CN102268432B - Orotate phosphoribosyltransferase promoter, application, construct and vector - Google Patents

Abstract

With the present invention, upstream sequence and downstream sequence of orotate phosphoribosyltransferase genomic DNA of rhodosporidium toruloides are amplified, biological information analysis and functional verification are performed for the amplified sequences to obtain the promoter and the terminator, wherein a target gene can be effectively expressed in the rhodosporidium toruloides through the promoter and terminator, such that the promoter and terminator can be applicable for the rhodosporidium toruloides genetic engineering operation and the strain improvement. The invention further relates to the DNA construct containing the components and the vector containing the components.

Description

Orotate phosphoribosyl transferase promotor and application and construct and carrier

Technical field

This patent belongs to gene engineering technology field, is specifically related to the red winter spore Yeast promoter of circle, terminator and uses thereof, comprises the necessary method for transformation of construction method of gene engineering strain etc.

Background technology

Microorganism is the most widely one of species of occurring in nature distribution, has remarkable biosynthesis ability, almost can synthesize organic chemicals all on the earth.Kind diversity and the genetic diversity of microorganism have determined its metabolism diversity.Compare with multicellular organism, although the pathways metabolism of microorganism is relatively simple, its chemical combination the production of material is efficient, quick, and in close relations with the daily productive life of the mankind.

Bacterial strain is used in natural production bacterial strain or environmental improvement as a certain chemical, and its specific production performance often is not optimization.How optimizing or change metabolism network and the expression regulation network of industrial strain, with accumulating rate or the directed quality of controlling the target product that improves the bio-based product, is focus and the difficult point of current biological technical field research.Whether in fact, can reach or surpass the chemical process level understanding, the development and utilization of microbial oil metabolic process, also be the key that improves the fermenting process economy.The progress of genome sequencing and genetic engineering technique is for understanding and the strain improvement of bacterial strain physiology characteristic provides than the more rational method of traditional induced-mutation technique.

The essence of metabolic engineering is to utilize recombinant DNA technology and other technology, on purpose changes existing metabolism and expression regulation network, understands better and utilize the pathways metabolism of cell.Metabolic engineering can be in cell and molecular level understanding and engineered cells process, and it not only can explain the cell physiological biochemical characteristic, but also can give the new proterties of starting strain and phenotype: (1) enlarges the substrate utilization scope; (2) produce original non-existent new compound; (3) enhancing is to the degradation capability of harmful toxic matter in the environment; (4) improve thalline to the adaptive faculty of environment; (5) generation of blocking-up or reduction byproduct; (6) (Bailey J E.Toward a science ofmetabolic engineering.Science.1991,252 (5013): 1668-1675 such as raising of metabolism products production speed and production performance; Aristidou A,

M.Metabolic engineering applications to renewable resource utilization.Curr.Opin.Biotechnol.2000,11 (2): 187-198).

The circle Rhodosporidium is in Basidiomycota heterothally type fungi, it is a kind of very important microorganism in the fermentation industry, can utilize the hexose and the pentose that come from biomass to be the important bio-based product of raw material production: microbial oil, grease can reach (the Ratledge C more than 60% of dry cell weight in the born of the same parents, WynnJ P.The biochemistry and molecular biology of lipid accumulation in oleaginousmicroorganisms.Adv.Appl.Microbiol.2002,51:1-51; Li Y, Zhao Z, Bai F.High-density cultivation of oleaginous yeast Rhodosporidium toruloides Y4infed-batch culture.Enzyme Microb.Technol.2007,41 (3): 312-317); Industrial enzymes or medicine synthetic usefulness enzyme such as phosphodiesterase, phenylalanine ammonia lyase (Hodgins D S.Yeastphenylalanine ammonia-lyase.Purification, properties, and the identification ofcatalytically essential dehydroalanine.J Biol.Chem.1971,246 (9): 2977-2985; Gilbert H J, Clarke I N, Gibson R K, et al.Molecular cloning of the phenylalanineammonia lyase gene from Rhodosporidium toruloides in Escherichia coli K-12.JBacteriol.1985,161 (1): 314-320), D amino-acid oxidase (Gadda G Negri A, PiloneM S.Reaction of phenylglyoxal with arginine groups in D-amino-acid oxidasefrom Rhodotorula gracilis.J Biol.Chem.1994,269 (27): 17809-17814; Liao G J, Lee Y J, Lee Y H, et al.Structure and expression of the D-amino-acid oxidasegene from the yeast Rhodosporidium toruloides.Biotechnol.Appl.Biochem.1998,27 (Pt 1): 55-61) etc.; β-carotene and exocellular polysaccharide; And in sewage disposal and bio-pharmaceuticals, have more widely and to use.Experimental result shows, this bacterium can utilize five-carbon sugar and hexose to be substrate simultaneously, good stress resistance, directly the accumulation grease take the maize straw acid hydrolysis liquid as carbon source can realize that biomass are to Efficient Conversion (Li Yonghong, the Liu Bo of bio-based product, Sun Yan, Deng. the screening of Oleaginous Yeasts for Broad-Spectrum Carbohydrates Assimilating Capacity. Chinese biological engineering magazine, 2005,25 (12): 39-44).

Although the red winter spore yeast of circle has good industrial production performance, simultaneously, the proteolytic enzyme heterogenous expression in circle red winter spore yeast source (the Pollegioni L that also succeeds, Molla G, Campaner S, Cloning, sequencing and expression in E.coli of a D-amino acid oxidase cDNA fromRhodotorula gracilis active on cephalosporin C.J Biotechnol.1997,58 (2): 115-123; Faulkner J D, Anson J G, Tuite M F, et al.High-level expression of thephenylalanine ammonia lyase-encoding gene from Rhodosporidium toruloidesin Saccharomyces cerevisiae and Escherichia coli using a bifunctionalexpression system.Gene.1994,143 (1): 13-20), but the red winter spore yeast of circle self lacks corresponding genetic operating system.Take the red winter spore yeast of circle as Host Strains, no matter be genetically engineered operation or the improvement of metabolic engineering bacterial strain, all be subject to the restriction of genetic operating system, be difficult to carry out the strain improvement of targeting.

Promotor is essential for genetic operating system.Therefore, if the red winter spore yeast of circle is carried out genetic manipulation, the promotor that acquisition can be worked in the red winter spore yeast of circle is the key link of this work.

Summary of the invention

In view of above-mentioned prior art bottleneck, main purpose of the present invention provides and is used in effective expression foreign gene in the red winter spore yeast of circle, and can be used for being undertaken by gene engineering promotor and the terminator of the improvement of Rhodosporidium toruloides kind.

For realizing purpose of the present invention, the inventor conducts in-depth research the expression conditions in the red winter spore yeast of circle, finds that ura5 is a constitutive expression gene, and its promotor is middle strong promoter.By experiment design in conjunction with methods such as degenerate pcr, chromosome walkings, has successfully obtained to comprise the dna fragmentation of effective promotor from the red winter spore yeast chromosomal dna of circle, finished thus the present invention.

Specifically, the present invention comprises following embodiment (A) to (H)

(A) the present invention relates to a kind of dna fragmentation with the red winter spore yeast transcripting promoter activity of circle, described dna fragmentation have the full sequence of dna sequence dna shown in SEQ ID NO:1 or comprise this dna sequence dna from 3 '-terminal 800bp with interior partial sequence, or have and to play 800bp with interior partial sequence hybridization with whole or its dna sequence dna 3 '-end of sequence shown in SEQ ID NO:1, and the sequence that keeps the transcripting promoter activity, or the deoxynucleoside acid sequence shown in the SEQ ID NO:1 carried out the replacement of one or more bases, disappearance, insertion or interpolation obtain, and have 50% above homology with sequence shown in the SEQ ID NO:1, and the sequence with promoter activity.

