CN110106095A - Aspergillus niger genetically engineered bacterium with calcium ion channel CchA gene inactivated, and construction method and application thereof - Google Patents
Aspergillus niger genetically engineered bacterium with calcium ion channel CchA gene inactivated, and construction method and application thereof Download PDFInfo
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- 108090000623 proteins and genes Proteins 0.000 title claims abstract description 47
- 108090000312 Calcium Channels Proteins 0.000 title claims abstract description 28
- 102000003922 Calcium Channels Human genes 0.000 title claims abstract description 28
- 241000894006 Bacteria Species 0.000 title claims abstract description 19
- 238000010276 construction Methods 0.000 title claims abstract description 7
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 claims abstract description 94
- 238000000855 fermentation Methods 0.000 claims abstract description 46
- 230000004151 fermentation Effects 0.000 claims abstract description 46
- 238000000034 method Methods 0.000 claims abstract description 23
- 238000003209 gene knockout Methods 0.000 claims abstract description 10
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- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- 241000233866 Fungi Species 0.000 description 2
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- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 1
- 229920000936 Agarose Polymers 0.000 description 1
- 241000228212 Aspergillus Species 0.000 description 1
- 239000002028 Biomass Substances 0.000 description 1
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 description 1
- 108010059892 Cellulase Proteins 0.000 description 1
- 238000007400 DNA extraction Methods 0.000 description 1
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 1
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- 102000004310 Ion Channels Human genes 0.000 description 1
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- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 1
- FEWJPZIEWOKRBE-UHFFFAOYSA-N Tartaric acid Natural products [H+].[H+].[O-]C(=O)C(O)C(O)C([O-])=O FEWJPZIEWOKRBE-UHFFFAOYSA-N 0.000 description 1
- 235000009754 Vitis X bourquina Nutrition 0.000 description 1
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- 239000011575 calcium Substances 0.000 description 1
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- 239000012362 glacial acetic acid Substances 0.000 description 1
- 108010087005 glusulase Proteins 0.000 description 1
- 238000000227 grinding Methods 0.000 description 1
- 239000002054 inoculum Substances 0.000 description 1
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- 229910052757 nitrogen Inorganic materials 0.000 description 1
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- 238000001556 precipitation Methods 0.000 description 1
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- 238000000926 separation method Methods 0.000 description 1
- 229910052708 sodium Inorganic materials 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- GEHJYWRUCIMESM-UHFFFAOYSA-L sodium sulfite Chemical compound [Na+].[Na+].[O-]S([O-])=O GEHJYWRUCIMESM-UHFFFAOYSA-L 0.000 description 1
- HPALAKNZSZLMCH-UHFFFAOYSA-M sodium;chloride;hydrate Chemical compound O.[Na+].[Cl-] HPALAKNZSZLMCH-UHFFFAOYSA-M 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 230000004763 spore germination Effects 0.000 description 1
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- 239000005720 sucrose Substances 0.000 description 1
- 150000008163 sugars Chemical class 0.000 description 1
- QAOWNCQODCNURD-UHFFFAOYSA-N sulfuric acid Substances OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 239000011975 tartaric acid Substances 0.000 description 1
- 235000002906 tartaric acid Nutrition 0.000 description 1
- 235000012976 tarts Nutrition 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 239000004753 textile Substances 0.000 description 1
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Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/37—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi
- C07K14/38—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi from Aspergillus
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- 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
- 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
- C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
- C12N15/90—Stable introduction of foreign DNA into chromosome
- C12N15/902—Stable introduction of foreign DNA into chromosome using homologous recombination
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
- C12P7/44—Polycarboxylic acids
- C12P7/48—Tricarboxylic acids, e.g. citric acid
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- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Genetics & Genomics (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Molecular Biology (AREA)
- General Health & Medical Sciences (AREA)
- Biotechnology (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- Microbiology (AREA)
- Mycology (AREA)
- Biophysics (AREA)
- Biomedical Technology (AREA)
- General Chemical & Material Sciences (AREA)
- Gastroenterology & Hepatology (AREA)
- Medicinal Chemistry (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Physics & Mathematics (AREA)
- Plant Pathology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses an Aspergillus niger genetically engineered bacterium with calcium ion channel CchA gene inactivated, a construction method and application thereof, and belongs to Aspergillus niger with a calcium ion channel CchA gene knocked out. The invention also discloses a construction method of the genetic engineering bacteria. Further discloses the application of the genetic engineering bacteria in the production of citric acid. According to the invention, by constructing the Aspergillus niger genetically engineered bacterium with the gene knock-out of the calcium ion channel CchA gene, the yield of a biological membrane is reduced and the hydrophobicity of the Aspergillus niger is reduced in the process of producing citric acid by immobilized fermentation, the agglomeration phenomenon of a carrier is reduced, the yield of citric acid and the sugar conversion efficiency are improved, and the method is suitable for industrial production.
Description
Technical field
The invention belongs to genetic engineering and microorganisms technical fields, and in particular to a kind of knockout calcium channel CchA gene
Aspergillus niger and its construction method and application.
Background technique
One of the organic acid that citric acid has the call as the whole world is widely used in the row such as food, medicine, daily use chemicals
Industry.Wherein, 75% citric acid is used for food industry, and acid, antioxidant, the pH being mainly used in food additives are adjusted
Agent is commonly used to the system of the food such as beverage, cake, grape wine, dairy products simultaneously because citric acid has mild frank tart flavour
It makes.In addition having 15% is that may be used as buffer, complexing agent, screening agent, metal builder, mordant dyeing for chemical industry and textile industry
Agent etc..There are also 10% citric acids to be used as anticoagulant, antiacid, corrigent, cosmetics and feed addictive etc.,
It is widely used in medical industry and animal husbandry.In recent years, since China's provision price generally goes up, it is following
It is exactly sharp rising for Citric Acid Production cost of material.Therefore how to improve citric acid fermentation method and further reduce the cost
Just at the entire citric acid industry in China concern the most.
Fermentation of Aspergillus niger produces one of the main path that citric acid is current industrial fermentation production of citric acid, is fixed by cell
The fermentation efficiency of aspergillus niger can be substantially improved in the method for change.Currently, there are mainly four types of the methods of cell fixation: investment,
Cross-linking method, covalent coupling method and absorption method.For filamentous fungi, absorption method is easy to operate, not needing of fixation procedure
The change of structure is learned, and is theoretically adapted to all filamentous fungis, promotion and application more conducively industrially.Absorption method
Principle is mainly the interaction force of the group and cell surface that rely on carrier surface, and then cell is promoted to be adsorbed on carrier surface
It is grown.And it can fall off from carrier after apoptosis, while active cell adsorbs up again, form one
Dynamic equilibrium, makes entire microenvironment form an efficient conversion system, and whole fermentation efficiency has obvious compared with free fermentation
Promotion.It being found in research process of the people to immobilization fermentation mechanism, microorganism can generally form biomembrane on carrier,
And it is formed by biomembrane difference and will lead to the performance of immobilization fermentation and very big difference occurs.It would therefore be desirable to life
Object film Forming Mechanism further understands, and could really solve the problems, such as immobilization difficulty in industrial application.
Summary of the invention
To solve low output when existing industrial fermentation of Aspergillus niger produces citric acid, the long problem of fermentation period, the present invention
Technical problems to be solved are to provide the aspergillus niger of one plant of calcium channel CchA gene defection type.
The present invention also technical problems to be solved are to provide the building side of above-mentioned production citric acid waste residue genetic engineering bacterium
Method.
The last technical problems to be solved of the present invention are to provide above-mentioned aspergillus niger and produce in citric acid in fermentation
Application.
In order to solve the above technical problems, the present invention adopts the following technical scheme:
The aspergillus niger of one plant of calcium channel CchA gene inactivation, it is utilized by double crossing over method
Hyg resistant gene replaces the genetic engineering bacterium of CchA Gene Partial sequence, and calcium channel CchA gene participates in mycelia main shaft
Polar growth produces the integrality of spore and cell wall.Cell wall integrity can influence the generation of biomembrane, therefore, calcium ion
Channel C chA gene can regulate and control the generation of biomembrane, to influence the yield of citric acid.
Wherein, the aspergillus niger is Aspergillus Niger 831 (A.niger 831),
For nucleotide sequence as shown in SEQ ID NO.1, the calcium ion is logical before the calcium channel CchA gene inactivates
Nucleotide sequence is as shown in SEQ ID NO.2 after road CchA gene inactivation.
A kind of construction method of the aspergillus niger of calcium channel CchA gene inactivation, includes the following steps:
(1) genomic DNA of aspergillus niger A.niger 831 is extracted;
(2) genome obtained using step (1) is template, with nucleotide shown in SEQ ID NO.3 and SEQ ID NO.4
Sequence is primer, amplifies the upstream homology arm of CchA gene;
The genome obtained using step (1) is template, with nucleotides sequence shown in SEQ ID NO.5 and SEQ ID NO.6
It is classified as primer, amplifies the downstream homology arm of CchA gene;
Using plasmid pan7-1 as template, arranged with nucleotides sequence shown in SEQ ID NO.7 and SEQ ID NO.8 as primer,
Amplify hyg resistance element;
By overlap PCR, with the upstream homology arm of CchA gene, the downstream homology arm of CchA gene, hyg resistance member
Part is template, is arranged with nucleotides sequence shown in SEQ ID NO.9 and SEQ ID NO.10 as primer, PCR amplification obtains clpp gene
Except segment;
(3) aspergillus niger protoplast is prepared;
(4) gene knockout recombinant fragment is imported in protoplast and carries out homologous recombination, obtain calcium channel CchA base
Because of the aspergillus niger of inactivation.
Wherein, the nucleotide sequence of the upper homology arm of CchA gene is as shown in SEQ ID NO.11, and CchA gene is similarly hereinafter
The nucleotide sequence of source arm as shown in SEQ ID NO.12, the nucleotide sequence of hyg resistance element as shown in SEQ ID NO.13,
The nucleotide sequence of gene knockout segment is as shown in SEQ ID NO.14.
