CN113502297B - Recombinant pichia pastoris for synthesizing guanosine diphosphate fucose, construction method and application thereof - Google Patents

Abstract

The invention discloses recombinant pichia pastoris for synthesizing guanosine diphosphate fucose, a construction method and application thereof, wherein the recombinant pichia pastoris is obtained by expressing fucose-1-guanosine phosphate transferase and fucose kinase in a genome of Pichia pastoris GS115. The strain capable of synthesizing the guanosine diphosphate fucose is obtained through modification, so that the guanosine diphosphate fucose can be efficiently synthesized. The construction method of the recombinant pichia pastoris is simple, convenient to use and has good application prospect.

Description

A recombinant Pichia pastoris that synthesizes guanosine diphosphate fucose and its construction method and application

technical field

The invention belongs to the technical field of genetic engineering, and specifically relates to a recombinant Pichia pastoris for synthesizing guanosine diphosphate fucose and its construction method and application.

Background technique

Guanylate diphosphate fucose (GDP-L-Fuc) is the nucleotide form of fucose (L-fucose), which provides fucosyl receptors for fucosylation reactions, and is used in living organisms It plays an important role in tissue growth, angiogenesis, adhesion of fertilized cells, inflammation and tumor metastasis. At the same time, oligosaccharides containing fucosyl groups are the main components of breast milk, and their functions involve a wide range of biological processes, such as preventing pathogen infection, improving immune system responses, and reducing inflammatory responses, etc. Cocosylated oligosaccharides are synthesized under the catalysis of fucosyltransferase using guanosine diphosphate fucose (GDP-L-fucose) as a fucosyl donor. Because of the huge demand due to the wide range of effects of fucosyl oligosaccharides, many pharmaceutical companies are trying to efficiently synthesize sufficient guanosine diphosphate fucose through chemical and biological methods. In chemical synthesis, guanosine diphosphate fucose is synthesized from L-fucopyranosyltetraacetic acid as the starting material, but chemical synthesis is accompanied by multiple protection and deprotection steps. This complexity makes chemical Synthesis is impractical for industrial applications. Biosynthesis is based on the discovery of two metabolic pathways for the synthesis of GDP-L-fucose in organisms: the salvage pathway and the de novo pathway, both of which occur in the cytoplasm. The biosynthesis of fucose guanosine diphosphate is shown in Figure 1.

The salvage pathway is found in the human metabolic pathway, exogenous fucose is transferred into the cell, ATP is consumed by fucose kinase and phosphorylated (EC2.7.1.52) to form fucose-1-phosphate ( Fuc-1-P). Fuc-1-P combines with guanosine triphosphate (GTP) to generate GDP-L- Fucose. The de novo synthesis pathway, which was first discovered in bacteria and then found in plants, mammals, and invertebrates, is a pathway in which GDP-fucose is dehydrated from GDP-mannose via guanosine diphosphate-mannose Enzyme (GMD, EC4.2.1.47) and guanosine diphosphate mannose epimerase (WCAG, EC1.1.1.271) catalyzed synthesis.

Pichia pastoris is widely used as an excellent host for exogenous protein expression, and its products have been certified by the FDA as "generally regarded as safe" (GRAS) safety level.

Therefore, how to use Pichia pastoris to efficiently synthesize fucose guanosine diphosphate through biological methods is still an urgent problem in this field.

Contents of the invention

The object of the present invention is to provide a construction method and application of recombinant Pichia pastoris for synthesizing guanosine diphosphate fucose. The constructed recombinant Pichia pastoris can efficiently synthesize guanosine diphosphate fucose.

The technical scheme that the present invention takes is:

The first aspect of the present invention provides a vector containing the expression sequence of fucose-1-phosphate guanosyltransferase and/or the expression sequence of fucose kinase.

In some embodiments of the present invention, the amino acid sequence of the fucose-1-phosphate guanosyltransferase is shown in SEQ ID NO.1, and the amino acid sequence of the fucose kinase is shown in SEQ ID NO.2 .

In some embodiments of the present invention, the fucose-1-phosphate guanosyltransferase is derived from the FPGT gene of wild boar (Susscrofa), and the fucose kinase gene is derived from the FCSK gene of wild boar (Sus scrofa).

In some embodiments of the present invention, the expression sequence of the fucose-1-phosphate guanosyltransferase is shown in SEQ ID NO.3, and the expression sequence of the fucose kinase is shown in SEQ ID NO.4 .

In some embodiments of the present invention, the sequence of the vector is shown in SEQ ID NO.5.

A second aspect of the present invention provides a recombinant cell comprising the expression vector described in the first aspect of the present invention, wherein the cell is a non-plant cell.

In some embodiments of the invention, the cell is Pichia pastoris.

In some preferred embodiments of the present invention, the cell is Pichia pastoris GS115.

The third aspect of the present invention provides the method for constructing the recombinant cell described in the second aspect of the present invention, comprising the following steps: transferring the vector described in the first aspect of the present invention into the cell.

In some embodiments of the present invention, the construction method is specifically to electrotransform the competent cells of Pichia pastoris GS115 with the vector described in the first aspect of the present invention.

The fourth aspect of the present invention provides the use of the vector described in the first aspect of the present invention or the recombinant cell described in the second aspect of the present invention.

In some preferred embodiments of the present invention, the application is specifically the application in the preparation of fucose guanosine diphosphate.

The fifth aspect of the present invention provides a method for preparing guanosine diphosphate fucose, which is produced by the recombinant cell described in the second aspect of the present invention.

In some embodiments of the present invention, the fermentation condition is that the recombinant cell seed solution is inserted into the fermentation medium with an inoculation amount of OD600 of 0.5-1.5, and cultured at 25-35°C and 200-300rpm for 100-140h.

In some preferred embodiments of the present invention, the fermentation condition is that the recombinant cell seed liquid is inserted into the fermentation medium with an inoculum amount of OD value of 1, and cultivated for 120 h at 30 ° C and 250 rpm.

The present invention also provides guanosine diphosphate fucose prepared by the method described in the fifth aspect of the present invention.

The beneficial effects of the present invention are:

The present invention provides a vector, which contains the expression sequence of fucose-1-phosphate guanosyltransferase and/or the expression sequence of fucose kinase. It also provides a recombinant cell, which is obtained by expressing fucose-1-phosphate guanylyltransferase and fucose kinase genes on the basis of Pichia pastoris GS115, through transformation to obtain fucoidan that can synthesize guanosine diphosphate Sugar recombinant strains. The construction method of the recombinant Pichia pastoris of the present invention is simple, easy to use, and has good application prospects.

Description of drawings

Figure 1 is a schematic diagram of the biosynthesis of fucose guanosine diphosphate.

Fig. 2 is a schematic diagram of synthesis of guanosine diphosphate fucose plasmid in Example 1 of the present invention.

Fig. 3 is an agarose gel electrophoresis diagram of PCR verification of yeast colonies expressing FPGT and FCSK genes transformed with plasmids in Example 1 of the present invention.

Figure 4 is a high performance liquid chromatogram in Example 2 of the present invention.

Fig. 5 is the mass spectrogram in embodiment 2 of the present invention.

Detailed ways

The concept of the present invention and the technical effects produced will be clearly and completely described below in conjunction with the embodiments, so as to fully understand the purpose, characteristics and effects of the present invention. Apparently, the described embodiments are only some of the embodiments of the present invention, rather than all of them. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without creative efforts belong to The protection scope of the present invention.

