CN110106095B - Aspergillus niger genetically engineered bacterium with calcium ion channel CchA gene inactivated, and construction method and application thereof - Google Patents

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

Aspergillus niger genetically engineered bacterium with calcium ion channel CchA gene inactivated, and construction method and application thereof

Technical Field

The invention belongs to the technical field of genetic engineering and microorganisms, and particularly relates to an Aspergillus niger genetically engineered bacterium for knocking out a calcium ion channel CchA gene, and a construction method and application thereof.

Background

Citric acid is one of the most demanded organic acids worldwide, and is widely used in industries such as food, medicine, daily chemicals and the like. Wherein, 75% of citric acid is used in food industry, mainly used in sour agent, antioxidant, pH regulator in food additive, meanwhile, since citric acid has mild and refreshing sour taste, it is widely used in the manufacture of food such as beverage, cake, wine, dairy products, etc. Another 15% is used in the chemical and textile industries and can be used as buffers, complexing agents, sequestering agents, metal builders, mordants, etc. Also 10% of citric acid is used as anticoagulant, antacid, taste corrigent, cosmetic and feed additive, etc., and is widely used in the pharmaceutical industry and animal husbandry. In recent years, due to the general rise of food prices in China, the cost of raw materials for producing citric acid is greatly increased. Therefore, how to improve the citric acid fermentation method and further reduce the cost becomes the most concerned problem in the whole citric acid industry in China.

The citric acid produced by fermenting aspergillus niger is one of the main ways for producing citric acid by current industrial fermentation, and the fermentation efficiency of aspergillus niger can be greatly improved by a cell immobilization method. At present, there are four main methods for cell immobilization: entrapment, cross-linking, covalent bonding, and adsorption. For filamentous fungi, the adsorption method is simple to operate, the chemical structure is not required to be changed in the fixing process, and the method is theoretically suitable for all filamentous fungi and is more beneficial to industrial popularization and application. The principle of the adsorption method mainly depends on the interaction force between the groups on the surface of the carrier and the surface of the cells, so that the cells are promoted to be adsorbed on the surface of the carrier for growth. And when the cells die, the cells fall off from the carrier, and simultaneously, the active cells are adsorbed to form dynamic balance, so that the whole microenvironment forms an efficient conversion system, and the overall fermentation efficiency is obviously improved compared with the free fermentation. In the process of research on the immobilized fermentation mechanism, microorganisms commonly form a biological membrane on a carrier, and the formed biological membrane is different, so that the performance of immobilized fermentation is greatly different. Therefore, we need further understanding of the mechanism of biofilm formation to really solve the problem of difficult industrial application of immobilization.

Disclosure of Invention

In order to solve the problems of low yield and long fermentation period in the prior industrial production of citric acid by fermenting aspergillus niger, the invention aims to solve the technical problem of providing a calcium ion channel CchA gene-deficient aspergillus niger genetically engineered bacterium.

The invention also aims to solve the technical problem of providing a construction method of the citric acid producing aspergillus niger genetically engineered bacterium.

The invention finally solves the technical problem of providing the application of the Aspergillus niger genetically engineered bacterium in the production of citric acid by fermentation.

In order to solve the technical problems, the invention adopts the following technical scheme:

a calcium ion channel CchA gene inactivated Aspergillus niger genetic engineering bacterium is a genetic engineering bacterium which replaces partial sequence of CchA gene by hyg resistance gene through a double exchange method, and the calcium ion channel CchA gene participates in the polar growth of hypha main axis, sporulation and the integrity of cell wall. The integrity of the cell wall can affect the production of biofilm, and therefore, the calcium channel CchA gene can regulate the production of biofilm, thereby affecting the production of citric acid.

Wherein the Aspergillus Niger is Aspergillus Niger831 (A. Niger831),

the nucleotide sequence of the calcium ion channel CchA gene before inactivation is shown in SEQ ID NO.1, and the nucleotide sequence of the calcium ion channel CchA gene after inactivation is shown in SEQ ID NO. 2.

