CN108676810A - By agriculture bacillus mediated using pyrithiamine as the aspergillus oryzae transformation system construction method of selection markers - Google Patents
By agriculture bacillus mediated using pyrithiamine as the aspergillus oryzae transformation system construction method of selection markers Download PDFInfo
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Abstract
The present invention provides a kind of by agriculture bacillus mediated using pyrithiamine as the aspergillus oryzae transformation system construction method of selection markers, and this method mainly includes the following steps that：(1) plant expressing vector is transformed into the binary expression vector containing prtA expression cassettes, contains GPDA promoters and GPF reporter genes；(2) binary vector conversion Agrobacterium AGL1；(3) lower spore is washed after 3.042,3 days in DPY medium culture wild types aspergillus oryzae；(4) spore co-cultures 48 hours with the Agrobacterium containing purposeful carrier；(5) spore after co-culturing is transferred to the CD Screening of Media containing cephalo and pyrithiamine；(6) transformant that screening and culturing medium grows out further is verified.The present invention establishes agriculture bacillus mediated using pyrithiamine as the transformation system of selection markers for the first time, and the transformation system that more original protoplast mediates reduces workload, improves transformation efficiency, lays a good foundation for aspergillus oryzae genetic manipulation.
Description
Technical field
The present invention relates to technical field of molecular biology, further to aspergillus oryzae transformation system constructing technology, specifically
Be related to it is a kind of by agriculture bacillus mediated using pyrithiamine as the aspergillus oryzae transformation system construction method of selection markers.
Background technology
Aspergillus oryzae not only has in the industries such as traditional food fermentation, flavouring and brewing as the fungi of aliment security level
Very long applicating history, and using also increasingly wide in the modern biotechnologies industry such as enzyme preparation, recombinant protein
It is general.Aspergillus oryzae genome has been sequenced completion in 2005, but due to aspergillus oryzae to G418, hygromycin, oligomycin, it is rich come it is mould
The common resistance drug such as element, phleomycin, kanamycins is insensitive, therefore its genetic manipulation is difficult so that aspergillus oryzae work(
Energy progression is slow.
The common transgenic method of fungi has protoplast to mediate and agriculture bacillus mediated two kinds.The former has culture difficulty
Greatly, regeneration frequency is low, the period is long and it is complicated for operation the shortcomings of so that its application have certain limitation.And the latter has operation
Simply, transformation efficiency is high, and T-DNA is inserted into the advantages that genome stablizes heredity.Although agriculture bacillus mediated transgenic technology
Succeed in other fungies such as yeast, monascus, aspergillus niger, but since aspergillus oryzae is currently available that resistance screening mark
The reason of remembering less and own growth characteristic, these conventional spore concentrations, co-culture the time and at a temperature of cannot achieve and turn
Gene.Currently, the use of Agrobacterium-mediated Transformation aspergillus oryzae being international research hot spot always, 2016 establish pass through Ura/ in the world for the first time
Uri auxotrophs are the aspergillus oryzae transformation system of selection markers, it is determined that spore concentration co-cultures the items such as time and temperature
Part so that being possibly realized property of Agrobacterium-mediated Transformation aspergillus oryzae.And it is not have to structure using pyrithiamine as the largest benefit of selection markers
Auxotroph aspergillus oryzae is built, directly carries out transgenosis by background of wild type.But the use of pyrithiamine is at present screening
The Agrobacterium transgenosis of label did not succeed, main cause may be co-culture the time and temperature it is improper, or
It is that binary vector does not utilize target gene conversion etc..Therefore, aspergillus oryzae transgenosis depends on protoplast mediation at present
Transgenic technology.And in aspergillus oryzae and it is difficult pyrithiamine as the report of the Agrobacterium transgenosis of selection markers at present.
Invention content
The present invention is directed to the technological deficiency for the prior art, it by agriculture bacillus mediated is sieve with pyrithiamine to provide a kind of
Select label aspergillus oryzae transformation system construction method, with solve in the prior art be directed to aspergillus oryzae transgenic method be difficult to by
Agrobacterium is come the technical issues of mediation.
