阅读说明：本技术 高产溶血磷脂酶黑曲霉重组表达菌株的构建及应用 (Construction and application of aspergillus niger recombinant expression strain for high-yield lysophospholipase ) 是由 赵正阳 于 2021-10-11 设计创作，主要内容包括：本发明公开了高表达溶血磷脂酶的黑曲霉重组菌株及其构建方法。一种构建重组黑曲霉表达菌的方法,构建包含溶血磷脂酶的重组表达盒,该重组表达盒为包含溶血磷脂酶基因序列、启动子、终止子、筛选标记、上下游同源序列等元件的基因片段。本发明通过基因工程的手段,构建了溶血磷脂酶的表达盒,并将其导入黑曲霉表达宿主菌,实现了溶血磷脂酶的高效分泌表达,获得了高表达溶血磷脂酶的菌株。高表达溶血磷脂酶的菌株液体发酵的上清液粗酶液表达水平可达2.88g/L,活性可达2345unit/ml,比酶活可达815unit/mg。(The invention discloses an Aspergillus niger recombinant strain for highly expressing lysophospholipase and a construction method thereof. A method for constructing recombinant Aspergillus niger expression bacteria is to construct a recombinant expression cassette containing lysophospholipase, wherein the recombinant expression cassette is a gene fragment containing lysophospholipase gene sequences, promoters, terminators, screening markers, upstream and downstream homologous sequences and other elements. The expression cassette of the lysophospholipase is constructed by means of genetic engineering and is introduced into an Aspergillus niger expression host bacterium, so that the high-efficiency secretory expression of the lysophospholipase is realized, and the bacterial strain with high expression of the lysophospholipase is obtained. The expression level of the supernatant crude enzyme liquid obtained by liquid fermentation of the bacterial strain with high expression of lysophospholipase can reach 2.88g/L, the activity can reach 2345unit/ml, and the specific enzyme activity can reach 815 unit/mg.)

1. A method for constructing an Aspergillus niger recombinant expression strain for producing lysophospholipase is characterized in that Aspergillus niger is taken as a host cell, and an expression cassette containing lysophospholipase genes is introduced into the host cell to obtain the recombinant expression strain; the lysophospholipase gene is selected from lysophospholipase genes from aspergillus, yeast or mucor.

2. The construction method according to claim 1, wherein the lysophospholipase gene is derived from Aspergillus niger, and has an amino acid sequence shown in SEQ ID No.1 and other sequences having homology of 90% or more with SEQ ID No. 1; the nucleotide sequence is shown in SEQ ID NO.8, and other sequences with homology of 90% or more with SEQ ID NO. 8.

3. The method of claim 1, wherein the expression cassette is a gene fragment comprising a lysophospholipase gene sequence, a promoter, a regulatory sequence linked to the 3' end of the promoter, a terminator, a selection marker, and upstream and downstream homologous sequences.

4. The method of claim 3, wherein the promoter is selected from the group consisting of an Aspergillus niger endogenous promoter and an exogenous promoter; the Aspergillus niger endogenous promoter is an Aspergillus niger glucoamylase promoter, a neutral amylase promoter, an acid amylase promoter or an alpha-glucosidase promoter; the exogenous promoter is an aspergillus oryzae neutral amylase promoter or a rhizopus saccharidase promoter; the control sequence connected with the 3 'end of the promoter is a leader sequence 5' UT.

5. The method of claim 1, wherein the expression cassette further comprises a signal peptide sequence selected from the group consisting of a glucoamylase signal peptide, an acid amylase signal peptide, an aspergillus niger phytase signal peptide, and an aspergillus oryzae TAKA amylase signal peptide, preferably a signal peptide encoded by the lysophospholipase gene sequence itself, having an amino acid sequence as shown in SEQ ID No.2, and other sequences having 90% or greater homology to SEQ ID No. 2. The nucleotide sequence is shown in SEQ ID NO.9, and other sequences with homology of 90% or more with SEQ ID NO. 9.

6. The method according to claim 3, wherein the terminator is obtained from the following genes for enzymes: aspergillus niger glucoamylase, Aspergillus oryzae TAKA amylase, Aspergillus nidulans anthranilate synthase, Aspergillus niger alpha-glucosidase, or Fusarium oxysporum trypsin-like protease.

7. The method of construction according to claim 3, wherein the selectable marker element is selected from the group consisting of a selectable marker and/or a counter-selectable marker; said selectable marker is selected from the group consisting of acetamidase amdS, ornithine carbamoyltransferase argB, glufosinate bar, acetyltransferase, hygromycin phosphotransferase hyg, nitrate reductase niaD, orotidine-5' -phosphate decarboxylase pyrG, sulfate adenyltransferase sC, anthranilate synthase trpC, or equivalents thereof, preferably used in a cell of Aspergillus is amdS, hyg of Aspergillus nidulans or Aspergillus oryzae; the counter selection marker is selected from the group consisting of a selectable marker for filamentous fungal host cells or hsvTK, preferably acetamidase amdS, orotidine-5' -phosphate decarboxylase pyrG.

8. The method according to any one of claims 1 to 8, wherein the expression cassette nucleotide sequence of the lysophospholipase gene is shown in SEQ ID No.7, and other sequences having 90% or more homology with SEQ ID No. 7.

9. The method of claim 1 or claim 3, wherein the expression cassette is introduced by conventional methods, randomly inserted into the genome of the host A.niger or site-directed integration into one or more loci of the host A.niger. The locus is selected from any one of glucoamylase gla, neutral amylase amya, neutral amylase amyb, acid amylase aa, alpha glucosidase agda or alpha glucosidase agdb.

10. The method according to claim 1, wherein the host is Aspergillus niger in which the glucoamylase gene, the fungal amylase gene and the acid amylase gene are deleted.

