CN108728477A - A kind of efficient Transpositional mutation system and construction method - Google Patents

A kind of efficient Transpositional mutation system and construction method Download PDF

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CN108728477A
CN108728477A CN201710270598.XA CN201710270598A CN108728477A CN 108728477 A CN108728477 A CN 108728477A CN 201710270598 A CN201710270598 A CN 201710270598A CN 108728477 A CN108728477 A CN 108728477A
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plasmid
gene
transpositional
promoter
mutation system
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CN108728477B (en
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周勉
朱锦祥
朱巧云
张元兴
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East China University of Science and Technology
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Abstract

The present invention relates to a kind of efficient Transpositional mutation system and construction methods.The present invention construct one kind can efficiently concentrating Transposon mutant, and between mutant strain a kind of repeated low double-mass model Transpositional mutation system and removable swivel base enzyme coding gene simple substance grain Transpositional mutation system.The transposon mutant system that the present invention obtains, which can screen, obtains phenotypic mutation strain, later, the genotype of mutant strain can be quickly determined by chromosome walking technology.

Description

A kind of efficient Transpositional mutation system and construction method
Technical field
The invention belongs to molecular biology field, more particularly it relates to a kind of efficient Transpositional mutation system and Construction method.
Background technology
Technology applied to yeast random mutation mainly has mutagenesis and ultraviolet mutagenesis, these traditional method of mutagenesis sieves Obtained mutant strain genotype is selected to be difficult to be identified.And transposon mutant label technique is a kind of according to transposons radom insertion The characteristic of mutagenesis and the method for mutagenesis of separable unknown gene to grow up.However the existing swivel base in yeast The problems such as subsystem generally existing transposition efficiency is low, insertion mutation randomness is not strong, operation is relatively complicated so that in yeast Transposon mutant system cannot get universal application, screening mutant strain and the work progress for studying corresponding gene function are slow Slowly.
Transposons is one section of moveable DNA fragmentation, it can jump to another position from a position of chromosome or plasmid It sets.When external source transposon integration enter certain unknown gene promoter region or coding region, so that gene mutation is inactivated and is generated A kind of new mutant phenotype, carrys out the gene of clonal anergy further according to the known array designed on transposons at this time, and in this, as Probe, clone obtains originally non-deactivated gene in wild type, to achieve the purpose that identify the unknown gene.Transposons is prominent Become the effective ways that label technique is decision-orientated study gene, and carries out the assignment of genes gene mapping and its functional study, especially one at present The prefered method of serial controlling gene research.The design basic principle of transposon tagging is：Only retain on transposons and completes swivel base The specific fragment of process, while selection markers (such as resistant gene) and reporter is added.The technology have quickly, it is accurate and The characteristics of with a certain phenotype correlation gene, is found in scale.
The genome sequence of Pichia pastoris has been disclosed, and is predicted out a gene more than 5000.It is wherein most Gene does not obtain experimental verification, and is noted as assuming that the gene of albumen just has more than 1600.For ease of quick, large area Genes with unknown function is studied on ground, and efficient swivel base insertion mutation system is established in Pichia pastoris will be with very high practical valence Value.
Invention content
The purpose of the present invention is to provide a kind of efficient Transpositional mutation system and construction methods.
In the first aspect of the present invention, a kind of double-mass model Transpositional mutation system is provided, using yeast cells as host cell, packet It includes：
(1) transposase expression cassette is integrated on the genome of yeast cells；With
(2) plasmid being free in yeast cells, the plasmid include：Transposable element；And the plasmid does not include yeast and can know Other replication site sequence.
In a preference, the transposase expression cassette includes (5 ' → 3 ')：Promoter, swivel base enzyme coding gene and Terminator；Preferably, the promoter is selected from：Induction type checks type promoter.
In another preferred example, the promoter is to check type promoter, including but not limited to selected from the group below to open Mover：Thiamine checks type promoter PpTHI11p or methionine checks type promoter PpMET3p；Or the promoter is Inducible promoter, including but not limited to promoter：Methanol inducible promoters PpAOX1p.
In another preferred example, the thiamine checks the nucleotide sequence such as SEQ ID of type promoter PpTHI11p NO:Shown in 5.
In another preferred example, the methionine checks the nucleotide sequence such as SEQ ID of type promoter PpMET3p NO:Shown in 6.
In another preferred example, the nucleotide sequence of the methanol inducible promoters PpAOX1p such as SEQ ID NO: Shown in 14.
In another preferred example, the transposase includes but is not limited to transposase selected from the group below：TcBaseCO V596A transposases, SBase100X transposases, piggyBac transposases.
In another preferred example, the amino acid sequence of the transposase such as SEQ ID NO:1 or SEQ ID NO:2 institutes Show；Or the nucleotide sequence of the encoding gene of the transposase such as SEQ ID NO:3 or SEQ ID NO:Shown in 4.
In another preferred example, the transposable element includes the transposon ends repeat region being operatively connected and screening Marker gene sequence.
In another preferred example, the transposon ends repeat region includes but is not limited to transposons selected from the group below Terminal repeat domain：(being operatively connected) TcBL/TcBR, (being operatively connected) SBL/SBR；Or the selection markers packet Include (but not limited to) selection markers selected from the group below：HIS4, blasticidin resistance gene ZeocinR, G-418 resistant genes kanR。
In another preferred example, the TcBL is 5 ' end inverted repeat region of TcBuster transposons；Preferably, its Nucleotide sequence such as SEQ ID NO:Shown in 7.
In another preferred example, the TcBR is 3 ' inverted terminal repeat sequence of TcBuster transposons；Preferably, its Nucleotide sequence such as SEQ ID NO:Shown in 8.
In another preferred example, the SBL is 5 ' end inverted repeat region of Sleeping beauty transposons；Compared with Goodly, nucleotide sequence such as SEQ ID NO:Shown in 9.
In another preferred example, the SBR is 3 ' end inverted repeat region of Sleeping beauty transposons；Compared with Goodly, nucleotide sequence such as SEQ ID NO:Shown in 10.
In another preferred example, the nucleotide sequence of the expression cassette of the HIS4 such as SEQ ID NO:Shown in 11.
In another aspect of this invention, the kit for building the double-mass model Transpositional mutation system, the examination are provided Agent box includes：(1) plasmid is helped, including swivel base enzyme coding gene, is integrated on the genome of yeast cells；(2) Free plasmid, the plasmid include：Transposable element；And the plasmid does not include the identifiable replication site sequence of yeast.
In another preferred example, in the kit further include the one or more of the following group：For plasmid linearization Enzyme, conversion reagent, yeast cells culture medium, recon indentifying substance, operation instructions.
In another aspect of this invention, the purposes that any double-mass model Transpositional mutation system in front is provided, for into Row gene mutation obtains the gene of mutation.
In another aspect of this invention, a kind of simple substance grain Transpositional mutation system is provided, using yeast cells as host cell, packet It includes：The plasmid being free in yeast cells, the plasmid include to be operatively connected：Transposase expression cassette, transposable element, virulence egg White expression cassette；And the plasmid includes yeast replication element.
In a preference, the amino acid sequence such as SEQ ID NO of the transposase:1 or SEQ ID NO:2 institutes Show；Or the nucleotide sequence of the encoding gene of the transposase such as SEQ ID NO:3 or SEQ ID NO:Shown in 4.
In another preferred example, the transposable element includes the transposon ends repeat region being operatively connected and screening Marker gene sequence；Preferably, the transposon ends repeat region includes but is not limited to transposons end selected from the group below Hold repeat region：(being operatively connected) TcBL/TcBR, (being operatively connected) SBL/SBR；Preferably, the screening mark Note includes but is not limited to selection markers selected from the group below：HIS4, blasticidin resistance gene ZeocinR, G-418 resistant genes kanR。
In another preferred example, the virulence protein includes but is not limited to：mazF.
In another preferred example, the nucleotide sequence of the mazF such as SEQ ID NO:Shown in 13.
In another preferred example, the virulence protein is mazF, and the virulence protein expression cassette includes strong induction type Promoter；Preferably, the strong inducible promoter is PpAOX1p promoters.
In another preferred example, the nucleotide sequence of the PpAOX1p promoters such as SEQ ID NO:Shown in 14.
In another preferred example, the reproduction element is PARS2 reproduction elements.
In another preferred example, the nucleotide sequence of the PARS2 reproduction elements such as SEQ ID NO:Shown in 12.
In another aspect of this invention, the kit for building the simple substance grain Transpositional mutation system, the examination are provided Agent box includes：Plasmid, the plasmid include to be operatively connected：Transposase expression cassette, transposable element, virulence protein expression cassette； And the plasmid includes yeast replication element.
In another preferred example, further include the following group in the kit for building simple substance grain Transpositional mutation system It is one or more：Conversion reagent, yeast cells culture medium, recon indentifying substance, operation instructions.
In another aspect of this invention, the purposes that any simple substance grain Transpositional mutation system in front is provided, for into Row gene mutation obtains the gene of mutation.
In a preference, the yeast cells includes but is not limited to yeast cells selected from the group below：Finish red ferment Mother cell, brewing yeast cell, candida cell, Hansenula yeast cell.
The other aspects of the present invention are apparent to those skilled in the art due to this disclosure 's.
Description of the drawings
Fig. 1, pBRZeo-TTcBaseCOV596A carrier figure.
Fig. 2, pBRZeo-MSBase100X carrier figure.
Fig. 3, pBRAmp-TcBHis carrier figure.
Fig. 4, pBRAmp-SBHis carrier figure.
Fig. 5, pTTcBHis4AmazFARS carrier figure.
Fig. 6, pMSBHis4AmazFARS carrier figure.
Fig. 7, strain is covered with histidine defect culture medium YND (His-) screening HIS4 genes.A, it is integrating and is expressing In the bacterial strain of TcBaseCO V596A transposases, the covering strain screened after pBRAmp-His plasmids is converted far less than conversion The covering strain screened after pBRAmp-TcBHis plasmids.B, in integrating and expressing the bacterial strain of SBase100X transposases, The covering strain screened also is considerably less than after conversion pBRAmp-TcBHis plasmids to screen and obtain after conversion pBRAmp-His plasmids Covering strain.
Two kinds of different Transposon mutants that Fig. 8, hiTail PCR amplification double-mass model system are collected.
A, the agarose gel electrophoresis figure run after the hiTail PCR of TcBuster Transposon mutants；
B, the agarose gel electrophoresis figure run after the hiTail PCR of Sleeping beauty Transposon mutants.
Fig. 9, GS115, GS115+1/100 GS115-GFP-His, GS115+1/10000 GS115-GFP-His and 1/ The growth curve of this four groups of bacterial strains of 10000 GS115-GFP-His.
Figure 10, flow cytomery expression quantity GFP reporter proteins bacterial strain and do not express the bacterial strain content of GFP albumen. When figure is 12 hours, 36 hours, 82 hours and 100 hours, GS115, GS115+1/100 GS115-GFP-His, GS115+1/ This four groups of bacterial strain composition detections of 10000 GS115-GFP-His and 1/10000 GS115-GFP-His.
