CN110079465A - It gives a report the aspergillus oryzae transformation system construction method of gene using phleomycin as selection markers/GFP - Google Patents
It gives a report the aspergillus oryzae transformation system construction method of gene using phleomycin as selection markers/GFP Download PDFInfo
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- CN110079465A CN110079465A CN201910396332.9A CN201910396332A CN110079465A CN 110079465 A CN110079465 A CN 110079465A CN 201910396332 A CN201910396332 A CN 201910396332A CN 110079465 A CN110079465 A CN 110079465A
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- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/14—Fungi; Culture media therefor
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/65—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression using markers
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/66—General methods for inserting a gene into a vector to form a recombinant vector using cleavage and ligation; Use of non-functional linkers or adaptors, e.g. linkers containing the sequence for a restriction endonuclease
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/10—Transferases (2.)
- C12N9/1025—Acyltransferases (2.3)
- C12N9/1029—Acyltransferases (2.3) transferring groups other than amino-acyl groups (2.3.1)
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- C12N2800/00—Nucleic acids vectors
- C12N2800/22—Vectors comprising a coding region that has been codon optimised for expression in a respective host
Abstract
It gives a report the aspergillus oryzae transformation system construction method of gene using phleomycin as selection markers/GFP, codon optimization is carried out to coding phleomycin resistant gene transacetylase blmB first, using gpdA as the promoter of gene expression, trpc is that terminator constructs blmB expression casette, it is again the double base recombinant vector blmB-pEX1 for carrying blmB gene expressed intact core and fluorescent reporter gene GFP by pEX1 vector modification, recombinant vector blmB-pEX1 is successively converted into GV3101 Agrobacterium competent cell, the bacterium solution of aspergillus oryzae spore and Agrobacterium is co-cultured again, carry out the aspergillus oryzae transgenosis of mediated by agriculture bacillus, it is screened after co-cultivation by phleomycin resistance screening and culturing medium, it finally chooses resistant transformants and expands culture, by fluorescence microscope Transformant false positive rate is effectively reduced in GFP successful expression, reduces screening operation amount, to overcome the problems, such as that resistance screening label is insufficient.
Description
Technical field
The present invention relates to technical field of molecular biology, more particularly to one kind to report by selection markers/GFP of phleomycin
Accuse the aspergillus oryzae transformation system construction method of gene.
Background technique
Aspergillus oryzae belongs to Deuteromycotina aspergillus, is a kind of bacterial strain that can produce complex enzyme, and the enzyme generated includes
Protease, amylase, carbohydrase, cellulase, phytase etc. have particularly significant in fermentation industries such as food, word material, brewings
Effect, using also very extensive in the production of soy sauce, rice wine, commercial enzyme and medical protein.It is production bacterial strain with aspergillus oryzae,
There is mature fermentation and post-processing technology, therefore frequently as gene expression, protein secretion research and heterologous protein expression
Ideal tools.But in actual production, the enzymatic productivity of aspergillus oryzae is not often good enough, by carrying out genetic engineering transformation to aspergillus oryzae
To improve its enzyme activity, it will improve production efficiency to a certain extent, reduce cost.
With the development of molecular biology technology, utilizing the research of technique for gene engineering building aspergillus oryzae genetic transformation system
It is paid more and more attention, and selecting suitable selection markers is to carry out the premise of genetic transformation, can reduce false positive rate, reduces sieve
Select workload.Genetic marker for filamentous fungi transformation and selection mainly has 3 classes: antibiotic-resistance marker, auxotroph mark
Note and the gene with chromogenic reaction, wherein antibiotic-resistance marker is dominant selection markers, when drug resistance gene is transferred to
After recipient cell, the latter can be made to grow under certain density drug, show drug resistance, due to most mycotrophy defects
Phenotypic marker is difficult to obtain, and the detection of reporter gene is relatively complicated, therefore antibiotic-resistance marker obtains because being easier in contrast
And detection is simple and more advantage, therefore application is relatively extensive.Phleomycin is a kind of antibiotic secreted by streptomyces verticillatus,
Eukaryon and prokaryotes DNA and RNA chain break can be caused so as to cause cell death, in aspergillus nidulans, aspergillus flavus, coarse arteries and veins
As resistance screening label (Austin et al., 1990) in a variety of fungi transgeneic procedures such as spore bacterium.
Aspergillus oryzae is one kind of filamentous fungi, all resistant to Multiple Classes of Antibiotics and fungicide, and existing oneself reports drug
The type of resistance marker is less, the arsenic pyridine thiamine resistant gene of current only ptrA coding and withering peaceful resist of becoming rusty for AoadhB coding
Property gene is applied (Suzuki et al., 2009) in aspergillus oryzae transformation system, although both selection markers effects
Preferably, but each selection markers has its advantage and disadvantage, its use is limited because arsenic pyridine thiamine is expensive, therefore develops more
Aspergillus oryzae resistance screening label will provide effective approach for aspergillus oryzae transgeneic procedure.Since aspergillus oryzae is to phleomycin
Resistance level is higher, does not succeed by the carry out transgenosis of selection markers of phleomycin, improves aspergillus oryzae to phleomycin
Sensibility facilitate using phleomycin realize aspergillus oryzae transgeneic procedure.