The aspergillus niger of above-mentioned calcium channel CchA gene inactivation is preparing applying at this in citric acid
Within the protection scope of invention.
Preferably, using the aspergillus niger of calcium channel CchA gene inactivation as fermentation strain, by solid
Surely change preparation of citric acid by fermentation.
Wherein, the immobilization fermentation is using porous fibrous material as entrapment media, and preparation of citric acid by fermentation is described
Porous fibrous material is activated carbon fibre.
Wherein, the configuration method of the fermentation medium of the immobilization fermentation is as follows:
The corn flour for taking 200g/L~300g/L tapioca starch, 200g/L-300g/L respectively, at 60 DEG C~70 DEG C, 1L hair
The α-amylase of 1~2mL is added in zymotic fluid, digests 35min~45min, then tapioca starch liquid and corn flour liquid are separately heated to up to 85
DEG C, the α-amylase of 1~2mL will be added in every 1L fermentation liquid, 35min~45min is digested, until the constant indigo plant of iodine solution, filters tapioca starch
Liquid obtains tapioca starch liquid supernatant, is added into tapioca starch liquid supernatant and the unfiltered corn flour that volume fraction is 2-10% is added
Liquid is uniformly mixed so as to obtain immobilization fermentation culture medium, and alpha-amylase is contained in the α-amylase, and the enzyme activity of the alpha-amylase is
60000~70000U/mL.
Wherein, the condition of culture of the immobilization fermentation is as follows: cultivation temperature be 30 DEG C~37 DEG C, incubation time be 72~
120h, revolving speed are 180~330rpm.
Wherein, the porous fibrous material is first pre-processed using the following method:
By 1~1.5h of soaking with sodium hydroxide of porous fibrous material 1M, then being rinsed with water to pH is neutrality, in 1M hydrochloric acid
1~1.5h of middle immersion, then being rinsed with water to pH is neutrality, drying to constant weight, obtains modified porous fibrous material.
Citric acid is produced using the aspergillus niger immobilization fermentation of above-mentioned calcium channel CchA gene inactivation
Method, specifically comprise the following steps:
(1) preparation of carrier: by the soaking with sodium hydroxide 1h of porous fibrous material 1M, after being cleaned with pure water, in 1M salt
1h is impregnated in acid, be with pure water rinsing to pH later it is neutral, place into 65 DEG C of baking ovens that drying to constant weight.It is identical to be cut into size
Carrier.
(2) it ferments: the aspergillus niger plate of activation being scraped with spore liquid is scraped, spore liquid is made.With
The inoculum concentration of 0.2% (volume fraction) is inoculated into fermented and cultured sterilized and containing 0.1g/100mL porous fibrous material
In base, fermentation obtains citric acid.
The fermentation medium, including following component: 200-250g/L corn flour, 200-250g/L tapioca starch.
Fermentation condition is as follows: cultivation temperature is 35 DEG C, incubation time 72-96h, and revolving speed is 200~250rpm.
The utility model has the advantages that
The present invention constructs the aspergillus niger gene work of one plant of calcium channel CchA gene delection by gene knockout means
Journey bacterium.The genetic engineering bacterium reduces aspergillus niger biofilm biomass during immobilization fermentation produces citric acid, and phenomenon of uniting mitigates,
The yield and sugar conversion ratio for improving citric acid, shorten fermentation period.
Detailed description of the invention
Fig. 1 is the electrophoretogram of 831 genome of aspergillus niger Aspergillus Niger;
Fig. 2 is PAN7-1 plasmid map;
Fig. 3 is the PCR electrophoretogram of CchA upstream region of gene homology arm, downstream homology arm, wherein M is the DNA of DL5000
Marker, swimming lane 1 are the upper homology arm of CchA, and swimming lane 3 is the lower homology arm of CchA, and swimming lane 5 is hyg resistance expression element;
Fig. 4 is the electrophoretogram of gene knockout segment, and it is gene knockout segment that wherein M, which is marker swimming lane 1,;
Fig. 5 is violet staining figure;
Fig. 6 is violet staining OD value disparity map;
Fig. 7 is the fermentation results of original aspergillus niger and aspergillus niger.
Fig. 8 is the biomembrane electron microscope for bacterium germination Aspergillus niger strain;
Fig. 9 is the biomembrane electron microscope of aspergillus niger;
Specific embodiment
Embodiment 1: the building of aspergillus niger calcium channel cchA clpp gene degerming.
(1) original aspergillus niger genome is extracted
Kit (the takara minibest plant genomic of Plant Genome is extracted using takara company
DNA extraction kit), specific prevention is as follows:
1. taking 1mL to be inoculated in 50mLDP culture medium the aspergillus niger spore liquid of scraping, in 35 DEG C, 250r/min is cultivated
15h;
2. collect mycelium pellet in 5000r/min centrifugation 5min, with brine twice after, the mycelium pellet of collection is used
It liquid nitrogen grinding 3 times, weighs the ground powder of 100mg and is added in the tube pipe of Buffer HS II that 500uL is added in advance
Mix, then be added 10uL RNase A, mixing fullys shake, in 56 DEG C water-bath 10 minutes.
3. being mixed well to 62.5uL Buffer KAC is added in step 2.5min, 12000rpm centrifugation are placed on ice
5min.Supernatant 600uL is taken, the Buffer GB of 600uL is added, mixes well.
4. Spin Column is placed in Collection Tube, solution is moved in Spin Column, 12000rpm from
Heart 1min abandons filtrate.
5. the Buffer WA of 500uL is added in Spin Column, 12000rpm is centrifuged 1min, abandons filtrate.
6. the Buffer WB of 700uL is added in Spin Column, 12000rpm is centrifuged 1min, abandons filtrate.
7. it is primary to repeat step 6.
8. Spin Column is placed on Collection Tube, 12000rpm, it is centrifuged 2min.
9. Spin Column is placed on new 1.5mL centrifuge tube, it is added in the centre of Spin Column film
65 DEG C of aqua sterilisas of 40uL, are stored at room temperature 1min.12000rpm is centrifuged 2min, eluted dna.It is measured by agarose gel electrophoresis
Aspergillus niger genome concentration is as shown in Figure 1.
Fig. 1 shows that the DNA marker that M is DL15000, No. 1 is the aspergillus niger genome extracted.
(2) with homology arm above and below round pcr amplification CchA gene.
1 PCR reaction system of table
Using original aspergillus niger genome as template, with CchA-up-F be upper primer, with CchA-up-R be lower primer (such as
Shown in SEQ ID NO.3 and SEQ ID NO.4) homology arm in amplification;With CchA-down-F for upper primer, with CchA-down-R
Lower homology arm is expanded for lower primer (as shown in SEQ ID NO.5 and SEQ ID NO.6).Reaction system is shown in Table 1, reaction condition:
98 DEG C of denaturation 10s, 55~65 DEG C of annealing 30s, 68 DEG C of extension 2min, above-mentioned steps repeat 30 times.PCR product benefit after the reaction was completed
It is quantitative with agarose gel electrophoresis, see Fig. 3.
(3) hyg resistance expression element is expanded
With PAN7-1 plasmid (PAN7-1 plasmid map is shown in that Fig. 2, nucleotide sequence are shown in SEQ ID NO.17) for template, with
CchA-hyg-F is upper primer, is lower primer (as shown in SEQ ID NO.7 and SEQ ID NO.8) amplification with CchA-hyg-R
Hyg resistance expression's element.Reaction system is shown in Table 1, reaction condition: 98 DEG C of denaturation 10s, 55-65 DEG C of annealing 30s, 68 DEG C extend
3min, above-mentioned steps repeat 30 times.PCR product is quantitative using agarose gel electrophoresis after the reaction was completed, sees Fig. 3.Wherein hyg is anti-
The nucleotide sequence of property Expression element is shown in SEQ ID NO:13.
Fig. 3 indicates that the DNA Marker that M is DL5000, No. 1 upper homology arm for CchA, No. 3 are homologous under CchA
Arm, No. 5 are hyg resistance expression element.
(4) amplification gene knocks out segment
Using CchA upstream and downstream homology arm and hyg resistance expression element as template, with CchA-F for upper primer, it is with CchA-R
Lower primer (as shown in SEQ ID NO.9 and SEQ ID NO.10) expands CchA gene knockout piece using Overlap round pcr
Section.Reaction system is shown in Table 1, reaction condition: 98 DEG C of denaturation 10s, 55-65 DEG C of annealing 30s, 68 DEG C of extension 7min, above-mentioned steps weight
It is 30 times multiple.PCR product is quantitative using agarose gel electrophoresis after the reaction was completed, sees Fig. 4.Wherein knock out the nucleotide sequence of segment
See SEQ ID NO:14.
Fig. 4 indicates that the DNA Marker that M is DL10000, No. 1 is gene knockout segment.
(5) preparation and conversion of aspergillus niger protoplast
1. aspergillus niger is inoculated into PDA plate, spore to be covered with adds 3mL to scrape spore buffer into plate, will with spreading rod
Spore scrapes, and is transferred in the 5mL centrifuge tube of sterilizing.
2. being inoculated with 0.5mL spore liquid to 50mL YPD culture medium, 35 DEG C, 250rpm cultivates 9-13h.
3. after spore germination, being filtered with Miracloth, mycelia is left.Preparing enzymolysis liquid, (Lysing enzyme, collapses
Routed enzyme, each 0.1g/10mL of glusulase, 400 μ L/10mL of cellulase), with asepsis injector filtration sterilization.
4. taking 2g mycelia into enzyme solution, 30 DEG C, 220rpm digests 30min.It is reduced to 150rpm to cultivate 4h revolving speed.
5. being filtered after the completion of enzymatic hydrolysis with filter paper, filtrate is taken, 4 DEG C, 5000rpm is centrifuged 10min.Supernatant is removed, 1mL 1M is added
Sorbierite (ice-water bath) is mixed with rifle pressure-vaccum, adds 15mL sorbierite, is centrifuged, is removed supernatant.Then it repeats primary.Remove
1mL Solution5 is added in supernatant, is mixed with rifle pressure-vaccum.