11. A lysophospholipase-producing aspergillus niger recombinant expression strain constructed according to the method of any of claims 1-10.

Technical Field

The invention belongs to the field of genetic engineering breeding, and relates to construction and application of a high-yield lysophospholipase Aspergillus niger recombinant expression strain.

Background

Lysophospholipase is a biocatalyst that can efficiently degrade lysolecithin. Lysolecithin is present in a variety of animals and plants, including corn, wheat, barley, humans, and the like. Its inventory in corn is particularly high. However, lysolecithin forms micelles in oil-water mixtures due to its strong oil-water amphiphilicity, greatly increasing the viscosity of the oil-water mixtures and changing the rheological properties thereof, which makes it impossible to perform processes such as filtration, stirring, mixing, salting out, dissolution, and the like. The lysolecithin can be efficiently and quickly degraded by using lysophospholipase, so that the lysolecithin loses the capability of forming micelles, and flocculent precipitates are formed and separated out under the characteristic conditions. Thereby greatly improving the rheological properties of the solution. It has wide application in industrial production. However, the relatively low expression level of the polypeptide leads to high price, which limits the popularization of the polypeptide in the industry. Therefore, there is an urgent need in the industry to develop a method for mass production of lysophospholipase.

Very few lysophospholipases have been studied, such as those from aspergillus niger, nematodes, and humans. And there is little research focused on improving the industrial production thereof. The production source of the enzyme is mainly microbial fermentation expression. However, the expression levels in current fermentations are relatively low. In particular, Aspergillus niger derived lysophospholipases, which are expressed in the Cloning and characterization of Two lipases and a lysophospholipases from Aspergillus niger, are very low, if not undetectable. Aspergillus niger is a very efficient industrial biofermentation strain used to express a number of industrially very demanding enzymes, such as saccharifying enzymes and the like. Therefore, the expression of lysophospholipase by Aspergillus niger is a potential industrial production method with high yield potential. However, the expression level of the currently reported lysophospholipase in the study using Aspergillus niger as a host is still low.

Disclosure of Invention

The invention provides a method for constructing a lysophospholipase recombinant Aspergillus niger expression strain.

The invention also provides a lysophospholipase recombinant expression vector.

The purpose of the invention can be realized by the following technical scheme:

a method for constructing recombinant Aspergillus niger expression bacteria is to construct a recombinant expression cassette containing lysophospholipase gene sequence, wherein the recombinant expression cassette is a gene segment containing lysophospholipase gene sequence, promoter, terminator, signal peptide, screening marker and other elements.

The lysophospholipase gene is a lysophospholipase gene derived from aspergillus or penicillium; preferably Aspergillus niger derived lysophospholipase gene; more preferably has an amino acid sequence shown as SEQ ID NO. 1; and has a nucleotide sequence shown as SEQ ID NO. 8; the host cell is Aspergillus niger.

The promoter may be an aspergillus niger endogenous promoter: such as Aspergillus niger glucoamylase promoter, neutral amylase promoter, acid amylase promoter, alpha-glucosidase promoter, etc.; it may also be an exogenous promoter: such as Aspergillus oryzae neutral amylase promoter, Rhizopus oryzae glucoamylase promoter; aspergillus niger glucoamylase promoter or Aspergillus niger neutral amylase promoter is preferred in the present invention.

Linked to the 3' end of the promoter may be regulatory sequences: such as a suitable leader sequence (5' UTR), a nontranslated region of an mRNA that is important for translation by the host cell, such as the Aspergillus oryzae neutral amylase and Aspergillus nidulans triose phosphate isomerase leader sequences.

For secretory expression of a specific protein, signal peptide sequence mediation is required, and commonly used signal peptide sequences in Aspergillus niger are glucoamylase signal peptide, acid amylase signal peptide, Aspergillus niger phytase signal peptide, Aspergillus oryzae TAKA amylase signal peptide, and in the present invention, a signal peptide encoded by the lysophospholipase gene sequence itself is used.

Preferred terminators are obtained from the genes for the following enzymes: aspergillus niger glucoamylase, Aspergillus oryzae TAKA amylase, Aspergillus nidulans anthranilate synthase, Aspergillus niger alpha-glucosidase, and Fusarium oxysporum trypsin-like protease.

The specific gene is linked to a promoter, a regulatory sequence, a signal peptide sequence and a terminator to form an expression cassette. Can be introduced into the A.niger genome by conventional methods, either randomly inserted into the genome or site-directed integration at one or more loci. Alternative loci are gla (glucoamylase), amya (neutral amylase), amyb (neutral amylase), aa (acid amylase), agda (alpha glucosidase), agdb (alpha glucosidase).

The expression cassette may preferably be linked to one or more selectable markers which allow for easy selection of transformed, transfected, transduced, or the like cells or strains. Selectable markers are genes whose products provide biocide or viral resistance, resistance to heavy metals, prototrophy to auxotrophs (prototrophy to autotrophs), and the like. Selectable markers for use in a filamentous fungal host cell include, but are not limited to, amdS (acetamidase), argB (ornithine carbamoyltransferase), bar (glufosinate) acetyltransferase, hyg (hygromycin phosphotransferase), niaD (nitrate reductase), pyrG (orotidine-5' -phosphate decarboxylase), sC (sulfate adenyltransferase), and trpC (anthranilate synthase) and equivalents thereof. Preferred for use in an Aspergillus cell is the amdS or hyg of Aspergillus nidulans (Aspergillus nidulans) or Aspergillus oryzae.

The expression cassette may preferably be linked to one or more counter-selectable markers (negative selection markers). Selectable markers for use in filamentous fungal host cells include, but are not limited to, amdS (acetamidase), pyrG (orotidine-5' -phosphate decarboxylase), hsvTK (herpes simplex virus thymidine kinase).

