CN101736025A - Electric shock transformation method for golden alga - Google Patents
Electric shock transformation method for golden alga Download PDFInfo
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- CN101736025A CN101736025A CN200910255769A CN200910255769A CN101736025A CN 101736025 A CN101736025 A CN 101736025A CN 200910255769 A CN200910255769 A CN 200910255769A CN 200910255769 A CN200910255769 A CN 200910255769A CN 101736025 A CN101736025 A CN 101736025A
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Abstract
The invention provides an electric shock transformation method for golden alga. The method improves transformation efficiency by carrying out suction filtration and collection, pretreatment, voltage input, plasmid concentration and electric shock of buffer solution, and is characterized in that the method comprises the following steps: (1) providing strains, exogenous DNA and electrotransformation buffer solution; (2) carrying out suction filtration and collection, and pretreatment before electrotransformation on the golden alga; (3) preparing plasmid and salmon sperm DNA concentration required by electrotransformation and setting conditions of electric shock; and (4) setting culture conditions after transformation. The electric shock transformation method reduces damages to transformed alga and greatly improves transformation efficiency.
Description
Technical field
The present invention relates to a kind of electric shock transformation method, specifically about a kind of electric shock transformation method to chrysophyceae (Isochrysissp.CCMM5001).
Background technology
Chrysophyceae is a kind of widely distributed Marine Planktonic unicellular algae, is the desirable bait that aquatic animal is grown seedlings.Recent studies show that chrysophyceae also contains abundant chlorophyll a, Chlorofucsin, β-Hu Luobusu, xenthophylls and unsaturated fatty acids isoreactivity material, especially DHA, EPA content is higher.Because individual little, growth and breeding is fast, and is nutritious, so chrysophyceae is the ideal material that carries out genetically engineered research.
Electric Study on Transformation to marine microalgae originates in nineteen ninety the earliest, and Matsunaga T utilizes electrotransformation that the pUSY02 plasmid has been transformed into synechococcus (Synechococcus sp).Till now, the little algae that has carried out gene transformation research mainly contains: anabena (Anabaena), beads algae (Nostoc), two-tube Fu Shi algae (Fremyelladiplosiphon), spirulina plalensis (Sirulina platensis), Dunaliella salina (Dunaliella sallina), Chlamydomonas reinhardtii (Chlamydomonas reinhardtii) etc.The method for transformation that adopts is also varied, as: particle bombardment, glass bead method, supersonic method, electrotransformation etc.Compare these several method for transformation, electricity transforms to have than remarkable advantages, simple to operate, cheap, and can reduce the influence of operation to the result.
The factor that influences the conversion of algae electric shock mainly contains electric shock voltage, plasmid concentration, electric shock damping fluid composition, algae kind state etc.The result of study of Brown shows that electric shock transforms back cell survival rate transformation efficiency when 50% left and right sides and reaches maximum value.The high capacitance low voltage can reduce the injury to frustule, improves cell survival rate, thereby improves transformation efficiency.Find in the experimentation that traditional electric shock damping fluid also is not suitable for chrysophyceae,, designed sucrose-HEPES damping fluid at the characteristics of chrysophyceae.Present relevant studies show that, logarithm algae kind survival rate in earlier stage generally will be higher than the algae kind in mid-term and later stage, but the algae kind in mid-term can obtain the highest transformation efficiency.Reason may be that the algae kind that is in mid-log phase is in the vigorous growth state, and environment is relatively more responsive to external world, and is stronger to the foreign gene receptivity.Be in the logarithmic growth algae kind in early stage, cell fission is less, and foreign gene is difficult for being integrated in the genome.
Summary of the invention
The purpose of this invention is to provide a kind of electric shocking method that utilizes foreign gene is transformed into the intravital novel method of chrysophyceae algae, collect, pre-treatment by suction filtration by chrysophyceae is carried out for this method, adopts suitable voltage, plasmid concentration, electric shock damping fluid to improve transformation efficiency.
The objective of the invention is to realize: developed a kind of electric shock transformation method to chrysophyceae by following technical scheme, this method has improved transformation efficiency by chrysophyceae being carried out suction filtration collection, pre-treatment, input voltage, plasmid concentration, electric shock damping fluid, and its feature comprises following steps:
(1) provide chrysophyceae algae strain Isochrysis sp.CCMM5001, exogenous DNA, electricity to change damping fluid;
(2) the chrysophyceae suction filtration is collected and the preceding pre-treatment of electricity conversion;
(3) electricity transforms plasmid, the concentration of salmon sperm dna, the electric shock condition enactment that needs;
(4) culture condition after the conversion;
Described exogenous DNA is the pCAMBIA1303 plasmid.