6. inhaling the protoplast of 100 μ L into 1.5mL sterile centrifugation tube, the clpp gene constructed in 10 μ L step (4) is added
Except segment mixes therewith.The Solution 4 of 50 μ L is added, is mixed, simultaneously timing 15-30min is placed on ice.
After 7.20min, 900uLSolution4 is added, turns upside down several times, is placed on room temperature and timing 15-30min.
After 15-30min, 6000rpm is centrifuged 5min, abandons 900uL supernatant, and it is 1mol/L that remaining thallus, which is coated on sucrose concentration, and tide is mould
Plain concentration is just to set culture in the PDA culture medium of 75mmol/L.Obtain transformant.
8. picking transformant carries out bacterium colony PCR verifying.The specific method is as follows for it: appropriate transformant being taken to be added to the bacterium of 50uL
It falls in PCR buffer (100mM/L Tris-Hcl, 10mM/L EDTA, 1M/L Kcl), 95 DEG C of water-bath 10min take 0.5uL to add
Enter PCR reaction system.PCR primer is hyg-F and hyg-R (as shown in SEQ ID NO.15 and SEQ IDNO.16), agarose electricity
Swimming display amplified band, expression convert successfully.
Embodiment 2: violet staining experiment
Original aspergillus niger (Aspergillus Niger 831) and genetic engineering bacterium are inoculated into PDA plate respectively, wait grow
Full spore, adds 3mL to scrape spore buffer into plate, scrapes spore with spreading rod, be transferred in the 5mL centrifuge tube of sterilizing, uses
Spore buffer constant volume is scraped to 2mL, obtains spore liquid.It is quantified with blood counting chamber and is diluted to 106A/mL continues thereafter with dilution
To 105, 104。
It is previously added the synthetic media of 1ml in 24 orifice plates, then takes 2uL to be inoculated into training the spore liquid of various concentration
It supports in base.Stationary culture 36h at 35 DEG C makes aspergillus niger form a film in orifice plate bottom.Culture medium is outwelled later, is rinsed 2 times with PBS
Afterwards, 0.1% violet staining 15min is added.Crystal violet is outwelled later, and after being rinsed 2 times with PBS, glacial acetic acid earthquake is added
30min is placed in instrument, and crystal violet is made to decolourize.Then observation and microplate reader detection OD570 are carried out.Violet staining figure and OD value
Disparity map is shown in Fig. 5 and Fig. 6.
2 biomembrane violet staining of table tests the OD value under different spore concentrations
Fig. 5 and Fig. 6 is the results show that Δ CchA bacterial strain is after decoloration, and purple color is obviously lighter than original bacteria, 105Concentration
The color of lower Δ CchA bacterial strain has been removed completely, and the OD value detected with microplate reader, data result is consistent with color.Show calcium
After ion channel CchA gene inactivation, biomembrane is to reduce.
Embodiment 3: genetic engineering bacterium immobilization fermentation experiment.
1. the preparation of porous fibrous material entrapment media
The soaking with sodium hydroxide 1h that porous fibrous material (activated carbon fibre) is used to 1M, after being cleaned with pure water, in 1M hydrochloric acid
Middle immersion 1h, be with pure water rinsing to pH later it is neutral, place into 65 DEG C of baking ovens that drying to constant weight.It is cut into the identical load of size
Body.
2. the preparation of fermentation medium
The corn flour of the tapioca starch and 200g/L-300g/L that weigh 200/L-300g/L respectively carries out in 75 DEG C of water-baths
Gelatinization, reaches 65 DEG C to tapioca starch liquid and corn flour liquid, adds the α-amylase of 1-2mL, and liquefy 40min, and water-bath is heated to later
95 DEG C, reach 85 DEG C to tapioca starch liquid and corn flour liquid, add the α-amylase of 1-2mL, liquefy 40min, until the constant indigo plant of iodine solution, mistake
Filter tapioca starch liquid obtains supernatant, and the unfiltered corn flour liquid that 2-10% is added makees feed back, mixes, every 100mL be dispensed into containing
In the 500mL conical flask of appropriate carrier.Sterilizing, it is cooling stand-by.
3. steps are as follows for fermentation process
(1) aspergillus niger frozen and original aspergillus niger spore are inoculated on PDA plate, constant incubator
35 DEG C are cultivated 4~5 days, to expire spore thereon.
(2) spore is scraped to obtain spore suspension with scraping spore buffer, takes and is transferred in right amount equipped with 100mL immobilization
In the 500mL conical flask of culture medium, 72-96h is cultivated in 30-35 DEG C, 250rpm shaking table, every 12h is sampled in fermentation process,
12000rpm is centrifuged 5min, by supernatant and precipitation and separation, measures remaining sugar concentration and lemon acid yield in supernatant.When residual sugar is dense
When degree is lower than 5g/L, fermentation ends.Citric acid wherein is surveyed using NaOH titration, measures total reducing sugar with DNS method.
Wherein NaOH titration are as follows: take sample 1mL (dilution a certain concentration) to be added in 250mL conical flask, be added simultaneously
50mL pure water is titrated with the NaOH of 0.1429M, and the amount of consumed NaOH is exactly the yield of citric acid.
DNS method are as follows:
The preparation of DNS: weighing 3,5- dinitrosalicylic acid 10g, is placed in about 600mL water, is gradually added into sodium hydroxide
10g, in 50 DEG C of water-baths magnetic agitation dissolve, sequentially add tartaric acid first sodium 200g,
Phenol 2g and anhydrous sodium sulfite 5g is cooled to room temperature, after all dissolving and clarifying with pure water constant volume
To 1000mL.It is stored in brown reagent bottle, is used after 7d is placed in dark place.
It marks bent production method: preparing a series of Standard for Sugars solution that concentration are 0.0-1.0g/L, each concentration takes respectively
0.5mL is added in 15mL centrifuge tube, and 0.5mL DNS solution is then added in each centrifuge tube,
Centrifuge tube is placed in boiling water bath after reacting 5min, is placed in cooling in ice water, 8mL is added in each centrifuge tube later
Pure water measures the light absorption value OD540 under wavelength 540nm after mixing, be control with 0.0g/L.The concentration of standard solution is vertical sit
Mark, light absorption value OD540 are that abscissa draws standard curve.
Sample measurement method: it after sample suitably dilutes, takes the 1mL concentrated sulfuric acid to be added in 10mL sample and is reacted in boiling water bath
After 15min, cooling, tune PH to neutrality is placed in ice water, and constant volume is being diluted to debita spissitudo, measurement method later to 100mL
Ibid.It is bent that different sugar corresponds to different marks.
Fig. 7 indicates the difference of Δ CchA bacterial strain and original strain under fermentation conditions, after the 96h that ferments as the result is shown, Δ
CchA strain fermentation produces the yield average out to 162.8g/L of citric acid, and original bacteria fermentation produces the yield average out to of citric acid
153.2g/L, Δ CchA bacterial strain lemon acid yield improve 6.27% compared with original bacteria.Aspergillus niger (Aspergillus
It Niger831 is) that a plant height produces citric acid bacterial strain, yield can increase on this basis, it may be said that be in aspergillus niger immobilization
It is fermented on the basis of producing citric acid and has made significant headway, be suitable for industrial production.
Embodiment 4:SEM observes biomembrane
Carrier after immobilization fermentation 72h is taken out, rinses 3 times mycelia for washing off absorption with PBS.It is put in -80 DEG C of refrigerators
It sets to be placed in freeze dryer overnight and be lyophilized.Carrier is adhesive on a dressing table by conduction again, 20mA 30s carries out metal spraying.Then
It takes in TM3000 and is observed.Biomembrane electron microscope is shown in Fig. 8 and Fig. 9.Fig. 8 shows the immobilization feelings of aspergillus niger original strain
Condition, Fig. 9 indicate the immobilization situation of Δ Cch bacterial strain.It can be found that aspergillus niger original bacteria formd on carrier it is multiple spherical
Object, these spheres connection film forming, while the aperture of carrier being blocked, affect the biography oxygen transfer of internal thallus.And Δ CchA
Bacterial strain, a small amount of biomembrane is formd in the corner of carrier and surface, is not influenced the biography oxygen transfer of internal thallus, is conducive to
Immobilization fermentation produces citric acid.