The preferable nucleotide sequence of the expression cassette is shown as SEQ ID NO. 7.

The expression cassette is introduced into the genome of a host Aspergillus niger by a conventional method and randomly inserted into the genome of the host Aspergillus niger or site-specific integrated into one or more loci of the host Aspergillus niger.

The gene locus is selected from glucoamylase gla, neutral amylase amya, neutral amylase amyb, acid amylase aa, alpha-glucosidase agda and alpha-glucosidase agdb.

The host is Aspergillus niger with the glucoamylase gene, the fungal amylase gene and the acid amylase gene being knocked out.

The recombinant expression strain of the aspergillus niger capable of producing lysophospholipase is constructed according to the method.

A recombinant expression vector comprises the expression cassette containing the lysophospholipase gene.

The invention relates to application of a lysophospholipase-producing Aspergillus niger recombinant expression strain in lysophospholipase production.

The invention has the advantages of

According to the invention, an expression cassette of lysophospholipase is constructed by means of genetic engineering and is introduced into an Aspergillus niger expression host bacterium, so that the high-efficiency secretory expression of lysophospholipase is realized, and the Aspergillus niger expression strain with high yield of lysophospholipase is obtained. By optimizing the fermentation conditions, the protein expression level of the crude enzyme liquid of the fermented supernatant can reach 2.88g/L, the activity can reach 2345unit/ml, and the specific enzyme activity can reach 815 unit/mg.

Drawings

The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the principles of the invention and not to limit the invention. In the drawings:

FIG. 1: a p-ZYAN01 plasmid map;

FIG. 2: p-ZYAN02-LPPL plasmid map.

Detailed Description

The preferred embodiments of the present invention will be described in conjunction with the accompanying drawings, and it will be understood that they are described herein for the purpose of illustration and explanation and not limitation.

Examples

Construction of p-ZYAN02-LPPL plasmid

The preparation of the plasmid mainly comprises the following two steps:

1. an intermediate plasmid p-ZYAN01 was prepared.

2. Linearizing intermediate plasmid p-ZYAN01, and integrating lysophospholipase gene expression cassette and upstream and downstream homologous fragments into p-ZYAN01 to form p-ZYAN02-LPPL plasmid

The intermediate plasmid p-ZYAN01 was prepared as follows:

the p-ZYAN01 is mainly composed of the following parts and necessary connecting sequences, or is composed of the following parts directly.

(1) A 2305bp fragment obtained after double digestion of pUC57 plasmid XbaI-PscI;

(2) the sequence of the hyg gene expression cassette is shown in SEQ ID NO. 3;

(3) amds expression cassette, sequence shown in SEQ ID No. 4.

The 2305bp fragment obtained by double digestion of pUC57 plasmid XbaI-PciI, the hyg gene expression cassette, and the amds expression cassette, the three gene fragment sequence fragments were recombined by Gibson Master Mix Kit (E2611, New England Biolabs) to obtain recombinant plasmid p-ZYAN01 (FIG. 1). Expression of the lysophospholipase expression cassette is integrated into the aspergillus niger glucoamylase locus for expression using a glucoamylase promoter and a glucoamylase terminator. Constructing lysophospholipase integration expression plasmid p-ZYAN 02-LPPL. The construction method of the integration plasmid is as follows: linearizing the p-ZYAN01 plasmid by a method commonly used in the industry; the Aspergillus niger gene fragment SEQ ID NO.5 is taken as a 5 'end homologous fragment, the Aspergillus niger gene fragment SEQ ID NO.6 is taken as a 3' end homologous fragment, and each fragment is 2000bp long. The linearized p-ZYAN01 vector, the homologous fragment and the lysophospholipase expression cassette fragment were recombined by GibsoMaster Mix Kit (E2611, New England Biolabs) to obtain an integration plasmid p-ZYAN02-LPPL, which contains a lysophospholipase expression cassette containing an Aspergillus niger glucoamylase promoter sequence, an Aspergillus niger-derived lysophospholipase sequence and an Aspergillus niger glucoamylase terminator sequence, and the sequences were confirmed by sequencing, and the plasmid map is shown in FIG. 2.

Integration of lysophospholipase expression cassette by transformation

The starting strain in this example is ZYAN05, which is obtained by knocking out glucoamylase gene, fungal amylase gene and acid amylase gene from conventional strains. The gene knockout/knock-in method in Aspergillus niger can be realized by referring to the technical method disclosed in the patent C N103937766A or CN 104962594A example. I.e., the method described with reference to Delmas (apple Environ Microbiol.2014,80(11):3484-7), et al. Specifically, circular DNA vectors are used, which contain the 5 'and 3' homologous sequences, a selectable marker, and an E.coli replication sequence. The circular vector is transferred into Aspergillus niger, and a recombinant strain is obtained by selection.

The protoplast transformation method is adopted to introduce the p-ZYAN02-LPPL plasmid into Aspergillus niger ZYAN05, and the specific operation steps are as follows: preparation of protoplast: aspergillus niger mycelia were cultured in a nutrient-rich TZ liquid medium (beef extract powder 0.8%, yeast extract 0.2%, peptone 0.5%, NaCl 0.2%, sucrose 3%, pH 5.8). The mycelium was filtered from the culture broth by mira-cloth (Calbiochem Co.) and washed with 0.7M NaCl (pH5.8), and after draining the mycelium was transferred to an enzymatic hydrolysate (pH5.8) containing 1% cellulase (Sigma), 1% helicase (Sigma) and 0.2% lywallzyme (Sigma) and enzymatically hydrolyzed at 30 ℃ at 65rpm for 3 hours. Then putting the enzymatic hydrolysate containing the protoplast on ice, filtering the enzymatic hydrolysate by using four layers of mirror paper, carrying out mild centrifugation on the obtained filtrate at 3000rpm and 4 ℃ for 10min, and then removing the supernatant; the protoplasts attached to the vessel wall were washed once with STC solution (1M DSorbitol, 50mM CaCl2, 10mM Tris, pH7.5) and finally the protoplasts were resuspended in the appropriate amount of STC solution.

