CN105524914A - Screening method of Aspergillus oryzae uracil nutritional deficient strains - Google Patents
Screening method of Aspergillus oryzae uracil nutritional deficient strains Download PDFInfo
- Publication number
- CN105524914A CN105524914A CN201511004106.XA CN201511004106A CN105524914A CN 105524914 A CN105524914 A CN 105524914A CN 201511004106 A CN201511004106 A CN 201511004106A CN 105524914 A CN105524914 A CN 105524914A
- Authority
- CN
- China
- Prior art keywords
- aspergillus oryzae
- substratum
- medium
- feso
- hpo
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N13/00—Treatment of microorganisms or enzymes with electrical or wave energy, e.g. magnetism, sonic waves
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- 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
- C12N1/02—Separating microorganisms from their culture media
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Zoology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Organic Chemistry (AREA)
- Genetics & Genomics (AREA)
- Wood Science & Technology (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Biomedical Technology (AREA)
- Medicinal Chemistry (AREA)
- Tropical Medicine & Parasitology (AREA)
- Virology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention belongs to the field of bioengineering, and discloses a screening method of Aspergillus oryzae uracil nutritional deficient strains. The method concretely comprises the following steps: mutagenizing Aspergillus oryzae spores; coating a medium 1 with mutagenic conidiophores; culturing at 30DEG C for 7-10d, and choosing anti-5-FOA single colonies; transferring the single colonies to the slant of a medium 2; collecting Aspergillus oryzae spores on the slant, and coating a medium 1 dish with the collected Aspergillus oryzae spores; respectively pointing the Aspergillus oryzae single colonies growing on the dish to a medium 3 and a medium 4; and collecting strains incapable of growing on the medium 3 and capable of growing on the medium 4, which are the Aspergillus oryzae uracil nutritional deficient strains. The method allows a large amount of the Aspergillus oryzae uracil nutritional deficient strains to be rapidly obtained, has strong genetic stability, and lays a technologic foundation for an Aspergillus oryzae strain conversion system.
Description
Technical field
The present invention relates to the method for a kind of aspergillus oryzae uracil auxotrophy screening, belong to technical field of bioengineering.
Background technology
Filamentous fungus aspergillus oryzae grows rapidly, is easy to cultivation, and has very high protein secreting ability, correctly can carry out various post translational processing, and similar with higher eucaryote.Aspergillus oryzae is again generally acknowledged food safety production bacterial strain, fermentation and post-processing technology maturation, therefore it except being used to produce except elementary, secondary metabolite and enzyme product in fermentation industry, usually become carry out genetic expression and protein excretion research ideal object in order to by channel genes aspergillus oryzae to be expressed, first need to set up a stable efficient gene conversion system.Set up aspergillus oryzae Transformation Systems mainly from the viewpoint of recipient cell, genetic transformation and selected marker selection etc.The hard cell walls of aspergillus oryzae is the major obstacle of genetic transformation, so generally using protoplastis as recipient cell when transforming, the material preparing protoplastis comprises young tender mycelia, conidium and sporidium.The method being generally used for aspergillus oryzae genetic transformation has PEG/CaCl2 method, electroporated and Gene Knock-out Mice etc., and wherein PEG/CaCl2 method is the most conventional because operation easier is little, protoplastis has Population feature that is large, that easily obtain homozygosity transformant as recipient cell; Though electric shocking method is easy and simple to handle, transformation efficiency is lower; The advantage of particle bombardment be operation rapidly, simple and acceptor material extensively and do not need to prepare protoplastis, but it is expensive, low conversion rate, mosaic are not easily got rid of, and therefore adopts the research of latter two method fewer for aspergillus oryzae.The selected marker that aspergillus oryzae transforms has 3 classes: nutrient defect type mark gene, drug resistant markers's gene and functional label gene, and wherein with the characteristic of its low background growth, more effective compared to other system using Orotidine-5 '-phosphate decarboxylase gene (pyrG) as selective marker and using the defect fungal strain of this gene, as the conversion system of acceptor, oneself is proved to be a kind of highly effective Transformation Systems to auxotroph complementary gene conversion system abroad.
Summary of the invention
The object of the invention is the screening method providing a kind of aspergillus oryzae uracil auxotrophy bacterial strain, can obtain a large amount of aspergillus oryzae auxotrophic strains fast, and genetic stability is strong, and the carrying out for aspergillus oryzae strain conversion system has established technical foundation.