Sequence table
<110>Nanjing University of Technology
The aspergillus niger and its construction method and application that<120>one plants of calcium channel CchA genes inactivate
<160> 16
<170> SIPOSequenceListing 1.0
<210> 1
<211> 6470
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 1
atggcttcaa gtagccatga ccgcggcccc gacgacgata acttccacat cgatcactca 60
atccccttgc aggatctctc atcgccagac catgaacgtg gaggcccagg tggtgttgcg 120
acaaggctgg gccgatcttt gaccggtagg ggtcggacag ggagaaacta tgagcgcata 180
gcggtggatt cacccgttga aacggccaat gcggcgggca atgcgcgccc gcatcctcac 240
atttcccatg aagaagatga agaagcaatc gaagatccgg aggcgtttgc tcaagcaact 300
tcgttcggtc tcagctttga tccctcgccc agtacctctc ttgccccgac gcatagtcga 360
gcaccctcca gcgtcgacct agataccgtg ccgctcgacg gagccgaaca ctacctcgcc 420
cacatcgaca cctacaatga caccgcacga ttgacggaaa cccaaaacgt ccagcccata 480
agcggagcat ccggctcaga caatgaacat cagaacgatc ggggcggcac aagatcggtt 540
caattccctg ctaccggcca ttcggggtca cgattgggtg atgatctaca caatctggag 600
gatggtttcg tggggagatc ccgcggggga agcaatgcag cggacaggtc gcgatcgctg 660
tccccctcgg cctcgggatc tgccttgttg cgggccagtt ctatgatgaa gtccatgtcc 720
cagcgtattg ttaaccttag taacgaacca gaagtggtag agcaatcaat cctcagggag 780
gaatcgcata agaatgcgcg aatggaagag cccccagtac ttccatcttt gccggactat 840
gcgcatgatg cgccgtctac cacttcattg aacagttcag ctcctagaga gaaactgccc 900
tcgagcaata aggcatggcg cggtattagc aatcctttga gagggaagtc gctgggtatc 960
ctaggcccag ataatgctct gcggatgtgg ttgtgtgata tactcgtcca cccgttcaca 1020
gaacccttta ttctgatcgt gatcgtggtc cagacaattt tgctgaccat tgaatctgca 1080
cgttctgtct ggaaatatcc ccgcgctgtc cgttggggag ccaatcccat ggattatccc 1140
tactttgcca tcttcattat ctacacgctc gagattatcg ccaaaatcct tgtgtcagga 1200
ttgatcatca accctgccga atacagcacc atagaccggt cgatgggctt caggaaggca 1260
gtggtcgaga aagggaagaa cctgatcata ccacagcggc aattctcggt gcgaaagtct 1320
gccatgtcag aacaacccca ggcctccatt atacgaactt ttacgggcgg cttgaatcag 1380
ttggagaatc aattggccga tgaccctctt cagaaaagtc gcgtacgact agctcatcgt 1440
gcattcctgc gacactcatt caatcggctc gatttcgtcg ccttagtatc gtactgggtc 1500
tccttcttcc tctcgatata tggagtcgaa actcgccagc agctctatgt tttcagcatg 1560
ttgagctgtc ttcgaattct tcgtctcttg aatctgacca atggtacttc tgtaagtaaa 1620
ccgccgaata ctcttagcca cgtcgttcac tgacatcaga gacaaggtta tcctccgcag 1680
tctcaagaaa gcagcaccct tgcttgcgca cgtagcgttc cttatcggct tcttctggct 1740
tctgtttgct atcgtcggca tccaaagctt caagtcaagc cttcggagaa cctgtgtctg 1800
gcttggcgtt gatggtgaaa gcgattacgc acagaatgat ccaaatggct ctttacagtt 1860
ttgtggaggc tacctcaatg cgaccactaa acaacaaatg ccatggattc agaaggataa 1920
tactccgagt tcgtcttctc cgaaaggcta catctgtcct gcaggctcta tttgtcttga 1980
aggagacaat ccctacaatg ggacgttaag ttttgacaac atcgtgaatt cgctggagct 2040
tgtgtttgta attatgagct cgaacacatt cactgaccta ctctactata ctgccgacag 2100
tgactatctc gcttcttctc ttttcttcat atgcgggctt ctcatattga gtttgtggat 2160
ggtcaatttg ttggttgcag tgatcaccca tgcttttcag gtcattcgag aggaaagtca 2220
gcgcagtgcc tttgccgtta agaagataga cacgactgaa agagaggatt tggcctctcg 2280
gaaggtcagt gcgatcaagc gtctttatga caagaccgaa tggctctggg tttgcatcat 2340
cattttcgat cttgtggtcc aggctctgag gagcgcttcc atgagcgacg atcgggccca 2400
gtttattgac accacagaac ttgttatgac tttcgtattc cttttcgaaa ttatcctacg 2460
atttgcctcg gattggcgca ccttccacaa aaaaacgcgg aattgggtcg atctgggcct 2520
cgtcataata acgtgcatca ttcagatccc ggcgatcaag cgtgaacgag catacgatgt 2580
ccttacgctc tttcaaattc ttcgagtata tcgcgtcgtg ctcgcattta aggtgaccag 2640
ggacctcatt atggttgtct ttcgtaatgc agttggtctg ctgaacctga tcttctttgt 2700
ctttctgatt accttccttg cttccatttt cgcaactcag ctcttccgtg gccagattcc 2760
agaggaagac gcagacggcg ataccataat catcaccttt tccgacatct acaactcttt 2820
tctcgggatg tatcaaatct tgtcaagcga aaattggacg gacatcttgt ataacgctac 2880
cacgtacaca gtgtcataca atacagcttg gatctctgcg gctttcttga tattatggtt 2940
tatcctggcc aacttcattg tcctgaacat gttcattgct gtcatccagg aaagctttga 3000
tgtctcggag gatgaaaaac ggcttcagca ggtcaaagcg ttcttgcagc agaaacaagt 3060
tagcatggcc tcgcagggga acttgtctct ttccaagatt tttaagctgg gcaaggactc 3120
gaaccggtat aaggaccctc ttgaccacgg cccagcggca ctggagatgt tactcaagga 3180
tgctgtcgtc caggagttcc ttgatgagga tgaaccaccc gagcatcggc caggagacaa 3240
cgtcccatta gaacagtcgg ccacggccga gacagctcag ccaggattct tctcgcggat 3300
ctggacgaag ttcaccacgt caatcatgcg tcgagagccg aaccctttct actccaagct 3360
ggatatcccg cgtacatttg atgagctaga cccaaggacg atggcgaagg aattcgtttc 3420
agcagctgag cagaggaaaa gggcccaacg agagtatctc atgcgacacc caaattacaa 3480
caagtcgctg tttatctttg cgccaaacca ccccgttcgg aagctatgcc agcgtatcgt 3540
ggggccgggg cgtggagttc aacgagtgga gggtgtagat ccctacaagc ctgtctggta 3600
tgcattttcc gcgttcattt acgcggccat cgtcgcaatg gtgttgctgg cctgcataac 3660
cacaccaatc taccagaaga atcactttac gacgaacagg gattggttta cctacactga 3720
catgggcttc gcggtcttgt tcaccataga agctatcatc aaagttatag cggatgggtt 3780
tttctggacg cctaatgcgt actttcgcgg ttcctggggc tttcttgatg gtgtggtttt 3840
gatcacgctc tggatcagcg tcggtggatc cctgttcgaa gattggggcg tcacccgagc 3900
gattggagct ttcaaggctc ttagggcttt gagactcttg aacgtcagtg atagcgctaa 3960
gaatactttc cattcagtga tcattgttgg aggatggaag gtcattgctg taagtcccgc 4020
cccctgttga gccccctaga ggcagaatct gatgcgcttg gcaggccgcg gctgtttcga 4080
tgagcttttt gatccccttc gctatctatg gagtaaatct attcgctgga cgaatggttt 4140
catgcaatga tggcgacatt tcaggaagcc tggatcagtg cattggtgag tacaagaaca 4200
cgcctttcaa ttgggatgtt ctttctccgc gagtggccga caattcttat tacgactttg 4260
acaactttgg cgattccctc ttcattctgt tccagattgt ctctcaagaa ggctggatcg 4320
acgtgcagga cagtgctatg agtattactg gtgtggatat gcagccgcag gactacgttg 4380
cgccggagaa tgggctcttc tttatcattt tcaacctgct tggtgccgtc ttcgtcctga 4440
cgctgttcgt atctgtgttc atgcggaact acacagaaga gactggtgta gcgtatctta 4500
ctgctgaaca gcgatcgtgg ctggaattga gaaagttgct ccggcaaatc tccccttcaa 4560
agcgctcctt tgacaataag agccgacaat ggaagatgtg gagttaccga gtcgccgtta 4620
agaaacatgg cccatgggcg agatgcgtga cattcatcct cacactccat ctgttgttgc 4680
tggtcttgga atactatccc gagccggatg tatgggatca gacccgaggt gagtagttct 4740
tcctatggcc ttgcaaacga gcttgttccc gctaatttag tacagagata atattctttg 4800
ccttcaactt tgtctacatt gctaatgttc tgatccgaat gcttggcttg ggttggcacc 4860
ggtttagccg gagttcatgg gatgtgtatt cgttgctctc tgtctccggg acgttcataa 4920
cgacgatttt gagattcgtc tcgtctagcc aggtgatcaa cgagctgaac aagctcttcc 4980
tggtttcgat cactcttctt attatacccc ggaacaacca gcttgaccag ctattcaaaa 5040
ccgccgcagc tagtctgacg tcgatcggaa acctcatcgc cacctggttt gtcctcttcc 5100
tagtgttcgc gatcgccatg aaccaggcct ttggtctcac gaagtttggt tcacaagaga 5160
ccgacaacct caacttccgc gatgttccca aggcgctggt gctgctcttc cggatgagtt 5220
gcggagaagg ctggaacgag atcatggaag attatgccac gatgagtcct ccgatgtgca 5280
cctacgatgg caactttttc ttagatgact gtggcagtgc gccatgggct cggacgctgt 5340
tcattgcttg gaatattatc agcatgtatc tcttcgtctc actgttcacg tccttgatct 5400
tcgagagctt ctcctacgtg tatcagaaga gcagtgggct ctatgcgatc agccgcgagg 5460
atatccgccg tttcaagcat gcgtgggcta catatgaccc ggacggaacc gggtatatca 5520
ccaaagaaca gttcccgcga ctgctgggag agctctcggg ggtgttttcg atgcggatct 5580
acgatggtga gttcactatc ggccaaatca tggaagaatg ccgggtggac aagcgcgact 5640
cgctgcttgc ccatcgcaga gtggtcgacg ggctagactt ggacaagatg gcccgaatcc 5700
ttcgacagat cccaacggac gtggtgcgca accgccggca acggctgaat gcgttctatg 5760
aggaggtgct tgtgtcggcg gacccggtgc gcggcatctc gttccactcc tgtctcatga 5820