High-performance liquid chromatography (HPLC) detection method: Agilent, UV detector, C18 column (250×4.6mm, 5μm), mobile phase A: 20mM pH6.0 triethylamine acetic acid buffer (TEAA), mobile phase B: acetonitrile , the flow rate is 0.35mL/min, the column temperature is 40°C, and the injection volume is 10μL.

The construction of embodiment 1 recombinant Pichia pastoris

According to the sequence of fucose-1-phosphate guanosine transferase (FPGT) and fucose kinase (FCSK) of wild boar [Sus scrofa (pig)] published on NCBI, in which fucose-1-phosphate guanosine transfer The Gene ID of the enzyme gene on NCBI: 100513664, its amino acid sequence is shown in SEQ ID NO.1; the Gene ID of the fucose kinase gene on NCBI: 100625448, its sequence is shown in SEQ ID NO.2, the above two Genes were optimized according to the codon bias of Pichia pastoris. The base sequence of the optimized fucose-1-phosphate guanosyltransferase is shown in SEQ ID NO.3, and the base sequence of fucose kinase is shown in SEQ ID NO.4; by enzyme cleavage and connection, A recombinant plasmid whose sequence is shown in SEQ ID NO.5 was constructed. The schematic diagram of the plasmid is shown in Figure 2.

in:

SEQ ID NO.5:

In SEQ ID NO.5, " _ " is the gene FPGT region, "~" is the FCSK region, and "..." is the promoter region.

The constructed recombinant plasmid was electrotransformed into competent cells of recombinant Pichia pastoris GS115, the amount of recombinant plasmid added was 800-1500ng, the electroporation conditions were: voltage 2.5kV, electroporation reagent 5.6ms, recovery at 30°C for 1.5h, and final coating. Concentration of 100mg/L bleomycin-resistant YPDZ plate, cultivated at 30°C for 96h, and selected several single colonies.

Through bleomycin resistance plate screening and colony PCR verification, it was confirmed that fucose-1-phosphate guanosyltransferase (FPGT) and fucose kinase (FCSK) were successfully integrated into the yeast genome. The results of agarose gel electrophoresis identification are shown in Figure 3, in which bands 1, 2, 3, 4, and 5 are colony samples, and M is Marker. It can be seen that there are special bands in the colony PCR verification, which proves that fucose-1-phosphate guanosyltransferase and fucokinase are successfully integrated in the Pichia pastoris GS115 genome. It was confirmed that the fucose-1-phosphate guanosyltransferase and fucokinase derived from wild boar were successfully expressed, and the recombinant Pichia pastoris GS115 was obtained.

Example 2 Fermentative production of fucose guanosine diphosphate

The recombinant Pichia pastoris in Example 1 is made into seed liquid, and the formula of seed liquid culture medium is: glycerol 10g/L, tryptone 10g/L, yeast powder 5g/L, NaCl 10g/L; Seed liquid preparation method is: Pick a single colony on a fresh plate and place it in the seed medium for 24 hours.

Insert the seed solution into the fermentation medium with an inoculation amount of OD value of 1. The formula of the fermentation medium is: peptone 20g/L, yeast powder 10g/L, 1.34% YNB; 10% 10×pH6.0 phosphate buffer culture medium at 30°C and 250rpm for 120h, adding 1% carbon source every 24h, the carbon source being methanol, and adding 1g/L fucose to the medium every 24h after fermentation for 48h until the end of fermentation.

After the fermentation, use high performance liquid chromatography to measure the guanosine diphosphate fucose in the fermented cells, the results are shown in Figure 4, and further confirmed by mass spectrometry, the results are shown in Figure 5, the guanosine diphosphate fucose The content is 289.8mg/L.

The above-mentioned specific embodiments have described the present invention in detail, but the present invention is not limited to the above-mentioned embodiments, within the scope of knowledge possessed by those of ordinary skill in the art, various changes can also be made without departing from the gist of the present invention. In addition, the embodiments of the present invention and the features in the embodiments can be combined with each other if there is no conflict.

SEQUENCE LISTING

<110> South China University of Technology

<120> A recombinant Pichia pastoris for synthesizing guanosine diphosphate fucose and its construction method and application

<130>

<160> 5

<170> PatentIn version 3.5

<210> 1

<211> 599

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Met Ala Ala Ala Ser Ala Pro Leu Gly Val Ser Leu Gln Glu Ala Thr

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Gln Arg Arg Leu Arg Arg Phe Ser Glu Leu Arg Gly Lys Pro Val Ala

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Ala Gly Glu Phe Trp Asp Ile Val Ala Ile Thr Ala Ala Asp Glu Lys

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Gln Glu Leu Ala Tyr Lys Gln Gln Leu Ser Glu Lys Leu Lys Lys Lys

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Glu Leu Pro Leu Gly Val Gln Tyr Leu Val Phe Val Asp Pro Ala Gly

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Ala Lys Ile Gly Asn Gly Gly Ser Thr Leu Cys Ala Leu Arg Cys Leu

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Glu Lys Leu Tyr Gly Asp Gln Trp Asn Ser Phe Thr Ile Leu Leu Ile

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His Ser Gly Gly Tyr Ser Gln Arg Leu Pro Asn Ala Ser Ala Leu Gly

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Lys Ile Phe Thr Ala Leu Pro Phe Gly Ser Pro Ile Tyr Gln Met Leu

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Glu Leu Lys Leu Ala Met Tyr Ile Asp Phe Pro Ser His Met Asn Pro

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Gly Ile Leu Val Thr Cys Ala Asp Asp Ile Glu Leu Tyr Ser Ile Gly

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Glu Ser Glu Phe Ile Arg Phe Asp Lys Pro Gly Phe Thr Ala Leu Ala

180 185 190

His Pro Ser Ser Leu Thr Val Gly Thr Thr His His Gly Val Phe Val Leu

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Glu Pro Phe Asn Arg Leu Glu Tyr Arg Asp Leu Glu Tyr Arg Cys Cys

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His Arg Phe Leu His Lys Pro Ser Ile Glu Met Met Tyr Lys Phe Asp

225 230 235 240

Ala Val Cys Arg Pro Gly Asn Phe Ser Gln Gln Asp Phe Gly Gly Gly

245 250 255

Asp Thr Pro Ser Leu Lys Leu Asp Pro Glu Tyr Val Tyr Thr Asp Ser

260 265 270

Val Phe Tyr Met Asp His Lys Thr Ala Lys Lys Leu Leu Ala Phe Tyr

275 280 285

Glu Lys Ile Asp Thr Leu Asn Cys Glu Ile Asp Ala Tyr Gly Asp Phe

290 295 300

Leu Gln Ala Leu Gly Pro Gly Ala Thr Val Glu Tyr Thr Arg Asn Thr

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Ser Asn Val Thr Lys Glu Glu Ser Glu Leu Val Asp Met Arg Gln Arg

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Ile Phe His Leu Leu Lys Gly Thr Pro Leu Asn Val Ile Val Leu Asn

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Asn Ser Lys Phe Tyr His Ile Gly Thr Thr Lys Glu Tyr Leu Phe His

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Phe Thr Ser Asp Ser Ser Leu Lys Ser Glu Leu Gly Leu Gln Ser Ile

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Ala Phe Ser Ile Phe Pro Ala Ile Pro Glu Tyr Ser Gly Asn Lys Ser

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Cys Ile Ile Gln Ser Ile Leu Asp Ser Arg Cys Ser Leu Ala Pro Gly

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Ser Val Val Glu Tyr Ser Arg Leu Gly Pro Asp Val Ser Val Gly Glu

