CN102102107A - Expression vector for large green alga bioreactor and transformation method thereof - Google Patents
Expression vector for large green alga bioreactor and transformation method thereof Download PDFInfo
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Abstract
The invention provides a vector capable of making an exogenous gene expressed stably and efficiently in a large green alga bioreactor and a method thereof for transformation and expression in a large green alga. In the invention, a protoplast of a large green alga is used as a receptor, the conventional vector of higher plants or a vector modified by a homologous recombination technique are fully utilized to construct a green fluorescent tracing and fusion protein gene-containing vector PSB-bar-ZsGeen for use in a large green alga bioreactor, and the vector PSB-bar-ZsGee is connected with a SV40 gene promoter, a resistance gene, a cytomegalovirus (CMV) promoter and the like in turn to realize the high-efficiency and stable expression of the exogenous gene in the large green alga.
Description
Technical field
The present invention relates to gene biological engineering field, the structure and the method for transformation thereof of the expression vector of particularly a kind of large transgenic green alga (Enteromorpha, feldspar water shield etc.) bio-reactor.
Background technology
Large-scale chlorella growth is very fast, goes the eutrophication effect good especially, and can effectively suppress the red tide generation, is present ideal marine ecology reparation person.Large-scale green alga also is a kind of more satisfactory " marine alga reactor ", can be used for producing important biomolecule active substance, the exploitation marine alga energy.China is one of the most flourishing country of algal culture in the world industry, and wherein sea-tangle, laver culture output account for No. 1 in the world and the 3rd respectively.Since the mid-90, the rise of the discovery of seaweed bio active substance and marine alga pharmaceutical technology is for new approach has been opened up in the marine alga comprehensive utilization.
Because the development of modern biotechnology, from the cell levels to the molecular level, carry out the hereditary mechanism research of kelp, realize the fast culture improved seeds, and utilize the fast numerous good seed of cell engineering, for the sustainable development that promotes traditional algal cultivation industry with set up novel aquaculture industry, significant.Though various in the world transgenic plant have produced huge economic benefit, the transgenic research integral body of marine alga is made slow progress.Chinese large-sized green alga transgenic research is almost blank, and mainly carries out in marine algas such as sea-tangle, laver, wakame.This is that marine alga transgenosis difficulty is bigger because marine alga is different with higher plant.Major cause has: 1) for a long time owing to do not find ideal resistance screening mark, greatly restricted the genetically modified development of marine alga; 2) also do not set up stable expression system, great majority can only reach the moment expression level.
Only sea-tangle obtains desirable screening marker gene now, has obtained big progress, the stably express report is arranged, and obtained vaccine transgenic Herba Zosterae marinae, hepatitis B virus surface antigen transgenic Herba Zosterae marinae etc. respectively.But compare with higher plant, the marine alga transgenic research is also very backward.At present, the technology of higher plant is used for reference in most researchs fully, utilize the organize stripping and slicing or the protoplastis of kelp to be transformation receptor, only realized the transient expression of foreign gene, in the new plant of protoplast regeneration, do not detect stabilized expression of exogenesis genes, because only used the promotor (CaMV35S) in a kind of higher plant source, thereby efficient is lower, kelp does not relate to as yet to antibiotic sensitivity test.
(green fluorescent protein is that a class is present in the intravital bioluminescent proteins of coelenterates such as jellyfish, hydra and coral GFP) to green fluorescent protein, is being subjected to ultraviolet or when blue-light excited, can emitting fluorescence.Because it can and carry out direct imaging under the situation of chemical staining at broken garland cells, is used widely in biological chemistry and cytobiology in recent years.The wild-type green fluorescent protein folds temperature influence, luminous a little less than; Expression frequency in vegetable cell is not high.By the transformation to its chromophore's amino acid, codon, promotor and gene order, the more stable susceptibility of green fluorescent protein fluorescence is higher, pair cell toxicity is littler, firing time is longer and can be by somatoscopy.