Green fluorescence egg GFP is derived from one of jellyfish native fluorescent proteins, and successful expression is raw in a variety of protokaryons
Object and eucaryote, it is easy to detect, it only needs ultraviolet light or blue light to excite, can directly be observed by fluorescence microscope.Because of GFP fluorescence
It is highly stable, it can express in real time in the living body, be the desirable fluorescent labelled protein for studying gene expression and albumen positioning in cell,
Therefore how GFP to be applied in the building of aspergillus oryzae transformation system and have become those skilled in the art's technology urgently to be resolved and ask
Topic.
Summary of the invention
Technical problem solved by the invention is to provide a kind of gives a report gene using phleomycin as selection markers/GFP
Aspergillus oryzae transformation system construction method, to solve the disadvantage in above-mentioned background technique.
Technical problem solved by the invention is realized using following technical scheme:
It gives a report the aspergillus oryzae transformation system construction method of gene using phleomycin as selection markers/GFP, specific steps
It is as follows:
1) blmB expression casette is constructed
1. carrying out codon optimization to coding phleomycin resistant gene transacetylase blmB, and to the coding after optimization
Phleomycin resistant gene transacetylase blmB carries out artificial synthesized blmB gene order, and sequence is shown in SEQ ID NO:1；
No.1 special primer blmB-F is set based on the DNA fragmentation of sequence SEQ ID NO:1 simultaneously:CTC GAG CAC CAT
GGT TAC TGA GCA TCC C and blmB-R:GGA TCCGCC TCA AAC TGG GGC AGA CAC carries out PCR amplification,
The overall length for cloning blmB gene, obtains blmB gene PCR product, and then blmB gene PCR product segment is subcloned to pGM-T and is carried
On body；
2. using restriction enzyme site Xho I and BamH I by after pGreen2 carrier double digestion, with the blmB after same double digestion
Genetic fragment is attached using T4DNA ligase, formed using gpdA as gene expression promoter (sequence is SEQ ID NO:
2), trpc is the blmB expression casette of terminator (sequence is SEQ ID NO:3), then uses No. two to blmB expression casette
Special primer (blmB box-F:GAA TTCGTG ACC GGT GAC TCT TTC TGG C, blmB box-R:ACT AGT
TCGAGT GGA GAT GTG GAG TGG G) carry out amplification complete b lmB expression casette；
2) building and Agrobacterium-mediated Transformation of blmB-pEX1 recombinant plasmid
It, will be after double digestion by T4DNA Ligase using restriction enzyme site Xho I and BamH I by pEX1 carrier double digestion
PEX1 carrier be connected with blmB expression casette obtained in step 1), be transferred to bacillus coli DH 5 alpha competent cell, and
The screening positive clone on the plate of kalamycin resistance afterwards, then pass through bacterium solution PCR rear extraction matter correct to positive colony verifying
Grain, to construct blmB-pEX1 recombinant plasmid；
3) recombinant plasmid transformed Agrobacterium
By blmB-pEX1 recombinant plasmid transformed Agrobacterium GV3101 competent cell, detailed process obtained in step 2)
It is as follows: a, Agrobacterium competent cell to be taken to melt on ice, and be equipped in the centrifuge tube of Agrobacterium competent cell and be added backward
The blmB-pEX1 recombinant plasmid of 100ng, ice bath 5min is placed on quick-frozen 5min in liquid nitrogen for the first time, then will be equipped with blmB-pEX1 weight
The centrifuge tube of group plasmid and Agrobacterium competent cell, which is quickly placed into 37 DEG C of water-baths, keeps 5min, secondary that centrifuge tube is put back to ice
Keep 5min in bath, sequentially add the LB liquid medium of 800 μ l antibiotic-frees, under the conditions of 28 DEG C of temperature shaken cultivation 2~
3h takes the 100 aforementioned bacterium solutions of μ l to be coated on the LB containing 100 μ g/ml kanamycins and 50 μ g/ml rifampins flat to promote thallus to recover
Culture 72h is inverted on plate, under the conditions of 28 DEG C of temperature, the bacterium colony grown is resistant transformants；B, positive gram of bacterium solution PCR verifying
It is grand, it the use of No. three specific primers is amplification GFP (GFP-F:ATG GTG AGC AAG GGC GAG G；GFP-F:CTT GTA
CAG CTC GTC CAT GCC) and blmB expression casette (blmB-F:ATG GTT ACT GAG CAT CCC；blmB-F:
CTC AAA CTG GGG CAG ACA C) specific primer, carry out respectively amplification verifying positive colony；
4) 3.042 strain spore of aspergillus oryzae is prepared
The spore inoculating of 3.042 strain of aspergillus oryzae is activated in PDA solid medium, is received after 3 days using sterile water washing
Collect spore suspension, 5000rpm centrifugal enrichment after then filtering spore suspension, then spore washed 2 times using sterile water,
It is counted under the microscope finally by blood cell counting plate, adds sterile water that spore concentration is adjusted to 107A/ml, obtains aspergillus oryzae
Spore suspension；
