CN109680008B - Genetic transformation method for haematococcus pluvialis - Google Patents
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Abstract
The invention relates to genetics, in particular to a novel genetic transformation method for haematococcus pluvialis. And (3) resuspending the enzymolyzed haematococcus pluvialis protoplast by using a protoplast washing solution, mixing the resuspension solution and exogenous DNA in different forms for incubation treatment, adding a protoplast transformation treatment solution I and a protoplast transformation treatment solution II in the incubation process to realize the transformation of genetic materials, and then washing and centrifuging by using the protoplast washing solution to obtain the transformed haematococcus pluvialis. The genetic transformation method of haematococcus pluvialis has the advantages of simple process operation, convenience, rapidness and repeatability, and compared with the traditional gene gun technology, the method has no special equipment requirement, small operation volume and easy realization of batch cell transformation.
Description
Technical Field
The invention relates to genetics, in particular to a novel genetic transformation method for haematococcus pluvialis.
Background
Astaxanthin is a red ketocarotenoid, not only can be used as a pigment source in the marine aquaculture industry, but also is widely applied to the fields of food, cosmetics, clinical research and the like due to the stronger antioxidant capacity than beta-carotene and vitamin E. Haematococcus pluvialis (Haematococcus pluvialis) is a unicellular green alga, is considered as a species with the strongest accumulation capacity and highest content of natural astaxanthin in nature, the accumulation amount can reach 7% of the dry weight of cells, is regarded as the best biological source of natural astaxanthin, and becomes another high-value economic microalgae following spirulina, dunaliella salina and the like, so that the research on the Haematococcus pluvialis has been widely concerned. At present, the culture of haematococcus pluvialis mainly depends on an open culture technology, and the problems of low biomass, strong environment dependence, complex culture conditions and the like exist, so that the commercial development of the haematococcus pluvialis is severely limited. The improvement of the property of haematococcus pluvialis by utilizing a genetic engineering method can possibly further improve the production efficiency and further popularize the application of the haematococcus pluvialis.
The current transformation methods commonly used by eukaryotic microalgae include a glass bead grinding transformation method, an agrobacterium tumefaciens mediated transformation method, an electric shock transformation method and a gene gun method. The bead milling method is to mix DNA, glass beads, polyethylene glycol and algae cells, and then to transfer exogenous DNA into the cells by using the glass beads to generate instantaneous holes on the cell membrane under the transient vortex oscillation, but the conversion method is only suitable for the conversion of few microalgae, such as chlamydomonas reinhardtii, dunaliella salina and the like with cell wall defects. The agrobacterium tumefaciens mediated transformation method is to realize transformation by utilizing agrobacterium tumefaciens with the capability of infecting plant growth, and Kumar (2004) completes the transformation of Chlamydomonas reinhardtii for the first time by utilizing the transformation method. The electroporation method is that the proper electric field intensity and pulse time are used to make the cell produce reversible electroporation to make the exogenous DNA enter the cell,
at present, the method is used for successfully completing transformation of the existing eukaryotic microalgae such as chlorella, chlamydomonas reinhardtii, dunaliella salina, phaeodactylum tricornutum and the like. The principle of the gene gun method is that gold powder or tungsten powder particles wrapped with exogenous DNA are bombarded at a certain pressure and randomly introduced into cells at a high speed, and the transformed Phaeodactylum tricornutum, clustered algae, chlorella and the like are successfully completed by the method.
The research on genetic transformation of haematococcus pluvialis starts late, the literature reports on the genetic transformation of haematococcus pluvialis at home and abroad are less, and the genetic transformation of haematococcus pluvialis is mostly realized by a particle gun method at present. Steinbrenner (2006) successfully introduced the modified Phytoene Dehydrogenase (PDS) gene into H.pluvialis using a particle gun technique. Guti é rrez (2012) realizes the effective integration of the exogenous expression module in the chloroplast genome by the particle gun method by using haematococcus pluvialis 16sDNA and 23sDNA as homologous double arms. Rev (2015) optimizes and modifies Phytoene Dehydrogenase (PDS), and the haematococcus pluvialis is transformed by a particle gun method, and researches show that the transformation efficiency of the haematococcus pluvialis is obviously superior to that reported before. In addition, Lushuxiu (2010) reports the application study of electric transformation in Haematococcus pluvialis, and proves the transient expression of exogenous genes in cells but does not obtain stable transformants.
