CN102220359B - Method for performing gene transformation of Schizochytrium aggregatum by medication by Agrobacterium tumefaciens - Google Patents
Method for performing gene transformation of Schizochytrium aggregatum by medication by Agrobacterium tumefaciens Download PDFInfo
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Abstract
The invention discloses a method for performing gene transformation of Schizochytrium aggregatum by medication by Agrobacterium tumefaciens, which comprises the following step: transforming Schizochytrium aggregatum protoplast by using recombinant Agrobacterium tumefaciens to obtain transgenic Schizochytrium aggregatum, wherein the recombinant Agrobacterium tumefaciens is obtained by transforming Agrobacterium tumefaciens with plasmids containing exogenous genes. In the method, expensive apparatuses are not used, the operation is easy, the repeatability is high, the transformation efficiency is high, and more than 100 transformants can be obtained from each 107 Schizochytrium aggregatum cells. The construction of the method lays a good foundation for improving the quality of Schizochytrium aggregatum by using transgenic technology and obtaining a new Schizochytrium aggregatum variety which grows quickly, has high lipa content and simple components. The method has a very important significance for the genetic improvement of Schizochytrium aggregatum.
Description
Technical field
The invention belongs to the genetically engineered field, relate to the method that the agriculture bacillus mediated counterincision kettle of a kind of usefulness algae carries out gene transformation.
Background technology
Docosahexenoic acid (Docosahexaenoic acid; be called for short DHA) be important ω-3 highly unsaturated fatty acids; having the brain cell of promotion grows; reducing blood-fat; hypoglycemic; vision protection, anticancer and improve the multiple important physiological function such as immunological competence, be subject to the extensive concern of medical profession and food circle.
The main source of commercialization DHA is fish oil, but the abyssal pelagic fishes resource-constrained, and DHA content is subjected to the impact in kind, season and geographical position of fish and unstable in the fish oil, can not satisfy the growing market requirement.Utilize DHA Production by Microorganism Fermentation to have the cycle weak point, lipid acid consists of simple, is not subjected to the restriction of raw material, is easy to the advantages such as purification, is expected to become effective source of high purity DHA.
Splitting the kettle algae is a kind of marine microalgae, belongs to the Stramenopiles guiding principle together with little algaes such as diatom, brown algas.Split the kettle algae and can utilize glucose to carry out the heterotrophism cultivation for carbon source, its vegetative cell carries out continuous binary division, and lipid acid forms simple, and DHA content is high, and separation and purification is simple, is the fine resource of obtaining natural high density DHA.
Genetic engineering technique is a kind of modern biotechnology of rising in recent years, and this technology has obtained some great achievements and obtained significant economic benefit in the breeding field, has obtained the good kind of many proterties.Utilize transgenic technology to improve to split the quality of kettle algae, acquisition fast growth, fat content height, component are simply split kettle algae new variety and will be had huge commercial advantage and wide market outlook, seem unusually important so utilize the modern genetic engineering technology to improve the quality of splitting the kettle algae.But the genetically engineered field of splitting at present the kettle algae still is in blank.Although splitting via Particle Bombardment Transformation and the electric shock transformation method of kettle algae sets up, but the recombination form of these method mediations is homologous recombination, therefore need to be from splitting clone's acquisition homologous fragment in the kettle frond, owing to lack the genome sequence column information that splits the kettle algae, limited greatly the application of above-mentioned two kinds of method for transformation.Therefore, development is new splits the non-homogeneous recombination method for transformation of kettle algae and will advance greatly the development of splitting kettle algae genetically engineered field.
Summary of the invention
The purpose of this invention is to provide the method that the agriculture bacillus mediated counterincision kettle of a kind of usefulness algae carries out gene transformation.
The invention provides a kind of method that transgenosis is split the kettle algae for preparing, comprise the steps: to split kettle algae protoplastis with the restructuring Agrobacterium-mediated Transformation, obtain transgenosis and split the kettle algae; Described restructuring Agrobacterium is that the Plasmid Transformation that will the contain foreign gene Agrobacterium that sets out obtains.
The described Agrobacterium that sets out can be agrobacterium strains LBA4404.
Describedly split the kettle algae and specifically can be and split kettle algae Schizochytrium sp.TIO1101 CGMCC No.4603.
The described kettle algae protoplast preparation method of splitting can comprise the steps:
(1) splits the kettle algae and be cultured to logarithmic phase described;
(2) collect the described cell that splits the kettle algae, with cellulase and helicase digestion, obtain to split kettle algae protoplastis.