A method for constructing Aspergillus niger genetically engineered bacteria with calcium ion channel CchA gene inactivated comprises the following steps:

(1) extracting genomic DNA of aspergillus niger a.niger 831;

(2) amplifying an upstream homology arm of the CchA gene by taking the genome obtained in the step (1) as a template and the nucleotide sequences shown in SEQ ID NO.3 and SEQ ID NO.4 as primers;

amplifying a downstream homology arm of the CchA gene by taking the genome obtained in the step (1) as a template and the nucleotide sequences shown in SEQ ID NO.5 and SEQ ID NO.6 as primers;

amplifying a hyg resistance element by taking the plasmid pan7-1 as a template and nucleotide sequences shown in SEQ ID NO.7 and SEQ ID NO.8 as primers;

performing PCR amplification by using an upstream homologous arm of a CchA gene, a downstream homologous arm of the CchA gene and a hyg resistance element as templates and nucleotide sequences shown in SEQ ID NO.9 and SEQ ID NO.10 as primers through overlap PCR to obtain a gene knockout fragment;

(3) preparing an Aspergillus niger protoplast;

(4) and (3) introducing the gene knockout recombinant fragment into the protoplast for homologous recombination to obtain the aspergillus niger genetically engineered bacterium with the calcium ion channel CchA gene inactivated.

Wherein, the nucleotide sequence of the upper homologous arm of the Ccha gene is shown as SEQ ID NO.11, the nucleotide sequence of the lower homologous arm of the Ccha gene is shown as SEQ ID NO.12, the nucleotide sequence of the hyg resistance element is shown as SEQ ID NO.13, and the nucleotide sequence of the gene knockout fragment is shown as SEQ ID NO. 14.

The application of the Aspergillus niger genetically engineered bacterium with the calcium ion channel CchA gene inactivated in the preparation of citric acid is within the protection scope of the invention.

Preferably, Aspergillus niger genetically engineered bacteria with calcium ion channel CchA gene inactivated are used as fermentation strains to prepare the citric acid through immobilized fermentation.

The citric acid is prepared by taking a porous fiber material as an immobilization medium through fermentation, wherein the porous fiber material is activated carbon fiber.

The preparation method of the fermentation medium for immobilized fermentation comprises the following steps:

respectively taking 200 g/L-300 g/L of cassava powder and 200 g/L0-300 g/L of corn powder, adding 1-2m L of liquefying enzyme into 1L of fermentation liquor at 60-70 ℃, carrying out enzymolysis for 35-45 min, respectively heating cassava powder liquid and corn powder liquid to 85 ℃, adding 1-2m L of liquefying enzyme into every 1L of fermentation liquor, carrying out enzymolysis for 35-45 min until the iodine solution does not turn blue, filtering the cassava powder liquid to obtain cassava powder liquid supernatant, adding 2-10% by volume of unfiltered corn powder liquid into the cassava powder liquid supernatant, and uniformly mixing to obtain an immobilized fermentation culture medium, wherein the liquefying enzyme contains α -amylase, and the enzyme activity of α -amylase is 60000-70000U/m L.

Wherein the culture conditions of the immobilized fermentation are as follows: the culture temperature is 30-37 ℃, the culture time is 72-120 h, and the rotating speed is 180-330 rpm.

Wherein, the porous fiber material is pretreated by the following method:

soaking the porous fiber material in 1M sodium hydroxide for 1-1.5 h, washing with water until the pH value is neutral, soaking in 1M hydrochloric acid for 1-1.5 h, washing with water until the pH value is neutral, and drying to constant weight to obtain the modified porous fiber material.

The method for producing citric acid by immobilized fermentation of aspergillus niger genetically engineered bacteria with the calcium ion channel CchA gene inactivated specifically comprises the following steps:

(1) preparation of the carrier: soaking porous fiber material in 1M sodium hydroxide for 1h, cleaning with pure water, soaking in 1M hydrochloric acid for 1h, washing with pure water until pH is neutral, and oven drying at 65 deg.C to constant weight. Cutting into carriers with the same size.

