CN1156579C - Lithium acetate mediated lentinus edodes gene transfer technology - Google Patents
Lithium acetate mediated lentinus edodes gene transfer technology Download PDFInfo
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- CN1156579C CN1156579C CNB011139218A CN01113921A CN1156579C CN 1156579 C CN1156579 C CN 1156579C CN B011139218 A CNB011139218 A CN B011139218A CN 01113921 A CN01113921 A CN 01113921A CN 1156579 C CN1156579 C CN 1156579C
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- protoplastis
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- lithium acetate
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Abstract
The present invention relates to a lithium acetate mediated mushroom gene transfer technology. A seepage stabilizer is added in an enzymolysis liquid of an acceptor protoplast; the pretreatment of an exogenous target gene uses lithium acetate as a mediated substance. Polyethylene glycol, lithium acetate, single chain carried DNA, exogenous plasmid DNA and sterilized water are orderly added in the protoplast to form a reaction liquid. After the reaction liquid is cultivated, centrifuged and separated from compounds, a converted protoplast is obtained. The converted protoplast is mixed with a culture medium to be cultivated in a rocking mode; then, the converted protoplast is moved onto a solid medium to be cultivated. The present invention has the advantages of high conversion efficiency, low conversion cost, simple required devices and conditions, and convenient popularization and generalization.
Description
Belong to technical field
The present invention relates to the edible mushrooms genetic engineering technique.
Background technology
In recent years, the research of edible mushrooms genetically engineered had obtained considerable progress, and a collection of goal gene is come out by the clone, but report relevant aspect the edible mushrooms genetic transformation is less, at home, the someone utilizes electric shocking method that total DNA of oyster cap fungus is imported purple spore and picks up the ears, and has obtained the conversion bacterial strain; Abroad, have to change hygromycin gene over to oyster cap fungus and Twospore Mushroom, the URAI gene changes Liu Songgu over to and solid gene (Frt1) changes Split-gill etc. over to.Above-mentioned trans-utilization mainly be calcium chloride/polyoxyethylene glycol (CaCl
2/ PEG) mediation protoplast transformation and electric shocking method.Aspect the mushroom transgenosis, a kind of transformation technology of restriction enzyme/polyoxyethylene glycol mediation that Japanese rock hand university sets up is only arranged, but the restriction enzyme that uses in this technology costs an arm and a leg, it is higher to transform cost.
Summary of the invention
The object of the present invention is to provide a kind of mushroom gene transfer technique of easy, economic, effective Lithium Acetate mediation.
The present invention includes the screening of the importing of preparation, external source goal gene of the extraction of external source goal gene and purifying, the primary physique of acceptor and conversion, protoplastis and identify four parts; Each process thes contents are as follows:
1, the extraction of external source goal gene and purge process adopt conventional " alkaline lysis " to extract plasmid DNA, plasmid DNA after the extraction adopts polyoxyethylene glycol precipitation classification purifying, detect plasmid DNA concentration and purity with ultraviolet absorption method, concentration requirement reaches 1 mg/ml, purity requirement reaches 0D260/OD280 〉=1.8,0D260/OD230 〉=2.0, and on 1% horizontal sepharose electrophoresis, detection plasmid size, it is stand-by to put into-20 ℃ of refrigerators then;
2, the preparation of acceptor protoplastis
For preventing that protoplastis from breaking, in the enzymolysis solution of protoplastis, add certain pressure, with concentration is that the N.F,USP MANNITOL of 0.4～0.8 mol or sucrose or Repone K are as permeating stablizer, in permeating stablizer, add lywallzyme, the lywallzyme enzymolysis solution of compound concentration 1.5～3%, get the Lentinus Edodes fungus pompon of cultivating 3～10 days, put into enzymolysis solution, under 20～35 ℃ of conditions, carry out enzymolysis, preparation acceptor protoplastis, purifying obtains the pure liquid of protoplastis after filtration, and acceptor protoplastis concentration is 10 in the pure liquid