tcctggccca ttacaacgtg atcagcgaca gcaagagtct gcgactggaa gagttcctgc 5880
gcagacgggc gcgactgcag cgcgtcgagg aaaccatccg tcggcagacg gtgatcggct 5940
tctttgacac actgtactgg tctcgcgagt tccggcgccg ggttgagcat aagaaatcgg 6000
cccggatgag tggtgttcct cagttctcgg tgccggagat ctttattgat gacggatctc 6060
acgatgagcc ggcggcggtc gaggggccac gagaacaagc acgcgacgcg cttaccggag 6120
aagaatcgtc gcagcagccg atgctgtccc cgacgtcccc gaccgggcga gccacccggt 6180
accagctgcc acccatcgat accagtccgc tgggtcggat ctctgtcctg aactccccgt 6240
cgacggaatg gtccagcatc agcccatcgc tgtcgcctct gcgggagcga gccggcacga 6300
cgtcgtcgta cggcagcgga ggtgatgtgc atgatgagca ggcaagccca gcgcattcgc 6360
ggcagaatag cgcgatgaac gtcaacgatg tgatgcagtc gctgggcgag tcggtatggg 6420
gagagagtat ccgacgcagc tttacacagc gacggcgatc gggggagtga 6470
<210> 2
<211> 5310
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 2
ctacacgctc gagattatcg ccaaaatcct tgtgtcagga ttgatcatca accctgccga 60
atacagcacc atagaccggt cgatgggctt caggaaggca gtggtcgaga aagggaagaa 120
cctgatcata ccacagcggc aattctcggt gcgaaagtct gccatgtcag aacaacccca 180
ggcctccatt atacgaactt ttacgggcgg cttgaatcag ttggagaatc aattggccga 240
tgaccctctt cagaaaagtc gcgtacgact agctcatcgt gcattcctgc gacactcatt 300
caatcggctc gatttcgtcg ccttagtatc gtactgggtc tccttcttcc tctcgatata 360
tggagtcgaa actcgccagc agctctatgt tttcagcatg ttgagctgtc ttcgaattct 420
tcgtctcttg aatctgacca atggtacttc tgtaagtaaa ccgccgaata ctcttagcca 480
cgtcgttcac tgacatcaga gacaaggtta tcctccgcag tctcaagaaa gcagcaccct 540
tgcttgcgca cgtagcgttc cttatcggct tcttctggct tctgtttgct atcgtcggca 600
tccaaagctt caagtcaagc cttcggagaa cctgtgtctg gcttggcgtt gatggtgaaa 660
gcgattacgc acagaatgat ccaaatggct ctttacagtt ttgtggaggc tacctcaatg 720
cgaccactaa acaacaaatg ccatggattc agaaggataa tactccgagt tcgtcttctc 780
cgaaaggcta catctgtcct gcaggctcta tttgtcttga aggagacaat ccctacaatg 840
ggacgttaag ttttgacaac atcgtgaatt cgctggagct tgtgtttgta attatgagct 900
cgaacacatt cactgaccta ctctactata ctgccgacag tgactatctc gcttcttctc 960
ttttcttcat atgcgggctt ctcatattga gtttgtggat ggtcaatttg ttggttgcag 1020
tgatcaccca tgcttttcag gtcattcgag aggaaagtca gcgcagtgcc tttgccgtta 1080
agaagataga cacgactgaa agagaggatt tggcctctcg gaaggtcagt gcgatcaagc 1140
gtctttatga caagaccgaa tggctctggg tttgcatcat cattttcgat cttgtggtcc 1200
aggctctgag gagcgcttcc atgagcgacg atcgggccca gtttattgac accacagaac 1260
ttgttatgac tttcgtattc cttttcgaaa ttatcctacg atttgcctcg gattggcgca 1320
ccttccacaa aaaaacgcgg aattgggtcg atctgggcct cgtcataata acgtgcatca 1380
ttcagatccc ggcgatcaag cgtgaacgag catacgatgt ccttacgctc tttcaaattc 1440
ttcgagtata tcgcgtcgtg ctcgcattta aggtgaccag ggacctcatt atggttgtct 1500
ttcgtaatgc agttggtctg ctgaacctga tcttctttgt ctttctgatt accttccttg 1560
cttccatttt cgcaactcag ctcttccgtg gccagattcc agaggaagac gcagacggcg 1620
ataccataat catcaccttt tccgacatct acaactcttt tctcgggatg tatcaaatct 1680
tgtcaagcga aaattggacg gacatcttgt ataacgctac cacgtacaca gtgtcataca 1740
atacagcttg gatctctgcg gctttcttga tattatggtt tatcctggcc aacttcattg 1800
tcctgaacat gttcattgct gtcatccagg aaagctttga tgtctcggag gatgaaaaac 1860
ggcttcagca ggtcaaagcg ttcttgcagc agaaacaagt tagcatggcc tcgcagggga 1920
acttgtctct ttccaagatt tttaagctgg gcaaggactc gaaccggtat aaggaccctc 1980
ttgaccacgg cccagcggca ctggagatgt tactcaagga tgctgtcgtc caggagttcc 2040
ttgatgagga tgaaccaccc gagcatcggc caggagacaa cgtcccatta gaacagtcgg 2100
ccacggccga gacagctcag ccaggattct tctcgcggat ctggacgaag ttcaccacgt 2160
caatcatgcg tcgagagccg aaccctttct actccaagct ggatatcccg cgtacatttg 2220
atgagctaga cccaaggacg atggcgaagg aattcgtttc agcagctgag cagaggaaaa 2280
gggcccaacg agagtatctc atgcgacacc caaattacaa caagtcgctg tttatctttg 2340
cgccaaacca ccccgttcgg aagctatgcc agcgtatcgt ggggccgggg cgtggagttc 2400
aacgagtgga gggtgtagat ccctacaagc ctgtctggta tgcattttcc gcgttcattt 2460
acgcggccat cgtcgcaatg gtgttgctgg cctgcataac cacaccaatc taccagaaga 2520
atcactttac gacgaacagg gattggttta cctacactga catgggcttc gcggtcttgt 2580
tcaccataga agctatcatc aaagttatag cggatgggtt tttctggacg cctaatgcgt 2640
actttcgcgg ttcctggggc tttcttgatg gtgtggtttt gatcacgctc tggatcagcg 2700
tcggtggatc cctgttcgaa gattggggcg tcacccgagc gattggagct ttcaaggctc 2760
ttagggcttt gagactcttg aacgtcagtg atagcgctaa gaatactttc cattcagtga 2820
tcattgttgg aggatggaag gtcattgctg taagtcccgc cccctgttga gccccctaga 2880
ggcagaatct gatgcgcttg gcaggccgcg gctgtttcga tgagcttttt gatccccttc 2940
gctatctatg gagtaaatct attcgctgga cgaatggttt catgcaatga tggcgacatt 3000
tcaggaagcc tggatcagtg cattggtgag tacaagaaca cgcctttcaa ttgggatgtt 3060
ctttctccgc gagtggccga caattcttat tacgactttg acaactttgg cgattccctc 3120
ttcattctgt tccagattgt ctctcaagaa ggctggatcg acgtgcagga cagtgctatg 3180
agtattactg gtgtggatat gcagccgcag gactacgttg cgccggagaa tgggctcttc 3240
tttatcattt tcaacctgct tggtgccgtc ttcgtcctga cgctgttcgt atctgtgttc 3300
atgcggaact acacagaaga gactggtgta gcgtatctta ctgctgaaca gcgatcgtgg 3360
ctggaattga gaaagttgct ccggcaaatc tccccttcaa agcgctcctt tgacaataag 3420
agccgacaat ggaagatgtg gagttaccga gtcgccgtta agaaacatgg cccatgggcg 3480
agatgcgtga cattcatcct cacactccat ctgttgttgc tggtcttgga atactatccc 3540
gagccggatg tatgggatca gacccgaggt gagtagttct tcctatggcc ttgcaaacga 3600
gcttgttccc gctaatttag tacagagata atattctttg ccttcaactt tgtctacatt 3660
gctaatgttc tgatccgaat gcttggcttg ggttggcacc ggtttagccg gagttcatgg 3720
gatgtgtatt cgttgctctc tgtctccggg acgttcataa cgacgatttt gagattcgtc 3780
tcgtctagcc aggtgatcaa cgagctgaac aagctcttcc tggtttcgat cactcttctt 3840
attatacccc ggaacaacca gcttgaccag ctattcaaaa ccgccgcagc tagtctgacg 3900
tcgatcggaa acctcatcgc cacctggttt gtcctcttcc tagtgttcgc gatcgccatg 3960
aaccaggcct ttggtctcac gaagtttggt tcacaagaga ccgacaacct caacttccgc 4020
gatgttccca aggcgctggt gctgctcttc cggatgagtt gcggagaagg ctggaacgag 4080
atcatggaag attatgccac gatgagtcct ccgatgtgca cctacgatgg caactttttc 4140
ttagatgact gtggcagtgc gccatgggct cggacgctgt tcattgcttg gaatattatc 4200
agcatgtatc tcttcgtctc actgttcacg tccttgatct tcgagagctt ctcctacgtg 4260
tatcagaaga gcagtgggct ctatgcgatc agccgcgagg atatccgccg tttcaagcat 4320
gcgtgggcta catatgaccc ggacggaacc gggtatatca ccaaagaaca gttcccgcga 4380
ctgctgggag agctctcggg ggtgttttcg atgcggatct acgatggtga gttcactatc 4440
ggccaaatca tggaagaatg ccgggtggac aagcgcgact cgctgcttgc ccatcgcaga 4500
gtggtcgacg ggctagactt ggacaagatg gcccgaatcc ttcgacagat cccaacggac 4560
gtggtgcgca accgccggca acggctgaat gcgttctatg aggaggtgct tgtgtcggcg 4620
gacccggtgc gcggcatctc gttccactcc tgtctcatga tcctggccca ttacaacgtg 4680
atcagcgaca gcaagagtct gcgactggaa gagttcctgc gcagacgggc gcgactgcag 4740
cgcgtcgagg aaaccatccg tcggcagacg gtgatcggct tctttgacac actgtactgg 4800
tctcgcgagt tccggcgccg ggttgagcat aagaaatcgg cccggatgag tggtgttcct 4860
cagttctcgg tgccggagat ctttattgat gacggatctc acgatgagcc ggcggcggtc 4920
gaggggccac gagaacaagc acgcgacgcg cttaccggag aagaatcgtc gcagcagccg 4980
atgctgtccc cgacgtcccc gaccgggcga gccacccggt accagctgcc acccatcgat 5040
accagtccgc tgggtcggat ctctgtcctg aactccccgt cgacggaatg gtccagcatc 5100
agcccatcgc tgtcgcctct gcgggagcga gccggcacga cgtcgtcgta cggcagcgga 5160
ggtgatgtgc atgatgagca ggcaagccca gcgcattcgc ggcagaatag cgcgatgaac 5220
gtcaacgatg tgatgcagtc gctgggcgag tcggtatggg gagagagtat ccgacgcagc 5280
tttacacagc gacggcgatc gggggagtga 5310
<210> 3