(2) And (3) fermenting, namely scraping the activated Aspergillus niger genetically engineered bacterium flat plate by using a sporulation liquid to prepare a spore liquid, inoculating the spore liquid into a sterilized fermentation culture medium containing 0.1g/100m L porous fiber material in an inoculation amount of 0.2 percent (volume fraction), and fermenting to obtain the citric acid.

The fermentation medium comprises the following components of 200-250 g/L corn flour and 200-250 g/L cassava flour.

The fermentation conditions were as follows: the culture temperature is 35 ℃, the culture time is 72-96h, and the rotating speed is 200-250 rpm.

Has the advantages that:

the invention constructs an Aspergillus niger genetically engineered bacterium with a calcium ion channel CchA gene deletion by a gene knockout means. The genetic engineering bacteria reduce the amount of biological membranes in the process of producing citric acid by Aspergillus niger through immobilized fermentation, reduce the phenomenon of cluster, improve the yield and the sugar conversion rate of the citric acid and shorten the fermentation period.

Drawings

FIG. 1 is an electrophoretogram of the genome of Aspergillus Niger 831;

FIG. 2 is a PAN7-1 plasmid map;

FIG. 3 is a PCR electrophoresis of the upstream and downstream homology arms of the Ccha gene, wherein M is DNAmarker D L5000, lane 1 is the upper homology arm of Ccha, lane 3 is the lower homology arm of Ccha, and lane 5 is the hyg resistance expression element;

FIG. 4 is an electrophoretogram of a knockout fragment, where M is marker lane 1;

FIG. 5 is a graph of crystal violet staining;

FIG. 6 is a graph showing the difference in OD values of crystal violet stains;

FIG. 7 shows the results of fermentation of original A.niger and A.niger genetically engineered bacteria.

FIG. 8 is an electron micrograph of a biofilm of the starting Aspergillus niger strain;

FIG. 9 is an electron microscope image of a biological membrane of Aspergillus niger genetically engineered bacteria;

Detailed Description

Example 1: construction of aspergillus niger calcium ion channel cchA gene knock-out bacteria.

Extraction of original Aspergillus niger genome

A kit for extracting a plant genome (takara minitest plant genomic DNA extraction kit) by takara corporation was used, and the following precautions were specifically taken:

1. inoculating 1m L of scraped Aspergillus niger spore liquid into 50m L DP culture medium, and culturing at 35 deg.C and 250r/min for 15 h;

2. centrifuging at 5000r/min for 5min to collect mycelium pellets, washing with normal saline twice, grinding the collected mycelium pellets with liquid nitrogen for 3 times, weighing 100mg of the ground powder, adding the powder into a tube with 500u L of Buffer HS II, mixing uniformly, adding 10u L of RNase A, shaking fully and mixing uniformly, and carrying out water bath at 56 ℃ for 10 minutes.

3. Adding 62.5u L Buffer KAC into step 2, mixing well, standing on ice for 5min, centrifuging at 12000rpm for 5min, collecting supernatant 600u L, adding 600u L Buffer GB, and mixing well.

4. The Spin Column was mounted on a Collection Tube, the solution was transferred to the Spin Column, centrifuged at 12000rpm for 1min, and the filtrate was discarded.

5. 500u L of Buffer WA WAs added to Spin Column, centrifuged at 12000rpm for 1min, and the filtrate WAs discarded.

6. 700u L of Buffer WB was added to Spin Column, centrifuged at 12000rpm for 1min, and the filtrate was discarded.

7. Repeat step 6 once.

8. Spin Column was mounted on a Collection Tube at 12000rpm and centrifuged for 2 min.

9. Spin Column was mounted on a new 1.5m L centrifuge tube, 40u L of sterilized water at 65 ℃ was added to the center of the Spin Column membrane, left to stand at room temperature for 1 min.12000rpm for 2min, and the dna was eluted.