Protoplasts are viable "naked cells" surrounded only by the cytoplasmic membrane. It has a high uptake capacity of exogenous DNA because it does not have a cell wall structure. Algal protoplast production work began in 1958, Fuhs successfully classified the protoplasts of Oscillatoria (Oscilla iaamoena) and subsequently succeeded in obtaining other algal protoplasts, and successful transformation of cells by protoplasts was achieved in many algal species to date. At present, Haematococcus pluvialis has a more complete protoplast preparation scheme and technology, and is prepared by using swimming cells under the condition of dawn (2016), and the yield of the protoplast can reach more than 80%. However, to date, there has been no report on the use of transformation based on H.pluvialis protoplasts.
The invention aims to provide a high-efficiency genetic transformation method for haematococcus pluvialis.
In order to achieve the purpose, the invention adopts the following technical scheme:
a genetic transformation method of haematococcus pluvialis comprises the steps of carrying out heavy suspension on zymolyzed haematococcus pluvialis protoplasts through a protoplast washing solution, mixing a heavy suspension with exogenous DNA in different forms for incubation treatment, adding a protoplast transformation treatment solution I and a protoplast transformation treatment solution II in the incubation process to realize transformation of genetic materials, and then washing and centrifuging through the protoplast washing solution to obtain the transformed haematococcus pluvialis.
And (3) resuspending the transformed haematococcus pluvialis in a cell recovery solution for recovery culture, and recovering the cultured cells to obtain a transformant for screening or gene expression detection.
The Haematococcus pluvialis is also called Haematococcus pluvialis or Haematococcus pluvialis, is a ubiquitous green alga, belongs to the order Volvicerus, the family Rhodococcus, and is classified by latin under the name Haematococcus pluvialis.
The haematococcus pluvialis protoplast can be obtained according to the prior art, and specifically comprises a protoplast cell obtained by treating single or mixed enzyme liquid such as protease, cellulase, hemicellulase and the like.
When the heavy suspension is mixed with exogenous DNA of different forms for incubation treatment, the protoplast conversion treatment solution I is firstly added and mixed uniformly, then the mixture is insulated in water bath at 30 ℃ for 25-35min, the mixture is shaken and mixed lightly every 5-10min in the insulation process, and after the protoplast conversion treatment solution I is insulated, the protoplast conversion treatment solution II is slowly added in batches at the temperature for treatment for 20-40 min, so that the conversion of genetic substances is realized; the addition amount of the protoplast conversion treatment liquid I is 1-3 times of the protoplast suspension volume of the enzymolyzed haematococcus pluvialis protoplast which is re-suspended by the protoplast washing liquid, and the addition amount of the protoplast conversion treatment liquid II is 1-4 times of the protoplast suspension volume of the enzymolyzed haematococcus pluvialis protoplast which is re-suspended by the protoplast washing liquid.
And slowly adding the protoplast transformation treatment solution II into the treatment system in batches, wherein the addition amount of the protoplast transformation treatment solution II is 1-2 times of the volume of the protoplast suspension of the enzymolyzed haematococcus pluvialis protoplast which is re-suspended by the protoplast washing solution every batch at intervals of 10-20 min.
The enzymolyzed haematococcus pluvialis protoplast is resuspended in a protoplast washing solutionThe cell density is 1-3 × 107And mixing the resuspension solution with different forms of exogenous DNA for incubation treatment, wherein the final concentration of the exogenous DNA in the incubation system is 5-30 ng/. mu.l.
The different forms of exogenous DNA include circular DNA, linearized DNA, fragments of PCR products, etc.
