1. A gene for coding alpha-agarase is characterized in that a nucleotide sequence for coding the alpha-agarase is shown as SEQ ID No. 1.

2. A recombinant vector carrying the gene of claim 1.

3. A recombinant cell carrying the gene of claim 1, or the recombinant vector of claim 2.

4. A recombinant Bacillus subtilis comprising the gene of claim 1 or the recombinant vector of claim 2.

5. The recombinant Bacillus subtilis according to claim 4 wherein Bacillus subtilis WB600, Bacillus subtilis WB800 or Bacillus subtilis 168 is the expression host.

6. The recombinant Bacillus subtilis of claim 4 or 5 wherein the expression vector is pHT01 plasmid, pHT43 plasmid or pHT304 plasmid.

7. A method for preparing setron tetrasaccharide or setron hexasaccharide is characterized by comprising the following steps: preparing the setron tetraose or the setron hexaose by fermenting the recombinant cell of claim 3 or the recombinant bacillus subtilis of any one of claims 4 to 6.

8. The method according to claim 7, wherein the recombinant cells or recombinant Bacillus subtilis are inoculated into a seed medium to prepare a seed solution, and the seed solution is inoculated into a fermentation medium to perform fermentation.

9. The method of claim 8, wherein the seed solution is inoculated into a fermentation medium according to the proportion of 2-4%, the seed solution is cultured for 60-72 hours at 37 ℃, and the fermentation liquid is centrifuged to take the supernatant, namely the fermentation liquid is fermented to prepare the agarotetraose or the agarohexaose.

10. Use of the gene of claim 1, or the recombinant vector of claim 2, or the recombinant cell of claim 3, or the recombinant bacillus subtilis of any one of claims 4 to 6, or the method of any one of claims 7 to 9 for the preparation of a product comprising setron tetraose or setron hexaose.

Background

Agar is a marine polysaccharide of economic value which is extracted from gelidium and other red algae. The hydrolyzed agar generates agar oligosaccharide with the polymerization degree of 2-20, has the advantages of good water solubility, high bioavailability and the like, and can be widely applied to the fields of food, medicines, cosmetics and the like.

The agar oligosaccharide has a plurality of unique medical health care functions, can be used as a raw material of functional foods, medicines and cosmetics, and is mainly expressed as follows: 1) the agar oligosaccharide has antioxidant effect, and can remove active oxygen free radicals directly or by improving activity of antioxidant enzymes, and also has oxidation inhibiting effect by chelating agar oligosaccharide with metal ions essential for free radicals. Therefore, the product can be applied to health-care food with the functions of resisting fatigue and the like. 2) The agar oligosaccharide can inhibit the generation of nitric oxide in macrophage and monocyte, and the content of prostaglandin E2 and the increase of cell active substance level of proinflammatory reaction, and has the functions of resisting tumor and improving immunity. Can be used in the production of anti-tumor, anti-inflammatory and antiviral medicines. 3) Researches find that the agar oligosaccharide has good moisturizing and whitening effects, can inhibit the activity of tyrosinase in melanoma cells, and has no toxicity to the cells. Therefore, it can be used as a high-quality raw material for cosmetics. 4) The agar oligosaccharide has certain antibacterial activity, and can inhibit bacterial growth. The agar oligosaccharide can be used as natural antiseptic in food, and can reduce food calorie. 5) In vivo and in vitro experiments prove that the new agaro-oligosaccharide can tolerate the action of digestive enzyme, improve the intestinal flora structure, promote the proliferation of bifidobacteria and lactobacilli in the intestinal tract and inhibit the growth of pathogenic bacteria. The antibacterial effect of the agar oligosaccharide with high polymerization degree is better than that of the agar oligosaccharide with low polymerization degree. Therefore, the agar oligosaccharide can be used as a raw material for developing a novel prebiotics preparation.

