CN107418967A - Uracil auxotrophy multiple-shaped nuohan inferior yeast and preparation method and application - Google Patents

Abstract

The invention discloses uracil auxotrophy multiple-shaped nuohan inferior yeast and preparation method and application.The preparation method of uracil auxotrophy multiple-shaped nuohan inferior yeast disclosed by the invention, including：Knock out the content of the phosphate decarboxylase gene of orotidine 5 ' or the DNA encoding the protein in multiple-shaped nuohan inferior yeast starting strain or inactivate the DNA encoding the protein, obtain uracil auxotrophy multiple-shaped nuohan inferior yeast；It is following M1 to knock out the phosphate decarboxylase gene of orotidine 5 ') and M2)：M1 the coding of the phosphate decarboxylase gene of orotidine 5 ') is made to terminate in advance；M2) exogenous dna fragment is inserted in the phosphate decarboxylase gene of orotidine 5 '.The uracil auxotrophy multiple-shaped nuohan inferior yeast inheritance stability that the present invention is built, and other growth characteristics do not have notable difference with wild-type strain, can be used as metabolic engineering or the chassis cell of synthetic biology transformation, and the cell factory of highly effective expressing recombinant protein.

Description

Uracil auxotrophy multiple-shaped nuohan inferior yeast and preparation method and application

Technical field

The present invention relates in biological technical field, uracil auxotrophy multiple-shaped nuohan inferior yeast and preparation method thereof is with answering With.

Background technology

Saccharomycete is that biological technical field applies very extensive microorganism, in large hair as unicellular eukaryote Very important effect is played in the production of ferment product, fine pharmaceutical chemicals and pharmaceutical grade protein.Hansenula polymorpha (Hansenula polymorpha) is a kind of methanotrophic yeast, can be using methanol as sole carbon source growth and inducible protein High efficient expression；Optimum growth temperature is 37 DEG C, is had to a variety of environment-stress such as heat stress, metal stress and Oxdative stress good Tolerance, therefore there is extensive adaptability to industrial environment；Exogenous DNA stabilization on chromosome, high copy are integrated and ensured The high efficiency of protein expression, stability and homogeneity；Protein secretion pathway and posttranslational modification mechanism are closer to height Deng eucaryote, the immunogenicity risk of recombinant protein is reduced；It is generally acknowledged safe microorganisms (Generally Recognized as safe, GRAS), obtain State Food and Drug Administration (CFDA) and approve；Some are at other Expression system can not realize that the high molecular weight protein, memebrane protein and complicated albumen of functional expression obtain in multiple-shaped nuohan inferior yeast Functional effective expression.Therefore multiple-shaped nuohan inferior yeast, which turns into, efficiently synthesizes recombinant protein medicine and other high-valued albumen the most Preferable Microbial cell factories.

Selection markers are important tools necessary to carrying out genetic modification to microbial strains.Multiple-shaped nuohan inferior yeast heredity changes The selection markers commonly used in making include antibiotic resistance screening and auxotrophy complementation.Compared with antibiotic resistance screens, There is the characteristics of biological safety is high, false positive rate is low by the auxotrophy selection markers having complementary functions, in food use and medicine Widely applied with having in the high efficient expression of recombinant protein.But the multiple-shaped nuohan inferior yeast auxotrophic strain used at present Majority derive from traditional physically or chemically mutagenesis, influences screening efficiency the defects of inheritance stability aspect；And original strain base This is the type strain of American Type Culture Collection center (American type culture collection, ATCC), It is restricted in commercial application.From source, exploitation has auxotrophy selection markers, inheritance stability multiple-shaped nuohan inferior yeast Bacterial strain, it is the advantage for giving full play to multiple-shaped nuohan inferior yeast in expression of recombinant proteins, realizes that commercial application is urgently to be resolved hurrily and ask Topic.

The content of the invention

The technical problems to be solved by the invention be how to prepare uracil auxotrophy multiple-shaped nuohan inferior yeast and how The marking protein in uracil auxotrophy multiple-shaped nuohan inferior yeast.

In order to solve the above technical problems, present invention firstly provides uracil auxotrophy multiple-shaped nuohan inferior yeast The preparation method of (Hansenula polymorpha).

The system of uracil auxotrophy multiple-shaped nuohan inferior yeast (Hansenula polymorpha) provided by the present invention Preparation Method, including：Gene or the reduction multiform Chinese are inferior in uracil route of synthesis in knockout multiple-shaped nuohan inferior yeast starting strain The content of DNA encoding the protein or go out the multiple-shaped nuohan inferior yeast in uracil route of synthesis described in yeast starting strain DNA encoding the protein inactivates in uracil route of synthesis described in bacterium germination strain, obtains the inferior ferment of the uracil auxotrophy multiform Chinese It is female；The multiple-shaped nuohan inferior yeast starting strain contains gene in the uracil route of synthesis.

The multiple-shaped nuohan inferior yeast starting strain can be multiple-shaped nuohan inferior yeast (Hansenula polymorpha) HP20110424, it is CGMCC in the preserving number of China Committee for Culture Collection of Microorganisms's common micro-organisms center No.7.89。

In the above method, gene can be orotidine -5 '-phosphate decarboxylase gene in the uracil route of synthesis.

In the above method, the orotidine -5 '-phosphate decarboxylase gene can be following A1) or A2) or A3) or A4)：

A1) coded sequence is the DNA molecular of sequence 1 in sequence table；

A2) coded sequence is the DNA molecular of the 634-1425 positions of sequence 1 in sequence table；

A3) and A1) or A2) nucleotide sequence that limits has 75% or more than 75% homogeneity, and the coding whey The DNA molecular of glycosides -5 '-phosphate decarboxylase；

A4) under strict conditions with A1) or the A2) nucleotide sequence hybridization that limits, and encode orotidine -5 '-phosphorus The DNA molecular of acid decarboxylase.

In the above method, gene can be following in uracil route of synthesis in the knockout multiple-shaped nuohan inferior yeast starting strain M1) and/or M2)：

M1 the coding of gene in the uracil route of synthesis) is made to terminate in advance；

M2) exogenous dna fragment is inserted in gene in the uracil route of synthesis.

M1) specifically can be by encoding base in orotidine -5 described in the multiple-shaped nuohan inferior yeast starting strain '-phosphate decarboxylase Because coded sequence 5-6 interdigits insertion adenylic acid realize, will the multiple-shaped nuohan inferior yeast starting strain it is described The sequence of orotidine -5 '-phosphate decarboxylase gene changes into the 620-1412 of sequence 2 by the 634-1425 positions of sequence 1 Position.

The insertion position of the exogenous dna fragment is the restriction enzyme identification of gene in the uracil route of synthesis Between sequence.The restriction enzyme can be Sac I.The insertion position of the exogenous dna fragment is the 1075- of sequence 2 The 1088-1089 interdigits of 1076 interdigits or sequence 1.The insertion of the exogenous dna fragment can cause the uracil to synthesize Gene function is lost in approach.

In an embodiment of the present invention, the sequence of the exogenous dna fragment is CYC1TT and its upstream and downstream sequence or sequence The 1434-5590 positions of sequence 5 in table.

In the above method, the insertion of the exogenous dna fragment can be completed by restriction enzyme.

In order to solve the above technical problems, present invention also offers the system of the uracil auxotrophy multiple-shaped nuohan inferior yeast Uracil auxotrophy multiple-shaped nuohan inferior yeast prepared by Preparation Method.

In order to solve the above technical problems, present invention also offers the method for express express target protein.

The method of express express target protein provided by the invention, including：The expression of encoding gene containing destination protein is carried Body is imported in the uracil auxotrophy multiple-shaped nuohan inferior yeast, is obtained recombinating multiple-shaped nuohan inferior yeast, is realized the purpose egg White expression.

In the method for above-mentioned express express target protein, the expression vector contains gene in the uracil route of synthesis.

In one embodiment of the invention, the destination protein is dextranase.

In order to solve the above technical problems, present invention also offers the uracil auxotrophy multiple-shaped nuohan inferior yeast in table Up to the application in protein.

In order to solve the above technical problems, present invention also offers a kind of reagent set for marking protein.

Reagent set provided by the present invention, carried by the uracil auxotrophy multiple-shaped nuohan inferior yeast and the expression Body forms.

Multiple-shaped nuohan inferior yeast (Hansenula polymorpha) HP20110424 falls within protection scope of the present invention, more Shape Hansenula yeast (Hansenula polymorpha) HP20110424 is common in China Committee for Culture Collection of Microorganisms The preserving number at microorganism center is CGMCC No.7.89.

The present invention is dashed forward using the multiple-shaped nuohan inferior yeast with independent intellectual property right as starting strain by the PCR genes mediated Become and the screening marker-free Gene silence of mazF-zeoR expression cassettes mediation constructs orotidine -5 '-phosphate decarboxylase function Missing, inheritance stability polymorpha strain, the bacterial strain removes, and there is nutrition caused by the blocking of uracil route of synthesis to lack Fall into outside phenotype, other growth characteristics do not have notable difference with wild-type strain, can be used as metabolic engineering or synthetic biology transformation Chassis cell, and the cell factory of highly effective expressing recombinant protein.

Biomaterial preservation explanation

The Classification And Nomenclature of biomaterial：Multiple-shaped nuohan inferior yeast (Hansenula polymorpha)

The strain number of biomaterial：HP20110424

Depositary institution's title of biomaterial：China Committee for Culture Collection of Microorganisms's common micro-organisms center

The depositary institution of biomaterial is referred to as：CGMCC

The depositary institution address of biomaterial：Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, Chinese Academy of Sciences microorganism Research institute, postcode：100101

The preservation date of biomaterial：On 05 02nd, 2017

The collection of biomaterial is registered on the books numbering：CGMCC No.7.89

Brief description of the drawings

Fig. 1 is wild-type strain multiple-shaped nuohan inferior yeast CGMCC 7.89 and the auxanogram of uracil-deficient bacterial strain compares.

Fig. 2 is the PCR checkings that reversely screening knocks out selection markers bacterial strain.Wherein, M is DNA molecular amount standard.

Fig. 3 is uracil auxotrophy polymorpha HP7.89-ura3 and wild type Hansenula polymorpha bacterium CGMCC 7.89 growth curve.

Embodiment

The present invention is further described in detail with reference to embodiment, the embodiment provided is only for explaining The bright present invention, the scope being not intended to be limiting of the invention.Experimental method in following embodiments, unless otherwise specified, it is Conventional method.Material used, reagent, instrument etc., unless otherwise specified, are commercially obtained in following embodiments. Quantitative test in following examples, it is respectively provided with and repeats to test three times, results averaged.