6Individual/more than the milliliter;
3, the importing of external source goal gene is carried out according to the following steps:
(1) get the pure liquid of acceptor protoplastis for preparing, carry out centrifugally, abandon supernatant liquor, again with concentration 0.6 mol N.F,USP MANNITOL washing-centrifugal more than 2 times, the composition of flush away substratum;
(2) concentration that has added mediation is that the Lithium Acetate (LiAc) of 0.1～0.5 mol mixes with the acceptor protoplastis, carry out centrifugal, absorb Lithium Acetate, Lithium Acetate with 0.1～0.5 mol mixes with the acceptor protoplastis again, adjust acceptor protoplastis concentration, require acceptor protoplastis number 10
6～10
9Between individual/milliliter;
(3) preparation external source delivery DNA, be used for improving transformation efficiency: strand is delivered DNA add the DNA damping fluid, concentration is 2 microgram milliliters, boils, and cools off in frozen water rapidly then, and is stand-by;
(4) get (2) protoplastis that obtains of step and carry out centrifugally, remove Lithium Acetate, obtain the pure protoplastis of pre-treatment;
(5) in pure protoplastis, add the polyoxyethylene glycol that concentration is 50～60% (weight/volume), Lithium Acetate, external source delivery DNA, exogenous plasmid dna and the sterilized water that concentration is 1.0～1.2 mol successively, constitute reaction solution, its volume ratio is: concentration is the polyoxyethylene glycol 50～60% of 50～60% (weight/volume), Lithium Acetate 10～15%, external source delivery DNA12～15%, plasmid DNA 0.1～2%/, all the other are sterilized water, make in the reaction solution acceptor protoplastis number 10
6～10
9Between individual/milliliter, plasmid DNA content 1.0～20 mcg/ml:
(6) the abundant mixing reaction solution of exerting oneself is cultivated down at 25～35 ℃ then;
(7) wave and culture 20～40 minutes in 35～45 ℃ of water-baths;
(8) reaction solution is centrifugal, then transformation mixture is absorbed the protoplastis after obtaining transforming;
(9) in MYG (maltose 0.5%, yeast powder 0.5%, glucose 1%, all the other be water) substratum, add sucrose, the sucrose concentration that makes substratum is 0.4～0.6 mol, protoplastis after transforming is mixed with substratum, wave and culture moved on the solid medium then and cultivates more than 20 hours.
3, transform the screening and the evaluation of primary physique
(1) identifies according to reporter gene: aforementioned (9) are centrifugal through the mixed solution of the protoplastis of wave and culture and substratum, absorb supernatant liquor, adding sucrose concentration is the MYG substratum and the X-Glux detection liquid of 0.4 mol, at 30～40 ℃ of following wave and culture more than 20 hours, cultivate more than 48 hours down at 2～10 ℃ again, the blue chromogen of inspection is given birth to physique under opticmicroscope then, calculates transformation efficiency;
(2) according to selecting the resistant maker gene screening: according to transforming the kalamycin resistance marker gene that plasmid carries, in the MYG substratum, add kantlex, form and select substratum, the protoplastis that above-mentioned (9) are cultivated is tiled on the selection substratum, cultivate down at 20～30 ℃, the bacterium colony that forms is carried out polymerase chain reaction (PCR) detect, to detecting positive strain, carry out molecular hybridization (Southern Blot) analysis, the bacterial strain that is positive is further cultivated utilization.
Advantage of the present invention is as follows:
1, improved transformation efficiency.Transformation efficiency of the present invention is about 10
4Transformant/microgram plasmid DNA, and calcium chloride/polyoxyethylene glycol (CaCl
2/ PEG) transformation efficiency of mediated method is 10
3Transformant/microgram plasmid DNA, the former is ten times of the latter.
2, the conversion cost is low.The present invention does not need special plant and instrument, the pulse instrument that electric shocking method is used, and homemade price is about 30,000 yuan, import price is about 100,000 yuan; Compare with restriction enzyme/polyoxyethylene glycol method, the cost of handling 1 milligram of required restriction enzyme of protoplastis is 5000 yuans, and the cost that the present invention handles with the required Lithium Acetate of protoplastis of amount only is 1 yuan.