5) Agrobacterium bacterium solution is prepared
Agrobacterium bacterium solution of the gained containing blmB-pEX1 recombinant plasmid in step 3) is inoculated in the LB Liquid Culture of 10ml
Base contains 100 μ g/ml kanamycins and 50 μ g/ml rifampins in LB liquid medium, and 200rpm is stayed overnight under the conditions of 28 DEG C of temperature
Shake bacterium；Drawing this step of 1ml LB liquid medium above-mentioned again adds the IM fluid nutrient medium mixing of 9ml to be placed on 28 DEG C of temperature strips
200rpm shakes bacterium 6h to OD under part600It is 0.7, obtains Agrobacterium bacterium solution；
6) Agrobacterium and aspergillus oryzae co-culture
The Agrobacterium bacterium solution and 80 μ l aspergillus oryzae spore suspensions for taking 80 μ l, are applied to IM solid culture after mixing
Base is protected from light culture under the conditions of 22 DEG C of temperature；After dark culture 60h, then CD solid medium is cooled to after non-scald on hand and is poured on this
Uniform fold on the IM solid medium of step, it is 50 DEG C~55 DEG C that CD solid medium, which is cooled to the temperature of non-scald on hand, herein,
Antibiotic is added at a temperature of this to fail；Culture 5-7 days is protected from light under the conditions of 30 DEG C of temperature, the bacterium colony grown is resistant transformants
Son；IM solid culture based formulas is as follows: the glucose of the MES of 40mM, 5mM, the glycerol of 0.5% (w/v), 200 μM of acetyl cloves
Ketone, the uridine of 0.05% (w/v), the uracil of 0.05% (w/v), the K of 0.205% (w/v)2HPO4, 0.145% (w/v)
KH2PO4, the NaCl of 0.015% (w/v), the MgSO of 0.05% (w/v)4·7H2O, the CaCl of 0.0068% (w/v)2·
2H2The O, (NH of 0.05% (w/v)4)2SO4, the FeSO of 0.00025% (w/v)4·7H2O, 1.8% agar powder；The training of CD solid
Feeding base contain the phleomycin of 30 μ g/ml, the cefotaxime of 300 μ g/ml, 0.007% (v/v) Triton X-100 and
The chlorpromazine of 0.1mM；
7) resistant transformants screening and identification
The resistant transformants obtained in picking step 6), which are seeded on the CD solid medium for be covered with glass slide, expands culture,
CD solid culture based formulas is as follows: the sucrose of 2% (w/v), the NaNO of 0.2% (w/v)3, the KH of 0.1% (w/v)2PO4,
The MgSO of 0.05% (w/v)4, the KCl of 0.05% (w/v), the NaCl of 0.05% (w/v), the FeSO of 0.002% (w/v)4,
The agar powder of 1.9% (w/v), PH 5.5, CD solid medium is containing the phleomycin of 30 μ g/ml, 0.007% (v/v)
The chlorpromazine of Triton X-100 and 0.1mM, regather mycelia, are then tried using the extracting genome DNA of Tiangeng biotech firm
Agent box extracts aspergillus oryzae mycelia genomic DNA, successively uses No. four special primer (blmB-F:ATG GTT ACT GAG CAT
CCC；BlmB-F:CTC AAA CTG GGG CAG ACA C) PCR amplification verifying is carried out to aspergillus oryzae mycelia genomic DNA, into
Whether verifying Insert Fragment is correct after one step passes through DNA sequencing, Insert Fragment process are as follows: the glass slide oblique cutting of sterilizing is entered culture
Base allows mycelia growth to climb up glass slide, and culture took out glass slide after 2-3 days, using fluorescence microscope exciting light 450~
Under 490nm blue light observe GFP fluorescence imaging, with intuitively detect the foreign gene being transferred to whether successful expression.
The utility model has the advantages that the present invention is established for the first time using phleomycin as resistance marker GFP, by fluorescence microscope in exciting light
The fluorescence imaging that GFP is observed under 450~490nm blue light, more intuitively detect the foreign gene that is transferred to whether successful expression, have
Effect reduces transformant false positive rate, reduces screening operation amount, overcomes the problems, such as that resistance screening label is insufficient in existing system.
Detailed description of the invention
Fig. 1 is that the BlmB expression casette expression core building in highly preferred embodiment of the present invention compares signal with electrophoresis result
Figure.
Fig. 2 is blmB-pEX1 recombinant plasmid map and electrophoresis single endonuclease digestion testing result in highly preferred embodiment of the present invention
Compare schematic diagram.
Fig. 3 is the resistant transformants structural schematic diagram in highly preferred embodiment of the present invention.
Fig. 4 is the GFP fluorescence imaging of the fluorescence microscope observation transformant mycelia in highly preferred embodiment of the present invention
Schematic diagram.
Attached drawing mark:
In Fig. 1, swimming lane 1,3,8 is marker, and swimming lane 2 is amplification blmB gene, and swimming lane 4 is the bis- enzymes of Xho I and BamH I
BlmB-pGM-T carrier is cut, swimming lane 5 is blmB-pGM-T vehicle Control, and swimming lane 6 is Xho I and BamH I double digestion pGreen load
Body, swimming lane 7 are pGreen vehicle Control；
In Fig. 2, swimming lane 1 is blmB-pEX1 recombinant vector, and swimming lane 2 is that single endonuclease digestion KpnI verifies recombinant vector.