Described electricity changes damping fluid sucrose-HEPES and consists of: 0.5-1.0mM sucrose, 0.5-1.0mMHEPES, PH6.5-8.0.
The needed pressure of described suction filtration is 0.01-0.03Mpa, and used filter membrane is: aperture 8 μ m, specification 50mm.
Described plasmid concentration is 7-10 μ g/ml, and the concentration of salmon sperm dna is: 4-6 μ g/ml.
Described electric shock condition is 1.4-2.5KV, and the burst length is 5-10ms.
Described conversion back cell dark recovery is cultivated 23-25h, adds kantlex, and light/dark 12/12h cultivates.
Positively effect of the present invention is to disclose a kind of electric shock transformation method to chrysophyceae, and this is the research of up to the present first chrysophyceae being shocked by electricity and transforming, and by the method that adopts suction filtration to collect, has reduced transforming the damage of frond, has greatly improved transformation efficiency.
Description of drawings:
Fig. 1 is the influences of different pulse field intensity to the chrysophyceae survival rate;
Fig. 2 is the influences of different growth conditions to the chrysophyceae survival rate
Embodiment:
The invention will be further described below in conjunction with example:
Chrysophyceae (Isochrysis sp.CCMM5001) (marine microalgae algae kind storehouse CCMM/KLMEE teacher Han Xiaotian of ocean institute of the Chinese Academy of Sciences provides) is adopted in experiment, is the foreign DNA of conversion with the plasmid pCAMBIA1303 (being provided by Shihezi Univ) with Totomycin (hptII) resistance.
Described electricity changes damping fluid sucrose-HEPES and consists of: 0.5mM sucrose, 0.5mMHEPES, PH6.5.
The needed pressure of described suction filtration is 0.01Mpa, and used filter membrane is: aperture 8 μ m, specification 50mm.
Described plasmid concentration is 7 μ g/ml, and the concentration of salmon sperm dna is: 4 μ g/ml.
Described electric shock condition is 1.6KV/cm, and the burst length is 5ms.
Described conversion back cell dark recovery is cultivated 23h, adds kantlex, and light/dark 12/12h cultivates.
Power taking swashs the conversion back and cultivates 5 days frustule 0.5mL, 6000r/min, centrifugal 2min, twice of distilled water flushing, add the gene contamination that DNaseI incubation 30min originates with the extracellular of degrading possible, extract DNA, carry out PCR with a pair of primer on the hygromycin gene and detect.The result shows that the chrysophyceae that has carried out electric conversion all has positive amplification.
Chrysophyceae (Isochrysis sp.CCMM5001) (marine microalgae algae kind storehouse CCMM/KLMEES teacher Han Xiaotian of ocean institute of the Chinese Academy of Sciences provides) is adopted in experiment, is the foreign DNA of conversion with the plasmid pCAMBIA1303 (being provided by Shihezi Univ) with Totomycin (hptII) resistance.
Described electricity changes damping fluid sucrose-HEPES and consists of: 0.8mM sucrose, 0.8mMHEPES, PH7.
The needed pressure of described suction filtration is 0.02Mpa, and used filter membrane is: aperture 8 μ m, specification 50mm.
Described plasmid concentration is 8 μ g/ml, and the concentration of salmon sperm dna is: 5 μ g/ml.
Described electric shock condition is 1.8KV/cm, and the burst length is 8ms.
Described conversion back cell dark recovery is cultivated 24h, adds kantlex, and light/dark 12/12h cultivates.
Power taking swashs the conversion back and cultivates 8 days frustule 0.5mL, 6000r/min, centrifugal 2min, twice of distilled water flushing, add the gene contamination that DNaseI incubation 30min originates with the extracellular of degrading possible, extract DNA, carry out PCR with a pair of primer on the hygromycin gene and detect.The result shows that the chrysophyceae that has carried out electric conversion all has positive amplification.
Chrysophyceae (Isochrysis sp.CCMM5001) (marine microalgae algae kind storehouse CCMM/KLMEES teacher Han Xiaotian of ocean institute of the Chinese Academy of Sciences provides) is adopted in experiment, is the foreign DNA of conversion with the plasmid pCAMBIA1303 (being provided by Shihezi Univ) with Totomycin (hptII) resistance.