3, method required equipment provided by the invention and condition are simple, and operating process is grasped easily, all can carry out under the condition of common laboratory, are convenient to penetration and promotion.
Embodiment
External source goal gene plasmid vector PGBI131SABC (Biological Technology Research Center, Chinese Academy of Agricultural Sciences provides) is transformed on the mushroom L26 (fungus institute of Jilin Agriculture University provides), this plasmid carries BT toxoprotein gene and cowpea trypsinase inhibin gene (CPTI), also has kalamycin resistance selectable marker gene and β-Pu Taotanggansuanmei reporter gene in addition.
1, the preparation of acceptor protoplastis
With concentration be 0.6 mol N.F,USP MANNITOL as permeating stablizer, preparation 2.5% lywallzyme enzymolysis solution is got " fragrant L26 " Lentinus Edodes fungus pompon of cultivating 5 days, add in the enzymolysis solution, under 30 ℃ of conditions, carry out enzymolysis, the preparation protoplastis, filter purifying, the protoplastis concentration behind the purifying is 10
6Individual/milliliter;
2, the importing of external source goal gene is carried out according to the following steps:
(1) get 30 milliliters of the pure liquid of the protoplastis for preparing, under 3000 rev/mins of conditions centrifugal 5 minutes, abandon supernatant liquor, wash 2 times with concentration 0.6 mol N.F,USP MANNITOL;
(2) adding 20 ml concns is that the Lithium Acetate (LiAc) of 0.1 mol mixes with protoplastis, under 20000 rev/mins of conditions centrifugal 15 seconds, absorb Lithium Acetate, again with the Lithium Acetate mixing protoplastis of 0.1 mol, adjust primary physique concentration, make the protoplastis number 3 * 10
6Individual/milliliter;
(3) preparation external source delivery DNA delivers DNA calf thymus peptide DNA with strand or salmon sperm dna adds in the TE damping fluid, and concentration is 2 mcg/ml, prepares 15 milliliters, boils 5 minutes, cools off in frozen water rapidly then, and is stand-by;
(4) it is centrifugal to get second 14 milliliters of the protoplastiss obtaining of step, removes Lithium Acetate;
(5) in protoplastis, add 60 milliliters of the polyoxyethylene glycol that concentration is 50% (weight/volume) successively, concentration is 10 milliliters of the Lithium Acetates of 1.0 mol, delivery DNA14 milliliter, concentration are 1 milliliter of 1 mg/ml PGBI131SABC plasmid DNA, all the other use sterilized water, and making its final volume is 100 milliliters;
(6) the abundant mixing mixed solution of exerting oneself was cultivated 30 minutes down at 30 ℃ then;
(7) wave and culture 30 minutes in 42 ℃ of water-baths;
(8) 8000 rev/mins centrifugal 15 seconds, then transformation mixture is absorbed;
(9) add sucrose in the MYG substratum, the sucrose concentration that makes substratum is 0.4 mol, and the protoplastis after transforming is mixed with substratum, and wave and culture 24 hours moves on the solid medium then and cultivates.Present embodiment can obtain about 10
6Individual conversion protoplastis with anti insect gene.