Specific embodiment
In order to be readily apparent from the technical means, the creative features, the aims and the efficiencies achieved by the present invention clearly, tie below
Specific embodiment is closed, the present invention is further explained.
It gives a report the aspergillus oryzae transformation system construction method of gene using phleomycin as selection markers/GFP, specific steps
It is as follows:
1) blmB expression casette is constructed
1. by codon using database (http://www.kazusa.or.jp/codon/) to coding phleomycin
Resistant gene transacetylase blmB carries out codon optimization, and commission Nanjing Jin Sirui company carries out artificial synthesized blmB gene sequence
Column, sequence are shown in SEQ ID NO:1, and blmB gene order is optimized according to the Preference that aspergillus oryzae codon uses；Together
When based on the DNA fragmentation of sequence SEQ ID NO:1 be arranged No.1 special primer blmB-F:CTC GAG CAC CAT GGT
TAC TGA GCA TCC C and blmB-R:GGA TCCGCC TCA AAC TGG GGC AGA CAC carries out PCR amplification, clone
The overall length of blmB gene, the size of gained blmB gene PCR product are 916bp (dashed part is restriction enzyme site sequence), then
BlmB gene PCR product segment is subcloned to pGM-T carrier；
SEQ ID NO:1
CACCATGGTTACTGAGCATCCCCGCGCTCATACTGCTCACCTGTGCACTGCTCGTCTCGAACTGACTCC
CCTTGATCCAGCCGCTGACGCTCGGCACTTGCATCACGCCTACGGCGATGAGGAAGTCATGCGTTGGTGGACCCGTC
CTGCTTGTGCTGACCCTGCTGAGACCGAACGCTATCTCACTTCCTGTGCTGCTGCTCCTGGTGCTCGTCTGTGGACC
ATCCGCGCCCCCGATGGAACTGTTCCTGGAATGGCTGGACTCCTGGGCGGTACTGATGTCCCAGGTCTTACTTGGCT
TTTGCGCCGTGACTCCTGGGGTCATGGATATGCTACTGAGGCTGCTGCTGCTGTTGTGGGACACGCCCTCGAAGATG
GAGGCCTGGACCGCGTGGAGGCTTGGATCGAAGCCGGAAATCGGCGCAGCCTTGCTGTTGCTGCACGCGTTGGACTT
ACTGAGCGTGCTCGTTTGGCCCAGCATTATCCACACCGTCCTGGCCCCCATGAAATGGTTGTCTTGGGCAAGGCTCG
GGCCGAGGAACCGCTTACCACTTTGGCTGTGATCACCGAGCTCCCTGTGCGTGATGTTGCTGCCACTCTTCGGTTGG
TTGAAGCAGCTCTGGGAGCTCGTACTGCTTTCGCTATCGGCGACCCTCCCGAGTTCGCTGAGGCTGCTCTTACTCCG
TGGTCTGCTGGACCTCGTTTCCGCTTGGCTGCTGTCCCAGGACCTGGTCCTGTCGAACCTGTGCGTCTCCATCTGGA
TGCTGCAGGTACTGCTGATTCTTTGCATCGTCGGGCTGTTGATGCTGGAGCTCGTGTTGATGGACCACCGGTGCGCC
GTCCTTGGGGCCGCTCCGAGTTCGTGATCACCCTCCCCGAGGGTCACGAACTGACTGTGTCTGCCCCAGTTTGAGGC
2. restriction enzyme site Xho I and BamH I shown in 1. by step) by after pGreen2 carrier in pairs digestion, with
BlmB genetic fragment after same double digestion is attached using T4DNA ligase, is formed using gpdA as the starting of gene expression
Sub (sequence is SEQ ID NO:2), the blmB expression casette that trpc is terminator (sequence is SEQ ID NO:3), then it is right
BlmB expression casette using No. two special primers (blmB box-F:GAA TTC GTG ACC GGT GAC TCT TTC TGG
C, blmB box-R:ACT AGTTCG AGT GGA GAT GTG GAG TGG G) amplification complete b lmB expression casette is carried out,
BlmB expression casette primer size 2400bp, blmB expression casette schematic diagram, the amplification of blmB expression casette and electrophoresis knot
Fruit, as shown in Figure 1；
SEQ ID NO:2
ACTAGTGTGACCGGTGACTCTTTCTGGCATGCGGAGAGACGGACGGACGCAGAGAGAAGGGCTGAGTAA
TAAGCGCCACTGCGCCAGACAGCTCTGGCGGCTCTGAGGTGCAGTGGATGATTATTAATCCGGGACCGGCCGCCCCT
CCGCCCCGAAGTGGAAAGGCTGGTGTGCCCCTCGTTGACCAAGAATCTATTGCATCATCGGAGAATATGGAGCTTCA
TCGAATCACCGGCAGTAAGCGAAGGAGAATGTGAAGCCAGGGGTGTATAGCCGTCGGCGAATAGCATGCCATTAACC
TAGGTACAGAAGTCCAATTGCTTCCGATCTGGTAAAAGATTCACGAGATAGTACCTTCTCCGAAGTAGGTAGAGCGA
GTACCCGGCGCGTAAGCTCCCTAATTGGCCCATCCGGCATCTGTAGGGCGTCCAAATATCGTGCCTCTCCTGCTTTG
CCCGGTGTATGAAACCGGAAAGGCCGCTCAGGAGCTGGCCAGCGGCGCAGACCGGGAACACAAGCTGGCAGTCGACC
CATCCGGTGCTCTGCACTCGACCTGCTGAGGTCCCTCAGTCCCTGGTAGGCAGCTTTGCCCCGTCTGTCCGCCCGGT
GTGTCGGCGGGGTTGACAAGGTCGTTGCGTCAGTCCAACATTTGTTGCCATATTTTCCTGCTCTCCCCACCAGCTGC
