination protein (antigen) and the specific binding of its antibody, such as
Western hybridization；3. the detection of biological activity.
4) the enzyme process detection of reporter gene, common reporter gene have: gus, no, ocs etc..
Certainly last transgenic plant must be transplanted in crop field, carry out field test, not only will also be to it to Ameliorative character
Its economical character is evaluated, the excellent strain of breeding Comprehensive Traits.A large amount of financial resources and manpower can be spent in this way, and can be spent
Take longer time.
Summary of the invention
Technical problem to be solved by the present invention lies in provide a kind of building side of high frequency zone transgenic plant carrier
Method.Hygromycin gene and red fluorescent gene are carried out the callus of chain rear rice transformation maturation embryonal induction by the method for the present invention
Tissue is added hygromycin in callus cell culture screening stage, detects in combination with red fluorescence, quickly and effectively
Screening.
In order to solve the above technical problems, the present invention provides a kind of construction method of high frequency zone transgenic plant carrier,
The following steps are included:
A, the acquisition of DsRed gene；
B, DsRed gene is connected on pCAMBIA1300 plant binary expression vector.
The step B further comprises:
The first step is expanded using the pGDR plasmid containing DsRED segment as template using primer, and DsRED piece is obtained
Section；
Second step, the processing of pCAMBIA1300 binary vector double digestion；
Third step, after the DsRED segment for introducing X ba I and BamH I restriction enzyme site is inserted into double digestion
On pCAMBIA1300 binary vector；
The fusion plasmid of pCAMBIA1300 carrier containing DsRED gene is imported into Agrobacterium by the 4th step, is formed
It can be used for the engineering bacteria agrobacterium strains EHA105 of mediated transformation plant cell.
In the first step, amplification system includes:
Distilled water constant volume is to 20 μ L.
In the first step, amplification program includes:
In the second step, pCAMBIA1300 binary vector double digestion processing digestion system includes:
Double digestion program:
37℃ 2h
70℃ 10min
4 DEG C of preservations.
In 4th step, the preparation of Agrobacterium competent cell includes:
Take the Agrobacterium of -70 DEG C of preservations in containing 50 μ g/ml rifampin plate streakings, 28 DEG C of cultures；
Picking single colonie is inoculated in 5ml YM fluid nutrient medium, 28 DEG C of shaken cultivation 12-16hr of 220rpm；
Take the switching of 2ml bacterium solution in 100ml YM fluid nutrient medium, 28 DEG C of 220rpm shaken cultivations to OD600=0.5；
It is transferred to sterile centrifugation tube, 5000rpm is centrifuged 5min, removes supernatant；
The CaCl2 solution of the 0.1M of 10ml pre-cooling is added, gently suspension cell, places 20min on ice；4 DEG C of 5000rpm from
Heart 5min, removes supernatant；
The CaCl2 solution of the 0.1M containing 15% glycerol of 4ml pre-cooling is added, gently suspends；
Agrobacterium suspension is sub-packed in sterile Eppendorf pipe, and every 200 μ l of pipe freezes in -70 DEG C.
In 4th step, convert Agrobacterium the step of include:
The Plasmid DNA of 0.8-1.2 μ g built is taken to be added in 200ml Agrobacterium competent cell, after mixing, ice bath
30min, -70 DEG C of placement 10min；
Again in 37 DEG C of water-bath 5min or 42 DEG C of water-bath 1min, then ice bath 2min, is added 800mlYM fluid nutrient medium 28
DEG C, 175rpm is coated on the YM plate containing 50 μ g/ml Kanamycin after shaking training 3hr；28 DEG C of cultures are to forming single colonie.
In order to solve the above technical problems, the present invention also provides a kind of, the high frequency zone transgenosis as described in aforementioned any one is planted
The transgenic plant carrier of the construction method building of strain vector.