(B) the present invention relates to a kind of dna fragmentation of justifying red winter spore yeast by oneself, described dna fragmentation has following feature: (1) dna sequence dna shown in SEQ ID NO:2 whole or comprise the partial sequence of this dna sequence dna 5 '-end; (2) or have can with sequence hybridization shown in (1) and keep sequence such as sequence activity as described in (1).

(C) the present invention relates to a kind of target gene of finishing at the dna molecular of the red winter spore yeast transcription initial sum Transcription Termination of circle, it has simultaneously such as sequence as described in (A) with such as sequence as described in (B), and be positioned at downstream such as sequence as described in (A) such as sequence as described in (B), be adjacent the dna fragmentation of 1-10000 Nucleotide.

(D) the present invention relates to a kind of DNA construct that target gene can be connected with (A)-(C) described any dna molecular, so that the recombinant DNA that target gene can be expressed in the red winter spore yeast of circle.Described target gene is encoding histone nucleic acid or antisense nucleic acid coding nucleic acid.

(E) the present invention relates to any one carrier in the described dna molecular of a kind of carrying (A)-(D).Described carrier can be plasmid vector or cosmid vector.

(F) the present invention relates to have changed over to such as (D) described dna molecular or such as spore yeast of red winter of circle or Rhodosporidium (Rhodosporidium) fungi of carrier as described in (E).

(G) pRtura5 promotor involved in the present invention is characterized in that: it derives from the red winter spore yeast of circle, can start the transcript and expression of goal gene in the red winter spore yeast of circle.

(H) the present invention relates to the dna fragmentation that coding has the active polypeptide of orotate phosphoribosyl transferase (orotatephosphoribosyl transferase), its cDNA sequence has the deoxynucleoside acid sequence shown in SEQ ID NO:3, its genomic dna has the deoxynucleoside acid sequence shown in the SEQ ID NO:5, and its aminoacid sequence is shown in SEQ ID NO:4.

Use and have the dna molecular of promoter activity and the DNA with transcription terminator activity among the present invention, the expression in the red winter spore yeast of circle of foreign gene or native gene be can realize, promotor, terminator and carrier for the red winter spore yeast of genetic engineering circle the invention provides.For the red winter spore yeast of circle has been opened a breeding new way, and therefore can provide spore yeast of red winter of the Novel circular with industrial use.

Description of drawings

Fig. 1 represents the result of the agarose gel electrophoresis of Rtura5 degenerate pcr product.

Fig. 2 represents the result of the agarose gel electrophoresis of pRtura5 promoter dna fragment.

Fig. 3 represents the result of the agarose gel electrophoresis of Rtura5t terminator dna fragmentation.

Fig. 4 represents the result of the agarose gel electrophoresis of Rtura5 promotor-ORF-terminator full length fragment.

Fig. 5 represents that the pRtura5 promotor starts the conformability expression of results of GFPuv in the red winter spore yeast ATCC 10788 of circle.

Fig. 6 represents that the pRtura5 promotor starts bleomycin resistant gene ble in the antibiosis expression of results of red winter spore yeast ATCC10788.

Fig. 7 represents that the pRtura5 promotor starts Geneticin resistant gene kanmx4 in the antibiosis expression of results of red winter spore yeast ATCC 10788.

Fig. 8 represents the cultivation results of the restructuring red winter spore yeast of circle on 5 '-FOA.

Fig. 9 is the structure iron of plasmid pura5gfp.

Figure 10 is the structure iron of plasmid pura5ble.

Figure 11 is the structure iron of plasmid pura5kan.

Figure 12 is the crystal structure model of the red winter spore yeast orotate phosphoribosyl transferase OPRTase of circle, and it shows that three-dimensional structure is correct.

The embodiment that this is bright

In this article, " promotor " refer to be identified by RNA polymerase, in conjunction with and the dna sequence dna of can promotor gene transcribing.Term " promotor " also can be regarded as: comprise 5 ' non-coding region, cis-acting elements (such as enhanser) and other nucleotide sequence that can be combined with transcription factor.

Existence or the intensity of promotor normally represent by promoter activity, its measuring method: the downstream of promotor as described in reporter gene (such as green fluorescent protein) is connected in, and this DNA construct transformed corresponding host cell, the examining report gene expression amount.If people observe the expression that is connected in described promotor downstream reporter gene, just can think that described promotor has activity in the host cell that it transforms.

In this article, " terminator " refers to provide on the karyomit(e) termination signal that RNA polymerase is separated with dna profiling and makes the section of DNA sequence of Transcription Termination.Can check that by the method such as Northern hybridization or RT-PCR the size of institute's transcribe rna confirms the existence of terminator.Or make the reporter gene effective expression determine the activity of terminator by " promotor-reporter gene-terminator " construct.

" the red winter spore yeast of circle " among the present invention comprises any diploid and the monoploid, wild type strain and the auxotrophic strain that belong to these " species "." Rhodosporidium fungi " among the present invention is not specifically limited, and its example comprises the fungi that belongs to this genus, except the red winter spore yeast of circle, such as Rhodosporidium azoricum, Rhodosporidium babjevae, Rhodosporidiumsphaerocarpum.

" goal gene " of the present invention comprises albumen coded sequence, sense-rna encoding sequence and the nuclease encoding sequence that can express in the red winter spore yeast of circle.The example of the albumen coded sequence that can express in the red winter spore yeast of circle comprises the nucleotide sequence that comes from the red winter spore yeast of circle, and is not limited to this.Goal gene of the present invention also comprises the albumen coded sequence that derives from other microorganism, plant and animal.

Promotor among the present invention has the whole of shown in SEQ ID NO:1 dna sequence dna or comprises the partial sequence of this dna sequence dna 3 '-end, or have can with sequence partial sequence hybridization and that keep the transcripting promoter activity of whole or its dna sequence dna 3 '-end of sequence shown in SEQ ID NO:1, or the deoxynucleoside acid sequence shown in the SEQ ID NO:1 carried out replacement, disappearance, insertion or the interpolation of one or more bases obtain, have 50% above homology and have the sequence of promoter activity with sequence shown in the SEQ ID NO:1.

Terminator among the present invention has such as the whole of the dna sequence dna shown in SEQ ID NO:2 or comprises the partial sequence of this dna sequence dna 5 '-end, or have can with sequence partial sequence hybridization and that keep the transcription terminator activity of whole or its dna sequence dna 5 '-end of sequence shown in SEQ ID NO:2, or the deoxynucleoside acid sequence shown in the SEQ ID NO:2 carried out replacement, disappearance, insertion or the interpolation of one or more bases obtain, have 50% above homology with sequence shown in the SEQ ID NO:2 and have the sequence of terminator activity.

The construct of the construct of the promotor-goal gene among the present invention, the construct of goal gene-terminator or promotor-goal gene-terminator, can directly or through carrier mediated conversion justify red winter spore yeast, so that destination gene expression, can the preferred plasmid carrier as the mediation carrier.

Promotor of the present invention can be separated from the red winter spore yeast of circle according to following aspect.