Another technical problem to be solved by the present invention is that conventional with its work of the aspergillus oryzae transgenic method of protoplast mediation
Work amount is larger, transformation efficiency is relatively low.
To realize that the above technical purpose, the present invention use following technical scheme：
By agriculture bacillus mediated using pyrithiamine as the aspergillus oryzae transformation system construction method of selection markers, including it is following
Step：
1) using I carriers of pPTR as template, with sequence for SEQ ID NO:1、SEQ ID NO:2 DNA fragmentation is held for primer
Row PCR amplification obtains ptrA expression cassettes；By restriction enzyme site EcoR I and Spe I by pEX1 carrier double digestions, by digestion products with
The ptrA expression cassettes connection, obtains pEX1-ptrA carriers；The pEX1-ptrA carriers are converted to the Agrobacterium of competence
AGL1 plants；
2) spore inoculating of 3.042 plants of aspergillus oryzae was collected into spore in PDA DPY medium cultures 3 days；It will collect
10min is centrifuged with the rotating speed of 4000rpm after object filtering, then is washed with distilled water 2 times, the mixture of spore and water is obtained, is adjusted
Wherein spore concentration is to 106A/mL is to get to spore suspension；
3) AGL1 plants of Agrobacterium after converting step 1) is seeded to 10mL while having kalamycin resistance and rifampin
In the LB culture mediums of resistance, with 28 DEG C of temperature, the rotating speed shaking flask culture of 200rpm；Take the 1mL culture solutions and 9mL IM liquid
Culture medium mixes, and then with 28 DEG C of temperature, the rotating speed shaking flask culture 6h of 200rpm, obtains bacterium solution；
4) spore suspension obtained by bacterium solution and 100 μ L steps 2) obtained by 100 μ L steps 3) is taken, is applied to is covered with glassine paper altogether
On IM solid mediums, in 22 DEG C, be protected from light culture 48h；
5) glassine paper is taken out, covers and is trained simultaneously containing the CD of 300 μ g/mL cefotaxime and 1 μ g/L pyrithiamines
It supports on base, 30 DEG C of 5~7d of culture；The bacterium colony that picking is grown, be seeded to it is another it is fresh, simultaneously contain 300 μ g/mL cefotaxime
On the CD culture mediums of 1 μ g/L pyrithiamines, culture, the bacterium colony grown is positive transformant.
Preferably, it is further comprising the steps of 6)：The positive transformant obtained by step 5) is taken, its DNA is extracted, uses GFP
Specific primer verifies whether it is positive transformant by PCR, while utilizing fluorescence microscope GFP.
Preferably, the length of ptrA expression cassettes described in step 1) is 2000bp.
Preferably, digestion products are attached with ptrA expression cassettes by infusion modes in step 1).
Preferably, step 3) the IM fluid nutrient mediums are prepared by the following method：40mM MES, 5mM grapes
Sugar, 0.5% (w/v) glycerine and 200 μM of acetosyringones are dissolved in MM salting liquids.
Preferably, MM salting liquids described in per 1L contain following component：K2HPO42.05g KH2PO41.45g NaCl
0.15g, MgSO4·7H2O 0.5g, CaCl2·6H2O 0.1g, (NH4)2SO40.5g, FeSO4·7H2O 0.0025g。
Preferably, in step 3), after the 1mL culture solutions are mixed with 9mL IM fluid nutrient mediums, OD600Value
It is 0.2~0.3.
Preferably, in step 3), after the shaking flask culture 6h, wherein OD600Value is 0.6~0.8.
Preferably, in step 6), the sequence of the GFP specific primers is respectively such as SEQ ID NO:3、SEQ ID NO:
Shown in 4.
In above technical scheme, used carrier pPTR I, pEX1, specifying information can be known by following documents：
Kubodera,T.,N.Yamashita and A.Nishimura(2000)."Pyrithiamine
Resistance Gene(ptrA)of Aspergillus oryzae:Cloning,Characterization and
Application as a Dominant Selectable Marker for Transformation."Bioscience
Biotechnology&Biochemistry 64(7):1416.
