CN108410870A - K marxianus promoter, secreting signal peptide and its preparation and application - Google Patents

K marxianus promoter, secreting signal peptide and its preparation and application Download PDF

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CN108410870A
CN108410870A CN201810153537.XA CN201810153537A CN108410870A CN 108410870 A CN108410870 A CN 108410870A CN 201810153537 A CN201810153537 A CN 201810153537A CN 108410870 A CN108410870 A CN 108410870A
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nucleotide sequence
primer
plasmid
pukdn132
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吕红
余垚
周峻岗
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Fudan University
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Abstract

The invention discloses a kind of K marxianus promoters of optimization, include at least：A) nucleotide sequence as shown in SEQ ID No.1；Or b) the nucleotide sequence as shown in SEQ ID No.2；Or c) the nucleotide sequence as shown in SEQ ID No.3.A kind of kluyveromyces marxianus secreting signal peptide of optimization is also disclosed, nucleotide sequence and a kind of assembly of optimization as shown in SEQ ID No.4 are included at least, includes at least the nucleotide sequence as shown in SEQ ID No.6.The invention also discloses the recombinant expression carriers containing above-mentioned K marxianus promoter and/or secreting signal peptide, genetic engineering bacterium and the preparation method and application thereof.The technical program can be obviously improved the ability of the high efficiency recombinant expressed foreign protein of kluyveromyces marxianus, to push kluyveromyces marxianus expression system to lay a good foundation in the application of industrial proteins manufacturing field.

Description

K marxianus promoter, secreting signal peptide and its preparation and application
Technical field
The present invention relates to technical field of bioengineering, and in particular to a kind of K marxianus promoter of optimization, Secreting signal peptide and combinations thereof body is carried containing the recombinant expression of the K marxianus promoter and/or secreting signal peptide Body, genetic engineering bacterium and the preparation method and application thereof.
Background technology
Kluyveromyces marxianus (Kluyveromyces marxianus, hereinafter referred KM) is homothallic half son Capsule bacterium yeast is located at Saccharomycetaceae Crewe dimension and belongs to.KM is usually in Yoghourt, koumiss, cheese, occurs in milk and sugarcane leaf. KM is a kind of yeast for the food-grade being commonly recognized.It has already been through the GRAS certifications of FDA, and quilt is gone back in the QPS certifications of European Union China defends planning commission and is approved as new raw-food material.Since its is safe, KM has in edible, the feeding and pharmaceutical protein field of production Advantageous advantage.But KM lacks efficient Expression element and mating expression vector at present, limits KM expression systems Development.
Invention content
In view of the drawbacks described above of the prior art, the present invention provides a kind of startups of the kluyveromyces marxianus of optimization Son, secreting signal peptide and combinations thereof body, the recombinant expression containing the K marxianus promoter and/or secreting signal peptide Carrier, genetic engineering bacterium and the preparation method and application thereof.Its specific technical solution is as follows：
The present invention provides a kind of K marxianus promoter of optimization in first aspect, and nucleotide sequence is extremely Include less：
A) nucleotide sequence as shown in SEQ ID No.1；Or
B) nucleotide sequence as shown in SEQ ID No.2；Or
C) nucleotide sequence as shown in SEQ ID No.3.
The present invention provides a kind of kluyveromyces marxianus secreting signal peptide of optimization, nucleotides sequence in second aspect Row include at least the nucleotide sequence as shown in SEQ ID No.4；Its amino acid sequence is included at least as shown in SEQ ID No.5 Amino acid sequence.
The present invention provides the K marxianus promoter and secreting signal peptide of a kind of optimization in the third aspect Assembly, nucleotide sequence include at least the nucleotide sequence as shown in SEQ ID No.6.
The present invention provides a kind of recombinant expression carrier in fourth aspect, and it includes the horses that the present invention is provided in first aspect The kluyveromyces marxianus secreting signal peptide that gram this kluyveromyces promoter and/or the present invention are provided in second aspect.
Preferably, above-mentioned recombinant expression carrier also includes kluyveromyces marxianus autonomously replicating sequence, Marx's Crewe Tie up yeast inulinase terminator gene order and auxotrophy riddled basins sequence.
It is highly preferred that above-mentioned auxotrophy riddled basins sequence is selected from URA3 genes, HIS3 genes and ADE2 genes One or more of.
Most preferably, above-mentioned auxotrophy riddled basins sequence is URA3 genes.
In preferred embodiment, the nucleotide sequence of above-mentioned recombinant expression carrier includes at least：
A), the nucleotide sequence as shown in SEQ ID No.22；Or
B), the nucleotide sequence as shown in SEQ ID No.23.
The present invention provides a kind of genetic engineering bacterium at the 5th aspect, and it includes marks that the present invention is provided in first aspect The kluyveromyces marxianus secreting signal peptide that this kluyveromyces promoter and/or the present invention are provided in second aspect.
The present invention also provides a kind of genetic engineering bacteriums, and it includes the recombinant expression loads that the present invention is provided in fourth aspect Body.
Preferably, the host strain of said gene engineering bacteria is yeast.
It is highly preferred that yeast includes at least kluyveromyces marxianus category yeast, Kluyveromyces lactis category yeast or wine Brewer yeast category yeast.
The present invention provides the application of above-mentioned K marxianus promoter at the 6th aspect, is used to improve external source The expression of albumen.
Preferably, foreign protein is feruloyl esterase.
The present invention provides the application of above-mentioned kluyveromyces marxianus secreting signal peptide at the 7th aspect, is used to improve Recombinant protein is from emiocytosis to extracellular efficiency.
The present invention provides the assembly of above-mentioned K marxianus promoter and secreting signal peptide in eighth aspect Application, be used to improve the expression of foreign protein, while improving recombinant protein from emiocytosis to extracellular efficiency.
The present invention provides the preparation method of above-mentioned K marxianus promoter, Marx's Crewe at the 9th aspect Yeast promoter is tieed up by carrying out coding mutation or deletion on the basis of kluyveromyces marxianus inulin enzyme promoters by structure It builds, includes at least：
A), by -351 thymidine deoxidation cores in the nucleotide sequence of kluyveromyces marxianus inulin enzyme promoters Nucleotide mutation is adenyl-deoxyribonucleotide；Or
B), 106 nucleotide bases between removal kluyveromyces marxianus inulin enzyme promoters -8bp to -115bp； Or
C), include the combinatorial mutagenesis of step a) and step b).
In preferred embodiment, above-mentioned preparation method specifically includes following steps：
Step 1, using the genome of kluyveromyces marxianus bacterium FIM-1 as template, utilize nucleotide sequence such as SEQ ID The primer I NU-R1PCR as shown in SEQ ID No.8 of primer I NU-F1 and nucleotide sequence shown in No.7 expands inulinase base Cause；Taq enzyme is added in the reaction system, and pcr amplification product is carried out to add A, obtains target fragment；Target fragment is recycled, with PMD18-T carriers are attached, and obtain the plasmid pMD18-T-INU containing kluyveromyces marxianus inulin enzyme promoters；
Step 2, using plasmid pMD18-T-INU as template, utilize nucleotide sequence primer as shown in SEQ ID No.9 INU Δ-F1 and nucleotide sequence the primer I NU Δ-R1 as shown in SEQ ID No.10 are by the sequence of inulin enzyme gene 79-1668bp Row removal, while multiple cloning sites are introduced between terminator codon after the 78bp of inulin enzyme gene, obtain plasmid pMD18-T-PINU-SP-MCS-TINU；
Step 3, with plasmid pMD18-T-PINU-SP-MCS-TINUFor template, nucleotide sequence such as SEQ ID No.11 are utilized Shown in primer I NU-F2 and nucleotide sequence the primer I NU-R2 as shown in SEQ ID No.12 expand PINU-SP-MCS-TINU Segment；
Step 4, using pUKD carriers as template, utilize nucleotide sequence primer pUKD-F1 as shown in SEQ ID No.14 And primer pUKD-R1 shown in nucleotide sequence such as SEQ ID No.15 removes the sites EcoRI between pKS and KD sequences, Obtain plasmid pUKD Δs E；Digestion is carried out to plasmid PUKD Δs E using EcoRI, recycles digestion products；
Step 5, by the P in step 3INU-SP-MCS-TINUSegment is connect with the digestion products in step 4, obtains plasmid pUKDN132；
Step 6, using plasmid pUKDN132 as template, using nucleotide sequence primer T as shown in SEQ ID No.16 (- 351) primer T (- 351) A-R will be on inulinase gene start codon as shown in SEQ ID No.17 for A-F and nucleotide sequence The thymidylic acid of trip 351 sports adenyl-deoxyribonucleotide, obtains plasmid pUKDN132-T (- 351) A, Including the nucleotide sequence as shown in SEQ ID No.1；Or
Step 7, using plasmid pUKDN132 as template, utilize nucleotide sequence primer UTR as shown in SEQ ID No.18 Δ A-F and nucleotide sequence the primer UTR Δs A-R as shown in SEQ ID No.19 are by inulinase gene start codon upstream Sequence removal between 8bp to upstream 115bp, obtains plasmid pUKDN132-UTR Δ A, including as shown in SEQ ID No.2 Nucleotide sequence；Or
Step 8, using plasmid pUKDN132-T (- 351) A obtained in step 6 as template, utilize nucleotide sequence such as SEQ The primer UTR Δs A-R as shown in SEQ ID No.19 of primer UTR Δs A-F and nucleotide sequence shown in ID No.18 is by inulin Sequence removal between enzyme gene upstream from start codon 8bp to upstream 115bp, obtains plasmid pUKDN132-T (- 351) A+ UTR Δ A, including the nucleotide sequence as shown in SEQ ID No.3.
Preferably, in above-mentioned steps 1, kluyveromyces marxianus bacterium FIM-1 is preserved in Chinese microorganism strain preservation pipe Reason committee common micro-organisms center, deposit number are CGMCC No.10621.
Preferably, in above-mentioned steps 2, multiple cloning sites include at least the site of SmaI, SpeI and NotI.
The present invention provides the preparation method of above-mentioned kluyveromyces marxianus secreting signal peptide, the preparation at the tenth aspect Method is included at least sports thymidylic acid by the deoxycytidylic acid of inulin enzyme gene the 29th.
In preferred embodiment, above-mentioned preparation method specifically includes following steps：Using plasmid pUKDN132 as template, utilize Nucleotide sequence primer P10L-F and nucleotide sequence primer as shown in SEQ ID No.21 as shown in SEQ ID No.20 The deoxycytidylic acid of inulin enzyme gene the 29th is sported thymidylic acid by P10L-R, and inulinase is made to believe The proline of number peptide the 10th becomes leucine.
Preferably, plasmid pUKDN132 is prepared according to the step 1-5 that the present invention is provided at the 9th aspect.
The present invention provides the combination of above-mentioned K marxianus promoter and secreting signal peptide in the tenth one side The preparation method of body comprising following steps：Using plasmid pUKDN132-T (- 351) A as template, nucleotide sequence such as SEQ is utilized The primer P10L-R as shown in SEQ ID No.21 of primer P10L-F and nucleotide sequence shown in ID No.20 is by inulinase base Because the 29th deoxycytidylic acid sports thymidylic acid, make the dried meat ammonia of inulinase signal peptide the 10th Acid becomes leucine.
Preferably, plasmid pUKDN132-T (- 351) A is prepared into according to the step 1-6 that the present invention is provided at the 9th aspect It arrives.