In recent years, the development scale of agar in China is small, products are mostly limited to agar and agar powder which are used as raw material outlets of chemical engineering, medicines and the like, the added value is low, and the environment is easily polluted. The method has important significance for reasonably utilizing agar resources, producing high-added-value products such as agar oligosaccharide and the like, preventing resource waste, increasing economic benefits, protecting the environment and the like. At present, two modes of acid hydrolysis and enzymolysis are mainly used for preparing agar oligosaccharides industrially, the acid hydrolysis method has high efficiency and high yield, but a large amount of byproducts are generated, and the problems of difficult separation and purification, potential safety hazard and the like of the agar oligosaccharides exist; the agarase enzymolysis method has the advantages of high catalytic efficiency, strong substrate specificity, mild reaction conditions and the like, and is a preferred mode for degrading agar polysaccharide. However, most of researches for degrading agar polysaccharide by using an enzymatic method are still in the laboratory research stage, and the main problems are that most of strains for producing agarase come from the sea, the enzyme production characters of the strains are unstable, the enzyme production amount is low, the cost for producing the agarase is high, the price is high, and the commercial application is difficult to realize. Therefore, an agarase gene with high hydrolytic activity and stable properties is found, and heterologous expression is realized to become the most economical and efficient method for producing the agarase.

Agarase is divided into alpha-agarase and beta-agarase, wherein the alpha-agarase hydrolyzes alpha-1, 3 glycosidic bonds to generate agaro-oligosaccharides, and the beta-agarase hydrolyzes beta-1, 4 glycosidic bonds to generate new agaro-oligosaccharides. Researches report that agarotetraose and agarohexaose have better antioxidant performance compared with other agaropectides, but because most of agarases discovered at present are beta-agarases, only the performances of four alpha-agarases are mined and reported, and the stability, the enzyme activity and the product specificity of the agarases are all to be improved, the large-scale industrial preparation and application of the agarotetraose and the agarohexaose are severely limited.

Researchers at home and abroad try to perform heterologous expression on agarase so as to greatly improve the yield of the agarase and break through the secretion limit of natural enzyme, while the existing research mainly focuses on the cloning expression of the agarase in escherichia coli, is mainly positioned in cytoplasm or periplasm space, and is not beneficial to the separation and purification of the agarase and the application of the agarase in the fields of food, medicine and the like. Even if the agarase is secreted and expressed in bacillus subtilis, the secretion amount is relatively low, so that a safer and more efficient agarase food-grade bacillus subtilis secretion expression system needs to be established and optimized.

Disclosure of Invention

In order to solve the problems of rare sources, low expression quantity, low product conversion rate, poor specificity and the like of alpha-agarase in the prior art, the invention provides a gene for coding the alpha-agarase, and the nucleotide sequence of the alpha-agarase is shown as SEQ ID No. 1.

In one embodiment of the invention, the gene of the alpha-agarase is obtained by optimization based on the alpha-agarase WT with the nucleotide sequence shown in SEQ ID NO. 2.

The invention also provides a recombinant vector carrying the gene of the alpha-agarase.

The invention also provides a gene carrying the alpha-agarase or a recombinant cell of the recombinant vector.

The invention also provides a recombinant bacillus subtilis which contains the gene or the recombinant vector.

In one embodiment of the invention, the recombinant Bacillus subtilis takes Bacillus subtilis WB600, Bacillus subtilis WB800 or Bacillus subtilis 168 as an expression host.

In one embodiment of the present invention, the recombinant Bacillus subtilis uses pHT01 plasmid, pHT43 plasmid or pHT304 plasmid as expression vector.

The invention also provides a method for preparing the agar tetrasaccharide or the agar hexasaccharide, which comprises the following steps: the recombinant cells or the recombinant bacillus subtilis are fermented to prepare alpha-agarase, and agarose solution is hydrolyzed by the alpha-agarase to obtain the agar tetrasaccharide or the agar hexasaccharide.

In one embodiment of the present invention, the method is: inoculating the recombinant cells or recombinant bacillus subtilis into a seed culture medium to prepare a seed solution, and inoculating the seed solution into a fermentation culture medium for fermentation.

In one embodiment of the invention, the seed solution is inoculated into a fermentation medium according to the proportion of 2-4% (v/v), the culture is carried out for 60-72 h at 37 ℃, and the fermentation liquor is centrifuged to obtain the supernatant, thus obtaining the alpha-agarase crude enzyme solution.