Nguyen,K.T.,Q.N.Ho,T.H.Pham,T.N.Phan and V.T.Tran(2016)."The
construction and use of versatile binary vectors carrying pyrG auxotrophic
marker and fluorescent reporter genes for Agrobacterium-mediated
transformation of Aspergillus oryzae."World J Microbiol Biotechnol 32(12):
204.
The present invention provides a kind of by agriculture bacillus mediated using pyrithiamine as the aspergillus oryzae transformation system of selection markers
Construction method, this method mainly include the following steps that：(1) plant expressing vector is transformed into the double base table containing prtA expression cassettes
Up to carrier, contain GPDA promoters and GPF reporter genes；(2) binary vector conversion Agrobacterium AGL1；(3) it is trained in DPY culture mediums
Breeding wild type aspergillus oryzae washes lower spore after 3.042,3 days；(4) spore co-cultures 48 hours with the Agrobacterium containing purposeful carrier；
(5) spore after co-culturing is transferred to the CD Screening of Media containing cephalo and pyrithiamine；(6) screening and culturing medium is grown
The transformant come is further verified.The present invention establishes for the first time agriculture bacillus mediated turns base using pyrithiamine as selection markers
Because of system, the transformation system of protoplast mediation more originally reduces workload, improves transformation efficiency, is aspergillus oryzae heredity
Operation is laid a good foundation.
Description of the drawings
Fig. 1 is the schematic diagram of pEX1-ptrA vector construction processes and corresponding PCR electrophoresis results figure；
Fig. 2 is the bacterium colony figure of the transformant grown on screening and culturing medium；
Fig. 3 is the bacterium colony figure that transformant is further cultivated using new screening and culturing medium；
Thalline figure observed by under being under the conditions of light field respectively in Fig. 4 and fluorescence microscope；Figure Green fluorescence be containing
There is the thalline of GFP.
Specific implementation mode
The specific implementation mode of the present invention will be described in detail below.In order to avoid excessive unnecessary details,
It will not be described in detail in following embodiment to belonging to well known structure or function.
Approximating language used in following embodiment can be used for quantitative expression, show in the feelings for not changing basic function
Quantity is allowed to have certain variation under condition.Therefore, it is not limited to this accurately with the modified numerical value of the language such as " about ", " left and right " institute
Numerical value itself.In some embodiments, the range for allowing its modified numerical value in positive and negative 10 (10%) " about " is indicated
Interior variation, for example, what " about 100 " indicated can be any numerical value between 90 to 110.In addition, " the about first numerical value arrives
In the statement of second value ", the first and second numerical value two values are at about corrected.In some cases, approximating language
It may be related with the precision of measuring instrument.
In addition to being defined, technical and scientific term used in following embodiment has and fields technology people of the present invention
The identical meanings that member is commonly understood by.
Embodiment 1
1, vector construction
1. from I carriers of pPTR with ptrA-F and ptrA-R primer amplification ptrA expression cassettes (ptrA-F：TGA CAT GAT TAC GAA TTC GGG CAA TTG ATT ACG GGA TCC and ptrA-R：GTC ACC GGT CAC ACT AGT ATG
GGG TGA CGA TGA GCC GC) amplified production size is 2000bp (dashed part is homologous recombination sequence)；2. passing through enzyme
PtrA expression cassettes are connected pEX1 vector constructions by enzyme site Pst I and Spe I by pEX1 carrier double digestions, by infusion modes
At pEX1-ptrA carriers；3. by pEX1-ptrA conversion Agrobacteriums AGL1.The principle of vector construction and the PCR electrophoresis results respectively walked
As shown in Figure 1.
2, aspergillus oryzae (3.042) spore prepares
Aspergillus oryzae (3.042) spore inoculating collected spore in PDA DPY medium cultures 3 days.Spore filters, then
4000rpm centrifuges 10min, and distillation washing 2 times, adjustment concentration is extremely：1*106A/ml, it is spare.