FIG. 1 shows DNA marker with M D L15000 and the extracted A.niger genome No. 1.

And secondly, amplifying the upper and lower homologous arms of the CchA gene by using a PCR technology.

TABLE 1 PCR reaction System

Amplifying an upper homologous arm by using an original Aspergillus niger genome as a template, using CchA-up-F as an upper primer and using CchA-up-R as a lower primer (shown as SEQ ID NO.3 and SEQ ID NO. 4); the lower homologous arm was amplified using CchA-down-F as the upper primer and CchA-down-R as the lower primer (shown in SEQ ID NO.5 and SEQ ID NO. 6). The reaction system is shown in Table 1, and the reaction conditions are as follows: denaturation at 98 ℃ for 10s, annealing at 55-65 ℃ for 30s, and extension at 68 ℃ for 2min, and repeating the steps for 30 times. After the reaction was completed, the PCR product was quantified by agarose gel electrophoresis, as shown in FIG. 3.

(III) amplification of hyg-resistant expression elements

Amplifying hyg resistance expression element with PAN7-1 plasmid (PAN7-1 plasmid map shown in figure 2, nucleotide sequence shown in SEQ ID NO.17) as template, CchA-hyg-F as upper primer, and CchA-hyg-R as lower primer (shown in SEQ ID NO.7 and SEQ ID NO. 8). The reaction system is shown in Table 1, and the reaction conditions are as follows: denaturation at 98 deg.C for 10s, annealing at 55-65 deg.C for 30s, and extension at 68 deg.C for 3min, and repeating the above steps for 30 times. After the reaction was completed, the PCR product was quantified by agarose gel electrophoresis, as shown in FIG. 3. Wherein the nucleotide sequence of the hyg resistance expression element is shown in SEQ ID NO: 13.

FIG. 3 shows DNA Marker M D L5000, upper homology arm of Ccha No.1, lower homology arm of Ccha No.3, and hyg resistance expression element No.5, in the case of DNA Marker D L.

(IV) amplification of knockout fragment

Using CchA upstream and downstream homology arms and hyg resistance expression element as template, CchA-F as upper primer, CchA-R as lower primer (shown in SEQ ID NO.9 and SEQ ID NO. 10), CchA gene knockout fragment was amplified by Overlap PCR technique. The reaction system is shown in Table 1, and the reaction conditions are as follows: denaturation at 98 deg.C for 10s, annealing at 55-65 deg.C for 30s, and extension at 68 deg.C for 7min, and repeating the above steps for 30 times. After completion of the reaction, the PCR product was quantified by agarose gel electrophoresis, as shown in FIG. 4. Wherein the nucleotide sequence of the knockout fragment is shown in SEQ ID NO: 14.

FIG. 4 shows a DNA Marker in which M is D L10000, and No.1 is a knockout fragment.

(V) preparation and transformation of Aspergillus niger protoplast

1. Aspergillus niger was inoculated into PDA plates and spores were grown, 3m L scrape buffer was added to the plates, the spores were scraped off with a spreading stick and transferred to sterilized 5m L centrifuge tubes.

2. Inoculating 0.5m L spore liquid to 50m L YPD medium, culturing at 35 deg.C and 250rpm for 9-13 h.

3. After the spores have germinated, they are filtered by Miracloth to leave hypha, and then enzymatic hydrolysate (L lysing enzyme, crashing enzyme, snailase each 0.1g/10m L, cellulase 400 mu L/10 m L) is prepared and sterilized by filtration with a sterile syringe.

4. 2g of mycelia was added to the enzyme solution and subjected to enzymolysis at 30 ℃ and 220rpm for 30 min. The rotation speed is reduced to 150rpm for 4h of culture.

5. After the enzymolysis is finished, filtering with filter paper, taking the filtrate, centrifuging at 4 ℃ and 5000rpm for 10min, removing the supernatant, adding 1M L1M sorbitol (ice water bath), blowing and sucking with a gun, mixing uniformly, adding 15M L sorbitol, centrifuging, removing the supernatant, repeating again, removing the supernatant, adding 1M L Solution5, blowing and sucking with a gun, and mixing uniformly.