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Asn Cys Ile Ile Ser Gly Ser His Ile Ile Thr Arg Ala Ile Leu Pro

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Ala Tyr Ser Phe Val Cys Ser Leu Ser Leu Lys Ile Asn Gly His Ile

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Lys Tyr Ser Thr Met Ala Cys Gly Val Gln Asp Asn Leu Lys Lys Asn

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Val Lys Thr Leu Ser Asp Val Lys Leu Leu Gln Phe Phe Gly Val Ser

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Leu Leu Ser Cys Leu Asp Val Trp Asn Leu Glu Val Thr Glu Glu Leu

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Phe Ser Gly Asn Lys Thr Cys Leu Ser Leu Trp Asn Ala Arg Ile Phe

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Pro Val Cys Ser Ser Leu Ser Asp Ser Val Ile Ala Ser Leu Lys Met

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Leu Asn Ala Val Gln Ser Lys Ser Val Phe Ser Leu Asn Asn Tyr Lys

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Leu Leu Ser Ile Glu Glu Met Leu Val Tyr Lys Asp Val Glu Asp Met

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Ile Thr Tyr Arg Glu Gln Ile Phe Leu Glu Ile Ala Leu Asn Arg Lys

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Gln Ser Val Leu Glu Thr Ser

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Met Glu Gln Pro Lys Gly Val Asp Trp Thr Val Ile Ile Leu Thr Cys

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Gln Tyr Lys Asp Ser Val Glu Val Phe Gln Lys Glu Leu Glu Ile Arg

20 25 30

Gln Lys Arg Glu Gln Ile Pro Ala Ser Thr Leu Leu Leu Ala Val Glu

35 40 45

Asp Pro Glu Val His Val Gly Ser Gly Gly Ala Thr Leu Asn Ala Leu

50 55 60

Leu Val Ala Ala Glu His Leu Ser Ala Arg Ala Gly Phe Thr Val Val

65 70 75 80

Thr Ser Asp Val Leu His Ser Ala Trp Ile Leu Ile Leu His Met Gly

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Arg Asp Phe Pro Phe Asp Asp Cys Gly Arg Ala Phe Thr Cys Leu Pro

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Val Glu Asn Pro Gln Ala Pro Val Glu Ala Val Val Cys Asn Leu Asp

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Cys Leu Leu Asp Ile Met Ser His Arg Leu Gly Pro Gly Ser Pro Pro

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Gly Val Trp Val Cys Ser Thr Asp Met Leu Leu Ser Val Pro Pro Asn

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Pro Gly Ile Asn Trp Asp Gly Phe Arg Gly Ala Arg Val Ile Ala Leu

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Pro Gly Ser Thr Ala Tyr Ala Arg Asn His Gly Val Tyr Leu Thr Asp

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Ser Gln Gly Phe Val Leu Asp Ile Tyr Tyr Gln Gly Thr Glu Ala Glu

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Ile Gln Arg Cys Ala Arg Pro Asp Gly Gln Val Pro Leu Val Ser Gly

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Ile Val Phe Phe Ser Val Glu Thr Ala Glu His Leu Leu Ala Thr His

225 230 235 240

Val Ser Pro Pro Leu Asp Ala Cys Thr Tyr Met Gly Leu Asp Ser Gly

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Ala Gln Pro Val Gln Leu Ser Leu Phe Phe Asp Ile Leu Leu Cys Met

260 265 270

Ala Arg Asn Val Arg Arg Glu Asp Phe Leu Val Gly Arg Pro Pro Glu

275 280 285

Met Gly Arg Gly Asp Met Glu Thr Glu Gly Tyr Leu Arg Gly Ala Arg

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Ala Glu Leu Trp Arg Glu Leu Arg Asp Gln Pro Leu Thr Leu Ala Tyr

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Val Pro Asp Gly Ser Tyr Asn Tyr Met Thr Asn Ser Ala Ser Glu Phe

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Leu His Ser Leu Thr Phe Pro Gly Ala Ser Gly Ala Gln Val Val His

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Ser Gln Val Glu Glu Gln Gln Leu Leu Gly Ala Gly Ser Ser Val Val

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Ser Cys Leu Leu Glu Gly Pro Val Gln Leu Gly Pro Gly Ser Val Leu

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Gln His Cys His Leu Arg Gly Pro Ile His Ile Gly Thr Gly Cys Phe

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Val Ser Gly Leu Asp Ala Ala Gln Ser Glu Ala Leu His Ser Leu Glu

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Leu His Asp Leu Val Leu Gln Gly His His Leu Gln Leu His Gly Ala

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Pro Ser Arg Ala Phe Thr Leu Val Gly Arg Leu Asp Ser Trp Glu Arg

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Gln Gly Ala Gly Thr Tyr Leu Asn Met Ser Trp Ser Lys Phe Phe Gln

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Lys Thr Gly Ile Arg Asp Trp Asp Leu Trp Asp Pro Asp Thr Pro Pro

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Thr Glu Arg Cys Leu Leu Ser Ala Arg Leu Phe Pro Val Leu His Pro

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Leu Arg Ala Leu Gly Pro Gln Asp Met Leu Trp Met Leu Asp Pro Gln

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Glu Asp Gly Gly Lys Ala Leu Arg Ala Trp Arg Asp Ser Trp Arg Leu

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Ser Trp Glu Gln Leu Gln Pro Cys Leu Asp Arg Ala Ala Thr Leu Ala

530 535 540

Ser Arg Arg Asp Leu Phe Phe Arg Gln Ala Leu His Lys Ala Arg His

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Val Leu Glu Ala Arg Gln Asp Leu Ser Leu Arg Pro Leu Ile Arg Ala

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Ala Val Arg Glu Gly Cys Pro Gly Pro Leu Met Ala Thr Leu Asp Gln

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Val Ala Ala Gly Ala Gly Asp Pro Gly Val Ala Ala Arg Ala Leu Ala

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Cys Val Ala Asp Ile Leu Gly Cys Met Ala Glu Gly Arg Gly Gly Leu

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Arg Ser Gly Pro Ala Ala Asn Pro Glu Trp Val Arg Pro Phe Ser Tyr

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Leu Glu Cys Gly Asp Leu Ala Gly Gly Val Glu Ala Leu Ala Gln Glu

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Arg Glu Lys Trp Leu Ser Arg Pro Ala Leu Leu Val Arg Ala Ala Arg

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His Tyr Glu Gly Ala Gly Gln Ile Leu Ile Arg Gln Ala Val Met Ser

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Ala Gln His Phe Val Ser Thr Glu Pro Val Glu Leu Pro Ala Pro Gly

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Gln Trp Val Val Ala Glu Cys Pro Ala Arg Val Asp Phe Ser Gly Gly

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Trp Ser Asp Thr Pro Pro Leu Ala Tyr Glu His Gly Gly Ala Val Leu

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Gly Leu Ala Val Arg Val Asp Gly Arg Arg Pro Ile Gly Ala Arg Ala

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Arg Arg Ile Pro Glu Pro Glu Leu Trp Leu Ala Val Gly Pro Gln His

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Asp Lys Met Ala Met Lys Ile Val Cys Arg Ser Leu Asp Asp Leu Gln

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Asp Tyr Cys Gln Pro His Ala Pro Gly Ala Leu Leu Lys Ala Ala Phe

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Ile Cys Ala Gly Ile Leu Ser Val His Ser Glu Leu Ser Leu Ser Glu

