Embodiment
Following examples are used for further illustrating content of the present invention, but should not be construed as limitation of the present invention, and without departing from the spirit and substance of the case in the present invention, the amendment do the inventive method, step or condition or replacement all belong to scope of the present invention.If specialize, the conventional means that technique means used in embodiment is well known to those skilled in the art.
Embodiment 1
The structure of A, homologous recombination plasmid
(1) material: aspergillus niger (
aspergillus niger), intestinal bacteria (
e.coli)dH5 α, Agrobacterium EHA105 and plasmid pWM1 are commercially produced product.Wherein, aspergillus niger is bought from ATCC, strain number ATCC10582; Agrobacterium EHA105 and plasmid pMW1 buys from Biovector company.
(2) synthesis of homologous recombination fragment and enzyme are cut
Synthesize the homologous recombination fragment (nucleotide sequence is as shown in SEQ ID NO.1) be made up of the back segment homologous sequence (SEQ ID NO.4) of leading portion homologous sequence (initiator codon that this sequence is possible is substituted to guarantee to transcribe from the initiator codon of double activated albumen by other base, and sequence is as shown in SEQ ID the NO.2)-Mierocrystalline cellulose excision enzyme of aspergillus niger heat shock protein gene and encoding sequence (SEQ ID the NO.3)-aspergillus niger heat shock protein gene of restriction endonuclease double activated albumen.
Add in this homologous recombination fragment of 100 μ L
sali enzyme liquid 2 μ L(TakaRa buys), 30 DEG C of enzymes cut 16h, and enzyme cuts rear 65 DEG C of water-bath 10min.
(3) extraction of pWM1 plasmid and enzyme are cut
Intestinal bacteria E containing pWM1 plasmid
.colidH5 α is (kantlex of 50 μ g/mL) 37 DEG C of shaking culture 12h in LB.Get 1.5mL thalline to manage in EP, with the centrifugal 3min of 4000rpm, abandon supernatant liquor.Add 0.1mL solution I (1% glucose, 50mM EDTA pH8.0,25mM Tris-HCl pH8.0) fully to mix.Add 0.2mL solution II (0.2mM NaOH, 1% SDS), overturn mixing gently, be placed in ice bath 5min.Add 0.15mL cooled solution III(5mol/L KAc, pH4.8), overturn mixing gently, ice bath 5min.With the centrifugal 20min of 10000rpm, get supernatant liquor in another new EP pipe.Add isopyknic primary isoamyl alcohol, after mixing, leave standstill 10min.Again with the centrifugal 20min of 10000rpm, abandon supernatant.Wash once with 70% ethanol 0.5mL, drain all liquid.After drying to be precipitated, be dissolved in 0.05mL TE damping fluid.
The pWM1 plasmid 50 μ L extracted adds
sali enzyme liquid 2 μ L(TakaRa buys), 30 DEG C of enzymes cut 16h, and enzyme cuts rear 65 DEG C of water-bath 10min.
(4) connect
The homologous recombination fragment 100 μ L that enzyme is cut buys with 5 μ L T4 DNA ligase(TakaRa with the pWM1 plasmid 20 μ L that enzyme is cut) 16 DEG C be connected 24h.
The preparation of B, competent escherichia coli cell
(1) by E
.colidH5 α is placed on LB substratum, incubated overnight at 37 DEG C.
(2) centrifugal bottle (250-500mL) that high-temperature sterilization is large is used in order to second day shaking flask.
(3) prepare several bottles of aqua sterilisas (total amount about 1.5 liters), be stored in refrigeration chamber and use in order to second day resuspension cell.
(4) transferase 10 .2-1mL overnight culture is to 20mL LB(or other nutritious substratum are housed) 100mL shaking flask.
At (5) 37 DEG C, thermal agitation cultivates 6 hours.
(6) nutrient solution OD is monitored
600value (cultivate and measure once per half an hour after 1 hour).
(7) OD is worked as
600when value reaches 0.5-1.0, from shaking table, take out shaking flask, be placed in cooled on ice 15 minutes.
(8) cell under 4 DEG C of 5000g centrifugal 15 minutes, abandons supernatant liquor.