6. And (3) sucking 100 mu L of protoplast into a 1.5m L sterilized centrifugal tube, adding the gene knockout fragment constructed in the step (IV) of 10 mu L, mixing uniformly, adding 50 mu L of Solution4, mixing uniformly, placing on ice and timing for 15-30 min.

After 7.20min, adding 900u L Solution4, turning upside down for several times, standing at room temperature for 15-30min, after 15-30min, centrifuging at 6000rpm for 5min, discarding 900u L supernatant, coating the residual thallus on PDA culture medium with sucrose concentration of 1 mol/L and hygromycin concentration of 75 mmol/L, and culturing at normal position to obtain transformant.

8. The specific method comprises the steps of adding a proper amount of transformant into 50u L colony PCR buffer solution (100 mM/L Tris-HCl, 10 mM/L EDTA and 1M/L Kcl), carrying out water bath at 95 ℃ for 10min, adding 0.5u L into a PCR reaction system, setting PCR primers hyg-F and hyg-R (shown as SEQ ID NO.15 and SEQ ID NO. 16), and displaying an amplification band by agarose electrophoresis to indicate that the transformation is successful.

Example 2: crystal violet staining test

Respectively inoculating original Aspergillus Niger (Aspergillus Niger831) and genetically engineered bacteria to PDA plate, adding 3m L spore-scraping buffer solution into the plate, scraping off the spores with a coating rod, transferring to sterilized 5m L centrifuge tube, diluting to 2m L with the spore-scraping buffer solution to obtain spore solution, quantifying with a blood counting chamber, and diluting to 10%⁶M L, then continuously diluting to 10⁵，10⁴。

Adding 1ml of synthetic culture medium into a 24-pore plate in advance, inoculating 2u L of spore liquid with different concentrations into the culture medium, standing and culturing at 35 ℃ for 36h to enable aspergillus niger to form a film at the bottom of the pore plate, pouring out the culture medium, washing for 2 times by PBS, adding 0.1% crystal violet for dyeing for 15min, pouring out the crystal violet, washing for 2 times by PBS, adding glacial acetic acid, placing for 30min in a shaking instrument to decolor the crystal violet, observing, and detecting by an enzyme labeling instrument, wherein the difference between the crystal violet dyeing graph and the OD value is shown in figures 5 and 6.

TABLE 2 OD values of biofilm crystal violet staining experiments at different spore concentrations

The results in FIGS. 5 and 6 show that the.DELTA.CchA strain was markedly lighter in purple color than the original strain after decolorization, at 10⁵The color of the strain Δ CchA at the concentration was removed and the data was consistent with the color as measured by OD using a microplate reader. Indicating that the biological membrane is reduced after the calcium ion channel CchA gene is inactivated.

Example 3: and (3) performing immobilized fermentation experiments on the genetically engineered bacteria.

1. Preparation of porous fiber material immobilized medium

Soaking porous fiber material (activated carbon fiber) in 1M sodium hydroxide for 1h, cleaning with pure water, soaking in 1M hydrochloric acid for 1h, washing with pure water until pH is neutral, and oven drying at 65 deg.C to constant weight. Cutting into carriers with the same size.

2. Preparation of fermentation Medium

Respectively weighing 200/L-300 g/L of cassava powder and 200 g/L-300 g/L of corn powder, gelatinizing in a 75 ℃ water bath kettle, adding 1-2m L of liquefying enzyme when the temperature of cassava powder liquid and corn powder liquid reaches 65 ℃, liquefying for 40min, then heating the water bath kettle to 95 ℃, adding 1-2m L of liquefying enzyme when the temperature of the cassava powder liquid and the corn powder liquid reaches 85 ℃, liquefying for 40min until the iodine solution does not turn blue, filtering the cassava powder liquid to obtain supernatant, adding 2-10% of unfiltered corn powder liquid as a return material, uniformly mixing, subpackaging every 100m L into 500m L conical bottles containing a proper amount of carriers, sterilizing and cooling for later use.