In one embodiment of the invention, the agarose solution with 0.25-2% (w/w) of hydrolysis is prepared by using alpha-agarase crude enzyme liquid obtained by fermentation or pure enzyme obtained by nickel column purification at 30-40 ℃ to prepare the agarose or the agarose.

The invention also provides the application of the gene, the recombinant vector, the recombinant cell, the recombinant bacillus subtilis or the method in preparing a product containing setron tetraose or setron hexaose.

Advantageous effects

(1) The invention provides alpha-agarase with a specific base sequence, and successfully adopts a food safe strain bacillus subtilis to carry out heterologous expression on the alpha-agarase, the method is safe and efficient, and can be applied to the production of food and medicines, and the catalytic activity of the expressed alpha-agarase crude enzyme liquid can reach 523.1U/mg by adopting the method.

(2) The alpha-agarase provided by the invention has better specific enzyme activity and thermal stability, and the substrate conversion rate is higher.

(3) The conversion rate and the product purity of the alpha-agarase are high, the conversion rate can reach about 53 percent through calculation, the yield of the agar tetrasaccharide reaches 0.34g/g, the yield of the agar hexasaccharide reaches 0.19g/g, the yield of the rare agar oligosaccharide can be effectively improved, the production and processing cost of the agar oligosaccharide is reduced, and therefore the alpha-agarase has industrial application value.

Drawings

FIG. 1: SDS-PAGE analysis of purified recombinant α -agarase, in lane 1: crude enzyme solution; lane 2: purified alpha-Aga_WT(ii) a Lane 3: purified alpha-Aga.

FIG. 2: relative enzyme activity data of alpha-agarase under different pH conditions.

FIG. 3: stability of the recombinant alpha-agarase under different pH conditions.

FIG. 4: performing HPLC (high performance liquid chromatography) atlas on a product obtained by hydrolyzing 0.25% (w/w) agarose solution by using the recombinant alpha-agarase alpha-Aga at different time; wherein, (A)1 h; (B)4 h; (C)12 h; (D) and (5) 24 h.

FIG. 5: performing HPLC (high performance liquid chromatography) atlas on a product obtained by hydrolyzing 0.5% (w/w) agarose solution by using the recombinant alpha-agarase alpha-Aga at different time; wherein, (A)1 h; (B)4 h; (C)12 h; (D) and (5) 24 h.

Detailed Description

The media involved in the following examples are as follows:

LB liquid medium: 1% (w/v) tryptone, 0.5% (w/v) yeast extract, 1% (w/v) sodium chloride, pH 7.0.

LB solid medium: 1.5(w/v) agar was added to the LB liquid medium.

TB liquid medium: 1.2% (w/v) tryptone, 2.4% (w/v) yeast extract, 0.4% (w/v) glycerol, 17mM KH₂PO₄，72mM K₂HPO₄，pH 6.0。

The detection methods referred to in the following examples are as follows:

the method for measuring the enzyme activity of the recombinant alpha-agarase comprises the following steps: Tris-HCl buffer (20mM, pH 7.0) is used to prepare 3g/L agarose substrate, 0.1mL enzyme solution diluted properly is added into 0.9mL substrate, reaction is carried out for 60min at 60 ℃, 1mL 3, 5-dinitrosalicylic acid (DNS) solution is added to stop enzymatic reaction, cooling is carried out on ice bath after boiling water bath is carried out for 5min, and the absorbance value is measured at 540nm and is compared with glucose standard curve.

Definition of enzyme activity: the amount of enzyme required to produce 1. mu. mol of reducing sugar (in terms of glucose) per minute was 1 enzyme activity unit (U).

The detection method of the agar tetrasaccharide or the agar hexasaccharide comprises the following steps: and (3) carrying out boiling water bath on the agarose solution hydrolyzed by the recombinant alpha-agarase for 30min to inactivate the enzyme, centrifuging (10,000 Xg, 10min), diluting, passing through a 0.22 mu m water system filter membrane, and analyzing the product by using High Performance Liquid Chromatography (HPLC). The HPLC conditions were as follows: sugar Pak I column (Waters, MA, USA), with a column temperature of 75 deg.C and a mobile phase of 50mg/L EDTA-CaNa₂The flow rate was 0.5 mL/min.