(9) with the frozen water resuspension cell of sterilizing.First use vortex instrument or pipette resuspension cell (several milliliters) in a small amount of volume, be then diluted with water to 2/3 volume of centrifuge tube.
(10) according to previous step repeated centrifugation, careful abandoning supernatant.
(11) according to the frozen water resuspension cell of previous step sterilizing.
(12) centrifugal, abandon supernatant liquor.
(13) with 10% glycerine resuspension cell after 20mL sterilizing, ice-cold.
(14) centrifugal according to previous step, careful abandoning supernatant (precipitation may be very loose).
(15) be 2-3mL with 10% glycerine resuspension cell to final volume.
(16) cell is loaded Eppendorf tube, in-80 DEG C of preservations by 150 μ L equal portions.
C, connection product conversion intestinal bacteria
(1) competent cell that step B of thawing is on ice obtained.
(2) every 100 μ L competent cells add the plasmid (the connection product of step A (4)) that 10 μ L connect, incubated on ice about 5 minutes.
(3) in transfer DNA/cell mixture to cooled 2mm electroporation container.
(4) add live conversion instrument, get out 300 μ L LB substratum.
(5) pulse (200 ohm, 25 μ Fd, 2.5 kilovolts) (testing time constant, should more than 3) is carried out to electroporation container.
(6) LB of 300 μ L is added immediately in electroporation container.
At (7) 37 DEG C, culturing cell 40 minutes to 1 hour is to restore.
(8) transitional cell is cultivated to containing on kantlex (50 μ g/mL) Selective agar medium, and the bacterium colony grown is the transformant containing homologous recombination plasmid (being connected into the pMW1 plasmid of homologous recombination fragment).
The extraction of D, homologous recombination plasmid
The transformant that step C obtains 37 DEG C of shaking culture 12h.Get 1.5mL bacterium liquid in EP pipe, with the centrifugal 3min of 4000rpm, abandon supernatant liquor.Add 0.1mL solution I (1% glucose, 50 mM EDTA pH 8.0,25mM Tris-HCl pH 8.0) fully to mix.Add 0.2mL solution II (0.2mM NaOH, 1% SDS), overturn mixing gently, be placed in ice bath 5min.Add 0.15mL cooled solution III(5mol/L KAc, pH4.8), overturn mixing gently, ice bath 5min.With the centrifugal 20min of 10000rpm, get supernatant liquor in another new EP pipe.Add isopyknic primary isoamyl alcohol, after mixing, leave standstill 10min.Again with the centrifugal 20min of 10000rpm, abandon supernatant.Wash once with 70% ethanol 0.5mL, drain all liquid.After drying to be precipitated, be dissolved in 0.05mL TE damping fluid.
E, homologous recombination Plastid transformation Agrobacterium
The competent preparation of Agrobacterium: the Agrobacterium EHA105 of fluid preservation coats on LB substratum, 28 DEG C of cultivations, by the time after single bacterium colony grows, picking list bacterium colony, be inoculated in the liquid nutrient medium of 5mL, 28 DEG C, 150r/m cultivates 24h, is inoculated in the fresh liquid nutrient medium of 5mL according to the inoculative proportion of 1:10,28 DEG C, 150r/m cultivates 24h.The thalline ice bath 40min cultivated, the centrifugal 5min of 5000r/m, with the 0.02mol/L CaCl of 10mL
2the resuspended thalline of thalline.The centrifugal 5min of 5000r/m again, thalline is suspended in the 0.02mol/L CaCl of 1mL
2solution, is positioned over preservation on ice.
The homologous recombination plasmid 1 μ L getting extraction mixes with the Agrobacterium competence suspension of 40 μ L, gently after shake mixing 5min, and rapid freezing 5min on liquid nitrogen, 37 DEG C of water-bath 5min.Add 1mL LB liquid medium, 28 DEG C shake 4h gently.The centrifugal 5min of thalline 5000r/m, abandoning supernatant, adds LB substratum 50 μ L, concussion Eddy diffusion liquid.Liquid spreading in containing on 50 μ g/mL kantlex LB flat boards, the colony inoculation grown in containing the LB liquid nutrient medium of 50 μ g/mL kantlex 28 DEG C, 150r/m cultivates 24h.