Plasmid pEBCMZC (Song PP, Liu S, Guo XN, Bai XJ, He XP, Zhang in following embodiments BR.Scarless gene deletion in methylotrophic Hansenula polymorpha by using mazF as counter-selectable marker.Anal Biochem,2015,468:66-74) public can be from applicant Place obtains the biomaterial, and the biomaterial is only attached most importance to used in the related experiment of duplicate invention, can not used as other purposes.

YEp352 carriers (Hill JE, Meyers AM, Koerner TJ, et al.A yeast/ in following embodiments E.coli shuttle vectors with multiple unique restriction sites.Yeast,1993,9: Saccharomyces cerevisiae URA3 genes 163-167) are carried, the public can obtain the biomaterial at applicant, and the biomaterial is only attached most importance to Used in the related experiment of duplicate invention, it can not be used as other purposes.

Antibiotic Zeocin in following embodiments is Invitrogen Products, catalog number R25001.

The various culture mediums used in embodiment：

YPD solid mediums：The culture medium is made up of solute and solvent；Solute is dusty yeast, peptone, glucose and fine jade Cosmetics, solvent are water；The concentration of solute is as follows：10g/L dusty yeasts, 20g/L peptones, 20g/L glucose and 10g/L agar Powder, natural pH.

YPM solid mediums：The culture medium is made up of solute and solvent；Solute is dusty yeast, peptone, methanol and agar Powder, solvent are water；The concentration of solute is as follows：10g/L dusty yeasts, 20g/L peptones, 10ml/L methanol and 10g/L agar powders, from Right pH.

SC solid mediums：The culture medium is made up of solute and solvent；Solute is yeast nitrogen culture medium (Yeast Nitrogen Base, YNB) and glucose, solvent be water；The concentration of solute is as follows：6.7g/L YNB, 10g/L glucose and 10g/L agar powders, natural pH.

SCM solid mediums：The culture medium is made up of solute and solvent；Solute is yeast nitrogen culture medium (Yeast Nitrogen Base, YNB) and methanol, solvent be water；The concentration of solute is as follows：6.7g/L YNB, 10ml/L methanol and 10g/L Agar powder, natural pH.

SCU solid mediums：The culture medium is made up of solute and solvent；Solute is yeast nitrogen culture medium (Yeast Nitrogen Base, YNB), glucose and uracil, solvent be water；The concentration of solute is as follows：6.7g/L YNB, 10g/L Portugals Grape sugar, 30mg/L uracils and 10g/L agar powders, natural pH.

The fluid nutrient medium of above culture medium is made by not adding agar powder therein, and remaining composition and concentration are homogeneous Together.

The structure of embodiment 1, uracil auxotrophy multiple-shaped nuohan inferior yeast HP7.89-ura3

The present embodiment, to go out bacterium germination, is constructed using multiple-shaped nuohan inferior yeast (Hansenula polymorpha) HP20110424 Uracil auxotrophy multiple-shaped nuohan inferior yeast, multiple-shaped nuohan inferior yeast (Hansenula polymorpha) HP20110424 is in The preserving number of state's Microbiological Culture Collection administration committee common micro-organisms center (abbreviation CGMCC) is CGMCC No.7.89, with Lower abbreviation multiple-shaped nuohan inferior yeast CGMCC 7.89.

First, for destroying orotidine -5 '-phosphate decarboxylase encoding gene URA3 construction of recombinant plasmid

(1) using primer URA3-2K-F and URA3-2K-R using multiple-shaped nuohan inferior yeast CGMCC 7.89 genomic DNA as Template enters performing PCR amplification, obtains the URA3 genes shown in sequence 1 in sequence table, the URA3 genes and the inferior ferment of the multiform Chinese in NCBI Female DL-1 URA3 and its flanking sequence uniformity is 95.93%.The URA3 genes are connected to cloning vector pEASY-blunt In simple, recombinant plasmid pEASY-URA3 is obtained.Wherein the 634-1425 positions of sequence 1 are the coded sequence of URA3 genes. Primer sequence is as follows：

URA3-2K-F：5’-AACCAGTGAATGAAAACGAAGAGC-3’；

URA3-2K-R：5’-GGTGAAGAAAACCGGGAAAAGAATT-3’.

(2) enter performing PCR amplification by template of pEASY-URA3 using primer URA3RH-F1 and URA3RH-R1, obtain DNA Fragment URA3-1, enter performing PCR amplification by template of pEASY-URA3 using primer URA3RH-F2 and URA3RH-R2, obtain DNA Fragment URA3-2, enter performing PCR amplification by template of URA3-1 and URA3-2 using primer URA3RH-F1 and URA3RH-R2, obtain DNA fragmentation shown in sequence 2.Recognition sequence containing a Sac I in sequence 2, the 1071-1076 positions of sequence 2 are Sac I Recognition sequence.The DNA fragmentation inserts a base A after the bit base of URA3 gene coded sequences the 5th, makes the of sequence 2 TATAA after the ATG of 620-622 positions sports continuous two terminator codon TAATAA, and the DNA fragmentation is named as into mURA3. MURA3 is connected in cloning vector pEASY-blunt simple, obtains recombinant plasmid pEASY-mURA3.The primer sequence Row are as follows：

URA3RH-F1：5’-AACGAAGAGCCCAACTACTACAAG-3’；

URA3RH-R1：5’-TCGCCCTTTCTCCATAAGATTTAtTACATGTTGATTATTATTCAGGGAAATGTTG- 3’；

URA3RH-F2：5’-CAACATTTCCCTGAATAATAATCAACATGTAaTAAATCTTATGGAGAAAGGGCGA- 3’；

URA3RH-R2：5’-GGTGAAGAAAACCGGGAAAAG-3’.

(3) enter performing PCR amplification by template of plasmid pEBCMZC using primer Ps-F and Ps-R, obtain shown in sequence 3 DNA fragmentation C-mazF-zeoR, the 4-9 positions and 3967-3972 positions of sequence 3 are Sac I recognition sequence, the of sequence 3 22-392 positions and the sequence that 3580-3950 positions are CYC1TT, the 432-2725 positions of sequence 3 are the virulent gene of methanol induction The sequence of mazF expression cassettes, the 432-1959 positions of sequence 3 are the sequence of MOX promoters, and the 2726-3950 positions of sequence 3 are The sequence of zeoR expression cassettes, the 3205-3579 positions of sequence 3 are zeoR sequence.The primer sequence is as follows：

Ps-F：5’-AGGGAGCTCATCCAATTGTGACACGTC-3 ' (underscore part is Sac I recognition sequence)；

Ps-R：5’-AGGGAGCTCGATGCCAGCAACGCG-3 ' (underscore part is Sac I recognition sequence).

By carrier bones of the C-mazF-zeoR after the DNA fragmentation after the digestions of Sac I and the digestions of pEASY-mURA3 Sac I Frame is connected, and obtains recombinant plasmid pmURA3-CMMZC, and C-mazF-zeoR is inserted into the SacI inside mURA3 in the recombinant plasmid Between recognition sequence, virulent gene mazF expression of the recombinant plasmid with zeocin resistance screenings mark zeoR and methanol induction Box.

2nd, for destroying the DNA fragmentations of URA3 genes

Using recombinant plasmid pmURA3-CMMZC as template, enter performing PCR amplification using primer pair URA3RH-F1 and P37, obtain The DNA fragmentation of 4536bp correct sequence, the DNA fragmentation include mURA3 5 ' terminal sequences, CYC1TT, mazF expression cassette and 5 ' terminal sequences of zeoR expression cassettes, are named as mUCMZ-1.

Using recombinant plasmid pmURA3-CMMZC as template, enter performing PCR amplification using primer pair ZEO-F and URA3RH-R2, obtain To the DNA fragmentation of 1515bp correct sequence, the DNA fragmentation includes 3 ' terminal sequences of zeoR expression cassettes and mURA3 3 ' end sequences Row, are named as ZmU-2.

259bp overlay region between DNA fragmentation mUCMZ-1 and ZmU-2 wherein be present, can be formed completely by recombinating ZeoR expression cassettes, conversion bacterial strain is set to show zeocin resistances.

Wherein, primer P37 is respectively positioned in zeoR gene order with ZEO-F sequences, and sequence is as follows：

P37：5’-CGTCCCGGAAGTTCGTGG-3’；

ZEO-F：5’-AAGTTGACCAGTGCCGTTCC-3’.

3rd, conversion of saccharomycetes and recombinant bacterial strain Screening and Identification

(1) DNA fragmentation mUCMZ-1 and ZmU-2 equimolar concentration are mixed, then takes the electricity conversion of 10 μ g hybrid dnas fragments Multiple-shaped nuohan inferior yeast CGMCC 7.89, bacterium solution is coated on YPD+zeocin solid mediums after conversion, in 37 DEG C of quiescent cultures 3-5d.The single bacterium colony grown is inoculated in 1mL sterilized waters, is stored at room temperature 2h, is then put respectively in SC solid mediums and YPD On solid medium, 37 DEG C of quiescent culture 48h, obtain the normal growth on YPD culture mediums and do not grown on SC culture mediums Convert bacterial strain, i.e. auxotrophic strain.Wherein, YPD+zeocin solid mediums are to add zeocin into YPD culture mediums Obtained zeocin concentration is 200 μ g/mL solid medium.

(2) auxotrophic strain and wild-type strain multiple-shaped nuohan inferior yeast CGMCC 7.89 obtained step (1) It is inoculated in respectively in 1mL sterilized waters, is stored at room temperature 2h, then respectively puts in SC, SCU, YPD and YPD+zeocin solid culture On base, 37 DEG C of quiescent culture 48h.As a result show, wild strain equal normal growth on SC, SCU, YPD culture medium, but in YPD+ It can not be grown on zeocin culture medium flat plates；The auxotrophic strain that step (1) obtains can not grow on SC culture mediums, But the normal growth on SCU, YPD and YPD+zeocin culture medium.Show that the auxotrophic strain that step (1) obtains is urine Pyrimidine synthesizes deficient strain, and is marked with zeocin resistance screenings.

Bacterial strain is identified：

According to multiple-shaped nuohan inferior yeast URA3 upstream and downstream flanking sequences, primer URA3UP-F and URA3DOWN- are designed and synthesized R, wherein primer URA3UP-F and URA3DOWN-R sequence are respectively on URA3 upstream and downstream flanking sequences, and each primer sequence is such as Under：

URA3UP-F：5’-AGTCAGACGAGGGTAAGGAA-3’；

URA3DOWN-R：5’-CGATGATCCTAGCCCTGTCG-3’.