As reporter gene, green fluorescent protein is in the expression of all succeeing of microorganism, animal, monocotyledons, dicotyledons.In the algae genetically engineered, GUS and lacZ are to detect by the chemical staining method as reporter gene, though its technology maturation, highly sensitive, but the tracking that all can't carry out the live body expression level detects, and green fluorescent protein is because its innate advantage has well solved this difficult problem.Obtained green fluorescent protein transient expression in laver, kelp gametophyte at present; Chlamydomonas reinhardtii chloroplast(id) atpA promotor is expressed in Dunaliella salina, but it is not successful as yet as reporter gene in large-scale green alga.Feldspar water shield (edge pipe Enteromorpha) growth not only can be used as ideal restoration of the ecosystem marine alga fast, and utilizes the pharmaceutical protein of its folding assembling with the exploitation complex construction, compares with Mammals, and the lower cycle of its cost is shorter.
Enteromorpha, being called feldspar water shield (Enteromorpha spp. or Ulva spp.) again is a kind of large-scale green alga, be commonly called as tongue bar, green laver etc., be the phycophyta of Chlorophyta Ulvales Ulvaceae Enteromorpha, common have chance with pipe Enteromorpha, flat Enteromorpha, bar Enteromorpha, intestines Enteromorpha etc.Abroad usually all and be a green laver (Ulva) with Enteromorpha and green laver.The price of Enteromorpha is the highest in Japanese marine alga market, and is also higher than laver price.China has also carried out Enteromorpha, reef film artificial culture in south at present.
Summary of the invention
Technical problem to be solved by this invention is that a kind of carrier that contains green fluorescence spike and antigen-4 fusion protein gene that is applied in the large-scale green alga bio-reactor is provided.The present invention is an acceptor with large-scale green alga protoplastis, make full use of the existing carrier of higher plant or use homologous recombination technique transformation carrier, made up the expression vector of large-scale green alga bio-reactor, with realize foreign gene can be in large-scale green alga efficient and stably express.
The another technical problem that will solve of the present invention is, the conversion expression method of above-mentioned expression vector is provided.
For solving the problems of the technologies described above, the technical scheme of employing is:
A kind of expression vector that is used for large-scale green alga bio-reactor is characterized in that described carrier is Psv-bar-ZsGreen, connects SV40 promotor, resistant gene, CMV promotor and MCS polyclone restriction site on it successively.
Described carrier merges the reporter gene that fluorescence protein gene ZsGreen is arranged in the MCS downstream.
Described resistant gene is the bar gene.
Before described SV40 promotor, be connected with at least one relaxed replication; Described carrier is the relaxed type carrier.
Also be connected with the Amp resistant gene in the described carrier.
The external source functional structure of can recombinating in MCS site gene; as antigen gene, vaccine, antibody, human growth factor, alexin etc.; therefore utilize this system to can be used as a kind of bio-reactor efficiently, be used for producing cytokine, hormone, monoclonal antibody, nutrient protein, vaccine, enzyme, various somatomedin and some other medicines and the employed raw material of industry or environmental protection department.
Described large-scale green alga is green laver green algas such as Enteromorpha, feldspar water shield.
A kind of method for transformation that is used for the expression vector of large-scale green alga bio-reactor is characterized in that, utilizes fluorescence protein gene ZsGreen to optimize transgenic method as reporter gene.
Described a kind of large transgenic green alga transforms expression method, comprising: 1. by the cell protoplast separation and purification of enzymolysis process to kelp, 2. the protoplastis as acceptor passes through the conversion medium pre-treatment before conversion; 3. improve transformation efficiency with the divalent cation material in transforming, complete soln keeps the osmotic pressure of protoplastis itself; 4. PEG6000 transforms final concentration at 14-23%; 5. cultivate adding cell walls fertile absorber in the regenerative process in the later stage, reduce the osmotic potential of solution simultaneously gradually.
A kind of large transgenic green alga transforms expression method, and it is bar-Basta (careless fourth phosphine) that pair cell transforms the marker combination of screening.
Described best enzymolysis prescription is that 2% cellulase mixes 2% macerozyme, and best enzymolysis pH value is 5.8, and the permeate agent mannitol concentration is 0.6M; Describedly be used for pretreated conversion medium and be: 0.7M N.F,USP MANNITOL, 15mM MgCl2; 0.1%MES transfers pH to 5.6 with KOH solution; The divalent cation of using in the described conversion is CaCl
2Solution; The cell walls fertile absorber of described adding is the dextran vitriolate of tartar.