Described step (1) is front carrying out, and can split the kettle algae and carries out preculture described.Described preculture specifically can be: split the kettle algae and be inoculated in the YPD substratum overnight incubation in 28 ℃ of shaking tables described.In the described step (1), described culture temperature can be 28 ℃.In the described step (1), can adopt specifically that the YPD culture medium culturing is described splits the kettle algae.
In the described step (2), can be by the described cell that splits the kettle algae of centrifugal collection.Described centrifugal parameter specifically can be: 4000rpm, 4 ℃ of centrifugal 5min.In the described step (2), collect described split carry out described digestion behind the kettle algae before, can be first wash described cell with the sterilized water of precooling.In the described step (2), the condition of described digestion specifically can be 28 ℃ of digestion 5-6 hour.In the described step (2), described cellulase and described helicase specifically can add by the mode of enzyme treatment solution; Described enzyme treatment solution is by solute and solvent composition; Described solvent is 20mM phosphate buffered saline buffer (pH5.8); Described solute and concentration thereof are as follows: 2% (mass percent) cellulase, 2% (mass percent) helicase and 0.7MKCl.
Describedly split kettle algae protoplastis with the restructuring Agrobacterium-mediated Transformation and can comprise the steps:
1. with Syringylethanone the restructuring Agrobacterium is induced in advance;
2. split kettle algae protoplastis and completing steps restructuring Agrobacterium is 1. cultivated altogether with described, obtain transgenosis and split the kettle algae.
Carry out described step 1. before, can be with described restructuring Agrobacterium in the YEB substratum, 28 ℃ of concussions are cultured to logarithmic phase, then centrifugal collection restructuring Agrobacterium thalline.Described step 1. in, described culture condition of inducing in advance can be 28 ℃ and cultivated 4-5 hour.1. described step can carry out in inducing culture IM.The concentration of described restructuring Agrobacterium in described inducing culture IM is OD600=0.6-0.8, and the concentration of described Syringylethanone in described inducing culture IM is 250umol/L.
Described step 2. in, described condition of cultivating altogether can be 28 ℃ of concussions and cultivated 10-18 hour, specifically can be 28 ℃ of concussions and cultivates 14 hours.2. described step can carry out in inducing culture IM.
The described method of splitting kettle algae protoplastis with the restructuring Agrobacterium-mediated Transformation also can comprise the step of the bacterium liquid after the common cultivation being carried out antibiotic-screening.Described resistance screening can adopt following condition: cultivated 2-3 days for 28 ℃.Microbiotic in the described antibiotic-screening specifically can be comprised of G418, penbritin and paraxin.Described antibiotic-screening preferably adopts the YPD screening culture medium to carry out.
Described inducing culture IM is comprised of water and solute; Described solute and the concentration in described inducing culture IM thereof are as follows: NaCl 0.15g/L, MgSO
47H
2O 0.25g/L, K
2HPO
42.28g/L, KH
2PO
41.36g/L, CaCl
2H
2O 0.078g/L, FeSO
40.0025g/L, (NH
4)
2SO
40.53g/L, glucose 1.98g/L, glycerine 0.54g/L, MES 7.808g/L; PH5.3.
Described YPD screening culture medium is comprised of YPD substratum, G418, penbritin and paraxin, and the concentration of G418 is 300mg/L, and the concentration of penbritin is 300mg/L, and the concentration of paraxin is 300mg/L.
Described YEB substratum is comprised of water and solute; Described solute and the concentration in described YEB substratum thereof are as follows: 5g/L beef extract, 1g/L yeast extract, 5g/L peptone, 5g/L sucrose, 0.5g/L MgSO
47H
2O; PH7.4.
Described YPD substratum is comprised of water and solute; Described solute and the concentration in described YPD substratum thereof are as follows: 10g/L yeast extract, 20g/L peptone, 20g/L glucose, 50% (volume percent) seawater; PH6.2-6.8.
Described foreign gene specifically can be at least a in the EGFP gene shown in the sequence 3 of the NPTII gene shown in the sequence 2 of the gus gene shown in the sequence 1 of sequence table, sequence table and sequence table.
When described foreign gene was described gus gene and described NPT II gene, the described plasmid that contains foreign gene was binary vector pCAMBIA2301.