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 3
cggcgatgac gacgagacta 20
<210> 4
<211> 40
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 4
ggtggaggcg gcggatttta agtcgtcaga gtgctaaccg 40
<210> 5
<211> 44
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 5
cccactccac atctccactc gactacacgc tcgagattat cgcc 44
<210> 6
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 6
gcgaggccat gctaacttgt 20
<210> 7
<211> 40
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 7
cggttagcac tctgacgact taaaatccgc cgcctccacc 40
<210> 8
<211> 44
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 8
ggcgataatc tcgagcgtgt agtcgagtgg agatgtggag tggg 44
<210> 9
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 9
cgaaccacaa ccacggagga 20
<210> 10
<211> 24
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 10
ttcatcctcc gagacatcaa agct 24
<210> 11
<211> 1956
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 11
cggcgatgac gacgagacta tggaagatgt tgaaggcgtg gaccatgacc cgaaccacaa 60
ccacggagga aacagcggag gagcaggccc ggatgcgact ggcggtgatg ttcgggcgga 120
aggccaagga tcgagggagg gtggtgccga tggtggaaga gcgtaggtac atatctcgac 180
agttttattt gtctctagtc ttgaacttta ctagccggga tattcaatat gctcggatat 240
tttggtcacg attataggcg ttcctttcca tgaattcaag ctaaccagca tcatttggct 300
cacagggccg ccgctgactc ggatccttca tctgtcgtgc agagcttcct gcagtcgttg 360
catgggggct cacggcaatc ccaagacccc gagagacctt tcactactct gcaagatcta 420
cttacaacct cgactacatt gcctttcctg gaatctgcgg acgaaaaagc ggttgacaat 480
ctcctgagct tcctgccacc atcgctgctt ctcctggcac aggacaatga tctcgacgaa 540
gccgcggcag ctgatcagga cccggacatt gctcaagccg ccctgggatc cctcgaactg 600
acgcaaaaga aagagattct gcgcaaagtt ctacactcac ctcaatttgc gcagagtcta 660
gcaagcttga cgatggccat tagggatggt ggattaccga gcatcagcga ggccctaaaa 720
attccggttg caaatggagg attcatgcgt agagggggag ttccattggg tggtggtgat 780
gctgttgaag cattccttca aggcgttcgg gatcatgtta aggattcggc tcaaggaaac 840
cgtatggaca ccgattgatt ggttccaaat agcaatgatt gtgttatgtt gacagggtcc 900
tttttttaac tttcttatct cttttttact attattatct tcagccagcc attctgtaac 960
tatccactga tgttttagaa atccattagc ggccttgcat ggatgtctca ttccctcctt 1020
agcagagaga gacttgtcat gggttatttt tggcatgagg tgggtggggt aaaaaggcca 1080
gtggccagta ctaggcagca tgataatact ggtaatctta ataccgttaa taataatgtg 1140
tcttggttgt ggcgtatggc ggactccgcc cgttaatctc aaacagaatc aagaattaaa 1200
aaagaaaaaa gtaggggaaa aaagtttccg actcgatgat ctcttcctcc accatggaaa 1260
aaaagaggtg ggacaggtat caaaggcatt gccgtatcgg gagtagacat aagtgatcct 1320
gtcagggacc aaagcaagtc aaatcagtcc gtactaagat acatcatggg gagggggcac 1380
gcatattccc ctcccgggcg cagcaggggg aaaatcccag cttcagaccc cgttccatcc 1440
cagagccatc catatccacc ctcaattatt ggccgtgtgt cgaacgaact cgacccggac 1500
cattcataag gctcggacgt ttagcaatca ctgggccaga gagcctggcc aatgggccat 1560
acagaagccg ccgctgccga atccgcccca gttttggccg ctttggcgcc gtccagccaa 1620
agctccgctg cgcataccga gcaataataa tcatcactgt gatctggaag tcgacacgtt 1680
tgggttcctt tccccatcat gcccgctgct tcttgacggc tgcctggctt gattggatcc 1740
ttgcccacgt ttccgcacgg ggtgccaccg ctaacttaac cagagttcgt cagccctttc 1800
gtactacttt aacttactta gggagtctgt cccatatttt ccccagcgcg cttttacttt 1860
gcctcggtcg tcaacgctcc ccagggccct tctgatcatc tctttttctt ctggaacagc 1920
ctgcgatgtc tgccttcggt tagcactctg acgact 1956
<210> 12
<211> 1914
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 12
ctacacgctc gagattatcg ccaaaatcct tgtgtcagga ttgatcatca accctgccga 60
atacagcacc atagaccggt cgatgggctt caggaaggca gtggtcgaga aagggaagaa 120
cctgatcata ccacagcggc aattctcggt gcgaaagtct gccatgtcag aacaacccca 180
ggcctccatt atacgaactt ttacgggcgg cttgaatcag ttggagaatc aattggccga 240
tgaccctctt cagaaaagtc gcgtacgact agctcatcgt gcattcctgc gacactcatt 300
caatcggctc gatttcgtcg ccttagtatc gtactgggtc tccttcttcc tctcgatata 360
tggagtcgaa actcgccagc agctctatgt tttcagcatg ttgagctgtc ttcgaattct 420
tcgtctcttg aatctgacca atggtacttc tgtaagtaaa ccgccgaata ctcttagcca 480
cgtcgttcac tgacatcaga gacaaggtta tcctccgcag tctcaagaaa gcagcaccct 540
tgcttgcgca cgtagcgttc cttatcggct tcttctggct tctgtttgct atcgtcggca 600
tccaaagctt caagtcaagc cttcggagaa cctgtgtctg gcttggcgtt gatggtgaaa 660
gcgattacgc acagaatgat ccaaatggct ctttacagtt ttgtggaggc tacctcaatg 720
cgaccactaa acaacaaatg ccatggattc agaaggataa tactccgagt tcgtcttctc 780
cgaaaggcta catctgtcct gcaggctcta tttgtcttga aggagacaat ccctacaatg 840
ggacgttaag ttttgacaac atcgtgaatt cgctggagct tgtgtttgta attatgagct 900
cgaacacatt cactgaccta ctctactata ctgccgacag tgactatctc gcttcttctc 960
ttttcttcat atgcgggctt ctcatattga gtttgtggat ggtcaatttg ttggttgcag 1020
tgatcaccca tgcttttcag gtcattcgag aggaaagtca gcgcagtgcc tttgccgtta 1080
agaagataga cacgactgaa agagaggatt tggcctctcg gaaggtcagt gcgatcaagc 1140
gtctttatga caagaccgaa tggctctggg tttgcatcat cattttcgat cttgtggtcc 1200
aggctctgag gagcgcttcc atgagcgacg atcgggccca gtttattgac accacagaac 1260
ttgttatgac tttcgtattc cttttcgaaa ttatcctacg atttgcctcg gattggcgca 1320
ccttccacaa aaaaacgcgg aattgggtcg atctgggcct cgtcataata acgtgcatca 1380
ttcagatccc ggcgatcaag cgtgaacgag catacgatgt ccttacgctc tttcaaattc 1440
ttcgagtata tcgcgtcgtg ctcgcattta aggtgaccag ggacctcatt atggttgtct 1500
ttcgtaatgc agttggtctg ctgaacctga tcttctttgt ctttctgatt accttccttg 1560
cttccatttt cgcaactcag ctcttccgtg gccagattcc agaggaagac gcagacggcg 1620
ataccataat catcaccttt tccgacatct acaactcttt tctcgggatg tatcaaatct 1680
tgtcaagcga aaattggacg gacatcttgt ataacgctac cacgtacaca gtgtcataca 1740
atacagcttg gatctctgcg gctttcttga tattatggtt tatcctggcc aacttcattg 1800
tcctgaacat gttcattgct gtcatccagg aaagctttga tgtctcggag gatgaaaaac 1860
ggcttcagca ggtcaaagcg ttcttgcagc agaaacaagt tagcatggcc tcgc 1914
<210> 13
<211> 2740
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 13
taaaatccgc cgcctccacc atttgtagaa aaatgtgacg aactcgtgag ctctgtacag 60
tgaccggtga ctctttctgg catgcggaga gacggacgga cgcagagaga agggctgagt 120
aataagccac tggccagaca gctctggcgg ctctgaggtg cagtggatga ttattaatcc 180
gggaccggcc gcccctccgc cccgaagtgg aaaggctggt gtgcccctcg ttgaccaaga 240
atctattgca tcatcggaga atatggagct tcatcgaatc accggcagta agcgaaggag 300
aatgtgaagc caggggtgta tagccgtcgg cgaaatagca tgccattaac ctaggtacag 360
aagtccaatt gcttccgatc tggtaaaaga ttcacgagat agtaccttct ccgaagtagg 420
tagagcgagt acccggcgcg taagctccct aattggccca tccggcatct gtagggcgtc 480
caaatatcgt gcctctcctg ctttgcccgg tgtatgaaac cggaaaggcc gctcaggagc 540
tggccagcgg cgcagaccgg gaacacaagc tggcagtcga cccatccggt gctctgcact 600
cgacctgctg aggtccctca gtccctggta ggcagctttg ccccgtctgt ccgcccggtg 660
tgtcggcggg gttgacaagg tcgttgcgtc agtccaacat ttgttgccat attttcctgc 720
tctccccacc agctgctctt ttcttttctc tttcttttcc catcttcagt atattcatct 780
tcccatccaa gaacctttat ttcccctaag taagtacttt gctacatcca tactccatcc 840
ttcccatccc ttattccttt gaacctttca gttcgagctt tcccacttca tcgcagcttg 900
actaacagct accccgcttg agcagacatc accatgcctg aactcaccgc gacgtctgtc 960
gagaagtttc tgatcgaaaa gttcgacagc gtctccgacc tgatgcagct ctcggagggc 1020
gaagaatctc gtgctttcag cttcgatgta ggagggcgtg gatatgtcct gcgggtaaat 1080
agctgcgccg atggtttcta caaagatcgt tatgtttatc ggcactttgc atcggccgcg 1140
ctcccgattc cggaagtgct tgacattggg gaattcagcg agagcctgac ctattgcatc 1200
tcccgccgtg cacagggtgt cacgttgcaa gacctgcctg aaaccgaact gcccgctgtt 1260
ctgcagccgg tcgcggaggc catggatgcg atcgctgcgg ccgatcttag ccagacgagc 1320
