CN101717783A - Universal expression vector for large transgenic green alga and transformation expression method thereof - Google Patents

Abstract

The present invention studies the universality of resistance selection markers and resistance selection marker genes of large green algae transgenes such as Enteromorpha, Ulva ulva, and reef membranes, and clones corresponding resistance selection marker genes. Utilize existing vectors of higher plants or apply homologous recombination technology to transform vectors; construct a general vector pSVB suitable for transgenic large green algae. Based on the cell regeneration system and gametoparthenogenesis system, the present invention utilizes the above-mentioned universal carrier for high-efficiency transformation and stable expression, and finally establishes a macroalgae transformation and expression system, so that foreign genes can be efficiently and stably expressed in macroalgae The expression will lay the foundation for the establishment of seaweed reactors and the production of important drugs and seaweed energy in the future.

Description

Universal expression vector of large transgenic green alga and conversion expression method thereof

Technical field

The present invention relates to gene biological engineering field, particularly universal expression vector and the stable conversion expression method thereof of the large transgenic green alga in gene field (Enteromorpha, feldspar water shield etc.).

Background technology

After the generation of the nineteen eighty-three first strain transgene tobacco, the research of various transgenic plant has obtained very big progress in the world at present, and has produced huge economic benefit.But the transgenic research work progress of algae is slow relatively, and 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.

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 the important biomolecule active substance, the exploitation marine alga energy.Various in the world transgenic plant have produced huge economic benefit at present, but the transgenic research integral body of marine alga is made slow progress.Chinese large-sized green alga transgenic research is almost blank at present, and mainly carries out in marine algas such as sea-tangle, laver, wakame.

Only sea-tangle obtains desirable screening marker gene at present, has obtained very 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.

" large-sized chlorophta chlorophyll body is as the application of the acceptor and the expression vector of foreign gene " of Inst of Huanghai Sea Marine Products, Chinese Academy of Aquatic Product Science, application number 200710113576.9; A kind of system that utilizes chloroplast(id) as the foreign gene expression of receptor is disclosed.

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

One of technical problem to be solved by this invention is the versatility by genetically modified resistance screening marker of large-scale green alga such as research Enteromorpha, feldspar water shield, reef film and resistance screening marker gene, and clone corresponding resistance screening marker gene, make full use of the existing carrier of higher plant on this basis or use homologous recombination technique transformation carrier; Construct a kind of universal expression vector that is suitable for the large transgenic green alga.

Another technical problem to be solved by this invention is to be material with large-scale green alga, on cell regeneration system and gamete monogenesis system basis, utilize the above-mentioned efficient conversion and the universal support of stably express, finally setting up kelp transforms and expression system, make foreign gene can be in large-scale green alga efficient and stably express, lay the foundation for setting up marine alga reactor, production important drugs and the marine alga energy from now on.

For solving the problems of the technologies described above, the technical scheme of employing is:

A kind ofly be used for large-scale green alga, particularly the genetically modified universal expression vector pSVB of green laver green alga Enteromorpha, feldspar water shield is connected with SV40 gene promoter, resistant gene, MCS polyclone restriction site expression cassette and NOS polyA terminator in turn on the described carrier pSVB.

Described universal expression vector is applicable to the gene transformation of marine algas such as large-scale green alga, particularly Enteromorpha, feldspar water shield, has the characteristic of efficient conversion and stably express.

Described universal expression vector; 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 herbicide resistance gene is the bar gene.

Described large-scale green alga is green laver green algas such as Enteromorpha, feldspar water shield.

A kind of large transgenic green alga transforms expression method, it is characterized in that,, transform with the above-mentioned universal expression vector pSVB that is used for large-scale green alga as acceptor with large-scale green alga protoplastis, filter out the large transgenic green alga of energy stably express at last with improvement PEG method.

Described a kind of large transgenic green alga transforms expression method, comprising: by the cell protoplast separation and purification of enzymolysis process to kelp.In the above-mentioned conversion expression method, find to obtain large-scale green alga, during as bar Enteromorpha or edge pipe Enteromorpha protoplastis, 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.

Cell by protoplast transformation mainly contains 3 kinds of different growth modes such as unicellular seedling, cell mass, sporocyst/gamocyst.