In Figure 11, quantitative fluorescent PCR, the DNA of this DNA in four of ACT1, SBL, SBR and SBasecopy~Ct standard curves.
Specific implementation mode
For the present inventor by a large amount of experiment and in-depth study, it based on yeast cells is host cell to construct a kind of Transpositional mutation system.The system is more specifically divided into double-mass model Transpositional mutation system and plasmid Transpositional mutation system.
An aspect of of the present present invention be constructed in yeast can efficiently concentrating Transposon mutant system, can by the system A large amount of Transposon mutant is simply and easily collected, this is real to the experiment of conditional filtering functional gene and transposons high-flux sequence It tests and all brings great convenience and help.
It is another aspect of the invention to provide the methods of structure Pichia pastoris Transposon mutant.The present invention passes through cleverly Experimental design not only finds that TcBuster and Sleeping beauty transposons is active in Pichia pastoris, also into one for the first time Step demonstrates integration target site of both transposons in Pichia pastoris, in addition also provides two kinds by the optimization of method The different transposon mutant system of feature is double-mass model system and single pUC pUC respectively.
Term
As used herein, " promoter " refers to a kind of nucleic acid sequence, is typically found in target gene code sequence The upstream (5 ' end) of row, can guide nucleic acid sequence to be transcribed into mRNA.Usually, promoter or promoter region provide RNA polymerizations The recognition site of other factors necessary to enzyme and correct starting transcription.Herein, the promoter or promoter region packet The active variants of promoter are included, which can be the variant that the allelic variant naturally occurred or non-natural occur. The variant includes substitution variants, Deletion variants and insert variation.
As used herein, " inducible promoter " can be as needed in specific cells growth phase or particular growth Under environment, "on" and "off" or "high" and " low " of rapid induction genetic transcription.It, can be by inducible promoter point according to source For naturally occurring promoter and artificial constructed promoter.
As used herein, described " checking type promoter " is a kind of promoter, generally in rich medium (such as YPD culture mediums) it is opened when being removed in culture medium or ruing out of corresponding repressor in checking state (or holddown) Mover will be activated." checking type promoter " can also sometimes be considered as the one kind of " inducible promoter ".
As used herein, " expression cassette " refer to include expression desired polypeptides (in the present invention for allogenic polypeptide or Mit1 polypeptides) needed for all necessary components gene expression system, usually it includes element：Promoter, coding polypeptide Gene order, terminator；Additionally alternative is including signal coding sequence etc..These elements are operatively connected.
As used herein, described " being operatively connected " or " operability be connected " refer to two or more nucleic acid regions or Functional space arrangement of nucleic acid sequence.Such as：Promoter region is placed in the certain bits relative to target gene nucleic acid sequence It sets so that the transcription of nucleic acid sequence is guided by the promoter region, to which promoter region is " operably connected " In the nucleic acid sequence.
As used herein, " element " refers to a series of useful functional nucleic acid of some expression for albumen Sequence, in of the invention, " element " is systematically built to form a kind of nucleic acid construct.The sequence of " element " Can also include their variant those of provided in the present invention, as long as these variants substantially remain the " member The function of part ".
As used herein, " yeast cells " includes：Pichia pastoris, brewing yeast cell, Candida, the Chinese Inferior yeast.
Double-mass model Transpositional mutation system
The present invention provides a kind of double-mass model Transpositional mutation system, which is that host is thin with yeast cells Born of the same parents, including：(1) transposase expression cassette is integrated on the genome of yeast cells；(2) matter being free in yeast cells Grain, the plasmid include：Transposable element；And the plasmid does not include the identifiable replication site sequence of yeast.
The characteristics of double-mass model Transpositional mutation system of the present invention, is, transposase coding base is incorporated on Yeast genome Cause；In addition, there is also a kind of cricoid donor plasmids to be free in yeast, which, which contains transposable element but do not include, ferment Female identifiable replication site sequence.
As the preferred embodiment of the present invention, the transposase expression cassette includes (5 ' → 3 ')：Promoter, transposase coding Gene and terminator；Preferably, the promoter is selected from：Induction type checks type promoter.
Prompt according to the present invention, those skilled in the art can choose suitable induction type or check type promoter, Therefore the promoter those of is not limited to illustrate in the embodiment of the present invention, other induction types with similar functions or checks Type promoter can also be included.As the present invention preferred embodiment, the promoter be check type promoter, including but It is not limited to promoter selected from the group below：Thiamine checks type promoter PpTHI11p or methionine checks type promoter PpMET3p.Some other available inducible promoter includes but not limited to promoter selected from the group below：Methanol evoked startup Sub- PpAOX1p.
As the preferred embodiment of the present invention, in double-mass model system of the present invention, the Transposon System can select From：TcBuster Transposon Systems or Sleeping beauty Transposon Systems.It is highly preferred that corresponding to TcBuster swivel bases Subsystem, using TcBaseCOV596A transposases；Corresponding to Sleeping beauty Transposon Systems, using SBase100X Transposase.TcBaseCOV596A and SBase100X is height optimization, the transposase with height transposition activity.Described turns The seat variant of enzyme, homologue or the like, as long as can retain transposase activity, can also be applied in the present invention.
The transposable element includes the transposon ends repeat region being operatively connected and riddled basins sequence.Make For the preferred embodiment of the present invention, the transposon ends repeat region includes but not limited to transposon ends weight selected from the group below Multiple region：The TcBL/TcBR being operatively connected, the SBL/SBR being operatively connected.
Selection markers can be selected according to actually required, it is however generally that, those skilled in the art are easily obtained properly Selection markers, therefore selection markers those of are not limited to specifically to enumerate in the present invention.As the present invention preferred embodiment, The selection markers include but not limited to selection markers selected from the group below：HIS4, blasticidin resistance gene ZeocinR, G- 418 resistant gene kanR。
The present invention also provides the kit for building the double-mass model Transpositional mutation system, wrapped in the kit It includes：(1) plasmid is helped, including swivel base enzyme coding gene, is integrated on the genome of yeast cells；(2) free plasmid, The plasmid includes：Transposable element；And the plasmid does not include the identifiable replication site sequence of yeast.In the kit also It may include：For the enzyme of plasmid linearization, conversion reagent, yeast cells culture medium, recon indentifying substance.In addition, described It may also include in kit：Operation instructions, consequently facilitating those skilled in the art use.
The present invention also provides the purposes of the double-mass model Transpositional mutation system to be dashed forward for carrying out gene mutation The gene of change.
The double-mass model Transpositional mutation system of the present invention can be applied to the startup that transposon integration is entered to certain unknown gene Subregion or coding region make the gene mutation inactivate and generate a kind of new mutant phenotype；Subsequently, it is designed according on transposons Known array carry out the gene of clonal anergy, and in this, as probe, clone obtains originally non-deactivated gene in wild type, To achieve the purpose that identify the unknown gene.
The double-mass model Transpositional mutation system of the present invention has the characteristics that repeatability is low, complexity is high, by addition further After optimizing enrichment method, also there is simple and quick concentration effect, it can be rich under conditions of not coated plate or painting a small amount of screen plate Collect plant mutant strains up to a million, the subsequent swivel base based on high-flux sequence is facilitated to be inserted into sequencing.
Simple substance grain Transpositional mutation system
The present invention provides a kind of simple substance grain Transpositional mutation system, the system using yeast cells as host cell, including：Trip From the plasmid in yeast cells, which includes to be operatively connected：Transposase expression cassette, transposable element, virulence protein table Up to box；And the plasmid includes yeast replication element.
The characteristics of simple substance grain Transpositional mutation system of the present invention, is that dissociated a kind of band yeast replication element in yeast Cyclic plasmid, and the plasmid simultaneously enzyme coding gene containing swivel base, transposable element and a kind of virulence protein expression cassette.
As the preferred embodiment of the present invention, in list pUC pUC of the present invention, the Transposon System can select From：TcBuster Transposon Systems or Sleeping beauty Transposon Systems.It is highly preferred that corresponding to TcBuster swivel bases Subsystem, using TcBaseCOV596A transposases；Corresponding to Sleeping beauty Transposon Systems, using SBase100X Transposase.TcBaseCOV596A and SBase100X is height optimization, the transposase with height transposition activity.Described turns The seat variant of enzyme, homologue or the like, as long as can retain transposase activity, can also be applied in the present invention.
As the preferred embodiment of the present invention, in the simple substance grain Transpositional mutation system, the transposable element includes behaviour The transposon ends repeat region and riddled basins sequence of the property made connection；Preferably, the transposon ends duplicate block Domain includes but not limited to transposon ends repeat region selected from the group below：It is operatively connected) TcBL/TcBR, it is operatively connected SBL/SBR.
Selection markers can be selected according to actually required, it is however generally that, those skilled in the art are easily obtained properly Selection markers, therefore selection markers those of are not limited to specifically to enumerate in the present invention.As the present invention preferred embodiment, The selection markers include but not limited to selection markers selected from the group below：HIS4, blasticidin resistance gene ZeocinR, G- 418 resistant gene kanR。
Further include virulence protein expression cassette in simple substance grain Transpositional mutation system of the present invention.The virulence protein It is a kind of mRNA interferases, the albumen in cell body can be inhibited to synthesize by cutting off the ACA sequences of single-stranded mRNA, to reach To the effect for killing cell.The virulence protein includes：mazF.The variant of the virulence protein, homologue or the like, As long as can retain identical active, can also be applied in the present invention.
As the preferred embodiment of the present invention, the virulence protein is mazF, also, in the virulence protein expression cassette In, further include strong inducible promoter；So as to express a large amount of mazF, ensure that the mazF for possessing sufficient dose in cell body comes Kill cell.Preferably, the strong inducible promoter is PpAOX1p promoters.
In the simple substance grain Transpositional mutation system, have the characteristics that the reproduction element of low copy number can be applied to the present invention In.Therefore, in the present invention, applicable reproduction element is not limited to those listed in the embodiment of the present invention.As the present invention Preferred embodiment, the reproduction element be PARS2 reproduction elements.
The present invention also provides the kit for building the simple substance grain Transpositional mutation system, wrapped in the kit It includes：Plasmid, the plasmid include to be operatively connected：Transposase expression cassette, transposable element, virulence protein expression cassette；And the plasmid Including yeast replication element.Further include in the described kit for building simple substance grain Transpositional mutation system one of the following group or It is multinomial：Conversion reagent, yeast cells culture medium, recon indentifying substance.In addition, may also include in the kit：It uses Specification, consequently facilitating those skilled in the art use.
The present invention also provides the purposes of the simple substance grain Transpositional mutation system to be dashed forward for carrying out gene mutation The gene of change.
The simple substance grain Transpositional mutation system of the present invention can be applied to the startup that transposon integration is entered to certain unknown gene Subregion or coding region make the gene mutation inactivate and generate a kind of new mutant phenotype；Subsequently, it is designed according on transposons Known array carry out the gene of clonal anergy, and in this, as probe, clone obtains originally non-deactivated gene in wild type, To achieve the purpose that identify the unknown gene.