F, agrobacterium mediation converted aspergillus niger
Aspergillus niger is inoculated in PDA medium slant, and cultivate 72h for 37 DEG C, with spore under the distilled water flushing of sterilizing, spore distilled water diluting is to 10
8individual spore/mL.Spore mixes with isopyknic Agrobacterium containing homologous recombination plasmid, adds Syringylethanone to 200 μm ol/L, and 30 DEG C of lucifuges cultivate 24h.Then spore being coated on dull and stereotyped upper (200g potato 1L water boil 30min from the beginning, filtered through gauze, adds 20g glucose, 20g Zulkovsky starch, 20g agar, 200 μm of ol cefotaximes, 200mg hygromycin B), cultivating to growing bacterium colony for 30 DEG C.
The bacterium colony grown is inoculated into (200g potato 1L water boil 30min from the beginning on inclined-plane respectively, filtered through gauze, add 20g glucose, 20g agar), cultivate 72h, with transfering loop scraping spore, be inoculated into (wheat bran: tap water=1:3(W/W) in bran mass for 30 DEG C), each 250mL triangular flask loads 30g bran mass, cultivate 4d for 30 DEG C, be then warmed up to 39 DEG C of insulation 12h, cool to 28 DEG C and cultivate 1d.Measure the cellulose enzyme activity of each transformant one by one, what enzyme work was high is transformant.Screening obtains the bacterial strain that a cellulose enzyme activity reaches 6.7U/g, is 1.2 times of starting strain 5.6U/g.The bacterial strain of screening is checked order further and confirms that the encoding gene of Mierocrystalline cellulose excision enzyme and restriction endonuclease double activated albumen has been incorporated into the position of aspergillus niger heat shock protein gene, show that the present invention expresses Mierocrystalline cellulose excision enzyme and restriction endonuclease double activated albumen structure cellulase superior strain successfully constructs.
By the inoculation of above-mentioned screening to (200g potato 1L water boil 30min from the beginning on inclined-plane, filtered through gauze, add 20g glucose, 20g agar), cultivate 72h for 30 DEG C, with transfering loop scraping spore, be inoculated into (wheat bran: tap water=1:3(W/W) in bran mass), 250mL triangular flask loads 30g bran mass, cultivates 5.5d for 28 DEG C.Measure enzyme to live as 5.9U/g, this explanation does not have heat shock, and it is high that cellulose enzyme activity does not have the enzyme of heat shock to live, and illustrates that heat shock expression strategy makes the enzyme of cellulase live and improves.
The measuring method of cellulose enzyme activity: get substratum 5.0g during fermentation ends, adds 0.05M citrate buffer solution 50mL, and 200r/m lixiviate 30min, filter paper filtering obtains enzyme liquid.Enzyme liberating reaction is carried out at 50 DEG C.The 0.05M citrate buffer solution of 10mL pH 5.0 is added in 25mL test tube, filter paper bar (0.5cm × 2cm) two panels, as substrate, adds fermented liquid centrifuged supernatant 2mL, 50 DEG C of insulation 30min in water-bath, then boil 5min with boiling water, measure the content of reducing sugar by DNS method.Enzyme is lived and is defined as: the per minute enzyme amount discharged required for 1 μm of ol reducing sugar is a Ge Meihuo unit U.