gggttcggcc cattcggacc gcaaggaatc ggtcaataca ctacatggcg tgatttcata 1380
tgcgcgattg ctgatcccca tgtgtatcac tggcaaactg tgatggacga caccgtcagt 1440
gcgtccgtcg cgcaggctct cgatgagctg atgctttggg ccgaggactg ccccgaagtc 1500
cggcacctcg tgcacgcgga tttcggctcc aacaatgtcc tgacggacaa tggccgcata 1560
acagcggtca ttgactggag cgaggcgatg ttcggggatt cccaatacga ggtcgccaac 1620
atcttcttct ggaggccgtg gttggcttgt atggagcagc agacgcgcta cttcgagcgg 1680
aggcatccgg agcttgcagg atcgccgcgg ctccgggcgt atatgctccg cattggtctt 1740
gaccaactct atcagagctt ggttgacggc aatttcgatg atgcagcttg ggcgcagggt 1800
cgatgcgacg caatcgtccg atccggagcc gggactgtcg ggcgtacaca aatcgcccgc 1860
agaagcgcgg ccgtctggac cgatggctgt gtagaagtac tcgccgatag tggaaaccga 1920
cgccccagca ctcgtccgag ggcaaaggaa tagagtagat gccgaccgcg ggatccactt 1980
aacgttactg aaatcatcaa acagcttgac gaatctggat ataagatcgt tggtgtcgat 2040
gtcagctccg gagttgagac aaatggtgtt caggatctcg ataagatacg ttcatttgtc 2100
caagcagcaa agagtgcctt ctagtgattt aatagctcca tgtcaacaag aataaaacgc 2160
gttttcgggt ttacctcttc cagatacagc tcatctgcaa tgcattaatg cattgactgc 2220
aacctagtaa cgccttncag gctccggcga agagaagaat agcttagcag agctattttc 2280
attttcggga gacgagatca agcagatcaa cggtcgtcaa gagacctacg agactgagga 2340
atccgctctt ggctccacgc gactatatat ttgtctctaa ttgtactttg acatgctcct 2400
cttctttact ctgatagctt gactatgaaa attccgtcac cagcncctgg gttcgcaaag 2460
ataattgcat gtttcttcct tgaactctca agcctacagg acacacattc atcgtaggta 2520
taaacctcga aatcanttcc tactaagatg gtatacaata gtaaccatgc atggttgcct 2580
agtgaatgct ccgtaacacc caatacgccg gccgaaactt ttttacaact ctcctatgag 2640
tcgtttaccc agaatgcaca ggtacacttg tttagaggta atccttcttt ctagaagtcc 2700
tcgtgtactg tgtaagcgcc cactccacat ctccactcga 2740
<210> 14
<211> 6502
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 14
cgaaccacaa ccacggagga aacagcggag gagcaggccc ggatgcgact ggcggtgatg 60
ttcgggcgga aggccaagga tcgagggagg gtggtgccga tggtggaaga gcgtaggtac 120
atatctcgac agttttattt gtctctagtc ttgaacttta ctagccggga tattcaatat 180
gctcggatat tttggtcacg attataggcg ttcctttcca tgaattcaag ctaaccagca 240
tcatttggct cacagggccg ccgctgactc ggatccttca tctgtcgtgc agagcttcct 300
gcagtcgttg catgggggct cacggcaatc ccaagacccc gagagacctt tcactactct 360
gcaagatcta cttacaacct cgactacatt gcctttcctg gaatctgcgg acgaaaaagc 420
ggttgacaat ctcctgagct tcctgccacc atcgctgctt ctcctggcac aggacaatga 480
tctcgacgaa gccgcggcag ctgatcagga cccggacatt gctcaagccg ccctgggatc 540
cctcgaactg acgcaaaaga aagagattct gcgcaaagtt ctacactcac ctcaatttgc 600
gcagagtcta gcaagcttga cgatggccat tagggatggt ggattaccga gcatcagcga 660
ggccctaaaa attccggttg caaatggagg attcatgcgt agagggggag ttccattggg 720
tggtggtgat gctgttgaag cattccttca aggcgttcgg gatcatgtta aggattcggc 780
tcaaggaaac cgtatggaca ccgattgatt ggttccaaat agcaatgatt gtgttatgtt 840
gacagggtcc tttttttaac tttcttatct cttttttact attattatct tcagccagcc 900
attctgtaac tatccactga tgttttagaa atccattagc ggccttgcat ggatgtctca 960
ttccctcctt agcagagaga gacttgtcat gggttatttt tggcatgagg tgggtggggt 1020
aaaaaggcca gtggccagta ctaggcagca tgataatact ggtaatctta ataccgttaa 1080
taataatgtg tcttggttgt ggcgtatggc ggactccgcc cgttaatctc aaacagaatc 1140
aagaattaaa aaagaaaaaa gtaggggaaa aaagtttccg actcgatgat ctcttcctcc 1200
accatggaaa aaaagaggtg ggacaggtat caaaggcatt gccgtatcgg gagtagacat 1260
aagtgatcct gtcagggacc aaagcaagtc aaatcagtcc gtactaagat acatcatggg 1320
gagggggcac gcatattccc ctcccgggcg cagcaggggg aaaatcccag cttcagaccc 1380
cgttccatcc cagagccatc catatccacc ctcaattatt ggccgtgtgt cgaacgaact 1440
cgacccggac cattcataag gctcggacgt ttagcaatca ctgggccaga gagcctggcc 1500
aatgggccat acagaagccg ccgctgccga atccgcccca gttttggccg ctttggcgcc 1560
gtccagccaa agctccgctg cgcataccga gcaataataa tcatcactgt gatctggaag 1620
tcgacacgtt tgggttcctt tccccatcat gcccgctgct tcttgacggc tgcctggctt 1680
gattggatcc ttgcccacgt ttccgcacgg ggtgccaccg ctaacttaac cagagttcgt 1740
cagccctttc gtactacttt aacttactta gggagtctgt cccatatttt ccccagcgcg 1800
cttttacttt gcctcggtcg tcaacgctcc ccagggccct tctgatcatc tctttttctt 1860
ctggaacagc ctgcgatgtc tgccttcggt tagcactctg acgacttaaa atccgccgcc 1920
tccaccattt gtagaaaaat gtgacgaact cgtgagctct gtacagtgac cggtgactct 1980
ttctggcatg cggagagacg gacggacgca gagagaaggg ctgagtaata agccactggc 2040
cagacagctc tggcggctct gaggtgcagt ggatgattat taatccggga ccggccgccc 2100
ctccgccccg aagtggaaag gctggtgtgc ccctcgttga ccaagaatct attgcatcat 2160
cggagaatat ggagcttcat cgaatcaccg gcagtaagcg aaggagaatg tgaagccagg 2220
ggtgtatagc cgtcggcgaa atagcatgcc attaacctag gtacagaagt ccaattgctt 2280
ccgatctggt aaaagattca cgagatagta ccttctccga agtaggtaga gcgagtaccc 2340
ggcgcgtaag ctccctaatt ggcccatccg gcatctgtag ggcgtccaaa tatcgtgcct 2400
ctcctgcttt gcccggtgta tgaaaccgga aaggccgctc aggagctggc cagcggcgca 2460
gaccgggaac acaagctggc agtcgaccca tccggtgctc tgcactcgac ctgctgaggt 2520
ccctcagtcc ctggtaggca gctttgcccc gtctgtccgc ccggtgtgtc ggcggggttg 2580
acaaggtcgt tgcgtcagtc caacatttgt tgccatattt tcctgctctc cccaccagct 2640
gctcttttct tttctctttc ttttcccatc ttcagtatat tcatcttccc atccaagaac 2700
ctttatttcc cctaagtaag tactttgcta catccatact ccatccttcc catcccttat 2760
tcctttgaac ctttcagttc gagctttccc acttcatcgc agcttgacta acagctaccc 2820
cgcttgagca gacatcacca tgcctgaact caccgcgacg tctgtcgaga agtttctgat 2880
cgaaaagttc gacagcgtct ccgacctgat gcagctctcg gagggcgaag aatctcgtgc 2940
tttcagcttc gatgtaggag ggcgtggata tgtcctgcgg gtaaatagct gcgccgatgg 3000
tttctacaaa gatcgttatg tttatcggca ctttgcatcg gccgcgctcc cgattccgga 3060
agtgcttgac attggggaat tcagcgagag cctgacctat tgcatctccc gccgtgcaca 3120
gggtgtcacg ttgcaagacc tgcctgaaac cgaactgccc gctgttctgc agccggtcgc 3180
ggaggccatg gatgcgatcg ctgcggccga tcttagccag acgagcgggt tcggcccatt 3240
cggaccgcaa ggaatcggtc aatacactac atggcgtgat ttcatatgcg cgattgctga 3300
tccccatgtg tatcactggc aaactgtgat ggacgacacc gtcagtgcgt ccgtcgcgca 3360
ggctctcgat gagctgatgc tttgggccga ggactgcccc gaagtccggc acctcgtgca 3420
cgcggatttc ggctccaaca atgtcctgac ggacaatggc cgcataacag cggtcattga 3480
ctggagcgag gcgatgttcg gggattccca atacgaggtc gccaacatct tcttctggag 3540
gccgtggttg gcttgtatgg agcagcagac gcgctacttc gagcggaggc atccggagct 3600
tgcaggatcg ccgcggctcc gggcgtatat gctccgcatt ggtcttgacc aactctatca 3660
gagcttggtt gacggcaatt tcgatgatgc agcttgggcg cagggtcgat gcgacgcaat 3720
cgtccgatcc ggagccggga ctgtcgggcg tacacaaatc gcccgcagaa gcgcggccgt 3780
ctggaccgat ggctgtgtag aagtactcgc cgatagtgga aaccgacgcc ccagcactcg 3840
tccgagggca aaggaataga gtagatgccg accgcgggat ccacttaacg ttactgaaat 3900
catcaaacag cttgacgaat ctggatataa gatcgttggt gtcgatgtca gctccggagt 3960
tgagacaaat ggtgttcagg atctcgataa gatacgttca tttgtccaag cagcaaagag 4020
tgccttctag tgatttaata gctccatgtc aacaagaata aaacgcgttt tcgggtttac 4080
ctcttccaga tacagctcat ctgcaatgca ttaatgcatt gactgcaacc tagtaacgcc 4140
ttncaggctc cggcgaagag aagaatagct tagcagagct attttcattt tcgggagacg 4200
agatcaagca gatcaacggt cgtcaagaga cctacgagac tgaggaatcc gctcttggct 4260
ccacgcgact atatatttgt ctctaattgt actttgacat gctcctcttc tttactctga 4320
tagcttgact atgaaaattc cgtcaccagc ncctgggttc gcaaagataa ttgcatgttt 4380
cttccttgaa ctctcaagcc tacaggacac acattcatcg taggtataaa cctcgaaatc 4440
anttcctact aagatggtat acaatagtaa ccatgcatgg ttgcctagtg aatgctccgt 4500
aacacccaat acgccggccg aaactttttt acaactctcc tatgagtcgt ttacccagaa 4560