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 PEG improved method, wherein, as protoplastis process conversion medium pre-treatment before conversion of acceptor; Improve transformation efficiency with other materials such as divalent cations in the conversion, complete soln keeps the osmotic pressure of protoplastis itself; PEG6000 transforms final concentration at 14-23%; Cultivate adding cell walls fertile absorber in the regenerative process in the later stage, reduce the osmotic potential of solution simultaneously gradually, to improve the transgenic plant regeneration ability.

Describedly be used for pretreated conversion medium and be 0.7M N.F,USP MANNITOL; 15mM MgCl2; 0.1%MES; Transfer 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.

Selective marker is expressed in large-scale green alga bar Enteromorpha cell transformation screens, studies show that, bar Enteromorpha protoplastis is insensitive to ampicillin, carbenicillin sodium, sulphuric acid kanamycin, G-418, Vetstrep, to the sensitivity of microbiotic paraxin and weedicide grass fourth phosphine Basta.Different development stage bar Enteromorpha is different to the susceptibility of paraxin and Basta, and the amount that paraxin needs is bigger.Results suggest bar gene might become the comparatively ideal selectable marker gene of Enteromorpha genetically engineered.

PEG method of the present invention has been simplified operation steps on conventional P EG method basis, saved the conversion cost, has reduced the probability of microbiological contamination.All reagent of present method all be with solution that the penetration of sea water gesture of protoplastis equates in carry out, conversion finishes, in the substratum that contains the cell walls fertile absorber, carry out, and progressively reduce the growth that original substratum osmotic pressure is beneficial to each stage of protoplastis, greatly reduce the toxic action of PEG itself to protoplasm somatocyte, increase the cell regeneration rate, improved transformation efficiency.

Because the present invention is with large-scale green alga, particularly the protoplastis of green laver green alga is the basis, is carrier with the universal support that makes up, and therefore the PEG method realization stabilized expression of exogenesis genes by improvement, has the following advantages:

1, large-scale chlorella growth speed is fast, is ideal restoration of the ecosystem person; Medicinal and the edibleness height of Enteromorpha, extensive in the coastal and Japanese consumption of China, can be used as the ideal bio-reactor.

2, utilize protoplastis as acceptor, can effectively avoid existing mosaic, can obtain the single foreign gene genetic stability of dominance.

3, made up foreign gene large-scale green alga, particularly a kind of universal support of stably express in the green laver green alga helps studying molecular biological research of kelp and stabilized expression of exogenesis genes method.

4, the PEG method is by under pharmaceutical chemicals polyoxyethylene glycol (PEG), poly-1-ornithine (Plo), poly-lysine, calcium phosphate and the high pH value condition, improve the permeability of protoplast membrane, induce protoplastis picked-up exogenous DNA molecule, impel between cytolemma and DNA and contact and adhesion, and disorder by the film surface charge and the identification between the interference cell, thereby be beneficial to intercellular fusion and foreign DNA enters protoplastis.The PEG method is to use the method for maximum direct metastatic genes at present, and it can break the restriction of Agrobacterium host range.The experimental cost of this method is low, and the result is stable, and good reproducibility does not especially need special instruments and equipment, is easy to promote.

Description of drawings

Fig. 1 universal expression vector plasmid of the present invention pSVB collection of illustrative plates.

The electrophoretogram of Fig. 2 expression vector plasmid pSVB; Wherein, Lane 1 is pSVB; Lane 2 is DL2000Marker.

Fig. 3 transforms the PCR product electrophorogram of cultivating green alga edge pipe Enteromorpha after 30 days, and wherein lane1 is unconverted Enteromorpha (edge pipe Enteromorpha) PCR product; Lane2 is a transgenosis edge pipe Enteromorpha PCR product; Lane3 is the plasmid contrast; Lane4: λ DNA/HindIII Marker.

Fig. 4 transforms the PCR product of cultivating green alga edge pipe Enteromorpha after 360 days, and wherein, lane1 is unconverted Enteromorpha (edge pipe Enteromorpha) PCR product; Lane2 is transgenosis Enteromorpha (edge pipe Enteromorpha) PCR product; Lane3 is 250bp DNA Ladder Marker.