Adding 10 μ l (concentration: 100ng/μ l) of circular p-ZYAN02-LPPL plasmid into 100 μ l of protoplast suspension, mixing, and standing at room temperature for 25 min; then adding 900 mul PEG solution into the mixture for 3 times, mixing evenly, and standing for 25min at room temperature; centrifuging at 3000rpm for 10min at normal temperature, discarding the supernatant, attaching protoplasts to the tube wall, and resuspending them in 1ml of STC solution. Mixing the suspension with a culture medium (acetamide 0.3%, sucrose 20%, agar 0.7%) previously cooled to about 45 deg.C, and plating; after the flat plate is solidified, putting the flat plate into an incubator at 34 ℃ for culture; after 24h, a layer of solid medium (agar 1% and the rest as above) containing 300 ng/. mu.l Hygromycin (Hygromycin) was spread on the plate, and the plate was further placed in an incubator at 34 ℃ for 4-5 days, after which transformants that had grown in the upper medium were called integrative transformants. Randomly selecting several integrative transformants, respectively subculturing the integrative transformants on a solid culture medium containing 300 ng/mu l of Hygromycin (Hygromycin), after culturing for 3 days at a constant temperature of 34 ℃, collecting mycelia, freezing the mycelia by using liquid nitrogen, grinding and crushing the mycelia, then extracting the genomic DNA of the integrative transformants by using a fungus genome extraction kit (Boy science and technology, Ltd. in Hangzhou), and finally performing PCR identification on the genomic DNA of the integrative transformants. Integration into the glucoamylase locus was confirmed by sequencing of the PCR product. And (4) sequencing the positive transformant by using a PCR product and then confirming to obtain a recombinant expression strain.

Liquid fermentation production of lysophospholipase by recombinant expression strain

Slant culture: inoculating one strain of the Aspergillus niger recombinant expression strain to a PDA solid inclined plane, and culturing at constant temperature of 35 ℃ for 60 h; and (3) shake flask culture: inoculating one strain of the strain obtained by slant culture into seed culture medium, and culturing at initial pH of 5.5 and 35 deg.C and shaking table rotation speed of 200rpm for about 110 h. The following table shows the enzyme production in 2 batches of 250ml shake flasks,

the slant culture medium is as follows: 20g of sucrose, 32 g of NaNO, 40.5 g of MgSO40, 0.5g of KCl, 40.01g of FeSO40, 41 g of K2HPO and 20g of agar, and the components are dissolved in 1000mL of water, and the pH is adjusted to 5.5,121 ℃ for sterilization for 20min for later use.

The shake flask seed culture medium is as follows: 200mL of wort and 5g of bean cake powder, adjusting the pH value to 5.5,121 ℃, and sterilizing for 20min for later use.

Meanwhile, the same shake flask culture is carried out by using unmodified aspergillus niger strains, and the supernatant is taken for detection, so that no lysophospholipase enzyme activity is detected. The fermentation supernatants of the engineered Aspergillus niger produced large amounts of lysophospholipase using SDS-PAGE testing of the supernatants of both species (test method is conventional in the industry) (FIG. 3A, arrow). Accordingly, the unmodified A.niger lysophospholipase species did not produce detectable lysophospholipase protein bands.

Meanwhile, in a control study of Cloning and characterization of Two lipases and a lysophospholipases from Aspergillus niger, the lysophospholipase activity in the supernatant of the A.niger engineered strain was only 14 units/ml.

Finally, it should be noted that: although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that changes may be made in the embodiments and/or equivalents thereof without departing from the spirit and scope of the invention. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Sequence listing

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atgtcaacaa gaataaaacg cgttttcggg tttacctctt ccagatacag ctcatctgca 1980