Using primer pair URA3UP-F and URA3DOWN-R respectively to multiple-shaped nuohan inferior yeast CGMCC 7.89 and step (1) The genomic DNA of obtained auxotrophic strain enters performing PCR amplification, is amplified from multiple-shaped nuohan inferior yeast CGMCC 7.89 2719bp DNA fragmentations, the DNA fragmentation is sequenced and shows its sequence as shown in sequence 4, the 544-2385 positions of sequence 4 Sequence and the URA3 gene orders shown in sequence 1 are completely the same；And the auxotrophic strain genome obtained from step (1) 6683bp DNA fragmentation is amplified, sequencing result shows its sequence as shown in sequence 5, the 558-1633 positions of sequence 5 The 1-1076 bit sequences of sequence and sequence 2 are completely the same, and the 1634-5590 positions of sequence 5 are C-mazF-zeo sequences, sequence 5 5597-6349 bit sequences and the 1077-1829 bit sequences of sequence 2 is completely the same.Therefore the nutrition that step (1) obtains Deficient strain is to identify sequence in mURA3 Sac I corresponding to the sequence of multiple-shaped nuohan inferior yeast CGMCC 7.89 URA3 genes The sequence that C-mazF-zeoR is obtained is inserted between row.Show URA3 positions on the auxotrophic strain genome that step (1) obtains C-mazF-zeoR, and the Partial Fragment of the mURA3 shown in 620-628 positions of the bacterial strain also containing sequence 2 are inserted in point, Cell is seted to produce uracil auxotrophic phenotype from synthesizing orotidine -5 '-phosphate decarboxylase, cell.Step (1) is obtained Auxotrophic strain be named as HP-ura3::MZ.

4th, reversely screening knocks out selection markers and obtains uracil-deficient type bacterial strain HP7.89-ura3

(1) by the uracil auxotrophy bacterial strain HP-ura3 of step 3::MZ is connected in 2ml YPD fluid nutrient mediums, 37 DEG C, 200rpm culture 18h, 5000rpm centrifugation 5min collection cells, sterilized water washed once, and add 2ml sterilized waters, room temperature After standing 2h, 100 μ L bacteria suspensions are taken to be coated on YPM solid mediums, 37 DEG C of quiescent cultures are to growing single bacterium colony.

Random four single bacterium colonies of picking (being respectively designated as Δ U1, Δ U2, Δ U3 and Δ U4) are inoculated in 1mL sterilized waters, After being stored at room temperature 2h, dibbling is on SC, SCU, YPD and YPD+zeocin solid medium respectively, while more with wild-type strain Shape Hansenula yeast CGMCC 7.89 and uracil-deficient type bacterial strain HP-ura3::MZ is compares, 37 DEG C of quiescent culture 48h.

As a result as shown in figure 1, all bacterial strains energy normal growth, but only bacterial strain HP-ura3 on YPD culture mediums::MZ It can be grown on YPD+zeocin culture mediums；Wild-type strain equal normal growth on SC, SCU culture medium, but bacterial strain HP- ura3::MZ and the single bacterium colony (Δ U1, Δ U2, Δ U3 and Δ U4) on YPM flat boards can not grow on SC culture mediums, The normal growth on the SCU culture mediums for the addition of uracil.Show HP-ura3::After MZ is cultivated on YPM culture mediums, due to first Alcohol-induced toxic protein MazF expression produces lethal effect to cell, and is only knocked out by the homologous recombination of CYC1TT mediations Zeocin resistance screenings mark could grow with the yeast cells after mazF expression cassettes, and show as uracil auxotrophy.

(2) according to multiple-shaped nuohan inferior yeast URA3 site nucleotide sequences, following primer is designed and synthesized：

URA3-mF：5’-CAGGCCGCAGAAGAAAGTAC-3’；

URA3HP-R：5’-TTAAGCAATGCGCCGCTTGTAAG-3’.

Extract Δ U1, Δ U2, Δ U3 and Δ U4 genomic DNA respectively, as template, using primer URA3-mF and URA3HP-R enters performing PCR amplification and above-mentioned bacterial strains is verified.Simultaneously respectively with wild-type strain CGMCC 7.89 and uracil Deficient strain HP-ura3::MZ genomes are control.

The electrophoresis detection result of pcr amplification product using primer URA3-mF and URA3HP-R from reverse as shown in Fig. 2 sieved Bacterial strain Δ U1 after choosing, Δ U2, Δ U3 and Δ U4 genome amplify about 800bp DNA fragmentations, from wild-type strain CGMCC 7.89 genome amplifications go out about 400bp DNA fragmentation, from bacterial strain HP-ura3::MZ genome amplifications go out about 4.4kb DNA pieces Section.Sequence analysis and comparison are carried out to PCR primer, the results showed that the PCR from bacterial strain Δ U1, Δ U2, Δ U3 and Δ U4 is produced Thing has completely the same nucleotide sequence, and they are compared with wild-type strain CGMCC 7.89 PCR primer, in the 57th core 405bp CYC1TT sequences are inserted behind thuja acid, with bacterial strain HP-ura3::MZ PCR primer is compared and has lacked mazF expression Box and zeocin resistance expression's boxes.The above results show, CYC1TT is inserted inside target gene URA3 by reversely screening to obtain The mutant strain of sequence, the screening marker-free for realizing URA3 destroy, and yeast cells is produced uracil auxotrophic phenotype.Will HP7.89- is named as by the uracil auxotrophy bacterial strain (i.e. Δ U1, Δ U2, Δ U3 and Δ U4) for reversely screening acquisition ura3。

Wild-type strain CGMCC 7.89 and uracil auxotrophy bacterial strain HP7.89-ura3 URA3 sites code sequence Row compare：

According to wild-type strain CGMCC 7.89 URA3 upstream from start codon about 200bp sequences, design and synthesize and draw Thing URA3ORF：5’-GTCCACCAAACTGGTGTAG-3’.

Respectively with wild-type strain CGMCC 7.89 and uracil auxotrophy bacterial strain HP7.89-ura3 genomic DNAs For template, enter performing PCR amplification using primer URA3ORF and URA3HP-R.

Go out 991bp DNA fragmentation (to this from wild-type strain genome amplification using primer URA3ORF and URA3HP-R The sequencing of DNA fragmentation shows that its sequence is sequence 6 in sequence table), from HP7.89-ura3 genome amplification go out 1397bp's DNA fragmentation (sequencing to the DNA fragmentation shows that its sequence is sequence 7 in sequence table).As a result show, with wild-type strain URA3 Coded sequence is compared, and the TATAA of HP7.89-ura3 URA3 coded sequences after the 620-622 positions ATG of sequence 2 is sported Continuous two terminator codon TAATAA；And insert CYC1TT at 454bp after the 620-622 positions ATG of sequence 2 (the 669-1039 positions of sequence 7) and its each 12bp and 22bp sequences of upstream and downstream.

5th, uracil-deficient type bacterial strain HP7.89-ura3 inheritance stability analysis

Uracil auxotrophy polymorpha strain HP7.89-ura3 is connected in 2ml YPD fluid nutrient mediums, 37 DEG C, 200rpm shaking table culture 20h, are transferred in fresh YPD medium, as above continuous passage culture 10 by 10% inoculum concentration It is secondary.The μ L of nutrient solution 200 of passage 1,5 and 10 time are taken respectively, carry out 10 times of gradient dilution.

Undiluted bacterium solution and each 100 μ L of different dilution factor bacterium solutions are respectively coated on SC, SCU and YPD culture medium flat plate, 37 DEG C of quiescent culture 48h, record colony growth situation on different flat boards.

As a result find all there is no colony growth on all SC flat boards, and bacterium colony normal growth on SCU and YPD flat boards, bacterium Fall number to improve and successively decrease with dilution factor, and the clump count under same treatment conditions on SCU and YPD flat boards does not have notable difference. Illustrate the uracil auxotrophy polymorpha strain HP7.89-ura3 inheritance stabilities of the present invention.

6th, wild type and uracil-deficient type bacterial strain HP7.89-ura3 growth characteristics compare

By wild type Hansenula polymorpha CGMCC 7.89 and uracil auxotrophy polymorpha HP7.89- Ura3 is inoculated in 2ml YPD fluid nutrient mediums respectively, 37 DEG C, 200rpm shaken cultivation 16h, by volume 10% inoculum concentration Transfer in 10ml YPD fluid nutrient mediums, 37 DEG C of shaking table shaken cultivations 16 hours；Somatic cells are collected by centrifugation, by cell nothing Bacterium is washed twice, and thalline is resuspended in 5ml sterilized waters and bacteria suspension is made；Bacteria suspension is transferred in SCU, YPD and YPM liquid respectively In culture medium, nutrient solution is set to originate OD₆₀₀About 0.15,37 DEG C, 200rpm shaking table shaken cultivation 36h, 2ml is during which sampled per 6h Determine OD₆₀₀, make growth curve.As a result as shown in figure 3, the uracil auxotrophy multiple-shaped nuohan inferior yeast that the present invention is built Bacterium (Hansenula polymorpha) HP7.89-ura3 and wild type Hansenula polymorpha bacterium CGMCC 7.89 are in growth characteristics Upper no notable difference.

The table of embodiment 2, dextranase in multiple-shaped nuohan inferior yeast (Hansenula polymorpha) HP7.89-ura3 Reach

First, the structure of expression vector

By the pMOXZ α-A (patent No.s：ZL200810101801.1) the DNA fragmentation between carrier EcoRI and XbaI recognition sequences This is replaced with up to saccharomyces oleaginosus glucanase coding gene (LSD1 genes, its sequence are the 1798-3624 positions of sequence 8), is obtained To recombinant vector, by the recombinant vector be named as pMOXZ α-LSD1, pMOXZ α-LSD1 can expressed sequence 8 1798-3624 This of LSD1 gene codes shown in position reaches saccharomyces oleaginosus dextranase.

Using recombinant vector pMOXZ α-LSD1 as template, enter performing PCR amplification using primer MOXp-F and AOX1TT-R, by sequence Arrange correct PCR primer and be named as DNA fragmentation 1, the sequence of DNA fragmentation 1 is sequence 8, and the 1-1511 positions of sequence 8 are methanol oxygen Change enzyme gene promoter MOXp sequences, the 1525-1791 positions of sequence 8 are alpha signal peptide sequence, the 1798-3624 positions of sequence 8 For LSD1 gene coded sequences, the 3695-4033 positions of sequence 8 are alcohol oxidase gene terminator AOX1TT sequences.

The primer sequence is as follows：

MOXp-F：5'-TCTTCGACGCGGAGAACGATCTCCT-3',

AOX1TT-R：5'-TCCGCACAAACGAAGGTCTC-3'.

DNA fragmentation 1 is inserted into the SmaI sites of YEp352 carriers, obtains recombinant vector, the recombinant vector is named For pMOXU α-LSD1.

2nd, conversion of saccharomycetes and recombinant bacterial strain screening

Using restriction enzyme SacII digested plasmid pMOXU α-LSD1, linearization plasmid pMOXU α-LSD1 are obtained；It is logical Cross electrotransformation (Bio-Rad Gene-Pulser instrument, 1.5kV, 50 μ F, 200 Ω, 3mSec) by linearization plasmid pMOXU α- Uracil auxotrophy polymorpha (Hansenula polymorpha) HP7.89- of LSD1 conversion embodiments 1 Ura3 (Host Strains HP7.89-ura3), transformed bacteria solution are coated on SC culture medium flat plates, and 37 DEG C of quiescent culture 3-5d are to forming list Bacterium colony.