Example 1: construction of recombinant Bacillus subtilis secretion expression system

The method comprises the following specific steps:

(1) alpha-agarase alpha-Aga with the chemical synthesis nucleotide sequence shown as SEQ ID NO.1 and alpha-agarase with the chemical synthesis nucleotide sequence shown as SEQ ID NO.2_WTα-Aga_WT(i.e., alpha-agarase prior to optimization);

the PCR amplification procedure involved therein was: pre-denaturation at 98 deg.C for 3min, denaturation at 98 deg.C for 30s, annealing at 60 deg.C for 30s, extension at 68 deg.C for 2min, cyclic amplification for 35 times, and final extension at 68 deg.C for 5 min.

(2) And (2) respectively connecting the alpha-agarase genes obtained in the step (1) with pHT01 plasmid vectors by using a homologous recombination method. The connecting system is as follows: 1. mu.L of purified agarase PCR fragment (50 ng/. mu.L), 2. mu.L of purified pHT01 plasmid vector PCR fragment (50 ng/. mu.L), 4. mu.L of 5 XCE II Buffer, 2. mu.L of Exnase II, ddH₂O11. mu.L. The homologous recombination conditions are as follows: incubating at 37 ℃ for 30 min; then, Escherichia coli E.coli JM109 is transformed, an LB solid culture medium with 5 mu g/mL kanamycin is coated, transformants are selected for sequencing and double enzyme digestion verification, and an expression vector containing alpha-agarase genes is obtained; transforming the expression vector into B.subtilis WB600 to obtain the genetic engineering bacteria B.subtilis WB600/pHT 01-alpha-Aga and B.subtilis WB600/pHT 01-alpha-Aga_WT。

Example 2: expression, separation and purification of recombinant alpha-agarase

The method comprises the following specific steps:

(1) respectively inoculating the single colony prepared in the example 1 into 50mL LB liquid culture medium containing 5 mug/mL kanamycin and 5 mug/mL gibberellin, and performing shake-flask culture at 37 ℃ and 200rpm for 8-12 h to prepare seed liquid;

respectively transferring the seed liquid obtained by the preparation to 50mL of TB liquid culture medium containing 5 mug/mL kanamycin and 5 mug/mL gibberellin according to the inoculum concentration of 4% (v/v), and performing shake-flask culture at 30 ℃ and 200rpm for 60-72 h to prepare fermentation liquid;

respectively taking 30mL of fermentation liquor with OD600 of 6.0, centrifuging at 4 ℃ and 10000rpm for 10min, taking supernatant, namely extracellular crude enzyme liquid, wherein the specific enzyme activity of the alpha-Aga crude enzyme liquid is as follows: 523.1U/mg; alpha-Aga_WTThe specific enzyme activity of the crude enzyme solution is as follows: 360.4U/mg.

It can be seen that the enzyme activity of the crude enzyme solution of the original enzyme is lower than that of the specific sequence provided by the invention.

(2) Adopting a nickel column to carry out the step (1) on the prepared recombinant alpha-agarase alpha-Aga and alpha-Aga_WTPurification was carried out with 500mM NaCl +50mM Tris-HCl +20mM imidazole in the equilibration solution (solution A, pH 7.5) and 500mM NaCl +50mM Tris-HCl +500mM imidazole in the eluent (solution B, pH 7.5). After cell sap is crushed and centrifuged, the pH of the supernatant is adjusted to 7.5, and the supernatant is filtered by a 0.45 mu m water system membrane to be purified. Balancing the ion column by using a buffer solution A with 5-6 column volumes at the flow rate of 2 mL/min; loading the sample at the flow rate of 2 mL/min; and then balancing the ion column by using 6-8 column volumes of buffer solution A, and then performing gradient elution by using 15% of buffer solution B at the flow rate of 2 mL/min. Collecting corresponding eluate according to the elution peak, placing in a dialysis bag, dialyzing overnight at 4 deg.C with 50mM Tris-HCl, pH 7.5, and then identifying by SDS-PAGE protein electrophoresis (the result is shown in FIG. 1) and enzyme activity determination. The calculated molecular weight of the recombinant alpha-agarase was reported to be 158.88kDa, consistent with the molecular weight estimated by SDS-PAGE.