In order to solve the above technical problems, invention further provides a kind of preparation methods of Agrobacterium competent cell, including
Following steps:
Take the Agrobacterium of -70 DEG C of preservations in containing 50 μ g/ml rifampin plate streakings, 28 DEG C of cultures；
Picking single colonie is inoculated in 5ml YM fluid nutrient medium, 28 DEG C of shaken cultivation 12-16hr of 220rpm；
Take the switching of 2ml bacterium solution in 100ml YM fluid nutrient medium, 28 DEG C of 220rpm shaken cultivations to OD600=0.5；
It is transferred to sterile centrifugation tube, 5000rpm is centrifuged 5min, removes supernatant；
The CaCl2 solution of the 0.1M of 10ml pre-cooling is added, gently suspension cell, places 20min on ice；4 DEG C of 5000rpm from
Heart 5min, removes supernatant；
The CaCl2 solution of the 0.1M containing 15% glycerol of 4ml pre-cooling is added, gently suspends；
Agrobacterium suspension is sub-packed in sterile Eppendorf pipe, and every 200 μ l of pipe freezes in -70 DEG C.
In order to solve the above technical problems, the present invention provides a kind of method for converting Agrobacterium again, comprising the following steps:
The Plasmid DNA of 0.8-1.2 μ g built is taken to be added in 200ml Agrobacterium competent cell, after mixing, ice bath
30min, -70 DEG C of placement 10min；
Again in 37 DEG C of water-bath 5min or 42 DEG C of water-bath 1min, then ice bath 2min, is added 800mlYM fluid nutrient medium 28
DEG C, 175rpm is coated on the YM plate containing 50 μ g/ml Kanamycin after shaking training 3hr；28 DEG C of cultures are to forming single colonie.
Beneficial effect of the present invention includes:
(1) it is reporter gene that the present invention provides one kind based on DsRED fluorescence protein gene and hygromycin is antibiotic sieve
The transformation of the engineered strain of choosing.
(2) the present invention also provides one kind can be observed that transgenosis whether successful one at plant transgene initial stage
Plant efficiently reporter gene detection method.Foundation is provided for subsequent efficient positive plant screening.
(3) it is reporter gene and hygromycin gene is that the present invention provides one kind based on DsRED fluorescence protein gene
The simultaneous engineered strain of screening-gene.
(4) the present invention also provides one kind can be with a kind of whether successful efficiently detection method of Double Selection transgenosis.
Detailed description of the invention
Fig. 1 is p CAMBIA1300 Vector map and hygromycin gene (HygR) figure described in the embodiment of the present invention；
Fig. 2 is that DsRED gene PCR described in the embodiment of the present invention expands detected through gel electrophoresis figure.
Specific embodiment
The present invention is described in detail below with reference to embodiment.To keep the objectives, technical solutions, and advantages of the present invention clearer, bright
Really, the present invention is described in more detail below, but the invention is not limited to these embodiments.
Hygromycin gene and red fluorescent gene (DsRed) gene are carried out chain rear rice transformation maturation by the present invention
The callus of embryonal induction is added hygromycin in callus cell culture screening stage, detects in combination with red fluorescence, fastly
Fast, effective screening.
For achieving the above object, the present invention is achieved by the following scheme:
A, the acquisition of DsRed gene；
Primer needed for DsRED gene magnification
DsRED-F5′TCTAGAGCCA TGGCCTCCTCCGAGAA-3′；
DsRED-R 5′-GGA TCC T TACAGGAACA GG TGGTGGC-3′
B, DsRed gene is connected on pCAMBIA1300 plant binary expression vector
The acquisition of each gene expression element and Connection Step include:
The first step is expanded using the pGDR plasmid containing DsRED segment as template using above-mentioned primer, and DsRED is obtained
Segment.Amplification system is as follows:
Distilled water constant volume is to 20 μ L
Amplification program:
Second step, it is as follows that pCAMBIA1300 binary vector double digestion handles digestion system:
Double digestion program:
37℃ 2h
70℃ 10min
4 DEG C of preservations
Third step, after the DsRED segment for introducing X ba I and BamH I restriction enzyme site is inserted into double digestion
On pCAMBIA1300 binary vector；
Linked system are as follows:
It is connected overnight under the conditions of 16 DEG C
The fusion plasmid of pCAMBIA1300 carrier containing DsRED gene is imported into Agrobacterium by the 4th step, is formed
It can be used for the engineering bacteria agrobacterium strains EHA105 of mediated transformation plant cell.