Carrier of the present invention has the following advantages:
1. this carrier has versatility, can insert any goal gene in the MCS site of ZsGreen upstream;
2. this carrier has double-promoter, the SV40 promotor is used to start selection markers, as resistant gene etc., introduce the CMV promotor and be used to start ZsGreen gene and expression of exogenous gene, thereby avoid goal gene long and influence the starting efficiency of promotor the selection markers gene;
3. utilize Bar gene and ZsGreen gene to carry out dual screening, can avoid false-positive appearance to greatest extent;
4.ZsGreen expression of gene both can be used as reporter gene and has been used to refer to and optimizes transgenic method, can carry out amalgamation and expression with foreign gene again, can be used as the indication of target protein, also can be used as significant notation and be used for purifying and produce later stage technologies such as albumen.
Description of drawings
Describe the present invention in detail below in conjunction with the drawings and specific embodiments;
Fig. 1 is a plasmid pZsGreenl-N1 collection of illustrative plates, and its molecular weight is 4.7kb.
Fig. 2 is a plasmid construction Psv-bar-ZsGreen electrophorogram.
Wherein, 2A:1,2 is the pZsGreen plasmid, and M is λ-Hind III digest;
2B:1,2 is the PCR product that contains the plasmid pZsGreen of Xba I joint; M is 250bp DNA LadderMarker;
2C:1 is the pMD20-T behind the connection PCR product, and M is 250bp DNALadder Marker;
2D:M1 is DL2000 DNA Marker, and 2 is that Xba I enzyme is cut back pMD20-T, and 3 is that Xba I enzyme is cut back pSV-bar-ZsGreen plasmid, and 4 is pSV-bar plasmid after Xba I enzyme is cut, and 5 is the pSV-bar-ZsGreen plasmid, and M6 is λ-Hind III DNA Marker;
2E:1 is the PCR product of pSV-bar-ZsGreen plasmid, and M is 250bp DNALadder Marker.
Fig. 3 is the structure schema of Psv-bar-ZsGreen.
Fig. 4 is feldspar water shield transfer-gen plant PCR electrophoresis detection figure.
Wherein 1 negative contrast, 2-4 is for transforming the PCR product of the total DNA of seedling, and M is 250bp DNALadder Marker.
Fig. 5 Psv-bar-ZsGreen expression vector is in feldspar water shield (edge pipe Enteromorpha) moment expression.
Chlorophyll sends red fluorescence under ultraviolet source excites, ZsGreen albumen sends green fluorescence, as shown by arrows.
(A.12 B.30 day expression of hour expression)
Embodiment
For technique means, creation characteristic that the present invention is realized, reach purpose and effect is easy to understand, below in conjunction with concrete diagram, further set forth the present invention.Following embodiment further describes the present invention, but described embodiment only is used to illustrate the present invention rather than restriction the present invention.
Molecular cloning method reference literature in the example " molecular cloning experiment guide " third edition, Huang Peitang etc. translate, Science Press, 2002 years.
1.1, as shown in Figure 1, design a pair of special primer according to pZsGeenl-N1 expression vector (buying company, about 4.7kb) in Clontech.5 ' end at upstream primer pZsGeenl P1 and downstream primer pZsGeen2 P2 adds that Xba I restriction enzyme site, its sequence comprise CMV immediate earlypromoter, enhanser zone, TATA box, start point, initiator codon, terminator codon (about 1500bp).
Primer sequence is as follows:
pZsGeen1?P1：5’-GTTATTAATAGTAATCAATTACGG-3’
pZsGeen1?P2：5’-TCTAGATCAGGGCAAGGCGGAGCCGG-3’
1.2 amplification PCR reaction system and condition are:
PCR reaction system (25 μ l):
Aseptic ultrapure water 17.1 μ l
10 * Buffer (contains Mg
2+) 2.5 μ l
dNTP?1μl
Each 1 μ l of primer (10ppm)
Template (0.8 μ g/ μ l) 2 μ l
Taq enzyme (TAKARA) 0.4 μ l (2.5U)
Pre-94 ℃ of 5min of sex change,
94℃?40s
58℃?30s
72℃?90s
Carry out 30 circulations altogether,
72 ℃ are extended 10min.