The present invention provides the preparation method of above-mentioned recombinant expression carrier at the 12nd aspect comprising following steps：
Step 1, using the genome of kluyveromyces marxianus bacterium FIM-1 as template, utilize nucleotide sequence such as SEQ ID The primer I NU-R1PCR as shown in SEQ ID No.8 of primer I NU-F1 and nucleotide sequence shown in No.7 expands inulinase base Cause；Taq enzyme is added in the reaction system, and pcr amplification product is carried out to add A, obtains target fragment；Target fragment is recycled, with PMD18-T carriers are attached, and obtain the plasmid pMD18-T-INU containing kluyveromyces marxianus inulin enzyme promoters；
Step 2, using plasmid pMD18-T-INU as template, utilize nucleotide sequence primer as shown in SEQ ID No.9 INU Δ-F1 and nucleotide sequence the primer I NU Δ-R1 as shown in SEQ ID No.10 are by the sequence of inulin enzyme gene 79-1668bp Row removal, while multiple cloning sites are introduced between terminator codon after the 78bp of inulin enzyme gene, obtain plasmid pMD18-T-PINU-SP-MCS-TINU；
Step 3, with plasmid pMD18-T-PINU-SP-MCS-TINUFor template, nucleotide sequence such as SEQ ID No.11 are utilized Shown in primer I NU-F2 and nucleotide sequence the primer I NU-R2 as shown in SEQ ID No.12 expand PINU-SP-MCS-TINU Segment；
Step 4, using pUKD carriers as template, utilize nucleotide sequence primer pUKD-F1 as shown in SEQ ID No.14 And primer pUKD-R1 shown in nucleotide sequence such as SEQ ID No.15 removes the sites EcoRI between pKS and KD sequences, Obtain plasmid pUKD Δs E；Digestion is carried out to plasmid PUKD Δs E using EcoRI, recycles digestion products；
Step 5, by the P in step 3INU-SP-MCS-TINUSegment is connect with the digestion products in step 4, obtains plasmid pUKDN132；
Step 6, using plasmid pUKDN132 as template, using nucleotide sequence primer T as shown in SEQ ID No.16 (- 351) primer T (- 351) A-R will be on inulinase gene start codon as shown in SEQ ID No.17 for A-F and nucleotide sequence The thymidylic acid of trip 351 sports adenyl-deoxyribonucleotide, obtains plasmid pUKDN132-T (- 351) A, Including the nucleotide sequence as shown in SEQ ID No.1；
Step 7, using plasmid pUKDN132-T (- 351) A obtained in step 6 as template, utilize nucleotide sequence such as SEQ The primer UTR Δs A-R as shown in SEQ ID No.19 of primer UTR Δs A-F and nucleotide sequence shown in ID No.18 is by inulin Sequence removal between enzyme gene upstream from start codon 8bp to upstream 115bp, obtains plasmid pUKDN132-T (- 351) A+ UTR Δ A, nucleotide sequence is as shown in SEQ ID No.22；Or
Step 8, using plasmid pUKDN132-T (- 351) A obtained in step 6 as template, utilize nucleotide sequence such as SEQ The primer P10L-R as shown in SEQ ID No.21 of primer P10L-F and nucleotide sequence shown in ID No.20 is by inulinase base Because the 29th deoxycytidylic acid sports thymidylic acid, plasmid pUKDN132-T (- 351) A+ is obtained P10L, nucleotide sequence is as shown in SEQ ID No.23.
Technical solution provided by the invention can be obviously improved the high efficiency recombinant expressed foreign protein of kluyveromyces marxianus Ability, to push kluyveromyces marxianus expression system to lay a good foundation in the application of industrial proteins manufacturing field.
It should be understood that within the scope of the present invention, above-mentioned each technical characteristic of the invention and have in below (eg embodiment) It can be combined with each other between each technical characteristic of body description, to form a new or preferred technical solution.As space is limited, exist This no longer tires out one by one states.So every do not depart from the equivalent or modification completed under principles of this disclosure, this hair is both fallen within The range of bright protection.
Below with reference to attached drawing, the invention will be further described, with absolutely prove the purpose of the present invention, technical characteristic and Technique effect.
Description of the drawings
Fig. 1 shows the recombinant expression carrier containing inulinase gene promoter and signal peptide in preferred embodiment of the present invention The schematic diagram of pUKDN132；
Fig. 2 shows T (- 351) A, UTR Δs A and P10L described in preferred embodiment of the present invention to be mutated in inulinase base Because of the position in promoter and signal peptide；
Fig. 3 shows T (- 351) A, UTR Δs A and P10L mutation described in preferred embodiment of the present invention and combinations thereof pair In the influence of the secreting, expressing of feruloyl esterase：On pUKDN132 carriers, the inulin enzyme promoters and letter of wild type or mutation The expression of number peptide driving feruloyl esterase；In the fermentation supernatant of transformant containing wild type carrier pUKDN132-EstE Ah The enzyme activity of Wei's acid esterase is defined as 1, and column indicates average value ± S.D (n=4)；
Fig. 4 shows shadow of T (- 351) A, UTR Δ A mutation described in preferred embodiment of the present invention for mRNA level in-site It rings：EstE in pUKDN132-EstE, pUKDN132-T (- 351) A-EstE and pUKDN132-UTR Δ A-EstE transformants MRNA is measured relative to the content of 18s rDNA；In transformant containing wild plasmid pUKDN132-EstE (WT) The relative amount of EstE mRNA is defined as 1, and column indicates average value ± S.D (n=4).
Fig. 5 shows shadow of the mutation of the P10L described in preferred embodiment of the present invention for feruloyl esterase secreting, expressing It rings；Contain pUKDN132-EstE-His6It is cultivated in YD culture mediums with the transformant of pUKDN132；It checks and accepts at the appointed time Collect supernatant cell；EstE-His in supernatant cell pyrolysis liquid is detected with Western6Content, histone H 3 is by as interior Ginseng.
Specific implementation mode
Before describing the present invention, it should be understood that the present invention is not limited to the specific method and experiment conditions, because this Class method and condition can change.It should also be understood that its purpose of the term as used herein is only that description specific embodiment, and And it is not intended to be restrictive, the scope of the present invention will be limited only by the claims which follow.
Embodiment 1, recombinant expression carrier containing kluyveromyces marxianus inulinase gene promoter and signal peptide Structure
According to the method for pastoris genomic dna extracts kit (Solarbio, D1900) specification, Marx gram is extracted Tie up the genome of saccharomycete FIM-1 in Shandong.The kluyveromyces marxianus bacterium is preserved in Chinese microorganism strain preservation conservator Meeting common micro-organisms center, deposit number are CGMCC No.10621 (depositary institution's codes：CGMCC, depositary institution address：North No. 3 China Committee for Culture Collection of Microorganisms's common micro-organisms centers of institute of the Chaoyang Districts Jing Shi North Star West Road 1, preservation date For on March 13rd, 2015, testing result：Survival, Classification And Nomenclature：Kluyveromyces marxianus Kluyveromyces marxianus)。
Using the genome of FIM-1 as template, primer I NU-F1 (sequence is as shown in SEQ ID No.7) and INU-R1 is utilized (sequence is as shown in SEQ ID No.8) expands inulin enzyme gene, including inulinase gene promoter (originates in initiation codon Swim 1136bp) inulin enzyme gene (1671bp) and inulinase gene terminator (842bp).The process of PCR is with reference to Phanta The description of product of Super Fidelity DNA Polymerase carries out (Vazyme, article No. P505-d1/d2/d3).PCR is completed Afterwards, 1 μ L Taq enzymes (Takara, R001Q) are added in reaction system, 72 DEG C of reaction 20min carry out PCR product to add A.Pass through Agarose electrophoretic analysis pcr amplification product obtains the target fragment of 3649bp.According to SanPrep pillar DNA glue reclaim reagents The specification operation of box (raw work, article No. B518131-0050), recycles PCR product.PCR product and pMD18-T are carried Body (Takara, 6011) is attached, and connection method is referring to product description.The plasmid of acquisition is named as pMD18-T-INU.
According to QuikChange II Site-Directed Mutagenesis Kit (Agilent, 200523) specification Method utilize primer I NU Δs-F1 (sequence is as shown in SEQ ID No.9) and INU Δs-R1 using pMD18-T-INU as template (sequence is as shown in SEQ ID No.10) removes the sequence of the 79-1668bp of inulin enzyme gene, while in inulin enzyme gene After 78bp multiple cloning sites are introduced between terminator codon (TGA).The multiple cloning sites include SmaI, SpeI and NotI Site.The plasmid of acquisition is named as pMD18-T-PINU-SP-MCS-TINU。
With pMD18-T-PINU-SP-MCS-TINUFor template, utilize primer I NU-F2 (sequence is as shown in SEQ ID No.11) P is expanded with INU-R2 (sequence is as shown in SEQ ID No.12)INU-SP-MCS-TINUSegment.The segment contains inulin enzyme gene Promoter (since upstream from start codon 1136bp, arriving upstream from start codon 1bp) (sequence such as SEQ ID No.13 institutes Show), the 1-78bp (sequence containing encoded signal peptide) of inulin enzyme gene, multiple cloning sites and inulinase terminator (842bp). PCR reactions use Phanta Super Fidelity DNA Polymerase.It is produced by agarose electrophoretic analysis PCR amplification Object obtains the P of 2150bpINU-SP-MCS-TINUSegment.Using SanPrep pillar DNA plastic recovery kits to PINU-SP- MCS-TINUSegment is recycled.
According to the operation instruction of QuikChange II Site-Directed Mutagenesis Kit, with pUKD carriers (Appl Microbiol Biotechnol(2003)62:387-391) it is template, utilizes primer pUKD-F1 (sequence such as SEQ Shown in ID No.14) and pUKD-R1 (sequence is as shown in SEQ ID No.15) by pKS sequences (Escherichia coli replication sequence and ammonia Benzyl resistant gene) between KD sequences (yeast autonomously replicating sequence) the sites EcoRI removal, acquisitions plasmid be pUKD Δs E.Profit Use EcoRI carries out digestion to pUKD Δs E, is recycled to digestion products using SanPrep pillar DNA plastic recovery kits.
Using Gibson Assembly traceless linkers system (NEB companies, article No. E2611S/L), by PINU-SP-MCS- The E of TINU segments and pUKD Δs EcoRI digestion products connect.The carrier of acquisition is pUKDN132, as shown in Figure 1.
Embodiment 2, the optimization to kluyveromyces marxianus inulinase gene promoter and signal peptide
According to the operation instruction of QuikChange II Site-Directed Mutagenesis Kit, with pUKDN132 For template, primer T (- 351) A-F (sequence is as shown in SEQ ID No.16) and primer T (- 351) A-R (sequence such as SEQ are utilized Shown in ID No.17) mutation is introduced in the promoter of inulinase, 351 T of upstream from start codon are sported into A, it should Mutation is in the binding site of potential transcription factor, as shown in Fig. 2, the carrier obtained is named as pUKDN132-T (- 351) A。
According to the operation instruction of QuikChange II Site-Directed Mutagenesis Kit, with pUKDN132 For template, primer UTR Δs A-F (sequence is as shown in SEQ ID No.18) and primer UTR Δs A-R (sequence such as SEQ ID are utilized Shown in No.19) sequence between upstream from start codon 8bp to upstream 115bp is removed, which is located at inulin enzyme gene In 5 ' UTR, as shown in Fig. 2, the carrier obtained is named as pUKDN132-UTR Δs A.
According to the operation instruction of QuikChange II Site-Directed Mutagenesis Kit, with pUKDN132 For template, primer P10L-F (sequence is as shown in SEQ ID No.20) and primer P10L-R (sequence such as SEQ ID No.21 are utilized It is shown) the 29th C of inulin enzyme gene be changed to T, which makes the 10th proline of inulinase signal peptide become bright Propylhomoserin.The carrier of acquisition is named as pUKDN132-P10L.
According to the operation instruction of QuikChange II Site-Directed Mutagenesis Kit, with pUKDN132- T (- 351) A is template, and using primer UTR Δs A-F (sequence is as shown in SEQ ID No.18) and primer UTR Δs A-R, (sequence is such as Shown in SEQ ID No.19) sequence between upstream from start codon 8bp to upstream 115bp is removed, which is located at inulin In 5 ' UTR of enzyme gene, as shown in Figure 2.The carrier of acquisition is named as pUKDN132-T (- 351) A+UTR Δ A, the carrier sequence Row are as shown in SEQ IDNo.22.
