CN110106095A - The aspergillus niger and its construction method and application that one plant of calcium channel CchA gene inactivates - Google Patents

Abstract

The aspergillus niger and its construction method that inactivate the invention discloses one plant of calcium channel CchA gene and application, it is the black-koji mould for knocking out calcium channel CchA gene.The invention also discloses the construction methods of said gene engineering bacteria.Further disclose application of the genetic engineering bacterium in production citric acid.The aspergillus niger that the present invention passes through building gene knockout calcium channel CchA gene, make the aspergillus niger reduction of biomembrane yield and hydrophobicity reduction during immobilization fermentation produces citric acid, alleviate the phenomenon of uniting of carrier, lemon acid yield and sugared transformation efficiency are improved, industrial production is suitable for.

Description

The aspergillus niger and its building of one plant of calcium channel CchA gene inactivation Method and application

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

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<211> 6470

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<213>artificial sequence (Artificial Sequence)

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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.