Example 3: optimum pH and pH stability of recombinant alpha-agarase

The most suitable pH and pH stability of the purified recombinant alpha-agarase alpha-Aga prepared in the embodiment 2 are respectively detected, and the specific steps are as follows:

(1) the method for measuring the optimal pH of the recombinant alpha-agarase comprises the following steps: respectively adopting 50mM sodium citrate buffer solution (pH 3.0-6.0), Tris-HCl buffer solution (pH 6.0-8.0) and glycine-NaOH buffer solution (pH 8.0-11.0) to prepare substrate solutions, measuring the enzyme activity of the recombinase under different pH conditions, and taking the highest enzyme activity as 100%, wherein the results are shown in Table 1 and figure 2.

Table 1: relative enzyme activity data under different pH conditions

(2) The method for measuring the pH stability of the recombinant alpha-agarase comprises the following steps: respectively adopting 50mM sodium citrate buffer solution (pH 3.0-6.0), Tris-HCl buffer solution (pH 6.0-8.0) and glycine-NaOH buffer solution (pH 8.0-11.0) to prepare substrate solution and diluted enzyme solution, standing at 4 ℃ for 6h, measuring the residual enzyme activity, and taking the enzyme activity of the enzyme solution without heat preservation as 100%, wherein the results are shown in Table 2 and figure 3.

Table 2: relative enzyme activity data after standing for 6h under different pH conditions

The optimal pH and pH stability of the recombinant α -agarase are shown in fig. 3. The enzyme shows the highest activity in Gly-NaOH buffer solution with the pH of 8.0, has the strongest stability at the pH of 6.0, keeps the relative activity of more than 60 percent in the range of pH 7.0-10.0, and has better pH stability.

Example 4: application of recombinant alpha-agarase

Due to the original enzyme alpha-Aga_WTThe enzyme activity of the alpha-Aga enzyme is far lower than that of the optimized alpha-Aga enzyme, so that the optimized alpha-Aga enzyme is adopted for the production and preparation of the agar oligosaccharide in the embodiment; the method comprises the following specific steps:

(1) 100g of agarose solutions were prepared at concentrations of 0.25% (w/w) and 0.5% (w/w), respectively.

(2) Placing agarose solutions with the concentrations of 0.25% (w/w) and 0.5% (w/w) in a water bath shaker at 40 ℃ for preheating for 10min to respectively obtain a reaction system 1 and a reaction system 2, and then respectively adding the pure enzyme solution prepared in the example 2 into the reaction systems 1 and 2; the addition amount is 10U/g, the reaction is carried out for 1-24 h in a water bath shaker at 40 ℃, and then the reaction is transferred to boiling water for inactivating enzyme for 20min to stop the reaction;

in this case, the enzyme solution was replaced with an equal amount of Tris-HCl buffer as a blank.

(3) After enzyme deactivation, centrifuging the reaction systems 1 and 2 for 10min at 10000rpm, respectively, taking supernate, passing through a water system membrane of 0.22 mu m to remove impurities, and analyzing the type and concentration of the agar oligosaccharide product in the reaction solution by using high performance liquid chromatography. The results are shown in Table 3:

table 3: the kind and concentration of the product under different substrate concentrations

As shown in fig. 4 (the yield of 0.25% of the substrate concentration of both the setron tetrose and the setron hexose) and fig. 5 (the yield of the substrate concentration of both the setron tetrose and the setron hexose), the main hydrolysis products are setron tetrose (a4) and setron hexose (a6), and the ratio of the setron tetrose is high; the total conversion can reach about 31% when the substrate concentration is 0.25%, and the total conversion can reach about 53% when the substrate concentration is 0.5%.

Comparative example 1:

the specific implementation manner is the same as that in examples 1-2, except that host bacteria are adjusted to be bacillus subtilis WB800 and BS168, and the results show that the specific enzyme activities of the prepared optimized recombinase alpha-agarase alpha-Aga crude enzyme solution are only respectively as follows: 216.8U/mg and 93.5U/mg.