The invention will be further described below in conjunction with drawings and Examples, will help those of ordinary skill in the art to understand the present invention, but not limit in any form the present invention.Synthetic and the examining order of all primers is then finished by Dalian TaKaRa company if no special instructions among the following embodiment.

Embodiment 1: the extraction of the red winter spore yeast ATCC 10788 total RNA of circle

With spore yeast R.toruloides of red winter of fresh circle ATCC 10788 (available from the biological product of USS collecting center, ATCC) by inclined plane inoculating in 50ml YEPD liquid nutrient medium, cultivate 24h in 30 ℃ of shaking tables, with 1: 50 volume ratio bacterium liquid is transferred to respectively in the 100ml YEPD liquid nutrient medium again, cultivates 12h in 30 ℃ of shaking tables and reach logarithmic phase.Under 4 ℃, the centrifugal 4min of 5000rpm collects thalline, with the rapid freezing thalline of liquid nitrogen, grinds broken wall.Use the TaKaRa RNAiso of company test kit, and extract total RNA according to its standard step.

RNA carries out 1.5% agarose gel electrophoresis, uses fluorescence-uv analyzer to observe and identifies, as seen two bands clearly.With the total RNA sample of ultraviolet/visible light spectrometer analysis, record OD ₂₆₀/ OD ₂₈₀=2.01, show that total RNA quality is fine.Total RNA sample is frozen in-80 ℃, for subsequent use.

Embodiment 2: the synthetic and ura5 degenerate pcr of red winter spore yeast ATCC 10788 cDNA the first chain of circle

Take the red winter spore yeast R.toruloides ATCC 10788 total RNA of circle as template, cDNA the first chain is synthesized in reverse transcription.At first, with the total RNA of 1.0 μ l (about 2 μ g), 1.0 μ l primer SMART IV:5 '-AAGCAGTGGTATCAACGCAGAGTGGCCATTACGGCCGGG-3 ' and 1.0 μ loligo dT-joint primer CDS III/3 ': 5 '-ATTCTAGAGGCCGAGGCGGCCGACATG-d (T) _30N-1N-3 ', (diethylpyrocarbonate is processed water 2.0 μ l DEPC processes water, available from Dalian TaKaRa company), join mixing in the PCR pipe, in 72 ℃ of insulation 2min, place immediately cooled on ice 2min, with 2.0 μ l, 5 * first strandbuffer, 1.0 μ l DTT (20mM), 1.0 μ l dNTP (10mM), 1.0 μ l powerscript reversetranscriptase (Clontech company) joins in the system mixing.In 42 ℃ of extension 60min, last 4 ℃ are finished reaction, are stored in-20 ℃, for subsequent use.

The orotate phosphoribosyl transferase aminoacid sequence of other source of species of announcing according to NCBI, obtain its relative conservative region by sequence alignment, and utilize this conservative region design to synthesize two degenerated primers, ura5-sense:5 '-ATGAG (AGCT) GC (AGCT) AC (AGCT) TCCTA (AGCT) GC-3 ' and ura5-anti:5 '-CTA (AGCT) T (CT) be AC (AG) CC (AGCT) CACT-3 ' (AG), take synthetic cDNA the first chain of reverse transcription as template, carry out the degenerate pcr amplification of Rtura5 gene, 10 * PCR damping fluid, 5.0 μ l, dNTPs (10mM) 1.0 μ l, ura5-sense primer (50mmol/l) 1.0 μ l, ura5-anti primer (50mmol/l) 1.0ul, rTaq enzyme (Dalian TakaRa) 0.5 μ l, synthetic cDNA the first chain template 1.0 μ l, ddH ₂O 40.5 μ l, in 94 ℃ of insulation 3min, then in 94 ℃ of 30s, 57 ℃ of 45s, 72 ℃ of 1min, 35 circulations, 72 ℃ of 10min, 4 ℃ are finished reaction.Amplified production carries out 1% (mass/volume concentration) agarose gel electrophoresis, observes the band (Fig. 1) about 0.8kb, utilizes DNA to reclaim test kit (available from the green skies), according to supplier's proposed steps purified pcr product.The method that the PCR product provides with reference to TaKaRa company is cloned into pMD18-T carrier (available from TaKaRa), be transformed into E.coli DH5 α competent cell, wherein competent cell is by Calcium Chloride Method (the molecular cloning experiment guide third edition, Pehanorm Brooker work, Huang Peitang etc. translate, and Science Press publishes) preparation.Select Amp resistance transformant and increase bacterium cultivation, plasmid extraction.The recombinant plasmid sample is delivered to TaKaRa company order-checking, and sequence results infers that the aminoacid sequence that analyzes through Blastp, turns out to be the orotate phosphoribosyl transferase sequence, shown in SEQ ID NO:4 sequence.Orotate phosphoribosyl transferase cDNA sequence is shown in SEQ ID NO:3 sequence.

The amplification of embodiment 3:Rtura5 gene genomic dna sequence

1.R.toruloides ATCC 10788 is (available from the biological product of USS collecting center, ATCC) extracting genome DNA adopts granulated glass sphere broken wall method (the fine works molecular biology experiment guide third edition the 13rd chapter, the work such as Ao Sibai, Yan Ziying etc. translate, Science Press publishes).The genomic dna for preparing utilizes Nanodrop 1000 to measure, and records OD ₂₆₀/ OD ₂₈₀=1.85, show that the genomic dna quality is fine.Concentration is 120ng/ μ l, totally 500 μ l, and genome DNA sample is frozen in-20 ℃, and is for subsequent use.

2. according to the orotate phosphoribosyl transferase cDNA sequence that obtains among the embodiment 2, design 1 pair of gene-specific primer, ura5-ORF-p1:5 '-ATGAGCGCCACGTCCTACGC-3 ' and ura5-ORF-p2:5 '-CTAGTTAACGCCCCACTTTGCG-3 ', take the circle red winter spore yeast ATCC 10788 genomic dna as template, carry out pcr amplification according to ordinary method, obtain the PCR product (figure slightly) of about 0.8kb.Pcr amplification product reclaims, is cloned into the pMD18-T carrier according to the operation steps of embodiment 2, and checks order, and obtains the dna sequence dna shown in sequence table SEQ ID NO:5.Through with embodiment 2 in the orotate phosphoribosyl transferase cDNA sequence alignment that obtains, confirm that this gene fragment is its orotate phosphoribosyl transferase genomic dna sequence, intronless.

Embodiment 4: chromosome walking obtains Rtura5 gene 5 ' flank sequence (promotor)

Present embodiment utilizes Genome Walking Kit (available from TaKaRa) to finish.

According to the ura5 genomic dna sequence that obtains among the embodiment 3, design 3 Specific Primer (gene-specific primer), ura5-SP1:5 '-AGCGACGAGGGGGATGCCCTTGT-3 ', ura5-SP2:5 '-GTTGAAGAAGTAGGGCGAGGAGC-3 ' and ura5-SP3:5 '-GAGAGCGGTCTCGATGATCGAG-3 ', as downstream primer, carry out following operation according to the test kit specification sheets.