3, Agrobacterium bacterium solution prepares
1. carrier of the conversion containing pEX1-ptrA plasmids is inoculated with, 10ml LB (containing Ka Na and rifampicin resistance), 28 DEG C,
It 200 turns, shakes bacterium and stays overnight；2. 1ml culture solutions is taken to add 9ml IM fluid nutrient mediums [40mM MES, 5mM glucose, 0.5% (w/v)
Glycerine and 200 μM of acetosyringones are dissolved in MM salting liquids；MM salting liquid formulas are (g/L)：K2HPO4,2.05g；KH2PO4,
1.45g；NaCl,0.15g；MgSO4.7H2O,0.5g；CaCl2.6H2O,0.1g；(NH4)2SO4,0.5g；FeSO4.7H2O,
0.0025g], OD600 is 0.2 to 0.3；3. mixed liquor is placed in 28 DEG C, 200 turns, shake bacterium 6 hours to OD600 be 0.6 to 0.8；
4, it co-cultures
Taking the spore suspension of 100 μ L bacterium solutions and 100 μ L, (concentration is about 1*106A/ml) it is coated onto on IM solid mediums (in advance
First spread glassine paper).Culture dish is placed in 22 DEG C, light culture 48 hours.
5, transformant screening and identification
1. glassine paper is transferred to screening and culturing (CD culture mediums+cefotaxime (300 μ g/ml)+pyrithiamine (1 μ
G/L), then glassine paper covers one layer of screening and culturing medium, and 30 DEG C are cultivated 5-7 days, and colonial morphology is as shown in Figure 2；2. picking is grown
Go out the bacterial plaque of culture medium to new screening and culturing basal growth, colonial morphology is as shown in Figure 3；3. it is special using GFP to extract transformant DNA
Specific primer PCR verifies whether as positive transformant (GFP-F:ATG GTG AGC AAG GGC GAG；GFP-R:TCA CTT
GTA CAG CTC GTC CAT GC)；4. fluorescence microscope GFP, thalli morphology is as shown in Figure 4 in microscope.
Embodiment 2
By agriculture bacillus mediated using pyrithiamine as the aspergillus oryzae transformation system construction method of selection markers, including it is following
Step：
1) using I carriers of pPTR as template, with sequence for SEQ ID NO:1、SEQ ID NO:2 DNA fragmentation is held for primer
Row PCR amplification obtains ptrA expression cassettes；By restriction enzyme site EcoR I and Spe I by pEX1 carrier double digestions, by digestion products with
The ptrA expression cassettes connection, obtains pEX1-ptrA carriers；The pEX1-ptrA carriers are converted to the Agrobacterium of competence
AGL1 plants；
2) spore inoculating of 3.042 plants of aspergillus oryzae was collected into spore in PDA DPY medium cultures 3 days；It will collect
10min is centrifuged with the rotating speed of 4000rpm after object filtering, then is washed with distilled water 2 times, the mixture of spore and water is obtained, is adjusted
Wherein spore concentration is to 106A/mL is to get to spore suspension；
3) AGL1 plants of Agrobacterium after converting step 1) is seeded to 10mL while having kalamycin resistance and rifampin
In the LB culture mediums of resistance, with 28 DEG C of temperature, the rotating speed shaking flask culture of 200rpm；Take the 1mL culture solutions and 9mL IM liquid
Culture medium mixes, and then with 28 DEG C of temperature, the rotating speed shaking flask culture 6h of 200rpm, obtains bacterium solution；
4) spore suspension obtained by bacterium solution and 100 μ L steps 2) obtained by 100 μ L steps 3) is taken, is applied to is covered with glassine paper altogether
On IM solid mediums, in 22 DEG C, be protected from light culture 48h；
5) glassine paper is taken out, covers and is trained simultaneously containing the CD of 300 μ g/mL cefotaxime and 1 μ g/L pyrithiamines
It supports on base, 30 DEG C of culture 5d；The bacterium colony that picking is grown, be seeded to it is another it is fresh, simultaneously contain 300 μ g/mL cefotaxime and 1
On the CD culture mediums of μ g/L pyrithiamines, culture, the bacterium colony grown is positive transformant；
6) positive transformant obtained by step 5) is taken, its DNA is extracted, verifying it by PCR using GFP specific primers is
No is positive transformant, while utilizing fluorescence microscope GFP.