atgcattaat gcattgactg caacctagta acgccttcag gctccggcga agagaagaat 2040

agcttagcag agctattttc attttcggga gacgagatca agcagatcaa cggtcgtcaa 2100

gagacctacg agactgagga atccgctctt ggctccacgc gactatatat ttgtctctaa 2160

ttgtactttg acatgctcct cttctttact ctgatagctt gactatgaaa attccgtcac 2220

cagccctggg ttcgcaaaga taattgcatg tttcttcctt gaactctcaa gcctacagga 2280

cacacattca tcgtaggtat aaacctcgaa atcattccta ctaagatggt atacaatagt 2340

aaccatggtt gcctagtgaa tgctccgtaa cacccaatac gccggccgaa acttttttac 2400

aactctccta tgagtcgttt acccagaatg cacaggtaca cttgtttaga ggtaatcctt 2460

ctttctagaa gtcctcgtgt actgtgtaag cgcccactcc acatctccac tcgag 2515

<210> 4

<211> 2724

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

ctagatctac gccaggaccg agcaagccca gatgagaacc gacgcagatt tccttggcac 60

ctgttgcttc agctgaatcc tggcaatacg agatacctgc tttgaatatt ttgaatagct 120

cgcccgctgg agagcatcct gaatgcaagt aacaaccgta gaggctgaca cggcaggtgt 180

tgctagggag cgtcgtgttc tacaaggcca gacgtcttcg cggttgatat atatgtatgt 240

ttgactgcag gctgctcagc gacgacagtc aagttcgccc tcgctgcttg tgcaataatc 300

gcagtgggga agccacaccg tgactcccat ctttcagtaa agctctgttg gtgtttatca 360

gcaatacacg taatttaaac tcgttagcat ggggctgata gcttaattac cgtttaccag 420

tgccgcggtt ctgcagcttt ccttggcccg taaaattcgg cgaagccagc caatcaccag 480

ctaggcacca gctaaaccct ataattagtc tcttatcaac accatccgct cccccgggat 540

caatgaggag aatgaggggg atgcggggct aaagaagcct acataaccct catgccaact 600

cccagtttac actcgtcgag ccaacatcct gactataagc taacacagaa tgcctcaatc 660

ctgggaagaa ctggccgctg ataagcgcgc ccgcctcgca aaaaccatcc ctgatgaatg 720

gaaagtccag acgctgcctg cggaagacag cgttattgat ttcccaaaga aatcggggat 780

cctttcagag gccgaactga agatcacaga ggcctccgct gcagatcttg tgtccaagct 840

ggcggccgga gagttgacct cggtggaagt tacgctagca ttctgtaaac gggcagcaat 900

cgcccagcag ttagtagggt cccctctacc tctcagggag atgtaacaac gccaccttat 960

gggactatca agctgacgct ggcttctgtg cagacaaact gcgcccacga gttcttccct 1020

gacgccgctc tcgcgcaggc aagggaactc gatgaatact acgcaaagca caagagaccc 1080

gttggtccac tccatggcct ccccatctct ctcaaagacc agcttcgagt caaggtacac 1140

cgttgcccct aagtcgttag atgtcccttt ttgtcagcta acatatgcca ccagggctac 1200

gaaacatcaa tgggctacat ctcatggcta aacaagtacg acgaagggga ctcggttctg 1260

acaaccatgc tccgcaaagc cggtgccgtc ttctacgtca agacctctgt cccgcagacc 1320

ctgatggtct gcgagacagt caacaacatc atcgggcgca ccgtcaaccc acgcaacaag 1380

aactggtcgt gcggcggcag ttctggtggt gagggtgcga tcgttgggat tcgtggtggc 1440

gtcatcggtg taggaacgga tatcggtggc tcgattcgag tgccggccgc gttcaacttc 1500

ctgtacggtc taaggccgag tcatgggcgg ctgccgtatg caaagatggc gaacagcatg 1560

gagggtcagg agacggtgca cagcgttgtc gggccgatta cgcactctgt tgagggtgag 1620

tccttcgcct cttccttctt ttcctgctct ataccaggcc tccactgtcc tcctttcttg 1680

ctttttatac tatatacgag accggcagtc actgatgaag tatgttagac ctccgcctct 1740

tcaccaaatc cgtcctcggt caggagccat ggaaatacga ctccaaggtc atccccatgc 1800

cctggcgcca gtccgagtcg gacattattg cctccaagat caagaacggc gggctcaata 1860

tcggctacta caacttcgac ggcaatgtcc ttccacaccc tcctatcctg cgcggcgtgg 1920

aaaccaccgt cgccgcactc gccaaagccg gtcacaccgt gaccccgtgg acgccataca 1980

agcacgattt cggccacgat ctcatctccc atatctacgc ggctgacggc agcgccgacg 2040

taatgcgcga tatcagtgca tccggcgagc cggcgattcc aaatatcaaa gacctactga 2100

acccgaacat caaagctgtt aacatgaacg agctctggga cacgcatctc cagaagtgga 2160

attaccagat ggagtacctt gagaaatggc gggaggctga agaaaaggcc gggaaggaac 2220

tggacgccat catcgcgccg attacgccta ccgctgcggt acggcatgac cagttccggt 2280

actatgggta tgcctctgtg atcaacctgc tggatttcac gagcgtggtt gttccggtta 2340

cctttgcgga taagaacatc gataagaaga atgagagttt caaggcggtt agtgagcttg 2400

atgccctcgt gcaggaagag tatgatccgg aggcgtacca tggggcaccg gttgcagtgc 2460

aggttatcgg acggagactc agtgaagaga ggacgttggc gattgcagag gaagtgggga 2520

agttgctggg aaatgtggtg actccatagc taataagtgt cagatagcaa tttgcacaag 2580

aaatcaatac cagcaactgt aaataagcgc tgaagtgacc atgccatgct acgaaagagc 2640

agaaaaaaac ctgccgtaga accgaagaga tatgacacgc ttccatctct caaaggaaga 2700

atcccttcag ggttgcgttt ccag 2724

<210> 5

<211> 2000

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