1.1 ^StThe PCR reaction

Genomic dna refining in the embodiment 3 carries out first round amplification as template.Reaction system 50 μ l:10 * LA PCR buffer II (Mg ²⁺Plus) 5.0 μ l, dNTPs (2.5mmol/l) 8.0 μ l, LA Taq archaeal dna polymerase (5U/ μ l, Dalian TakaRa) 1.0 μ l, AP 1Primer (100 μ mol/l, Dalian TakaRa) 1.0 μ l, ura5-SP1 (10 μ mol/l) 1.0 μ l, R.toruloides ATCC 10788 genomic dna templates (120ng/ μ l) 1.0 μ l, ddH ₂O adds to 50 μ l.Reaction conditions: carry out first the high specific reaction of 5 high temperature anneal temperature, then carry out the low specific reaction of 1 utmost point low temperature thermal oxidation; Then carry out hot asymmetric PCR: the high specific reaction of 2 high annealing temperatures (65 ℃) and the low specific reaction alternate cycles of 1 low temperature thermal oxidation (44 ℃), totally 15 times.Design parameter is as follows: 94 ℃ of 1min, 98 ℃ of 1min; 94 ℃ of 30s, 65 ℃ of 1min, 72 ℃ of 2min, totally 5 circulations; 94 ℃ of 30s, 25 ℃ of 3min, 72 ℃ of 2min; 94 ℃ of 30s, 65 ℃ of 1min, 72 ℃ of 2min, 94 ℃ of 30s, 65 ℃ of 1min, 72 ℃ of 2min, 94 ℃ of 30s, 44 ℃ of 1min, 72 ℃ of 2min, totally 15 circulations; 72 ℃ of 10min finish reaction.

2.2 ^NdThe nest-type PRC reaction

Reaction system 50 μ l:10 * LA PCR buffer II (Mg ²⁺Plus) 5.0 μ l, dNTPs (2.5mmol/l) 8.0 μ l, LA Taq archaeal dna polymerase (5U/ μ l, Dalian TakaRa) 1.0 μ l, AP1 Primer (100 μ mol/l, Dalian TakaRa) 1.0 μ l, 1 ^StPCR reaction product 1.0 μ l, ura5-SP2 (10 μ mol/l) 1.0 μ l, ddH ₂O adds to 50 μ l.Reaction conditions: 94 ℃ of 30s, 65 ℃ of 1min, 72 ℃ of 2min, 94 ℃ of 30s, 65 ℃ of 1min, 72 ℃ of 2min, 94 ℃ of 30s, 44 ℃ of 1min, 72 ℃ of 2min, totally 15 circulations; 72 ℃ of 10min finish reaction.

3.3 ^RdThe nest-type PRC reaction

Reaction system 50 μ l:10 * LA PCR buffer II (Mg ²⁺Plus) 5.0 μ l, dNTPs (2.5mmol/l) 8.0 μ l, LA Taq archaeal dna polymerase (5U/ μ l, Dalian TakaRa) 1.0 μ l, AP1 Primer (100 μ mol/l, Dalian TakaRa) 1.0 μ l, 2 ^NdNest-type PRC reaction product 1.0 μ l, ura5-SP3 (10 μ mol/l) 1.0 μ l, ddH ₂O adds to 50 μ l.Reaction conditions: 94 ℃ of 30s, 65 ℃ of 1min, 72 ℃ of 2min, 94 ℃ of 30s, 65 ℃ of 1min, 72 ℃ of 2min, 94 ℃ of 30s, 44 ℃ of 1min, 72 ℃ of 2min, totally 15 circulations; 72 ℃ of 10min finish reaction.

3 ^RdThe nest-type PRC reaction product is cut the purpose band behind 1% (mass/volume concentration) agarose gel electrophoresis, utilize dna fragmentation gel-purified test kit (available from the green skies) to carry out purifying.Dna fragmentation behind the purifying inserts pMD18-T carrier (available from TakaRa company) through the TA clone, transforms DH5 α competent cell; Wherein competent cell is by Calcium Chloride Method (Huang Peitang etc. translate for the molecular cloning experiment guide third edition, Pehanorm Brooker work, and Science Press publishes) preparation.Select Amp resistance transformant and increase bacterium cultivation, plasmid extraction.The recombinant plasmid sample is delivered to the order-checking of TaKaRa company, obtains the dna sequence dna shown in SEQ ID NO:1, turns out to be the pRtura5 gene order of expection.

Sequence number: 1 (SEQ ID NO:1)

Sequence length: 1000bp

Sequence type: DNA

Source: circle red winter spore yeast (Rhodosporidium toruloides)

1 TGCCGCCTAT CTGTTAAAAC TCAATGGATA CAATGCAAAG TCGGCTCGCG CCCTCTTCTC

61 CCGTTCCCCC GTACCTAGAC ATGCGTCGTG AGTGCTTCCC TCCGTCATGC AACCGTCGCA

121 AGCTCCTCGC TCCGTCGTCG CTGCTGAAGC TGCTCCTGCG CAATCCGCTG CGCGGCGGGT

181 ACCTCGCCTC GCTCCCTTTC CGCCGCCCGA ATCGCCTGTG TGAGCCTGTT CCTCCGAGAC

241 ACGACCATCG TCTTGGGATT GGCTTGTGGT ACGGGCAGAG GGCTGGTCTC TGCAATTGAG

301 CAAGCGAGCC GAGGCAGCGA TGGTCAGTTC CGGTCGCAGA CCGAGGCTCC AGGGAAGGGA

361 CGGCGGGACG CACTGATGAC GCCGGCCTGA ATGAGCAGCT TCTCAAAGCG CTTTGTCGGC

421 AGCGCACCCT GGCTGAGCCA GTACCGGACC CGCTCCAGGT TCCACTCGAC CTTCTTCTGC

481 CCGACCTTGG CGACGCCCGT GTTGGGCGTG TGCTGCCGTG GTCCCCACTC CTTCGGGTCC

541 CTGACCTGAC CGTTTGGCGA GACCGACGGC GCTGGCACGA CGACCGGTCG CGGGTTGTAC

601 GAACCGAGGA GCTCGAGGGG TTTGGCGGTC TGGCGCTGCG GCGCGCGGAT GGCGACGAGC

661 GAGTAGACGG GGTTGTTGCG CGTGCAGTGG TGGCGCGCGA GGCGCAATCG GACCGACATC

721 TCGTGCGCTG GCGGAGAGCG AGTGTGCGAG GACGAGGAAA AGGAGCGGGA GGAGGAGGGT

781 TGGGAGCGAA CCACCTCGTG CAGCTGGACT GGACGCGCTA GACGACTGCC AGACTCGCTA

841 TACTGCTCTT TACATCCGCT AGAATCCTGC ACCCGCTCGA ACCAGCTCCT CCCACCGTCA

901 GTTCGCCTCC CCCTCTCATC CTCATCTCTC AAAGGACTTC CTCGCTCGCA ACTCGCTGGA

961 GGGAGCTGCA AGACTGACAC GAGGTCGAAA ACACCGTCAG

Embodiment 5: chromosome walking obtains Rtura5 gene 3 ' flank sequence (terminator)

Present embodiment also is to utilize Genome Walking Kit (available from TaKaRa) to finish.

According to the ura5DNA sequence that obtains among the embodiment 3, design 3 Specific Primer (gene-specific primer), be respectively ura5-SP11:5 '-CACGACGAGGACGTGATATCGGCT-3 ', ura5-SP22:5 '-GAGGGCGGTCAGACGGCGGGTATT-3 ' and ura5-SP33:5 '-CGTCGTCAAGATGCGCGACATCGTT-3 ', as upstream primer, carry out the operation of 3 ' flank chromosome walking according to the test kit specification sheets, remove Specific Primer respectively by ura5-SP1, ura5-SP2, ura5-SP3 is replaced by ura5-SP11 successively, ura5-SP22, outside the ura5-SP33, the other the same as in Example 4.