When described foreign gene was the EGFP gene, the described plasmid that contains foreign gene was recombinant plasmid pCAMBIA2301-EGFP; Described recombinant plasmid pCAMBIA2301-EGFP can be in the multiple clone site of binary vector pCAMBIA2301 and inserts the recombinant plasmid that the TEF1-EGFP-CYC1 fragment obtains.HindIII and BamHI restriction enzyme site that described recombinant plasmid pCAMBIA2301-EGFP specifically can be at binary vector pCAMBIA2301 insert the recombinant plasmid that described TEF1-EGFP-CYC1 fragment obtains.Described TEF1-EGFP-CYC1 fragment comprises the transcription terminator CYC1 shown in the sequence 5 of the EGFP gene shown in the sequence 3 of the constitutive promoter TEF1 shown in the sequence 4 of sequence table, sequence table and sequence table successively from upstream to downstream.Described TEF1-EGFP-CYC1 fragment specifically can be the small segment that obtains with restriction enzyme HindIII and BamHI double digestion recombinant plasmid pBS-TEF1-EGFP-CYC1; Described recombinant plasmid pBS-TEF1-EGFP-CYC1 specifically can be: take cloning vector pBluescript II SK+ as skeleton plasmid, the EGFP gene shown in the sequence 3 of insertion sequence table between EcoRI and PstI site, the constitutive promoter TEF1 shown in the sequence 4 of insertion sequence table between HindIII and EcoRI site, the transcription terminator CYC1 shown in the sequence 5 of insertion sequence table between PstI and BamHI site.
More than the transgenosis that obtains of arbitrary described method split the kettle algae and also belong to protection scope of the present invention.
The invention provides a kind of efficient, stable, easy method of splitting the gene transformation of kettle algae.The present invention to be splitting kettle algae protoplastis as acceptor material, transforms by the Agrobacterium LBA4404 mediate foreign gene and splits the kettle algae, utilized cleverly to split kettle algae and Agrobacterium LBA4404 the different characteristics of antibiotic susceptibility have been finished the screening that transgenosis is split the kettle algae.The present invention does not need to relate to expensive instrument, easy handling, and good reproducibility, and transformation efficiency is high, and per 10
7Individual frond cell can obtain the transformant more than 100.The quality of utilizing transgenic technology to improve to split the kettle algae that is established as of the present invention obtains that fast growth, fat content are high, component simply splits kettle algae new variety and have laid a good foundation.The genetic improvement tool of the inventive method counterincision kettle algae is of great significance.
Description of drawings
Fig. 1 is the structural representation of binary vector pCAMBIA2301.
Fig. 2 is the phenotypic evaluation result that transgenosis is split the kettle algae.
Fig. 3 is the insertion situation that PCR checking transgenosis is split NPT II gene in the kettle algae.
Fig. 4 is the insertion situation that PCR checking transgenosis is split gus gene in the kettle algae.
Fig. 5 is the building process synoptic diagram of recombinant plasmid pBS-EGFP.
Fig. 6 is the building process synoptic diagram of recombinant plasmid pBS-TEF1-EGFP-CYC1.
Fig. 7 is the building process synoptic diagram of recombinant plasmid pCAMBIA2301-EGFP.
Fig. 8 is the insertion situation that PCR checking transgenosis is split foreign gene EGFP in the kettle algae.
Fig. 9 is the southern results of hybridization that transgenosis is split the kettle algae.
Figure 10 is that Western blot detects the expression that transgenosis is split foreign gene EGFP in the kettle algae.
Embodiment
Following embodiment is convenient to understand better the present invention, but does not limit the present invention.Experimental technique among the following embodiment if no special instructions, is ordinary method.Used test materials among the following embodiment if no special instructions, is and purchases available from routine biochemistry reagent shop.Quantitative test in following examples all arranges repeated experiments three times, results averaged.The MES Chinese is 2-(N-morpholino) ethane sulfonic acid one water.
Plasmid pEGFP-N1:Clontech, Catalog Number #6085-1.
Cloning vector pBluescript II SK+:Stratagene, Catalog Number #212205..
Plasmid pGAPZ α A:Invitrogen, Catalog Number V205-20.
Binary vector pCAMBIA2301:CAMBIA, Canberra, Australia..
Agrobacterium strains LBA4404:Invitrogen, Catalog Number 18313-015..
Enzyme treatment solution: by solute and solvent composition; Described solvent is 20mM phosphate buffered saline buffer (pH5.8); Described solute and concentration thereof are as follows: 2% (mass percent) cellulase, 2% (mass percent) helicase and 0.7M KCl.