TCTTTTCTTTTCTCTTTCTTTTCCCATCTTCAGTATATTCATCTTCCCATCCAAGAACCTTTATTTCCCCTAAGTAA
GTACTTTGCTACATCCATACTCCATCCTTCCCATCCCTTATTCCTTTGAACCTTTCAGTTCGAGCTTTCCCACTTCA
TCGCAGCTTGACTAACAGCTACCCCGCTTGAGCAGACATCACCCTCGAG
SEQ ID NO:3
GGATCC(BamHI)ACTTAACGTTACTGAAATCATCAAACAGCTTGACGAATCTGGATATAAGATCGTT
GGTGTCGATGTCAGCTCCGGAGTTGAGACAAATGGTGTTCAGGATCTCGATAAGATACGTTCATTTGTCCAAGCAGC
AAAGAGTGCCTTCTAGTGATTTAATAGCTCCATGTCAACAAGAATAAAACGCGTTTCGGGTTTACCTCTTCCAGATA
CAGCTCATCTGCAATGCATTAATGCATTGGACCTCGCAACCCTAGTACGCCCTTCAGGCTCCGGCGAAGCAGAAGAA
TAGCTTAGCAGAGTCTATTTTCATTTTCGGGAGACGAGATCAAGCAGATCAACGGTCGTCAAGAGACCTACGAGACT
GAGGAATCCGCTCTTGGCTCCACGCGACTATATATTTGTCTCTAATTGTACTTTGACATGCTCCTCTTCTTTACTCT
GATAGCTTGACTATGAAAATTCCGTCACCAGCCCCTGGGTTCGCAAAGATAATTGCACTGTTTCTTCCTTGAACTCT
CAAGCCTACAGGACACACATTCATCGTAGGTATAAACCTCGAAAATCATTCCTACTAAGATGGGTATACAATAGTAA
CCATGCATGGTTGCCTAGTGAATGCTCCGTAACACCCAATACGCCGGCCGAAACTTTTTTACAACTCTCCTATGAGT
CGTTTACCCAGAATGCACAGGTACACTTGTTTAGAGGTAATCCTTCTTTCTAGAAGTCCTCGTGTACTGTGTAAGCG
CCCACTCCACATCTCCACTCGACCTGCAGG
2) building and Agrobacterium-mediated Transformation of blmB-pEX1 recombinant plasmid
It, will be after double digestion by T4DNA Ligase using restriction enzyme site Xho I and BamH I by pEX1 carrier double digestion
PEX1 carrier be connected with blmB expression casette obtained in step 1), be transferred to bacillus coli DH 5 alpha competent cell, and
The screening positive clone on the plate of kalamycin resistance afterwards, then pass through bacterium solution PCR rear extraction matter correct to positive colony verifying
Grain, to construct blmB-pEX1 recombinant plasmid, the principle of construction of recombinant vector and the PCR electrophoresis result respectively walked are as shown in Figure 2；
3) recombinant plasmid transformed Agrobacterium
By blmB-pEX1 recombinant plasmid transformed Agrobacterium GV3101 competent cell, detailed process obtained in step 2)
It is as follows: a, Agrobacterium competent cell to be taken to melt on ice, and be equipped in the centrifuge tube of Agrobacterium competent cell and be added backward
The blmB-pEX1 recombinant plasmid of 100ng, ice bath 5min is placed on quick-frozen 5min in liquid nitrogen for the first time, then will be equipped with blmB-pEX1 weight
The centrifuge tube of group plasmid and Agrobacterium competent cell, which is quickly placed into 37 DEG C of water-baths, keeps 5min, secondary that centrifuge tube is put back to ice
Keep 5min in bath, sequentially add the LB liquid medium of 800 μ l antibiotic-frees, under the conditions of 28 DEG C of temperature shaken cultivation 2~
3h takes the 100 aforementioned bacterium solutions of μ l to be coated on the LB containing 100 μ g/ml kanamycins and 50 μ g/ml rifampins flat to promote thallus to recover
Culture 72h is inverted on plate, under the conditions of 28 DEG C of temperature, the bacterium colony grown is resistant transformants；B, positive gram of bacterium solution PCR verifying
It is grand, it the use of No. three specific primers is amplification GFP (GFP-F:ATG GTG AGC AAG GGC GAG G；GFP-F:CTT GTA
CAG CTC GTC CAT GCC) and blmB expression casette (blmB-F:ATG GTT ACT GAG CAT CCC；blmB-F:
CTC AAA CTG GGG CAG ACA C) specific primer, carry out respectively amplification verifying positive colony；
4) 3.042 strain spore of aspergillus oryzae is prepared
The spore inoculating of 3.042 strain of aspergillus oryzae is activated in PDA solid medium, is received after 3 days using sterile water washing
Collect spore suspension, 5000rpm centrifugal enrichment after then filtering spore suspension, then spore washed 2 times using sterile water,
It is counted under the microscope finally by blood cell counting plate, adds sterile water that spore concentration is adjusted to 107A/ml, obtains aspergillus oryzae