3 ^RdThe nest-type PRC reaction product utilizes dna fragmentation gel-purified test kit (available from the green skies) to carry out purifying, inserts pMD18-T carrier (available from TakaRa company) through the TA clone, transforms DH5 α competent cell; Wherein competent cell is by Calcium Chloride Method (Huang Peitang etc. translate for the molecular cloning experiment guide third edition, Pehanorm Brooker work, and Science Press publishes) preparation.Select Amp resistance transformant and increase bacterium cultivation, plasmid extraction.The recombinant plasmid sample is delivered to the order-checking of TaKaRa company, obtains the dna sequence dna shown in SEQ ID NO:2, turns out to be the Rtura5t gene order of expection.

Embodiment 6:Rtura5 promotor-open reading frame-terminator full-length gene obtains

According to the promotor and the terminator sequence that obtain among embodiment 4 and the embodiment 5, the redesign pair of primers carries out the amplification of Rtura5 " promotor-open reading frame-terminator " full-length gene.pRtura5t-p1：5’-TGCCGCCTATCTGTTAAAACTCAATG-3’，pRtura5t-p2：5’-GCCATTCAATCTGTTCAGCCACCC-3’。The R.toruloides genomic dna of preparation carries out pcr amplification as template in the embodiment 3.PCR system (50 μ l): 10 * Speed buffer, 5.0 μ l, dNTPs (10mmol/l) 1.0 μ l, upstream primer Rtura5-p1 (10 μ mol/l) 2.0 μ l, downstream primer Rtura5-p2 (10 μ mol/l) 2.0 μ l, SpeedSTAR ^TMHS archaeal dna polymerase (amplification rate is fast, and 1kb/10s is available from Dalian TaKaRa company) 0.5 μ l, genomic dna template (120ng/ μ l) 2 μ l, ddH ₂O adds to 50 μ l.Reaction conditions: 98 ℃ of 1min, 98 ℃ of 10s, 65 ℃ of 1.0min, 35 circulations, 72 ℃ of 10min, 4 ℃ are finished reaction.The PCR product utilizes PCR fragment purification test kit (available from the green skies) to carry out purifying after 1% (mass/volume concentration) agarose gel electrophoresis analysis.Fragment is inserted pMD18-T carrier (available from TakaRa company) through the TA clone, transforms DH5 α competent cell; Wherein competent cell is by Calcium Chloride Method (Huang Peitang etc. translate for the molecular cloning experiment guide third edition, Pehanorm Brooker work, and Science Press publishes) preparation.Select Amp resistance transformant and increase bacterium cultivation, plasmid extraction.The recombinant plasmid sample is delivered to the order-checking of TaKaRa company, obtains the dna sequence dna shown in SEQ ID NO:6, turns out to be the pRtura5t full length sequence of expection, this recombinant vectors called after T-pRtura5t.

Embodiment 7: the structure of the red winter spore yeast specificity egfp expression box ura5gfp of circle

The structure utilization of the red winter spore yeast specificity egfp expression box of ura5gfp circle be RF clone (Van den Ent, F., Lowe, J., 2006.RF cloning:A restriction-free method forinserting target genes into plasmids.J.Biochem.Biophys.Methods 67:67-74; Yang F, Zhang S, Tang W, Zhao Z, 2008.Identification of theorotidine-5 '-monophosphate decarboxylase gene of the oleaginous yeastRhodosporidium toruloides.Yeast 25 (9): method 623-630).

The amplification of pRtura5t full length sequence and clone see embodiment 6.

1. the acquisition of green fluorescent protein encoding gene GFPuv

Take pGFPuv plasmid (available from BD Biosciences) as template, utilize pair of primers gfp-p1:5 '-ATGAGTAAAGGAGAAGAACT-3 ' and gfp-p2:5 '-TCATTTGTAGAGCTCATCCAT-3 ', carry out pcr amplification.System (50 μ l): 5 * Primebuffer, 10.0 μ l, dNTPs (2.5mmol/l) 4.0 μ l, upstream primer gfp-p1 (10 μ mol/l) 2.0 μ l, downstream primer gfp-p2 (10 μ mol/l) 2.0 μ l, PrimeSTAR ^TMHS archaeal dna polymerase (Dalian TakaRa) 1.0 μ l, pGFPuv plasmid (120ng/ μ l) 1 μ l, ddH ₂O adds to 50 μ l.Reaction conditions: 95 ℃ of 3min, 98 ℃ of 8s, 49 ℃ of 15s, 72 ℃ of 1min, 35 circulations, 72 ℃ of 10min, 4 ℃ are finished reaction.The PCR reaction product utilizes dna fragmentation glue to reclaim the purification kit purifying, utilize the taq archaeal dna polymerase to carry out dna fragmentation 3 ' end and add A, system (50 μ l): 10 * PCRbuffer, 5.0 μ l, dNTPs (2.5mmol/l) 4.0 μ l, GFPuv dna fragmentation 30 μ l behind the purifying, ddH ₂O adds to 50 μ l.Reaction conditions: 72 ℃ of 30min, 4 ℃ are finished reaction.The GFPuv dna fragmentation that 3 ' end adds behind the A utilizes dna fragmentation glue to reclaim the purification kit purifying, is cloned into the pMD18-T carrier, send the order-checking of TaKaRa company, obtains the dna sequence dna shown in SEQ ID NO:7, turns out to be the GFPuv gene order of expection.

2. reference literature method (Van den Ent, F., Lowe, J., 2006.RF cloning:Arestriction-free method for inserting target genes into plasmids.J.Biochem.Biophys.Methods 67:67-74), design RF clone primer: ura5-gfp-p1:5 '-CACGAGGTCGAAAACACCGTCAG atgagtaaaggagaagaact-3 ' and ura5-gfp-p2:5 '-GGAACTGCCCAGACGCACTTGTAT ctatttgtagagctcatccat-3 ' (wherein original ura5ORF flank sequence is complementary in capitalization partial sequence and the pRtura5t cloning vector, and lowercase partial sequence and green fluorescent protein GFPuv ORF are complementary).

3.RF I reaction system and flow process: the GFPuvTA cloning vector that makes up in the present embodiment action-item 1 utilizes ura5gfp-p1 and ura5gfp-p2 to be primer as template, carries out the RF first round and increases.System (50 μ l): 5 * Prime buffer, 10.0 μ l, dNTPs (2.5mmol/l) 4.0 μ l, upstream primer (10 μ mol/l) 2.0 μ l, downstream primer (10 μ mol/l) 2.0 μ l, PrimeSTAR ^TMHS archaeal dna polymerase (Dalian TakaRa) 1.0 μ l, T-GFPuv plasmid (100ng/ μ l) 1 μ l, ddH ₂O adds to 50 μ l.Reaction conditions: 95 ℃ of 3min, 98 ℃ of 8s, 49 ℃ of 15s, 72 ℃ of 1min, 30 circulations, 72 ℃ of 10min, 4 ℃ are finished reaction.RF I reaction product utilizes dna fragmentation glue to reclaim the purification kit purifying, and-20 ℃ save backup.