According to the operation instruction of QuikChange II Site-Directed Mutagenesis Kit, with pUKDN132- T (- 351) A is template, utilizes primer P10L-F (sequence is as shown in SEQ ID No.20) and primer P10L-R (sequence such as SEQ Shown in ID No.21) the 29th C of inulin enzyme gene be changed to T, which makes the 10th dried meat ammonia of inulinase signal peptide Acid becomes leucine.The carrier of acquisition is named as pUKDN132-T (- 351) A+P10L, the carrier sequence such as SEQ IDNo.23 It is shown.
Embodiment 3 realizes ferulic acid using the promoter and signal peptide of kluyveromyces marxianus inulinase gene mutation The secreting, expressing of esterase
The pUKDN112-EstE (number of patent application 201711086961.9) that laboratory is preserved using SpeI and NotI into Row digestion.The digestion products of 754bp are recycled using SanPrep pillar DNA plastic recovery kits.The segment contains EstE genes Coded sequence.
Using SpeI and NotI to pUKDN132, pUKDN132-T (- 351) A, pUKDN132-UTR Δ A, pUKDN132- P10L, pUKDN132-T (- 351) A+UTR Δ A and pUKDN132-T (- 351) A+P10L carries out digestion respectively, utilizes SanPrep Pillar DNA plastic recovery kits recycle digestion products.It is said according to DNA ligation Kit Ver.2.1 (Takara, 6022) Bright book operation, by pUKDN132, pUKDN132-T (- 351) A, pUKDN132-UTR Δ A, pUKDN132-P10L, pUKDN132- The digestion products of T (- 351) A+UTR Δ A and pUKDN132-T (- 351) A+P10L are connected with the digestion products of EstE respectively It connects.The plasmid of acquisition be named as pUKDN132-EstE, pUKDN132-T (- 351) A-EstE, pUKDN132-UTR Δ A-EstE, PUKDN132-P10L-EstE, pUKDN132-T (- 351) A+UTR Δ A-EstE and pUKDN132-T (- 351) A+P10L- EstE。
The Yeast expression host strain that the present invention uses derives from kluyveromyces marxianus bacterium Fim-1, is preserved in China Microbiological Culture Collection administration committee common micro-organisms center, deposit number are CGMCC No.10621 (depositary institution's codes： CGMCC, depositary institution address：Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3 China Committee for Culture Collection of Microorganisms is general Logical microorganism center, the deposit date is on March 13rd, 2015, testing results：Survival, Classification And Nomenclature：Kluyveromyces marxianus Kluyveromyces marxianus), it is knocked out except URA3 genes by the method for homologous recombination, and utilize and contain 5- fluorine wheys The YPD screenings of sour (1.5g/L) obtain the expressive host bacterium of uracil-deficient, are named as kluyveromyces marxianus Fim- 1ura3Δ.Using lithium acetate transformation method (World Journal of Microbiology＆Biotechnology 16:653- 654,2000), by pUKDN132-EstE, pUKDN132-T (- 351) A-EstE, pUKDN132-UTR Δ A-EstE, PUKDN132-P10L-EstE, pUKDN132-T (- 351) A+UTR Δ A-EstE and pUKDN132-T (- 351) A+P10L-EstE It is transferred to respectively in Fim-1ura3 Δs.Product after conversion is coated on SD tablets, and (0.67% without amino acid yeast nitrogen, 2% Portugal Grape sugar, 2% agar), tablet is placed into 30 DEG C of constant incubator cultures, is cultivated 2-4 days until Clone formation.
4 different clones are selected from every group of conversion, be inoculated in equipped with 50ml YD culture mediums (2% yeast extract, 4% glucose) in, 30 DEG C, 220rpm culture 72h after collect supernatant, carry out the enzyme activity determination of feruloyl esterase.Assay method and Enzyme activity definition carries out (number of patent application 201711086961.9) with reference to patent.PUKDN132-T (- 351) A-EstE transformants Supernatant enzyme activity is 2.4 times of wild plasmid pUKDN132-EstE enzyme activity, the supernatant of pUKDN132-UTR Δ A-EstE transformants Enzyme activity is 2.1 times of wild plasmid, and the supernatant enzyme activity of pUKDN132-P10L-EstE transformants is the 5.1 of wild plasmid Times.The result illustrates that the mutation of the inulin enzyme promoters and signal peptide of the present invention can effectively promote inulin enzyme promoters and signal The secreting, expressing ability of peptide driving.
The enzyme activity for being combined with pUKDN132-T (- 351) A+UTR Δs A-EstE of T (- 351) A and UTR Δs A mutation is wild 3.5 times of type plasmid are higher than the enzyme activity of individual T (- 351) A and UTR Δ A mutant, show that the two mutation put forward enzyme activity Rising has synergistic effect.The enzyme activity for being combined with pUKDN132-T (- 351) A+P10L-EstE of T (- 351) A and P10L mutation is wild 5.9 times of raw type plasmid, are higher than the enzyme activity of individual T (- 351) A and P10L mutant, show that the two mutation put forward enzyme activity Rising also has synergistic effect.
Embodiment 4, T (- 351) A and UTR Δs A are mutated the influence to mRNA level in-site
From the transformant of pUKDN132-EstE, pUKDN132-T (- 351) A-EstE and pUKDN132-UTR Δs A-EstE Middle 4 clones of picking respectively, are inoculated in 3mL SD culture solutions (0.67% without amino acid yeast nitrogen, 2% glucose), 30 DEG C, 220rpm overnight incubations.The dilution of overnight bacterium is inoculated in the YD culture mediums of 50ml, starting OD is made600It is 0.2.30℃、 Cell is collected after 220rpm cultures 9h.According to ZR Fungal/Bacterial RNA MiniPrep specifications The method of (Zymoresearch companies, R2014), extracted total RNA.According to PrimeScript RT (Takara companies, RR037A) the method for specification, by RNA reverse transcriptions at cDNA.According to SYBR Premix Ex TaqII (Takara companies, RR820A) the method for specification carries out quantitative PCR using LightCycler 480II Real-Time PCR systems (Roche) Analysis.The primer for expanding EstE genes is EstE-F (sequence is as shown in SEQ ID No.24) and primer EstE-R (sequence such as SEQ Shown in ID No.25).The primer for expanding internal reference 18s rDNA is 18S-F (sequence is as shown in SEQ ID No.26) and primer 18S- R (sequence is as shown in SEQ ID No.27).Calculate EstE mRNA's by calculating the ratio of EstE mRNA and 18s rDNA Relative amount.The relative amount of EstE mRNA in pUKDN132-EstE transformants is defined as 1, then (- 351) pUKDN132-T The phase of EstE mRNA in the transformant that the relative amount of EstE mRNA is 2.0, pUKDN132-UTR Δs A-EstE in A transformants It is 22 to content.The result illustrates that T (- 351) A and UTR Δs A mutation of the present invention can significantly improve driven gene MRNA level in-site.
Embodiment 5, P10L are mutated the influence for protein secretion
According to the operation instruction of QuikChange II Site-Directed Mutagenesis Kit, with pUKDN132- EstE and pUKDN132-P10L-EstE is template, utilizes primer HIS6-F (sequence is as shown in SEQ ID No.28) and primer N-terminal addition coding Hiss of the HIS6-R (sequence is as shown in SEQ ID No.29) in EstE genes6The sequence of label.The load of acquisition Body is named as pUKDN132-EstE-His respectively6And pUKDN132-P10L-EstE-His6。
According to the method for embodiment 3, by pUKDN132-EstE-His6And pUKDN132-P10L-EstE-His6It is transferred to Fim-1ura3Δ.By pUKDN132-EstE-His6And pUKDN132-P10L-EstE-His6Transformant is inoculated in 3mL SD trainings In nutrient solution, 30 DEG C, 220rpm overnight incubations.The dilution of overnight bacterium is inoculated in the YD culture mediums of 50ml, starting OD is made600For 0.2.30 DEG C, 220rpm cultures 9h, for 24 hours, cell and supernatant are collected after 48h and 72h.It is split with Western detection supernatant cells Solve the EstE-His in liquid6, antibody anti-His6(Abmart companies, M30111).The group in cell pyrolysis liquid is detected simultaneously As a contrast, antibody is anti-H3.1 (Abmart companies, P30266) to albumen H3.The method of Western is according to " molecular cloning Experiment guide " illustrates to carry out.As shown in figure 5, pUKDN132-P10L-EstE-His6EstE-His in transformant supernatant6's Content is significantly more than pUKDN132-EstE-His6.It is consistent with this, pUKDN132-P10L-EstE-His6Transformant EstE-His in cell pyrolysis liquid6Content be considerably less than pUKDN132-EstE-His6.The result illustrates that P10L mutation can be with Effectively facilitate the secretion of EstE albumen.
The preferred embodiment of the present invention has been described in detail above.It should be appreciated that the ordinary skill of this field is without wound The property made labour, which according to the present invention can conceive, makes many modifications and variations.Therefore, all technician in the art Pass through the available technology of logical analysis, reasoning, or a limited experiment on the basis of existing technology under this invention's idea Scheme, all should be in the protection domain being defined in the patent claims.