Single bacterium colony on SC flat boards is transferred in 1ml sterilized waters, stands 2-4h at ambient temperature, correspond to respectively dibbling in On SC, SCU and YPD flat board, 37 DEG C are cultivated 48 hours, using Host Strains HP7.89-ura3 as control.As a result Host Strains HP7.89- Ura3 can not grow on SC flat boards, can only be grown on SCU and YPD flat boards, and convert pMOXU α-LSD1 single bacterium colony upper Equal energy normal growth on each culture medium flat plate is stated, illustrates that linearization plasmid pMOXU α-LSD1 successful conversions enter Host Strains HP7.89- Ura3, the uracil synthesis defect for the saccharomyces cerevisiae URA3 gene complementations Host Strains that linearization plasmid carries, will contain linear The recombinant bacterium for changing plasmid pMOXU α-LSD1 is named as HP-LSD1.

3rd, assay for dextranase activity

By Host Strains HP7.89-ura3 and 5 recombinant bacterium HP-LSD1 single bacterium colonies (HP-LSD1-1~HP- of random picking LSD1-5) it is connected to respectively in 5ml YPD culture mediums, 37 DEG C, 200rpm culture 20h, cell is collected by centrifugation, sterile washing is twice Afterwards, cell is resuspended in 10ml YPM culture mediums, 37 DEG C, 200rpm progress Fiber differentiation 72h, methanol is added to methanol per 12h Concentration of volume percent be 1%.Per 24h sample, by 3,5- dinitro bigcatkin willows acid system determine zymotic fluid in and yeast cells Interior dextranase activity (Kang HK, Kim SH, Park JY, Jin XJ, Oh DK, Kang SS, Kim D.Cloning and characterization of a dextranase gene from Lipomyces starkeyi and its expression in Saccharomyces cerevisiae.Yeast,2005,22:1239-1248).Total dextranase Activity is the summation of enzyme activity in intracellular enzyme activity and zymotic fluid.Specific method is as follows：

The preparation of solution：15mg glucose accurately is weighed, is dissolved in distilled water, obtains final concentration of 1mg/ml glucose Reservoir；7.16g disodium hydrogen phosphates accurately are weighed, are dissolved in distilled water, adjust pH to 5.5 with citric acid, distilled water is settled to 1000ml, obtain citrate-phosphate disodium hydrogen buffer solution (50mM, pH5.5)；2g glucan T-70 accurately are weighed, are dissolved in lemon In acid-disodium hydrogen phosphate buffer solution, constant volume to 100ml；8g sodium hydroxides accurately are weighed, are dissolved in distilled water, constant volume arrives 100ml, make final concentration of 2mol/L；1g 3 accurately is weighed, 5- dinitrosalicylic acids (DNS), is dissolved in 20ml sodium hydroxide solutions In 50ml distilled water, 30g Rochelle salts are added, after dissolving plus water is settled to 100ml, obtains DNS solution, is keeping away Saved backup in light closed container.

Glucose standard curve：The glucose reservoir that concentration is 1mg/ml is suitably diluted, obtaining concentration respectively is 0th, 0.1,0.3,0.5,0.7,0.9 and 1.0mg/ml glucose solution, each 1ml of above-mentioned glucose solution is taken in test tube, point Not Jia Ru 1ml DNS solution, after mixing, 5min is boiled in boiling water bath, after being cooled to room temperature, adds distilled water to be settled to 5ml, with not Reaction solution containing glucose is control, determines 540nm absorbance value (OD₅₄₀), using concentration of glucose as abscissa, OD₅₄₀For Ordinate, standard curve is drawn, obtains concentration of glucose and OD₅₄₀Between functional relation：Concentration of glucose (mg/ml)= (OD₅₄₀+0.0317)/1.4602。

Dextranase activity determines in zymotic fluid：Different time points take nutrient solution 5ml, 6000rpm centrifugation 5min, receive respectively Collect supernatant and yeast cells；0.5ml supernatants are taken wherein to be added in contrast test tube and reaction tube in reaction tube respectively 2% dextran solution of 37 DEG C of preheatings of 0.5ml；Control tube and reaction tube are incubated 30min at 37 DEG C, are then respectively adding 1ml DNS solution, after mixing, 0.5ml 2% dextran solution is added in control tube；Boiling water bath boils 5min；It is cooled to After room temperature, control tube and reaction tube are separately added into distilled water and are settled to 5ml；Returned to zero with control tube, measure reaction tube OD₅₄₀, foundation Standard curve functional expression calculates concentration of reduced sugar in reaction solution.

Cell endoglucanase activity determines：The yeast cells of foregoing collected after centrifugation, aqueous suspension is distilled with 5ml, 6000rpm centrifuges 5min, and yeast cells is resuspended in into citrate-phosphate hydrogen in the ratio of every gram of wet cell 10ml buffer solution In disodium buffer solution, -80 DEG C of freeze overnights；Naturally after melting, 0.5ml bacteria suspensions are taken to be added simultaneously in 1.5ml centrifuge tubes The a diameter of 0.45mm of 25mg bead, vortex oscillation 4 times, 1min, ice bath 1min is vibrated every time；4 DEG C, 6000rpm centrifugations 10min, collect supernatant；Dextranase activity in supernatant is determined by preceding method.

Enzyme activity is defined as：Under above-mentioned reaction condition, catalysis glucan hydrolysis per minute is produced equivalent to 1 μm of ol glucose Enzyme amount needed for reduced sugar is an enzyme-activity unit (IU).

As a result as shown in table 1, dextranase activity is not detected in host's fermented liquid, but in recombinant bacterium HP- Dextranase activity is detected in LSD1 zymotic fluid.After Fiber differentiation 72h, total dextranase activity is up to Dextranase enzyme activity is 720IU/L in 2050IU/L, wherein zymotic fluid.

The above results show uracil auxotrophy multiple-shaped nuohan inferior yeast HP7.89-ura3 of the dextranase in the present invention In obtain functional expression.

Dextranase activity and fermentation ends total enzyme activity in table 1, different strains zymotic fluid

<110>Institute of Microorganism, Academia Sinica, university of the Chinese Academy of Sciences

<120>Uracil auxotrophy multiple-shaped nuohan inferior yeast and preparation method and application