Comparative example 2:

the specific implementation manner is the same as that in examples 1-2, except that the expression vectors are adjusted to pHT43 plasmid and pHT304 plasmid, and the results show that the specific activities of the prepared optimized recombinant enzyme alpha-agarase alpha-Aga crude enzyme solution are only respectively as follows: 130.2U/mg and 174.9U/mg.

Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.

SEQUENCE LISTING

<110> university of south of the Yangtze river

<120> application of alpha-agarase gene and coding enzyme thereof

<130> BAA200990A

<160> 2

<170> PatentIn version 3.3

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tttgttggga ccagtgtttc atctggaccc aatatagaag tgcttgttaa tgtaaatggt 240

acatggcaaa gtcaaggctc ggttgctgtt ccttatggta gctgggatga ctttcaatca 300

ctcacgccat ctcatacagt gtcattacca gtgggtactt cgactgttcg cttgttggcg 360

gtaggctcaa cttggcaatg gaacttagaa tcatttaggt taactcaagt ttcatccgtt 420

gaacctgttg gtgatgcaga taatgacggc gtctatgata atcaagattt atgccctaat 480

accccatcag gtgtcacagt tgacaataat ggttgtcaaa ttaatggagg cacagatccg 540

gttggagaat cctttgtcat ccagatggaa gcttttgatt caactggtag tgatgattca 600

agagcacaag gtgtggttat tggtgagcgt ggctacccac aagacaagca tacggttgtt 660

gatagtgtcc aaactactga ttgggtggat tatagtatta atttcccatc ttctggcaac 720

tacagcatat ctatgttggc atcagggcaa acctcgcatg caaccgccgt attatttatt 780

gatggcactg aaattaatga agtaccagtt catacaggta atcaagccga ttttgaagat 840

tttcaactaa ctagttctgt atatgttgca gctggcaccc atactgtacg agttcaggcg 900

caaagctcta ctggcgagtt tagttggtta tggtttggtg atacattaac tttcactaac 960

ttagacggtg acgatggtag tgatgggggc aatggctcgc ctacacaaga tgcggataac 1020

gatggtgtgt tggatagctc agattcttgt ccaaatacgc ctataggtga acctgcagat 1080

gtgactggct gtagtgcatc gcaattagat gatgacaatg atggtgtttc aaataatgtc 1140

gatcaatgtc ctaatacagt tgctggtaca gaggtaggcg ctgatggctg tgaagttatt 1200

attgataacg acacagataa tgatggtgtt ttaaataatg tcgaccagtg cccaaatacg 1260

ccaccaggcg caactgtaga ctcaaatgga tgtgaagtaa catttgctga tacagataat 1320

gacggcatag aagattcgca agatttttgc ccgaacacac cagcaggtga agcggtcaat 1380

aattcaggtt gtggtgagtc gcaattagat gccgataatg atggcgttac aaacaatata 1440

gaccaatgtc caaatacacc agcgggtaca caagttgatg cgtcaggttg tgagatcgac 1500

aatggtggtg agccaggtga tagttattat cacaatggtc aaggtttatt gtttggtcgt 1560

gtagatggcg caactaactt tgtaggtgaa gaaggctatg ttgccaaccc tgataactat 1620

gatgtgacaa ccgatttact cgagaccgat gatgacatta gaggaaactc tactgaagta 1680

ttccgtggtg agatttatga cgcagatggt catatttctt tctatgaaca tatagatgat 1740

agcgttcgtc tatatatcga cggtcaactc gtgctttcaa atgatagctg ggaaaactcg 1800

tctcaaactc cagatttaag cctcacacct ggttggcata attttgagtt aagacttggt 1860

aacgccgacg gtggcagtgg tgctgtgagt ggcatcggtt ttggtataga tgttgacggt 1920

ggcacaaact ttgttcaccc aagtaattta agccctagta tgtttagatc gagcggccaa 1980