Wherein, the length of ptrA expression cassettes described in step 1) is 2000bp；Digestion products are expressed with ptrA in step 1)
Box is attached by infusion modes；Step 3) the IM fluid nutrient mediums are prepared by the following method：
40mM MES, 5mM glucose, 0.5% (w/v) glycerine and 200 μM of acetosyringones are dissolved in MM salting liquids；MM salt is molten described in per 1L
Liquid contains following component：K2HPO42.05g KH2PO41.45g, NaCl 0.15g, MgSO4·7H2O 0.5g, CaCl2·6H2O
0.1g, (NH4)2SO40.5g, FeSO4·7H2O 0.0025g；In step 3), the 1mL culture solutions are trained with 9mL IM liquid
After supporting base mixing, OD600Value is 0.3；In step 3), after the shaking flask culture 6h, wherein OD600Value is 0.8；Step 6)
In, the sequence of the GFP specific primers is respectively such as SEQ ID NO:3、SEQ ID NO:Shown in 4.
Embodiment 3
By agriculture bacillus mediated using pyrithiamine as the aspergillus oryzae transformation system construction method of selection markers, including it is following
Step：
1) using I carriers of pPTR as template, with sequence for SEQ ID NO:1、SEQ ID NO:2 DNA fragmentation is held for primer
Row PCR amplification obtains ptrA expression cassettes；By restriction enzyme site EcoR I and Spe I by pEX1 carrier double digestions, by digestion products
It is connect with the ptrA expression cassettes, obtains pEX1-ptrA carriers；The pEX1-ptrA carriers are converted to the agriculture bar of competence
AGL1 plants of bacterium；
2) spore inoculating of 3.042 plants of aspergillus oryzae was collected into spore in PDA DPY medium cultures 3 days；It will collect
10min is centrifuged with the rotating speed of 4000rpm after object filtering, then is washed with distilled water 2 times, the mixture of spore and water is obtained, is adjusted
Wherein spore concentration is to 106A/mL is to get to spore suspension；
3) AGL1 plants of Agrobacterium after converting step 1) is seeded to 10mL while having kalamycin resistance and rifampin
In the LB culture mediums of resistance, with 28 DEG C of temperature, the rotating speed shaking flask culture of 200rpm；Take the 1mL culture solutions and 9mL IM liquid
Culture medium mixes, and then with 28 DEG C of temperature, the rotating speed shaking flask culture 6h of 200rpm, obtains bacterium solution；
4) spore suspension obtained by bacterium solution and 100 μ L steps 2) obtained by 100 μ L steps 3) is taken, is applied to is covered with glassine paper altogether
On IM solid mediums, in 22 DEG C, be protected from light culture 48h；
5) glassine paper is taken out, covers and is trained simultaneously containing the CD of 300 μ g/mL cefotaxime and 1 μ g/L pyrithiamines
It supports on base, 30 DEG C of culture 7d；The bacterium colony that picking is grown, be seeded to it is another it is fresh, simultaneously contain 300 μ g/mL cefotaxime and 1
On the CD culture mediums of μ g/L pyrithiamines, culture, the bacterium colony grown is positive transformant.
The embodiments of the present invention have been described in detail above, but content is only the preferred embodiment of the present invention,
It is not intended to limit the invention.All all any modification, equivalent and improvement etc. done in the application range of the present invention, should all
It is included within protection scope of the present invention.