<160> 8

<170> PatentIn version 3.5

<210> 1

<211> 1842

<212> DNA

<213>Artificial sequence

<220>

<223>

<400> 1

aaccagtgaa tgaaaacgaa gagcccaact actacaagct aatacggcat cccgtggaca 60

ttaagagttt gggtaaagct attaggacgg gtgaaataaa gtcattcgac gagttggaat 120

ttgagcttca actgatgttc agcaacgcaa ttatgtacaa cgatatgaat cagacagaaa 180

cttacaaatg gacgattgaa atgatggaag aaacgcaaaa cttgttgagc ctattccggg 240

aatcatccag caattaaagc tcattaattg ccgcctctca tctggttcat caacatctgg 300

ttctgttcct ggaatcttaa accaactctt tcggagtgct tgaggaactt ttcagcgcac 360

ttgttcaaac atgtttcttc tctagagcta agtttgttgg aggtgaagtc gttgacgcaa 420

tcattaaaac atctgtccac caaactggtg tagagctgca atgttagttt tcagaaggcg 480

ttcacttact ctcatgaagt cattcatttg cttctgctcc acaattttct ggaattcttg 540

ctgttcttta tagttgagtt gatccatggt gtatcaaaaa taaactaatg aattatcgaa 600

aaattttcaa catttccctg aataataatc aacatgtata aatcttatgg agaaagggcg 660

aagtctcacc catctaaggt cgccagcaga ctacttaatt tgatggaatc caagcaaaca 720

aacctctgcg cttctgtgga tgtgactaaa actcaggaat tattggagct tcttgataaa 780

ctgggccctt acatctgcct tgtcaaaact catattgaca tagtagagga cttctcttat 840

gaacacacca ttttaccatt acaaggactt gcaaagaaac acaacttcat gatttttgaa 900

gacagaaagt ttgctgatat aggaaacaca gtcaaactac agtataaggg aggaatttat 960

cgaacatcca agtgggccga tatcacgaat gcacacggag tgactggcgc aggaattgtt 1020

gaaggtctta aacaggccgc agaagaaagt acagatgagc cacgtgggct tttgatgctt 1080

gctgagctct cttcaaaggg atcattagct accggtgagt atactcaaaa aactgtggaa 1140

atagcgaaaa gcgataaaga atttgtcatt ggatttattg cacagagaga catgggaggt 1200

cgtgaggaag gctttgactg gctgatcatg actccaggag ttggtttaga tgataaaggt 1260

gattctctgg gccaacagta cagaactgtt gatgaagtga tgcaaacagg aaccgatgtc 1320

attatcgttg gaagaggttt attcggaaaa ggaagagatc ctgaagtgga agggaagaga 1380

tacagaaatg ctgggtggga agcttacaag cggcgcattg cttaacggct ttcagttcta 1440

tatacatcgt caaaattgat tttcgctaaa atgctgacgg gatatttcga atacgaaaag 1500

cccaatagaa gtcgcgggaa tactagtgaa ggacgatctg aaaaagcctc tatagcagaa 1560

gccaaagagg gagaggtgag attttcgcca tcgttgtaga ataatatcaa atgtctcaat 1620

gtaagaatta tagtagagtt tgaagaagcg acgccgatcc agttgatgtg cttcgttgta 1680

gagatctaga gattcgagtc tagtcaggtg gaccttttga attttattga gtggatattg 1740

gattgcaagc agtgtagttg cggcggaagc tccagcaaga aggataaacg tcaaattcaa 1800

agctttgaaa aatcttgaat tcttttcccg gttttcttca cc 1842

<210> 2

<211> 1829

<212> DNA

<213>Artificial sequence

<220>

<223>

<400> 2

aacgaagagc ccaactacta caagctaata cggcatcccg tggacattaa gagtttgggt 60

aaagctatta ggacgggtga aataaagtca ttcgacgagt tggaatttga gcttcaactg 120

atgttcagca acgcaattat gtacaacgat atgaatcaga cagaaactta caaatggacg 180

attgaaatga tggaagaaac gcaaaacttg ttgagcctat tccgggaatc atccagcaat 240

taaagctcat taattgccgc ctctcatctg gttcatcaac atctggttct gttcctggaa 300

tcttaaacca actctttcgg agtgcttgag gaacttttca gcgcacttgt tcaaacatgt 360

ttcttctcta gagctaagtt tgttggaggt gaagtcgttg acgcaatcat taaaacatct 420

gtccaccaaa ctggtgtaga gctgcaatgt tagttttcag aaggcgttca cttactctca 480

tgaagtcatt catttgcttc tgctccacaa ttttctggaa ttcttgctgt tctttatagt 540

tgagttgatc catggtgtat caaaaataaa ctaatgaatt atcgaaaaat tttcaacatt 600

tccctgaata ataatcaaca tgtaataaat cttatggaga aagggcgaag tctcacccat 660

ctaaggtcgc cagcagacta cttaatttga tggaatccaa gcaaacaaac ctctgcgctt 720

ctgtggatgt gactaaaact caggaattat tggagcttct tgataaactg ggcccttaca 780

tctgccttgt caaaactcat attgacatag tagaggactt ctcttatgaa cacaccattt 840

taccattaca aggacttgca aagaaacaca acttcatgat ttttgaagac agaaagtttg 900

ctgatatagg aaacacagtc aaactacagt ataagggagg aatttatcga acatccaagt 960

gggccgatat cacgaatgca cacggagtga ctggcgcagg aattgttgaa ggtcttaaac 1020

aggccgcaga agaaagtaca gatgagccac gtgggctttt gatgcttgct gagctctctt 1080

caaagggatc attagctacc ggtgagtata ctcaaaaaac tgtggaaata gcgaaaagcg 1140

ataaagaatt tgtcattgga tttattgcac agagagacat gggaggtcgt gaggaaggct 1200

ttgactggct gatcatgact ccaggagttg gtttagatga taaaggtgat tctctgggcc 1260

aacagtacag aactgttgat gaagtgatgc aaacaggaac cgatgtcatt atcgttggaa 1320

gaggtttatt cggaaaagga agagatcctg aagtggaagg gaagagatac agaaatgctg 1380

ggtgggaagc ttacaagcgg cgcattgctt aacggctttc agttctatat acatcgtcaa 1440

aattgatttt cgctaaaatg ctgacgggat atttcgaata cgaaaagccc aatagaagtc 1500

gcgggaatac tagtgaagga cgatctgaaa aagcctctat agcagaagcc aaagagggag 1560

aggtgagatt ttcgccatcg ttgtagaata atatcaaatg tctcaatgta agaattatag 1620

tagagtttga agaagcgacg ccgatccagt tgatgtgctt cgttgtagag atctagagat 1680

tcgagtctag tcaggtggac cttttgaatt ttattgagtg gatattggat tgcaagcagt 1740

gtagttgcgg cggaagctcc agcaagaagg ataaacgtca aattcaaagc tttgaaaaat 1800

cttgaattct tttcccggtt ttcttcacc 1829

<210> 3

<211> 3975

<212> DNA

<213>Artificial sequence

<220>

<223>

<400> 3

agggagctca tccaattgtg acacgtccga cggcggccca cgggtcccag gcctcggaga 60

tccgtccccc ttttcctttg tcgatatcat gtaattagtt atgtcacgct tacattcacg 120

ccctcccccc acatccgctc taaccgaaaa ggaaggagtt agacaacctg aagtctaggt 180

ccctatttat ttttttatag ttatgttagt attaagaacg ttatttatat ttcaaatttt 240

tctttttttt ctgtacagac gcgtgtacgc atgtaacatt atactgaaaa ccttgcttga 300

gaaggttttg ggacgctcga aggctttaat ttgcaagctg gagaccaaca tgtgagcaaa 360

aggccagcaa aaggccagga accgtaaaaa ggaagggcaa ttctgcagat atccatcaca 420

ctggcggccg ctcatgcatg agatcagatc ttcgacgcgg agaacgatct cctcgagctg 480

ctcgcggatc agcttgtggc ccggtaatgg aaccaggccg acgcgacgct ccttgcggac 540

cacggtggct ggcgagccca gtttgtgaac gaggtcgttt agaacgtcct gcgcaaagtc 600

cagtgtcaga tgaatgtcct cctcggacca attcagcatg ttctcgagca gccatctgtc 660

tttggagtag aagcgtaatc tctgctcctc gttactgtac cggaagaggt agtttgcctc 720

gccgcccata atgaacaggt tctctttctg gtggcctgtg agcagcgggg acgtctggac 780

ggcgtcgatg aggcccttga ggcgctcgta gtacttgttc cgtcgctgta gccggccgcg 840

gtgacgatac ccacatagag gtccttggcc attagtttga tgaggtgggg caggatgggc 900

gactcggcat cgaaattttt gccgtcgtcg tacagtgtga tgtcaccatc gaatgtaatg 960

agctgcagct tgcgatctcg gatggttttg gaatggaaga accgcgacat ctccaacagc 1020

tgggccgtgt tgagaatgag ccggacgtcg ttgaacgagg gggccacaag ccggcgtttg 1080

ctgatggcgc ggcgctcgtc ctcgatgtag aaggcctttt ccagaggcag tctcgtgaag 1140

aagctgccaa cgctcggaac cagctgcacg agccgagaca attcgggggt gccggctttg 1200

gtcatttcaa tgttgtcgtc gatgaggagt tcgaggtcgt ggaagatttc cgcgtagcgg 1260

cgttttgcct cagagtttac catgaggtcg tccactgcag agatgccgtt gctcttcacc 1320

gcgtacagga cgaacggcgt ggccagcagg cccttgatcc attctatgag gccatctcga 1380

cggtgttcct tgagtgcgta ctccactctg tagcgactgg acatctcgag actgggcttg 1440

ctgtgctgga tgcaccaatt aattgttgcc gcatgcatcc ttgcaccgca agtttttaaa 1500

acccactcgc tttagccgtc gcgtaaaact tgtgaatctg gcaactgagg gggttctgca 1560

gccgcaaccg aacttttcgc ttcgaggacg cagctggatg gtgtcatgtg aggctctgtt 1620

tgctggggta gcctacaacg tgaccttgcc taaccggacg gcgctaccca ctgctgtctg 1680

tgcctgctac cagaaaatca ccagagcagc agagggccga tgtggcaact ggtggggtgt 1740

cggacaggct gtttctccac agtgcaaatg cgggtgaacc ggccagaaag taaattctta 1800

tgctaccgtg cagcgactcc gacatcccca gtttttgccc tacttgatca cagatggggt 1860

cagcgctgcc gctaagtgta cccaaccgtc cccacacggt ccatctataa atactgctgc 1920

cagtgcacgg tggtgacatc aatctaaagt acaaaaacaa agcttaggaa gtctggtaat 1980

ggtaagccga tacgtacccg atatgggcga tctgatttgg gttgattttg acccgacaaa 2040

aggtagcgag caagctggac atcgtccagc tgttgtcctg agtcctttca tgtacaacaa 2100

caaaacaggt atgtgtctgt gtgttccttg tacaacgcaa tcaaaaggat atccgttcga 2160

agttgtttta tccggtcagg aacgtgatgg cgtagcgtta gctgatcagg taaaaagtat 2220

cgcctggcgg gcaagaggag caacgaagaa aggaacagtt gccccagagg aattacaact 2280

cattaaagcc aaaattaacg tactgattgg gtagtctaga acaaaaactc atctcagaag 2340

aggatctgaa tagcgccgtc gaccatcatc atcatcatca ttgagtttta gccttagaca 2400

tgactgttcc tcagttcaag ttgggcactt acgagaagac cggtcttgct agattctaat 2460

caagaggatg tcagaatgcc atttgcctga gagatgcagg cttcattttt gatacttttt 2520

tatttgtaac ctatatagta taggattttt tttgtcattt tgtttcttct cgtacgagct 2580

tgctcctgat cagcctatct cgcagctgat gaatatcttg tggtaggggt ttgggaaaat 2640

cattcgagtt tgatgttttt cttggtattt cccactcctc ttcagagtac agaagattaa 2700

gtgagacctt cgtttgtgcg gatcccccac acaccatagc ttcaaaatgt ttctactcct 2760

tttttactct tccagatttt ctcggactcc gcgcatcgcc gtaccacttc aaaacaccca 2820

agcacagcat actaaatttt ccctctttct tcctctaggg tgtcgttaat tacccgtact 2880

aaaggtttgg aaaagaaaaa agagaccgcc tcgtttcttt ttcttcgtcg aaaaaggcaa 2940