Spore suspension；
5) Agrobacterium bacterium solution is prepared
Agrobacterium bacterium solution of the gained containing blmB-pEX1 recombinant plasmid in step 3) is inoculated in the LB Liquid Culture of 10ml
Base contains 100 μ g/ml kanamycins and 50 μ g/ml rifampins in LB liquid medium, and 200rpm is stayed overnight under the conditions of 28 DEG C of temperature
Shake bacterium；Drawing this step of 1ml LB liquid medium above-mentioned again adds the IM fluid nutrient medium mixing of 9ml to be placed on 28 DEG C of temperature strips
200rpm shakes bacterium 6h to OD under part600It is 0.7, obtains Agrobacterium bacterium solution；
6) Agrobacterium and aspergillus oryzae co-culture
The Agrobacterium bacterium solution and 80 μ l aspergillus oryzae spore suspensions for taking 80 μ l, are applied to IM solid culture after mixing
Base is protected from light culture under the conditions of 22 DEG C of temperature；After dark culture 60h, then CD solid medium is cooled to after non-scald on hand and is poured on this
Uniform fold on the IM solid medium of step, it is 50 DEG C~55 DEG C that CD solid medium, which is cooled to the temperature of non-scald on hand, herein,
Antibiotic is added at a temperature of this to fail；Culture 5-7 days is protected from light under the conditions of 30 DEG C of temperature, the bacterium colony grown is resistant transformants
Son, colonial morphology are as shown in Figure 3；IM solid culture based formulas is as follows: the glucose of the MES of 40mM, 5mM, 0.5% (w/v)
Glycerol, 200 μM of acetosyringones, the uridine of 0.05% (w/v), the uracil of 0.05% (w/v), 0.205% (w/v)
K2HPO4, the KH of 0.145% (w/v)2PO4, the NaCl of 0.015% (w/v), the MgSO of 0.05% (w/v)4·7H2O,
The CaCl of 0.0068% (w/v)2·2H2The O, (NH of 0.05% (w/v)4)2SO4, the FeSO of 0.00025% (w/v)4·7H2O,
1.8% agar powder；CD solid medium contains the phleomycin of 30 μ g/ml, the cefotaxime of 300 μ g/ml, 0.007% (v/
V) chlorpromazine of Triton X-100 and 0.1mM；
7) resistant transformants screening and identification
The resistant transformants obtained in picking step 6), which are seeded on the CD solid medium for be covered with glass slide, expands culture,
CD solid culture based formulas is as follows: the sucrose of 2% (w/v), the NaNO of 0.2% (w/v)3, the KH of 0.1% (w/v)2PO4,
The MgSO of 0.05% (w/v)4, the KCl of 0.05% (w/v), the NaCl of 0.05% (w/v), the FeSO of 0.002% (w/v)4,
The agar powder of 1.9% (w/v), PH 5.5, CD solid medium is containing the phleomycin of 30 μ g/ml, 0.007% (v/v)
The chlorpromazine of Triton X-100 and 0.1mM, regather mycelia, are then tried using the extracting genome DNA of Tiangeng biotech firm
Agent box extracts aspergillus oryzae mycelia genomic DNA, successively uses No. four special primer (blmB-F:ATG GTT ACT GAG CAT
CCC；BlmB-F:CTC AAA CTG GGG CAG ACA C) PCR amplification verifying is carried out to aspergillus oryzae mycelia genomic DNA, into
Whether verifying Insert Fragment is correct after one step passes through DNA sequencing, Insert Fragment process are as follows: the glass slide oblique cutting of sterilizing is entered culture
Base allows mycelia growth to climb up glass slide, and culture took out glass slide after 2-3 days, using fluorescence microscope exciting light 450~
The fluorescence imaging that GFP is observed under 490nm blue light, more intuitively detect the foreign gene that is transferred to whether successful expression, such as Fig. 4 institute
Show, transformant false positive rate is effectively reduced, reduce screening operation amount, overcomes resistance screening in existing system and mark insufficient ask
Topic.