Sequence table
<110>Jiangxi Science ＆ Technology Normal University
<120>By agriculture bacillus mediated using pyrithiamine as the aspergillus oryzae transformation system construction method of selection markers
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 39
<212> DNA
<213>Artificial sequence (Artificial sequence)
<400> 1
tgacatgatt acgaattcgg gcaattgatt acgggatcc 39
<210> 2
<211> 38
<212> DNA
<213>Artificial sequence (Artificial sequence)
<400> 2
gtcaccggtc acactagtat ggggtgacga tgagccgc 38
<210> 3
<211> 18
<212> DNA
<213>Artificial sequence (Artificial sequence)
<400> 3
atggtgagca agggcgag 18
<210> 4
<211> 23
<212> DNA
<213>Artificial sequence (Artificial sequence)
<400> 4
tcacttgtac agctcgtcca tgc 23
Claims (9)
1. by agriculture bacillus mediated using pyrithiamine as the aspergillus oryzae transformation system construction method of selection markers, it is characterised in that
Include the following steps：
1) using I carriers of pPTR as template, with sequence for SEQ ID NO:1、SEQ ID NO:2 DNA fragmentation is that primer executes PCR
Amplification, obtains ptrA expression cassettes；By restriction enzyme site EcoR I and Spe I by pEX1 carrier double digestions, by digestion products with it is described
PtrA expression cassettes connect, and obtain pEX1-ptrA carriers；The pEX1-ptrA carriers are converted to the Agrobacterium AGL1 of competence
Strain；
2) spore inoculating of 3.042 plants of aspergillus oryzae was collected into spore in PDA DPY medium cultures 3 days；By gleanings mistake
10min is centrifuged with the rotating speed of 4000rpm after filter, then is washed with distilled water 2 times, obtains the mixture of spore and water, adjustment is wherein
Spore concentration is to 106A/mL is to get to spore suspension；
3) AGL1 plants of Agrobacterium after converting step 1) is seeded to 10mL while having kalamycin resistance and rifampicin resistance
LB culture mediums in, with 28 DEG C of temperature, the rotating speed shaking flask culture of 200rpm；Take the 1mL culture solutions and 9mL IM Liquid Cultures
Base mixes, and then with 28 DEG C of temperature, the rotating speed shaking flask culture 6h of 200rpm, obtains bacterium solution；
4) spore suspensions obtained by bacterium solutions obtained by 100 μ L steps 3) and 100 μ L steps 2) are taken, be applied to altogether be covered with glassine paper IM it is solid
On body culture medium, in 22 DEG C, be protected from light culture 48h；
5) glassine paper is taken out, covers the CD culture mediums containing 300 μ g/mL cefotaxime and 1 μ g/L pyrithiamines simultaneously
On, 30 DEG C of 5~7d of culture；The bacterium colony that picking is grown, be seeded to it is another it is fresh, simultaneously contain 300 μ g/mL cefotaxime and 1 μ
On the CD culture mediums of g/L pyrithiamines, culture, the bacterium colony grown is positive transformant.
2. it is according to claim 1 by agriculture bacillus mediated using pyrithiamine as the aspergillus oryzae transformation system of selection markers
Construction method, it is characterised in that it is further comprising the steps of 6)：The positive transformant obtained by step 5) is taken, its DNA is extracted, is used
GFP specific primers verify whether it is positive transformant by PCR, while utilizing fluorescence microscope GFP.
3. it is according to claim 1 by agriculture bacillus mediated using pyrithiamine as the aspergillus oryzae transformation system of selection markers
Construction method, it is characterised in that the length of ptrA expression cassettes described in step 1) is 2000bp.
4. it is according to claim 1 by agriculture bacillus mediated using pyrithiamine as the aspergillus oryzae transformation system of selection markers
Construction method, it is characterised in that digestion products are attached with ptrA expression cassettes by infusion modes in step 1).
5. it is according to claim 1 by agriculture bacillus mediated using pyrithiamine as the aspergillus oryzae transformation system of selection markers
Construction method, it is characterised in that step 3) the IM fluid nutrient mediums are prepared by the following method：The Portugals 40mM MES, 5mM
Grape sugar, 0.5% (w/v) glycerine and 200 μM of acetosyringones are dissolved in MM salting liquids.