taaaaatttt tatcacgttt ctttttcttg aaattttttt ttttagtttt tttctctttc 3000

agtgacctcc attgatattt aagttaataa acggtcttca atttctcaag tttcagtttc 3060

atttttcttg ttctattaca acttttttta cttcttgttc attagaaaga aagcatagca 3120

atctaatcta agggcggtgt tgacaattaa tcatcggcat agtatatcgg catagtataa 3180

tacgacaagg tgaggaacta aaccatggcc aagttgacca gtgccgttcc ggtgctcacc 3240

gcgcgcgacg tcgccggagc ggtcgagttc tggaccgacc ggctcgggtt ctcccgggac 3300

ttcgtggagg acgacttcgc cggtgtggtc cgggacgacg tgaccctgtt catcagcgcg 3360

gtccaggacc aggtggtgcc ggacaacacc ctggcctggg tgtgggtgcg cggcctggac 3420

gagctgtacg ccgagtggtc ggaggtcgtg tccacgaact tccgggacgc ctccgggccg 3480

gccatgaccg agatcggcga gcagccgtgg gggcgggagt tcgccctgcg cgacccggcc 3540

ggcaactgcg tgcacttcgt ggccgaggag caggactgac acgtccgacg gcggcccacg 3600

ggtcccaggc ctcggagatc cgtccccctt ttcctttgtc gatatcatgt aattagttat 3660

gtcacgctta cattcacgcc ctccccccac atccgctcta accgaaaagg aaggagttag 3720

acaacctgaa gtctaggtcc ctatttattt ttttatagtt atgttagtat taagaacgtt 3780

atttatattt caaatttttc ttttttttct gtacagacgc gtgtacgcat gtaacattat 3840

actgaaaacc ttgcttgaga aggttttggg acgctcgaag gctttaattt gcaagctgga 3900

gaccaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct 3960

ggcatcgagc tccct 3975

<210> 4

<211> 2719

<212> DNA

<213>Artificial sequence

<220>

<223>

<400> 4

agtcagacga gggtaaggaa attacaaagg ttcttcaaga tctctcggat aaccagaaga 60

aagacggcca gattgtttta gatgaagaac agttggagaa aactttggaa atggccaaag 120

gtgacgcaca agaacctgaa gagaacgtgg acgataatga cgcagacaaa gagatcgatg 180

aaggtgtcga gcccgaggca gaatcatcgg ctcaatctca accggtcact gctttggaaa 240

aaagcagaac agagccatcg gaaacgcatt ccgaacctca agaaggccca gcgccaagct 300

ccgagttgaa tgtagaatca caaccccaaa cgaaattgga atccaagtcg gcttctaaag 360

aaataccatc gccaactcca gaacctgcta ccccatccac gccagcaagt ggagatgaag 420

aacgtgggag caagcgacgc ggatccgaaa gcgtcccccc tggttgtact aagcgcttta 480

atgctctttc tgcacaactt ctcacacaga tatcatccaa ccggtttgca tccatgttca 540

tgcaaccagt gaatgaaaac gaagagccca actactacaa gctaatacgg catcccgtgg 600

acattaagag tttgggtaaa gctattagga cgggtgaaat aaagtcattc gacgagttgg 660

aatttgagct tcaactgatg ttcagcaacg caattatgta caacgatatg aatcagacag 720

aaacttacaa atggacgatt gaaatgatgg aagaaacgca aaacttgttg agcctattcc 780

gggaatcatc cagcaattaa agctcattaa ttgccgcctc tcatctggtt catcaacatc 840

tggttctgtt cctggaatct taaaccaact ctttcggagt gcttgaggaa cttttcagcg 900

cacttgttca aacatgtttc ttctctagag ctaagtttgt tggaggtgaa gtcgttgacg 960

caatcattaa aacatctgtc caccaaactg gtgtagagct gcaatgttag ttttcagaag 1020

gcgttcactt actctcatga agtcattcat ttgcttctgc tccacaattt tctggaattc 1080

ttgctgttct ttatagttga gttgatccat ggtgtatcaa aaataaacta atgaattatc 1140

gaaaaatttt caacatttcc ctgaataata atcaacatgt ataaatctta tggagaaagg 1200

gcgaagtctc acccatctaa ggtcgccagc agactactta atttgatgga atccaagcaa 1260

acaaacctct gcgcttctgt ggatgtgact aaaactcagg aattattgga gcttcttgat 1320

aaactgggcc cttacatctg ccttgtcaaa actcatattg acatagtaga ggacttctct 1380

tatgaacaca ccattttacc attacaagga cttgcaaaga aacacaactt catgattttt 1440

gaagacagaa agtttgctga tataggaaac acagtcaaac tacagtataa gggaggaatt 1500

tatcgaacat ccaagtgggc cgatatcacg aatgcacacg gagtgactgg cgcaggaatt 1560

gttgaaggtc ttaaacaggc cgcagaagaa agtacagatg agccacgtgg gcttttgatg 1620

cttgctgagc tctcttcaaa gggatcatta gctaccggtg agtatactca aaaaactgtg 1680

gaaatagcga aaagcgataa agaatttgtc attggattta ttgcacagag agacatggga 1740

ggtcgtgagg aaggctttga ctggctgatc atgactccag gagttggttt agatgataaa 1800

ggtgattctc tgggccaaca gtacagaact gttgatgaag tgatgcaaac aggaaccgat 1860

gtcattatcg ttggaagagg tttattcgga aaaggaagag atcctgaagt ggaagggaag 1920

agatacagaa atgctgggtg ggaagcttac aagcggcgca ttgcttaacg gctttcagtt 1980

ctatatacat cgtcaaaatt gattttcgct aaaatgctga cgggatattt cgaatacgaa 2040

aagcccaata gaagtcgcgg gaatactagt gaaggacgat ctgaaaaagc ctctatagca 2100

gaagccaaag agggagaggt gagattttcg ccatcgttgt agaataatat caaatgtctc 2160

aatgtaagaa ttatagtaga gtttgaagaa gcgacgccga tccagttgat gtgcttcgtt 2220

gtagagatct agagattcga gtctagtcag gtggaccttt tgaattttat tgagtggata 2280

ttggattgca agcagtgtag ttgcggcgga agctccagca agaaggataa acgtcaaatt 2340

caaagctttg aaaaatcttg aattcttttc ccggttttct tcaccatgaa aaagccttga 2400

gcgccaatat tgataccact gtataactgc ttttgttttg tggaacccct ggtttttcac 2460

aagctcgaat actgaaaagt agaatgcaaa tccaaggctt tcctttatga aatttaaact 2520

aaagcctgca aatattccaa ctaaaccaat ttgttttagt ttgtataaag catactccca 2580

catgctcttc tgtttaccgc tgacaagctc tgagtaattc gatcgggcgt aaagtgcatc 2640

tagcggtgac gcagccacgc tagcagcagc tcccgcaaca aaaccagctt tgaaggtgtc 2700

gacagggcta ggatcatcg 2719

<210> 5

<211> 6683

<212> DNA

<213>Artificial sequence

<220>

<223>

<400> 5

agtcagacga gggtaaggaa attacaaagg ttcttcaaga tctctcggat aaccagaaga 60

aagacggcca gattgtttta gatgaagaac agttggagaa aactttggaa atggccaaag 120

gtgacgcaca agaacctgaa gagaacgtgg acgataatga cgcagacaaa gagatcgatg 180

aaggtgtcga gcccgaggca gaatcatcgg ctcaatctca accggtcact gctttggaaa 240

aaagcagaac agagccatcg gaaacgcatt ccgaacctca agaaggccca gcgccaagct 300

ccgagttgaa tgtagaatca caaccccaaa cgaaattgga atccaagtcg gcttctaaag 360

aaataccatc gccaactcca gaacctgcta ccccatccac gccagcaagt ggagatgaag 420

aacgtgggag caagcgacgc ggatccgaaa gcgtcccccc tggttgtact aagcgcttta 480

atgctctttc tgcacaactt ctcacacaga tatcatccaa ccggtttgca tccatgttca 540

tgcaaccagt gaatgaaaac gaagagccca actactacaa gctaatacgg catcccgtgg 600

acattaagag tttgggtaaa gctattagga cgggtgaaat aaagtcattc gacgagttgg 660

aatttgagct tcaactgatg ttcagcaacg caattatgta caacgatatg aatcagacag 720

aaacttacaa atggacgatt gaaatgatgg aagaaacgca aaacttgttg agcctattcc 780

gggaatcatc cagcaattaa agctcattaa ttgccgcctc tcatctggtt catcaacatc 840

tggttctgtt cctggaatct taaaccaact ctttcggagt gcttgaggaa cttttcagcg 900

cacttgttca aacatgtttc ttctctagag ctaagtttgt tggaggtgaa gtcgttgacg 960

caatcattaa aacatctgtc caccaaactg gtgtagagct gcaatgttag ttttcagaag 1020

gcgttcactt actctcatga agtcattcat ttgcttctgc tccacaattt tctggaattc 1080

ttgctgttct ttatagttga gttgatccat ggtgtatcaa aaataaacta atgaattatc 1140

gaaaaatttt caacatttcc ctgaataata atcaacatgt aataaatctt atggagaaag 1200

ggcgaagtct cacccatcta aggtcgccag cagactactt aatttgatgg aatccaagca 1260

aacaaacctc tgcgcttctg tggatgtgac taaaactcag gaattattgg agcttcttga 1320

taaactgggc ccttacatct gccttgtcaa aactcatatt gacatagtag aggacttctc 1380

ttatgaacac accattttac cattacaagg acttgcaaag aaacacaact tcatgatttt 1440

tgaagacaga aagtttgctg atataggaaa cacagtcaaa ctacagtata agggaggaat 1500

ttatcgaaca tccaagtggg ccgatatcac gaatgcacac ggagtgactg gcgcaggaat 1560

tgttgaaggt cttaaacagg ccgcagaaga aagtacagat gagccacgtg ggcttttgat 1620

gcttgctgag ctcatccaat tgtgacacgt ccgacggcgg cccacgggtc ccaggcctcg 1680

gagatccgtc ccccttttcc tttgtcgata tcatgtaatt agttatgtca cgcttacatt 1740

cacgccctcc ccccacatcc gctctaaccg aaaaggaagg agttagacaa cctgaagtct 1800

aggtccctat ttattttttt atagttatgt tagtattaag aacgttattt atatttcaaa 1860

tttttctttt ttttctgtac agacgcgtgt acgcatgtaa cattatactg aaaaccttgc 1920

ttgagaaggt tttgggacgc tcgaaggctt taatttgcaa gctggagacc aacatgtgag 1980

caaaaggcca gcaaaaggcc aggaaccgta aaaaggaagg gcaattctgc agatatccat 2040

cacactggcg gccgctcatg catgagatca gatcttcgac gcggagaacg atctcctcga 2100

gctgctcgcg gatcagcttg tggcccggta atggaaccag gccgacgcga cgctccttgc 2160

ggaccacggt ggctggcgag cccagtttgt gaacgaggtc gtttagaacg tcctgcgcaa 2220

agtccagtgt cagatgaatg tcctcctcgg accaattcag catgttctcg agcagccatc 2280

tgtctttgga gtagaagcgt aatctctgct cctcgttact gtaccggaag aggtagtttg 2340

cctcgccgcc cataatgaac aggttctctt tctggtggcc tgtgagcagc ggggacgtct 2400

ggacggcgtc gatgaggccc ttgaggcgct cgtagtactt gttccgtcgc tgtagccggc 2460

cgcggtgacg atacccacat agaggtcctt ggccattagt ttgatgaggt ggggcaggat 2520

gggcgactcg gcatcgaaat ttttgccgtc gtcgtacagt gtgatgtcac catcgaatgt 2580

aatgagctgc agcttgcgat ctcggatggt tttggaatgg aagaaccgcg acatctccaa 2640

cagctgggcc gtgttgagaa tgagccggac gtcgttgaac gagggggcca caagccggcg 2700

tttgctgatg gcgcggcgct cgtcctcgat gtagaaggcc ttttccagag gcagtctcgt 2760

gaagaagctg ccaacgctcg gaaccagctg cacgagccga gacaattcgg gggtgccggc 2820

tttggtcatt tcaatgttgt cgtcgatgag gagttcgagg tcgtggaaga tttccgcgta 2880