The YPD substratum: 10g/L yeast extract, 20g/L peptone, 20g/L glucose, 50% (volume percent) seawater, all the other are water; PH6.2-6.8.
YEB substratum: 5g/L beef extract, 1g/L yeast extract, 5g/L peptone, 5g/L sucrose, 0.5g/L MgSO
47H
2O, all the other are water; PH7.4.
Inducing culture IM: formed by water and solute; Described solute and concentration thereof are as follows: NaCl 0.15g/L, MgSO
47H
2O 0.25g/L, K
2HPO
42.28g/L, KH
2PO
41.36g/L, CaCl
2H
2O 0.078g/L, FeSO
40.0025g/L, (NH
4)
2SO
40.53g/L, glucose 1.98g/L, glycerine 0.54g/L, MES 7.808g/L; PH5.3.
YPD screening culture medium: formed by YPD substratum, G418, penbritin and paraxin; The concentration of G418 is 300mg/L, and the concentration of penbritin is 300mg/L, and the concentration of paraxin is 300mg/L.
One, splits kettle algae protoplast preparation
Split kettle algae Schizochytrium sp.TIO1101 and be preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center (abbreviation CGMCC on 02 23rd, 2011, address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica, postcode 100101), preserving number is CGMCC No.4603.Split kettle algae Schizochytrium sp.TIO1101 CGMCC No.4603 abbreviation and split kettle algae TIO1101.
The step of preparation protoplastis is as follows:
(1) will split kettle algae TIO1101 and be inoculated in the YPD substratum, overnight incubation in 28 ℃ of shaking tables.
(2) transfer in the fresh liquid YPD substratum of 50ml 28 ℃ according to the inoculum size of 1% (volume ratio) and be cultured to logarithmic phase next day.
(3) the 4 ℃ of centrifugal 5min of rotating speed with 4000rpm collect frustule, utilize the sterilized water washing frustule of precooling once to add afterwards 10ml enzyme treatment solution, digestion is 5-6 hour in 28 ℃ of shaking tables, the state that the kettle algae forms protoplastis is split in microscopic examination, can stop digestion when the kettle frustule is converted into protoplastis when splitting more than 90%.
Two, the gene transformation of kettle algae is split in the Agrobacterium LBA4404 mediation
The structure collection of illustrative plates of binary vector pCAMBIA2301 is seen Fig. 1.Binary vector pCAMBIA2301 contains alpha-glucosidase gene (gus gene is shown in the sequence 1 of sequence table) and G418 resistant gene (NPT II gene is shown in the sequence 2 of sequence table); Wherein gus gene can be expressed β-glucuronidase as reporter gene in splitting the kettle algae, and render transgenic splits kettle algae generation color reaction in the situation that substrate exists; NPT II gene is given transgenosis and is split the kettle algae to the resistance of G418 as selectable marker gene.
Concrete operation step is as follows:
1, binary vector pCAMBIA2301 is transformed agrobacterium strains LBA4404, obtain the Agrobacterium of recombinating.
2, picking restructuring Agrobacterium is to liquid YEB substratum, 28 ℃ of concussions are cultured to logarithmic phase, centrifugal collection thalline, the concentration of utilizing the resuspended dilution of inducing culture IM to adjust Agrobacterium is OD600=0.6-0.8, and to add final concentration be the Syringylethanone of 250umol/L, induced in advance 4-5 hour for 28 ℃.
3, the protoplastis of centrifugal collection step 1 preparation, resuspended with inducing culture IM after, add in the bacterium liquid that step 2 obtains, 14 hours (adopt 10-18 hour all can) be infected in 28 ℃ of concussions.
4, the frustule that step 3 is obtained and the centrifugal collection of thalline mixed culture are coated in the solid YPD screening culture medium, cultivate 2-3 days for 28 ℃.
5, the clone of the transformant in the picking solid YPD screening culture medium.
6, postsearch screening and phenotypic evaluation
With streak culture in solid YPD screening culture medium after the transformant clone cultivation that obtains, split kettle algae algae strain (splitting kettle algae TIO1101) as negative control with wild-type simultaneously, photo sees that (A1 to A7 is that transgenosis is split the strain of kettle algae algae to Fig. 2; WT is for splitting kettle algae TIO1101).Transgenosis is split kettle algae algae strain A1-A7 and all can be screened in the solid culture at YPD and grow, and wild-type is split the strain of kettle algae algae and then can not be grown.The result shows, has obtained transgenosis and has split the strain of kettle algae algae.