And after the conversion heat preservation treatment, washing by a protoplast washing solution, and centrifuging for 5-10min by 400-1000g to obtain the transformed haematococcus pluvialis.
The components of the protoplast washing liquid are 0.1-0.5mol of mannitol in each liter of water, 50mg of magnesium sulfate heptahydrate, 20mg of calcium chloride dihydrate, 1000mg of sodium nitrate and 1.2mg of sodium ethylene diamine tetracetate;
the protoplast transformation solution I is 40-60% (m/V) polyethylene glycol (PEG) and 50-100mmol Ca per liter of water2+；
The protoplast transformation treating solution II is 0.05-1mol Tris-HCl (pH 7.8) and 0.2-1mmol CaCl per liter water20.1-0.5mol sorbitol or mannitol.
The average molecular weight of the polyethylene glycol PEG is PEG4000, PEG6000 or PEG 8000.
The transformed haematococcus pluvialis is resuspended in cell recovery solution, and is cultured for 24-36h in dark recovery, and then is cultured in 5-15 mu mol m-2s-1Recovering for 24-36h under weak light, and recovering the cultured cells to obtain transformants for screening or gene expression detection.
The cell recovery solution comprises 1000mg of sodium nitrate 500-1000mg per liter of water, 50-100mg of magnesium sulfate heptahydrate, 20-40mg of calcium chloride dihydrate, 20-50mg of potassium dihydrogen phosphate and 20-50mg of sodium carbonate.
The invention has the advantages that:
the transformation method realizes the successful introduction of the exogenous DNA by carrying out simple chemical treatment on the haematococcus pluvialis protoplast, has simple operation, convenience and rapidness, and has no special equipment requirement, small operation volume and easy realization of mass cell transformation compared with the traditional technology.
Detailed description of the invention
The present invention is further illustrated by the following specific examples, which should be construed as merely illustrative, and not limitative of the remainder of the disclosure.
The following protoplast cells can be obtained by the prior art, and specifically, the protoplast cells can be obtained by treating Haematococcus pluvialis with a single or mixed enzyme solution such as protease, cellulase, hemicellulase, etc.
Example 1
Centrifuging at 800g for 10min to collect zooblast of Haematococcus pluvialis (NIES 144), and performing enzymolysis to prepare protoplast. Collecting zymolyzed protoplast cells, re-suspending the protoplast with a protoplast washing solution, and collecting the re-suspended protoplast cells at a ratio of 4-8 × 106Adding 3-5 mu g of annular exogenous DNA into each cell, sucking a protoplast transformation treatment solution I with 2 times volume of protoplast suspension resuspended in a protoplast washing solution of the haematococcus pluvialis protoplast after enzymolysis, treating the haematococcus pluvialis protoplast for 30min at the temperature of 30 ℃, stirring every 10min during the treatment to uniformly mix the treatment solution, adding a protoplast treatment solution II with 2 times volume of protoplast suspension resuspended in the protoplast washing solution of the haematococcus pluvialis protoplast after enzymolysis every 20min after the treatment of the transformation treatment solution I, adding the protoplast treatment solution II for 2 times, uniformly mixing after adding to realize the transformation of genetic materials, centrifugally washing the haematococcus pluvialis protoplast for 3 times by the protoplast washing solution after mixing, and resuspending the protoplast in a cell recovery solution after washing, so that the protoplast is recovered in the cell recovery solution for 24 hours in a dark state, and recovered for 24 hours in a weak light state. After 48h recovery, plates were plated and screened on resistant plates, with an average of 1-2X 10 per plate5Cells, at 25 ℃ with a continuous light intensity of 60. mu. mol m-2s-1Culturing under the condition. A total of 75 clones were obtained with a DNA conversion rate of about 4.76X 10 per μ g-6。
Example 2