Single pUC pUC can solve double-mass model system makes mutant strain because there is swivel base enzyme coding gene in vivo There are unstable inhereditary features.For this purpose, the transposase that the present inventor demonstrates single pUC pUC using quantitative fluorescent PCR encodes Gene is substantially removed.Another feature of single pUC pUC is the simplicity of operation, it is only necessary to use several different cultures instead Base can collect a large amount of mutant strain.
According to above-mentioned, the present invention construct one kind can efficiently concentrating Transposon mutant, and repeatability is low between mutant strain Double-mass model Transpositional mutation system；Also construct a kind of simple substance grain Transpositional mutation system of removable swivel base enzyme coding gene.This hair The phenotypic mutation strain that bright obtained transposon mutant screening system obtains can quickly be determined by chromosome walking technology and is mutated The genotype of strain, solves the problems, such as that the genotype of conventional ultra-violet mutagenic fungi is difficult to identify.
Present invention will be further explained below with reference to specific examples.It should be understood that these embodiments are merely to illustrate the present invention Rather than it limits the scope of the invention.In the following examples, the experimental methods for specific conditions are not specified, usually according to conventional strip Part such as J. Pehanorm Brookers etc. is write, Molecular Cloning:A Laboratory guide, the third edition, Science Press, the condition described in 2002, or According to the normal condition proposed by manufacturer.
Material
Used toolenzyme is purchased from NEB (Beijing) Co., Ltd, and specific reaction condition and the method used are joined Examine product manual.
Following commercialization plasmid and bacterial strain are used for gene cloning and protein expression：Plasmid pGAPZ α A, plasmid PPIC3.5k, Escherichia coli Top10, Pichi strain GS115 are purchased from Invitrogen companies.
YPD culture mediums：2% peptone, 1% yeast powder, 2% glucose, 2% agar powder；YNB culture mediums：0.67% YNB；MGY culture mediums：1% glycerine, 0.67%YNB；YND fluid nutrient mediums：1% glucose, 0.67%YNB.
When preparing the above culture medium, 115 DEG C of high pressure sterilization 20min of glucose, methanol adds when in use.Other ingredients 121 DEG C of high pressure sterilization 20min.Solid medium adds 2% agar powder.
The transposase gene TcBase of transposons TcBuster applied in the present inventionCOV596A derives from pGALS- TcBusterCO V596APlasmid, transposable element repeat end sequence and derive from URA3::actin intron::TcBuster- ClonNat plasmids (Li X, Ewis H, Hice RH, Malani N, Parker N, Zhou L, Feschotte C, Bushman FD,Atkinson PW,Craig NL.A resurrected mammalian hAT transposable element and a closely related insect element are highly active in human cell culture.Proc Natl Acad Sci U S A.2013Feb 5；110(6):E478-87), the two plasmids are provided by Graig NL.The swivel base Son from insect red flour beetle (Tribolium cAstaneum), belong to the Buster class transposons of hAT superfamilies.HAT turns Stand is characterized in：(1) target site that 8bp is generated in transposition event repeats；(2) the short opposing end of 5-27bp repeats； (3) most of hAT classes transposons are less than 4kb.According to the literature, TcBaseCOThe open reading frame length of V596A is 1911bp.The both ends transposable element TcBHis in the present invention respectively contain one section of inverted repeats TIR, respectively TcBL328, The bases longs of TcBR250, real inverted repeat are 16bp.In addition it will produce one section of 8bp at the target site of insertion just To repetitive sequence.TIR has been internally embedded the expression cassette of the histidinol dehydrogenase enzyme coding gene HIS4 of one section of Pichia pastoris.
The transposase SBase100X of the transposons Sleeping beauty applied derives from pCMV (CAT) T7-SB100 Plasmid (M á t é s L1, Chuah MK, Belay E, Jerchow B, Manoj N, Acosta-Sanchez A, Grzela DP, Schmitt A,Becker K,Matrai J,Ma L,Samara-Kuko E,Gysemans C,Pryputniewicz D, Miskey C,Fletcher B,VandenDriessche T,Ivics Z,Izsvák Z.Molecular evolution of a novel hyperactive Sleeping Beauty transposase enables robust stable gene transfer in vertebrates.Nat Genet.2009Jun；41(6):753-61), transposable element repeats end sequence From pT2-shp53/GFP4 plasmids, the two plasmids are purchased to Addgene (http://www.addgene.org/).This The open reading frame length of SBase100X transposases in invention is 1023bp；The both ends transposable element SBHis respectively contain one section Inverted repeats TIR, respectively 5 ' the end inverted repeat region ends SBL and 3 ' inverted repeat region SBR are really reversed heavy Multiple bases longs are 27bp.In addition it will produce the direct repetitive sequence of one section of 2bp at the target site of insertion；Inside TIR It is embedded in the expression cassette of the histidinol dehydrogenase enzyme coding gene HIS4 of one section of Pichia pastoris.
Primer such as table 1 applied in the embodiment of the present invention.
The primer that table 1, the present invention use
The content of present invention is further described below in conjunction with embodiment.It will be understood to those of skill in the art that below Embodiment be merely to illustrate the present invention, and should not be taken as limiting the scope of the invention.
Embodiment 1, the structure for helping plasmid pBRZeo-TTcBaseCOV596A and pBRZeo-MSBase100X
Pichia pastoris total genomic dna is extracted, pGALS-TcBuster is extractedCO V596A(it is obtained from Johns Hopkins University Medical college), pCMV (CAT) T7-SB100 (be obtained from Addgene) and pPICZ α A plasmids.
Using Pichia pastoris total genomic dna as template, thiamine is amplified with primer THI11p2-amp-F and THI11p-R Check type THI11p promoter fragments PpTHI11p-amp；
Methionine, which is amplified, with primer MET3p-amp-F and MET3p-R checks type promoter PpMET3p-amp segments.
With pGALS-TcBusterCO V596AFor template, with primer TcBase-thi11p-F and TcBase-aox1tt-R from Amplification obtains TcBaseCOV596A coding DNA segments TcBase-thi11p/aox1tt in the Plasmid DNA.
Using pCMV (CAT) T7-SB100 plasmids as template, expanded with primer SBase-met3p-F and SBase-aox1tt-R Obtain SBase100X encoding gene segments SBase-met3p/aox1tt.
Using pPICZ α plasmids as template, amplifies tape copy initiation site with primer pG-A1-F and pBR-ampr2-R and win The AOX1TT-Zeocin-pBR322-amp segments of Lay mycin resistant gene expression cassette Zeocin.
Use the seamless integration kit ClonExpress MultiS of Vazyme Biotech Nuo Weizan biotech firms One Step Cloning Kit, by segment PpTHI11p-amp, segment TcBase-thi11p/aox1tt and segment AOX1TT- Zeocin-pBR322-amp seamless integrations connection cyclization, obtains pBRZeo-TTcBaseCOV596A plasmids (Fig. 1).
By PpMET3p-amp segments, SBase-met3p/aox1tt segments and AOX1TT-Zeocin-pBR322-amp pieces Section is seamlessly connected and obtains pBRZeo-MSBase100X (Fig. 2).
Two kinds of connection liquid are converted respectively into Escherichia coli Top10 competence, it is solid with the LB for being added to ampicillin Body Screening of Media goes out positive transformant, and further amplification cultivation, extracts plasmid.
The structure of embodiment 2, donor plasmid pBRAmp-TcBHis and pBRAmp-SBHis
Extract pPIC3.5k plasmids and URA3::actin intron::TcBuster-ClonNat plasmids (are obtained from John suddenly Pu Jinsi University Medical Colleges).
Using pPIC3.5k plasmids as template, expand to obtain with primer pG-A2-F and Amp-L1-R respectively multiple with Escherichia coli The pBR322-Amp segments of element processed amplify HIS4 expression casette segments with His-starg-F and His-starg-R His4-arg。
With URA3::actin intron::TcBuster-ClonNat plasmids are template, with primer TcBL-amp-F/ TcBL-arg4-R, TcBR-au-F/TcBR-pbr322-R are expanded respectively obtains two end sequences of TcBuster transposable elements TcBL-amp/arg segments and TcBR-pbr322 segments.
Plasmid pT2-shp53/GFP4 (being obtained from Addgene) is extracted and using the plasmid as template, with primer SBL-amp-F/ SBL-arg4-R, SBR-arg4-F/SBR-pbr322-R are expanded respectively obtains two ends of Sleeping beauty transposable elements Sequence SBL-amp/arg segments and SBR-pbr322 segments.
By pBR322-Amp segments, TcBL-amp/arg segments, His4-arg segments and seamless group of TcBR-pbr322 segments Dress up pBRAmp-TcBHis plasmids (Fig. 3).
By pBR322-Amp segments, SBL-amp/arg segments, His4-arg segments and SBR-pbr322 segment seamless integrations At pBRAmp-SBHis plasmids (Fig. 4).
The structure of embodiment 3, plasmid pTTcBHis4AMazFARS and pMSBHis4AMazFARS
Using Pichia pastoris genome as template, primer PARS2-aox1tt-F/PARS2-R and PpAOX1p-F/ are used respectively PpAOX1p-R expands to obtain PARS2-aox1tt and PpAOX1p segments.
Using the genome of Escherichia coli Top10 as template, amplified with primer MazF-aox1p-F and MazF-aox1tt-R MazF-aox1p/aox1tt segments with toxin gene mazF.
Using pPICZ α plasmids as template, primer TEF1p-pars2-F/pBR322-R and pG-A1-F/ are used respectively AOX1TT-A1-R expands to obtain Zeocin-pBR322-pars2 segments and AOX1TT segments.
Next in the above-mentioned several plasmids built, using pBRZeo-TTcBaseCOV596A plasmids as template, with drawing Object THI11p2-aox1p-F and AOX1TT-A1-R is amplifiable to obtain the PpTHI11p-TcBase- of the expression cassette containing TcBase AOX1TT-aox1p segments.Using plasmid pBRZeo-MSBase100X as template, with primer MET3p-aox1p-F and AOX1TT- A1-R expands to obtain the PpMET3p-SBase-AOX1TT-aox1p segments of the expression cassette containing SBase；With plasmid pBRAmp-TcBHis For template, TcBuster transposable elements TcBTcBL- is obtained with primer TcBL-pbr322-F and TcBR-aox1tt-F are amplifiable His4-TcBR-pbr322/aox1tt segments；Using plasmid pBRAmp-SBHis as template, with primer SBL-pbr322-F and SBR- Aox1tt-F is amplifiable to obtain SB transposable element SBL-His4-SBR-pbr322/aox1tt segments.
By PARS2-aox1tt segments, PpAOX1p segments, MazF-aox1p/aox1tt segments, Zeocin-pBR322- Pars2 segments, AOX1TT segments, PpTHI11p-TcBase-AOX1TT-aox1p segments and TcBTcBL-His4-TcBR- Pbr322/aox1tt segments seamless integration is at pTTcBHis4AMazFARS plasmids (Fig. 5).