SEQUENCE LISTING
<110> Hubei University Of Technology
<120> mono-kind expresses the method for Mierocrystalline cellulose excision enzyme and restriction endonuclease double activated albumen structure cellulase high-yield
<130> 1
<160> 4
<170> PatentIn version 3.5
<210> 1
<211> 2296
<212> DNA
<213> Artificial Sequence
<220>
<223> homologous recombination fragment
<400> 1
ccgtcgactt gtctgccgag actttcgagt tccaggctga gatctctcag ctcctctccc 60
tcatcatcaa cactgtctac tccaacaagg agattttcct gcgtgagctg atctccaacg 120
cctccgatgc tctggacaag attcgctatg agtccctgtc cgacccctcc aagctcgact 180
cgggcaagga tctccgcatc gacctgatcc ccaacgccga ggccaagacc ctcaccatcc 240
gtgataccgg tatcggtatg accaaggctg taagtttttc tttcctatca ctccccgctc 300
tttcttctgc cattgagcgg taccctctta cgattagagt gaattctgac tttgtgggct 360
ttttttagga cctcatcaac aacctcggta ccattgctcg ctctggtact aagcagttca 420
tggaggctct ctccgctggt gctgatatct ccttgaatcc gtgatatttc atcaaaggaa 480
ttgattaagg aaatgaattt cggttggaat ttaggtaata ctatggatgc tcaatgtatt 540
gaatacttaa attatgaaaa ggatcaaact gcttcagaaa cttgctgggg taatccaaag 600
actactgaag atatgttcaa ggttttaatc gacaaccaat ttaatgtctt ccgtattcca 660
actacttggt ctggtcactt cggtgaagct ccagattata agattgatga aaaatggtta 720
aagagagttc atgaagttgt tgattatcca tacaagaacg gagcatttgt tatcttaaat 780
cttcatcatg aaacctggaa tcatgccttc tctgaaactc ttgatacagc caaggaaatt 840
ttagaaaaga tctggtctca aattgctgaa gaatttaagg attatgatga acacttaatc 900
ttcgaaggat taaacgaacc aagaaagaat gatactccag ttgaatggac tggtggtgat 960
caagaaggtt gggatgctgt taatgctatg aatgctgttt tcttaaagac tgttcgtagt 1020
gctggtggta ataatccaaa gcgtcatctt atgattccac catatgctgc tgcttgtaat 1080
gaaaactcat tcaacaactt tatcttccca gaagatgatg ataaggttat tgcttctgtt 1140
catgcctatg ctccatacaa ctttgcctta aataacggtg aaggagctgt tgataagttt 1200
gatgcagctg gtaagagaga tcttgaatgg aacattaatt taatgaagaa gagatttgtt 1260
gatcaaggta ttccaatgat tcttggtgaa tatggtgcta tgaaccgtga caatgaagaa 1320
gatcgtgcta cttgggctga attctacatg gaaaaggtta ctgctatggg agttccacaa 1380
atctggtggg ataatggtgt cttcgaaggt actggtgaac gttttggtct tcttgatcgt 1440
aagaacttaa agattgttta tccaactatt gttgctgctt tacaaaaggg tagaggttta 1500
gaagttaatg ttgttcatgc tattgaaaaa gaaacagagg aataagtatg cccgagcacc 1560
agaagcagat ctactacatc accggcgagt ccatcaaggc cgttgccaag tctcccttcc 1620
tggacagcct caagcagaag aacttcgagg ttctgttcct ggttgacccc attgacgagt 1680
acgccttcac tcagctgaag gagttcgacg gcaagaagct ggtcgacatc accaaggact 1740
tcgagcttga ggagtccgag gaggagaagg ctgagcgcga gaaggaggag aaggagttcg 1800
agggtctcgc caagagcctc aagaacatcc tcggcgacaa ggtcgagaag gtcgttgtct 1860
cccacaagct cgttggctct ccttgcgcca tccgtaccgg ccagttcggt tggtccgcta 1920
acatggagcg tatcatgaag gcccaggccc tccgtgacac ctccatgagc tcttacatgt 1980
cctccaagaa gactttcgag atctctccca agtcctctat catcaaggag ctccgcaaga 2040
aggttgaggc cgatggcgag ggtgaccgca ctgtcaagtc catcactcag ctgctcttcg 2100
agacctctct cctggtctcc ggtttcacca ttgaggagcc cgccagcttc gctgagcgca 2160
tccacaagct cgtctccctt ggtctgaaca tcgacgagga ggctgagacc accgaggaga 2220
aggctgctga ggaggctgcc cctgccgccg ccgctgccga gagctccatg gaggaggttg 2280
actaaccgtc gacttt 2296
<210> 2
<211> 456
<212> DNA
<213> Artificial Sequence
<220>