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Gln Leu Leu Cys Thr Phe Gly Gly Gly Phe Glu Leu Gln Thr Trp Ser

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Glu Leu Pro His Gly Ser Gly Leu Gly Thr Ser Ser Ile Leu Ala Gly

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Ala Ala Leu Ala Ala Leu Gln Arg Val Ala Gly Arg Ala Val Gly Thr

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Glu Ala Leu Ile His Ala Val Leu His Leu Glu Gln Val Leu Thr Thr

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Gly Gly Gly Trp Gln Asp Gln Val Gly Gly Leu Met Pro Gly Ile Lys

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Val Gly Arg Ser Arg Ala Gln Leu Pro Leu Lys Val Glu Val Glu Glu

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Ile Thr Val Pro Ala Gly Phe Val Gln Lys Leu Asn Asp His Leu Leu

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Leu Val Tyr Thr Gly Lys Thr Arg Leu Ala Arg Asn Leu Leu Gln Asp

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Val Leu Arg Ser Trp Tyr Ala Arg Leu Pro Pro Val Val Gln Asn Ala

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His Asn Leu Val Gln Gln Thr Glu Glu Cys Ala Glu Ala Phe Arg Gln

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Gly Ser Leu Pro Arg Leu Gly Gln Cys Leu Thr Ser Tyr Trp Glu Gln

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Lys Lys Leu Met Ala Pro Gly Cys Glu Pro Leu Ala Val Arg His Met

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Met Asp Ala Leu Ala Pro His Val His Gly Gln Ser Leu Ala Gly

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Ala Gly Gly Gly Gly Phe Leu Tyr Leu Leu Thr Lys Glu Pro Arg

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Gln Lys Glu Ala Leu Glu Ala Val Leu Ala Lys Thr Glu Gly Leu

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Gly Asn Tyr Ser Val His Leu Val Glu Val Asp Thr Gln Gly Leu

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Ser Leu Gln Leu Leu Gly Thr Glu Thr Ser Ser Thr Gly Cys Ser Phe