Sequence table
<110>Fudan University
<120>K marxianus promoter, secreting signal peptide and its preparation and application
<130> 2018
<160> 29
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1136
<212> DNA
<213>Artificial sequence ()
<400> 1
cacaaacaca aacacaaaca caaaaacgct aaattatgca cacaagggcc ggcggggctg 60
ccggaaaaaa aaagggaaaa atacacagac gagcgcgcac agatggggtt accactgcaa 120
gttacaagtt gcaagttgca cgctggaatc agaattggaa tcagaattgg aattggaatt 180
agaattagaa ttaaacttgg ggtagccacg ggaacgggat aactcaggaa tcgctcgcag 240
gcgtctccgt ctaggcaatc ccaaggtaag cctaggcact cccacagggg aaagaacggt 300
tgaaggcaaa gtagtgctaa caattggtaa cgaatggtaa caagtgtgtc cgtctccacc 360
tgacatttgc tagagctggg gattccacat tcttgtgctc tgaattctca aaccgaaatg 420
gggcgttgtt accccaggta tccggttgta gttggcactg gggatggaaa aaaatgatgt 480
tgatgttgag ttagttgggt tgagtcaatt agtgcgtgaa agtatcacca cttttgtcat 540
ccggcgtttc tgtgcgaatc acacacacac acacagttta ttggagcact tgtttctggc 600
gtattcgtaa ttgttctgcg gtgcggttct gtgtgcattt ttcctggggt gtctgccgca 660
cctactcatc acccacgccg tgggtttgag ccatggcgga ggtacgactg actggctgcc 720
tgcctgcctg actgactgcc tgactgcagg aaaagagggt ttcgaaggaa aaacttttcc 780
tgtgtaaatc cggccgtgcg ccgctgctcc aaaatccacc ttcatgagaa ggagtttgaa 840
aaaacaaaaa aattcacata taaaaagcgt atctcgagat ctcaaagtct cccttgaatc 900
gtgtttgcca gttgtaactc atcctttatt cttctattct atctctctct ttccttcccc 960
taatcagcaa ttaaatccgg ggtaaggaag aattactact gtgtgtaacg gttatatttc 1020
gttttttatt ttttttttcc attgccatag agaaagaaaa aaaaaaaaaa gagagtttgt 1080
gaagatcttc cattcgaatc ccataagtga cacatttaat tttttttttg ttagat 1136
<210> 2
<211> 1028
<212> DNA
<213>Artificial sequence ()
<400> 2
cacaaacaca aacacaaaca caaaaacgct aaattatgca cacaagggcc ggcggggctg 60
ccggaaaaaa aaagggaaaa atacacagac gagcgcgcac agatggggtt accactgcaa 120
gttacaagtt gcaagttgca cgctggaatc agaattggaa tcagaattgg aattggaatt 180
agaattagaa ttaaacttgg ggtagccacg ggaacgggat aactcaggaa tcgctcgcag 240
gcgtctccgt ctaggcaatc ccaaggtaag cctaggcact cccacagggg aaagaacggt 300
tgaaggcaaa gtagtgctaa caattggtaa cgaatggtaa caagtgtgtc cgtctccacc 360
tgacatttgc tagagctggg gattccacat tcttgtgctc tgaattctca aaccgaaatg 420
gggcgttgtt accccaggta tccggttgta gttggcactg gggatggaaa aaaatgatgt 480
tgatgttgag ttagttgggt tgagtcaatt agtgcgtgaa agtatcacca cttttgtcat 540
ccggcgtttc tgtgcgaatc acacacacac acacagttta ttggagcact tgtttctggc 600
gtattcgtaa ttgttctgcg gtgcggttct gtgtgcattt ttcctggggt gtctgccgca 660
cctactcatc acccacgccg tgggtttgag ccatggcgga ggtacgactg actggctgcc 720
tgcctgcctg actgactgcc tgactgcagg aaaagagggt ttcgaaggaa aaacttttcc 780
tgtgttaatc cggccgtgcg ccgctgctcc aaaatccacc ttcatgagaa ggagtttgaa 840
aaaacaaaaa aattcacata taaaaagcgt atctcgagat ctcaaagtct cccttgaatc 900
gtgtttgcca gttgtaactc atcctttatt cttctattct atctctctct ttccttcccc 960
taatcagcaa ttaaatccgg ggtaaggaag aattactact gtgtgtaacg gttatatttc 1020
ggttagat 1028
<210> 3
<211> 1028
<212> DNA
<213>Artificial sequence ()
<400> 3
cacaaacaca aacacaaaca caaaaacgct aaattatgca cacaagggcc ggcggggctg 60
ccggaaaaaa aaagggaaaa atacacagac gagcgcgcac agatggggtt accactgcaa 120
gttacaagtt gcaagttgca cgctggaatc agaattggaa tcagaattgg aattggaatt 180
agaattagaa ttaaacttgg ggtagccacg ggaacgggat aactcaggaa tcgctcgcag 240
gcgtctccgt ctaggcaatc ccaaggtaag cctaggcact cccacagggg aaagaacggt 300
tgaaggcaaa gtagtgctaa caattggtaa cgaatggtaa caagtgtgtc cgtctccacc 360
tgacatttgc tagagctggg gattccacat tcttgtgctc tgaattctca aaccgaaatg 420
gggcgttgtt accccaggta tccggttgta gttggcactg gggatggaaa aaaatgatgt 480
tgatgttgag ttagttgggt tgagtcaatt agtgcgtgaa agtatcacca cttttgtcat 540
ccggcgtttc tgtgcgaatc acacacacac acacagttta ttggagcact tgtttctggc 600
gtattcgtaa ttgttctgcg gtgcggttct gtgtgcattt ttcctggggt gtctgccgca 660
cctactcatc acccacgccg tgggtttgag ccatggcgga ggtacgactg actggctgcc 720
tgcctgcctg actgactgcc tgactgcagg aaaagagggt ttcgaaggaa aaacttttcc 780
tgtgtaaatc cggccgtgcg ccgctgctcc aaaatccacc ttcatgagaa ggagtttgaa 840
aaaacaaaaa aattcacata taaaaagcgt atctcgagat ctcaaagtct cccttgaatc 900
gtgtttgcca gttgtaactc atcctttatt cttctattct atctctctct ttccttcccc 960
taatcagcaa ttaaatccgg ggtaaggaag aattactact gtgtgtaacg gttatatttc 1020
ggttagat 1028
<210> 4
<211> 78
<212> DNA
<213>Artificial sequence ()
<400> 4
atgaagttag catactccct cttgcttcta ttggcaggag tcagtgcttc agtgatcaat 60
tacaagagag acggtgac 78
<210> 5
<211> 26
<212> PRT
<213>Artificial sequence ()
<400> 5
Met Lys Leu Ala Tyr Ser Leu Leu Leu Leu Leu Ala Gly Val Ser Ala
1 5 10 15
Ser Val Ile Asn Tyr Lys Arg Asp Gly Asp
20 25
<210> 6
<211> 1214
<212> DNA
<213>Artificial sequence ()
<400> 6
cacaaacaca aacacaaaca caaaaacgct aaattatgca cacaagggcc ggcggggctg 60
ccggaaaaaa aaagggaaaa atacacagac gagcgcgcac agatggggtt accactgcaa 120
gttacaagtt gcaagttgca cgctggaatc agaattggaa tcagaattgg aattggaatt 180
agaattagaa ttaaacttgg ggtagccacg ggaacgggat aactcaggaa tcgctcgcag 240
gcgtctccgt ctaggcaatc ccaaggtaag cctaggcact cccacagggg aaagaacggt 300
tgaaggcaaa gtagtgctaa caattggtaa cgaatggtaa caagtgtgtc cgtctccacc 360
tgacatttgc tagagctggg gattccacat tcttgtgctc tgaattctca aaccgaaatg 420
gggcgttgtt accccaggta tccggttgta gttggcactg gggatggaaa aaaatgatgt 480
tgatgttgag ttagttgggt tgagtcaatt agtgcgtgaa agtatcacca cttttgtcat 540
ccggcgtttc tgtgcgaatc acacacacac acacagttta ttggagcact tgtttctggc 600
gtattcgtaa ttgttctgcg gtgcggttct gtgtgcattt ttcctggggt gtctgccgca 660
cctactcatc acccacgccg tgggtttgag ccatggcgga ggtacgactg actggctgcc 720
tgcctgcctg actgactgcc tgactgcagg aaaagagggt ttcgaaggaa aaacttttcc 780
tgtgtaaatc cggccgtgcg ccgctgctcc aaaatccacc ttcatgagaa ggagtttgaa 840
aaaacaaaaa aattcacata taaaaagcgt atctcgagat ctcaaagtct cccttgaatc 900
gtgtttgcca gttgtaactc atcctttatt cttctattct atctctctct ttccttcccc 960
taatcagcaa ttaaatccgg ggtaaggaag aattactact gtgtgtaacg gttatatttc 1020
gttttttatt ttttttttcc attgccatag agaaagaaaa aaaaaaaaaa gagagtttgt 1080
gaagatcttc cattcgaatc ccataagtga cacatttaat tttttttttg ttagatatga 1140
agttagcata ctccctcttg cttctattgg caggagtcag tgcttcagtg atcaattaca 1200
agagagacgg tgac 1214
<210> 7
<211> 28
<212> DNA
<213>Artificial sequence ()
<400> 7
cacaaacaca aacacaaaca caaaaacg 28
<210> 8
<211> 29
<212> DNA
<213>Artificial sequence ()
<400> 8
tagaatgttg gtcagatgtg atgtacacc 29
<210> 9
<211> 107
<212> DNA
<213>Artificial sequence ()
<400> 9
ggagtcagtg cttcagtgat caattacaag agagacggtg accccgggac tagtgcggcc 60
gcttaaggcc gcaagctttg atctgatctg cttactttac taacgac 107
<210> 10
<211> 107
<212> DNA
<213>Artificial sequence ()
<400> 10
gtcgttagta aagtaagcag atcagatcaa agcttgcggc cttaagcggc cgcactagtc 60
ccggggtcac cgtctctctt gtaattgatc actgaagcac tgactcc 107
<210> 11
<211> 55
<212> DNA
<213>Artificial sequence ()
<400> 11
tgccgattcg cacgctgcaa ccgcggcaca aacacaaaca caaacacaaa aacgc 55
<210> 12
<211> 54
<212> DNA
<213>Artificial sequence ()
<400> 12
atggtctttc caatcagaat tcgaccgatc ctagaatgtt ggtcagatgt gatg 54
<210> 13
<211> 1136
<212> DNA
<213>Artificial sequence ()
<400> 13
cacaaacaca aacacaaaca caaaaacgct aaattatgca cacaagggcc ggcggggctg 60
ccggaaaaaa aaagggaaaa atacacagac gagcgcgcac agatggggtt accactgcaa 120
gttacaagtt gcaagttgca cgctggaatc agaattggaa tcagaattgg aattggaatt 180
agaattagaa ttaaacttgg ggtagccacg ggaacgggat aactcaggaa tcgctcgcag 240
gcgtctccgt ctaggcaatc ccaaggtaag cctaggcact cccacagggg aaagaacggt 300
tgaaggcaaa gtagtgctaa caattggtaa cgaatggtaa caagtgtgtc cgtctccacc 360
tgacatttgc tagagctggg gattccacat tcttgtgctc tgaattctca aaccgaaatg 420
gggcgttgtt accccaggta tccggttgta gttggcactg gggatggaaa aaaatgatgt 480
tgatgttgag ttagttgggt tgagtcaatt agtgcgtgaa agtatcacca cttttgtcat 540
ccggcgtttc tgtgcgaatc acacacacac acacagttta ttggagcact tgtttctggc 600
gtattcgtaa ttgttctgcg gtgcggttct gtgtgcattt ttcctggggt gtctgccgca 660
cctactcatc acccacgccg tgggtttgag ccatggcgga ggtacgactg actggctgcc 720
tgcctgcctg actgactgcc tgactgcagg aaaagagggt ttcgaaggaa aaacttttcc 780
tgtgttaatc cggccgtgcg ccgctgctcc aaaatccacc ttcatgagaa ggagtttgaa 840
aaaacaaaaa aattcacata taaaaagcgt atctcgagat ctcaaagtct cccttgaatc 900
gtgtttgcca gttgtaactc atcctttatt cttctattct atctctctct ttccttcccc 960
taatcagcaa ttaaatccgg ggtaaggaag aattactact gtgtgtaacg gttatatttc 1020
gttttttatt ttttttttcc attgccatag agaaagaaaa aaaaaaaaaa gagagtttgt 1080
gaagatcttc cattcgaatc ccataagtga cacatttaat tttttttttg ttagat 1136
<210> 14
<211> 69
<212> DNA
<213>Artificial sequence ()
<400> 14
gtcacgacgt tgtaaaacga cggccagtgc caagcttgca tgcatcacta atgaaaagca 60
tacgacgcc 69
<210> 15
<211> 69
<212> DNA
<213>Artificial sequence ()
<400> 15
ggcgtcgtat gcttttcatt agtgatgcat gcaagcttgg cactggccgt cgttttacaa 60
cgtcgtgac 69
<210> 16
<211> 30
<212> DNA
<213>Artificial sequence ()
<400> 16
aaacttttcc tgtgtaaatc cggccgtgcg 30
<210> 17
<211> 30
<212> DNA
<213>Artificial sequence ()
<400> 17
cgcacggccg gatttacaca ggaaaagttt 30
<210> 18
<211> 63
<212> DNA
<213>Artificial sequence ()
<400> 18
gaattactac tgtgtgtaac ggttatattt cggttagata tgaagttagc atactccctc 60
ttg 63
<210> 19
<211> 63
<212> DNA
<213>Artificial sequence ()
<400> 19
caagagggag tatgctaact tcatatctaa ccgaaatata accgttacac acagtagtaa 60
ttc 63
<210> 20
<211> 40
<212> DNA
<213>Artificial sequence ()
<400> 20
agcatactcc ctcttgcttc tattggcagg agtcagtgct 40
<210> 21
<211> 40
<212> DNA
<213>Artificial sequence ()
<400> 21
agcactgact cctgccaata gaagcaagag ggagtatgct 40