By PARS2-aox1tt segments, PpAOX1p segments, MazF-aox1p/aox1tt segments Zeocin-pBR322- Pars2 segments, AOX1TT segments, PpMET3p-SBase-AOX1TT-aox1p segments and SBL-His4-SBR-pbr322/ Aox1tt segments seamless integration is at pMSBHis4AMazFARS plasmids (Fig. 6).
Embodiment 4, double-mass model genealogy of law system collect Transposon mutant
TcBuster Transposon mutants are enriched with：Using Nde I restriction enzymes in plasmid pBRZeo- After being linearized after the promoter THI11p of TTcBaseCOV596A, which is gone in GS115 competence, YPD cultures Base recovery culture is applied to the YPD solid mediums for being added to bleomycin after 2 hours, picking Partial Conversion after culture 2~3 days Its genome is cultivated and extracted, the transformant of the enzyme coding gene of swivel base containing TcBaseCOV596A is verified with primer PCR, obtains sun Property transformant GS115-pBRZeo-TTcBaseCOV596A.Positive transformant is seeded to 100mL EMM cultures, initial inoculation Concentration is about 0.3OD600, culture to 1.6OD600GS115-pBRZeo-TTcBaseCOV596A competence is prepared when left and right.Then By pBRAmp-TcBHis cyclic plasmids electrotransformation to GS115-pBRZeo-TTcBaseCOV596A competence bacterial strains, while with not PBRAmp-His plasmids with transposable element are applied to shortage group ammonia after 3 hours as a contrast, using YND medium cultures transformant In the YND solid culture plates of acid, culture picking Partial Conversion culture and extracts its genome as subsequent authentication after 2~3 days With.
SB Transposon mutants are enriched with：After Sac I restriction enzyme plasmids pBRZeo-SBase100X, by this Plasmid electricity is gone in GS115 competence, and YPD culture mediums recovery culture is applied to the YPD solids training for being added to bleomycin after 2 hours Base is supported, culture picking Partial Conversion culture and extracts its genome after 2~3 days, and PCR verifies the volume of transposase containing SBase100X The transformant of code gene, obtains positive transformant GS115-pBRZeo-MSBase100X.Positive transformant is seeded to 100mL YND is cultivated, and initial inoculation concentration is about 0.3OD600, culture to 1.6OD600GS115-pBRZeo- is prepared when left and right MSBase100X competence.Then the pBRAmp-His cyclic plasmids by pBRAmp-SBHis and as a contrast distinguish electrotransformation extremely GS115-pBRZeo-MSBase100X competence is applied to after 3 hours using YND medium cultures transformant and lacks histidine In YND solid culture plates, culture 2~3 days after picking Partial Conversion culture and extract its genome as subsequent authentication use.
Embodiment 5, hiTail-PCR verification Transposon mutants and integration site analysis
The postgenome for extracting Transposon mutant to be verified then uses primer TcBR- if it is verification TcB mutant strains SP1 and LAD1-3 as first round primer, using TcBR-SP2 and LAD0 as the second wheel primer pair target gene group template into (method is referring to Yao-Guang Liu and Yuanling Chen.2007.High-efficiency for the hot asymmetric PCR of row thermal asymmetric interlaced PCR for amplification of unknown flanking sequences.BioTechniques.43:649-656).If it is verification SB swivel base strains, then SBR-SP1 and LAD1-1 is used As first round primer, using SBR-SP2 and LAD0 as the second wheel primer.
20 plants of TcB mutant strains of the random picking of the present inventor and 20 plants of SB mutant strains, (such as Fig. 8) is surveyed after hiTail-PCR Then sequence sequencing result and the sequencing of GS115 full genomes is compared, analysis result is as shown in table 2 and table 3.20 plants of TcB Mutant strain is randomly dispersed in the different loci of four chromosome of Pichia pastoris, and integrates target site and meet document report The base of centre two of the feature of NNNTANNN, i.e., 8 base target sites of most mutant strains is all TA, other Base comparisons At random, in addition to MT3# mutant strain particular points, intermediate two bases are TG.And there are 19 plants of difference positions in 20 plants of SB mutant strains to be verified In the different loci of full-length genome, in addition one plant of bacterial strain is still located at the inside of pBRAmp-SBHis plasmid sequences, does not occur to turn Seat (false positive mutant strain caused by pBRAmp-SBHis plasmid homologous recombinations to genome, behind can analyze).19 plants of swivel bases The integration site of mutant strain shows that swivel base target site of the SB transposons in Pichia pastoris is TA sequences, this and document report It matches.In addition the present inventors have noted that the event ratio for being integrated into gene internal has been more than 50%, imply that the transposons has Conducive to screening function gene mutation strain.
The transposition integration site information of table 2, TcBuster double-mass model systems
The transposition integration site information of table 3, Sleeping beauty double-mass model systems
The apparent transposition activity analysis of embodiment 6, double-mass model system
Although the plasmid without Pichia pastoris reproduction element will not exist in Pichia pastoris body too long, due to cyclic annular matter The probability that grain has meeting certain is integrated into Pichia pastoris genome (Cregg JM.DNA-mediated transformation.Methods Mol Biol.2007；389:27-42), this just makes double-mass model system collect Transpositional mutation There are certain false positives when strain.As shown in fig. 7, control board has still grown dozens of transformant, it accounts for TcB experimental group bacterial strains 3% or so, account for 10% or so of SB experimental group bacterial strains.This false positive ratio is acceptable, and the present inventor still may be used With by the transformant number in statistical experiment group come the apparent transposition activity of general estimation double-mass model system.1OD600TcB Electricity turns liquid (about 5106Strain bacterial strain) apply YND (his-) plate after can grow 1400 or so transformants, thus estimate that TcB's is apparent Swivel base rate is 2.8 × 10-4, this can be described as considerable efficiency, because the efficiency of electrotransformation is 10 in Pichia pastoris-2It is left The right side (Cregg JM.DNA-mediated transformation.Methods Mol Biol.2007；389:27-42), it goes out The influence of electrotransformation, transposition efficiencies of the TcBaseCOV596A in Pichia pastoris is about 10-2.Same method, the present inventor estimate The apparent transposition efficiency for calculating SB swivel bases is about 4.2 × 10-5。
The Transposon mutant that embodiment 7, liquid enrichment double-mass model system are collected
The advantages of double-mass model system is that transposition event is completed in a short period of time, the positive grown on YND screen plates Transformant generally is independent Transposon mutant, wherein can to collect complexity in this way high by such the present inventor Mutation library.However, apparent transposition efficiency existing for double-mass model system is not high, especially SB transposons；It subsequently to collect up to a million Mutant strain be used for high-flux sequence, if coated plate-board-washing is used to collect, it would be desirable to larger amount of reagent and labour.
In view of the bacterial strain for the donor plasmid for not being transferred to the expression casette containing HIS4, or it is not that turning for transposition integration occurs Beggar can not grow in the YND fluid nutrient mediums for lacking histidine, and the present inventor attempts first a large amount of electricity and turns donor plasmid, then It is gone to be enriched with the transposon mutant strain for having covered his with YND fluid nutrient mediums.In order to study the feasibility of this scheme, explore simultaneously Mutant strain accounting is more than 50% time after liquid enrichment culture, and the present inventor is in advance with having covered his while being transferred to GFP reports The GS115-GFP-His bacterial strains for accusing albumen (being used for flow cytomery quantity) remove simulation Transposon mutant.With specific reference to The ratio of GS115-GFP-His bacterial strain mixing wild strains GS115 is different, can be divided into following four groups of experiments：
A) 15OD is taken600GS115 inoculations are in 50mL YND culture mediums.
B) 15OD is taken600GS115 bacterial strains and 15/100OD600GS115-GFP-His inoculations are in 50mL YND culture mediums.
C) 15OD is taken600GS115 bacterial strains and 15/10000OD600GS115-GFP-His inoculations are cultivated in 50mL YND Base.
D) 15/10000OD is taken600GS115-GFP-His inoculations are in 50mL YND culture mediums.
Above four groups of bacterial strains are put in 30 DEG C of 200rpm shaking tables and are cultivated, it is dense in suitable time sampling survey bacterium, to describe Growth curve is prepared.In addition, the present inventor was at 12 hours, 36 hours, 82 hours and 100 hours, this four time points additionally take Sample is used for the number of flow cytomery cell.Growth curve as shown in figure 9, control group GS115 due to being histidine nutrition Deficiency, slow-growing in YND, culture only has 3.8OD to concentration at 100 hours600Left and right, and no longer increase.And it is another It is that his covers strain to compare strain GS115-GFP-His, since inoculum concentration is extremely low, so being grown before 30 hours very slow, but is entered pair Growth rate is exceedingly fast after the number phase, and enters stationary phase at 60 hours, and concentration is more than 20OD600.And it is mixed with GS115 and GS115- The speed of growth of two kinds of GFP-His experiment strains between two control strains, especially ratio close to Transposon mutant c groups, The speed of growth before about 42 hours is not distinguished significantly with GS115, but apparent quickening is grown after 42 hours, and 100 hours bacterium are dense to reach 10OD600Left and right.Dense according to control strain GS115 maximum bacterium is 3.8OD600Left and right, the present inventor's estimation exist 100 hours, the GS115-GFP-His bacterial strains accounting of c groups should be more than 60%.Further, the present inventor passes through more accurate Flow cytometry experiment obtains (Figure 10), when incubation time reaches 100 hours, GS115-GFP-His initial concentration accountings About 10-2Accounting of the b groups at 100 hours can reach 90% or more, and it is 10 to originate accounting-3C groups at 100 hours Accounting also reached 82%, reached considerable concentration effect.
So far, the present inventor can remove the Transposon mutant that double-mass model method is collected by this liquid concentration method, be subsequent high pass The experiment that measurement sequence builds million mutant strain ranks is prepared.
Embodiment 8, double-mass model system collect Transposon mutant
The disadvantage of double-mass model system is to save transposase in Pichia pastoris body.TcBaseCOV596A and Even if SBase100X checks type promoter PpTHI1p in thiamine respectively and methionine checks the PpMET3p controls of type promoter Under, added in culture corresponding repressor also can not completely the same transposase activity, also checking condition really in experiment Under detect swivel base phenomenon.This show double-mass model system collect Transposon mutant there are unstable hidden danger, after this will give Continuous screening function gene makes troubles.
To solve this problem, the present inventor devises a kind of single pUC pUC (plasmid is as illustrated in Figures 5 and 6).The system There are several features：
A) transposase and transposable element design meet two conditions of swivel base generation in the same plasmid.
B) above-mentioned plasmid carries Pichia pastoris reproduction element PARS2 so that the plasmid has in Pichia pastoris body self The function of duplication ensure that the expression time of transposase.
C) there is also the inducible expression boxes of virulence protein mazF for above-mentioned plasmid.The expression cassette is in PpAOX1p promoters Guaranteed, the bacterial strain with mazF expression cassette plasmids under the conditions of methanol will be unable to growth (Yang J, Jiang W, Yang S.mazF as a counter-selectable marker for unmarked genetic modification of Pichia pastoris.FEMS Yeast Res.2009Jun；9(4):600-9).