3. The fermentation method comprises the following steps

(1) Inoculating the frozen aspergillus niger genetically engineered bacteria and the original aspergillus niger spores onto a PDA (personal digital assistant) plate, culturing for 4-5 days at the constant temperature of 35 ℃ in a constant temperature incubator, and filling the spores on the plate.

(2) Scraping spores with a spore scraping buffer solution to obtain a spore suspension, transferring a proper amount of the spore suspension into a 500m L conical flask filled with 100m L immobilized culture medium, culturing for 72-96h at 30-35 ℃ in a shaking table at 250rpm, sampling every 12h in the fermentation process, centrifuging at 12000rpm for 5min, separating the supernatant from the precipitate, and determining the residual sugar concentration and the citric acid yield in the supernatant.

Wherein the NaOH titration method comprises adding a sample of 1M L (diluted to a certain concentration) into a 250M L conical flask, simultaneously adding 50M L pure water, and titrating with 0.1429M NaOH, wherein the consumed NaOH amount is the yield of citric acid.

The DNS method comprises the following steps:

the DNS is prepared by weighing 10g of 3, 5-dinitrosalicylic acid, placing in L m water of about 600m, gradually adding 10g of sodium hydroxide, magnetically stirring and dissolving in 50 ℃ water bath, sequentially adding 200g of sodium methyl tartrate, and,

2g of phenol and 5g of anhydrous sodium sulfite, cooling to room temperature after all the phenol and the anhydrous sodium sulfite are dissolved and clarified, and fixing the volume by pure water

To 1000m L, the product is stored in a brown reagent bottle and used after being placed in the dark for 7 days.

The preparation method of the standard yeast comprises preparing a series of sugar standard solutions with concentration of 0.0-1.0 g/L, adding 0.5m L into 15m L centrifuge tubes, adding 0.5m L DNS solution into each centrifuge tube,

placing the centrifugal tubes in a boiling water bath for reaction for 5min, placing the centrifugal tubes in ice water for cooling, then adding 8m L pure water into each centrifugal tube, uniformly mixing, measuring the light absorption value OD540 under the wavelength of 540nm, taking 0.0 g/L as a reference, taking the concentration of the standard solution as an ordinate, and taking the light absorption value OD540 as an abscissa to draw a standard curve.

The sample measurement method comprises the steps of adding 1m L concentrated sulfuric acid into a 10m L sample after the sample is properly diluted, reacting in a boiling water bath for 15min, placing in ice water for cooling, adjusting the pH to be neutral, fixing the volume to 100m L, and then diluting to the proper concentration, wherein the measurement method is the same as the above, and different sugars correspond to different standard yeasts.

FIG. 7 shows the difference between the Δ CchA strain and the original strain under fermentation conditions, and the results show that after 96h of fermentation, the yield of citric acid produced by fermentation of the Δ CchA strain is 162.8 g/L on average, the yield of citric acid produced by fermentation of the original strain is 153.2 g/L on average, and the yield of citric acid produced by the Δ CchA strain is 6.27% higher than that of the original strain.

Example 4: SEM observation of biofilm

The immobilized carrier after fermentation for 72h was taken out, washed 3 times with PBS to remove adsorbed mycelia. The mixture was placed in a freezer at-80 ℃ overnight and then lyophilized in a lyophilizer. And the carrier is adhered on a point mirror table through conductive glue, and 20mA 30s is subjected to gold spraying. Then, the sample was taken into TM3000 for observation. The electron micrographs of the biofilm are shown in FIGS. 8 and 9. FIG. 8 shows immobilization of the A.niger original strain, and FIG. 9 shows immobilization of the. DELTA.Cch strain. It was found that the Aspergillus niger original bacteria formed a plurality of spheres on the carrier, and these spheres connected to form a film and simultaneously blocked the pore size of the carrier, which affected the oxygen and mass transfer of the inner bacteria. The delta CchA strain forms a small amount of biomembranes at the corners and on the surface of the carrier, does not influence the oxygen and mass transfer of the internal bacteria, and is favorable for producing citric acid by immobilized fermentation.