Centrifuging at 800g for 5-10min to collect Haematococcus pluvialis (SCCAP K-0084) motile cells, and performing enzymolysis to obtain protoplast. Resuspending the enzymolyzed protoplast cell in a protoplast washing solution, and taking the protoplast cell 3-6X 106Adding 3-5 μ g of annular exogenous DNA into the cells, sucking the protoplast transformation treatment solution with 2 times volume of protoplast suspension obtained by resuspending the enzymolyzed haematococcus pluvialis protoplast with a protoplast washing solutionAnd I, treating the haematococcus pluvialis protoplast at the temperature of 30 ℃ for 30min, uniformly mixing the haematococcus pluvialis protoplast and the protoplast treatment solution II every 10min, adding the protoplast treatment solution II with 2.5 times of the volume of the protoplast suspension re-suspended by the protoplast washing solution after enzymolysis at the temperature of 30 ℃, adding the protoplast treatment solution II twice in total to realize the transformation of genetic materials, centrifugally washing the transformed haematococcus pluvialis protoplast washing solution for 3 times, recovering the cell recovery solution for 24 hours in a dark state, and recovering the cell recovery solution for 24 hours in a weak light state. After 48h recovery, plates were plated and 103 clones were obtained after selection in resistant plates with a DNA conversion rate of about 11.4X 10 per μ g-6。
Example 3
Centrifuging at 500g for 5-10min, collecting Haematococcus pluvialis (SCCAP NIES144) motile cells, and performing enzymolysis to obtain protoplast. Taking protoplast cell 4-6X 106Adding 3-6 mu g of linearized exogenous DNA into each cell, then sucking 1-time volume of protoplast transformation treatment liquid I of the protoplast suspension of the haematococcus pluvialis protoplast subjected to enzymolysis and resuspended in the protoplast washing liquid, treating for 30min at 30 ℃, uniformly mixing every 10min, adding 1.5-time volume of protoplast transformation treatment liquid II of the haematococcus pluvialis protoplast subjected to enzymolysis and resuspended in the protoplast washing liquid at 30 ℃ every 10min, and adding twice in total. The transformation of genetic materials is realized, after the transformation, the genetic materials are centrifugally washed by a protoplast washing solution for 3 times, and then the genetic materials are recovered for 36 hours in a cell recovery solution in dark and 24 hours in weak light. After 48h recovery, plates were plated and 134 clones were obtained after selection in resistant plates with a DNA conversion rate of about 9.57X 10 per μ g-6。
Example 4
Centrifuging at 700g for 10min to collect Haematococcus pluvialis (SCCAP K-0084) motile cells, and performing enzymolysis to prepare protoplast. Resuspending the enzymolyzed protoplast cells with a protoplast washing solution, and taking the protoplast cells at a ratio of 3X 106Adding 3 μ g of exogenous DNA as PCR product fragment, adding 1 times of protoplast transformation treating solution I of protoplast suspension obtained by resuspending the zymolyzed haematococcus pluvialis protoplast with a protoplast washing solution, treating at 30 deg.C for 30min, and mixing uniformly every 10 min. Mixing, adding zymolytic haematococcus pluvialis protoplast at 30 deg.C every 10min, and performing protoplastThe protoplast treatment solution II was added three times in total to 1 volume of the protoplast heavy suspension resuspended in the plastid wash solution. The transformation of genetic materials is realized, after the transformation, the genetic materials are centrifugally washed for 3 times by a protoplast washing solution, and then the genetic materials are recovered for 24 hours in a dark state and 36 hours in a weak light state in a cell recovery solution. After 48h recovery, plates were plated and 148 clones were obtained after selection in resistant plates with a DNA conversion rate of about 16.4X 10 per μ g-6。
The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art should be able to cover the technical solutions and the inventive concepts of the present invention within the scope of the present invention.