Specific method is that the present inventor turns pTTcBHis4AMazFARS plasmids or pMSBHis4AMazFARS plasmid electricity Enter GS115 competence, be coated in the YPD culture plates for being added to bleomycin after recovery, 30 DEG C are cultivated 3 days or so, and then picking turns It is cultivated in beggar to the YPD for be added to bleomycin, waits for culture to OD600For 5-10 when, first use Tiangeng biochemical technology (Beijing) The small extraction reagent kit of yeast plasmid (DP112) of Co., Ltd extracts plasmid, then using swivel base enzyme coding gene verify primer into Row PCR verifications.Twice of the bacterial strain being verified with sterile water wash, by a concentration of 0.5OD of initial inoculation600Inoculum concentration inoculation To the induced expression for carrying out transposase in secondary medium.TcB transposons uses EMM inducing cultures, and SB transposons uses YND inducing cultures.During this, transposase can promote the generation of transposition event after being expressed, simultaneously because plasmid Unstability (Cregg JM.DNA-mediated transformation.Methods Mol Biol.2007；389:27- 42), part bacterial strain can there is a phenomenon where plasmid loss in incubation.When two level incubation time was by 24 hours, bacterial strain is basic Come into stationary phase.Bacterial strain is cleaned with aqua sterilisa again, and is seeded in methanol medium YNM and carries out induction virulence protein The expression of mazF, induction time are 20 hours or more.This process can remove the bacterial strain with plasmid, due to transposase expression cassette On plasmid, it is also achieved that the rejecting of swivel base enzyme coding gene.Further, it is selected by YND screen of the painting without histidine Transposon mutant.
The copy number of transposable element and transposase in embodiment 9, the single pUC pUC of quantitative fluorescent PCR verification
Quantitative fluorescent PCR can be detected in Pichia pastoris gene copy number (method referring to Xuan, Y.J., X.S.Zhou, W.W.Zhang,X.Zhang,Z.W.Song,and Y.X.Zhang.2009.An upstream activation sequence controls the expression of AOX1gene in Pichia pastoris.FEMS Yeast Res.9:1271– 1282) it is, that further whether verification transposase gene has been removed, while analyzing the copy of transposable element in single pUC pUC Number, the present inventor go detection SBase100X genes and SBHis transposable elements to copy by taking SB transposons as an example, with quantitative fluorescent PCR Shellfish number.The experimental results showed that (Figure 11), reference gene ACT1, SBL and SBR in transposable element, transposase gene SBase100X Mark song linear coefficient R2All reach 0.999 or more, has illustrated that the logarithm of the copy number of these DNA and Ct values have very Good linear relationship.The copy number of DNA can be derived by Ct value results.Final result is as shown in table 4, and SBL and SBR's copies Shellfish number illustrates that transposable element is mainly present in the form of single copy in yeast 1 or so；And the copy number of SBase100X genes It is very low, almost 0.001, it is contemplated that experiment it is error, may infer that most SB Transposon mutants are free of SBase100X genes.
Table 4, the mono- pUC pUCs of Sleeping beauty should quantitative PCR Ct values
All references mentioned in the present invention is incorporated herein by reference, independent just as each document It is incorporated as with reference to such.In addition, it should also be understood that, after reading the above teachings of the present invention, those skilled in the art can To be made various changes or modifications to the present invention, such equivalent forms equally fall within model defined by the application the appended claims It encloses.
Sequence table
<110>East China University of Science
<120>A kind of efficient Transpositional mutation system and construction method
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Met Met Leu Asn Trp Leu Lys Ser Gly Lys Leu Glu Ser Gln Ser Gln
1 5 10 15
Glu Gln Ser Ser Cys Tyr Leu Glu Asn Ser Asn Cys Leu Pro Pro Thr
20 25 30
Leu Asp Ser Thr Asp Ile Ile Gly Glu Glu Asn Lys Ala Gly Thr Thr
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Ser Arg Lys Lys Arg Lys Tyr Asp Glu Asp Tyr Leu Asn Phe Gly Phe
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Thr Trp Thr Gly Asp Lys Asp Glu Pro Asn Gly Leu Cys Val Ile Cys
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Glu Gln Val Val Asn Asn Ser Ser Leu Asn Pro Ala Lys Leu Lys Arg
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His Leu Asp Thr Lys His Pro Thr Leu Lys Gly Lys Ser Glu Tyr Phe
100 105 110
Lys Arg Lys Cys Asn Glu Leu Asn Gln Lys Lys His Thr Phe Glu Arg
115 120 125
Tyr Val Arg Asp Asp Asn Lys Asn Leu Leu Lys Ala Ser Tyr Leu Val
130 135 140
Ser Leu Arg Ile Ala Lys Gln Gly Glu Ala Tyr Thr Ile Ala Glu Lys
145 150 155 160
Leu Ile Lys Pro Cys Thr Lys Asp Leu Thr Thr Cys Val Phe Gly Glu
165 170 175
Lys Phe Ala Ser Lys Val Asp Leu Val Pro Leu Ser Asp Thr Thr Ile
180 185 190
Ser Arg Arg Ile Glu Asp Met Ser Tyr Phe Cys Glu Ala Val Leu Val
195 200 205
Asn Arg Leu Lys Asn Ala Lys Cys Gly Phe Thr Leu Gln Met Asp Glu
210 215 220
Ser Thr Asp Val Ala Gly Leu Ala Ile Leu Leu Val Phe Val Arg Tyr
225 230 235 240
Ile His Glu Ser Ser Phe Glu Glu Asp Met Leu Phe Cys Lys Ala Leu
245 250 255
Pro Thr Gln Thr Thr Gly Glu Glu Ile Phe Asn Leu Leu Asn Ala Tyr
260 265 270
Phe Glu Lys His Ser Ile Pro Trp Asn Leu Cys Tyr His Ile Cys Thr
275 280 285
Asp Gly Ala Lys Ala Met Val Gly Val Ile Lys Gly Val Ile Ala Arg
290 295 300
Ile Lys Lys Leu Val Pro Asp Ile Lys Ala Ser His Cys Cys Leu His
305 310 315 320
Arg His Ala Leu Ala Val Lys Arg Ile Pro Asn Ala Leu His Glu Val
325 330 335
Leu Asn Asp Ala Val Lys Met Ile Asn Phe Ile Lys Ser Arg Pro Leu
340 345 350
Asn Ala Arg Val Phe Ala Leu Leu Cys Asp Asp Leu Gly Ser Leu His
355 360 365
Lys Asn Leu Leu Leu His Thr Glu Val Arg Trp Leu Ser Arg Gly Lys
370 375 380
Val Leu Thr Arg Phe Trp Glu Leu Arg Asp Glu Ile Arg Ile Phe Phe
385 390 395 400
Asn Glu Arg Glu Phe Ala Gly Lys Leu Asn Asp Thr Ser Trp Leu Gln
405 410 415
Asn Leu Ala Tyr Ile Ala Asp Ile Phe Ser Tyr Leu Asn Glu Val Asn
420 425 430
Leu Ser Leu Gln Gly Pro Asn Ser Thr Ile Phe Lys Val Asn Ser Arg
435 440 445
Ile Asn Ser Ile Lys Ser Lys Leu Lys Leu Trp Glu Glu Cys Ile Thr
450 455 460
Lys Asn Asn Thr Glu Cys Phe Ala Asn Leu Asn Asp Phe Leu Glu Thr
465 470 475 480
Ser Asn Thr Ala Leu Asp Pro Asn Leu Lys Ser Asn Ile Leu Glu His
485 490 495
Leu Asn Gly Leu Lys Asn Thr Phe Leu Glu Tyr Phe Pro Pro Thr Cys
500 505 510
Asn Asn Ile Ser Trp Val Glu Asn Pro Phe Asn Glu Cys Gly Asn Val
515 520 525
Asp Thr Leu Pro Ile Lys Glu Arg Glu Gln Leu Ile Asp Ile Arg Thr
530 535 540
Asp Thr Thr Leu Lys Ser Ser Phe Val Pro Asp Gly Ile Gly Pro Phe
545 550 555 560
Trp Ile Lys Leu Met Asp Glu Phe Pro Glu Ile Ser Lys Arg Ala Val
565 570 575
Lys Glu Leu Met Pro Phe Val Thr Thr Tyr Leu Cys Glu Lys Ser Phe
580 585 590
Ser Val Tyr Ala Ala Thr Lys Thr Lys Tyr Arg Asn Arg Leu Asp Ala
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Glu Asp Asp Met Arg Leu Gln Leu Thr Thr Ile His Pro Asp Ile Asp
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Met Gly Lys Ser Lys Glu Ile Ser Gln Asp Leu Arg Lys Arg Ile Val
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Ala Val Pro Arg Ser Ser Val Gln Thr Ile Val Arg Lys Tyr Lys His
35 40 45
His Gly Thr Thr Gln Pro Ser Tyr Arg Ser Gly Arg Arg Arg Val Leu
50 55 60
Ser Pro Arg Asp Glu Arg Thr Leu Val Arg Lys Val Gln Ile Asn Pro
65 70 75 80
Arg Thr Thr Ala Lys Asp Leu Val Lys Met Leu Glu Glu Thr Gly Thr
85 90 95
Lys Val Ser Ile Ser Thr Val Lys Arg Val Leu Tyr Arg His Asn Leu
100 105 110
Lys Gly His Ser Ala Arg Lys Lys Pro Leu Leu Gln Asn Arg His Lys
115 120 125
Lys Ala Arg Leu Arg Phe Ala Thr Ala His Gly Asp Lys Asp Arg Thr
130 135 140
Phe Trp Arg Asn Val Leu Trp Ser Asp Glu Thr Lys Ile Glu Leu Phe
145 150 155 160
Gly His Asn Asp His Arg Tyr Val Trp Arg Lys Lys Gly Glu Ala Cys
165 170 175
Lys Pro Lys Asn Thr Ile Pro Thr Val Lys His Gly Gly Gly Ser Ile
180 185 190
Met Leu Trp Gly Cys Phe Ala Ala Gly Gly Thr Gly Ala Leu His Lys
195 200 205
Ile Asp Gly Ile Met Asp Ala Val Gln Tyr Val Asp Ile Leu Lys Gln
210 215 220