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<210> 3

<211> 1800

<212>DNA

<213> Artificial sequence

<400> 3

atggctgctg cttcagctcc attgggagtt tccttgcaag aagctactca aagaagattg 60

agaagatttt ccgaattgag aggtaagcca gttgctgctg gtgaattttg ggatattgtt 120

gctattactg ctgctgatga aaagcaagaa ttggcttaca agcaacaatt gtccgaaaag 180

ttgaagaagaaga aggaattgcc attgggagtt caatacttgg tttttgttga tccagctggt 240

gctaagattg gtaacggagg tagcactttg tgtgctttga gatgtttgga aaagttgtac 300

ggtgatcaat ggaactcctt tactattttg ttgattcata gcgggggata ctcccaaaga 360

ttgcctaacg cttcagcttt gggtaagatt tttactgctt tgccatttgg ttcacctatt 420

taccaaatgt tggaattgaa gttggctatg tacattgatt ttccatcaca tatgaaccct 480

ggtattttgg ttacttgtgc tgatgatatt gaattgtact ccattggaga atccgaattt 540

attagatttg ataagccagg atttactgct ttggctcatc catcatcctt gactgttggt 600

actactcatg gagtttttgt tttggaacca tttaacagat tggaatacag agattggaa 660

tacagatgtt gtcatagatt tttgcataag ccatctattg aaatgatgta caagtttgat 720

gctgtttgta gacctggtaa cttttcccaa caagattttg gtggaggtga tactccatcc 780

ttgaagctag atccagaata cgtttacact gattccgttt tttacatgga tcataagact 840

gctaagaagt tgttggcttt ttacgaaaag attgatactt tgaactgtga aattgatgct 900

tacggtgatt ttttgcaagc tttgggacca ggagctactg ttgaatacac tagaaacact 960

tctaacgtta ctaaggaaga atccgaattg gttgatatga gacaaagaat ttttcatttg 1020

ttgaagggta ctccattgaa cgttattgtt ttgaacaact ctaagtttta ccatattggt 1080

actactaagg aatacttgtt tcattttact tctgattctt ccttgaagtc cgaattggga 1140

ttgcaatcaa ttgctttttc tatttttcca gctattccag aatactcagg taacaagtca 1200

tgtattattc aatctatttt ggattctaga tgttccttgg ctccaggatc agttgttgaa 1260

tactctagat tgggacctga tgtttccgtt ggtgaaaact gtattatttc aggttcacat 1320

attattacta gagctatttt gccagcttac tcctttgttt gttccttgtc cttgaagatt 1380

aacggacata ttaagtactc aactatggct tgtggagttc aagataactt gaagaagaac 1440

gttaagactt tgtccgatgt taagttgttg caattttttg gagtttcctt gttgtcctgt 1500

ttggatgttt ggaacttgga agttactgaa gaattgtttt caggtaacaa gacttgtttg 1560

tccttgtgga acgctagaat ttttccagtt tgttcttcct tgtccgattc cgttattgct 1620

tccttgaaga tgttgaacgc tgttcaatct aagtcagttt tttctttgaa caactacaag 1680

ttgttgtcaa ttgaagaaat gttggtttac aaggatgttg aagatatgat tacttacaga 1740

gaacaaattt ttttggaaat tgctttgaac agaaagcaat ccgttttgga aacttcttaa 1800

<210> 4

<211> 3252

<212>DNA

<213> Artificial sequence

<400> 4

atggaacaac ctaagggagt tgattggact gttattattt tgacttgtca atacaaggat 60

tccgttgaag tttttcaaaa ggaattggaa attagacaaa agagagaaca aattccagct 120

tctactttgt tgttggctgt tgaagatcct gaagttcatg ttggtagtgg aggagctact 180

ttgaacgctt tgttggttgc tgctgaacat ttgtccgcta gagctggatt tactgttgtt 240

acttccgatg ttttgcattc cgcttggatt ttgattttgc atatgggtag agattttcca 300

tttgatgatt gtggtagagc ttttacttgt ttgccagttg aaaacccaca agctccagtt 360

gaagctgttg tttgtaactt ggattgtttg ttggatatta tgtcacatag attgggacca 420

ggatcacctc caggagtttg ggtttgttct actgatatgt tgttgtcagt tccacctaac 480

ccaggttatta actgggatgg atttagaggt gctagagtta ttgctttgcc aggaagcact 540

gcttacgcta gaaaccatgg agtttacttg actgattccc aaggatttgt tttggatatt 600

tactaccaag gaactgaagc tgaaattcaa agatgtgcta gacctgatgg tcaagttcca 660

ttggtttccg gtattgtttt tttttccgtt gaaactgctg aacatttgtt ggctactcat 720

gtttccccac cattggatgc ttgtacttac atggattgg attcaggtgc tcaaccagtt 780

caattgtcat tgttttttga tattttgttg tgtatggcta gaaacgttag aagagaagat 840

tttttggttg gtagacccacc agaaatgggt agaggtgata tggaaactga aggttacttg 900

agaggtgcta gagctgaatt gtggagagaa ttgagagatc aaccattgac tttggcttac 960

gttcctgatg gttcttacaa ctacatgact aactccgctt cagaattttt gcattccttg 1020

acttttccag gtgcttccgg tgctcaagtt gttcattccc aagttgaaga acaacaattg 1080

ttgggtgctg gttcttccgt tgtttcctgt ttgttggaag gaccagttca attgggacca 1140

ggttccgttt tgcaacattg tcatttgaga ggacctattc atattggtac tggatgtttt 1200

gtttcgggct tggatgctgc tcaatccgaa gctttgcatt ccttggaatt gcatgatttg 1260

gttttgcaag gtcatcattt gcaattgcat ggtgctccat ccagagcttt tactttggtt 1320

ggtagattgg attcctggga aagacaaggt gctggtactt acttgaatat gtcctggtct 1380

aagttttttc aaaagactgg tattatagagat tgggatttgt gggaccctga tactcctcca 1440

actgaaagat gtttgttgtc cgctagattg tttccagttt tgcatccatt gagagctttg 1500

ggaccacaag atatgttgtg gatgttagat cctcaagaag atggaggtaa ggctttgaga 1560

gcttggagag attcatggag attgtcatgg gaacaattgc aaccatgttt ggatagagct 1620

gctactttgg cttctagaag agatttgttt tttagacaag ctttgcataa ggctcgccat 1680

gttttggaag ctagacaaga tttgtccttg agaccattga ttagagctgc tgttagagaa 1740

ggatgtccag gtccatgat ggctactttg gatcaagttg ctgctggagc tggtgatcca 1800

ggagttgctg ctagagcttt ggcttgtgtt gctgatattt tgggatgtat ggctgaaggt 1860

agaggtggtt tgagatcagg acctgctgct aacccagaat gggttagacc attttcttac 1920

ttggaatgtg gtgatttggc tggaggagtt gaagctttgg ctcaagaaag agaaaagtgg 1980

ttgtctagac ctgctttgtt ggttagagct gctagacatt acgaaggtgc tggacaaatt 2040

ttgattagac aagctgttat gtccgctcaa cattttgttt ctactgaacc agttgaattg 2100

ccagctccag gtcaatgggt tgttgctgaa tgtccagcta gagttgattt ttcaggagga 2160

tggtccgata ctccaccatt ggcttacgaa catggaggtg ctgttttggg attggctgtt 2220

agagttgatg gtagaagacc tattggtgct agagctagac gtattccaga accagaattg 2280

tggttggctg ttggacctca acatgataag atggctatga agattgtttg tagatccttg 2340

gatgatttgc aagattactg tcaacctcat gctccaggag ctttgttgaa ggctgctttt 2400

atttgtgctg gtattttgtc cgttcattca gaattgtcct tgtccgaaca attgttgtgt 2460

acttttggag gaggatttga attgcaaact tggtccgaat tgccacatgg ttcaggattg 2520

ggtacttctt ctattttggc tggtgctgct ttggctgctt tgcaaagagt tgctggtaga 2580

gctgttggta ctgaagcttt gattcatgct gttttgcatt tggaacaagt tttgactact 2640

ggaggaggat ggcaagatca agttggagga ttgatgccag gtattaaggt tggaagatcc 2700

cgtgcgcaat tgccattgaa ggttgaagtt gaagaaatta ctgttcctgc tggttttgtt 2760

caaaagttga acgatcattt gttgttggtt tacactggta agactagatt ggctagaaac 2820

ttgttgcaag atgttttgag atcctggtac gctagattgc caccagttgt tcaaaacgct 2880

cataacttgg ttcaacaaac tgaagaatgt gctgaagctt ttagacaagg ttccttgcca 2940

agattgggtc aatgtttgac ttcttactgg gaacaaaaga agttgatggc tccaggatgt 3000

gaaccattgg ctgttagaca tatgatggat gctttggctc ctcatgttca tggtcaatcc 3060

ttggctggtg ctggtggagg aggttttttg tacttgttga ctaaggaacc tagacaaaag 3120

gaagctttgg aagctgtttt ggctaagact gaaggattgg gtaactactc agttcatttg 3180