Concrete technical scheme:
A screening method for aspergillus oryzae uracil auxotrophy bacterial strain, comprises the steps:
(1) be 5 × 10 by 5mL total quantity
7it is that the plate of 6cm carries out ultraviolet mutagenesis that the individual fresh aspergillus oryzae spore by 5 μm of syringe filters is placed in diameter; Mutagenic condition is: mutagenesis wavelength 254nm, mutagenesis height 16cm, mutation time 1.5min, magnetic stirring apparatus rotating speed 400r/min;
(2) conidium of mutagenesis is coated on substratum 1;
Described substratum 1 compound method is: glucose 20g, NaNo
32g, K
2hPo
41g, FeSo
40.1g, Kcl0.5g, MgSo
4.7H
2o0.5g, Tritionx-1000.07g, 5-fluororotic acid (5-FOA) 1.5g, uridine 2.4g, agar 20g, be dissolved in 1000mL water, pH6.6, and 115 DEG C, 20min sterilizing is for subsequent use;
(3) under 30 DEG C of conditions, cultivate 7 ~ 10d, pick out single bacterium colony that can resist 5-FOA; By single colony lift to substratum 2 inclined-plane;
Described substratum 2 compound method is: glucose 20g, NaNo
32g, K
2hPo
41g, FeSo
40.1g, Kcl0.5g, MgSo
4.7H
2o0.5g, Tritionx-1000.07g, inositol 2g, 5-FOA1.5g, uridine 2.4g, agar 20g, be dissolved in 1000mL water, pH6.6, and 115 DEG C, 20min sterilizing is for subsequent use;
(4) collect the aspergillus oryzae spore on inclined-plane, be coated on substratum 1 plate;
(5) by aspergillus oryzae list bacterium colony that plate grows respectively to point on substratum 3 and substratum 4; Collect and can not grow on substratum 3, on substratum 4, the bacterial strain of growth is aspergillus oryzae uracil auxotrophy bacterial strain;
The compound method of described substratum 3 is: glucose 20g, NaNo
32g, K
2hPo
41g, FeSo
40.1g, Kcl0.5g, MgSo
4.7H
2o0.5g, agar 20g, be dissolved in 1000mL water, pH6.6, and 115 DEG C, 20min sterilizing is for subsequent use;
The compound method of described substratum 4 is: glucose 20g, NaNo
32g, K
2hPo
41g, FeSo
40.1g, Kcl0.5g, MgSo
4.7H
2o0.5g, uridine 2.4g, agar 20g, be dissolved in 1000mL water, pH6.6, and 115 DEG C, 20min sterilizing is for subsequent use.
Step (4) collects the aspergillus oryzae spore on inclined-plane, after 3 μm of needle tubing strainers, is coated on substratum 1 plate
Beneficial effect of the present invention: adopt the inventive method, a large amount of aspergillus oryzae uracil auxotrophy bacterial strains can be obtained fast, and genetic stability is strong, and the carrying out for aspergillus oryzae strain conversion system has established technical foundation.
Accompanying drawing explanation
The aspergillus oryzae uracil auxotrophy bacterial strain of cultivation after 4 days that Fig. 1 the present invention obtains.
Embodiment
As no specific instructions, various raw material of the present invention all can be obtained by market sale; Or the ordinary method according to this area prepares.Unless otherwise defined or described herein, technical term used herein and scientific words and those skilled in the art the same meaning be familiar with, any method similar or impartial to described content and material all can be applicable in the present invention in addition.
Below in conjunction with specific embodiment, set forth the present invention further.These embodiments are only not used in for illustration of the present invention and limit scope of the present invention.The experimental technique of unreceipted actual conditions in embodiment, measures according to national standard usually below.If there is no corresponding national standard, then according to general national standard, normal condition or carry out according to the condition that manufacturers advises.