6. it is according to claim 5 by agriculture bacillus mediated using pyrithiamine as the aspergillus oryzae transformation system of selection markers
Construction method, it is characterised in that MM salting liquids contain following component described in per 1L：K2HPO42.05g KH2PO41.45g NaCl
0.15g, MgSO4·7H2O 0.5g, CaCl2·6H2O 0.1g, (NH4)2SO40.5g, FeSO4·7H2O 0.0025g。
7. it is according to claim 1 by agriculture bacillus mediated using pyrithiamine as the aspergillus oryzae transformation system of selection markers
Construction method, it is characterised in that in step 3), after the 1mL culture solutions are mixed with 9mL IM fluid nutrient mediums, OD600
Value is 0.2~0.3.
8. it is according to claim 1 by agriculture bacillus mediated using pyrithiamine as the aspergillus oryzae transformation system of selection markers
Construction method, it is characterised in that in step 3), after the shaking flask culture 6h, wherein OD600Value is 0.6~0.8.
9. it is according to claim 2 by agriculture bacillus mediated using pyrithiamine as the aspergillus oryzae transformation system of selection markers
Construction method, it is characterised in that in step 6), the sequence of the GFP specific primers is respectively such as SEQ ID NO:3、SEQ ID
NO:Shown in 4.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110079465A (en) * | 2019-05-14 | 2019-08-02 | 江西农业大学 | It gives a report the aspergillus oryzae transformation system construction method of gene using phleomycin as selection markers/GFP |
| CN110592073A (en) * | 2019-09-25 | 2019-12-20 | 江西科技师范大学 | Method for directionally genetically modifying aspergillus oryzae gene based on CRISPR technology |
| CN113201555A (en) * | 2021-04-01 | 2021-08-03 | 云南师范大学 | Construction method of binary vector containing eGFP marker and hygromycin resistance |
-
2018
- 2018-05-29 CN CN201810526455.5A patent/CN108676810A/en active Pending
Non-Patent Citations (6)
| Title |
|---|
| KHUYEN THI NGUYEN ET AL.: "The construction and use of versatile binary vectors carrying pyrG auxotrophic marker and fluorescent reporter genes for Agrobacterium-mediated transformation of Aspergillus oryzae", 《WORLD J MICROBIOL BIOTECHNOL》 * |
| TAKAFUMI KUBODERA ET AL.: "Pyrithiamine resistance gene (ptrA) of Aspergillus oryzae: Cloning, Characterization and Application as a Dominant Selectable Marker for Transformation", 《BIOSCI. BIOTECHNOL. BIOCHEM.》 * |
| TAKAFUMI KUBODERA ET AL.: "Transformation of Aspergillus sp. and Trichoderma reesei Using the Pyrithiamine Resistance Gene ( ptrA) of Aspergillus oryzae", 《BIOSCI. BIOTECHNOL. BIOCHEM.》 * |
| 崔华等: "以吡啶硫胺素抗性基因为选择标记的红曲菌原生质体转化研究", 《安徽农业科学》 * |
| 崔翠: "米黑根毛霉脂肪酶基因在曲霉中的表达研究", 《中国硕士论文全文数据库基础科学辑》 * |
| 陈发忠等: "米曲霉AS3.800基因工程转化体系的构建", 《轻工科技》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110079465A (en) * | 2019-05-14 | 2019-08-02 | 江西农业大学 | It gives a report the aspergillus oryzae transformation system construction method of gene using phleomycin as selection markers/GFP |
| CN110592073A (en) * | 2019-09-25 | 2019-12-20 | 江西科技师范大学 | Method for directionally genetically modifying aspergillus oryzae gene based on CRISPR technology |
| CN113201555A (en) * | 2021-04-01 | 2021-08-03 | 云南师范大学 | Construction method of binary vector containing eGFP marker and hygromycin resistance |
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