4.RF II reaction: 5 * Prime buffer, 10.0 μ l, dNTPs (2.5mmol/l) 4.0 μ l, T-pRtura5t plasmid (100ng/ μ l) the 1.0 μ l that make up among the embodiment 6, RFI reaction product in the present embodiment abovementioned steps (100ng/ μ l) 5.0 μ l, PrimeSTAR ^TMHS archaeal dna polymerase (Dalian TakaRa) 1.0 μ l, ddH ₂O adds to 50 μ l.Reaction conditions: 95 ℃ of 3min, 68 ℃ of 12min, 95 ℃ of 30s afterwards, 65 ℃ of 45s (1 ℃/cyc), 68 ℃ of 12min, next 15 circulations are carried out taking turns: 95 ℃ of 30s, 55 ℃ of 45s again, 68 ℃ of 12min, 20 circulations, 72 ℃ of 10min, 4 ℃ are finished reaction.

5.DpnI digestion and electric shock transform: get 8 μ l RF II reaction product add behind 1 μ l DpnI (available from New England Biolabs) and the 1 μ l DpnI buffer mixing 37 ℃ act on 120min and remove former T-pRtura5t plasmid after, get respectively 2 μ l electric shock and transform DH5 α competent cell, competent cell is by standard method preparation (the molecular cloning experiment guide third edition, Pehanorm Brooker work, Huang Peitang etc. translate, Science Press publishes), electric shock Transformation Parameters: 2200-2500V, 400 Ω, 25 μ F, 0 ℃.Select Amp resistance transformant and increase bacterium cultivation, plasmid extraction, and utilize RF I reaction the primer ura5gfp-p1 and ura5gfp-p2 to carry out bacterium colony PCR and identify, identify that positive recombinant vectors send TaKaRa to check order, obtain the ura5gfp expression cassette that 5 ' end and 3 ' end are respectively ura5 promotor and ura5 terminator, simultaneously, this recombinant vectors called after pura5gfp.GFPuv ORF sequence is shown in SEQ ID NO:7; Complete ura5gfp expression cassette is shown in SEQ ID NO:8.

Embodiment 8: utilize the ura5gfp expression cassette to carry out the Rtura5 gene knockout egfp expression of holding concurrently

1.Rtura5gfp knock out the preparation of box

The pura5gfp carrier that makes up take embodiment 7 is as template, and the oligonucleotide sequence pRtura5t-p1 in the embodiment 6 and pRtura5t-p2 carry out a large amount of preparations that Rtura5gfp knocks out box as primer.PCR system (500 μ l): 10 * Speed buffer, 50.0 μ l, dNTPs (10mmol/l) 10.0 μ l, upstream primer (10 μ mol/l) 20.0 μ l, downstream primer (10 μ mol/l) 20.0 μ l, SpeedSTAR ^TMHS archaeal dna polymerase (Dalian TaKaRa company) 5.0 μ l, genomic dna template (120ng/ μ l) 15.0 μ l, ddH ₂O adds to 500 μ l.Reaction conditions: 98 ℃ of 1min, 98 ℃ of 10s, 65 ℃ of 60s, 35 circulations, 72 ℃ of 10min, 4 ℃ are finished reaction.The PCR product utilizes PCR fragment purification test kit (available from the green skies) to carry out purifying after 1% (mass/volume concentration) agarose gel electrophoresis analysis.Dna fragmentation concentration behind the purifying is at 380ng/ μ l, totally 50 μ l, and-20 ℃ save backup.

2. the red winter spore yeast ATCC 10788 competent cells preparation of monoploid circle

The preparation of R.toruloides np11 competent cell: R.toruloides ATCC 10788 (available from the biological product ATCC of collecting center of USS) bacterial strain is chosen colony inoculation 10ml YEPD substratum (glucose 20.0g/l, yeast extract 10.0g/l, peptone 20.0g/l, pH 6.0), 30 ℃, 200rpm cultivates 20h; 1: 50 ratio of culture fresh YEPD substratum of transferring, 100ml (500ml Erlenmeyer flask, liquid amount 100ml), 30 ℃, 200rpm cultivates 6-9h, and the OD value reaches 0.6-1.2; Culture ice bath 10-30min, 4 ℃, the centrifugal 5min of 4000rpm abandons supernatant; 0 ℃ of aseptic Milli-Q washes 1 time; 0 ℃ of 1mol/l sorbyl alcohol washs 2 times; Ice bath, for subsequent use.

3. justifying the electric shock conversion of red winter spore yeast ATCC 10788 and the PCR of transformant identifies

The electric shock that Rtura5gfp knocks out box transforms: get 100 μ l R.toruloides ATCC, 10788 competent cells, add Rtura5gfp and knock out box 5 μ l (altogether 2 μ g), ice bath 5min behind the mixing, move in the electric shock cup that is chilled in advance 0 ℃, voltage 0.8-2.0 kilovolt, resistance 200 Ω, electric capacity 25 μ F, time 4-10ms; Add immediately 1ml YEPD after the electric shock, 30 ℃ of incubation 1-2h; Coating YEPD is dull and stereotyped, and 10 μ l/ are dull and stereotyped, cultivates 28-36h for 30 ℃; Choose one by one mono-clonal and utilize fluorescent microscope to carry out microscopy, the fluorescence photograph of positive recombinant ATCC 10788 Δ ura5::gfp as shown in Figure 5.

The red winter spore yeast ATCC 10788 Δ ura5::gfp YEPD of 3 strains restructuring circle cultivate the culture of 24h, utilize physiological saline (0.9%NaCl damping fluid) to carry out doubling dilution, select its 10 ^-3, 10 ^-4, 10 ^-5, 10 ^-6Four extent of dilution pipette respectively 10 μ l bacterium liquid in the SC solid medium that contains 5 '-FOA (5 '-fluororotic acid is available from Shanghai Jinhe Biotechnology Co., Ltd) (0.2%5 '-FOA, glucose 70g/L, (NH ₄) ₂SO ₄0.1g/L, yeast powder 0.75g/L, KH ₂PO ₄0.4g/L, MgSO ₄7H ₂O1.5g/L, pH 6.0) flat board, treat that liquid-absorbent is complete, be inverted in 30 ℃ and cultivated 2 days, find that the red winter spore yeast ATCC 10788 Δ ura5::gfp of restructuring circle possess 5 '-FOA resistance, the red winter spore yeast ATCC 10788 of control strain circle is then without colony growth.

Above presentation of results, Rtura5gfp knock out box (expression cassette) when starting the conformability of green fluorescent protein in the red winter spore yeast of circle and expressing, and the orotidine-5′-phosphate decarboxylase of can deactivation integrating position ura5 genes encoding is active.Like this be designed with the genetic manipulation that is beneficial to the later stage.

Embodiment 9: the structure of the red winter spore yeast specificity bleomycin resistance expression box ura5ble of circle

The structure of the red winter spore yeast specificity bleomycin expression cassette of ura5ble circle is the method for utilizing the RF clone equally.The amplification of pRtura5t full length sequence and clone see embodiment 6.

1. reference literature method (Van den Ent, F., Lowe, J., 2006.RF cloning:Arestriction-free method for inserting target genes into plasmids.J.Biochem.Biophys.Methods 67:67-74), design RF clone primer: ura5-ble-p1:5 '-CACGAGGTCGAAAACACCGTCAG atggccaagttgaccagtgccgtt-3 ' and ura5-ble-p2:5 '-GGAACTGCCCAGACGCACTT GTATctagtcctgctcctcggccacg-3 ' (wherein original ura5ORF flank sequence is complementary in capitalization partial sequence and the pRtura5t cloning vector, and the lowercase part is complementary with bleomycin resistant gene ble ORF).