Embodiment 1
(1) be 5 × 10 by 5mL total quantity
7it is that the plate of 6cm carries out ultraviolet mutagenesis that the individual fresh aspergillus oryzae spore by 5 μm of syringe filters is placed in diameter, mutagenic condition (mutagenesis wavelength 254nm, mutagenesis height 16cm, mutation time 1.5min, magnetic stirring apparatus rotating speed 400r/min);
(2) conidium of mutagenesis is coated on substratum 1;
Described substratum 1 compound method is: glucose 20g, NaNo
32g, K
2hPo
41g, FeSo
40.1g, Kcl0.5g, MgSo
4.7H
2o0.5g, Tritionx-1000.07g, 5-FOA1.5g, uridine 2.4g, agar 20g, add in 1000mL water, pH6.6,115 DEG C, 20min sterilizing;
(3) under 30 DEG C of conditions, cultivate 7 ~ 10d, pick out single bacterium colony that can resist 5-FOA; By single colony lift to substratum 2 inclined-plane;
Described substratum 2 compound method is: glucose 20g, NaNo
32g, K
2hPo
41g, FeSo
40.1g, Kcl0.5g, MgSo
4.7H
2o0.5g, Tritionx-1000.07g, inositol 2g, 5-FOA1.5g, uridine 2.4g, agar 20g, add in 1000mL water, pH6.6,115 DEG C, 20min sterilizing;
(4) collect the aspergillus oryzae spore on inclined-plane, after 3 μm of needle tubing strainers, be coated on substratum 1 plate;
(5) by aspergillus oryzae list bacterium colony that plate grows respectively to point on substratum 3 and substratum 4; Collect and can not grow on substratum 3, on substratum 4, the bacterial strain of growth is aspergillus oryzae uracil auxotrophy bacterial strain;
The compound method of described substratum 3 is: glucose 20g, NaNo
32g, K
2hPo
41g, FeSo
40.1g, Kcl0.5g, MgSo
4.7H
2o0.5g, agar 20g, add in 1000mL water, pH6.6,115 DEG C, 20min sterilizing;
The compound method of described substratum 4 is: glucose 20g, NaNo
32g, K
2hPo
41g, FeSo
40.1g, Kcl0.5g, MgSo
4.7H
2o0.5g, uridine 2.4g, agar 20g, add in 1000mL water, pH6.6,115 DEG C, 20min sterilizing.
The aspergillus oryzae uracil auxotrophy bacterial strain cultivated 4 days is shown in Fig. 1.
The inventive method can obtain uracil auxotrophy aspergillus oryzae strain fast; Still keep uracil auxotrophy proterties after the uracil auxotrophy aspergillus oryzae that the present invention obtains goes down to posterity through 20 times, there is higher genetic stability.
Claims (2)
1. a screening method for aspergillus oryzae uracil auxotrophy bacterial strain, is characterized in that comprising the steps:
(1) be 5 × 10 by 5mL total quantity
7it is that the plate of 6cm carries out ultraviolet mutagenesis that the individual fresh aspergillus oryzae spore by 5 μm of syringe filters is placed in diameter; Mutagenic condition is: mutagenesis wavelength 254nm, mutagenesis height 16cm, mutation time 1.5min, magnetic stirring apparatus rotating speed 400r/min;
(2) conidium of mutagenesis is coated on substratum 1;
Described substratum 1 compound method is: glucose 20g, NaNo
32g, K
2hPo
41g, FeSo
40.1g, Kcl0.5g, MgSo
4.7H
2o0.5g, Tritionx-1000.07g, 5-fluororotic acid 1.5g, uridine 2.4g, agar 20g, be dissolved in 1000mL water, pH6.6, and 115 DEG C, 20min sterilizing is for subsequent use;
(3) under 30 DEG C of conditions, cultivate 7 ~ 10d, pick out single bacterium colony that can resist 5-FOA; By single colony lift to substratum 2 inclined-plane;
Described substratum 2 compound method is: glucose 20g, NaNo
32g, K
2hPo
41g, FeSo
40.1g, Kcl0.5g, MgSo
4.7H
2o0.5g, Tritionx-1000.07g, inositol 2g, 5-fluororotic acid 1.5g, uridine 2.4g, agar 20g, be dissolved in 1000mL water, pH6.6, and 115 DEG C, 20min sterilizing is for subsequent use;
(4) collect the aspergillus oryzae spore on inclined-plane, be coated on substratum 1 plate;
(5) by aspergillus oryzae list bacterium colony that plate grows respectively to point on substratum 3 and substratum 4; Collect and can not grow on substratum 3, on substratum 4, the bacterial strain of growth is aspergillus oryzae uracil auxotrophy bacterial strain;
The compound method of described substratum 3 is: glucose 20g, NaNo
32g, K
2hPo
41g, FeSo
40.1g, Kcl0.5g, MgSo
4.7H
2o0.5g, agar 20g, be dissolved in 1000mL water, pH6.6, and 115 DEG C, 20min sterilizing is for subsequent use;
The compound method of described substratum 4 is: glucose 20g, NaNo
32g, K
2hPo
41g, FeSo
40.1g, Kcl0.5g, MgSo
4.7H
2o0.5g, uridine 2.4g, agar 20g, be dissolved in 1000mL water, pH6.6, and 115 DEG C, 20min sterilizing is for subsequent use.