Embodiment

Following embodiment further describes the present invention, but described embodiment only is used to illustrate the present invention rather than restriction the present invention.

Below in conjunction with embodiment, to being used for foreign gene, be described in further detail, but and do not mean that and limit the scope of the invention in large-scale green alga (Enteromorpha, feldspar water shield etc.) stable conversion expression method.Molecular cloning method reference literature in the example " molecular cloning experiment guide " third edition, Huang Peitang etc. translate, Science Press, 2002 years.

The structure of the universal expression vector Psv-bar of embodiment 1 large transgenic green alga

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 in Promega company, 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 the Psv-bar universal support.

The screening of the conversion selective marker of embodiment 2 large-scale green algas

By screening, weedicide grass fourth phosphine Basta 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 the about week age of Basta of 12.5 μ g/ml concentration can all cause death the Enteromorpha seedling.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.Enteromorpha protoplastis by protoplast transformation mainly contains 3 kinds of different growth modes such as unicellular seedling, cell mass, sporocyst/gamocyst.

Embodiment 4 improves PEG method transformation system

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 screening in the VSE nutrient solution (proportion 1.020) that contains 5ppm Basta over to.1 seedling of can regenerating after each cell (or protoplastis) was cultivated through 7-14 days.

VSE nutrient solution storage liquid prescription (adding the 1mL storage liquid in the time spent 1L sea water medium):

Na ₂HPO ₄·12H 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

Embodiment 5 bar gene fragment orders are analyzed and sequence alignment

Extraction has transformed the total DNA of feldspar water shield (edge pipe Enteromorpha) of plasmid pSVB, with this DNA is template, carry out PCR, electrophoresis detection PCR product, DNA marker is respectively the 250bp DNA Ladder Marker available from sky root λ DNA/HindIII marker and TAKARA company, and glue reclaims product by the order-checking of the precious biotech firm in Dalian.

Embodiment 6 Southern blotting detect

Adopt with EcoI and the total DNA of HindIII (available from TAKARA company) double digestion transgenosis feldspar water shield (edge pipe Enteromorpha), the digoxigenin labeled digested plasmid prepares probe and hybridizes.

6.1 random priming label probe

1) HindIII plasmid PSVB (37 ℃ are spent the night):

2) electrophoresis reclaims the 555bp fragment (containing complete bar gene order) that enzyme is cut;

3) 100 ℃ place mixture of ice and water after water-bath x10 minute at once;

4) add 2 μ l ViaL5 (six nuclear former times acid mixtures), 10 μ l Vial6 (dNTP mark mixture)+1 μ lVial7 (Klenow enzyme)+1 μ l ultrapure water to 20 μ l, 37 ℃ of water-baths are spent the night;

5) EDTA (pH8.0) of adding 0.8 μ l 0.5mol/L is the 20mmol/L termination reaction to final concentration, and the LiCI and the 60 μ l pre-cooled ethanols that add 2 μ l 4mol/L spend the night in-20 ℃ of precipitations;

6) 4 ℃ centrifugal 14Kx15 minute, inhale and to remove supernatant, 70% ethanol that adds the precooling new system is washed one time, it is heavy molten to add 50 μ l TE ,-20 ℃ of preservations are standby.

6.2 hybridization

I) agarose electrophoresis glue is put into culture dish, the NaOH that gets the 0.25mol/L of an amount of volume soaks, vibration;

2) with 0.5 X TBE solution (time spent dilutes with 5 X TBE storage liquid) rinse, balance running gel;

3) cut 2 of filter paper according to the size of running gel, 1 of Hybond membrane is reinstated 0.5X T BE solution soaking balance together with fiber mat (fiber pad);

4) clean electric transfer device post-erection plate, put into fiber mat, filter paper, running gel, film, filter paper, fiber mat successively, do not go up switches set after compressing and dress up one, press gel → transfer film (negative pole → positive pole) direction and insert in the transfer groove from negative pole to positive pole;

5) pour the 0.5X TBE buffer of 4 ℃ of precoolings into, put into Cooling Unit, 80V electricity transfer printing 1 hour;

6) play plate, film is transferred to glass culture dish, 120 ℃ of roasting films 30 minutes;