With PCR product (about 1500bp) behind agarose gel electrophoresis, reclaim the DNA product that test kit (available from TaKaRa company) reclaims PCR with DNA FragmentPurification Kit Ver.2.0 glue.
1.3 with the purifying fragment that obtains, be cloned on the pMD20-T carrier, (buy biotech firm, 2736bp), and transform TOP10 competence bacteria (buying biotech firm) in TIANGEN in TaKaRa.Behind the selection culture medium culturing 18h of new system, carry out blue hickie screening.
1.4 the amplification of picking mono-clonal is with its called after pMD20-T-18; Sample presentation carries out determined dna sequence, determines that the correct also forward of sequence clone inserts the bacterium colony of pMD20-T.The bacterium colony amplification back that order-checking is correct is standby with MiniBEST Plasmid Purification Kit Ver.2.0 test kit (available from TaKaRa company) extracting plasmid.
1.5 the building process of PSV-bar plasmid
Initial carrier is that psv-β-Galactosidase Control buys the Promega company in the U.S., and this carrier is about 6820bp, and this carrier is the coding region of lacZ at 710-3755.Utilize the restriction enzyme site HindIII and the EcoR I enzyme at these gene two ends to cut with standby after this gene elmination.
Utilize the existing plasmid vector PUC-bar in laboratory, its character is identical with commercially available character.[wherein, the PUC plasmid is purchased the company in Promega, adopts conventional molecule experimental technique to be built into plasmid vector PUC-bar with synthetic bar gene (sequence number FJ858786.1 or FJ826509.1)].
From above-mentioned plasmid vector PUC-bar, clone the bar gene, described bar gene, total length is 555bp.Cloning used primer is: upstream primer 5 '-3 ' GCACCATCGTCAACCACTA;
Downstream primer 5 '-3 ' CAGAAACCCACGTCATGC.
Above-mentioned HindIII is inserted at the bar gene two ends that clone respectively be connected restriction enzyme site with EcoR I and cut standby carrier segments with above-mentioned enzyme and be connected, obtain Psv-bar universal support (4191bp).
1.6 cut the plasmid of PSV-bar plasmid and forward insertion pMD20-T with XbaI37 ℃ of enzyme; Reclaim the purpose fragment with 3 times of volume raw spirits, 0.1 times of volume 4mol/L ClLi precipitation.The purpose fragment is connected the back of spending the night transforms DH5 α competence bacteria with PSV-bar, sample presentation order-checking and screening positive clone, with its called after PSV-bar-ZsGeen, molecular weight is 5730bp, primer sequence is:
Test?bar+pZsG1：GCACCATCGTCAACCACTACATC
Test2?pAs1500：GTACATGAAGGCGGCGGACAAG
Whole building processs of Psv-bar-ZsGreen carrier are seen Fig. 3.
The screening of the conversion selective marker of embodiment 2 large-scale green algas
By screening, weedicide Basta (careless fourth phosphine) all has the very strong effect of killing livestock to bar Enteromorpha spore and seedling, wherein the Basta of 5 μ g/ml concentration can all kill bar Enteromorpha spore in 3 days, and about week age can all cause death the Enteromorpha seedling under the 12.5 μ g/ml concentration.Subsequent experimental shows that the result is equally applicable to other kind Enteromorphas such as edge pipe Enteromorpha.
3 pairs of bar Enteromorphas of embodiment and edge pipe Enteromorpha cell protoplast separation and purification condition are optimized
When discovery obtained bar Enteromorpha and edge pipe Enteromorpha protoplastis with enzyme process, best enzymolysis prescription was that 2% cellulase mixes 2% macerozyme, and best enzymolysis pH value is 6.5, and the permeate agent mannitol concentration is 0.6M.Observe the Enteromorpha cell by the protoplastis cultivation and mainly contain 3 kinds of different growth modes such as unicellular seedling, cell mass, sporocyst/gamocyst.