Three, changing effect is identified (PCR checking foreign gene splits integration in the kettle algae genome in transgenosis)
Split kettle algae A1-A6 (to split kettle algae TIO1101 as negative control with transgenosis respectively, WT) genomic dna is template, the primer that uses NPT II-F and NPT II-R to form detects NPT II gene to PCR, and the primer that uses GUS-F and GUS-R to form detects gus gene to PCR.
NPT?II-F：5’-TCACTGAAGCGGGAAGGGACT-3’；
NPT?II-R：5’-GCGGCGATACCGTAAAGCAC-3’。
GUS-F：5’-GACTCGTCCGTCCTGTAGAAACC-3’；
GUS-R：5’-AAAGTCCCGCTAGTGCCTTGTC-3’。
The PCR reaction conditions: 95 ℃ of denaturation 5min, then through 30 circulations (94 ℃ of 30s, 56 ℃ of 30s, 72 ℃ of 1min), 72 ℃ of 10min.
Get 5 μ l PCR products and carry out 1% agarose gel electrophoresis, the EB observation of dyeing.
The results are shown in Figure 3 and Fig. 4.The result shows: split in the genome of kettle algae A1-A6 in transgenosis and all can obtain the purpose fragment (about 500bp) of NPTII gene and the purpose fragment (about 750bp) of gus gene by pcr amplification; In the genome that splits kettle algae TIO1101, there is not the purpose band.Illustrate that foreign gene has successfully inserted transgenosis and split in the genome of kettle algae.Per 10
7Individual frond cell can obtain 100 above transgenosiss and split the strain of kettle algae algae.
Embodiment 2, the agriculture bacillus mediated gene transformation (EGFP gene) of splitting the kettle algae
One, the structure of recombinant plasmid pCAMBIA2301-EGFP
1, the clone of green fluorescence protein gene (EGFP)
(1) take plasmid pEGFP-N1 as template, the primer that forms with EGFP-F and EGFP-R obtains pcr amplification product (green fluorescence protein gene claims again the EGFP gene, shown in the sequence 3 of sequence table) to carrying out pcr amplification.
EGFP-F：5’-GGAATTCATGGTGAGCAAGGGCGA-3’；
EGFP-R：5’-AACTGCAGTTACTTGTACAGCTCGTC-3’。
The PCR reaction conditions: 95 ℃ of denaturation 5min, then through 30 circulations (94 ℃ of 30s, 58 ℃ of 30s, 72 ℃ of 1min), 72 ℃ of 10min.
(2) with restriction enzyme EcoRI and PstI double digestion pcr amplification product, reclaim enzyme and cut product.
(3) with restriction enzyme EcoRI and PstI double digestion cloning vector pBluescript II SK+, reclaim carrier framework (about 3000bp).
(4) enzyme of step (2) is cut product and is connected 3 with step) carrier framework connect, obtain recombinant plasmid pBS-EGFP.
The structural representation of recombinant plasmid pBS-EGFP is seen Fig. 5.
2, the structure of egfp expression module
(1) take plasmid pGAPZ α A as template, the primer that forms with TEF-F and TEF-R obtains pcr amplification product (constitutive promoter TEF1 is shown in the sequence 4 of sequence table) to carrying out pcr amplification.
TEF-F：5’-CCCAAGCTTCCCACACACCATAGCTTC-3’；
TEF-R：5’-GGAATTCGGTTTAGTTCCTCACCTT-3’。
The PCR reaction conditions: 95 ℃ of denaturation 5min, then through 30 circulations (94 ℃ of 30s, 56 ℃ of 30s, 72 ℃ of 1min), 72 ℃ of 10min.
(2) with restriction enzyme HindIII and EcoRI double digestion pcr amplification product, reclaim enzyme and cut product.
(3) with restriction enzyme HindIII and EcoRI double digestion recombinant plasmid pBS-EGFP, reclaim carrier framework (about 3800bp).
(4) enzyme of step (2) is cut product and is connected 3 with step) carrier framework connect, obtain recombinant plasmid pBS-TEF1-EGFP.
(5) take plasmid pGAPZ α A as template, the primer that forms with CYC1-F and CYC1-R obtains pcr amplification product (transcription terminator CYC1 is shown in the sequence 5 of sequence table) to carrying out pcr amplification.