gttgtggttg atccaatttt accaccacca ggcggaattt atattcagct agaagacttt 2040

gacgaaacag gtactgttgg ccgagtcgcg agcgatccaa acgatggctt tgttaaaggt 2100

gattcaaacg ttggttgggt taccaatgga gattggggta aataccacaa tgtattttta 2160

gaagctggta catatagagc attcattact gtatcaacac ctgcgggtgg tagttatggt 2220

gcacgtgttg atatagacgg cgaacctttt gcttggggat attttgatag cacaggtggc 2280

tgggatatcg cagcagaata tgagttgtat ggtggtgatt tagtcgtaga aagcactggt 2340

aaccatacgc tgcatattga agctgttggt gggtcagatt ggcagtggag tggcgactta 2400

gtgcgtttag ctaaagtgaa cgacagtaca gtcaaacaac ctcgtgtcta caatccaaac 2460

gaacatcttg ttgctgaaat cgaagggcct gcaactggtt tacaatattt aaaaacacca 2520

gtggaaattc cattagccaa taaggtatta aaatctgacg tttggtatac ctacccgcaa 2580

aaccgaaacc tagtcgttga cggtgacaca ccatacgctg actttggcgc aacgggtgcc 2640

ttctggggac atccacctga acacgatttc tacgatgata ctgtgatcat ggattgggcg 2700

gttaatgtcg ttgatgattt ccaaagtgaa gggtttgaat atactgcacg cggtgaattc 2760

gactggggtt atggctggtt tactgagttt acgactaatc cgcagccaca ttatgttcaa 2820

accttggatg gccgtaatgt acgtatgaca ttcatgggct atttaagcca tgacggttat 2880

aacaacaatt ggttaagtaa tcacagccca gcgtttgtac ctttcatgaa gtcacaggtt 2940

gatcaaatct taaaagcgaa cccggataag ttgatgtttg atacacaaac taactcaaca 3000

cgttcaactg atatgcgtga ctttggtggg gatttctctc cttatgcgat ggaaaatttc 3060

cgagtttggt tgtcgaagaa atacagctat gcagaactaa gcgctatggg tattaacgat 3120

attactacct ttgactataa gcagcactta ctggatgcag gtgtaactca tacatcatgg 3180

tcaaatgcag gagatagact cgaaggcaat attccaatgc ttgaagattt tatctacttt 3240

aatcgtgacg tttggaacca aaaatttgct gaagtattag attacattcg ccagcaaaga 3300

cctaatattg aaatcggtgc cagtacacac ctatttgaat cacgtggcta tgtatttaat 3360

gagaatatta ccttcttatc gggagagctt aatttaggtg caagaaccag tatttcagaa 3420

ttaccaacta atattcttgt acacttaaaa ggtgcgcaag cagttgataa aaccttggct 3480

tacttcccat atccgtggga gtttgatgaa ttacgtctac aaaatgcgcc tagatttggt 3540

cgtggttggg ttgctcaagc ttatgcttat ggtggcttat tctctattcc tgctaatgtg 3600

tgggtaggtg gtgaagtatt tacttggtca cctggtgctg ataactatcg cgatatctat 3660

caatttgtcc gtgcacaagc taacttattc gatggttata cctcatacgc gaaagctggt 3720

tatgttcacg ctatgttctc atcaatgaaa gctggtttta ttgatggtgg caaccaagtt 3780

caatcaagcg tgaaaatttt aactgaagat aatatcaatt ttgacatgtt agtgtttggt 3840

gatgcgggat atcctgttgt accacgccag gctgattttg ataaatttga gcatattttc 3900

tacgacggtg atctaaacta cttaacgact gagcaaaaag cagtattgga tgcacaaggt 3960

agtaaggttc gtcacattgg tcaacgcggt tctttagctg gtttacagat taatgtaagc 4020

attaatggta gtgtatctaa cgaaactgtg tctgctgtat ctcgtattca tgaaacagac 4080

tcaacggcac cttatgtagt acacctgatt aatcgtccgt ttgcgggtgg ggtaacacca 4140

atactgaaca atgttgaagt ggcaattcct gcaagctatt ttcctgaagg agtaacctca 4200

gcgaaattac acttaccaga tggaacaagt tcaaccgtcg cggtttcgac caatgcgaat 4260

ggcgatgctg ttgtatctgt tagtaatctt gaagtttggg gtattttaga attagctcac 4320

tga 4323