In above technical scheme, involved blmB gene information, pEX1, pEX2 carrier information can by consult under
State document acquisition:
1、Austin,B.,Hall,R.M.,Tyler,B.M.Optimized vectors and selection for
transformation of Neurospora crassa and Aspergillus nidulans to bleomycin and
phleomycin resistance.Gene.1990,93:157-162；
2、Sugiyama,M.,Kumagai,T.,Shionoya,M.,Kimura,E.,Davies,
J.E.Inactivation of bleomycin by an N-acetyltransferase in the bleomycin-
producing strain Streptomyces verticillus.FEMS Microbiol.Lett.,1994,121:81-
85；
3、Nguyen,K.T.,Ho,Q.N.,Do,L.T.B.X.,Mai,L.T.D.,Pham,D.,Tran,H.T.T.,Le,
D.H.,Nguyen,H.Q.,and Tran,V.T.A new and efficient approach for construction
of uridine/uracil auxotrophic mutants in the filamentous fungus Aspergillus
oryzae using Agrobacterium tumefaciens-mediated transformation.World
J.Microbiol.Biotechnol.,2017,33:107。
Claims (10)
1. giving a report the aspergillus oryzae transformation system construction method of gene using phleomycin as selection markers/GFP, feature exists
In, the specific steps are as follows:
1) blmB expression casette is constructed
1. carrying out codon optimization to coding phleomycin resistant gene transacetylase blmB, and to the rotten grass of coding after optimization
Mycin resistant gene transacetylase blmB carries out artificial synthesized blmB gene order, and sequence is shown in SEQ ID NO:1；Simultaneously
No.1 special primer is set based on the DNA fragmentation of sequence SEQ ID NO:1 and carries out PCR amplification, clones the complete of blmB gene
It is long, blmB gene PCR product is obtained, then blmB gene PCR product segment is subcloned to pGM-T carrier；
2. using restriction enzyme site Xho I and BamH I by after pGreen2 carrier double digestion, with the blmB gene after same double digestion
Segment is attached using T4 DNA ligase, and being formed by the promoter of gene expression, trpc of gpdA is the blmB of terminator
Expression casette, then amplification complete b lmB expression casette is carried out using No. two special primers to blmB expression casette；
2) building and Agrobacterium-mediated Transformation of blmB-pEX1 recombinant plasmid
It, will be after double digestion by T4 DNA Ligase using restriction enzyme site Xho I and BamH I by pEX1 carrier double digestion
PEX1 carrier is connected with blmB expression casette obtained in step 1), bacillus coli DH 5 alpha competent cell is transferred to, with structure
Build blmB-pEX1 recombinant plasmid；
3) recombinant plasmid transformed Agrobacterium
By blmB-pEX1 recombinant plasmid transformed Agrobacterium GV3101 competent cell obtained in step 2)；
4) 3.042 strain spore of aspergillus oryzae is prepared
The spore inoculating of 3.042 strain of aspergillus oryzae is activated in PDA solid medium, spore is collected using sterile water washing after 3 days
Sub- suspension, 5000rpm centrifugal enrichment after then filtering spore suspension, then using sterile water to spore washing 2 times, finally
It is counted under the microscope by blood cell counting plate, adds sterile water that spore concentration is adjusted to 107A/ml obtains aspergillus oryzae spore
Suspension；
5) Agrobacterium bacterium solution is prepared
Agrobacterium bacterium solution of the gained containing blmB-pEX1 recombinant plasmid in step 3) is inoculated in the LB liquid medium of 10ml,
Contain 100 μ g/ml kanamycins and 50 μ g/ml rifampins in LB liquid medium, 200rpm shakes overnight under the conditions of 28 DEG C of temperature
Bacterium；Drawing this step of 1ml LB liquid medium above-mentioned again adds the IM fluid nutrient medium mixing of 9ml to be placed on 28 DEG C of temperature conditions
Lower 200rpm shakes bacterium 6h to OD600It is 0.7, obtains Agrobacterium bacterium solution；
6) Agrobacterium and aspergillus oryzae co-culture
The Agrobacterium bacterium solution and 80 μ l aspergillus oryzae spore suspensions for taking 80 μ l, are applied to IM solid medium after mixing,
Culture is protected from light under the conditions of 22 DEG C of temperature；After dark culture 60h, CD solid medium is cooled to the IM that this step is poured on after non-scald on hand
Uniform fold on solid medium, it is 50 DEG C~55 DEG C, at a temperature of this that CD solid medium, which is cooled to the temperature of non-scald on hand, herein
Antibiotic, which is added, to fail；Culture 5-7 days is protected from light under the conditions of 30 DEG C of temperature, the bacterium colony grown is resistant transformants；
7) resistant transformants screening and identification
The resistant transformants obtained in picking step 6), which are seeded on the CD solid medium for be covered with glass slide, expands culture, then receives
Collect mycelia, then extracts aspergillus oryzae mycelia genomic DNA using the genome DNA extracting reagent kit of Tiangeng biotech firm, successively
PCR amplification verifying is carried out using No. four primer pair aspergillus oryzae mycelia genomic DNAs, further by verifying after DNA sequencing
Whether Insert Fragment is correct, finally by the fluorescence imaging of fluorescence microscope GFP, intuitively to detect the external source base being transferred to
Because of expression status.