ctaccaatgc tctcgaggat tgcctgaaca ttgacattcg gcgtccggcc gggaccaccg 60

cggactcgaa gctgcctgtg ctggtctgga tctttggcgg aggctttgaa cttggttcaa 120

aggcgatgta tgatggtaca acgatggtat catcgtcgat agacaagaac atgcctatcg 180

tgtttgtagc aatgaattat cgcgtgggag gtttcgggtt cttgcccgga aaggagatcc 240

tggaggacgg gtccgcgaac ctagggctcc tggaccaacg ccttgccctg cagtgggttg 300

ccgacaacat cgaggccttt ggtggagacc cggacaaggt gacgatttgg ggagaatcag 360

caggagccat ttccgttttt gatcagatga tcttgtacga cggaaacatc acttacaagg 420

ataagccctt gttccggggg gccatcatgg actccggtag tgttgttccc gcagaccccg 480

tcgatggggt caagggacag caagtatatg atgcggtagt ggaatctgca ggctgttcct 540

cttctaacga caccctagct tgtctgcgtg aactagacta caccgacttc ctcaatgcgg 600

caaactccgt gccaggcatt ttaagctacc attctgtggc gttatcatat gtgcctcgac 660

cggacgggac ggcgttgtcg gcatcaccgg acgttttggg caaagcaggg aaatatgctc 720

gggtcccgtt catcgtgggc gaccaagagg atgaggggac cttattcgcc ttgtttcagt 780

ccaacattac gacgatcgac gaggtggtcg actacctggc ctcatacttc ttctatgacg 840

ctagccgaga gcagcttgaa gaactagtgg ccctgtaccc agacaccacc acgtacgggt 900

ctccgttcag gacaggcgcg gccaacaact ggtatccgca atttaagcga ttggccgcca 960

ttctcggcga cttggtcttc accattaccc ggcgggcatt cctctcgtat gcagaggaaa 1020

tctcccctga tcttccgaac tggtcgtacc tggcgaccta tgactatggc accccagttc 1080

tggggacctt ccacggaagt gacctgctgc aggtgttcta tgggatcaag ccaaactatg 1140

cagctagttc tagccacacg tactatctga gctttgtgta tacgctggat ccgaactcca 1200

accgggggga gtacattgag tggccgcagt ggaaggaatc gcggcagttg atgaatttcg 1260

gagcgaacga cgccagtctc cttacggatg atttccgcaa cgggacatat gagttcatcc 1320

tgcagaatac cgcggcgttc cacatctgat gccattggcg gaggggtccg gacggtcagg 1380

aacttagcct tatgagatga atgatggacg tgtctggcct cggaaaagga tatatgggga 1440

tcatgatagt actagccata ttaatgaagg gcatatacca cgcgttggac ctgcgttata 1500

gcttcccgtt agttatagta ccatcgttat accagccaat caagtcacca cgcacgaccg 1560

gggacggcga atccccggga attgaaagaa attgcatccc aggccagtga ggccagcgat 1620

tggccacctc tccaaggcac agggccattc tgcagcgctg gtggattcat cgcaatttcc 1680

cccggcccgg cccgacaccg ctataggctg gttctcccac accatcggag attcgtcgcc 1740

taatgtctcg tccgttcaca agctgaagag cttgaagtgg cgagatgtct ctgcaggaat 1800

tcaagctaga tgctaagcga tattgcatgg caatatgtgt tgatgcatgt gcttcttcct 1860

tcagcttccc ctcgtgcaga tgaggtttgg ctataaattg aagtggttgg tcggggttcc 1920

gtgaggggct gaagtgcttc ctccctttta gacgcaactg agagcctgag cttcatcccc 1980

agcatcatta cacctcagca 2000

<210> 6

<211> 2000

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

acaatcaatc catttcgcta tagttaaagg atggggatga gggcaattgg ttatatgatc 60

atgtatgtag tgggtgtgca taatagtagt gaaatggaag ccaagtcatg tgattgtaat 120

cgaccgacgg aattgaggat atccggaaat acagacaccg tgaaagccat ggtctttcct 180

tcgtgtagaa gaccagacag acagtccctg atttaccctt gcacaaagca ctagaaaatt 240

agcattccat ccttctctgc ttgctctgct gatatcactg tcattcaatg catagccatg 300

agctcatctt agatccaagc acgtaattcc atagccgagg tccacagtgg agcagcaaca 360

ttccccatca ttgctttccc caggggcctc ccaacgacta aatcaagagt atatctctac 420

cgtccaatag atcgtcttcg cttcaaaatc tttgacaatt ccaagagggt ccccatccat 480

caaacccagt tcaataatag ccgagatgca tggtggagtc aattaggcag tattgctgga 540

atgtcggggc cagttggccc ggtggtcatt ggccgcctgt gatgccatct gccactaaat 600

ccgatcattg atccaccgcc cacgaggcgc gtctttgctt tttgcgcggc gtccaggttc 660

aactctctct gcagctccag tccaacgctg actgactagt ttacctactg gtctgatcgg 720

ctccatcaga gctatggcgt tatcccgtgc cgttgctgcg caatcgctat cttgatcgca 780

accttgaact cactcttgtt ttaatagtga tcttggtgac ggagtgtcgg tgagtgacaa 840

ccaacatcgt gcaagggaga ttgatacgga attgtcgctc ccatcatgat gttcttgccg 900

gctttgttgg ccctattcgt gggatgcgat gccctcgctg tgcagcagca ggtactgctg 960

gatgaggagc catcggtctc tgcacgcaaa cccaacttcc tcttcattct cacggatgat 1020

caggatctcc ggatgaattc tccggcgtat atgccgtata cgcaggcgag aatcaaggaa 1080

aagggtaccg agttcttgaa ccatttcgtc actaccgcgc tttgctgtcc gtcgcgcgtg 1140

agtctttgga cgggaagaca ggctcataat actaatgtga cggatgtgaa cccgccttat 1200

ggtatggaca ctgcttcgat cggtcttgat tcttcagcgt ggttacaatt gctaatgcgg 1260

cataggcgga taccccaaat tcgtcgctca aggcttcaac gaaaacttcc tccccgtttg 1320