The preparation of Agrobacterium competent cell
Take the Agrobacterium of -70 DEG C of preservations in containing 50 μ g/ml rifampin plate streakings, 28 DEG C of cultures.
Picking single colonie is inoculated in 5ml YM fluid nutrient medium, 28 DEG C of shaken cultivation 12-16hr of 220rpm.
Take the switching of 2ml bacterium solution in 100ml YM fluid nutrient medium, 28 DEG C of 220rpm shaken cultivations to OD600=0.5.
It is transferred to sterile centrifugation tube, 5000rpm is centrifuged 5min, removes supernatant.
The CaCl2 solution of the 0.1M of 10ml pre-cooling is added, gently suspension cell, places 20min on ice.4 DEG C of 5000rpm from
Heart 5min, removes supernatant.
The CaCl2 solution of the 0.1M containing 15% glycerol of 4ml pre-cooling is added, gently suspends.
Agrobacterium suspension is sub-packed in sterile Eppendorf pipe, and every 200 μ l of pipe freezes in -70 DEG C.
Convert Agrobacterium
The above-mentioned Plasmid DNA built of 1 μ g or so is taken to be added in 200ml Agrobacterium competent cell, after mixing, ice
Bathe 30min, -70 DEG C of placement 10min.Again in 37 DEG C of water-bath 5min or 42 DEG C of water-bath 1min, then ice bath 2min, is added
28 DEG C of 800mlYM fluid nutrient medium, 175rpm is coated on the YM plate containing 50 μ g/ml Kanamycin after shaking training 3hr.28 DEG C of trainings
It supports to form single colonie.
The above is only several embodiments of the present invention, not any type of limitation is done to the present invention, although this hair
It is bright to be disclosed as above with preferred embodiment, however be not intended to limit the invention, any person skilled in the art, it is not taking off
In the range of technical solution of the present invention, a little variation or modification are made using the technology contents of the disclosure above and is equal to
Case study on implementation is imitated, is belonged in technical solution of the present invention protection scope.
Claims (10)
1. a kind of construction method of high frequency zone transgenic plant carrier, which comprises the following steps:
A, the acquisition of DsRed gene；
B, DsRed gene is connected on pCAMBIA1300 plant binary expression vector.
2. the construction method of high frequency zone transgenic plant carrier according to claim 1, which is characterized in that the step B
Further comprise:
The first step is expanded using the pGDR plasmid containing DsRED segment as template using primer, and DsRED segment is obtained；
Second step, the processing of pCAMBIA1300 binary vector double digestion；
Third step, the pCAMBIA1300 after the DsRED segment for introducing X ba I and BamH I restriction enzyme site to be inserted into double digestion
On binary vector；
The fusion plasmid of pCAMBIA1300 carrier containing DsRED gene is imported into Agrobacterium by the 4th step, and formation can be with
Engineering bacteria agrobacterium strains EHA105 for mediated transformation plant cell.
3. the construction method of high frequency zone transgenic plant carrier according to claim 2, which is characterized in that the first step
In, amplification system includes:
4. the construction method of high frequency zone transgenic plant carrier according to claim 2, which is characterized in that the first step
In, amplification program includes:
5. the construction method of high frequency zone transgenic plant carrier according to claim 2, which is characterized in that the second step
In, pCAMBIA1300 binary vector double digestion processing digestion system includes:
6. the construction method of high frequency zone transgenic plant carrier according to claim 2, which is characterized in that the 4th step
In, the preparation of Agrobacterium competent cell includes:
Take the Agrobacterium of -70 DEG C of preservations in containing 50 μ g/ml rifampin plate streakings, 28 DEG C of cultures；
Picking single colonie is inoculated in 5ml YM fluid nutrient medium, 28 DEG C of shaken cultivation 12-16hr of 220rpm；
Take the switching of 2ml bacterium solution in 100ml YM fluid nutrient medium, 28 DEG C of 220rpm shaken cultivations to OD600=0.5；
It is transferred to sterile centrifugation tube, 5000rpm is centrifuged 5min, removes supernatant；
The CaCl2 solution of the 0.1M of 10ml pre-cooling is added, gently suspension cell, places 20min on ice；4 DEG C of 5000rpm centrifugations
5min removes supernatant；
The CaCl of the 0.1M containing 15% glycerol of 4ml pre-cooling is added2Solution gently suspends；
Agrobacterium suspension is sub-packed in sterile Eppendorf pipe, and every 200 μ l of pipe freezes in -70 DEG C.