His Leu Lys Thr Ser Val Arg Lys Leu Lys Leu Gly Arg Lys Trp Val
225 230 235 240
Phe Gln His Asp Asn Asp Pro Lys His Thr Ser Lys Val Val Ala Lys
245 250 255
Trp Leu Lys Asp Asn Lys Val Lys Val Leu Glu Trp Pro Ser Gln Ser
260 265 270
Pro Asp Leu Asn Pro Ile Glu Asn Leu Trp Ala Glu Leu Lys Lys Arg
275 280 285
Val Arg Ala Arg Arg Pro Thr Asn Leu Thr Gln Leu His Gln Leu Cys
290 295 300
Gln Glu Glu Trp Ala Lys Ile His Pro Asn Tyr Cys Gly Lys Leu Val
305 310 315 320
Glu Gly Tyr Pro Lys Arg Leu Thr Gln Val Lys Gln Phe Lys Gly Asn
325 330 335
Ala Thr Lys Tyr
340
<210> 3
<211> 1911
<212> DNA
<213>Artificial sequence
<220>
<221> misc_feature
<223>The coding nucleotide sequence of TcBaseCO V596A transposases
<400> 3
atgatgctga attggctgaa aagcggtaaa cttgagtcac agtcccaaga gcagagcagt 60
tgctaccttg agaacagtaa ttgtctgcca cccactctgg actccacgga tattattggt 120
gaggaaaaca aagctggcac cacaagtcgg aagaagagga agtatgacga ggactacctg 180
aactttgggt ttacctggac aggcgataag gatgaaccca atggactctg cgtgatttgc 240
gagcaggttg tgaacaacag ttcactgaat cccgccaagc ttaaacggca tctcgacaca 300
aagcacccta cactgaaggg gaaaagtgag tacttcaaaa ggaagtgcaa cgagctgaat 360
caaaagaaac ataccttcga aagatatgtg cgcgacgaca acaaaaatct gcttaaggca 420
agctatctgg taagccttcg gatcgccaaa cagggagaag cctatacaat tgctgagaag 480
ttgattaaac cctgcacaaa agacctgacg acctgtgtct ttggtgagaa atttgcctct 540
aaggtagacc tggttccttt gtccgacacc actatcagcc gacgcataga ggacatgagt 600
tatttctgtg aagccgtgct ggtcaaccgc ttgaaaaacg ccaagtgcgg attcactttg 660
cagatggacg agtccaccga cgtagcaggc ctggctattc ttctcgtctt cgtgaggtac 720
atccacgagt cttctttcga ggaggatatg ctgttctgta aagctctgcc tacccagact 780
accggggaag aaatctttaa tctcctcaat gcctactttg agaaacacag catcccatgg 840
aacctctgct atcacatttg cactgacggg gcaaaagcta tggttggagt catcaaaggg 900
gtgattgcga gaatcaaaaa gctcgtccct gacataaagg cgagccattg ttgccttcac 960
cgacatgctc tcgctgtaaa gcgcataccg aatgcccttc acgaggttct gaatgacgca 1020
gtcaagatga tcaattttat taagtcccga ccactcaatg caagagtgtt tgccctgttg 1080
tgcgatgacc ttggaagtct ccataaaaac ctgctcctcc acactgaggt gaggtggttg 1140
tcacggggaa aggttctgac tcgattctgg gagctgcgcg atgagattag gattttcttt 1200
aacgagcgag aattcgccgg gaaactgaac gacacaagtt ggttgcagaa tctcgcgtac 1260
attgcagaca tcttcagcta cctgaatgaa gtcaacctca gcttgcaggg gccaaattcc 1320
accatcttca aggtcaacag caggattaac tctatcaaaa gtaagctgaa actgtgggag 1380
gaatgtatta cgaaaaacaa cactgaatgc ttcgctaacc ttaatgactt ccttgagaca 1440
tccaatactg ccttggatcc caatcttaag tctaatatcc tggagcatct gaacggcctt 1500
aaaaacacct tcctggagta cttcccacct acctgcaaca acatcagctg ggtggaaaat 1560
cctttcaacg agtgcggaaa cgttgataca ttgcctatta aagaaaggga acagctgatc 1620
gatattagga cagataccac actgaagtcc tcattcgtgc ccgacggcat tgggcctttc 1680
tggatcaagc tcatggatga gttccctgag atctccaagc gcgctgtgaa agaacttatg 1740
ccatttgtga ctacatacct ttgtgaaaaa tctttttctg tatacgctgc gactaagacc 1800
aaataccgga accgactcga cgccgaagat gatatgcggc tgcagttgac cacaattcac 1860
cctgatatag ataatttgtg taacaacaag caggcccaga aatctcactg a 1911
<210> 4
<211> 1023
<212> DNA
<213>Artificial sequence
<220>
<221> misc_feature
<223>The coding nucleotide sequence of SBase100X transposases
<400> 4
atgggaaaat caaaagaaat cagccaagac ctcagaaaaa gaattgtaga cctccacaag 60
tctggttcat ccttgggagc aatttccaaa cgcctggcgg taccacgttc atctgtacaa 120
acaatagtac gcaagtataa acaccatggg accacgcagc cgtcataccg ctcaggaagg 180
agacgcgttc tgtctcctag agatgaacgt actttggtgc gaaaagtgca aatcaatccc 240
agaacaacag caaaggacct tgtgaagatg ctggaggaaa caggtacaaa agtatctata 300
tccacagtaa aacgagtcct atatcgacat aacctgaaag gccactcagc aaggaagaag 360
ccactgctcc aaaaccgaca taagaaagcc agactacggt ttgcaactgc acatggggac 420
aaagatcgta ctttttggag aaatgtcctc tggtctgatg aaacaaaaat agaactgttt 480
ggccataatg accatcgtta tgtttggagg aagaaggggg aggcttgcaa gccgaagaac 540
accatcccaa ccgtgaagca cgggggtggc agcatcatgt tgtgggggtg ctttgctgca 600
ggagggactg gtgcacttca caaaatagat ggcatcatgg acgccgtgca gtatgtggat 660
atattgaagc aacatctcaa gacatcagtc aggaagttaa agcttggtcg caaatgggtc 720
ttccaacacg acaatgaccc caagcatact tccaaagttg tggcaaaatg gcttaaggac 780
aacaaagtca aggtattgga gtggccatca caaagccctg acctcaatcc tatagaaaat 840
ttgtgggcag aactgaaaaa gcgtgtgcga gcaaggaggc ctacaaacct gactcagtta 900
caccagctct gtcaggagga atgggccaaa attcacccaa attattgtgg gaagcttgtg 960
gaaggctacc cgaaacgttt gacccaagtt aaacaattta aaggcaatgc taccaaatac 1020
tag 1023
<210> 5
<211> 1089
<212> DNA
<213>Artificial sequence
<220>
<221> misc_feature
<223>The nucleotide sequence of promoter PpTHI11p
<400> 5
caagttccgg tacaagcgtg ctaatatctc caagtgtatc taacgggtct gataatattt 60
gttcaattgc gcaagtcaag cagagtttaa tcttttcagc ttcatcgtca gtgatatttc 120
tcagcccaca gaccaagtca actttggaat ctaacaacct tgttcttaca atgttagaac 180
tcttaagtcg catgccatga tcttcaagct gaattttgtg aaggaggtca aaccccacaa 240
tggcatctag ttgtttagaa tacatgcctt cgacaagtgt ttgagtgtcc aaaatcaaga 300
gctcaaaatt attgaatttg tctgccaata acgccgtaaa ttgattagtg tccagcccac 360
caacaatagg agcacctata gttaattttt cagataaatt taagttatca aggtaaagga 420
gctctaagtt taccccttcc aacagggtta tttgagaact caataaattg ttgaattcaa 480
aaccaattgt ctttgaattc tccactggag cttccttgct gaaattgatt ttgataccat 540
tggcatcaaa gagacccgta tgataactcc ataaaaaggg gagatgatag gccttaaatt 600
catcgttaat ctgcaaattt attcctgaca tgtctttgta aatagttata gttcagaaac 660
tggaattgag ctcaaaaaac tggaatcgag cggatatttg aagattgatg ccttactcat 720
gaattgattg ataagagctc cgtgattcac tctgtcaatg attacccctc tcctacccga 780
tttgggactt tttcttcagt cttggggact ttttttcata tgacttgacc ttgctttccc 840
aatagggaag gactcaccca tggatgatta agtttggatt actcgtttag gaaatagtag 900
ccatgaatca atttgaatca taccatcatg aaatagggtt aggctgtaaa tgcctcaaaa 960
atggctcttg aggctggatt tttgggtatt ggaatgttgg tagcaattgg tataaaaggc 1020
catttgtatt tcactttttt gtccttcata ctttactctt ctcaactttg gaaacttcaa 1080
taaatcatc 1089
<210> 6
<211> 993
<212> DNA
<213>Artificial sequence
<220>
<221> misc_feature
<223>The nucleotide sequence of promoter PpMET3p
<400> 6
ttcaggcaac aggaccctcc ggaagaattt tgaagggtga cgtattggca tatctgggca 60
aaatttcacc cgaattgaat gtcaaaattt ccgagtacat taataacaag tctcacttag 120
atctgtctaa gatacaaatc agggaaacca aggcaagcga acaatctcca tcatcttcag 180
gagataaacc agccaaacct gaaaaacctg ctaagaaaga gcctctaaag atcgagaagg 240
aattgacact atctcaagct tacagtaagg aggccctgca aagattatta actattgcag 300
aacaacatgc atacagtgcc aagctgtacc aagaggattc agaagtgatt gatccactgt 360
ttgaggagat cgttgctcca cctagaaatg ccgaacgatt caaaacacaa tttatcgtca 420
cgccctcaga caatagtgtt tccacattga agcttacctt attagtcaac gagagtatat 480
tggatgccaa gcaaagagct cagttattcc ttgatgaagt caaggatcaa ttgacgcaag 540
ataatggtgg cgtttcgtcg tctccccaac ttgaagagtt attctgagtt gcaacaagtc 600
taagtagtaa gtaattaaac catcatgatc ctatgatcgt gatcattcat taaagcacgg 660
tgtggcaatt attgctaggg agatcgtcac tgtatggtgg cagaattatc tctacaagat 720
gtctcaaagt ccccacaaag cttggaccct ctcatctgta atgcattttc ctgtaactcc 780
ccttagccac acgtcaaggg ctctgaatcc gttgaaaagc tgtggcgtct gccaccttta 840
acgtcttcat gagggatgtg cacgtgatat tgtctttccc ttctctaaag cttcgaaaaa 900
aacgcatctc aatgcgagaa gcagatcgat atatataaag aactagtcca ttgaaagatc 960
tctcaatttc actggaaacc aactcagaaa gaa 993
<210> 7
<211> 328
<212> DNA
<213>Artificial sequence
<220>
<221> misc_feature
<223>TcBuster transposons 5' terminal region TcBL sequences
<400> 7
cagtgttctt caacctgtgt tccgcggaac cctagggttc cacccaaagg ctttcggggt 60
tccgcgagtc attgcttcaa ttcgagagac gtcggccgcg ccgctcttca gaatgcacat 120
gcgtcaatcg gagtttcatg ttgaaacatg ttatccattc gcatagttga cttacactgc 180
acttaacctt aattttcaaa aatatgtaac tgtacttgtg gtcgtagttt tgttgttgtt 240
ttaggtttag acaagcaaag gtaagttaac ttacagtttt aaaataaatt gtattttgtt 300
tgatcctaac ctagaatcgt tcagaaat 328
<210> 8
<211> 250
<212> DNA
<213>Artificial sequence
<220>
<221> misc_feature
<223>TcBuster transposons 3' terminal region TcBR sequences
<400> 8
tttttatttt ttattttata tattatttta tttggaatat ttatcttttt gtttaaagtt 60
tgtttaaata aatgcaaatt taaatcttat ttagagtttt tattaccaaa gcacgggctc 120
accttgttcg taacaagtca acgcagctgt ccctaaaatc tcatctgggt gtattactaa 180
atgaagggtt ccataaaaaa aaatatctcg acaaagggtt ccgccggatg gcaaaggttg 240
aagaacactg 250
<210> 9
<211> 227
<212> DNA
<213>Artificial sequence
<220>
<221> misc_feature
<223>Sleeping Beauty transposons 5' terminal region SBL sequences
<400> 9
cagttgaagt cggaagttta catacactta agttggagtc attaaaactc gtttttcaac 60
tactccacaa atttcttgtt aacaaacaat agttttggca agtcagttag gacatctact 120