2. according to claim 1 give a report the aspergillus oryzae transformation system of gene using phleomycin as selection markers/GFP
Construction method, which is characterized in that in step 1), No.1 special primer is blmB-F:CTC GAG CAC CAT GGT TAC TGA
GCA TCC C and blmB-R:GGA TCC GCC TCA AAC TGG GGC AGA CAC。
3. according to claim 1 give a report the aspergillus oryzae transformation system of gene using phleomycin as selection markers/GFP
Construction method, which is characterized in that in step 1), No. two special primers are blmB box-F:GAA TTC GTG ACC GGT GAC
TCT TTC TGG C and blmB box-R:ACT AGTTCG AGT GGA GAT GTG GAG TGG G。
4. according to claim 1 give a report the aspergillus oryzae transformation system of gene using phleomycin as selection markers/GFP
Construction method, which is characterized in that in step 3), the tool of blmB-pEX1 recombinant plasmid transformed Agrobacterium GV3101 competent cell
Body process is as follows: a, Agrobacterium competent cell being taken to melt on ice, and is equipped with the centrifuge tube of Agrobacterium competent cell backward
The middle blmB-pEX1 recombinant plasmid that 100ng is added, ice bath 5min is placed on quick-frozen 5min in liquid nitrogen for the first time, then blmB- will be housed
The centrifuge tube of pEX1 recombinant plasmid and Agrobacterium competent cell, which is quickly placed into 37 DEG C of water-baths, keeps 5min, secondary by centrifuge tube
It puts back to and keeps 5min in ice bath, sequentially add the LB liquid medium of 800 μ l antibiotic-frees, training is vibrated under the conditions of 28 DEG C of temperature
2~3h is supported to promote thallus to recover, the 100 aforementioned bacterium solutions of μ l is taken to be coated on containing 100 μ g/ml kanamycins and 50 μ g/ml rifampins
LB plate on, under the conditions of 28 DEG C of temperature be inverted culture 72h, the bacterium colony grown is resistant transformants；B, bacterium solution PCR verifying sun
Property clone, using No. three specific primers be expand GFP and blmB expression casette specific primer, carry out respectively expand test
Demonstrate,prove positive colony.
5. according to claim 4 give a report the aspergillus oryzae transformation system of gene using phleomycin as selection markers/GFP
Construction method, which is characterized in that No. three specific primers include that GFP specific primer and blmB expression casette specificity are drawn
Object, GFP specific primer are GFP-F:ATG GTG AGC AAG GGC GAG G and GFP-F:CTT GTA CAG CTC GTC
CAT GCC, blmB expression casette specific primer is blmB-F:ATG GTT ACT GAG CAT CCC and blmB-F:CTC
AAA CTG GGG CAG ACA C。
6. according to claim 1 give a report the aspergillus oryzae transformation system of gene using phleomycin as selection markers/GFP
Construction method, which is characterized in that in step 6), IM solid culture based formulas is as follows: the glucose of the MES of 40mM, 5mM, 0.5%
(w/v) glycerol, 200 μM of acetosyringones, the uridine of 0.05% (w/v), the uracil of 0.05% (w/v), 0.205%
(w/v) K2HPO4, the KH of 0.145% (w/v)2PO4, the NaCl of 0.015% (w/v), the MgSO of 0.05% (w/v)4·7H2O,
The CaCl of 0.0068% (w/v)2·2H2The O, (NH of 0.05% (w/v)4)2SO4, the FeSO of 0.00025% (w/v)4·7H2O,
1.8% agar powder；CD solid medium contains the phleomycin of 30 μ g/ml, the cefotaxime of 300 μ g/ml, 0.007% (v/
V) chlorpromazine of Triton X-100 and 0.1mM.
7. according to claim 1 give a report the aspergillus oryzae transformation system of gene using phleomycin as selection markers/GFP
Construction method, which is characterized in that in step 7), CD solid culture based formulas is as follows: the sucrose of 2% (w/v), 0.2% (w/v's)
NaNO3, the KH of 0.1% (w/v)2PO4, the MgSO of 0.05% (w/v)4, the KCl of 0.05% (w/v), 0.05% (w/v's)
NaCl, the FeSO of 0.002% (w/v)4, the agar powder of 1.9% (w/v), PH 5.5；CD solid medium contains the corruption of 30 μ g/ml
Oxamycin, 0.007% (v/v) Triton X-100 and 0.1mM chlorpromazine.
8. according to claim 1 give a report the aspergillus oryzae transformation system of gene using phleomycin as selection markers/GFP
Construction method, which is characterized in that in step 7), No. four special primers be blmB-F:ATG GTT ACT GAG CAT CCC and
blmB-F:CTC AAA CTG GGG CAG ACA C。
9. according to claim 1 give a report the aspergillus oryzae transformation system of gene using phleomycin as selection markers/GFP
Construction method, which is characterized in that in step 7), Insert Fragment process are as follows: the glass slide oblique cutting of sterilizing is entered into culture medium, allows mycelia
Glass slide is climbed up in growth, and culture took out glass slide after 2-3 days.
10. according to claim 1 give a report the aspergillus oryzae Transgenics of gene using phleomycin as selection markers/GFP
It is construction method, which is characterized in that in step 7), observe GFP under exciting light 450~490nm blue light using fluorescence microscope
Fluorescence imaging.
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