CN104651389A - Liposome-mediated agaricus bisporus gill transformation method - Google Patents

Abstract

The invention relates to a liposome-mediated agaricus bisporus gill transformation method. The method specifically comprises the following steps: (1), picking up fruit bodies of tender mushroom with unbroken gills, cleaning the surfaces of the fruit bodies, collecting gill tissues and chopping into blocks; (2), uniformly mixing liposome and plasmids, and standing for 15-60 min at 5 DEG C below zero to 5 DEG C; (3), diluting products of the step (2) in sterile ultrapure water for 1000 times, mixing the solution with the gill tissue blocks uniformly, and standing for 80-120 min at room temperature; (4), adding the products of the step (3) to an RCM solid medium, and culturing for 8-12 days at room temperature; (5), transferring germinated agaricus bisporus gill tissue blocks to a hygromycin-containing PDA flat board, and culturing for 10-20 days at room temperature; and (6), cutting off newly grown hypha blocks, transplanting the hypha blocks to a PDA slope to culture, subculturing for 2-5 times, transplanting to the hygromycin-containing PDA flat board, thus obtaining transformers, which are hypha blocks capable of growing regularly. The liposome-mediated agaricus bisporus gill transformation method has the advantages of simplicity and convenience in operation, high transformation rate, stable transformers and the like.

Description

A kind of method of liposome-mediated transfection Twospore Mushroom lamella

Technical field

The invention belongs to lipofection technical field, be specifically related to a kind of method of liposome-mediated transfection Twospore Mushroom lamella.

Background technology

Twospore Mushroom ( agaricus bisporus) also known as white mushroom, mushroom, Agaricus campestris, be cultivate in the world the most extensively, turnout and the maximum edible mushrooms of consumption, output accounts for 3/4 of world's edible mushrooms ultimate production.But the genetic transformation of Twospore Mushroom lacks simple and effective method always.Have protoplastis electrotransformation, protoplastis PEG mediated transformation method, the particle bombardment of Twospore Mushroom genetic transforming method successively report bombard the mycelium of Twospore Mushroom and sporophore and agriculture bacillus mediated lamella conversion method, and foreign gene is proceeded in Twospore Mushroom bacterial strain.The shortcomings such as these method ubiquity complex operations, device complexity, low conversion rate, transformant are unstable.Some method needs to prepare protoplastis, and preparation process is complicated, as the protoplast transformation that protoplastis electrotransformation and PEG mediate.Some method does not need to prepare protoplastis, but device is complicated, it is high to transform cost, as Gene Knock-out Mice.The transformation efficiency of some method is unstable, and transformation efficiency, by the impact of many factors, is subject to the impact of the many factors such as agrobacterium strains type, plasmid vector type and match condition between the two, medium component and inductive condition as Agrobacterium-mediated Transformation method transformation efficiency.

Summary of the invention

The object of the invention is to overcome prior art deficiency, and provide a kind of method of liposome-mediated transfection Twospore Mushroom lamella, the method is simple and efficient to handle, transformation efficiency is high, transformant inheritance stability, does not need complex appts.

For achieving the above object, the present invention adopts following technical scheme:

A method for liposome-mediated transfection Twospore Mushroom lamella, it comprises the following steps:

1) pluck the tender Twospore Mushroom sporophore of children that lamella does not break, behind clean surface, collect lamella tissue and be cut into block, obtaining lamella tissue block;

2) by liposome Lipofectamine ^tM2000 and plasmid pBHg-dsACO mix, place 15-60 min in-5-5 DEG C;

3) by step 2) the aseptic ultrapure water of product dilutes 1000 times, then mixes with step 1) lamella tissue block, and room temperature (25 ± 5 DEG C) places 80-120 min;

4) step 3) products therefrom is joined in RCM solid medium, cultivate 8-12d for 20-30 DEG C;

5) the Twospore Mushroom lamella tissue block of sprouting is transferred on the PDA flat board containing Totomycin, cultivate 10-20d for 20-30 DEG C;

6) the mycelia block cutting new growth is transplanted to PDA inclined-plane and is cultivated, Secondary Culture like this 2-5 time, then is transferred on the PDA flat board containing Totomycin, can normal growth be transformant.Transformant can be inoculated into conventional PDA inclined-plane to carry out cultivating and preserving.