7) 68 ℃ prehybridization I hour, hybridization solution (5X SSC, blocking reagent, 1% (w/v), N-lauroylsarcosine, 0.1% (w/v); SDS, 0.02% (w/v)) the 20ml/100cm2 film;

8) discard prehybridization solution, add 3ml hybridization solution and 6 μ l sex change probes, hybridized X6 hour for 68 ℃;

9) 50ml washing lotion (2XSSC, SDS0.1% (w/V))/100cm2 washes film 2 times, 2 * 5 minutes under the room temperature;

10) wash film 2 times, 2 * 15 minutes with 50ml washing lotion (0.1XSSC, SDS 0.1% (w/v))/100cm2 under 68 ℃ of temperature:

11) use buffer 1 (pH 7.5 for maleic acid 0.1M, NaCl 0.15M) balance 1 minute under the room temperature;

12) discard buffer 1, add 100ml buffer2/100cm2 film (blocking stock solution does 1/10 dilution by buffer1, i.e. blocking reagent, 1% (w/v)) sealing 30 minutes;

13) discard confining liquid, add 20ml buffer2-antibody/100cm2 film, antibody concentration was 150mU/ml (1: 5000), in conjunction with 30 minutes;

14) discard buffer 2-antibodies liquid, add 100ml buffer 1/100cm2 and wash film 2 times, 2 * 15 minutes;

15) discard buffer 1, add 20ml buffer 3 (Tris-HCI 100mmol, NaCl 100mmol, MgCl ₂50mmol)/100cm2 balance 2 minutes;

16) discard bufer3, add the colour developing of 10ml colour developing liquid (Buffer 3 10ml, NBT solution 45 μ l, X-phosphate-solution 35 μ l) black out.

17) abandon colour developing liquid, TE buffer rinsing three times is taken a picture, and dry sealing is kept in Dark Place.

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. universal expression vector pSVB who is used for the large transgenic green alga, it is characterized in that, connect SV40 gene promoter, resistant gene, MCS polyclone restriction site expression cassette and NOS polyA terminator on the described universal expression vector pSVB successively.

2. a kind of universal expression vector that is used for the large transgenic green alga according to claim 1 is characterized in that, reorganization has the external source functional gene in described MCS site, as antigen gene, vaccine, antibody, human growth factor, alexin; Described resistant gene is the bar gene; Described large-scale green alga is the green laver green alga.

3. a large transgenic green alga transforms expression method, it is characterized in that, as acceptor, use the universal expression vector pSVB of large transgenic green alga to transform with large-scale green alga protoplastis, filter out the large transgenic green alga of energy stably express at last with improvement PEG method.

4. a kind of large transgenic green alga according to claim 3 transforms expression method, it is characterized in that, by the cell protoplast separation and purification of enzyme process to kelp.

5. a kind of large transgenic green alga according to claim 4 transforms expression method, it is characterized in that described 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.

6. a kind of large transgenic green alga according to claim 3 transforms expression method, it is characterized in that the described protoplastis that is used to transform is unicellular seedling, cell mass, 3 kinds of growth modes of sporocyst/gamocyst.

7. a kind of large transgenic green alga according to claim 3 transforms expression method, it is characterized in that, it is bar gene-careless fourth phosphine Basta that pair cell transforms the marker combination of screening.

8. a kind of large transgenic green alga according to claim 3 transforms expression method, it is characterized in that, and described PEG improved method, wherein, as protoplastis process conversion medium pre-treatment before conversion of acceptor; Improve transformation efficiency with the divalent cation material in the conversion, complete soln keeps the osmotic pressure of protoplastis itself; PEG6000 transforms final concentration at 14-23%; Cultivate adding cell walls fertile absorber in the regenerative process in the later stage, reduce the osmotic potential of solution simultaneously gradually.

9. a kind of large transgenic green alga according to claim 8 transforms expression method, it is characterized in that, describedly is used for pretreated conversion medium and is: 0.7M N.F,USP MANNITOL; 15mM MgCl2; 0.1%MES; Transfer 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.

10. transform expression method according to each described a kind of large transgenic green alga of claim 3-9, it is characterized in that described large-scale green alga is the green laver green alga.

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