gttgaagttg atactcaagg attgtccttg caattgttgg gaactgaaac ttctactgga 3240

tgttcctttt aa 3252

<210> 5

<211> 13774

<212>DNA

<213> Artificial sequence

<400> 5

tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg 60

gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg 120

ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag 180

cgtggcgctt tctcaatgct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc 240

caagctgggc tgtgtgcacg aacccccccgt tcagcccgac cgctgcgcct tatccggtaa 300

ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg 360

taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc 420

taactacggc tacactagaa ggacagtatt tggtatctgc gctctgctga agccagttac 480

cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg gtagccggtg 540

gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt 600

tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg 660

tcatgagatc agatcttttt tgtagaaatg tcttggtgtc ctcgtccaat caggtagcca 720

tctctgaaat atctggctcc gttgcaactc cgaacgacct gctggcaacg taaaattctc 780

cggggtaaaa cttaaatgtg gagtaatgga accagaaacg tctcttccct tctctctcct 840

tccaccgccc gttaccgtcc ctaggaaatt ttactctgct ggagagcttc ttctacggcc 900

cccttgcagc aatgctcttc ccagcattac gttgcgggta aaacggaggt cgtgtacccg 960

acctagcagc ccagggatgg aaaagtcccg gccgtcgctg gcaataatag cgggcggacg 1020

catgtcatga gattattgga aaccaccaga atcgaatata aaaggcgaac acctttccca 1080

atttggttt ctcctgaccc aaagacttta aatttaattt atttgtccct atttcaatca 1140

attgaacaac tatatggctg ctgcttcagc tccattggga gtttccttgc aagaagctac 1200

tcaaagaaga ttgagaagat tttccgaatt gagaggtaag ccagttgctg ctggtgaatt 1260

ttgggatatt gttgctatta ctgctgctga tgaaaagcaa gaattggctt acaagcaaca 1320

attgtccgaa aagttgaaga agaaggaatt gccattggga gttcaatact tggtttttgt 1380

tgatccagct ggtgctaaga ttggtaacgg aggtagcact ttgtgtgctt tgagatgttt 1440

ggaaaagttg tacggtgatc aatggaactc ctttactatt ttgttgattc atagcggggg 1500

atactcccaa agattgccta acgcttcagc tttgggtaag atttttactg ctttgccatt 1560

tggttcacct atttaccaaa tgttggaatt gaagttggct atgtacattg attttccatc 1620

acatatgaac cctggtattt tggttacttg tgctgatgat attgaattgt actccattgg 1680

agaatccgaa tttattagat ttgataagcc aggatttact gctttggctc atccatcatc 1740

cttgactgtt ggtactactc atggagtttt tgttttggaa ccatttaaca gattggaata 1800

cagagatttg gaatacagat gttgtcatag atttttgcat aagccatcta ttgaaatgat 1860

gtacaagttt gatgctgttt gtagacctgg taacttttcc caacaagatt ttggtggagg 1920

tgatactcca tccttgaagc tagatccaga atacgtttac actgattccg ttttttacat 1980

ggatcataag actgctaaga agttgttggc tttttacgaa aagattgata ctttgaactg 2040

tgaaattgat gcttacggtg attttttgca agctttggga ccaggagcta ctgttgaata 2100

cactagaaac acttctaacg ttactaagga agaatccgaa ttggttgata tgagacaaag 2160

aatttttcat ttgttgaagg gtactccatt gaacgttatt gttttgaaca actctaagtt 2220

ttaccatatt ggtactacta aggaatactt gtttcatttt acttctgatt cttccttgaa 2280

gtccgaattg ggattgcaat caattgcttt ttctattttt ccagctattc cagaatactc 2340

aggtaacaag tcatgtatta ttcaatctat tttggattct agatgttcct tggctccagg 2400

atcagttgtt gaatactcta gattgggacc tgatgtttcc gttggtgaaa actgtattat 2460

ttcaggttca catattatta ctagagctat tttgccagct tactcctttg tttgttcctt 2520

gtccttgaag attaacggac atattaagta ctcaactatg gcttgtggag ttcaagataa 2580

cttgaagaag aacgttaaga ctttgtccga tgttaagttg ttgcaatttt ttggagtttc 2640

cttgttgtcc tgtttggatg tttggaactt ggaagttact gaagaattgt tttcaggtaa 2700

caagacttgt ttgtccttgt ggaacgctag aatttttcca gtttgttctt ccttgtccga 2760

ttccgttatt gcttcccttga agatgttgaa cgctgttcaa tctaagtcag ttttttcttt 2820

gaacaactac aagttgttgt caattgaaga aatgttggtt tacaaggatg ttgaagatat 2880

gattacttac agagaacaaa tttttttgga aattgctttg aacagaaagc aatccgtttt 2940

ggaaacttct taagaacaaa aactcatctc agaagaggat ctgaatagcg ccgtcgacca 3000

tcatcatcat catcattgag ttttagcctt agacatgact gttcctcagt tcaagttggg 3060

cacttacgag aagaccggtc ttgctagatt ctaatcaaga ggatgtcaga atgccatttg 3120

cctgagagat gcaggcttca tttttgatac ttttttattt gtaacctata tagtatagga 3180

ttttttttgt cattttgttt cttctcgtac gagcttgctc ctgatcagcc tatctcgcag 3240

ctgatgaata tcttgtggta ggggtttggg aaaatcattc gagtttgatg tttttcttgg 3300

tatttcccac tcctcttcag agtacagaag attaagtgag aacgcgtaga tcttttttgt 3360

agaaatgtct tggtgtcctc gtccaatcag gtagccatct ctgaaatatc tggctccgtt 3420

gcaactccga acgacctgct ggcaacgtaa aattctccgg ggtaaaactt aaatgtggag 3480

taatggaacc agaaacgtct cttcccttct ctctccttcc accgcccgtt accgtcccta 3540

ggaaatttta ctctgctgga gagcttcttc tacggccccc ttgcagcaat gctcttccca 3600

gcattacgtt gcgggtaaaa cggaggtcgt gtacccgacc tagcagccca gggatggaaa 3660

agtcccggcc gtcgctggca ataatagcgg gcggacgcat gtcatgagat tattggaaac 3720

caccagaatc gaatataaaa ggcgaacacc tttcccaatt ttggtttctc ctgacccaaa 3780

gactttaaat ttaatttatt tgtccctatt tcaatcaatt gaacaactat ttcgaaacga 3840

ggaattcatg gaacaaccta agggagttga ttggactgtt attattttga cttgtcaata 3900

caaggattcc gttgaagttt ttcaaaagga attggaaatt agacaaaaga gagaacaaat 3960

tccagcttct actttgttgt tggctgttga agatcctgaa gttcatgttg gtagtggagg 4020

agctactttg aacgctttgt tggttgctgc tgaacatttg tccgctagag ctggatttac 4080

tgttgttact tccgatgttt tgcattccgc ttggattttg attttgcata tgggtagaga 4140

ttttccattt gatgattgtg gtagagcttt tacttgtttg ccagttgaaa accccacaagc 4200

tccagttgaa gctgttgttt gtaacttgga ttgtttgttg gatattatgt cacatagatt 4260

gggaccagga tcacctccag gagtttgggt ttgttctact gatatgttgt tgtcagttcc 4320

acctaaccca ggtattaact gggatggatt tagaggtgct agagttatg ctttgccagg 4380

aagcactgct tacgctagaa accatggagt ttacttgact gattcccaag gatttgtttt 4440

ggatattac taccaaggaa ctgaagctga aattcaaaga tgtgctagac ctgatggtca 4500

agttccattg gtttccggta ttgtttttttttccgttgaa actgctgaac atttgttggc 4560

tactcatgtt tccccaccat tggatgcttg tacttacatg ggattggatt caggtgctca 4620

accagttcaa ttgtcattgt tttttgatat tttgttgtgt atggctagaa acgttagaag 4680

agaagatttt ttggttggta gaccaccaga aatgggtaga ggtgattgg aaactgaagg 4740

ttacttgaga ggtgctagag ctgaattgtg gagagaattg agagatcaac cattgacttt 4800

ggcttacgtt cctgatggtt cttacaacta catgactaac tccgcttcag aatttttgca 4860

ttccttgact tttccaggtg cttccggtgc tcaagttgtt cattcccaag ttgaagaaca 4920

acaattgttg ggtgctggtt cttccgttgt ttcctgtttg ttggaaggac cagttcaatt 4980