Concrete, described step 2) in by 1 μ L liposome Lipofectamine ^tM2000 and 2-3 μ g plasmid pBHg-dsACO mix.

RCM solid medium in described step 4) consists of: Tryptones 2.0 g, yeast extract 2.0 g, MgSO ₄7H ₂o 0.5 g, K ₂hPO ₄0.46 g, KH ₂pO ₄1 g, glucose 20 g, agar 20 g, N.F,USP MANNITOL 109.3 g, distilled water 1000 mL, pH 7.0.

Described step 5) and 6) in PDA flat board containing Totomycin concentration be 20-100 μ g/mL.

Compared to the prior art, Twospore Mushroom liposome-mediated transfection lamella method of the present invention has easy and simple to handle, quick, and transformation efficiency is high, transformant inheritance stability, and does not need to prepare protoplastis, does not need the advantages such as complex appts.

Accompanying drawing explanation

Fig. 1 is the physical map of plasmid pBHg-dsACO;

Fig. 2 is the transformant of Twospore Mushroom; CK:AS2796 starting strain; Arrow place is the transformant grown on multiple sieve resistance plate;

Fig. 3 is the PCR checking of transformant; A is hph gene PCR result; B is ACO gene PCR result; In figure, M:DNA Marker; WT: starting strain AS2796; P: plasmid pBHg-dsACO; 1-5, transformant;

Fig. 4 is the Semi quantitative PCR analysis of transformant ACO gene; WT: starting strain AS2796; 1-5: transformant; *, difference reaches conspicuous level (p<0.01) compared with WT;

Fig. 5 is transformant ACO enzyme activity determination result; WT: starting strain AS2796; 1-5: transformant; WT:*, difference reaches conspicuous level (p<0.05) compared with WT;

Fig. 6 is transformant ethylene synthase flow measurement result; WT: starting strain AS2796; 1-5: transformant; WT:*, difference reaches conspicuous level (p<0.05) compared with WT.

Embodiment

The present invention is further illustrated by the following examples, but protection scope of the present invention is not limited thereto.

embodiment 1

1 experiment material

1.1 bacterial strain

Twospore Mushroom CGMCC NO.0214, from China General Microbiological culture presevation administrative center.

1.2 plasmid

Plasmid pBHg, purchased from American Sylvan company.Plasmid pBHg-dsACO by pBHg transform form, containing hygromycin gene ( hph) expression cassette and Twospore Mushroom 1-amino-cyclopropane-1-carboxylic acid (ACC) oxidase gene ( aCO) expression cassette of partial sequence double-stranded RNA (ds RNA), its physical map is shown in Fig. 1, wherein the sequence of S-ACO is: GAACCCACCCAGAACCTCTTAGGCCGTTCCTATCTGAAATCGACGATTTCTCGGAT CAAATCCACTCTGATATAATTAACACAATTCTTCGCCTCATCGCAATGAGCTTGGA GCTAGATGAAGATTACTTCATCCAAATGCATGATCGTTCTGCGAACGCAGAAACAT TCCTCCGGTTTGTGAATTATTTCCCTCATCCAGAAGAAGAAGAGAATAAATCCGGC GGAGTCTGGTTGAAAGGACATACTG, the sequence of R-ACO is: ACCTAGCAGCCGAACATCATCGGGGGACAGTCCTTAAGTTAACGGAGAGGTTATAA CTGTTCTTGAGGTAAGAGTTGCACGAACTGGGTGAAGGTAACGGGTAGAAATCAGT AATAGACCTCCCTACGATGACCGACCGACTTATCGTTGCAATCGCAGTATTTCAGT CATACAGGAAAGTTGGTCTGAGGCGGCCTAAATAAGAGAAGAAGAAGACCTACTCC CTTTATTAAGTGTTTGGCCTCCTTACAAAGACGCAAGCGTCTTGCTAGTACGTAAA CCTACTTCATTAGAAGTAGATCGAGGTTCGAGTAACGCTACTCCGCTTCTTAACAC AATTAATATAGTCTCACCTAAACTAGGCTCTTTAGCAGCTAAAGTCTATCCTTGCC GGATTCTCCAAGACCCACCCAAG.Concrete remodeling method is this area routine techniques, can see document (Smith, N. A., Singh, S. P., Wang, M. B., Stoutjesdijk, P. A., Green, A. G., & Waterhouse, P. M. (2000). Gene expression:Total silencing by intron-spliced hairpin RNAs. Nature, 407 (6802), 319-320).Wherein, preferably plasmid pBHg-dsACO is built as follows:

Plasmid pBHg-dsACO is by Twospore Mushroom aCOthe multiple clone site place of the expression cassette insertion plasmid pBHg of gene C DS partial sequence double-stranded RNA (dsRNA) builds and forms.Twospore Mushroom aCOgene C DS partial sequence dsRNA expression cassette is cut enzyme by four DNA fragmentations through enzyme and is got continuously, and four DNA fragmentations are Twospore Mushroom glycerol-3-phosphate gene respectively gpdpromotor, Twospore Mushroom aCOgene C DS part forward sequence S-ACO, Twospore Mushroom aCOthe terminator sequence T35S of gene C DS part reverse sequence R-ACO and CaMV35S gene.Both S-ACO and R-ACO have the tumor-necrosis factor glycoproteins of 248 bp. gpdthe primer that the pcr amplification of promotor uses is GPD-U, GPD-D(table 1), template is plasmid pBHg, pcr amplification condition: 94 DEG C of denaturation 5 min, 94 DEG C of 40 s, 55 DEG C of 1 min, 72 DEG C of 1 min totally 30 circulation, and 72 DEG C extend 10 min.The primer that amplification S-ACO and R-ACO uses is S-ACO-U, S-ACO-D and R-ACO-U, R-ACO-D, according to Twospore Mushroom AS2796 aCOgene cDNA sequence (GeneBank accession number JQ314344.1) designs, and template is that the total serum IgE of Twospore Mushroom AS2796 is through adopting the cDNA of MMLV Reverse Transcriptase 1st-Strand cDNA Synthesis Kit synthesis.Pcr amplification condition: 94 DEG C of denaturation 5 min, 94 DEG C of 40 s, 56 DEG C of 1 min, 72 DEG C of 1 min totally 30 circulation, 72 DEG C extend 10 min.The primer that T35S amplification uses is T35S-U, T35S-D(table 1), template is plasmid pBHg, pcr amplification condition: 94 DEG C of denaturation 5 min, 94 DEG C of 40 s, 55 DEG C of 1 min, 72 DEG C of 1 min totally 30 circulation, and 72 DEG C extend 10 min.

Use KpnI single endonuclease digestion gpdpromotor and R-ACO fragment, by ApaLI single endonuclease digestion T35S and S-ACO fragment, purifying connects respectively with T4 ligase enzyme after reclaiming gpdpromotor and R-ACO fragment and T35S and S-ACO fragment.Connecting product for template with enzyme, is primer with GPD-U, R-ACO-D, pcr amplification gpdpromotor+R-ACO fragment is primer with T35S-D and S-ACO-D, pcr amplification S-ACO+T35S fragment, pcr amplification condition all: 94 DEG C of denaturation 5 min, 94 DEG C of 40 s, 55 DEG C of 1 min, 72 DEG C of 1 min totally 30 circulation, 72 DEG C extend 10 min.PCR primer purifying reclaims, and respectively will gpdpromotor+R-ACO fragment is connected with pMD19-T simple carrier with S-ACO+T35S fragment, proceeds to e. colijM109, extracts after cultivating and obtains two recombinant plasmids.Two recombinant plasmid SalI/ NcoI double digestions respectively, purifying reclaims and contains gpdlarge fragment after the plasmid enzyme restriction of promotor+R-ACO fragment, containing the small segment after the plasmid enzyme restriction of S-ACO+T35S, and to connect with T4 ligase enzyme, obtain cloning vector pMD19-dsACO.PMD19-dsACO is proceeded to e. colijM109, extracts cloning vector pMD19-dsACO after cultivating propagation.With SalI/BamHI double digestion plasmid pBHg and pMD19-dsACO, reclaim large fragment and small segment respectively, with T4 ligase enzyme, large and small fragment carried out enzyme and connect, obtain expression plasmid pBHg-dsACO(Fig. 1), be transformed into e. coliin HA105, after multiplication culture, extract plasmid pBHg-dsACO, for transforming Twospore Mushroom lamella.