1. protoplastis is suspended in (0.7M N.F,USP MANNITOL in the conversion medium; 15mM MgCl2; 0.1%MES; Transfer pH to 5.6 with KOH solution), adjusting density is 2 * 10
6/ ml gets 0.5ml and places centrifuge tube (10ml).2. add 5 μ L salmon sperm dnas (10mg/ml), left standstill behind the mixing gently 10～15 minutes.3. add 50 μ L plasmid DNA (0.5mg/ml), left standstill behind the mixing gently 10～15 minutes.4. (PEG-6000 is dissolved in 0.1MCa (NO to add isopyknic 40%PEG solution
3)
2And in the 0.6M mannitol solution, transfer pH to 8.0 with KOH solution, and making the PEG final concentration is 20%, mixing immediately, and room temperature was placed 30 minutes, vibrated once in a while.5. added 1～2ml 0.2M CaCl every 3～5 minutes
2Solution progressively is diluted to till the 10ml.6. centrifugal collection protoplastis is suspended in the 5ml protoplast culture medium and cultivates.7. transformation experiment group and negative control group feldspar water shield (the edge pipe Enteromorpha) cell that will cultivate after 5 days change in the nutrient solution and screen.1 seedling of can regenerating after each cell (or protoplastis) was cultivated through 7-14 days.
The total DNA of plant after adopting the extraction of LiCl method to transform one month, and set up negative control group and blank group.Negative control group is not add plasmid DNA in the transformation system, and culture condition is identical with test group.
The design Auele Specific Primer is
Test?DNA+pZsG1：5’-GGCACGGACTTGGCCTTGTA-3’，
Test?DNA+pZsG2：5’-TGGGACCGCTCCTTCCTGTT-3’
Bar gene fragment with the long 296bp that increases.The PCR reaction system is 25 μ L, and reaction parameter is: 94 ℃ of sex change 40s, and 60 ℃ of annealing 30s, 72 ℃ are extended 45s, 30 circulations of coamplification.The amplification back is detected with 1.0% agarose gel electrophoresis.
Embodiment 6 feldspar water shields (edge pipe Enteromorpha) transfer-gen plant fluoroscopic examination
After transforming 48h, draw protoplastis at random and observe with OLYMPAS IX71 fluorescence inverted microscope.Change transformation experiment group and negative control group feldspar water shield (the edge pipe Enteromorpha) cell of cultivation after 5 days in the VSE nutrient solution (proportion 1.015) that contains 5ppm Totomycin screening, culture condition is that intensity of illumination is 72 μ molm-2s-1,20 ℃ of temperature, periodicity of illumination is 12D:12L, and pressure is cultivated after three months and observed with OLYMPASIX71 fluorescence inverted microscope.
Wherein, VSE nutrient solution storage liquid prescription is (adding the 1mL storage liquid in the time spent 1L sea water medium):
Na
2HPO4·12H
2O 10.75g/L
NaNO
3 42.5g/L
Na
2EDTA 3.72g/L
MnCl
2·4H
2O 0.0198g/L
FeSO
4·7H
2O 0.0278g/L
Vitamin V B
10.2g/L
Vitamin V B
120.01g/L
Vitamin H 0.001g/L
The result shows: the protoplastis behind a small amount of conversion of the absorption 12h, under fluorescent microscope, observe, blue-light excited, transform the green fluorescence that successful protoplastis presents (figure A) and transform pigment soma such as the interior chloroplast(id) of successful cell and disturb, can present the red fluorescence background.Observe after remaining protoplastis continued to be cultured to 72h, find that protoplastis germinates, wherein transform the obvious green fluorescence that is successfully.After one week the protoplastis of growing is transferred in the 250ml Erlenmeyer flask inflation and adds basta and continue pressure and cultivate, cultivate and under fluorescent microscope, observe after one month, transform successful feldspar water shield (edge pipe Enteromorpha) and developed into seedling, after blue-light excited, whole frond all presents green, and negative control group presents the redness of background all the time, shows that the feldspar water shield (edge pipe Enteromorpha) that transforms by the PEG method has obtained to express.