Claims (8)
1. A genetic transformation method of haematococcus pluvialis is characterized by comprising the following steps: resuspending the enzymolyzed haematococcus pluvialis protoplast by using a protoplast washing solution, mixing the resuspension solution with exogenous DNA in different forms for incubation treatment, adding a protoplast transformation treatment solution I and a protoplast transformation treatment solution II in the incubation process to realize the transformation of genetic materials, and then washing and centrifuging by using the protoplast washing solution to obtain the transformed haematococcus pluvialis;
the components of the protoplast washing liquid are 0.1-0.5mol of mannitol in each liter of water, 50mg of magnesium sulfate heptahydrate, 20mg of calcium chloride dihydrate, 1000mg of sodium nitrate and 1.2mg of sodium ethylene diamine tetracetate;
the protoplast transformation treating solution I is 40-60% (m/V) polyethylene glycol PEG and 50-100mmol Ca per liter of water2+；
The protoplast transformation treating solution II is 0.05-1mol of Tris-HCl and 0.2-1mmol of CaCl in each liter of water20.1-0.5mol sorbitol or mannitol; the Tris-HCl pH was 7.8.
2. The method of genetic transformation of Haematococcus pluvialis according to claim 1, wherein: the transformed haematococcus pluvialis is resuspended in a cell recovery solution for recovery culture, and cells after culture are recovered, so that a transformant for screening or gene expression detection can be obtained;
the cell recovery solution comprises 1000mg of sodium nitrate 500-1000mg per liter of water, 50-100mg of magnesium sulfate heptahydrate, 20-40mg of calcium chloride dihydrate, 20-50mg of potassium dihydrogen phosphate and 20-50mg of sodium carbonate.
3. The method of genetic transformation of Haematococcus pluvialis according to claim 1, wherein: when the heavy suspension is mixed with exogenous DNA of different forms for incubation treatment, the protoplast conversion treatment solution I is firstly added and mixed uniformly, then the mixture is insulated in water bath at 30 ℃ for 25-35min, the mixture is shaken and mixed lightly every 5-10min in the insulation process, and after the protoplast conversion treatment solution I is insulated, the protoplast conversion treatment solution II is slowly added in batches at the temperature for treatment for 20-40 min, so that the conversion of genetic substances is realized; the addition amount of the protoplast conversion treatment liquid I is 1-3 times of the protoplast suspension volume of the enzymolyzed haematococcus pluvialis protoplast which is re-suspended by the protoplast washing liquid, and the addition amount of the protoplast conversion treatment liquid II is 1-4 times of the protoplast suspension volume of the enzymolyzed haematococcus pluvialis protoplast which is re-suspended by the protoplast washing liquid.
4. The method of genetic transformation of Haematococcus pluvialis according to claim 3, wherein:
and slowly adding the protoplast transformation treatment solution II into the treatment system in batches, wherein the addition amount of the protoplast transformation treatment solution II is 1-2 times of the volume of the protoplast suspension of the enzymolyzed haematococcus pluvialis protoplast which is re-suspended by the protoplast washing solution every batch at intervals of 10-20 min.
5. The method of genetic transformation of Haematococcus pluvialis according to claim 1, wherein: the enzymolyzed haematococcus pluvialis protoplast is resuspended in a protoplast washing solution until the cell density is 1-3 multiplied by 107And mixing the resuspension solution with different forms of exogenous DNA for incubation treatment, wherein the final concentration of the exogenous DNA in the incubation system is 5-30 ng/. mu.l.
6. The method of genetic transformation of Haematococcus pluvialis according to claim 1, wherein: after heat preservation treatment, the transformation is washed by a protoplast washing solution and centrifuged for 5-10min at 400-1000g to obtain the transformed haematococcus pluvialis.
7. The method of genetic transformation of Haematococcus pluvialis according to claim 1, wherein: the polyethylene glycol PEG is selected from PEG4000, PEG6000 or PEG 8000.
8. The method of genetic transformation of Haematococcus pluvialis according to claim 2, wherein: the transformed haematococcus pluvialis is resuspended in cell recovery solution, and is cultured for 24-36h in dark recovery, and then is cultured in 5-15 mu mol m-2s-1Recovering for 24-36h under weak light, and recovering the cultured cells to obtain transformants for screening or gene expression detection.
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| CN105647810A (en) * | 2016-04-08 | 2016-06-08 | 中国科学院青岛生物能源与过程研究所 | Culture method of haematococcus pluvialis swarm cells and method for preparing protoplast |
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