1.3 substratum

PDA solid medium: potato 200 g(peeling stripping and slicing adds water boil 20 min four layers of filtered through gauze and gets filtrate), agar 20 g, glucose 20 g, distilled water 1000 mL.

PD liquid nutrient medium: potato 200 g(peeling stripping and slicing adds water boil 20 min four layers of filtered through gauze and gets filtrate), glucose 20 g, distilled water 1000 mL.

RCM solid medium: Tryptones 2.0 g, yeast extract 2.0 g, MgSO ₄7H ₂o 0.5 g, K ₂hPO ₄0.46 g, KH ₂pO ₄1 g, glucose 20 g, agar 20 g, N.F,USP MANNITOL 109.3 g, distilled water 1000 mL, pH 7.0.

1.4 liposome

Liposome Lipofectamine ^tM2000 purchased from invitorgen company.

2.1 experimental technique

A method for liposome-mediated transfection Twospore Mushroom lamella, it comprises the following steps:

1) pluck the tender mushroom fruitbody of children that lamella does not break, behind clean surface, collect lamella tissue and be cut into thin block, obtain lamella and organize thin block, organize thin block to join in the aseptic centrifuge tube of 50 mL lamella;

2) by 1 μ L liposome Lipofectamine ^tM2000 and 2.5 μ g plasmid pBHg-dsACO mix, and place 30 min for 0 DEG C;

3) by step 2) the aseptic ultrapure water of product dilutes 1000 times, is then transferred to lamella is housed organizes in the centrifuge tube of thin block and (organize thin block to be advisable to flood lamella), and mixing, places 100 min for 25 DEG C;

4) step 3) products therefrom is joined in RCM solid medium, be down flat plate, be inverted for 25 DEG C and cultivate 10d;

5) the Twospore Mushroom lamella tissue block of sprouting is transferred on the PDA flat board containing 30 μ g/mL Totomycin, cultivate 2 weeks for 25 DEG C;

6) the mycelia block cutting new growth is transplanted to conventional PDA inclined-plane and is cultivated, Secondary Culture like this 3 times, then is transferred on the PDA flat board containing 30 μ g/mL Totomycin, can normal growth be transformant (see figure 2).

The PCR qualification of 2.2 transformants

Choose 5 transformants grown fine, be transferred in PDA liquid nutrient medium, shake-flask culture.Collect mycelia, adopt CTAB method to extract the genomic dna of transformant mycelia, with the genomic dna extracted for template, use hphgene and aCOthe Auele Specific Primer of the CDS of gene carries out pcr amplification checking.

hphgene amplification primer is hph-F(5 '-CTATTCCTTTGCCCTCGG-3 ') and hph-R(5 '-ATGAAAAAGCCTGAA CTCACC-3 ').Amplification condition: 94 DEG C of denaturation 5 min, 94 DEG C of 40 s, 50 DEG C of 1 min, 72 DEG C of 1 min totally 30 circulation, 72 DEG C extend 10 min.

aCOthe CDS amplimer of gene is T4-F(5 '-GAACCCACCCAGAACCTC-3 ') and T4-R(5 '-ATGTTCGGCTGTTAGGTA-3 ').Amplification condition: 94 DEG C of denaturation 5 min, 94 DEG C of 45 s, 52 DEG C of 1 min, 72 DEG C of 30 s totally 30 circulation, 72 DEG C extend 10 min.