gctgcagtcc gccggttaca atacctacta cacggggaag ctgttcaact cgcacagtgt 1380

cgctacctat aacgcgccct ttgtgaacgg tttcaatggc tccgacttcc tcctcgaccc 1440

ccacacatat tcctactgga atgcgacata ccagcgaaac catgagcctc cgcggagtta 1500

cgagggacaa tatactacgg atgtgatgaa ggagaaggca tcgggattgt tggcagatgc 1560

gctggacagt gacgcgccat tcttcctgac ggtcgcgccg atcgcaccgc acacgaacat 1620

cgatgtggag gggctgagcg gtgcgggtgg accgaagatg acagagccgc tgcctgcacc 1680

gagacatgcg catttgtttg ctgatgcaaa ggtgccgcgg acgcctaatt tcaatccgga 1740

caaggtgtgt gatatcctga cacagtggtg gggacgggca ctgacaagag taggattctg 1800

gtgcggggtg gatccaaacc atggaactac agaaccagac cgtcatcgac tacgaagacc 1860

atctttatcg ccagcgtctg cgcactttgc aagccgtcga tgagatggtg gatgcgctga 1920

tcacgcagct ggaagaaagt gggcagatcg acaataccta catcatttac agtgctgata 1980

acggctacca cattggccat 2000

<210> 7

<211> 3913

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

tgtacagggg cataaaatta cgcactaccc gaatcgatag aactactcat ttttatatag 60

aagtcagaat tcatggtgtt ttgatcattt taaattttta tatggcgggt ggtgggcaac 120

tcgcttgcgc gggcaactcg cttaccgatt acgttagggc tgatatttac gtaaaaatcg 180

tcaagggatg caagaccaaa ccgttaaatt tccggagtca acagcatcca agcccaagtc 240

cttcacggag aaaccccagc gtccacatca cgagcgaagg accacctcta ggcatcggac 300

gcaccatcca attagaagca gcaaagcgaa acagcccaag aaaaaggtcg gcccgtcggc 360

cttttctgca acgctgatca cgggcagcga tccaaccaac accctccaga gtgactaggg 420

gcggaaattt atcgggatta atttccactc aaccacaaat cacagtcgtc cccggtattg 480

tcctgcagac ggcaatttaa cggcttctgc gaatcgcttg gattccccgc ccctggccgt 540

agagcttaaa gtatgtccct tgtcgatgcg atgtatcaca acataaattt ttatatggcg 600

ggtggtgggc aactcgcttg cgcgggcaac tcgcttaccg attacgttag ggctgatatt 660

tacgtaaaaa tcgtcaaggg atgcaagacc aaaccgttaa atttccggag tcaacagcat 720

ccaagcccaa gtccttcacg gagaaacccc agcgtccaca tcacgagcga aggaccacct 780

ctaggcatcg gacgcaccat ccaattagaa gcagcaaagc gaaacagccc aagaaaaagg 840

tcggcccgtc ggccttttct gcaacgctga tcacgggcag cgatccaacc aacaccctcc 900

agagtgacta ggggcggaaa tttatcggga ttaatttcca ctcaaccaca aatcacagtc 960

gtccccggta ttgtcctgca gacggcaatt taacggcttc tgcgaatcgc ttggattccc 1020

cgcccctggc cgtagagctt aaagtatgtc ccttgtcgat gcgatgtatc acaacataaa 1080

tttttatatg gcgggtggtg ggcaactcgc ttgcgcgggc aactcgctta ccgattacgt 1140

tagggctgat atttacgtaa aaatcgtcaa gggatgcaag accaaaccgt taaatttccg 1200

gagtcaacag catccaagcc caagtccttc acggagaaac cccagcgtcc acatcacgag 1260

cgaaggacca cctctaggca tcggacgcac catccaatta gaagcagcaa agcgaaacag 1320

cccaagaaaa aggtcggccc gtcggccttt tctgcaacgc tgatcacggg cagcgatcca 1380

accaacaccc tccagagtga ctaggggcgg aaatttatcg ggattaattt ccactcaacc 1440

acaaatcaca gtcgtccccg gtattgtcct gcagacggca atttaacggc ttctgcgaat 1500

cgcttggatt ccccgcccct ggccgtagag cttaaagtat gtcccttgtc gatgcgatgt 1560

atcacaacat ataaatactg gcaagggatg ccatgcttgg agtttccaac tcaatttacc 1620

tctatccaca cttctcttcc ttcctcaatc ctctatatac acaactgggg atccttcacc 1680

atgaagttca atgcactctt aacgaccctc gcggcgctgg ggtatatcca aggaggcgcc 1740

gcggttccta caaccgtcga cctcacatat gcagacatat cacctcgcgc actggataat 1800

gcccctgatg gttatacccc gagcaatgta tcctgtcctg caaacagacc gacgattcgc 1860

agcgcgtcaa ccctgtcatc gaacgagacg gcatgggtgg acgtccggcg taagcagact 1920

gtctcagcga tgaaagacct tttcggccat atcaacatga gctcatttga cgctatttcg 1980

tacatcaaca gccattcatc aaatatcacc aacataccca acatcggtat tgccgtgtcc 2040

ggcggtggct acagagccct gaccaacggc gcgggagcac tcaaggcatt cgacagtcga 2100

acggaaaact caacccataa tggacagctc ggtggtcttc tgcagtcagc cacatacctg 2160

tccggtctct ccggaggtgg ctggctcctg ggctcaatct acatcaacaa cttcaccacc 2220

gtctccaatc tgcaaaccta caaagagggc gaagtctggc agttccagaa ttcaatcacg 2280

aaaggcccaa agaccaacgg cttgcaggct tgggatacag ccaagtacta ccgcgatctg 2340

gccaaggtgg tcgctggcaa gaaggacgcg ggcttcaaca cttccttcac ggactactgg 2400

ggtcgcgcac tctcctacca gctgattaac gcgaccgacg gaggcccagg ctacacctgg 2460

tcatcgatcg ctttaaccca ggacttcaag aacggaaaca tgcccatgcc gctccttgtc 2520

gccgacggcc gcaacccagg cgagacccta atcggcagca actcgaccgt gtatgagttc 2580