<210> 22
<211> 11280
<212> DNA
<213>Artificial sequence ()
<400> 22
cgtaatcatg tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa 60
catacgagcc ggaagcataa agtgtaaagc ctggggtgcc taatgagtga gctaactcac 120
attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca 180
ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgct cttccgcttc 240
ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc 300
aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc 360
aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag 420
gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc 480
gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt 540
tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct 600
ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg 660
ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct 720
tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat 780
tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg 840
ctacactaga agaacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa 900
aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt 960
ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc 1020
tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt 1080
atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta 1140
aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat 1200
ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac 1260
tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc gagacccacg 1320
ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag 1380
tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt 1440
aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt 1500
gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt 1560
tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt 1620
cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct 1680
tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa ccaagtcatt 1740
ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac gggataatac 1800
cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt cggggcgaaa 1860
actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc gtgcacccaa 1920
ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa caggaaggca 1980
aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca tactcttcct 2040
ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga 2100
atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa aagtgccacc 2160
tgacgtctaa gaaaccatta ttatcatgac attaacctat aaaaataggc gtatcacgag 2220
gccctttcgt ctcgcgcgtt tcggtgatga cggtgaaaac ctctgacaca tgcagctccc 2280
ggagacggtc acagcttgtc tgtaagcgga tgccgggagc agacaagccc gtcagggcgc 2340
gtcagcgggt gttggcgggt gtcggggctg gcttaactat gcggcatcag agcagattgt 2400
actgagagtg caccataaaa ttgtaaacgt taatattttg ttaaaattcg cgttaaattt 2460
ttgttaaatc agctcatttt ttaaccaata ggccgaaatc ggcaaaatcc cttataaatc 2520
aaaagaatag cccgagatag ggttgagtgt tgttccagtt tggaacaaga gtccactatt 2580
aaagaacgtg gactccaacg tcaaagggcg aaaaaccgtc tatcagggcg atggcccact 2640
acgtgaacca tcacccaaat caagtttttt ggggtcgagg tgccgtaaag cactaaatcg 2700
gaaccctaaa gggagccccc gatttagagc ttgacgggga aagccggcga acgtggcgag 2760
aaaggaaggg aagaaagcga aaggagcggg cgctagggcg ctggcaagtg tagcggtcac 2820
gctgcgcgta accaccacac ccgccgcgct taatgcgccg ctacagggcg cgtactatgg 2880
ttgctttgac gtatgcggtg tgaaataccg cacagatgcg taaggagaaa ataccgcatc 2940
aggcgccatt cgccattcag gctgcgcaac tgttgggaag ggcgatcggt gcgggcctct 3000
tcgctattac gccagctggc gaaaggggga tgtgctgcaa ggcgattaag ttgggtaacg 3060
ccagggtttt cccagtcacg acgttgtaaa acgacggcca gtgccaagct tgcatgcatc 3120
actaatgaaa agcatacgac gcctgcgtct gacatgcact cattctgaag aagattctgg 3180
gcgcgtttcg ttctcgtttt cctctgtata ttgtactctg gtggacaatt tgaacataac 3240
gtctttcacc tcgccattct caataatggg ttccaattct atccaggtag cggttaattg 3300
acggtgctta agccgtatgc tcactctaac gctaccgttg tccaaacaac ggaccccttt 3360
gtgacgggtg taagacccat catgaagtaa aacatctcta acggtatgga aaagagtggt 3420
acggtcaagt ttcctggcac gagtcaattt tccctcttcg tgtagatcag aggctatata 3480
catgccgagg tattcgatca ctctacgatg acggtctgtt agctcaacaa cttcttctaa 3540
atgctccata accgtaacgt aagaagcata actgtcaata ctgaagtcat cccagtttat 3600
tggtgctcct gttgaacagt catccactat atgttcgaat agcccaggat cacgaggagg 3660
tcctacaaac ggatacggta cagtcttctt tttatagtct gcaaattcta gaatagcatt 3720
ttttatccaa tagtgtcgaa tcgtcctggc cgttctaccg ataaaggatc caatgtgatt 3780
attagctcca ctacacgata tgttaagttt gatcgatgtc ttgttaacaa acgctaaact 3840
caagttcggc atttccaaca gcgagaagaa atcatcaatt ccatcggcta tctcttgata 3900
agtcattaga tcatatacct tctcgggatg tcgttgagtt actttatgac tagaaatctt 3960
caggttatca tcaacgtaat tgttctccaa tagctctgga gagggacata acaatacttt 4020
gattttttcc atggcctgga cttgtttccg taggaaatac ttgttctttt gtagacgttc 4080
catgatgagt ttgtatacct ctgctggaga tatccattct agatctttga tataagtttg 4140
gtatggtaaa gagttgattt tgtaggacac gtaaatctgc gctagataag tacattgtgc 4200
aaatgcctct ggtacttcgt aagacccatg ctgcgtaatt atagtattat tgagtggatc 4260
ataagcgttg tactcgtttt tgaatttaaa actgtctaat aaggccctgt aaatctctct 4320
gacttgttgt acacctttct gctcttcggg actgagatcg gatagcaatg gagcagcagt 4380
ttctgagctt tctgatgggg ctgacatggc agatgcctat tcaatgctgc cttttgtttg 4440
ggaggttatg aaatgcatct gtttacattg tatgtaatac ccttactagg caatgttata 4500
agcaaaaatc ctttgatcac atggaatatc actttatacg tgttgaaata tgcaaaaaaa 4560
cagtccccct gagctcaggg ggtggtttac gcttttgagg ctcagcagcg cgaattctct 4620
cttggggctg aagtgaaatt taaaaaagtc gcttgaggct cagccggaat tataaaacat 4680
cacctgagtc ttgagagcgc tttcactcac ctgaggctca gctgaaattt caaaaagtca 4740
cttgagccca gaaggagtgt ttcaccccct gaggctataa cgttcgttat tttaatacct 4800
aaataaacaa aaatatatgg tacaggaacg cgaggcaacg cgccgataca gggtcaatgg 4860
gtacacgaga gggtgacact aggcgtagaa agtcattagt ataaaataca gtggtatata 4920
gtagatattt agtttgtttt ccttttcttt ttctccaaaa cgatatcaga catttgtctg 4980
ataatgaagc attatcagac aaatgtctga tatcgttttt caataataat atacatcatc 5040
acaaaacaaa caaacatagc atcgcaagcc ccatcatgcc accaccgtcc gctgtgatcg 5100
caactcatgt ttccggcggt attctgcaat gaattggaga acctcgtctg agataattcc 5160
atgccattgt tcgaacaact ggaggctagg atgagctgag aaggattgag cgaccaagcg 5220
cggacttgac ggtgggctga gtggtgggct accagggctg ttaccctcct cttcaagtag 5280
ctcctcgcga gataaaggtt tattagaagg atccttcaaa acatatattt cactgcccaa 5340
tggggcttcc ttgtaaaaac ctgatataaa ggcaaataca cggtcatcta cagtcacact 5400
accatgactg tagtgtgatc tagccactgc actttgaatt tcacggtccc cagcccaatt 5460
gcccagtgag gagacatatt ttcccttctc agatctcgat aaaaaggtcg ccattaaatg 5520
tcttccaaaa tgagacttcg ggccgttcca gatcttgaag actggttcat cgacatgctg 5580
ggtaagaaac ctagagaacg ttctggccaa tgactctggt aaaaactgat gagtttgatt 5640
agttggtcta ttactggata ctgttttttc aataggcgag caaacacgca aatagtcgta 5700
taatgatatc agaagatcgc aatcaccatt cacaggatag aagttaacgt accgttcagt 5760
tctgcttttc gtttctgtca cagtagcacg cacaattggg cccagaaatg aattgttgta 5820
gatctcaaaa gtccttggat ctagattctt cagatcgctg tatctgcagc aatttccaac 5880
agctcccaga agtagcaatc ggtattccgc tcgttttgta gtggttacgc aggactgatc 5940
gaagaagcag gcaatcctgg agacaatctt ccaaatatct ttttcttttg acagaatatt 6000
agtgaattgt aatccaacca tagaagcatc gtatttatgt gtttcctcgt agcgatcaaa 6060
caaggaaact tcttgatttt taaatgggct aacaacaacc ttgtaagaag gcaatgctga 6120
ttcgatatcc ttttgcagag actctgtctt tcttagtcta acagtgaatt tgataatttt 6180
gtcatcctta tcaaaagaca gagatttgcc aattgcgctc ttgtaagagc ggtaggtatt 6240
gattttcatc tcgcgtcgga tagatagcga ctgcattgtc aagatagaga atagggacgc 6300
cagcttattt ctaatttctt tcgcatttat attaaaggtg tcagattcca gaatttcatt 6360
aatttcatta gcacactgat gaggtgtgag gtgagcagcc tccgcaaagg tagacatagg 6420
ggcattggtt ggaggccttt gaggtaccac tagagtgctg caaacatagc accgttcgag 6480
actttaaaat cttcagtttt aaaattatga aaaaaaacat cgtcctgagt tgaaacggtc 6540
gtttcaacct ccgtgtacag aaagatacat agcatatggc aagctgcacg cagcgtaaac 6600
atgccggaca actgtcattt cgtcagatca gttgatctac tctctgtgat actgcttcgt 6660
ttgtccacgg aggtcggact aactctcacc acgcttccac ggcattcgaa agaactaata 6720
ttgtatcatt gtacatatga ggaacacgca gttgaactga gcaaaccagg actcaggaaa 6780
gcaggaggta agtgctcgct tttcgtggat ccagaggaac gtgaaaattc gccttctcct 6840
cctataccgc cgtatcagat atcagagatg ccccttcatg aacttctcga gtcaggcaat 6900
gctaaattgg ttccaaatcc cgagtttgat ctaactgatc cagacgactt tcataagtgt 6960
ttctcggtca cctattcagc attatcttta atggtaccat atctgcccag agctgctcta 7020
aaggctgctc gagtgttttg taaagatcat tcaatattaa caacggatat gcttgatttg 7080
aattatcttg aagagctaat tgagttctca aaggaaactg tgaacaaaat cccagctaga 7140
atccctatag aggacatgct tctcgagcgg ggatatgtgc taccatgggt tcatggtggt 7200
acagtgaagg gaggaaagct actgaccccc aacgattgat tctttaccga atcattgcat 7260
aattcattgc ataattcatt gcagaatacc gccggaaaca tgagttgcga tcacagcgga 7320
cggtggtggc atgatggggc ttgcgatgct atgtttgttt gttttgtgat gatgtatatt 7380
attattgaaa aacgatatca gacatttgtc tgataatgct tcattatcag acaaatgtct 7440
gatatcgttt tggagaaaaa gaaaaggaaa acaaactaaa tatctactat ataccactgt 7500
attttatact aatgactttc tacgcctagt gtcaccctct cgtgtaccca ttgaccctgt 7560
atcggcgcgt tgcctcgcgt tcctgtacca tatatttttg tttatttagg tattaaaatt 7620
tactttcctc atacaaatat taaattcacc aaacttctca aaaactaatt attcgtagtt 7680
acaaactcta ttttacaatc acgtttattc aaccattcta catccaataa ccaaaatgcc 7740
catgtacctc tcagcgaagt ccaacggtac tgtccaatat tctcattaaa tagtctttca 7800
tctatatatc agaaggtaat tataattaga gatttcgaat cattaccgtg ccgattcgca 7860