Get 5 μ l PCR primer 1% agarose gel electrophoresis respectively to detect, result shows (see figure 3): all can amplify hphgene (product size is 750 bp) and aCOgene product (product size is 416 bp).

The Semi quantitative PCR analysis of 2.3 transformant ACO gene expression amounts

Random selecting 5 transformants, adopt Trizol method to extract the total serum IgE of transformant mycelia.The AMV Reverse Transcriptase test kit utilizing precious biotechnology (Dalian) company limited to provide carries out reverse transcription, synthesis cDNA first chain.

aCOprimer set for amplification is T4-F(5 '-GAACCCACCCAGAACCTC-3 ') and T4-R(5 '-ATGTTCGGCTGTTAGGTA-3 ').Reference gene GAPDH amplimer is GAPDH-F(5 '-TCACGCCACCACCGCTACTCAA-3 ') and GAPDH-R(5 '-CGGGCTTCTCAAGACGAACAACAA-3 '), amplified production size is respectively 416 bp and 200 bp.Amplification condition is: 94 DEG C of denaturation 5 min, 94 DEG C of 30 s, 52 DEG C of 30 S, 72 DEG C of 45 S totally 26 circulation, and 72 DEG C extend 10 min.

Get 5 μ l PCR primer 1% agarose gel electrophoresis to detect, take a picture with gel imaging system and use the gray-scale value of each electrophoretic band of Gel Base/Gel Blot Support software analysis.Result shows (see figure 4): 5 transformants aCOgene expression dose significantly reduces, and reduces 47-74% than starting strain.

2.4 transformant ACO oxydase enzyme assaies

By 5 of random selecting transformants, be transferred in PD liquid nutrient medium, shake-flask culture 20 d.Collected by centrifugation mycelium pellet, 20 mL test tubes put into by the Twospore Mushroom mycelium pellet sample getting 1g fresh, then add 3 mL Mops damping fluids (100 mmol/L, pH7.2), mixing; Each transformant does 6 repetitions.To as adding 50 μ L sterilized waters in the test tube of blank.Then add 50 μ L in all the other test tubes containing 50mmol/L ACC(1-amino-cyclopropane-1-carboxylic acid) the aqueous solution, and immediately with soft rubber ball sealing, and add CO with syringe ₂make the CO in test tube ₂volume about reaches 5%.Then test tube is placed in 26 DEG C of water-baths and reacts 30 min.Extract the invisible spectro gaseous sample of 1 mL with airtight pin, use gas chromatograph for determination ethylene emanation, thus calculate acc oxidase activity.With 1h every milligram albumen under 26 DEG C of conditions produce ethene receive rub number be expressed as 1 enzyme activity unit (U).

Result shows (see figure 5): compared with starting strain, and the ACO enzyme activity of 5 transformants significantly reduces, and enzyme activity reduces 68-86%.

2.5 transformant ethylene synthase flow measurements

The starting strain of Twospore Mushroom and transformant are inoculated in PD liquid nutrient medium respectively, in 25 DEG C, cultivate under 160 r/min conditions, 6 repetitions are done in often kind of process.When cultivating 20 d, open sealed membrane and aseptically blow 4 h(in order to remove ethene), after sealing cultivation 8 h, utilize ethylene content in gas chromatography determination each sample, sample size is 1 mL.GC conditions: Agilent 7890 type gas chromatograph; Chromatographic column: HP-2 55%(capillary column) Phenyl Met hyl Siloxane Capillary 3010 m × 320 μm × 0.125 μm of nominal, fid detector, column temperature 100 DEG C; Hydrogen ion flame detector (FID), detector temperature 150 DEG C; Carrier gas N ₂flow velocity 50 mL/min, combustion gas H ₂flow velocity 50 mL/min, air velocity 400 mL/min, retention time is 3.0 min.Enter 1 mL ethene sample by sample introduction needle to measure on chromatographic instrument, the sample introduction flow velocity of gas sample is 115 mL/min, replication 6 times.