The leading portion homologous sequence of <223> aspergillus niger heat shock protein gene
<400> 2
ccgtcgactt gtctgccgag actttcgagt tccaggctga gatctctcag ctcctctccc 60
tcatcatcaa cactgtctac tccaacaagg agattttcct gcgtgagctg atctccaacg 120
cctccgatgc tctggacaag attcgctatg agtccctgtc cgacccctcc aagctcgact 180
cgggcaagga tctccgcatc gacctgatcc ccaacgccga ggccaagacc ctcaccatcc 240
gtgataccgg tatcggtatg accaaggctg taagtttttc tttcctatca ctccccgctc 300
tttcttctgc cattgagcgg taccctctta cgattagagt gaattctgac tttgtgggct 360
ttttttagga cctcatcaac aacctcggta ccattgctcg ctctggtact aagcagttca 420
tggaggctct ctccgctggt gctgatatct ccttga 456
<210> 3
<211> 1089
<212> DNA
<213> Artificial Sequence
<220>
The encoding sequence of <223> Mierocrystalline cellulose excision enzyme and restriction endonuclease double activated albumen
<400> 3
atccgtgata tttcatcaaa ggaattgatt aaggaaatga atttcggttg gaatttaggt 60
aatactatgg atgctcaatg tattgaatac ttaaattatg aaaaggatca aactgcttca 120
gaaacttgct ggggtaatcc aaagactact gaagatatgt tcaaggtttt aatcgacaac 180
caatttaatg tcttccgtat tccaactact tggtctggtc acttcggtga agctccagat 240
tataagattg atgaaaaatg gttaaagaga gttcatgaag ttgttgatta tccatacaag 300
aacggagcat ttgttatctt aaatcttcat catgaaacct ggaatcatgc cttctctgaa 360
actcttgata cagccaagga aattttagaa aagatctggt ctcaaattgc tgaagaattt 420
aaggattatg atgaacactt aatcttcgaa ggattaaacg aaccaagaaa gaatgatact 480
ccagttgaat ggactggtgg tgatcaagaa ggttgggatg ctgttaatgc tatgaatgct 540
gttttcttaa agactgttcg tagtgctggt ggtaataatc caaagcgtca tcttatgatt 600
ccaccatatg ctgctgcttg taatgaaaac tcattcaaca actttatctt cccagaagat 660
gatgataagg ttattgcttc tgttcatgcc tatgctccat acaactttgc cttaaataac 720
ggtgaaggag ctgttgataa gtttgatgca gctggtaaga gagatcttga atggaacatt 780
aatttaatga agaagagatt tgttgatcaa ggtattccaa tgattcttgg tgaatatggt 840
gctatgaacc gtgacaatga agaagatcgt gctacttggg ctgaattcta catggaaaag 900
gttactgcta tgggagttcc acaaatctgg tgggataatg gtgtcttcga aggtactggt 960
gaacgttttg gtcttcttga tcgtaagaac ttaaagattg tttatccaac tattgttgct 1020
gctttacaaa agggtagagg tttagaagtt aatgttgttc atgctattga aaaagaaaca 1080
gaggaataa 1089
<210> 4
<211> 751
<212> DNA
<213> Artificial Sequence
<220>
The back segment homologous sequence of <223> aspergillus niger heat shock protein gene
<400> 4
gtatgcccga gcaccagaag cagatctact acatcaccgg cgagtccatc aaggccgttg 60
ccaagtctcc cttcctggac agcctcaagc agaagaactt cgaggttctg ttcctggttg 120
accccattga cgagtacgcc ttcactcagc tgaaggagtt cgacggcaag aagctggtcg 180
acatcaccaa ggacttcgag cttgaggagt ccgaggagga gaaggctgag cgcgagaagg 240
aggagaagga gttcgagggt ctcgccaaga gcctcaagaa catcctcggc gacaaggtcg 300
agaaggtcgt tgtctcccac aagctcgttg gctctccttg cgccatccgt accggccagt 360
tcggttggtc cgctaacatg gagcgtatca tgaaggccca ggccctccgt gacacctcca 420
tgagctctta catgtcctcc aagaagactt tcgagatctc tcccaagtcc tctatcatca 480
aggagctccg caagaaggtt gaggccgatg gcgagggtga ccgcactgtc aagtccatca 540
ctcagctgct cttcgagacc tctctcctgg tctccggttt caccattgag gagcccgcca 600
gcttcgctga gcgcatccac aagctcgtct cccttggtct gaacatcgac gaggaggctg 660
agaccaccga ggagaaggct gctgaggagg ctgcccctgc cgccgccgct gccgagagct 720
ccatggagga ggttgactaa ccgtcgactt t 751