cgctgcaacc gcggcacaaa cacaaacaca aacacaaaaa cgctaaatta tgcacacaag 7920
ggccggcggg gctgccggaa aaaaaaaggg aaaaatacac agacgagcgc gcacagatgg 7980
ggttaccact gcaagttaca agttgcaagt tgcacgctgg aatcagaatt ggaatcagaa 8040
ttggaattgg aattagaatt agaattaaac ttggggtagc cacgggaacg ggataactca 8100
ggaatcgctc gcaggcgtct ccgtctaggc aatcccaagg taagcctagg cactcccaca 8160
ggggaaagaa cggttgaagg caaagtagtg ctaacaattg gtaacgaatg gtaacaagtg 8220
tgtccgtctc cacctgacat ttgctagagc tggggattcc acattcttgt gctctgaatt 8280
ctcaaaccga aatggggcgt tgttacccca ggtatccggt tgtagttggc actggggatg 8340
gaaaaaaatg atgttgatgt tgagttagtt gggttgagtc aattagtgcg tgaaagtatc 8400
accacttttg tcatccggcg tttctgtgcg aatcacacac acacacacag tttattggag 8460
cacttgtttc tggcgtattc gtaattgttc tgcggtgcgg ttctgtgtgc atttttcctg 8520
gggtgtctgc cgcacctact catcacccac gccgtgggtt tgagccatgg cggaggtacg 8580
actgactggc tgcctgcctg cctgactgac tgcctgactg caggaaaaga gggtttcgaa 8640
ggaaaaactt ttcctgtgta aatccggccg tgcgccgctg ctccaaaatc caccttcatg 8700
agaaggagtt tgaaaaaaca aaaaaattca catataaaaa gcgtatctcg agatctcaaa 8760
gtctcccttg aatcgtgttt gccagttgta actcatcctt tattcttcta ttctatctct 8820
ctctttcctt cccctaatca gcaattaaat ccggggtaag gaagaattac tactgtgtgt 8880
aacggttata tttcggttag atatgaagtt agcatactcc ctcttgcttc cattggcagg 8940
agtcagtgct tcagtgatca attacaagag agacggtgac cccgggacta gtgcggccgc 9000
ttaaggccgc aagctttgat ctgatctgct tactttacta acgacaaaaa aaaatcaaaa 9060
aaaaaaaaac aatcagtcct tctcttctta cgatatgata tgattaaatg atgctatgaa 9120
atcatcttct tcttaacttt cttaaatctt acgcgtcact tactctatat acccgtttag 9180
ctttgcctgg tcacagcgac attttatata agtgtacgta ttttcttttt ttttttaaaa 9240
atttctattc taaccttaga aaagtgccct ttaaaccagc tgtcctggca ctatatcttt 9300
atcatgtgcc ggtcgctttc cctttccgtt tcccttttcc tttcaattgg tggcctggaa 9360
ttccgaactc attttcgcat ctgaaactaa ttctcgaaac ctttaacatc aaacaattga 9420
aaagatcatc atcaccagaa ataagaaaaa gatcaacaca acagctaata acagtacgaa 9480
agaaagatcg ctcgagtgaa aaggcagcca agaaaggtca ttcgatttgg gtctagactg 9540
attatagaca taccaattgc actcagtaag aaaatgagtt tcaaatttga cgatgacggt 9600
gtggtaaaag aatttcacgg caacaccatc atatgccata ttcctcaaca aaccgaattc 9660
ttcaacaaat tgttggactt ctaccgtttt gcgaaacgac tttccttcta cgacaagatc 9720
accctacttc ctccttcaag ctaccacgtt acgatcatga attgctgcca cgaacacgat 9780
cgttctgagg gccactggcc caaaggaatc gatccggaca caagcatgct gcggtgtaca 9840
tcacatctga ccaacattct aggatcggtc gaattctgat tggaaagacc attctgcttt 9900
acttttagag catcttggtc ttctgagctc attatacctc aatcaaaact gaaattaggt 9960
gcctgtcacg gctctttttt tactgtacct ttgacttcct ttcttatttc caaggatgct 10020
catcacaata cgcttctaga tctattatgc attataatta atagttgtag ctacaaaagg 10080
taaaagaaag tccggggcag gcaacaatag aaatcggcaa aaaaaactac agaaatacta 10140
agagcttctt ccccattcag tcatcgcatt tcgaaacaag aggggaatgg ctctggctag 10200
ggaactaacc accatcgact gactctatgc actaaccacg tgactacata tatgtgatcg 10260
tttttaacat ttttcaaagg ctgtgtgtct ggctgtttcc attaattttc actgattaag 10320
cagtcatatt gaatctgagc tcatcaccaa caagaaattc taccgtaaaa gtgtaaaagt 10380
tcgtttaaat catttgtaaa ctggaacagc aagaggaagt atcatcagct agccccataa 10440
actaatcaaa ggaggatgtc gactaagagt tactcggaaa gagcagctgc tcatagaagt 10500
ccagttgctg ccaagctttt aaacttgatg gaagagaaga agtcaaactt atgtgcttct 10560
cttgatgttc gtaaaacagc agagttgtta agattagttg aggttttggg tccatatatc 10620
tgtctattga agacacatgt agatatcttg gaggatttca gctttgagaa taccattgtg 10680
ccgttgaagc aattagcaga gaaacacaag tttttgatat ttgaagacag gaagtttgcc 10740
gacattggga acactgttaa attacaatac acgtctggtg tataccgtat cgccgaatgg 10800
tctgatatca ccaatgcaca cggtgtgact ggtgcgggca ttgttgctgg tttgaagcaa 10860
ggtgccgagg aagttacaaa agaacctaga gggttgttaa tgcttgccga gttatcgtcc 10920
aaggggtctc tagcgcacgg tgaatacact cgtgggaccg tggaaattgc caagagtgat 10980
aaggactttg ttattggatt tattgctcaa aacgatatgg gtggaagaga agagggctac 11040
gattggttga tcatgacgcc aggtgttggt cttgatgaca aaggtgatgc tttgggacaa 11100
caatacagaa ctgtggatga agttgttgcc ggtggatcag acatcattat tgttggtaga 11160
ggtcttttcg caaagggaag agatcctgta gtggaaggtg agagatacag aaaggcggga 11220
tgggacgctt acttgaagag agtaggcaga tccgcttaag aggggtaccg agctcgaatt 11280
<210> 23
<211> 11388
<212> DNA
<213>Artificial sequence ()
<400> 23
cgtaatcatg tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa 60
catacgagcc ggaagcataa agtgtaaagc ctggggtgcc taatgagtga gctaactcac 120
attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca 180
ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgct cttccgcttc 240
ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc 300
aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc 360
aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag 420
gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc 480
gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt 540
tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct 600
ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg 660
ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct 720
tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat 780
tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg 840
ctacactaga agaacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa 900
aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt 960
ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc 1020
tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt 1080
atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta 1140
aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat 1200
ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac 1260
tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc gagacccacg 1320
ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag 1380
tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt 1440
aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt 1500
gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt 1560
tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt 1620
cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct 1680
tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa ccaagtcatt 1740
ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac gggataatac 1800
cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt cggggcgaaa 1860
actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc gtgcacccaa 1920
ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa caggaaggca 1980
aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca tactcttcct 2040
ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga 2100
atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa aagtgccacc 2160
tgacgtctaa gaaaccatta ttatcatgac attaacctat aaaaataggc gtatcacgag 2220
gccctttcgt ctcgcgcgtt tcggtgatga cggtgaaaac ctctgacaca tgcagctccc 2280
ggagacggtc acagcttgtc tgtaagcgga tgccgggagc agacaagccc gtcagggcgc 2340
gtcagcgggt gttggcgggt gtcggggctg gcttaactat gcggcatcag agcagattgt 2400
actgagagtg caccataaaa ttgtaaacgt taatattttg ttaaaattcg cgttaaattt 2460
ttgttaaatc agctcatttt ttaaccaata ggccgaaatc ggcaaaatcc cttataaatc 2520
aaaagaatag cccgagatag ggttgagtgt tgttccagtt tggaacaaga gtccactatt 2580
aaagaacgtg gactccaacg tcaaagggcg aaaaaccgtc tatcagggcg atggcccact 2640
acgtgaacca tcacccaaat caagtttttt ggggtcgagg tgccgtaaag cactaaatcg 2700
gaaccctaaa gggagccccc gatttagagc ttgacgggga aagccggcga acgtggcgag 2760
aaaggaaggg aagaaagcga aaggagcggg cgctagggcg ctggcaagtg tagcggtcac 2820
gctgcgcgta accaccacac ccgccgcgct taatgcgccg ctacagggcg cgtactatgg 2880
ttgctttgac gtatgcggtg tgaaataccg cacagatgcg taaggagaaa ataccgcatc 2940
aggcgccatt cgccattcag gctgcgcaac tgttgggaag ggcgatcggt gcgggcctct 3000
tcgctattac gccagctggc gaaaggggga tgtgctgcaa ggcgattaag ttgggtaacg 3060
ccagggtttt cccagtcacg acgttgtaaa acgacggcca gtgccaagct tgcatgcatc 3120
actaatgaaa agcatacgac gcctgcgtct gacatgcact cattctgaag aagattctgg 3180
gcgcgtttcg ttctcgtttt cctctgtata ttgtactctg gtggacaatt tgaacataac 3240
gtctttcacc tcgccattct caataatggg ttccaattct atccaggtag cggttaattg 3300
acggtgctta agccgtatgc tcactctaac gctaccgttg tccaaacaac ggaccccttt 3360
gtgacgggtg taagacccat catgaagtaa aacatctcta acggtatgga aaagagtggt 3420
acggtcaagt ttcctggcac gagtcaattt tccctcttcg tgtagatcag aggctatata 3480
catgccgagg tattcgatca ctctacgatg acggtctgtt agctcaacaa cttcttctaa 3540
atgctccata accgtaacgt aagaagcata actgtcaata ctgaagtcat cccagtttat 3600
tggtgctcct gttgaacagt catccactat atgttcgaat agcccaggat cacgaggagg 3660
tcctacaaac ggatacggta cagtcttctt tttatagtct gcaaattcta gaatagcatt 3720
ttttatccaa tagtgtcgaa tcgtcctggc cgttctaccg ataaaggatc caatgtgatt 3780
attagctcca ctacacgata tgttaagttt gatcgatgtc ttgttaacaa acgctaaact 3840
caagttcggc atttccaaca gcgagaagaa atcatcaatt ccatcggcta tctcttgata 3900
agtcattaga tcatatacct tctcgggatg tcgttgagtt actttatgac tagaaatctt 3960
caggttatca tcaacgtaat tgttctccaa tagctctgga gagggacata acaatacttt 4020
gattttttcc atggcctgga cttgtttccg taggaaatac ttgttctttt gtagacgttc 4080
catgatgagt ttgtatacct ctgctggaga tatccattct agatctttga tataagtttg 4140
gtatggtaaa gagttgattt tgtaggacac gtaaatctgc gctagataag tacattgtgc 4200
aaatgcctct ggtacttcgt aagacccatg ctgcgtaatt atagtattat tgagtggatc 4260
ataagcgttg tactcgtttt tgaatttaaa actgtctaat aaggccctgt aaatctctct 4320
gacttgttgt acacctttct gctcttcggg actgagatcg gatagcaatg gagcagcagt 4380
ttctgagctt tctgatgggg ctgacatggc agatgcctat tcaatgctgc cttttgtttg 4440
ggaggttatg aaatgcatct gtttacattg tatgtaatac ccttactagg caatgttata 4500