Result shows (see figure 6): compared with starting strain, and the ethylene yield of 5 transformants significantly reduces, and ethylene yield reduces 27-48%.

2.6 Dual culture times are on the impact of lamella germination rate

1) pluck the tender mushroom fruitbody of children that lamella does not break, behind clean surface, collect lamella tissue and be cut into thin block, obtain lamella and organize thin block, organize thin block to join in the aseptic centrifuge tube of 50 mL lamella;

2) by 1 μ L liposome Lipofectamine ^tM2000 and 2.5 μ g plasmid pBHg-dsACO mix, and place 30 min for 0 DEG C;

3) by step 2) the aseptic ultrapure water of product dilutes 1000 times, then be transferred to and lamella is housed organizes in the centrifuge tube of thin block and (organize thin block to be advisable to flood lamella), mixing, place 20 min, 40 min, 60 min, 80 min, 100 min, 120 min for 25 DEG C, totally six time gradients are with comparing;

4) step 3) products therefrom is joined in RCM solid medium, be down flat plate, be inverted cultivation one week for 25 DEG C, calculate the sprouting number of lamella block.

The lamella block that sprouting number/participation of germination rate=lamella block transforms is total.The results are shown in Table 2.Along with the increase of Dual culture time, the germination rate of Twospore Mushroom lamella tissue constantly raises.After the Dual culture time reaches 100 min, extend the Dual culture time again, germination rate raises less, considers the ratio of Dual culture time and germination rate, determines the Dual culture time with 100 min for the best.

conclusion

As can be seen from the above results, Twospore Mushroom liposome-mediated transfection lamella method of the present invention has the advantages such as easy and simple to handle, transformation efficiency is high, transformant is stable.

Claims (4)

1. a method for liposome-mediated transfection Twospore Mushroom lamella, is characterized in that, comprises the following steps:

1) pluck the tender Twospore Mushroom sporophore of children that lamella does not break, behind clean surface, collect lamella tissue and be cut into block, obtaining lamella tissue block;

2) by liposome Lipofectamine ^tM2000 and plasmid pBHg-dsACO mix, place 15-60 min in-5-5 DEG C;

3) by step 2) the aseptic ultrapure water of product dilutes 1000 times, then mixes with step 1) lamella tissue block, and room temperature places 80-120 min;

4) step 3) products therefrom is joined in RCM solid medium, cultivate 8-12d for 20-30 DEG C;

5) the Twospore Mushroom lamella tissue block of sprouting is transferred on the PDA flat board containing Totomycin, cultivate 10-20d for 20-30 DEG C;

6) the mycelia block cutting new growth is transplanted to PDA inclined-plane and is cultivated, Secondary Culture like this 2-5 time, then is transferred on the PDA flat board containing Totomycin, can normal growth be transformant.

2. the method for liposome-mediated transfection Twospore Mushroom lamella as claimed in claim 1, is characterized in that, described step 2) in by 1 μ L liposome Lipofectamine ^tM2000 and 2-3 μ g plasmid pBHg-dsACO mix.

3. the method for liposome-mediated transfection Twospore Mushroom lamella as claimed in claim 1, it is characterized in that, the RCM solid medium in described step 4) consists of: Tryptones 2.0 g, yeast extract 2.0 g, MgSO ₄7H ₂o 0.5 g, K ₂hPO ₄0.46 g, KH ₂pO ₄1 g, glucose 20 g, agar 20 g, N.F,USP MANNITOL 109.3 g, distilled water 1000 mL, pH 7.0.

4. the method for liposome-mediated transfection Twospore Mushroom lamella as claimed in claim 1, is characterized in that, described step 5) and 6) in PDA flat board be 20-100 μ g/mL containing Totomycin concentration.