aacccctggg aattcggcag ttttgatccg tccatcttcg gcttcgctcc cctcgaatac 2640

ctcggatcct actttgagaa cggcgaagtc ccatccagcc gatcctgcgt ccgcggcttc 2700

gataacgcag gcttcgtcat gggaacctcc tccagtctct tcaaccaatt catcctgaag 2760

ctcaacacca ccgacatccc atcaaccctc aaaacggtca tcgccagcat cctagaagaa 2820

ctaggcgacc gcaacgacga catcgccatc tactctccca accccttcta cgggtaccgc 2880

aacgcgacag tttcatacga aaagaccccg gacctgaacg tcgtcgacgg tggcgaagac 2940

aaacagaacc tccccctcca tcctctcatc caacccgccc gcaacgtgga cgtcatcttc 3000

gccgtcgact cctcagccag tacctcggac aactggccca acggaagtcc tctcgtcgcg 3060

acttacgaac gtagtctcaa ctcaaccggt atcggaaacg gcaccgcgtt ccctagcatc 3120

ccggacaaga gcaccttcat taacctgggc ttgaacaccc gtccgacttt cttcggctgc 3180

aatagttcca atatcacagg ccatgcaccc ctggttgtct acctccccaa ctacccctac 3240

acaaccctct ccaacaagtc gaccttccag ctcaagtacg agatcttgga gcgtgatgag 3300

atgatcacca atggctggaa cgtggttact atgggtaatg gatcaaggaa gtcttacgag 3360

gattggccga cttgtgcggg ctgcgctatt ctgagtcgct cgtttgatcg gactaatacc 3420

caggtgccgg atatgtgctc gcagtgtttt gacaagtatt gctgggatgg aacgaggaat 3480

agtacgacgc cggcggcgta tgagccgaag gtattgatgg ctagtgcggg tgtgaggggt 3540

atttcgatgt cgaggttggt tttgggtctc tttccggtgg tggttggggt ttggatgatg 3600

taactcgaga tctagagggt gactgacacc tggcggtaga caatcaatcc atttcgctat 3660

agttaaagga tggggatgag ggcaattggt tatatgatca tgtatgtagt gggtgtgcat 3720

aatagtagtg aaatggaagc caagtcatgt gattgtaatc gaccgacgga attgaggata 3780

tccggaaata cagacaccgt gaaagccatg gtctttcctt cgtgtagaag accagacaga 3840

cagtccctga tttacccttg cacaaagcac tagaaaatta gcattccatc cttctctgct 3900

tgctctgctg ata 3913

<210> 8

<211> 1857

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

gttcctacaa ccgtcgacct cacatatgca gacatatcac ctcgcgcact ggataatgcc 60

cctgatggtt ataccccgag caatgtatcc tgtcctgcaa acagaccgac gattcgcagc 120

gcgtcaaccc tgtcatcgaa cgagacggca tgggtggacg tccggcgtaa gcagactgtc 180

tcagcgatga aagacctttt cggccatatc aacatgagct catttgacgc tatttcgtac 240

atcaacagcc attcatcaaa tatcaccaac atacccaaca tcggtattgc cgtgtccggc 300

ggtggctaca gagccctgac caacggcgcg ggagcactca aggcattcga cagtcgaacg 360

gaaaactcaa cccataatgg acagctcggt ggtcttctgc agtcagccac atacctgtcc 420

ggtctctccg gaggtggctg gctcctgggc tcaatctaca tcaacaactt caccaccgtc 480

tccaatctgc aaacctacaa agagggcgaa gtctggcagt tccagaattc aatcacgaaa 540

ggcccaaaga ccaacggctt gcaggcttgg gatacagcca agtactaccg cgatctggcc 600

aaggtggtcg ctggcaagaa ggacgcgggc ttcaacactt ccttcacgga ctactggggt 660

cgcgcactct cctaccagct gattaacgcg accgacggag gcccaggcta cacctggtca 720

tcgatcgctt taacccagga cttcaagaac ggaaacatgc ccatgccgct ccttgtcgcc 780

gacggccgca acccaggcga gaccctaatc ggcagcaact cgaccgtgta tgagttcaac 840

ccctgggaat tcggcagttt tgatccgtcc atcttcggct tcgctcccct cgaatacctc 900

ggatcctact ttgagaacgg cgaagtccca tccagccgat cctgcgtccg cggcttcgat 960

aacgcaggct tcgtcatggg aacctcctcc agtctcttca accaattcat cctgaagctc 1020

aacaccaccg acatcccatc aaccctcaaa acggtcatcg ccagcatcct agaagaacta 1080

ggcgaccgca acgacgacat cgccatctac tctcccaacc ccttctacgg gtaccgcaac 1140

gcgacagttt catacgaaaa gaccccggac ctgaacgtcg tcgacggtgg cgaagacaaa 1200

cagaacctcc ccctccatcc tctcatccaa cccgcccgca acgtggacgt catcttcgcc 1260

gtcgactcct cagccagtac ctcggacaac tggcccaacg gaagtcctct cgtcgcgact 1320

tacgaacgta gtctcaactc aaccggtatc ggaaacggca ccgcgttccc tagcatcccg 1380

gacaagagca ccttcattaa cctgggcttg aacacccgtc cgactttctt cggctgcaat 1440

agttccaata tcacaggcca tgcacccctg gttgtctacc tccccaacta cccctacaca 1500

accctctcca acaagtcgac cttccagctc aagtacgaga tcttggagcg tgatgagatg 1560

atcaccaatg gctggaacgt ggttactatg ggtaatggat caaggaagtc ttacgaggat 1620

tggccgactt gtgcgggctg cgctattctg agtcgctcgt ttgatcggac taatacccag 1680

gtgccggata tgtgctcgca gtgttttgac aagtattgct gggatggaac gaggaatagt 1740

acgacgccgg cggcgtatga gccgaaggta ttgatggcta gtgcgggtgt gaggggtatt 1800

tcgatgtcga ggttggtttt gggtctcttt ccggtggtgg ttggggtttg gatgatg 1857

<210> 9

<211> 63

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 9

atgaagttca atgcactctt aacgaccctc gcggcgctgg ggtatatcca aggaggcgcc 60

gcg 63