gggaccaggt tccgttttgc aacattgtca tttgagagga cctattcata ttggtactgg 5040

atgttttgtt tcgggcttgg atgctgctca atccgaagct ttgcattcct tggaattgca 5100

tgatttggtt ttgcaaggtc atcatttgca attgcatggt gctccatcca gagcttttac 5160

tttggttggt agattggatt cctgggaaag acaaggtgct ggtacttact tgaatatgtc 5220

ctggtctaag ttttttcaaa agactggtat tagagattgg gatttgtggg accctgatac 5280

tcctccaact gaaagatgtt tgttgtccgc tagattgttt ccagttttgc atccattgag 5340

agctttggga ccacaagata tgttgtggat gttagatcct caagaagatg gaggtaaggc 5400

tttgagagct tggagagatt catggagatt gtcatgggaa caattgcaac catgtttgga 5460

tagagctgct actttggctt ctagaagaga tttgtttttt agacaagctt tgcataaggc 5520

tcgccatgtt ttggaagcta gacaagattt gtccttgaga ccattgatta gagctgctgt 5580

tagagaagga tgtccaggtc cattgatggc tactttggat caagttgctg ctggagctgg 5640

tgatccagga gttgctgcta gagctttggc ttgtgttgct gatattttgg gatgtatggc 5700

tgaaggtaga ggtggtttga gatcaggacc tgctgctaac ccagaatggg ttagaccatt 5760

ttcttacttg gaatgtggtg atttggctgg aggagttgaa gctttggctc aagaaagaga 5820

aaagtggttg tctagacctg ctttgttggt tagagctgct agacattacg aaggtgctgg 5880

acaaattttg attagacaag ctgttatgtc cgctcaacat tttgtttcta ctgaaccagt 5940

tgaattgcca gctccaggtc aatgggttgt tgctgaatgt ccagctagag ttgatttttc 6000

aggaggatgg tccgatactc caccattggc ttacgaacat ggaggtgctg ttttgggatt 6060

ggctgttaga gttgatggta gaagacctat tggtgctaga gctagacgta ttccagaacc 6120

agaattgtgg ttggctgttg gacctcaaca tgataagatg gctatgaaga ttgtttgtag 6180

atccttggat gatttgcaag attackgtca acctcatgct ccaggagctt tgttgaaggc 6240

tgcttttatt tgtgctggta ttttgtccgt tcattcagaa ttgtccttgt ccgaacaatt 6300

gttgtgtact tttggaggag gatttgaatt gcaaacttgg tccgaattgc cacatggttc 6360

aggattgggt acttcttcta ttttggctgg tgctgctttg gctgctttgc aaagagttgc 6420

tggtagagct gttggtactg aagctttgat tcatgctgtt ttgcatttgg aacaagtttt 6480

gactactgga ggaggatggc aagatcaagt tggaggattg atgccaggta ttaaggttgg 6540

aagatcccgt gcgcaattgc cattgaaggt tgaagttgaa gaaattactg ttcctgctgg 6600

ttttgttcaa aagttgaacg atcatttgtt gttggtttac actggtaaga ctagattggc 6660

tagaaacttg ttgcaagatg ttttgagatc ctggtacgct agattgccac cagttgttca 6720

aaacgctcat aacttggttc aacaaactga agaatgtgct gaagctttta gacaaggttc 6780

cttgccaaga ttgggtcaat gtttgacttc ttactgggaa caaaagaagt tgatggctcc 6840

aggatgtgaa ccattggctg ttagacatat gatggatgct ttggctcctc atgttcatgg 6900

tcaatccttg gctggtgctg gtggaggagg ttttttgtac ttgttgacta aggaacctag 6960

acaaaaggaa gctttggaag ctgttttggc taagactgaa ggattgggta actactcagt 7020

tcatttggtt gaagttgata ctcaaggatt gtccttgcaa ttgttgggaa ctgaaacttc 7080

tactggatgt tccttttaag cggccgccag cttgggcccg aacaaaaact catctcagaa 7140

gaggatctga atagcgccgt cgaccatcat catcatcatc attgagtttt agccttagac 7200

atgactgttc ctcagttcaa gttgggcact tacgagaaga ccggtcttgc tagattctaa 7260

tcaagaggat gtcagaatgc catttgcctg agagatgcag gcttcatttt tgatactttt 7320

ttatttgtaa cctatatagt ataggatttt ttttgtcatt ttgtttcttc tcgtacgagc 7380

ttgctcctga tcagcctatc tcgcagctga tgaatatctt gtggtagggg tttgggaaaa 7440

tcattcgagt ttgatgtttt tcttggtatt tcccactcct cttcagagta cagaagatta 7500

agtgagacct tcgtttgtgc ggatccatga catttccctt gctacctgca tacgcaagtg 7560

ttgcagagtt tgataattcc ttgagtttgg taggaaaagc cgtgtttccc tatgctgctg 7620

accagctgca caacctgatc aagttcactc aatcgactga gcttcaagtt aatgtgcaag 7680

ttgagtcatc cgttacagag gaccaatttg aggagctgat cgacaacttg ctcaagttgt 7740

acaataatgg tatcaatgaa gtgattttgg acctagattt ggcagaaaga gttgtccaaa 7800

ggatgatccc aggcgctagg gttatctata ggaccctggt tgataaagtt gcatccttgc 7860

ccgctaatgc tagtatcgct gtgccttttt cttctccact gggcgatttg aaaagtttca 7920

ctaatggcgg tagtagaact gtttatgctt tttctgagac cgcaaagttg gtagatgtga 7980

cttccactgt tgcttctggt ataatcccca ttattgaatgc tcggcaattg actactgaat 8040

acgaactttc tgaagatgtc aaaaagttcc ctgtcagtga aattttgttg gcgtctttga 8100

ctactgaccg ccccgatggt ctattcacta ctttggtggc tgactcttct aattactcgt 8160

tgggcctggt gtactcgtcc aaaaagtcta ttccggaggc tataaggaca caaactggag 8220

tctaccaatc tcgtcgtcac ggtttgtggt ataaaggtgc tacatctgga gcaactcaaa 8280

agttgctggg tatcgaattg gattgtgatg gagactgctt gaaatttgtg gttgaacaaa 8340

caggtgttgg tttctgtcac ttggaacgca cttcctgttt tggccaatca aagggtctta 8400

gagccatgga agccaccttg tgggatcgta agagcaatgc tccagaaggt tcttatacca 8460

aacggttattgacgacgaa gttttgttga acgctaaaat tagggaggaa gctgatgaac 8520

ttgcagaagc taaatccaag gaagatatag cctgggaatg tgctgactta ttttattttg 8580

cattagttag atgtgccaag tacggtgtga cgttggacga ggtggagaga aacctggata 8640

tgaagtccct aaaggtcact agaaggaaag gagatgccaa gccaggatac accaaggaac 8700

aacctaaaga agaatccaaa cctaaagaag tcccttctga aggtcgtatt gaattgtgca 8760

aaattgacgt ttctaaggcc tcctcacaag aaattgaaga tgcccttcgt cgtcctatcc 8820

agaaaacgga acagattatg gaattagtca aaccaattgt cgacaatgtt cgtcaaaatg 8880

gtgacaaagc ccttttagaa ctaactgcca agtttgatgg agtcgctttg aagacacctg 8940

tgttagaagc tcctttccca gaggaactta tgcaattgcc agataacgtt aagagagcca 9000

ttgatctctc tatagataac gtcaggaaat tccatgaagc tcaactaacg gagacgttgc 9060

aagttgagac ttgccctggt gtagtctgct ctcgttttgc aagacctatt gagaaagttg 9120

gcctctatat tcctggtgga accgcaattc tgccttccac ttccctgatg ctgggtgttc 9180

ctgccaaagt tgctggttgc aaagaaattg tttttgcatc tccacctaag aaggatggta 9240

cccttacccc agaagtcatc tacgttgccc acaaggttgg tgctaagtgt atcgtgctag 9300

caggaggcgc ccaggcagta gctgctatgg cttacggaac agaaactgtt cctaagtgtg 9360

acaaaatatt tggtccagga aaccagttcg ttactgctgc caagatgatg gttcaaaatg 9420

acacatcagc cctgtgtagt attgacatgc ctgctgggcc ttctgaagtt ctagttattg 9480

ctgataaata cgctgatcca gatttcgttg cctcagacct tctgtctcaa gctgaacatg 9540

gtattgattc ccaggtgatt ctgttggctg tcgatatgac agacaaggag cttgccagaa 9600

ttgaagatgc tgttcacaac caagctgtgc agttgccaag ggttgaaatt gtacgcaagt 9660

gtattgcaca ctctacaacc ctatcggttg caacctacga gcaggctttg gaaatgtcca 9720

atcagtacgc tcctgaacac ttgatcctgc aaatcgagaa tgcttcttct tatgttgatc 9780

aagtacaaca cgctggatct gtgtttgttg gtgcctactc tccagagagt tgtggagatt 9840

actcctccgg taccaaccac actttgccaa cgtacggata tgcccgtcaa tacagcggag 9900

ttaacactgc aaccttccag aagttcatca cttcacaaga cgtaactcct gagggactga 9960

aacatattgg ccaagcagtg atggatctgg ctgctgttga aggtctagat gctcaccgca 10020

atgctgttaa ggttcgtatg gagaaactgg gacttattta aggatcctac cgttcgtata 10080