gcggcgtttt gcctcagagt ttaccatgag gtcgtccact gcagagatgc cgttgctctt 2940

caccgcgtac aggacgaacg gcgtggccag caggcccttg atccattcta tgaggccatc 3000

tcgacggtgt tccttgagtg cgtactccac tctgtagcga ctggacatct cgagactggg 3060

cttgctgtgc tggatgcacc aattaattgt tgccgcatgc atccttgcac cgcaagtttt 3120

taaaacccac tcgctttagc cgtcgcgtaa aacttgtgaa tctggcaact gagggggttc 3180

tgcagccgca accgaacttt tcgcttcgag gacgcagctg gatggtgtca tgtgaggctc 3240

tgtttgctgg ggtagcctac aacgtgacct tgcctaaccg gacggcgcta cccactgctg 3300

tctgtgcctg ctaccagaaa atcaccagag cagcagaggg ccgatgtggc aactggtggg 3360

gtgtcggaca ggctgtttct ccacagtgca aatgcgggtg aaccggccag aaagtaaatt 3420

cttatgctac cgtgcagcga ctccgacatc cccagttttt gccctacttg atcacagatg 3480

gggtcagcgc tgccgctaag tgtacccaac cgtccccaca cggtccatct ataaatactg 3540

ctgccagtgc acggtggtga catcaatcta aagtacaaaa acaaagctta ggaagtctgg 3600

taatggtaag ccgatacgta cccgatatgg gcgatctgat ttgggttgat tttgacccga 3660

caaaaggtag cgagcaagct ggacatcgtc cagctgttgt cctgagtcct ttcatgtaca 3720

acaacaaaac aggtatgtgt ctgtgtgttc cttgtacaac gcaatcaaaa ggatatccgt 3780

tcgaagttgt tttatccggt caggaacgtg atggcgtagc gttagctgat caggtaaaaa 3840

gtatcgcctg gcgggcaaga ggagcaacga agaaaggaac agttgcccca gaggaattac 3900

aactcattaa agccaaaatt aacgtactga ttgggtagtc tagaacaaaa actcatctca 3960

gaagaggatc tgaatagcgc cgtcgaccat catcatcatc atcattgagt tttagcctta 4020

gacatgactg ttcctcagtt caagttgggc acttacgaga agaccggtct tgctagattc 4080

taatcaagag gatgtcagaa tgccatttgc ctgagagatg caggcttcat ttttgatact 4140

tttttatttg taacctatat agtataggat tttttttgtc attttgtttc ttctcgtacg 4200

agcttgctcc tgatcagcct atctcgcagc tgatgaatat cttgtggtag gggtttggga 4260

aaatcattcg agtttgatgt ttttcttggt atttcccact cctcttcaga gtacagaaga 4320

ttaagtgaga ccttcgtttg tgcggatccc ccacacacca tagcttcaaa atgtttctac 4380

tcctttttta ctcttccaga ttttctcgga ctccgcgcat cgccgtacca cttcaaaaca 4440

cccaagcaca gcatactaaa ttttccctct ttcttcctct agggtgtcgt taattacccg 4500

tactaaaggt ttggaaaaga aaaaagagac cgcctcgttt ctttttcttc gtcgaaaaag 4560

gcaataaaaa tttttatcac gtttcttttt cttgaaattt ttttttttag tttttttctc 4620

tttcagtgac ctccattgat atttaagtta ataaacggtc ttcaatttct caagtttcag 4680

tttcattttt cttgttctat tacaactttt tttacttctt gttcattaga aagaaagcat 4740

agcaatctaa tctaagggcg gtgttgacaa ttaatcatcg gcatagtata tcggcatagt 4800

ataatacgac aaggtgagga actaaaccat ggccaagttg accagtgccg ttccggtgct 4860

caccgcgcgc gacgtcgccg gagcggtcga gttctggacc gaccggctcg ggttctcccg 4920

ggacttcgtg gaggacgact tcgccggtgt ggtccgggac gacgtgaccc tgttcatcag 4980

cgcggtccag gaccaggtgg tgccggacaa caccctggcc tgggtgtggg tgcgcggcct 5040

ggacgagctg tacgccgagt ggtcggaggt cgtgtccacg aacttccggg acgcctccgg 5100

gccggccatg accgagatcg gcgagcagcc gtgggggcgg gagttcgccc tgcgcgaccc 5160

ggccggcaac tgcgtgcact tcgtggccga ggagcaggac tgacacgtcc gacggcggcc 5220

cacgggtccc aggcctcgga gatccgtccc ccttttcctt tgtcgatatc atgtaattag 5280

ttatgtcacg cttacattca cgccctcccc ccacatccgc tctaaccgaa aaggaaggag 5340

ttagacaacc tgaagtctag gtccctattt atttttttat agttatgtta gtattaagaa 5400

cgttatttat atttcaaatt tttctttttt ttctgtacag acgcgtgtac gcatgtaaca 5460

ttatactgaa aaccttgctt gagaaggttt tgggacgctc gaaggcttta atttgcaagc 5520

tggagaccaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt 5580

tgctggcatc gagctctctt caaagggatc attagctacc ggtgagtata ctcaaaaaac 5640

tgtggaaata gcgaaaagcg ataaagaatt tgtcattgga tttattgcac agagagacat 5700

gggaggtcgt gaggaaggct ttgactggct gatcatgact ccaggagttg gtttagatga 5760

taaaggtgat tctctgggcc aacagtacag aactgttgat gaagtgatgc aaacaggaac 5820

cgatgtcatt atcgttggaa gaggtttatt cggaaaagga agagatcctg aagtggaagg 5880

gaagagatac agaaatgctg ggtgggaagc ttacaagcgg cgcattgctt aacggctttc 5940

agttctatat acatcgtcaa aattgatttt cgctaaaatg ctgacgggat atttcgaata 6000

cgaaaagccc aatagaagtc gcgggaatac tagtgaagga cgatctgaaa aagcctctat 6060

agcagaagcc aaagagggag aggtgagatt ttcgccatcg ttgtagaata atatcaaatg 6120

tctcaatgta agaattatag tagagtttga agaagcgacg ccgatccagt tgatgtgctt 6180

cgttgtagag atctagagat tcgagtctag tcaggtggac cttttgaatt ttattgagtg 6240

gatattggat tgcaagcagt gtagttgcgg cggaagctcc agcaagaagg ataaacgtca 6300

aattcaaagc tttgaaaaat cttgaattct tttcccggtt ttcttcacca tgaaaaagcc 6360

ttgagcgcca atattgatac cactgtataa ctgcttttgt tttgtggaac ccctggtttt 6420

tcacaagctc gaatactgaa aagtagaatg caaatccaag gctttccttt atgaaattta 6480

aactaaagcc tgcaaatatt ccaactaaac caatttgttt tagtttgtat aaagcatact 6540

cccacatgct cttctgttta ccgctgacaa gctctgagta attcgatcgg gcgtaaagtg 6600

catctagcgg tgacgcagcc acgctagcag cagctcccgc aacaaaacca gctttgaagg 6660

tgtcgacagg gctaggatca tcg 6683

<210> 6

<211> 991

<212> DNA

<213>Artificial sequence

<220>

<223>

<400> 6

gtccaccaaa ctggtgtaga gctgcaatgt tagttttcag aaggcgttca cttactctca 60

tgaagtcatt catttgcttc tgctccacaa ttttctggaa ttcttgctgt tctttatagt 120

tgagttgatc catggtgtat caaaaataaa ctaatgaatt atcgaaaaat tttcaacatt 180

tccctgaata ataatcaaca tgtataaatc ttatggagaa agggcgaagt ctcacccatc 240

taaggtcgcc agcagactac ttaatttgat ggaatccaag caaacaaacc tctgcgcttc 300

tgtggatgtg actaaaactc aggaattatt ggagcttctt gataaactgg gcccttacat 360

ctgccttgtc aaaactcata ttgacatagt agaggacttc tcttatgaac acaccatttt 420

accattacaa ggacttgcaa agaaacacaa cttcatgatt tttgaagaca gaaagtttgc 480

tgatatagga aacacagtca aactacagta taagggagga atttatcgaa catccaagtg 540

ggccgatatc acgaatgcac acggagtgac tggcgcagga attgttgaag gtcttaaaca 600

ggccgcagaa gaaagtacag atgagccacg tgggcttttg atgcttgctg agctctcttc 660

aaagggatca ttagctaccg gtgagtatac tcaaaaaact gtggaaatag cgaaaagcga 720

taaagaattt gtcattggat ttattgcaca gagagacatg ggaggtcgtg aggaaggctt 780

tgactggctg atcatgactc caggagttgg tttagatgat aaaggtgatt ctctgggcca 840

acagtacaga actgttgatg aagtgatgca aacaggaacc gatgtcatta tcgttggaag 900

aggtttattc ggaaaaggaa gagatcctga agtggaaggg aagagataca gaaatgctgg 960

gtgggaagct tacaagcggc gcattgctta a 991

<210> 7

<211> 1397

<212> DNA

<213>Artificial sequence

<220>

<223>

<400> 7

gtccaccaaa ctggtgtaga gctgcaatgt tagttttcag aaggcgttca cttactctca 60

tgaagtcatt catttgcttc tgctccacaa ttttctggaa ttcttgctgt tctttatagt 120

tgagttgatc catggtgtat caaaaataaa ctaatgaatt atcgaaaaat tttcaacatt 180

tccctgaata ataatcaaca tgtaataaat cttatggaga aagggcgaag tctcacccat 240

ctaaggtcgc cagcagacta cttaatttga tggaatccaa gcaaacaaac ctctgcgctt 300

ctgtggatgt gactaaaact caggaattat tggagcttct tgataaactg ggcccttaca 360

tctgccttgt caaaactcat attgacatag tagaggactt ctcttatgaa cacaccattt 420

taccattaca aggacttgca aagaaacaca acttcatgat ttttgaagac agaaagtttg 480

ctgatatagg aaacacagtc aaactacagt ataagggagg aatttatcga acatccaagt 540

gggccgatat cacgaatgca cacggagtga ctggcgcagg aattgttgaa ggtcttaaac 600

aggccgcaga agaaagtaca gatgagccac gtgggctttt gatgcttgct gagctcatcc 660

aattgtgaca cgtccgacgg cggcccacgg gtcccaggcc tcggagatcc gtcccccttt 720

tcctttgtcg atatcatgta attagttatg tcacgcttac attcacgccc tccccccaca 780

tccgctctaa ccgaaaagga aggagttaga caacctgaag tctaggtccc tatttatttt 840

tttatagtta tgttagtatt aagaacgtta tttatatttc aaatttttct tttttttctg 900

tacagacgcg tgtacgcatg taacattata ctgaaaacct tgcttgagaa ggttttggga 960

cgctcgaagg ctttaatttg caagctggag accaacatgt gagcaaaagg ccagcaaaag 1020

gccaggaacc gtaaaaaggc cgcgttgctg gcatcgagct ctcttcaaag ggatcattag 1080

ctaccggtga gtatactcaa aaaactgtgg aaatagcgaa aagcgataaa gaatttgtca 1140

ttggatttat tgcacagaga gacatgggag gtcgtgagga aggctttgac tggctgatca 1200

tgactccagg agttggttta gatgataaag gtgattctct gggccaacag tacagaactg 1260

ttgatgaagt gatgcaaaca ggaaccgatg tcattatcgt tggaagaggt ttattcggaa 1320

aaggaagaga tcctgaagtg gaagggaaga gatacagaaa tgctgggtgg gaagcttaca 1380

agcggcgcat tgcttaa 1397

<210> 8

<211> 4033

<212> DNA

<213>Artificial sequence

<220>

<223>

<400> 8

tcttcgacgc ggagaacgat ctcctcgagc tgctcgcgga tcagcttgtg gcccggtaat 60

ggaaccaggc cgacgcgacg ctccttgcgg accacggtgg ctggcgagcc cagtttgtga 120

acgaggtcgt ttagaacgtc ctgcgcaaag tccagtgtca gatgaatgtc ctcctcggac 180

caattcagca tgttctcgag cagccatctg tctttggagt agaagcgtaa tctctgctcc 240

tcgttactgt accggaagag gtagtttgcc tcgccgccca taatgaacag gttctctttc 300

tggtggcctg tgagcagcgg ggacgtctgg acggcgtcga tgaggccctt gaggcgctcg 360

tagtacttgt tccgtcgctg tagccggccg cggtgacgat acccacatag aggtccttgg 420