agcaaaaatc ctttgatcac atggaatatc actttatacg tgttgaaata tgcaaaaaaa 4560
cagtccccct gagctcaggg ggtggtttac gcttttgagg ctcagcagcg cgaattctct 4620
cttggggctg aagtgaaatt taaaaaagtc gcttgaggct cagccggaat tataaaacat 4680
cacctgagtc ttgagagcgc tttcactcac ctgaggctca gctgaaattt caaaaagtca 4740
cttgagccca gaaggagtgt ttcaccccct gaggctataa cgttcgttat tttaatacct 4800
aaataaacaa aaatatatgg tacaggaacg cgaggcaacg cgccgataca gggtcaatgg 4860
gtacacgaga gggtgacact aggcgtagaa agtcattagt ataaaataca gtggtatata 4920
gtagatattt agtttgtttt ccttttcttt ttctccaaaa cgatatcaga catttgtctg 4980
ataatgaagc attatcagac aaatgtctga tatcgttttt caataataat atacatcatc 5040
acaaaacaaa caaacatagc atcgcaagcc ccatcatgcc accaccgtcc gctgtgatcg 5100
caactcatgt ttccggcggt attctgcaat gaattggaga acctcgtctg agataattcc 5160
atgccattgt tcgaacaact ggaggctagg atgagctgag aaggattgag cgaccaagcg 5220
cggacttgac ggtgggctga gtggtgggct accagggctg ttaccctcct cttcaagtag 5280
ctcctcgcga gataaaggtt tattagaagg atccttcaaa acatatattt cactgcccaa 5340
tggggcttcc ttgtaaaaac ctgatataaa ggcaaataca cggtcatcta cagtcacact 5400
accatgactg tagtgtgatc tagccactgc actttgaatt tcacggtccc cagcccaatt 5460
gcccagtgag gagacatatt ttcccttctc agatctcgat aaaaaggtcg ccattaaatg 5520
tcttccaaaa tgagacttcg ggccgttcca gatcttgaag actggttcat cgacatgctg 5580
ggtaagaaac ctagagaacg ttctggccaa tgactctggt aaaaactgat gagtttgatt 5640
agttggtcta ttactggata ctgttttttc aataggcgag caaacacgca aatagtcgta 5700
taatgatatc agaagatcgc aatcaccatt cacaggatag aagttaacgt accgttcagt 5760
tctgcttttc gtttctgtca cagtagcacg cacaattggg cccagaaatg aattgttgta 5820
gatctcaaaa gtccttggat ctagattctt cagatcgctg tatctgcagc aatttccaac 5880
agctcccaga agtagcaatc ggtattccgc tcgttttgta gtggttacgc aggactgatc 5940
gaagaagcag gcaatcctgg agacaatctt ccaaatatct ttttcttttg acagaatatt 6000
agtgaattgt aatccaacca tagaagcatc gtatttatgt gtttcctcgt agcgatcaaa 6060
caaggaaact tcttgatttt taaatgggct aacaacaacc ttgtaagaag gcaatgctga 6120
ttcgatatcc ttttgcagag actctgtctt tcttagtcta acagtgaatt tgataatttt 6180
gtcatcctta tcaaaagaca gagatttgcc aattgcgctc ttgtaagagc ggtaggtatt 6240
gattttcatc tcgcgtcgga tagatagcga ctgcattgtc aagatagaga atagggacgc 6300
cagcttattt ctaatttctt tcgcatttat attaaaggtg tcagattcca gaatttcatt 6360
aatttcatta gcacactgat gaggtgtgag gtgagcagcc tccgcaaagg tagacatagg 6420
ggcattggtt ggaggccttt gaggtaccac tagagtgctg caaacatagc accgttcgag 6480
actttaaaat cttcagtttt aaaattatga aaaaaaacat cgtcctgagt tgaaacggtc 6540
gtttcaacct ccgtgtacag aaagatacat agcatatggc aagctgcacg cagcgtaaac 6600
atgccggaca actgtcattt cgtcagatca gttgatctac tctctgtgat actgcttcgt 6660
ttgtccacgg aggtcggact aactctcacc acgcttccac ggcattcgaa agaactaata 6720
ttgtatcatt gtacatatga ggaacacgca gttgaactga gcaaaccagg actcaggaaa 6780
gcaggaggta agtgctcgct tttcgtggat ccagaggaac gtgaaaattc gccttctcct 6840
cctataccgc cgtatcagat atcagagatg ccccttcatg aacttctcga gtcaggcaat 6900
gctaaattgg ttccaaatcc cgagtttgat ctaactgatc cagacgactt tcataagtgt 6960
ttctcggtca cctattcagc attatcttta atggtaccat atctgcccag agctgctcta 7020
aaggctgctc gagtgttttg taaagatcat tcaatattaa caacggatat gcttgatttg 7080
aattatcttg aagagctaat tgagttctca aaggaaactg tgaacaaaat cccagctaga 7140
atccctatag aggacatgct tctcgagcgg ggatatgtgc taccatgggt tcatggtggt 7200
acagtgaagg gaggaaagct actgaccccc aacgattgat tctttaccga atcattgcat 7260
aattcattgc ataattcatt gcagaatacc gccggaaaca tgagttgcga tcacagcgga 7320
cggtggtggc atgatggggc ttgcgatgct atgtttgttt gttttgtgat gatgtatatt 7380
attattgaaa aacgatatca gacatttgtc tgataatgct tcattatcag acaaatgtct 7440
gatatcgttt tggagaaaaa gaaaaggaaa acaaactaaa tatctactat ataccactgt 7500
attttatact aatgactttc tacgcctagt gtcaccctct cgtgtaccca ttgaccctgt 7560
atcggcgcgt tgcctcgcgt tcctgtacca tatatttttg tttatttagg tattaaaatt 7620
tactttcctc atacaaatat taaattcacc aaacttctca aaaactaatt attcgtagtt 7680
acaaactcta ttttacaatc acgtttattc aaccattcta catccaataa ccaaaatgcc 7740
catgtacctc tcagcgaagt ccaacggtac tgtccaatat tctcattaaa tagtctttca 7800
tctatatatc agaaggtaat tataattaga gatttcgaat cattaccgtg ccgattcgca 7860
cgctgcaacc gcggcacaaa cacaaacaca aacacaaaaa cgctaaatta tgcacacaag 7920
ggccggcggg gctgccggaa aaaaaaaggg aaaaatacac agacgagcgc gcacagatgg 7980
ggttaccact gcaagttaca agttgcaagt tgcacgctgg aatcagaatt ggaatcagaa 8040
ttggaattgg aattagaatt agaattaaac ttggggtagc cacgggaacg ggataactca 8100
ggaatcgctc gcaggcgtct ccgtctaggc aatcccaagg taagcctagg cactcccaca 8160
ggggaaagaa cggttgaagg caaagtagtg ctaacaattg gtaacgaatg gtaacaagtg 8220
tgtccgtctc cacctgacat ttgctagagc tggggattcc acattcttgt gctctgaatt 8280
ctcaaaccga aatggggcgt tgttacccca ggtatccggt tgtagttggc actggggatg 8340
gaaaaaaatg atgttgatgt tgagttagtt gggttgagtc aattagtgcg tgaaagtatc 8400
accacttttg tcatccggcg tttctgtgcg aatcacacac acacacacag tttattggag 8460
cacttgtttc tggcgtattc gtaattgttc tgcggtgcgg ttctgtgtgc atttttcctg 8520
gggtgtctgc cgcacctact catcacccac gccgtgggtt tgagccatgg cggaggtacg 8580
actgactggc tgcctgcctg cctgactgac tgcctgactg caggaaaaga gggtttcgaa 8640
ggaaaaactt ttcctgtgta aatccggccg tgcgccgctg ctccaaaatc caccttcatg 8700
agaaggagtt tgaaaaaaca aaaaaattca catataaaaa gcgtatctcg agatctcaaa 8760
gtctcccttg aatcgtgttt gccagttgta actcatcctt tattcttcta ttctatctct 8820
ctctttcctt cccctaatca gcaattaaat ccggggtaag gaagaattac tactgtgtgt 8880
aacggttata tttcgttttt tatttttttt ttccattgcc atagagaaag aaaaaaaaaa 8940
aaaagagagt ttgtgaagat cttccattcg aatcccataa gtgacacatt taattttttt 9000
tttgttagat atgaagttag catactccct cttgcttcta ttggcaggag tcagtgcttc 9060
agtgatcaat tacaagagag acggtgaccc cgggactagt gcggccgctt aaggccgcaa 9120
gctttgatct gatctgctta ctttactaac gacaaaaaaa aatcaaaaaa aaaaaaacaa 9180
tcagtccttc tcttcttacg atatgatatg attaaatgat gctatgaaat catcttcttc 9240
ttaactttct taaatcttac gcgtcactta ctctatatac ccgtttagct ttgcctggtc 9300
acagcgacat tttatataag tgtacgtatt ttcttttttt ttttaaaaat ttctattcta 9360
accttagaaa agtgcccttt aaaccagctg tcctggcact atatctttat catgtgccgg 9420
tcgctttccc tttccgtttc ccttttcctt tcaattggtg gcctggaatt ccgaactcat 9480
tttcgcatct gaaactaatt ctcgaaacct ttaacatcaa acaattgaaa agatcatcat 9540
caccagaaat aagaaaaaga tcaacacaac agctaataac agtacgaaag aaagatcgct 9600
cgagtgaaaa ggcagccaag aaaggtcatt cgatttgggt ctagactgat tatagacata 9660
ccaattgcac tcagtaagaa aatgagtttc aaatttgacg atgacggtgt ggtaaaagaa 9720
tttcacggca acaccatcat atgccatatt cctcaacaaa ccgaattctt caacaaattg 9780
ttggacttct accgttttgc gaaacgactt tccttctacg acaagatcac cctacttcct 9840
ccttcaagct accacgttac gatcatgaat tgctgccacg aacacgatcg ttctgagggc 9900
cactggccca aaggaatcga tccggacaca agcatgctgc ggtgtacatc acatctgacc 9960
aacattctag gatcggtcga attctgattg gaaagaccat tctgctttac ttttagagca 10020
tcttggtctt ctgagctcat tatacctcaa tcaaaactga aattaggtgc ctgtcacggc 10080
tcttttttta ctgtaccttt gacttccttt cttatttcca aggatgctca tcacaatacg 10140
cttctagatc tattatgcat tataattaat agttgtagct acaaaaggta aaagaaagtc 10200
cggggcaggc aacaatagaa atcggcaaaa aaaactacag aaatactaag agcttcttcc 10260
ccattcagtc atcgcatttc gaaacaagag gggaatggct ctggctaggg aactaaccac 10320
catcgactga ctctatgcac taaccacgtg actacatata tgtgatcgtt tttaacattt 10380
ttcaaaggct gtgtgtctgg ctgtttccat taattttcac tgattaagca gtcatattga 10440
atctgagctc atcaccaaca agaaattcta ccgtaaaagt gtaaaagttc gtttaaatca 10500
tttgtaaact ggaacagcaa gaggaagtat catcagctag ccccataaac taatcaaagg 10560
aggatgtcga ctaagagtta ctcggaaaga gcagctgctc atagaagtcc agttgctgcc 10620
aagcttttaa acttgatgga agagaagaag tcaaacttat gtgcttctct tgatgttcgt 10680
aaaacagcag agttgttaag attagttgag gttttgggtc catatatctg tctattgaag 10740
acacatgtag atatcttgga ggatttcagc tttgagaata ccattgtgcc gttgaagcaa 10800
ttagcagaga aacacaagtt tttgatattt gaagacagga agtttgccga cattgggaac 10860
actgttaaat tacaatacac gtctggtgta taccgtatcg ccgaatggtc tgatatcacc 10920
aatgcacacg gtgtgactgg tgcgggcatt gttgctggtt tgaagcaagg tgccgaggaa 10980
gttacaaaag aacctagagg gttgttaatg cttgccgagt tatcgtccaa ggggtctcta 11040
gcgcacggtg aatacactcg tgggaccgtg gaaattgcca agagtgataa ggactttgtt 11100
attggattta ttgctcaaaa cgatatgggt ggaagagaag agggctacga ttggttgatc 11160
atgacgccag gtgttggtct tgatgacaaa ggtgatgctt tgggacaaca atacagaact 11220
gtggatgaag ttgttgccgg tggatcagac atcattattg ttggtagagg tcttttcgca 11280
aagggaagag atcctgtagt ggaaggtgag agatacagaa aggcgggatg ggacgcttac 11340
ttgaagagag taggcagatc cgcttaagag gggtaccgag ctcgaatt 11388
<210> 24
<211> 22
<212> DNA
<213>Artificial sequence ()
<400> 24
agagcgacgg caagtttgag ga 22
<210> 25
<211> 24
<212> DNA
<213>Artificial sequence ()
<400> 25
cagcaggtga tagtggaatg agtg 24
<210> 26
<211> 24
<212> DNA
<213>Artificial sequence ()
<400> 26
tagttggtgg agtgatttgt ctgc 24
<210> 27
<211> 22
<212> DNA
<213>Artificial sequence ()
<400> 27
ctcgctggct ccgtcagtgt ag 22
<210> 28
<211> 62
<212> DNA
<213>Artificial sequence ()
<400> 28
atgcttgagc agattgctaa acaccatcat caccaccatt aagcggccgc ttaaggccgc 60
aa 62
<210> 29
<211> 62
<212> DNA
<213>Artificial sequence ()
<400> 29
ttgcggcctt aagcggccgc ttaatggtgg tgatgatggt gtttagcaat ctgctcaagc 60
at 62

Claims (27)

1. a kind of K marxianus promoter of optimization, which is characterized in that the K marxianus promoter Nucleotide sequence include at least：
A) nucleotide sequence as shown in SEQ ID No.1；Or
B) nucleotide sequence as shown in SEQ ID No.2；Or
C) nucleotide sequence as shown in SEQ ID No.3.