Claims (3)
1, the mushroom gene transfer technique of Lithium Acetate mediation, adopt conventional " alkaline lysis " to extract the plasmid DNA of external source goal gene, plasmid DNA after the extraction adopts PEG precipitation classification purifying, detect plasmid DNA concentration and purity with ultraviolet absorption method, concentration requirement reaches 1 mg/ml, purity requirement reaches 0D260/0D280 〉=1.8, OD260/OD230 〉=2.0, and on 1% horizontal sepharose electrophoresis, detection plasmid size, it is stand-by to put into-20 ℃ of refrigerators then, it is characterized in that its method also comprises:
The preparation of A, acceptor protoplastis
With concentration is that the N.F,USP MANNITOL of 0.4～0.8 mol or sucrose or Repone K are as permeating stablizer, add lywallzyme at permeating stablizer, the lywallzyme enzymolysis solution of compound concentration 1.5～3%, get the Lentinus Edodes fungus pompon of cultivating 3～10 days, put into enzymolysis solution, under 20～35 ℃ of conditions, carry out enzymolysis, preparation acceptor protoplastis, purifying makes acceptor protoplastis concentration 10 after filtration
6Individual/more than the milliliter;
The importing of B, external source goal gene is carried out according to the following steps:
(1) get the pure liquid of acceptor protoplastis for preparing, carry out centrifugally, abandon supernatant liquor, again with concentration 0.6 mol N.F,USP MANNITOL washing-centrifugal more than 2 times, the composition of flush away substratum;
(2) adding concentration is that the Lithium Acetate of 0.1～0.5 mol mixes with the acceptor protoplastis, carry out centrifugally, absorb Lithium Acetate, the Lithium Acetate with 0.1～0.5 mol mixes with the acceptor protoplastis again, adjust acceptor protoplastis concentration, require acceptor protoplastis number 10
6～10
9Between individual/milliliter;
(3) preparation external source delivery DNA: strand is delivered DNA add in the damping fluid, concentration is 2 mcg/ml, boils, and cools off in frozen water rapidly then, and is stand-by;
(4) get (2) protoplastis that obtains of step and carry out centrifugally, remove Lithium Acetate, obtain the pure protoplastis of pre-treatment;
(5) in pure protoplastis, add the polyoxyethylene glycol that concentration is 50～60% (weight/volume), Lithium Acetate, external source delivery DNA, exogenous plasmid dna and the sterilized water that concentration is 1.0～1.2 mol successively, constitute reaction solution, its volume ratio is: concentration is the polyoxyethylene glycol 50～60% of 50～60% (weight/volume), Lithium Acetate 10～15%, external source delivery DNA12～15%, plasmid DNA 0.1～2%/, all the other are sterilized water, make in the reaction solution acceptor protoplastis number 10
6～10
9Between individual/milliliter, plasmid DNA content 1.0～20 mcg/ml:
(6) abundant mixing reaction solution is cultivated down at 25～35 ℃ then;
(7) wave and culture 20～40 minutes in 35～45 ℃ of water-baths;
(8) reaction solution is centrifugal, then transformation mixture is absorbed the protoplastis after obtaining transforming;
(9) add sucrose in the MYG substratum, the sucrose concentration that makes substratum is 0.4～0.6 mol, and the protoplastis after transforming is mixed with substratum, and wave and culture moved on the solid medium then and cultivates more than 20 hours.
2, according to the mushroom gene transfer technique of the said Lithium Acetate mediation of claim 1, it is characterized in that permeating stablizer is a N.F,USP MANNITOL.
3,, it is characterized in that strand delivery DNA is calf thymus peptide DNA or salmon sperm dna according to the mushroom gene transfer technique of the said Lithium Acetate mediation of claim 1.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB011139218A CN1156579C (en) | 2001-04-29 | 2001-04-29 | Lithium acetate mediated lentinus edodes gene transfer technology |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB011139218A CN1156579C (en) | 2001-04-29 | 2001-04-29 | Lithium acetate mediated lentinus edodes gene transfer technology |
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| CN1384202A CN1384202A (en) | 2002-12-11 |
| CN1156579C true CN1156579C (en) | 2004-07-07 |
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| CNB011139218A Expired - Fee Related CN1156579C (en) | 2001-04-29 | 2001-04-29 | Lithium acetate mediated lentinus edodes gene transfer technology |
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| CN107828815A (en) * | 2017-10-09 | 2018-03-23 | 中国农业科学院烟草研究所 | A kind of Saccharomyces cerevisiae transformant method and its application for being used for batch and converting |
| CN108467870A (en) * | 2018-04-08 | 2018-08-31 | 上海市农业科学院 | A kind of method of mushroom genetic transformation |
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