More than show and described ultimate principle of the present invention, principal character and advantage of the present invention.The technician of the industry should understand; the present invention is not restricted to the described embodiments; that describes in the foregoing description and the specification sheets just illustrates principle of the present invention; the present invention also has various changes and modifications without departing from the spirit and scope of the present invention, and these changes and improvements all fall in the claimed scope of the invention.The claimed scope of the present invention is defined by appending claims and equivalent thereof.
Claims (10)
1. an expression vector that is used for large-scale green alga bio-reactor is characterized in that, described carrier is PSV-bar-ZsGeen, connects SV40 promotor, resistant gene, CMV promotor and MCS polyclone restriction site on it successively.
2. a kind of expression vector that is used for large-scale green alga bio-reactor according to claim 1 is characterized in that, merge in described MCS downstream reporter gene ZsGreen.
3. a kind of expression vector that is used for large-scale green alga bio-reactor according to claim 1 is characterized in that described resistant gene is the bar gene.
4. a kind of expression vector that is used for large-scale green alga bio-reactor according to claim 1 is characterized in that, is connected with the sub-ori of at least one relaxed replication before the described SV40 promotor, and described carrier is the relaxed type carrier.
5. a kind of expression vector that is used for large-scale green alga bio-reactor according to claim 1 is characterized in that, also is connected with the Amp resistant gene in the described carrier.
6. according to each described a kind of expression vector that is used for large-scale green alga bio-reactor of claim 1-5, it is characterized in that described large-scale green alga is the green laver green alga.
7. a method for transformation that is used for the expression vector of large-scale green alga bio-reactor is characterized in that, utilizes fluorescence protein gene ZsGreen to optimize transgenic method as reporter gene.
8. method for transformation according to claim 7 is characterized in that, comprise the steps: 1. by the cell protoplast separation and purification of enzymolysis process kelp, 2. as the protoplastis of acceptor before conversion through the conversion medium pre-treatment; 3. improve transformation efficiency with the divalent cation material in transforming, complete soln keeps the osmotic pressure of protoplastis itself; 4. PEG6000 transforms final concentration at 14-23%; 5. cultivate adding cell walls fertile absorber in the regenerative process in the later stage, reduce the osmotic potential of solution simultaneously gradually.
9. method for transformation according to claim 7 is characterized in that, it is bar-Basta grass fourth phosphine that pair cell transforms the marker combination of screening.
10. method for transformation according to claim 8 is characterized in that, described best enzymolysis prescription is that 2% cellulase mixes 2% macerozyme, and best enzymolysis pH value is 5.8, and the permeate agent mannitol concentration is 0.6M; Described conversion medium is: 0.7M N.F,USP MANNITOL, 15mM MgCl2; 0.1%MES transfers pH to 5.6 with KOH solution; The divalent cation of using in the described conversion is CaCl
2Solution; Described cell walls fertile absorber is the dextran vitriolate of tartar.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102337217A (en) * | 2011-09-22 | 2012-02-01 | 天津师范大学 | Method for constructing chlorella bioreactor |
| CN106520766A (en) * | 2016-05-31 | 2017-03-22 | 中国科学院海洋研究所 | Seaweed endogenesis constructive promoter and application thereof |
| CN109694878A (en) * | 2018-12-29 | 2019-04-30 | 中国海洋大学 | A kind of kelp molecular breeding method based on protoplast |
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2010
- 2010-01-29 CN CN2010101032849A patent/CN102102107A/en active Pending
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102337217A (en) * | 2011-09-22 | 2012-02-01 | 天津师范大学 | Method for constructing chlorella bioreactor |
| CN102337217B (en) * | 2011-09-22 | 2014-03-05 | 天津师范大学 | Method for constructing chlorella bioreactor |
| CN106520766A (en) * | 2016-05-31 | 2017-03-22 | 中国科学院海洋研究所 | Seaweed endogenesis constructive promoter and application thereof |
| CN106520766B (en) * | 2016-05-31 | 2019-12-10 | 中国科学院海洋研究所 | Seaweed endogenous constitutive promoter and application thereof |
| CN109694878A (en) * | 2018-12-29 | 2019-04-30 | 中国海洋大学 | A kind of kelp molecular breeding method based on protoplast |
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Application publication date: 20110622 |