ttgtgcatga cacaagtcat ttttccaaca attgtttaca gacagattat ttcacttata 180
attcactgta tcacaattcc agtgggtcag aagtttacat acactaa 227
<210> 10
<211> 228
<212> DNA
<213>Artificial sequence
<220>
<221> misc_feature
<223>Sleeping Beauty transposons 3' terminal region SBR sequences
<400> 10
ttgagtgtat gtaaacttct gacccactgg gaatgtgatg aaagaaataa aagctgaaat 60
gaatcattct ctctactatt attctgatat ttcacattct taaaataaag tggtgatcct 120
aactgaccta agacagggaa tttttactag gattaaatgt caggaattgt gaaaaagtga 180
gtttaaatgt atttggctaa ggtgtatgta aacttccgac ttcaactg 228
<210> 11
<211> 3209
<212> DNA
<213>Artificial sequence
<220>
<221> misc_feature
<223>HIS4 expression casette sequences
<400> 11
tggatgctgt aggcataggc ttggttatgc cggtactgcc gggcctcttg cgggatatcg 60
tccattccga cagcatcgcc agtcactatg gcgtgctgct agcgctatat gcgttgatgc 120
aatttctatg cgcacccgtt ctcggagcac tgtccgaccg ctttggccgc cgcccagtcc 180
tgctcgcttc gctacttgga gccactatcg actacgcgat catggcgacc acacccgtcc 240
tgtggatcta tcgaatctaa atgtaagtta aaatctctaa ataattaaat aagtcccagt 300
ttctccatac gaaccttaac agcattgcgg tgagcatcta gaccttcaac agcagccaga 360
tccatcactg cttggccaat atgtttcagt ccctcaggag ttacgtcttg tgaagtgatg 420
aacttctgga aggttgcagt gttaactccg ctgtattgac gggcatatcc gtacgttggc 480
aaagtgtggt tggtaccgga ggagtaatct ccacaactct ctggagagta ggcaccaaca 540
aacacagatc cagcgtgttg tacttgatca acataagaag aagcattctc gatttgcagg 600
atcaagtgtt caggagcgta ctgattggac atttccaaag cctgctcgta ggttgcaacc 660
gatagggttg tagagtgtgc aatacacttg cgtacaattt caacccttgg caactgcaca 720
gcttggttgt gaacagcatc ttcaattctg gcaagctcct tgtctgtcat atcgacagcc 780
aacagaatca cctgggaatc aataccatgt tcagcttgag acagaaggtc tgaggcaacg 840
aaatctggat cagcgtattt atcagcaata actagaactt cagaaggccc agcaggcatg 900
tcaatactac acagggctga tgtgtcattt tgaaccatca tcttggcagc agtaacgaac 960
tggtttcctg gaccaaatat tttgtcacac ttaggaacag tttctgttcc gtaagccata 1020
gcagctactg cctgggcgcc tcctgctagc acgatacact tagcaccaac cttgtgggca 1080
acgtagatga cttctggggt aagggtacca tccttcttag gtggagatgc aaaaacaatt 1140
tctttgcaac cagcaacttt ggcaggaaca cccagcatca gggaagtgga aggcagaatt 1200
gcggttccac caggaatata gaggccaact ttctcaatag gtcttgcaaa acgagagcag 1260
actacaccag ggcaagtctc aacttgcaac gtctccgtta gttgagcttc atggaatttc 1320
ctgacgttat ctatagagag atcaatggct ctcttaacgt tatctggcaa ttgcataagt 1380
tcctctggga aaggagcttc taacacaggt gtcttcaaag cgactccatc aaacttggca 1440
gttagttcta aaagggcttt gtcaccattt tgacgaacat tgtcgacaat tggtttgact 1500
aattccataa tctgttccgt tttctggata ggacgacgaa gggcatcttc aatttcttgt 1560
gaggaggcct tagaaacgtc aattttgcac aattcaatac gaccttcaga agggacttct 1620
ttaggtttgg attcttcttt aggttgttcc ttggtgtatc ctggcttggc atctcctttc 1680
cttctagtga cctttaggga cttcatatcc aggtttctct ccacctcgtc caacgtcaca 1740
ccgtacttgg cacatctaac taatgcaaaa taaaataagt cagcacattc ccaggctata 1800
tcttccttgg atttagcttc tgcaagttca tcagcttcct ccctaatttt agcgttcaac 1860
aaaacttcgt cgtcaaataa ccgtttggta taagaacctt ctggagcatt gctcttacga 1920
tcccacaagg tggcttccat ggctctaaga ccctttgatt ggccaaaaca ggaagtgcgt 1980
tccaagtgac agaaaccaac acctgtttgt tcaaccacaa atttcaagca gtctccatca 2040
caatccaatt cgatacccag caacttttga gttgctccag atgtagcacc tttataccac 2100
aaaccgtgac gacgagattg gtagactcca gtttgtgtcc ttatagcctc cggaatagac 2160
tttttggacg agtacaccag gcccaacgag taattagaag agtcagccac caaagtagtg 2220
aatagaccat cggggcggtc agtagtcaaa gacgccaaca aaatttcact gacagggaac 2280
tttttgacat cttcagaaag ttcgtattca gtagtcaatt gccgagcatc aataatgggg 2340
attataccag aagcaacagt ggaagtcaca tctaccaact ttgcggtctc agaaaaagca 2400
taaacagttc tactaccgcc attagtgaaa cttttcaaat cgcccagtgg agaagaaaaa 2460
ggcacagcga tactagcatt agcgggcaag gatgcaactt tatcaaccag ggtcctatag 2520
ataaccctag cgcctgggat catcctttgg acaactcttt ctgccaaatc taggtccaaa 2580
atcacttcat tgataccatt attgtacaac ttgagcaagt tgtcgatcag ctcctcaaat 2640
tggtcctctg taacggatga ctcaacttgc acattaactt gaagctcagt cgattgagtg 2700
aacttgatca ggttgtgcag ctggtcagca gcatagggaa acacggcttt tcctaccaaa 2760
ctcaaggaat tatcaaactc tgcaacactt gcgtatgcag gtagcaaggg aaatgtcata 2820
cttgaagtcg gacagtgagt gtagtcttga gaaattctga agccgtattt ttattatcag 2880
tgagtcagtc atcaggagat cctctacgcc ggacgcatcg tggccgacct gcaggggggg 2940
ggggggcgct gaggtctgcc tcgtgaagaa ggtgttgctg actcatacca ggcctgaatc 3000
gccccatcat ccagccagaa agtgagggag ccacggttga tgagagcttt gttgtaggtg 3060
gaccagttgg tgattttgaa cttttgcttt gccacggaac ggtctgcgtt gtcgggaaga 3120
tgcgtgatct gatccttcaa ctcagcaaaa gttcgattta ttcaacaaag ccgccgtccc 3180
gtcaagtcag cgtaatgctc tgccagtgt 3209
<210> 12
<211> 393
<212> DNA
<213>Artificial sequence
<220>
<221> misc_feature
<223>Reproduction element PARS2 sequences
<400> 12
tcgaacatag tccgtccccg ggggaagatt tattgtctca aaaggtcaat ttcatatttt 60
atatgcattc aatacttatt tattattaat ttagcttgac tacgatgcat ataattttaa 120
ttttatttta aattatatac tgaggtaaga gtataactct aaacctaata aatatataat 180
taattatacg caatagttaa accatagatt aattacaact aatcctttcg tactaagttg 240
taatccttta ttgacatttc cctaaagcag atagaaacca tactgtctca cgacgtatta 300
aacccaactc acgtaacctt ttaattgacg aacagtcaaa cccttatcag cgtgtgctac 360
caataggata ggttgagtcg acatcgagga tcc 393
<210> 13
<211> 336
<212> DNA
<213>Artificial sequence
<220>
<221> misc_feature
<223>The nucleotide sequence of virulence protein mazF
<400> 13
atggtaagcc gatacgtacc cgatatgggc gatctgattt gggttgattt tgacccgaca 60
aaaggtagcg agcaagctgg acatcgtcca gctgttgtcc tgagtccttt catgtacaac 120
aacaaaacag gtatgtgtct gtgtgttcct tgtacaacgc aatcaaaagg atatccgttc 180
gaagttgttt tatccggtca ggaacgtgat ggcgtagcgt tagctgatca ggtaaaaagt 240
atcgcctggc gggcaagagg agcaacgaag aaaggaacag ttgccccaga ggaattacaa 300
ctcattaaag ccaaaattaa cgtactgatt gggtaa 336
<210> 14
<211> 942
<212> DNA
<213>Artificial sequence
<220>
<221> misc_feature
<223>The nucleotide sequence of promoter PpAOX1p
<400> 14
cgagatctaa catccaaaga cgaaaggttg aatgaaacct ttttgccatc cgacatccac 60
aggtccattc tcacacataa gtgccaaacg caacaggagg ggatacacta gcagcagacc 120
gttgcaaacg caggacctcc actcctcttc tcctcaacac ccacttttgc catcgaaaaa 180
ccagcccagt tattgggctt gattggagct cgctcattcc aattccttct attaggctac 240
taacaccatg actttattag cctgtctatc ctggcccccc tggcgaggtt catgtttgtt 300
tatttccgaa tgcaacaagc tccgcattac acccgaacat cactccagat gagggctttc 360
tgagtgtggg gtcaaatagt ttcatgttcc ccaaatggcc caaaactgac agtttaaacg 420
ctgtcttgga acctaatatg acaaaagcgt gatctcatcc aagatgaact aagtttggtt 480
cgttgaaatg ctaacggcca gttggtcaaa aagaaacttc caaaagtcgg cataccgttt 540
gtcttgtttg gtattgattg acgaatgctc aaaaataatc tcattaatgc ttagcgcagt 600
ctctctatcg cttctgaacc ccggtgcacc tgtgccgaaa cgcaaatggg gaaacacccg 660
ctttttggat gattatgcat tgtctccaca ttgtatgctt ccaagattct ggtgggaata 720
ctgctgatag cctaacgttc atgatcaaaa tttaactgtt ctaaccccta cttgacagca 780
atatataaac agaaggaagc tgccctgtct taaacctttt tttttatcat cattattagc 840
ttactttcat aattgcgact ggttccaatt gacaagcttt tgattttaac gacttttaac 900
gacaacttga gaagatcaaa aaacaactaa ttattcgaaa cg 942
<210> 15
<211> 39
<212> DNA
<213>Primer
<400> 15
aataacagtt attattcgca agttccggta caagcgtgc 39
<210> 16
<211> 23
<212> DNA
<213>Primer
<400> 16
catgatgatt tattgaagtt tcc 23
<210> 17
<211> 39
<212> DNA
<213>Primer
<400> 17
aaacttcaat aaatcatcat gatgctgaat tggctgaaa 39
<210> 18
<211> 41
<212> DNA
<213>Primer
<400> 18
atgtctaagg ctaaaactta tcagtgagat ttctgggcct g 41
<210> 19
<211> 23
<212> DNA
<213>Primer
<400> 19
taagttttag ccttagacat gac 23
<210> 20
<211> 39
<212> DNA
<213>Primer
<400> 20
cgaataataa ctgttattca tgaccaaaat cccttaacg 39
<210> 21
<211> 38
<212> DNA
<213>Primer
<400> 21
aataacagtt attattcgtt caggcaacag gaccctcc 38
<210> 22
<211> 23
<212> DNA
<213>Primer
<400> 22
catttctttc tgagttggtt tcc 23
<210> 23
<211> 36
<212> DNA
<213>Primer
<400> 23
ccaactcaga aagaaatggg aaaatcaaaa gaaatc 36
<210> 24
<211> 34
<212> DNA
<213>Primer
<400> 24
tctaaggcta aaacttagta tttggtagca ttgc 34
<210> 25
<211> 18
<212> DNA
<213>Primer
<400> 25
tgtgagcaaa aggccagc 18
<210> 26
<211> 26
<212> DNA
<213>Primer
<400> 26
cgaataataa ctgttatttt tcagtg 26
<210> 27
<211> 37
<212> DNA
<213>Primer
<400> 27
ataacagtta ttattcgact ttaggccagt gttcttc 37
<210> 28
<211> 36
<212> DNA
<213>Primer
<400> 28
ggaccttctc gcagaatttc tgaacgattc taggtt 36
<210> 29
<211> 35
<212> DNA
<213>Primer
<400> 29
attctgcgag aaggtcctgg atgctgtagg catag 35
<210> 30
<211> 37
<212> DNA
<213>Primer
<400> 30
cttcgtttgt gcggatcaca ctggcagagc attacgc 37
<210> 31
<211> 34
<212> DNA
<213>Primer
<400> 31
gatccgcaca aacgaagtgt gcagaaaagt tcct 34