2.RF I reaction system and flow process: take pPICZ α A plasmid (available from Invitrogen company) as template, utilize ura5ble-p1 and ura5ble-p2 to be primer, carry out the RF first round and increase.System (50 μ l): 5 * Prime buffer, 10.0 μ l, dNTPs (2.5mmol/l) 4.0 μ l, upstream primer (10 μ mol/l) 2.0 μ l, downstream primer (10 μ mol/l) 2.0 μ l, PrimeSTAR ^TMHS archaeal dna polymerase (Dalian TakaRa) 1.0 μ l, pPICZ α A plasmid (100ng/ μ l) 1 μ l, ddH ₂O adds to 50 μ l.Reaction conditions: 95 ℃ of 3min, 98 ℃ of 8s, 49 ℃ of 15s, 72 ℃ of 1min, 30 circulations, 72 ℃ of 10min, 4 ℃ are finished reaction.RF I reaction product utilizes dna fragmentation glue to reclaim the purification kit purifying, and-20 ℃ save backup.

3.RF II reaction: 5 * Prime buffer, 10.0 μ l, dNTPs (2.5mmol/l) 4.0 μ l, T-pRtura5t plasmid (100ng/ μ l) the 1.0 μ l that make up among the embodiment 6, RFI reaction product in the present embodiment abovementioned steps (100ng/ μ l) 5.0 μ l, PrimeSTAR ^TMHS archaeal dna polymerase (Dalian TakaRa) 1.0 μ l, ddH ₂O adds to 50 μ l.Reaction conditions: 95 ℃ of 3min, 68 ℃ of 12min, 95 ℃ of 30s afterwards, 65 ℃ of 45s (1 ℃/cyc), and 68 ℃ of 12min, 15 circulations, next carry out again taking turns: 95 ℃ of 30s, 55 ℃ of 45s, 68 ℃ of 12min, 20 circulations, 72 ℃ of 10min, 4 ℃ are finished reaction.

4.DpnI digestion and electric shock transform: get 8 μ l RF II reaction product add behind 1 μ l DpnI (available from New England Biolabs) and the 1 μ l DpnI buffer mixing 37 ℃ act on 120min and remove former T-pRtura5t plasmid after, get respectively 2 μ l electric shock and transform DH5 α competent cell, competent cell is by standard method preparation (the molecular cloning experiment guide third edition, Pehanorm Brooker work, Huang Peitang etc. translate, Science Press publishes), electric shock Transformation Parameters: 2200-2500V, 400 Ω, 25 μ F, 0 ℃.Select Amp resistance transformant and increase bacterium cultivation, plasmid extraction, and utilize RF I reaction the primer ura5ble-p1 and ura5ble-p2 to carry out bacterium colony PCR and identify, identify that positive recombinant vectors send TaKaRa to check order, obtain the ura5ble expression cassette that 5 ' end and 3 ' end are respectively ura5 promotor and ura5 terminator, simultaneously, this recombinant vectors called after pura5ble.Ble ORF sequence is shown in SEQ ID NO:9; Complete ura5ble expression cassette is shown in SEQ ID NO:10.

Embodiment 10: utilize the ura5ble expression cassette to carry out the Rtura5 gene knockout bleomycin resistance expression that holds concurrently

1.Rtura5ble knock out the preparation of box

The pura5ble carrier that makes up take embodiment 9 is as template, and the oligonucleotide sequence pRtura5t-p1 in the embodiment 6 and pRtura5t-p2 carry out a large amount of preparations that Rtura5ble knocks out box as primer.PCR system (500 μ l): 10 * Speed buffer, 50.0 μ l, dNTPs (10mmol/l) 10.0 μ l, upstream primer (10 μ mol/l) 20.0 μ l, downstream primer (10 μ mol/l) 20.0 μ l, SpeedSTAR ^TMHS archaeal dna polymerase (Dalian TaKaRa company) 5.0 μ l, genomic dna template (120ng/ μ l) 15.0 μ l, ddH ₂O adds to 500 μ l.Reaction conditions: 98 ℃ of 1min, 98 ℃ of 10s, 65 ℃ of 60s, 35 circulations, 72 ℃ of 10min, 4 ℃ are finished reaction.

The PCR product utilizes PCR fragment purification test kit (available from the green skies) to carry out purifying after 1% (mass/volume concentration) agarose gel electrophoresis analysis.Dna fragmentation concentration behind the purifying is at 300ng/ μ l, totally 50 μ l, and-20 ℃ save backup.

2. the red winter spore yeast ATCC 10788 competent cells preparation of monoploid circle

The preparation of R.toruloides np11 competent cell is with the action-item 3 among the embodiment 8.

3. justifying the electric shock conversion of red winter spore yeast ATCC 10788 and the PCR of transformant identifies

The electric shock that Rtura5ble knocks out box transforms: get 100 μ l R.toruloides ATCC, 10788 competent cells, add Rtura5ble and knock out box 10 μ l (altogether 3 μ g), move in the electric shock cup that is chilled in advance 0 ℃ behind the mixing, voltage 0.8-2.0 kilovolt, resistance 200 Ω, electric capacity 25 μ F, time 4-10ms; Add immediately 1ml YEPD after the electric shock, 30 ℃ of incubation 1-2h; Coating contains the YEPD flat board of 20 μ g/ml Zeocin (available from Invitrogen company), and 200 μ l/ are dull and stereotyped, cultivates 2-10d, has approximately 100 transformants to occur successively for 30 ℃.

3 Zeocin resistances of picking transformant is cultivated 24h in the YEPD substratum, culture utilizes physiological saline (0.9%NaCl damping fluid) to carry out doubling dilution, select its 10 ^-3, 10 ^-4, 10 ^-5, 10 ^-6Four extent of dilution pipette respectively 10 μ l bacterium liquid spottings in the SC solid medium that contains 5 '-FOA (available from Shanghai Jinhe Biotechnology Co., Ltd) (0.2%5 '-FOA, glucose 70g/L, (NH ₄) ₂SO ₄0.1g/L, yeast powder 0.75g/L, KH ₂PO ₄0.4g/L, MgSO ₄7H ₂O 1.5g/L, pH 6.0) flat board, after liquid-absorbent is complete, be inverted cultivation 3 days in 30 ℃, find that the red winter spore yeast ATCC 10788 Δ ura5::ble of restructuring circle possess 5 '-FOA resistance, the red winter spore yeast ATCC10788 of control strain circle is then without colony growth.

Above presentation of results, Rtura5ble knock out box (expression cassette) when starting the bleomycin resistant gene conformability is expressed in the red winter spore yeast of circle, and the orotidine-5′-phosphate decarboxylase of can deactivation integrating position ura5 genes encoding is active.Like this be designed with the genetic manipulation that is beneficial to the later stage.

Embodiment 11: the structure of the red winter spore yeast specificity Geneticin resistance expression box ura5kan of circle

The structure of the red winter spore yeast specificity Geneticin resistance expression box of ura5kan circle is the method for utilizing the RF clone equally.The amplification of pRtura5t full length sequence and clone see embodiment 6.

1. reference literature method (Van den Ent, F., Lowe, J., 2006.RF cloning:Arestriction-free method for inserting target genes into plasmids.J.Biochem.Biophys.Methods 67:67-74), design RF clone primer: ura5-kan-p1:5 '-CACGAGGTCGAAAACACCGTCAG atgggtaaggaaaagactcacgt-3 ' and ura5-kan-p2:5 '-GGAACTGCCCAGACGCACTT GTATttagaaaaactcatcgagcatc-3 ' (wherein original ura5ORF flank sequence is complementary in capitalization partial sequence and the pRtura5t cloning vector, and lowercase partial sequence and Geneticin resistant gene kanmx4ORF are complementary).