Described electricity changes damping fluid sucrose-HEPES and consists of: 1.0mM sucrose, 1.0mMHEPES, PH8.
The needed pressure of described suction filtration is 0.03Mpa, and used filter membrane is: aperture 8 μ m, specification 50mm.
Described plasmid concentration is 10 μ g/ml, and the concentration of salmon sperm dna is: 6 μ g/ml.
Described electric shock condition is 2.0KV/cm, and the burst length is 10ms.
Described conversion back cell dark recovery is cultivated 25h, adds kantlex, and light/dark 12/12h cultivates.
Power taking swashs the conversion back and cultivates 12 days frustule 0.5mL, 6000r/min, centrifugal 2min, twice of distilled water flushing, add the gene contamination that DNaseI incubation 30min originates with the extracellular of degrading possible, extract DNA, carry out PCR with a pair of primer on the hygromycin gene and detect.The result shows that the chrysophyceae that has carried out electric conversion all has positive amplification.
Those of ordinary skill in the art can understand, and in protection scope of the present invention, makes amendment for the foregoing description, and it all is possible adding and replacing, and it does not all exceed protection scope of the present invention.
Claims (7)
1. electric shock transformation method to chrysophyceae, this method has improved transformation efficiency by chrysophyceae being carried out suction filtration collection, pre-treatment, input voltage, plasmid concentration, electric shock damping fluid, and its feature comprises following steps:
(1) provide chrysophyceae algae strain Isochrysis sp.CCMM5001, exogenous DNA, electricity to change damping fluid;
(2) the chrysophyceae suction filtration is collected and the preceding pre-treatment of electricity conversion;
(3) preparation of the concentration of the plasmid of electricity conversion needs, salmon sperm dna, electric shock condition enactment;
(4) culture condition after the conversion;
2. the electric shock transformation method to chrysophyceae according to claim 1 is characterized in that: described exogenous DNA is the pCAMBIA1303 plasmid.
3. the electric shock transformation method to chrysophyceae according to claim 1 is characterized in that: described electricity changes damping fluid sucrose-HEPES and consists of: 0.5-1.0mM sucrose, 0.5-1.0mMHEPES, PH6.5-8.0.
4. the electric shock transformation method to chrysophyceae according to claim 1 is characterized in that: the needed pressure of described suction filtration is 0.01-0.03Mpa, and used filter membrane is: aperture 8 μ m, specification 50mm.
5. the electric shock transformation method to chrysophyceae according to claim 1 is characterized in that: described plasmid concentration is 7-10 μ g/ml, and the concentration of salmon sperm dna is: 4-6 μ g/ml.
6. the electric shock transformation method to chrysophyceae according to claim 1 is characterized in that: described electric shock condition is 1.4-2.5KV, and the burst length is 5-10ms.
7. the electric shock transformation method to chrysophyceae according to claim 1 is characterized in that: described conversion back cell dark recovery is cultivated 23-25h, adds kantlex, and light/dark 12/12h cultivates.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2014027995A1 (en) * | 2012-08-12 | 2014-02-20 | Life Technologies Corporation | Molecular biology tools for algal engineering |
| US9018013B2 (en) | 2011-08-12 | 2015-04-28 | Life Technologies Corporation | Molecular biology tools for algal engineering |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN1354792A (en) * | 1999-05-28 | 2002-06-19 | 爱尔基因泰克株式会社 | Biosynthesis of foreign proteins using transformed microalgae |
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Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1354792A (en) * | 1999-05-28 | 2002-06-19 | 爱尔基因泰克株式会社 | Biosynthesis of foreign proteins using transformed microalgae |
Non-Patent Citations (4)
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| 曹军平: "莱茵衣藻外源基因高效转化系统的建立及金藻转化方法的研究", 《中国农业科学院论文集》 * |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US9018013B2 (en) | 2011-08-12 | 2015-04-28 | Life Technologies Corporation | Molecular biology tools for algal engineering |
| US10280428B2 (en) | 2011-08-12 | 2019-05-07 | Life Technologies Corporation | Molecular biology tools for algal engineering |
| WO2014027995A1 (en) * | 2012-08-12 | 2014-02-20 | Life Technologies Corporation | Molecular biology tools for algal engineering |
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Application publication date: 20100616 |