2. screening method according to claim 1, is characterized in that step (4) collects the aspergillus oryzae spore on inclined-plane, after 3 μm of needle tubing strainers, is coated on substratum 1 plate.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201511004106.XA CN105524914A (en) | 2015-12-29 | 2015-12-29 | Screening method of Aspergillus oryzae uracil nutritional deficient strains |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201511004106.XA CN105524914A (en) | 2015-12-29 | 2015-12-29 | Screening method of Aspergillus oryzae uracil nutritional deficient strains |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN105524914A true CN105524914A (en) | 2016-04-27 |
Family
ID=55767443
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201511004106.XA Pending CN105524914A (en) | 2015-12-29 | 2015-12-29 | Screening method of Aspergillus oryzae uracil nutritional deficient strains |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN105524914A (en) |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2006345817A (en) * | 2005-06-20 | 2006-12-28 | Gekkeikan Sake Co Ltd | New mutant of aspergillus oryzae and selected marker |
| CN104004760A (en) * | 2014-06-12 | 2014-08-27 | 华东理工大学 | Foreign protein expression device used for secretory expression in Aspergillus oryzae cells and Aspergillus oryzae genetically engineered bacteria |
-
2015
- 2015-12-29 CN CN201511004106.XA patent/CN105524914A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2006345817A (en) * | 2005-06-20 | 2006-12-28 | Gekkeikan Sake Co Ltd | New mutant of aspergillus oryzae and selected marker |
| CN104004760A (en) * | 2014-06-12 | 2014-08-27 | 华东理工大学 | Foreign protein expression device used for secretory expression in Aspergillus oryzae cells and Aspergillus oryzae genetically engineered bacteria |
Non-Patent Citations (3)
| Title |
|---|
| CHRISTOPHE DENFERT等: "Attenuated Virulence of Uridine-Uracil Auxotrophs of Aspergillus fumigatus", 《INFECTION AND IMMUNITY》 * |
| 王连会等: "香菇尿嘧啶营养缺陷型菌株的筛选与分子验证", 《上海农业学报》 * |
| 邢来君等: "米曲霉两株营养缺陷型原生质体的形成和再生的条件实验", 《真菌学报》 * |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN103931450B (en) | A kind of method of paddy field aquaculture rice fragrance of a flower cray | |
| CN102630490B (en) | Artificial cultivation method for cordyceps militaris mycelium for increasing cordycepin content | |
| CN105087455A (en) | Genetic engineering strain for producing phenazine-1-carboxylic acid and application of genetic engineering strain | |
| CN102440147A (en) | Production technology of edible mushroom liquid spawn by adopting reducing process | |
| CN111500489B (en) | Bacillus coagulans and application thereof in tea planting | |
| US10612007B2 (en) | Method for increasing citric acid production by Aspergillus niger fermentation | |
| CN102796673A (en) | Feruloyl esterase production strain and method for producing feruloyl esterase by using same | |
| CN103031265B (en) | Corynebacterium glutamicum mutant strain and application thereof in production of L-leucine by fermentation method | |
| CN107365715A (en) | A kind of preparation method and application of the Candida glycerolgenesis engineering bacteria of the benzyl carbinol of high yield 2 | |
| KR20160026550A (en) | Microorganism extract using the effective microorganisms and functionality microorganism and the production method thereof | |
| CN107119001B (en) | The production method of genetically engineered hydrogenlike silicon ion and preparation method thereof and farnesol | |
| CN102766663B (en) | Preparation method of active polysaccharides from phellinus linteus | |
| CN102031226A (en) | Aspergillus niger strain and application thereof | |
| CN101861794A (en) | Method for producing liquid strain of cordyceps militaris | |
| CN101709297A (en) | Mutagenic screening method of strain mortierella alpina for generating arachidonic acid | |
| CN105524914A (en) | Screening method of Aspergillus oryzae uracil nutritional deficient strains | |
| CN110106098B (en) | Saccharomyces cerevisiae engineering strain for high yield of pyruvic acid and fermentation method thereof | |
| CN104480167A (en) | Method for preparing amino-oligosaccharin by fermentation method | |
| CN105462868A (en) | Method for improving yield and production intensity of pyruvic acid | |
| CN105463038A (en) | Method for producing 4-Acetylantroquinonol B through fermentation | |
| CN106244614B (en) | The structure of Trichoderma harzianum engineered strain with strong parasitic broad-spectrum fungi and its application | |
| CN108546652A (en) | The screening technique of the general bacterium mutant strain PA11ZM1 of one plant of camel thorn and its application | |
| CN108794170A (en) | A kind of Resistant nutrient solution and preparation method thereof for cotton planting seedling stage | |
| CN109536534A (en) | The method for preparing lycopene | |
| CN103184259A (en) | Method and fermentation culturing medium used for producing natamycin |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20160427 |
|
| RJ01 | Rejection of invention patent application after publication |