7. the construction method of high frequency zone transgenic plant carrier according to claim 2, which is characterized in that the 4th step
In, convert Agrobacterium the step of include:
The Plasmid DNA of 0.8-1.2 μ g built is taken to be added in 200ml Agrobacterium competent cell, after mixing, ice bath
30min, -70 DEG C of placement 10min；
Again in 37 DEG C of water-bath 5min or 42 DEG C of water-bath 1min, then ice bath 2min, is added 28 DEG C of 800mlYM fluid nutrient medium,
175rpm is coated on the YM plate containing 50 μ g/ml Kanamycin after shaking training 3hr；28 DEG C of cultures are to forming single colonie.
8. what a kind of construction method of high frequency zone transgenic plant carrier as described in any one of claim 1-7 constructed turns base
Because of plant carrier.
9. a kind of preparation method of Agrobacterium competent cell, which comprises the following steps:
Take the Agrobacterium of -70 DEG C of preservations in containing 50 μ g/ml rifampin plate streakings, 28 DEG C of cultures；
Picking single colonie is inoculated in 5ml YM fluid nutrient medium, 28 DEG C of shaken cultivation 12-16hr of 220rpm:
Take the switching of 2ml bacterium solution in 100ml YM fluid nutrient medium, 28 DEG C of 220rpm shaken cultivations to OD600=0.5；
It is transferred to sterile centrifugation tube, 5000rpm is centrifuged 5min, removes supernatant；
The CaCl2 solution of the 0.1M of 10ml pre-cooling is added, gently suspension cell, places 20min on ice；4 DEG C of 5000rpm centrifugations
5min removes supernatant；
The CaCl of the 0.1M containing 15% glycerol of 4ml pre-cooling is added2Solution gently suspends；
Agrobacterium suspension is sub-packed in sterile Eppendorf pipe, and every 200 μ 1 of pipe freezes in -70 DEG C.
10. a kind of method for converting Agrobacterium, which comprises the following steps:
The Plasmid DNA of 0.8-1.2 μ g built is taken to be added in 200ml Agrobacterium competent cell, after mixing, ice bath
30min, -70 DEG C of placement 10min；
Again in 37 DEG C of water-bath 5min or 42 DEG C of water-bath 1min, then ice bath 2min, is added 28 DEG C of 800mlYM fluid nutrient medium,
175rpm is coated on the YM plate containing 50 μ g/ml Kanamycin after shaking training 3hr；28 DEG C of cultures are to forming single colonie.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US7060876B2 (en) * | 1992-07-07 | 2006-06-13 | Japan Tobacco Inc. | Method for transforming monocotyledons |
| CN108977458A (en) * | 2018-07-05 | 2018-12-11 | 青岛袁策集团有限公司 | Plant transgene cell preparation method |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US7060876B2 (en) * | 1992-07-07 | 2006-06-13 | Japan Tobacco Inc. | Method for transforming monocotyledons |
| CN108977458A (en) * | 2018-07-05 | 2018-12-11 | 青岛袁策集团有限公司 | Plant transgene cell preparation method |
Non-Patent Citations (1)
| Title |
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| 张蕾等: "《植物发育生物学实验指导》", 31 October 2010, 武汉大学出版社 * |
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