2. a kind of kluyveromyces marxianus secreting signal peptide of optimization, which is characterized in that the kluyveromyces marxianus point The nucleotide sequence of pil signal peptide includes at least the nucleotide sequence as shown in SEQ ID No.4.
3. a kind of kluyveromyces marxianus secreting signal peptide of optimization, which is characterized in that the kluyveromyces marxianus point The amino acid sequence of pil signal peptide includes at least the amino acid sequence as shown in SEQ ID No.5.
4. a kind of K marxianus promoter of optimization and the assembly of secreting signal peptide, which is characterized in that described group Fit nucleotide sequence includes at least the nucleotide sequence as shown in SEQ ID No.6.
5. a kind of recombinant expression carrier, which is characterized in that the recombinant expression carrier includes mark according to claim 1 This kluyveromyces promoter and/or kluyveromyces marxianus secreting signal peptide according to claim 2.
6. recombinant expression carrier according to claim 5, which is characterized in that the recombinant expression carrier also includes Marx Kluyveromyces autonomously replicating sequence, kluyveromyces marxianus inulinase terminator gene order and auxotrophy selection markers Gene order.
7. recombinant expression carrier according to claim 6, which is characterized in that the auxotrophy riddled basins sequence Selected from one or more of URA3 genes, HIS3 genes and ADE2 genes.
8. recombinant expression carrier according to claim 7, which is characterized in that the auxotrophy riddled basins sequence For URA3 genes.
9. recombinant expression carrier according to claim 5, which is characterized in that the nucleotide sequence of the recombinant expression carrier It includes at least：
A), the nucleotide sequence as shown in SEQ ID No.22；Or
B), the nucleotide sequence as shown in SEQ ID No.23.
10. a kind of genetic engineering bacterium, which is characterized in that the genetic engineering bacterium includes Marx according to claim 1 Kluyveromyces promoter and/or kluyveromyces marxianus secreting signal peptide according to claim 2.
11. a kind of genetic engineering bacterium, which is characterized in that the genetic engineering bacterium includes according to any one of claim 5-9 The recombinant expression carrier.
12. the genetic engineering bacterium according to claim 10 or 11, which is characterized in that the host strain of the genetic engineering bacterium is Yeast.
13. genetic engineering bacterium according to claim 12, which is characterized in that the yeast is tieed up including at least Marx's Crewe Saccharomyces cerevisiae, Kluyveromyces lactis category yeast or saccharomyces cerevisiae category yeast.
14. the application of K marxianus promoter according to claim 1, which is characterized in that the Marx Kluyveromyces promoter is used to improve the expression of foreign protein.
15. application according to claim 14, which is characterized in that the foreign protein is feruloyl esterase.
16. the application of kluyveromyces marxianus secreting signal peptide according to claim 2 or 3, which is characterized in that described Kluyveromyces marxianus secreting signal peptide is for improving recombinant protein from emiocytosis to extracellular efficiency.
17. the application of assembly according to claim 4, which is characterized in that the assembly is for improving foreign protein Expression, while improving recombinant protein from emiocytosis to extracellular efficiency.
18. the preparation method of K marxianus promoter according to claim 1, which is characterized in that the horse Gram this kluyveromyces promoter by carried out on the basis of kluyveromyces marxianus inulin enzyme promoters coding mutation or It deletes and builds, include at least：
A), by -351 thymidine deoxidation cores in the nucleotide sequence of the kluyveromyces marxianus inulin enzyme promoters Nucleotide mutation is adenyl-deoxyribonucleotide；Or
B) 106 nucleotide bases between the kluyveromyces marxianus inulin enzyme promoters -8bp to -115bp, are removed； Or
C), include the combinatorial mutagenesis of step a) and step b).
19. preparation method according to claim 18, which is characterized in that the preparation method specifically includes following steps：
Step 1, using the genome of kluyveromyces marxianus bacterium FIM-1 as template, utilize nucleotide sequence such as SEQ ID No.7 Shown in primer I NU-F1 and nucleotide sequence the primer I NU-R1PCR as shown in SEQ ID No.8 expand inulin enzyme gene； Taq enzyme is added in reaction system, and pcr amplification product is carried out to add A, obtains target fragment；The target fragment is recycled, with PMD18-T carriers are attached, and obtain the plasmid pMD18-T-INU containing kluyveromyces marxianus inulin enzyme promoters；
Step 2, using the plasmid pMD18-T-INU as template, utilize nucleotide sequence primer as shown in SEQ ID No.9 INU Δ-F1 and nucleotide sequence the primer I NU Δ-R1 as shown in SEQ ID No.10 are by the sequence of inulin enzyme gene 79-1668bp Row removal, while multiple cloning sites are introduced between terminator codon after the 78bp of inulin enzyme gene, obtain plasmid pMD18-T-PINU-SP-MCS-TINU；
Step 3, with the plasmid pMD18-T-PINU-SP-MCS-TINUFor template, nucleotide sequence such as SEQ ID No.11 are utilized Shown in primer I NU-F2 and nucleotide sequence the primer I NU-R2 as shown in SEQ ID No.12 expand PINU-SP-MCS-TINU Segment；
Step 4, using pUKD carriers as template, utilize nucleotide sequence primer pUKD-F1 and core as shown in SEQ ID No.14 Primer pUKD-R1 shown in nucleotide sequence such as SEQ ID No.15 removes the sites EcoRI between pKS and KD sequences, obtains Plasmid pUKD Δs E；Digestion is carried out to the plasmid PUKD Δs E using EcoRI, recycles digestion products；
Step 5, by the P in step 3INU-SP-MCS-TINUSegment is connect with the digestion products in step 4, obtains matter Grain pUKDN132；
Step 6, using the plasmid pUKDN132 as template, using nucleotide sequence primer T as shown in SEQ ID No.16 (- 351) primer T (- 351) A-R will be on inulinase gene start codon as shown in SEQ ID No.17 for A-F and nucleotide sequence The thymidylic acid of trip 351 sports adenyl-deoxyribonucleotide, obtains plasmid pUKDN132-T (- 351) A, Including the nucleotide sequence as shown in SEQ ID No.1；Or
Step 7, using the plasmid pUKDN132 as template, utilize nucleotide sequence primer UTR as shown in SEQ ID No.18 Δ A-F and nucleotide sequence the primer UTR Δs A-R as shown in SEQ ID No.19 are by inulinase gene start codon upstream Sequence removal between 8bp to upstream 115bp, obtains plasmid pUKDN132-UTR Δ A, including as shown in SEQ ID No.2 Nucleotide sequence；Or
Step 8, using plasmid pUKDN132-T (- 351) A obtained in step 6 as template, utilize nucleotide sequence such as SEQ The primer UTR Δs A-R as shown in SEQ ID No.19 of primer UTR Δs A-F and nucleotide sequence shown in ID No.18 is by inulin Sequence removal between enzyme gene upstream from start codon 8bp to upstream 115bp, obtains plasmid pUKDN132-T (- 351) A+ UTR Δ A, including the nucleotide sequence as shown in SEQ ID No.3.
20. preparation method according to claim 19, which is characterized in that in step 1, the kluyveromyces marxianus bacterium FIM-1 is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, deposit number CGMCC No.10621。
21. preparation method according to claim 19, which is characterized in that in step 2, the multiple cloning sites include at least The site of SmaI, SpeI and NotI.
22. the preparation method of kluyveromyces marxianus secreting signal peptide according to claim 2, which is characterized in that institute It states preparation method and includes at least and the deoxycytidylic acid of inulin enzyme gene the 29th is sported into thymidine Acid.
23. preparation method according to claim 22, which is characterized in that the preparation method specifically includes following steps： Using plasmid pUKDN132 as template, nucleotide sequence primer P10L-F and nucleotide sequence as shown in SEQ ID No.20 are utilized The deoxycytidylic acid of inulin enzyme gene the 29th is sported thymus gland by the primer P10L-R as shown in SEQ ID No.21 Fudr acid, makes the proline of inulinase signal peptide the 10th become leucine.
24. preparation method according to claim 23, which is characterized in that the plasmid pUKDN132 is according to claim 19 The step 1-5 is prepared.
25. the preparation method of assembly according to claim 4, which is characterized in that the preparation method includes following step Suddenly：Using plasmid pUKDN132-T (- 351) A as template, nucleotide sequence primer P10L-F as shown in SEQ ID No.20 is utilized With nucleotide sequence primer P10L-R as shown in SEQ ID No.21 by the cytimidine deoxyribonucleoside of inulin enzyme gene the 29th Acid mutation is thymidylic acid, and the proline of inulinase signal peptide the 10th is made to become leucine.
26. preparation method according to claim 25, which is characterized in that plasmid pUKDN132-T (- 351) A according to Step 1-6 described in claim 19 is prepared.
27. the preparation method of recombinant expression carrier according to claim 9, which is characterized in that the preparation method includes Following steps：
Step 1, using the genome of kluyveromyces marxianus bacterium FIM-1 as template, utilize nucleotide sequence such as SEQ ID No.7 Shown in primer I NU-F1 and nucleotide sequence the primer I NU-R1 PCR amplification inulin enzyme genes as shown in SEQ ID No.8； Taq enzyme is added in the reaction system, and pcr amplification product is carried out to add A, obtains target fragment；The target fragment is recycled, with PMD18-T carriers are attached, and obtain the plasmid pMD18-T-INU containing kluyveromyces marxianus inulin enzyme promoters；
Step 2, using the plasmid pMD18-T-INU as template, utilize nucleotide sequence primer as shown in SEQ ID No.9 INU Δ-F1 and nucleotide sequence the primer I NU Δ-R1 as shown in SEQ ID No.10 are by the sequence of inulin enzyme gene 79-1668bp Row removal, while multiple cloning sites are introduced between terminator codon after the 78bp of inulin enzyme gene, obtain plasmid pMD18-T-PINU-SP-MCS-TINU；
Step 3, with the plasmid pMD18-T-PINU-SP-MCS-TINUFor template, nucleotide sequence such as SEQ ID No.11 are utilized Shown in primer I NU-F2 and nucleotide sequence the primer I NU-R2 as shown in SEQ ID No.12 expand PINU-SP-MCS-TINU Segment；
Step 4, using pUKD carriers as template, utilize nucleotide sequence primer pUKD-F1 and core as shown in SEQ ID No.14 Primer pUKD-R1 shown in nucleotide sequence such as SEQ ID No.15 removes the sites EcoRI between pKS and KD sequences, obtains Plasmid pUKD Δs E；Digestion is carried out to the plasmid PUKD Δs E using EcoRI, recycles digestion products；
Step 5, by the P in step 3INU-SP-MCS-TINUSegment is connect with the digestion products in step 4, obtains matter Grain pUKDN132；
Step 6, using the plasmid pUKDN132 as template, using nucleotide sequence primer T as shown in SEQ ID No.16 (- 351) primer T (- 351) A-R will be on inulinase gene start codon as shown in SEQ ID No.17 for A-F and nucleotide sequence The thymidylic acid of trip 351 sports adenyl-deoxyribonucleotide, obtains plasmid pUKDN132-T (- 351) A, Including the nucleotide sequence as shown in SEQ ID No.1；
Step 7, using plasmid pUKDN132-T (- 351) A obtained in step 6 as template, utilize nucleotide sequence such as SEQ The primer UTR Δs A-R as shown in SEQ ID No.19 of primer UTR Δs A-F and nucleotide sequence shown in ID No.18 is by inulin Sequence removal between enzyme gene upstream from start codon 8bp to upstream 115bp, obtains plasmid pUKDN132-T (- 351) A+ UTR Δ A, nucleotide sequence is as shown in SEQ ID No.22；Or
Step 8, using plasmid pUKDN132-T (- 351) A obtained in step 6 as template, utilize nucleotide sequence such as SEQ The primer P10L-R as shown in SEQ ID No.21 of primer P10L-F and nucleotide sequence shown in ID No.20 is by inulinase base Because the 29th deoxycytidylic acid sports thymidylic acid, plasmid pUKDN132-T (- 351) A+ is obtained P10L, nucleotide sequence is as shown in SEQ ID No.23.
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