CYC1-F：5’-AACTGCAGCACGTCCGACGGCGGCC-3’；
CYC1-R：5’-CGGGATCCAGCTTGCAAATTAAAGCCT-3’。
The PCR reaction conditions: 95 ℃ of denaturation 5min, then through 30 circulations (94 ℃ of 30s, 56 ℃ of 30s, 72 ℃ of 1min), 72 ℃ of 10min.
(6) with restriction enzyme PstI and BamHI double digestion pcr amplification product, reclaim enzyme and cut product.
(7) with restriction enzyme PstI and BamHI double digestion recombinant plasmid pBS-TEF1-EGFP, reclaim carrier framework (about 4300bp).
(8) enzyme of step (6) is cut product and is connected 7 with step) carrier framework connect, obtain recombinant plasmid pBS-TEF1-EGFP-CYC1.
The structural representation of recombinant plasmid pBS-TEF1-EGFP-CYC1 is seen Fig. 6.
3, the structure of recombinant plasmid pCAMBIA2301-EGFP
(1) with restriction enzyme HindIII and BamHI double digestion recombinant plasmid pBS-TEF1-EGFP-CYC1, reclaims enzyme and cut product (the expression module TEF1-EGFP-CYC1 of EGFP gene).
(2) with restriction enzyme HindIII and BamHI double digestion binary vector pCAMBIA2301, reclaim carrier framework.
(3) enzyme of step (1) is cut product and is connected 2 with step) carrier framework connect, obtain recombinant plasmid pCAMBIA2301-EGFP.
The structural representation of recombinant plasmid pCAMBIA2301-EGFP is seen Fig. 7.
Two, the gene transformation of kettle algae is split in the Agrobacterium LBA4404 mediation
1, splits kettle algae protoplast preparation
2, the gene transformation of kettle algae is split in the Agrobacterium LBA4404 mediation
Replace binary vector pCAMBIA2301 with recombinant plasmid pCAMBIA2301-EGFP, the step 2 of the other the same as in Example 1 obtains transgenosis and splits the strain of kettle algae algae.
3, changing effect is identified
(1) PCR checking foreign gene splits integration in the kettle algae genome in transgenosis
Split kettle algae (algae strain E1-E6 with transgenosis respectively; To split kettle algae TIO1101 as negative control, genomic dna WT) is template, and the primer that uses EGFP-F and EGFP-R to form detects EGFP gene (egfp gene) to PCR.
EGFP-F：5’-GGAATTCATGGTGAGCAAGGGCGAG-3’；
EGFP-R：5’-AACTGCAGTTACTTGTACAGCTCGTC-3’。
The PCR reaction conditions: 95 ℃ of denaturation 5min, then through 30 circulations (94 ℃ of 30s, 56 ℃ of 30s, 72 ℃ of 1min), 72 ℃ of 10min.
Get 5 μ l PCR products and carry out 1% agarose gel electrophoresis, the EB observation of dyeing.
The results are shown in Figure 8.The result shows: split in the genome of kettle algae E1-E6 all the purpose fragment (about 700bp) that can pcr amplification obtains the EGFP gene in transgenosis; In the genome that splits kettle algae TIO1101, there is not the purpose band.Illustrate that foreign gene has successfully inserted transgenosis and split in the genome of kettle algae.
(2) Southern hybridization checking transgenosis is split the copy number of foreign gene in the kettle algae
Respectively transgenosis is split kettle algae (algae strain E1-E6; Splitting kettle algae TIO1101 as negative control, genomic dna WT) carries out southern hybridization (adopt the egfp gene fragment of digoxigenin labeled as probe, probe such as the sequence 3 of sequence table from shown in 5 ' terminal the 5th to 668 Nucleotide).
The results are shown in Figure 9.The result shows that it is all positive that resulting transgenosis is split the kettle algae, and foreign gene is single copy radom insertion.
(3) Western blot detects foreign gene and splits expression in the kettle algae in transgenosis
Respectively transgenosis is split kettle algae (algae strain E1-E6; To split kettle algae TIO1101 as negative control, WT) with obtaining protein liquid after the protein lysate cracking.Various protein liquids are carried out Western blot (primary antibodie is the antibody of EGFP).