ccattagttt gatgaggtgg ggcaggatgg gcgactcggc atcgaaattt ttgccgtcgt 480

cgtacagtgt gatgtcacca tcgaatgtaa tgagctgcag cttgcgatct cggatggttt 540

tggaatggaa gaaccgcgac atctccaaca gctgggccgt gttgagaatg agccggacgt 600

cgttgaacga gggggccaca agccggcgtt tgctgatggc gcggcgctcg tcctcgatgt 660

agaaggcctt ttccagaggc agtctcgtga agaagctgcc aacgctcgga accagctgca 720

cgagccgaga caattcgggg gtgccggctt tggtcatttc aatgttgtcg tcgatgagga 780

gttcgaggtc gtggaagatt tccgcgtagc ggcgttttgc ctcagagttt accatgaggt 840

cgtccactgc agagatgccg ttgctcttca ccgcgtacag gacgaacggc gtggccagca 900

ggcccttgat ccattctatg aggccatctc gacggtgttc cttgagtgcg tactccactc 960

tgtagcgact ggacatctcg agactgggct tgctgtgctg gatgcaccaa ttaattgttg 1020

ccgcatgcat ccttgcaccg caagttttta aaacccactc gctttagccg tcgcgtaaaa 1080

cttgtgaatc tggcaactga gggggttctg cagccgcaac cgaacttttc gcttcgagga 1140

cgcagctgga tggtgtcatg tgaggctctg tttgctgggg tagcctacaa cgtgaccttg 1200

cctaaccgga cggcgctacc cactgctgtc tgtgcctgct accagaaaat caccagagca 1260

gcagagggcc gatgtggcaa ctggtggggt gtcggacagg ctgtttctcc acagtgcaaa 1320

tgcgggtgaa ccggccagaa agtaaattct tatgctaccg tgcagcgact ccgacatccc 1380

cagtttttgc cctacttgat cacagatggg gtcagcgctg ccgctaagtg tacccaaccg 1440

tccccacacg gtccatctat aaatactgct gccagtgcac ggtggtgaca tcaatctaaa 1500

gtacaaaaac aaagcttcga aacgatgaga tttccttcaa tttttactgc tgttttattc 1560

gcagcatcct ccgcattagc tgctccagtc aacactacaa cagaagatga aacggcacaa 1620

attccggctg aagctgtcat cggttactca gatttagaag gggatttcga tgttgctgtt 1680

ttgccatttt ccaacagcac aaataacggg ttattgttta taaatactac tattgccagc 1740

attgctgcta aagaagaagg ggtatctctc gagaaaagag aggctgaagc tgaattcatg 1800

acattaatct acgtgccttc aatatttaca atggtcccct caatcacacg gattgtactg 1860

gttaacattc tgttggcgac gttggttttg ggagctgcag tccttccacg agacaacaga 1920

actgtttgcg ggagtcaact ctgcacatgg tggcacgact ccggcgagat aaacaccggt 1980

actcctgtac aggcaggaaa cgttcgacaa tcccgaaagt actctgtcca tgtgagcctg 2040

gcagaccgta accaattcta cgactctttc gtatatgaat cgatacctag gaacggcaat 2100

ggcagaattt attctcccac cgacccacct aacagcaata cattgaatag tagcattgac 2160

gacggtatat caatcgaacc atctctcggc atcaacatgg cttggtccca gttcgaatat 2220

agacgagatg tcgacattaa gattactaca atcgatggct caatattgga tggccctttg 2280

gacattgtta ttcggccgac ttctgttaag tactcagtca aaagatgtgt gggtggtatc 2340

attattagag tcccttatga tcccaatggt cgaaaattct ctgttgagtt aaagagtgac 2400

ctttacagtt acctctccga cggttcgcaa tatgtgacct ctggagggag cgtggttggt 2460

gtggagccaa aaaatgccct ggtgatcttt gccagccctt tcttgccacg ggatatggtt 2520

cctcatatga caccacacga cacccagaca atgaagccgg gcccaatcaa taatggggac 2580

tggggttcaa agcctatact ctacttcccg cctggcgtat actggatgaa cgaggatacc 2640

tctggtaacc ccgggaagct cggctcaaat catatgcggc tggatcccaa tacctactgg 2700

gtccatctag ccccaggagc ctatgtgaaa ggagccattg agtatttcac gaagcaaaat 2760

ttctatgcaa cgggtcatgg cgttctctca ggtgagaact atgtttatca ggccaatgca 2820

gctgataact actatgccgt caagagtgat ggcacaagct tgagaatgtg gtggcacaac 2880

aaccttggag gcggtcaaac atggttttgc atggggccca ccattaatgc accgccgttt 2940

aatacgatgg acttcaacgg aaactctaat atttccagcc ggattagtga ctataagcag 3000

gttggcgctt attttttcca aacagacgga ccggagatct acgaggacag tgttgtccat 3060

gacgtcttct ggcatgttaa tgatgatgcc atcaagacat attattccgg agcttcaatt 3120

tcacgagcaa ccatctggag gtgtcacaat gacccgatca tacagatggg ctggacgtca 3180

cgaaatctca ccggaatcag cattgataac ctgcacgtca tccacacgag atatttcaaa 3240

tctgaaacag tggttccttc agcaatcatt ggagcgtctc cattctacgc aagtggaatg 3300

actgttgatc ccagcgagtc catcagcatg accatctcta acgtggtgtg tgagggtcta 3360

tgcccctcac tgttccgtat cactccgctt cagagctaca acaaccttgt tgtcaagaac 3420

gtggcctttc ccgatggact gcagacaaat ccaatcggaa taggagagag cattatacca 3480

gcagcttccg gctgtacaat ggacttggaa atcacaaact ggaccgtcaa aggacaaaaa 3540

gtcaccatgc aaaactttca gtccgggtca cttggccagt tcgatatcga tggttcatac 3600

tggggtcaat ggtccataaa ctaatctaga acaaaaactc atctcagaag aggatctgaa 3660

tagcgccgtc gaccatcatc atcatcatca ttgagtttgt agccttagac atgactgttc 3720

ctcagttcaa gttgggcact tacgagaaga ccggtcttgc tagattctaa tcaagaggat 3780

gtcagaatgc catttgcctg agagatgcag gcttcatttt tgatactttt ttatttgtaa 3840

cctatatagt ataggatttt ttttgtcatt ttgtttcttc tcgtacgagc ttgctcctga 3900

tcagcctatc tcgcagctga tgaatatctt gtggtagggg tttgggaaaa tcattcgagt 3960

ttgatgtttt tcttggtatt tcccactcct cttcagagta cagaagatta agtgagacct 4020

tcgtttgtgc gga 4033

Claims (10)

1. the preparation method of uracil auxotrophy multiple-shaped nuohan inferior yeast, including：Knock out in multiple-shaped nuohan inferior yeast starting strain Gene or gene in uracil route of synthesis is reduced described in the multiple-shaped nuohan inferior yeast starting strain in uracil route of synthesis The content of encoding proteins matter makes described in the multiple-shaped nuohan inferior yeast starting strain gene code egg in uracil route of synthesis White matter inactivates, and obtains uracil auxotrophy multiple-shaped nuohan inferior yeast；The multiple-shaped nuohan inferior yeast starting strain contains the urine Gene in pyrimidine synthesis pathway.

2. according to the method for claim 1, it is characterised in that：The multiple-shaped nuohan inferior yeast starting strain is the inferior ferment of the multiform Chinese Mother (Hansenula polymorpha) HP20110424, it is in China Committee for Culture Collection of Microorganisms's common micro-organisms The preserving number at center is CGMCC No.7.89.

3. method according to claim 1 or 2, it is characterised in that：In the uracil route of synthesis gene be orotidine- 5 '-phosphate decarboxylase gene.

4. according to the method for claim 3, it is characterised in that：The orotidine -5 '-phosphate decarboxylase gene is as follows A1) or A2) or A3) or A4)：

A1) coded sequence is the DNA molecular of sequence 1 in sequence table；

A2) coded sequence is the DNA molecular of the 634-1425 positions of sequence 1 in sequence table；

A3) and A1) or A2) nucleotide sequence that limits has 75% or more than 75% homogeneity, and the coding orotidine- The DNA molecular of 5 '-phosphate decarboxylase；

A4) under strict conditions with A1) or the A2) nucleotide sequence hybridization that limits, and encode orotidine -5 '-phosphoric acid and take off The DNA molecular of carboxylic acid；

And/or

Gene is following M1 in uracil route of synthesis in the knockout multiple-shaped nuohan inferior yeast starting strain) and/or M2)：

M1 the coding of gene in the uracil route of synthesis) is made to terminate in advance；

M2) exogenous dna fragment is inserted in gene in the uracil route of synthesis.

5. according to the method for claim 4, it is characterised in that：M1) by the multiple-shaped nuohan inferior yeast starting strain institute The 5-6 interdigits insertion adenylic acid for stating orotidine -5 '-phosphate decarboxylase encoding gene coded sequence is realized；

And/or the restriction enzyme that the insertion position of the exogenous dna fragment is gene in the uracil route of synthesis is known Between other sequence.

6. the uracil auxotrophy multiple-shaped nuohan inferior yeast that in claim 1-5 prepared by any described method.

7. the method for express express target protein, including：The expression vector of encoding gene containing destination protein is imported into claim 6 In described uracil auxotrophy multiple-shaped nuohan inferior yeast, obtain recombinating multiple-shaped nuohan inferior yeast, realize the destination protein Expression.

8. according to the method for claim 7, it is characterised in that：The expression vector contains any institute in claim 1-4 State gene in uracil route of synthesis.

9. application of the uracil auxotrophy multiple-shaped nuohan inferior yeast in marking protein described in claim 6.

10. it is following 1) or 2)：

1) reagent set, as in the uracil auxotrophy multiple-shaped nuohan inferior yeast described in claim 6 and claim 8 or 9 Described expression vector composition；

2) multiple-shaped nuohan inferior yeast (Hansenula polymorpha) HP20110424, it is in Chinese microorganism strain preservation management The preserving number of committee's common micro-organisms center is CGMCC No.7.89.