tgcacaggta cacttgttta gaggtaatcc ttctttctag aagtcctcgt gtactgtgta 4620
agcgcccact ccacatctcc actcgactac acgctcgaga ttatcgccaa aatccttgtg 4680
tcaggattga tcatcaaccc tgccgaatac agcaccatag accggtcgat gggcttcagg 4740
aaggcagtgg tcgagaaagg gaagaacctg atcataccac agcggcaatt ctcggtgcga 4800
aagtctgcca tgtcagaaca accccaggcc tccattatac gaacttttac gggcggcttg 4860
aatcagttgg agaatcaatt ggccgatgac cctcttcaga aaagtcgcgt acgactagct 4920
catcgtgcat tcctgcgaca ctcattcaat cggctcgatt tcgtcgcctt agtatcgtac 4980
tgggtctcct tcttcctctc gatatatgga gtcgaaactc gccagcagct ctatgttttc 5040
agcatgttga gctgtcttcg aattcttcgt ctcttgaatc tgaccaatgg tacttctgta 5100
agtaaaccgc cgaatactct tagccacgtc gttcactgac atcagagaca aggttatcct 5160
ccgcagtctc aagaaagcag cacccttgct tgcgcacgta gcgttcctta tcggcttctt 5220
ctggcttctg tttgctatcg tcggcatcca aagcttcaag tcaagccttc ggagaacctg 5280
tgtctggctt ggcgttgatg gtgaaagcga ttacgcacag aatgatccaa atggctcttt 5340
acagttttgt ggaggctacc tcaatgcgac cactaaacaa caaatgccat ggattcagaa 5400
ggataatact ccgagttcgt cttctccgaa aggctacatc tgtcctgcag gctctatttg 5460
tcttgaagga gacaatccct acaatgggac gttaagtttt gacaacatcg tgaattcgct 5520
ggagcttgtg tttgtaatta tgagctcgaa cacattcact gacctactct actatactgc 5580
cgacagtgac tatctcgctt cttctctttt cttcatatgc gggcttctca tattgagttt 5640
gtggatggtc aatttgttgg ttgcagtgat cacccatgct tttcaggtca ttcgagagga 5700
aagtcagcgc agtgcctttg ccgttaagaa gatagacacg actgaaagag aggatttggc 5760
ctctcggaag gtcagtgcga tcaagcgtct ttatgacaag accgaatggc tctgggtttg 5820
catcatcatt ttcgatcttg tggtccaggc tctgaggagc gcttccatga gcgacgatcg 5880
ggcccagttt attgacacca cagaacttgt tatgactttc gtattccttt tcgaaattat 5940
cctacgattt gcctcggatt ggcgcacctt ccacaaaaaa acgcggaatt gggtcgatct 6000
gggcctcgtc ataataacgt gcatcattca gatcccggcg atcaagcgtg aacgagcata 6060
cgatgtcctt acgctctttc aaattcttcg agtatatcgc gtcgtgctcg catttaaggt 6120
gaccagggac ctcattatgg ttgtctttcg taatgcagtt ggtctgctga acctgatctt 6180
ctttgtcttt ctgattacct tccttgcttc cattttcgca actcagctct tccgtggcca 6240
gattccagag gaagacgcag acggcgatac cataatcatc accttttccg acatctacaa 6300
ctcttttctc gggatgtatc aaatcttgtc aagcgaaaat tggacggaca tcttgtataa 6360
cgctaccacg tacacagtgt catacaatac agcttggatc tctgcggctt tcttgatatt 6420
atggtttatc ctggccaact tcattgtcct gaacatgttc attgctgtca tccaggaaag 6480
ctttgatgtc tcggaggatg aa 6502
<210> 15
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 15
ttcgacagcg tctccgacct 20
<210> 16
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 16
acacagccat cggtccagac 20
Claims (10)
1. the aspergillus niger of one plant of calcium channel CchA gene inactivation, which is characterized in that calcium ion in the bacterial strain
Channel C chA gene inactivation.
2. the aspergillus niger of calcium channel CchA gene inactivation according to claim 1, which is characterized in that
The aspergillus niger is Aspergillus Niger 831.
3. the aspergillus niger of calcium channel CchA gene inactivation according to claim 1, which is characterized in that
The calcium channel CchA gene, nucleotide sequence is as shown in SEQ ID NO.1, after calcium channel CchA gene inactivation
Nucleotide sequence as shown in SEQ ID NO.2.
4. the construction method of the aspergillus niger of calcium channel CchA gene inactivation according to claim 1,
It is characterized by comprising the following steps:
(1) genomic DNA of aspergillus niger Aspergillus Niger 831 is extracted;
(2) genomic DNA obtained using step (1) is template, with nucleotide shown in SEQ ID NO.3 and SEQ ID NO.4
Sequence is primer, amplifies the upstream homology arm of CchA gene;
The genome obtained using step (1) as template, with nucleotides sequence shown in SEQ ID NO.5 and SEQ ID NO.6 column be
Primer amplifies the downstream homology arm of CchA gene;
It using plasmid pan7-1 as template, is arranged with nucleotides sequence shown in SEQ ID NO.7 and SEQ ID NO.8 as primer, amplification
Hyg resistance element out;
By overlap PCR, it is with the upstream homology arm of CchA gene, the downstream homology arm of CchA gene, hyg resistance element
Template is arranged with nucleotides sequence shown in SEQ ID NO.9 and SEQ ID NO.10 as primer, and PCR amplification obtains gene knockout piece
Section;
(3) aspergillus niger protoplast is prepared;
(4) gene knockout recombinant fragment is imported in protoplast and carries out homologous recombination, obtain the mistake of calcium channel CchA gene
Aspergillus niger living.
5. the aspergillus niger of any calcium channel CchA gene inactivation of claims 1 to 3 is in preparation lemon
Application in acid.
6. application according to claim 5, which is characterized in that the aspergillus niger gene inactivated with calcium channel CchA gene
Engineering bacteria is fermentation strain, prepares citric acid by immobilization fermentation.
7. application according to claim 6, which is characterized in that the immobilization fermentation is using porous fibrous material as admittedly
Surely change medium, preparation of citric acid by fermentation, the porous fibrous material is activated carbon fibre.
8. application according to claim 6, which is characterized in that the configuration method of the fermentation medium of the immobilization fermentation
It is as follows:
The corn flour for taking 200g/L~300g/L tapioca starch, 200g/L-300g/L respectively, at 60 DEG C -70 DEG C, every 1L fermentation liquid
The α-amylase of 1~2mL is added, digests 35min~45min, then tapioca starch liquid and corn flour liquid are separately heated to up to 85 DEG C, often
The α-amylase of 1~2mL is added in 1L fermentation liquid, digests 35min~45min, until the constant indigo plant of iodine solution, filters tapioca starch liquid, obtain wooden
Potato powder liquid supernatant is added and is added into tapioca starch liquid supernatant the unfiltered corn flour liquid that volume fraction is 2-10%, mixes
Immobilization fermentation culture medium is obtained, contains alpha-amylase in the α-amylase, the enzyme activity of the alpha-amylase is 60000~
70000U/mL。
9. application according to claim 6, which is characterized in that the condition of culture of the immobilization fermentation is as follows: culture temperature
Degree is 28 DEG C~37 DEG C, and incubation time is that 72~120h revolving speed is 180~330rpm.
10. application according to claim 7, which is characterized in that the porous fibrous material first carries out using the following method
Pretreatment:
By 1~1.5h of soaking with sodium hydroxide of porous fibrous material 1M, then being rinsed with water to pH is neutrality, is soaked in 1M hydrochloric acid
1~1.5h is steeped, then being rinsed with water to pH is neutrality, drying to constant weight, obtains modified porous fibrous material.
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CN111088173A (en) * | 2019-12-26 | 2020-05-01 | 南京高新工大生物技术研究院有限公司 | Aspergillus niger genetically engineered bacterium and construction method and application thereof |
CN111088173B (en) * | 2019-12-26 | 2021-08-20 | 南京高新工大生物技术研究院有限公司 | Aspergillus niger genetically engineered bacterium and construction method and application thereof |
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