gcatacatta tacgaagtta taacatccaa agacgaaagg ttgaatgaaa cctttttgcc 10140

atccgacatc cacaggtcca ttctcacaca taagtgccaa acgcaacagg aggggtaca 10200

ctagcagcag accgttgcaa acgcaggacc tccactcctc ttctcctcaa cacccacttt 10260

tgccatcgaa aaaccagccc agttattggg cttgattgga gctcgctcat tccaattcct 10320

tctattaggc tactaacacc atgactttat tagcctgtct atcctggccc ccctggcgag 10380

gttcatgttt gtttatttcc gaatgcaaca agctccgcat tacacccgaa catcactcca 10440

gatgagggct ttctgagtgtggggtcaaat agtttcatgt tccccaaatg gcccaaaact 10500

gacagtttaa acgctgtctt ggaacctaat atgacaaaag cgtgatctca tccaagatga 10560

actaagtttg gttcgttgaa atgctaacgg ccagttggtc aaaaagaaac ttccaaaagt 10620

cggcataccg tttgtcttgt ttggtattga ttgacgaatg ctcaaaaata atctcattaa 10680

tgcttagcgc agtctctcta tcgcttctga accccggtgc acctgtgccg aaacgcaaat 10740

ggggaaacac ccgctttttg gatgattatg cattgtctcc aattgtatg cttccaagat 10800

tctggtggga atactgctga tagcctaacg ttcatgatca aaatttaact gttctaaccc 10860

ctacttgaca gcaatatata aacagaagga agctgccctg tcttaaacct ttttttttat 10920

catcattatt agcttacttt cataattgcg actggttcca attgacaagc ttttgatttt 10980

aacgactttt aacgacaact tgagaagatc aaaaaacaac taattattcg aaacggaatt 11040

gtgagcggat aacaaaggat gtccaattta ctgaccgtac accaaaattt gcctgcatta 11100

ccggtcgatg caacgagtga tgaggttcgc aagaacctga tggacatgtt cagggatcgc 11160

caggcgtttt ctgagcatac ctggaaaatg cttctgtccg tttgccggtc gtgggcggca 11220

tggtgcaagt tgaataaccg gaaatggttt cccgcagaac ctgaagatgt tcgcgattat 11280

cttctatatc ttcaggcgcg cggtctggca gtaaaaacta tccagcaaca tttgggccag 11340

ctaaacatgc ttcatcgtcg gtccgggctg ccacgaccaa gtgacagcaa tgctgtttca 11400

ctggttatgc ggcgcatccg aaaagaaaac gttgatgccg gtgaacgtgc aaaacaggct 11460

ctagcgttcg aacgcactga tttcgaccag gttcgttcac tcatggaaaa tagcgatcgc 11520

tgccaggata tacgtaatct ggcatttctg gggattgctt ataacaccct gttacgtata 11580

gccgaaattg ccaggatcag ggttaaagat atctcacgta ctgacggtgg gagaatgtta 11640

atccatattg gcagaacgaa aacgctggtt agcaccgcag gtgtagagaa ggcacttagc 11700

ctgggggtaa ctaaactggt cgagcgatgg atttccgtct ctggtgtagc tgatgatccg 11760

aataactacc tgttttgccg ggtcagaaaa aatggtgttg ccgcgccatc tgccaccagc 11820

cagctatcaa ctcgcgccct ggaagggatt tttgaagcaa ctcatcgatt gatttacggc 11880

gctaaggatg actctggtca gagatacctg gcctggtctg gacacagtgc ccgtgtcgga 11940

gccgcgcgag atatggcccg cgctggagtt tcaataccgg agatcatgca agctggtggc 12000

tggaccaatg taaatattgt catgaactat atccgtaccc tggatagtga aacaggggca 12060

atggtgcgcc tgctggaaga tggcgattag cgaacaaaaa ctcatctcag aagaggatct 12120

gaatagcgcc gtcgaccatc atcatcatca tcattgagtt ttagccttag acatgactgt 12180

tcctcagttc aagttgggca ccttacgagaa gaccggtctt gctagattct aatcaagagg 12240

atgtcagaat gccatttgcc tgagagatgc aggcttcatt tttgatactt ttttatttgt 12300

aacctatata gtataggatt ttttttgtca ttttgtttct tctcgtacga gcttgctcct 12360

gatcagccta tctcgcagct gatgaatatc ttgtggtagg ggtttgggaa aatcattcga 12420

gtttgatgtt tttcttggta tttcccactc ctcttcagag tacagaagat taagtgagac 12480

cttcgtttgt gcggaaccccc cacacaccat agcttcaaaa tgtttctact ccttttttac 12540

tcttccagat tttctcggac tccgcgcatc gccgtaccac ttcaaaacac ccaagcacag 12600

catactaaat tttccctctt tcttccctcta gggtgtcgtt aattacccgt actaaaggtt 12660

tggaaaagaaaaaagagacc gcctcgtttc tttttcttcg tcgaaaaagg caataaaaat 12720

ttttatcacg tttctttttc ttgaaatttt tttttttagt ttttttctct ttcagtgacc 12780

tccattgata tttaagttaa taaacggtct tcaatttctc aagtttcagt ttcatttttc 12840

ttgttctatt acaacttttt ttacttcttg ttcattagaa agaaagcata gcaatctaat 12900

ctaaggggcg gtgttgacaa ttaatcatcg gcatagtata tcggcatagt ataatacgac 12960

aaggtgagga actaaaccat ggccaagttg accagtgccg ttccggtgct caccgcgcgc 13020

gacgtcgccg gagcggtcga gttctggacc gaccggctcg ggttctcccg ggacttcgtg 13080

gaggacgact tcgccggtgt ggtccgggac gacgtgaccc tgttcatcag cgcggtccag 13140

gaccaggtgg tgccggacaa caccctggcc tgggtgtggg tgcgcggcct ggacgagctg 13200

tacgccgagt ggtcggaggt cgtgtccacg aacttccggg acgcctccgg gccggccatg 13260

accgagatcg gcgagcagcc gtgggggcgg gagttcgccc tgcgcgaccc ggccggcaac 13320

tgcgtgcact tcgtggccga ggagcaggac tgacacgtcc gacggcggcc cacgggtccc 13380

aggcctcgga gatccgtccc ccttttcctt tgtcgatatc atgtaattag ttatgtcacg 13440

cttacattca cgccctcccc ccacatccgc tctaaccgaa aaggaaggag ttagacaacc 13500

tgaagtctag gtccctattt atttttttat agttatgtta gttattaagaa cgttatttt 13560

atttcaaatt tttcttttttttctgtacag ataacttcgt atagcataca ttatacgaac 13620

ggtaacgcgt gtacgcatgt aacattatac tgaaaacctt gcttgagaag gttttgggac 13680

gctcgaaggc tttaatttgc aagctggaga ccaacatgtg agcaaaaggc cagcaaaagg 13740

ccaggaaccg taaaaaggcc gcgttgctgg cgtt 13774

Claims (8)

1. A recombinant Pichia cell, characterized in that, the recombinant cell comprises a carrier, and the carrier contains an expression sequence of fucose-1-phosphate guanosyltransferase and an expression sequence of fucokinase; The expression sequence of the fucose-1-phosphate guanosyltransferase is shown in SEQ ID NO.3, and the expression sequence of the fucose kinase is shown in SEQ ID NO.4. 2. The recombinant Pichia cell according to claim 1, wherein the amino acid sequence of the fucose-1-phosphate guanosyltransferase is as shown in SEQ ID NO.1, and the fucose kinase The amino acid sequence of is shown in SEQ ID NO.2. 3. The recombinant Pichia cell according to claim 1, wherein the sequence of the vector is shown in SEQ ID NO.5. 4. The recombinant Pichia cell according to claim 1, wherein the cell is Pichia GS115. 5. A method for constructing the recombinant Pichia cell according to any one of claims 1 to 4, comprising the following steps: transferring the vector according to any one of claims 1 to 4 into the Pichia cell. 6. The use of the recombinant Pichia pastoris cell according to any one of claims 1 to 4 in the preparation of guanosine diphosphate fucose. 7. A method for preparing guanosine diphosphate fucose, which is produced by fermentation of the recombinant Pichia pastoris cell according to any one of claims 1-4. 8. The method according to claim 7, characterized in that, the fermentation condition is that the seed liquid of the recombinant Pichia pastoris cell is inserted into the fermentation medium with an OD value of 0.5 to 1.5, at 25 to 35° C. , Cultivate for 100-140 hours at 200-300 rpm.