The results are shown in Figure 10.The result shows that transgenosis is split among the kettle algae E1-E6 all can successfully detect the EGFP protein expression, and wild-type is split in the kettle algae and then can not be detected.Splitting in the kettle algae in transgenosis and having obtained expression of success of foreign gene EGFP is described.Per 10
7Individual frond cell can obtain 100 above transgenosiss and split the strain of kettle algae algae.
Claims (8)
1. one kind prepares the method that transgenosis is split the kettle algae, comprises the steps: to split kettle algae protoplastis with the restructuring Agrobacterium-mediated Transformation, obtains transgenosis and splits the kettle algae; Described restructuring Agrobacterium is that the Plasmid Transformation Agrobacterium that will contain foreign gene obtains; Described Agrobacterium is agrobacterium strains LBA4404; Describedly split the kettle algae for splitting kettle algae (Schizochytrium sp.) TIO1101 CGMCC No.4603;
Describedly split kettle algae protoplastis with the restructuring Agrobacterium-mediated Transformation and comprise the steps:
1. with Syringylethanone the restructuring Agrobacterium is induced in advance;
2. split kettle algae protoplastis and completing steps restructuring Agrobacterium is 1. cultivated altogether with described, obtain transgenosis and split the kettle algae;
Described step 1. in, described culture condition of inducing in advance is 28 ℃ and cultivated 4-5 hour; 1. described step carries out in inducing culture IM, and the concentration of described restructuring Agrobacterium in described inducing culture IM is OD600=0.6-0.8, and the concentration of described Syringylethanone in described inducing culture IM is 250umol/L;
Described step 2. in, described condition of cultivating altogether is that 28 ℃ of concussions were cultivated 10-18 hour; 2. described step carries out in inducing culture IM;
Described inducing culture IM is comprised of water and solute; Described solute and the concentration in described inducing culture IM thereof are as follows: NaCl 0.15g/L, MgSO
47H
2O 0.25g/L, K
2HPO
42.28g/L, KH
2PO
41.36g/L, CaCl
2H
2O 0.078g/L, FeSO
40.0025g/L, (NH
4)
2SO
40.53g/L, glucose 1.98g/L, glycerine 0.54g/L, MES 7.808g/L; PH5.3.
2. the method for claim 1, it is characterized in that: the described kettle algae protoplast preparation method of splitting comprises the steps:
(1) splits the kettle algae and be cultured to logarithmic phase described;
(2) collect the described cell that splits the kettle algae, with cellulase and helicase digestion, obtain to split kettle algae protoplastis.
3. method as claimed in claim 1 or 2, it is characterized in that: described method also comprises the step of the bacterium liquid after the common cultivation being carried out antibiotic-screening.
4. method as claimed in claim 3, it is characterized in that: the microbiotic in the described antibiotic-screening is comprised of G418, penbritin and paraxin.
5. method as claimed in claim 1 or 2 is characterized in that: described foreign gene is at least a in the EGFP gene shown in the sequence 3 of the NPT II gene shown in the sequence 2 of the gus gene shown in the sequence 1 of sequence table, sequence table and sequence table.
6. method as claimed in claim 5, it is characterized in that: described foreign gene is described gus gene and described NPTII gene; The described plasmid that contains foreign gene is binary vector pCAMBIA2301.
7. method as claimed in claim 5, it is characterized in that: described foreign gene is the EGFP gene; The described plasmid that contains foreign gene is recombinant plasmid pCAMBIA2301-EGFP; Described recombinant plasmid pCAMBIA2301-EGFP inserts the TEF1-EGFP-CYC1 fragment in the multiple clone site of binary vector pCAMBIA2301 to obtain; Described TEF1-EGFP-CYC1 fragment comprises the transcription terminator CYC1 shown in the sequence 5 of the EGFP gene shown in the sequence 3 of the constitutive promoter TEF1 shown in the sequence 4 of sequence table, sequence table and sequence table successively from upstream to downstream.
8. the transgenosis that arbitrary described method obtains in the claim 1 to 7 is split the kettle algae.
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| Cheng RU-Bin et al..Establishment of transgene expression system for the marine microalga Schizochytrium by 18S rDNA-targeted homologous recombination.《World J Microbiol Biotechnol》.2010, * |
| Metz JG et al..Biochemical characterization of polyunsaturated fatty acid synthesis in Schizochtrium: Release of the products as free fatty acids..《Plant Physiology and Biochemistry》.2009, * |
| 方卫国 等.根癌农杆菌介导真菌遗传转化的研究进展.《中国生物工程杂志》.2002,第22卷(第5期), * |
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