Sequence listing

<110> Nanjing university of industry

<120> Aspergillus niger genetically engineered bacterium with calcium ion channel CchA gene inactivated, and construction method and application thereof

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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 tacctcaatg720

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 attttcctgc720

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 (6)

1. The application of Aspergillus niger genetically engineered bacteria with calcium ion channel CchA gene inactivated in preparing citric acid by immobilized fermentation is characterized in that the Aspergillus niger isAspergillusNiger 831；

The nucleotide sequence of the calcium ion channel CchA gene is shown in SEQ ID NO.1, and the nucleotide sequence of the calcium ion channel CchA gene after inactivation is shown in SEQ ID NO. 2;

the immobilized fermentation is to prepare the citric acid by fermenting with porous fiber material as an immobilized medium.

2. The application of claim 1, wherein the Aspergillus niger genetically engineered bacterium is constructed according to the following method:

(1) extraction of Aspergillus nigerAspergillusGenomic DNA of Niger 831;

(2) amplifying an upstream homologous arm of the CchA gene by taking the genomic DNA obtained in the step (1) as a template and taking the nucleotide sequences shown in SEQ ID NO.3 and SEQ ID NO.4 as primers;

amplifying a downstream homology arm of the CchA gene by taking the genome obtained in the step (1) as a template and the nucleotide sequences shown in SEQ ID NO.5 and SEQ ID NO.6 as primers;

amplifying a hyg resistance element by taking the plasmid pan7-1 as a template and nucleotide sequences shown in SEQ ID NO.7 and SEQ ID NO.8 as primers;

performing PCR amplification by using an upstream homologous arm of a CchA gene, a downstream homologous arm of the CchA gene and a hyg resistance element as templates and nucleotide sequences shown in SEQ ID NO.9 and SEQ ID NO.10 as primers through overlap PCR to obtain a gene knockout fragment;

(3) preparing an Aspergillus niger protoplast;

(4) and (3) introducing the gene knockout recombinant fragment into the protoplast for homologous recombination to obtain the Aspergillus niger genetically engineered bacterium with the calcium ion channel CchA gene inactivated, wherein the nucleotide sequence of the calcium ion channel CchA gene inactivated is shown as SEQ ID No. 2.

3. The use according to claim 1, wherein the immobilized fermentation is cultured under the following conditions: the culture temperature is 28-37 ℃, the culture time is 72-120 h, and the rotating speed is 180-330 rpm.

4. The use according to claim 3, wherein the fermentation medium for immobilized fermentation is prepared by:

respectively taking 200 g/L-300 g/L of cassava powder and 200 g/L0-300 g/L of corn powder, adding 1-2m L of liquefying enzyme into every 1L of fermentation liquor at the temperature of 60-70 ℃, carrying out enzymolysis for 35-45 min, respectively heating cassava powder liquid and corn powder liquid to 85 ℃, adding 1-2m L of liquefying enzyme into every 1L of fermentation liquor, carrying out enzymolysis for 35-45 min until the iodine solution does not turn blue, filtering the cassava powder liquid to obtain cassava powder liquid supernatant, adding 2-10% by volume of unfiltered corn powder liquid into the cassava powder liquid supernatant, and uniformly mixing to obtain an immobilized fermentation culture medium, wherein the liquefying enzyme contains α -amylase, and the enzyme activity of α -amylase is 60000-70000U/m L.

5. Use according to claim 1, wherein the porous fibrous material is activated carbon fiber.

6. Use according to claim 5, wherein the porous fibrous material is pre-treated by:

soaking the porous fiber material in 1M sodium hydroxide for 1-1.5 h, washing with water until the pH value is neutral, soaking in 1M hydrochloric acid for 1-1.5 h, washing with water until the pH value is neutral, and drying to constant weight to obtain the modified porous fiber material.

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