<210> 32
<211> 28
<212> DNA
<213>Primer
<400> 32
caagcttttt attttttatt ttatatat 28
<210> 33
<211> 38
<212> DNA
<213>Primer
<400> 33
ctggcctttt gctcacaaag tataaagcag tgttcttc 38
<210> 34
<211> 37
<212> DNA
<213>Primer
<400> 34
ataacagtta ttattcgaat tggagctcgg atcccta 37
<210> 35
<211> 37
<212> DNA
<213>Primer
<400> 35
ggaccttctc gcagaatcta tggctcgtac tctatag 37
<210> 36
<211> 37
<212> DNA
<213>Primer
<400> 36
gatccgcaca aacgaagaga tctagcttgt ggaaggc 37
<210> 37
<211> 37
<212> DNA
<213>Primer
<400> 37
ctggcctttt gctcacagac tctagctaga ggatccc 37
<210> 38
<211> 37
<212> DNA
<213>Primer
<400> 38
ttggatgtta gatctcgcaa gttccggtac aagcgtg 37
<210> 39
<211> 18
<212> DNA
<213>Primer
<400> 39
ggatccgcac aaacgaag 18
<210> 40
<211> 35
<212> DNA
<213>Primer
<400> 40
ttcgtttgtg cggatcctcg aacatagtcc gtccc 35
<210> 41
<211> 19
<212> DNA
<213>Primer
<400> 41
ggatcctcga tgtcgactc 19
<210> 42
<211> 31
<212> DNA
<213>Primer
<400> 42
tcgacatcga ggatccccca cacaccatag c 31
<210> 43
<211> 22
<212> DNA
<213>Primer
<400> 43
gatctcatgc atgaccaaaa tc 22
<210> 44
<211> 37
<212> DNA
<213>Primer
<400> 44
tcatgcatga gatcctttag gccagtgttc ttcaacc 37
<210> 45
<211> 48
<212> DNA
<213>Primer
<400> 45
gaagaacact gctttatact tgtgcttttt ttgttggatc cgcacaaa 48
<210> 46
<211> 36
<212> DNA
<213>Primer
<400> 46
taattattcg aaacgatggt aagccgatac gtaccc 36
<210> 47
<211> 36
<212> DNA
<213>Primer
<400> 47
tgtctaaggc taaaacttac ccaatcagta cgttaa 36
<210> 48
<211> 21
<212> DNA
<213>Primer
<400> 48
cgagatctaa catccaaaga c 21
<210> 49
<211> 20
<212> DNA
<213>Primer
<400> 49
cgtttcgaat aattagttgt 20
<210> 50
<211> 39
<212> DNA
<213>Primer
<400> 50
tttggatgtt agatctcgtt caggcaacag gaccctccg 39
<210> 51
<211> 39
<212> DNA
<213>Primer
<400> 51
ggtcatgcat gagatcaatt ggagctcgga tccctatac 39
<210> 52
<211> 35
<212> DNA
<213>Primer
<400> 52
ccaacaaaaa aagcacgact ctagctagag gatcc 35
<210> 53
<211> 28
<212> DNA
<213>Primer
<220>
<221> misc_feature
<222> (20)..(20)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (22)..(24)
<223> n is a, c, g, or t
<400> 53
acgatggact ccagaggcvn vnnnggaa 28
<210> 54
<211> 29
<212> DNA
<213>Primer
<220>
<221> misc_feature
<222> (21)..(21)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (23)..(25)
<223> n is a, c, g, or t
<400> 54
acgatggact ccagaggcvv nvnnnccaa 29
<210> 55
<211> 24
<212> DNA
<213>Primer
<400> 55
aaagcacggg ctcaccttgt tcgt 24
<210> 56
<211> 25
<212> DNA
<213>Primer
<400> 56
tgtccctaaa atctcatctg ggtgt 25
<210> 57
<211> 25
<212> DNA
<213>Primer
<400> 57
gagtgtatgt aaacttctga cccac 25
<210> 58
<211> 25
<212> DNA
<213>Primer
<400> 58
gtgatcctaa ctgacctaag acagg 25
<210> 59
<211> 18
<212> DNA
<213>Primer
<400> 59
acgatggact ccagaggc 18

Claims (18)

1. a kind of double-mass model Transpositional mutation system, which is characterized in that using yeast cells as host cell, including：
(1) transposase expression cassette is integrated on the genome of yeast cells；With
(2) plasmid being free in yeast cells, the plasmid include：Transposable element；And the plasmid is identifiable not comprising yeast Replication site sequence.
2. double-mass model Transpositional mutation system as described in claim 1, which is characterized in that the transposase expression cassette includes： Promoter, swivel base enzyme coding gene and terminator；Preferably, the promoter is selected from：Induction type checks type promoter.
3. double-mass model Transpositional mutation system as claimed in claim 2, which is characterized in that the promoter is to check type startup Son, including promoter selected from the group below：Thiamine checks type promoter PpTHI11p or methionine checks type promoter PpMET3p；Or
The promoter is inducible promoter, including promoter：Methanol inducible promoters PpAOX1p.
4. double-mass model Transpositional mutation system as described in claim 1, which is characterized in that the transposase includes being selected from the group Transposase：TcBaseCOV596A transposases, SBase100X transposases, piggyBac transposases.
5. double-mass model Transpositional mutation system as described in claim 1, which is characterized in that the amino acid sequence of the transposase Such as SEQ ID NO:1 or SEQ ID NO:Shown in 2；Or the nucleotide sequence of the encoding gene of the transposase such as SEQ ID NO:3 or SEQ ID NO:Shown in 4.
6. double-mass model Transpositional mutation system as described in claim 1, which is characterized in that the transposable element includes operability The transposon ends repeat region and riddled basins sequence of connection.
7. double-mass model Transpositional mutation system as claimed in claim 6, which is characterized in that the transposon ends repeat region Including transposon ends repeat region selected from the group below：TcBL/TcBR, SBL/SBR；Or
The selection markers include selection markers selected from the group below：HIS4, blasticidin resistance gene ZeocinR, G-418 is anti- Property gene kanR。
8. the kit for building double-mass model Transpositional mutation system described in claim 1, which is characterized in that in the kit Including：
(1) plasmid is helped, including swivel base enzyme coding gene, is integrated on the genome of yeast cells；With
(2) free plasmid, the plasmid include：Transposable element；And the plasmid does not include the identifiable replication site sequence of yeast.
9. the purposes of any double-mass model Transpositional mutation systems of claim 1-7 is mutated for carrying out gene mutation Gene.
10. a kind of simple substance grain Transpositional mutation system, which is characterized in that using yeast cells as host cell, including：It is free in yeast Plasmid in cell, the plasmid include to be operatively connected：Transposase expression cassette, transposable element, virulence protein expression cassette；And The plasmid includes yeast replication element.
11. simple substance grain Transpositional mutation system as claimed in claim 10, which is characterized in that the amino acid sequence of the transposase Row such as SEQ ID NO:1 or SEQ ID NO:Shown in 2；Or the nucleotide sequence of the encoding gene of the transposase such as SEQ ID NO:3 or SEQ ID NO:Shown in 4.
12. simple substance grain Transpositional mutation system as claimed in claim 10, which is characterized in that the transposable element includes operation Property connection transposon ends repeat region and riddled basins sequence；Preferably, the transposon ends repeat region Including transposon ends repeat region selected from the group below：TcBL/TcBR, SBL/SBR；Preferably, the selection markers include Selection markers selected from the group below：HIS4, blasticidin resistance gene ZeocinR, G-418 resistant genes kanR。
13. simple substance grain Transpositional mutation system as claimed in claim 10, which is characterized in that the virulence protein includes being selected from The virulence protein of the following group：mazF.
14. simple substance grain Transpositional mutation system as claimed in claim 13, which is characterized in that the virulence protein is mazF, The virulence protein expression cassette includes strong inducible promoter；Preferably, the strong inducible promoter opens for PpAOX1p Mover.
15. simple substance grain Transpositional mutation system as claimed in claim 10, which is characterized in that the reproduction element is PARS2 Reproduction element.
16. the kit for building simple substance grain Transpositional mutation system according to any one of claims 10, which is characterized in that the kit Include：Plasmid, the plasmid include to be operatively connected：Transposase expression cassette, transposable element, virulence protein expression cassette；And it should Plasmid includes yeast replication element.
17. the purposes of any simple substance grain Transpositional mutation systems of claim 10-15 is obtained for carrying out gene mutation The gene of mutation.
The simple substance as described in 18. double-mass model Transpositional mutation system or claim 10-15 as described in claim 1-7 is any are any Grain Transpositional mutation system, which is characterized in that the yeast cells includes yeast cells selected from the group below：Pichia pastoris, Brewing yeast cell, candida cell, Hansenula yeast cell.
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