2.RF I reaction system and flow process: take pFA6-kanmx4 plasmid (available from EUROSCARF) as template, utilize ura5kan-p1 and ura5kan-p2 to be primer, carry out the RF first round and increase.System (50 μ l): 5 * Prime buffer, 10.0 μ l, dNTPs (2.5mmol/l) 4.0 μ l, upstream primer (10 μ mol/l) 2.0 μ l, downstream primer (10 μ mol/l) 2.0 μ l, PrimeSTAR ^TMHS archaeal dna polymerase (Dalian TakaRa) 1.0 μ l, pFA6-kanmx4 plasmid (110ng/ μ l) 1 μ l, ddH ₂O adds to 50 μ l.Reaction conditions: 95 ℃ of 3min, 98 ℃ of 8s, 49 ℃ of 15s, 72 ℃ of 1min, 30 circulations, 72 ℃ of 10min, 4 ℃ are finished reaction.RF I reaction product utilizes dna fragmentation glue to reclaim the purification kit purifying, and-20 ℃ save backup.

3.RF II reaction: 5 * Prime buffer, 10.0 μ l, dNTPs (2.5mmol/l) 4.0 μ l, T-pRtura5t plasmid (100ng/ μ l) the 1.0 μ l that make up among the embodiment 6, RFI reaction product in the present embodiment abovementioned steps (100ng/ μ l) 5.0 μ l, PrimeSTAR ^TMHS archaeal dna polymerase (Dalian TakaRa) 1.0 μ l, ddH ₂O adds to 50 μ l.Reaction conditions: 95 ℃ of 3min, 68 ℃ of 12min, 95 ℃ of 30s afterwards, 65 ℃ of 45s (1 ℃/cyc), and 68 ℃ of 12min, 15 circulations, next carry out again taking turns: 95 ℃ of 30s, 55 ℃ of 45s, 68 ℃ of 12min, 20 circulations, 72 ℃ of 10min, 4 ℃ are finished reaction.

4.DpnI digestion and electric shock transform: get 8 μ l RF II reaction product add behind 1 μ l DpnI (available from New England Biolabs) and the 1 μ l DpnI buffer mixing 37 ℃ act on 120min and remove former T-pRtura5t plasmid after, get respectively 2 μ l electric shock and transform DH5 α competent cell, competent cell is by standard method preparation (the molecular cloning experiment guide third edition, Pehanorm Brooker work, Huang Peitang etc. translate, Science Press publishes), electric shock Transformation Parameters: 2200-2500V, 400 Ω, 25 μ F, 0 ℃.Select Amp resistance transformant and increase bacterium cultivation, plasmid extraction, and utilize RF I reaction the primer ura5kan-p1 and ura5kan-p2 to carry out bacterium colony PCR and identify, identify that positive recombinant vectors send TaKaRa to check order, obtain the ura5kan expression cassette that 5 ' end and 3 ' end are respectively ura5 promotor and ura5 terminator, simultaneously, this recombinant vectors called after pura5kan.Kanmx ORF sequence is shown in SEQID NO:11; Complete ura5kan expression cassette is shown in SEQ ID NO:12.

Embodiment 12: utilize the ura5kan expression cassette to carry out the Rtura5 gene knockout Geneticin resistance expression that holds concurrently

1.Rtura5kan knock out the preparation of box

The pura5kan carrier that makes up take embodiment 11 is as template, and the oligonucleotide sequence pRtura5t-p1 in the embodiment 6 and pRtura5t-p2 carry out a large amount of preparations that Rtura5kan knocks out box as primer.PCR system (500 μ l): 10 * Speed buffer, 50.0 μ l, dNTPs (10mmol/l) 10.0 μ l, upstream primer (10 μ mol/l) 20.0 μ l, downstream primer (10 μ mol/l) 20.0 μ l, SpeedSTAR ^TMHS archaeal dna polymerase (Dalian TaKaRa company) 5.0 μ l, genomic dna template (120ng/ μ l) 15.0 μ l, ddH ₂O adds to 500 μ l.Reaction conditions: 98 ℃ of 1min, 98 ℃ of 10s, 65 ℃ of 60s, 35 circulations, 72 ℃ of 10min, 4 ℃ are finished reaction.

The PCR product utilizes PCR fragment purification test kit (available from the green skies) to carry out purifying after 1% (mass/volume concentration) agarose gel electrophoresis analysis.Dna fragmentation concentration behind the purifying is at 300ng/ μ l, totally 40 μ l, and-20 ℃ save backup.

2. the red winter spore yeast ATCC 10788 competent cells preparation of monoploid circle

The preparation of R.toruloides np11 competent cell is with the action-item 3 among the embodiment 8.

3. justifying the electric shock conversion of red winter spore yeast ATCC 10788 and the PCR of transformant identifies

The electric shock that Rtura5kan knocks out box transforms: get 100 μ l R.toruloides ATCC, 10788 competent cells, add Rtura5kan and knock out box 10 μ l (altogether 3.2 μ g), ice bath 5min behind the mixing, move in the electric shock cup that is chilled in advance 0 ℃, voltage 0.8-2.0 kilovolt, resistance 200 Ω, electric capacity 25 μ F, time 4-10ms; Add immediately 1ml YEPD after the electric shock, 30 ℃ of incubation 1-2h; Coating contains the YEPD flat board of 50 μ g/mlG418 (available from Beijing boat ancient cooking vessel state), and 200 μ l/ are dull and stereotyped, cultivates 2-10d, has approximately 60 transformants to occur successively for 30 ℃.

3 G418 resistances of picking transformant is cultivated 24h in the YEPD substratum, culture utilizes physiological saline (0.9%NaCl damping fluid) to carry out doubling dilution, select its 10 ^-3, 10 ^-4, 10 ^-5, 10 ^-6Four extent of dilution pipette respectively 10 μ l bacterium liquid spottings in the SC solid medium that contains 5 '-FOA (available from Shanghai Jinhe Biotechnology Co., Ltd) (0.2%5 '-FOA, glucose 70g/L, (NH ₄) ₂SO ₄0.1g/L, yeast powder 0.75g/L, KH ₂PO ₄0.4g/L, MgSO ₄7H ₂O 1.5g/L, pH 6.0) flat board, after liquid-absorbent is complete, be inverted cultivation 3 days in 30 ℃, find that the red winter spore yeast ATCC 10788 Δ ura5::kan of restructuring circle possess 5 '-FOA resistance, the red winter spore yeast ATCC10788 of control strain circle is then without colony growth.

Above presentation of results, Rtura5kan knock out box (expression cassette) when starting the Geneticin resistant gene conformability is expressed in the red winter spore yeast of circle, and the orotidine-5′-phosphate decarboxylase of ura5 genes encoding of can deactivation integrating the position is active.Like this be designed with the genetic manipulation that is beneficial to the later stage.

(not of the same race) determination of activity in the genus of embodiment 13:pRtura5 promotor and Rtura5t terminator

Also be that the expression cassettes such as ura5gfp, ura5ble, ura5kan are in the functional verification of R.babjevae.

Utilize the 26SrDNA gene as integration site at this, make each integration expression box 5 ' end carry the 26SrDNA homologous recombination arm of the 500bp that has an appointment by merging PCR method, carry out respectively ura5gfp expression cassette, ura5ble expression cassette, the ura5kan expression cassette conformability in R.babjevae and express.

1. the R.babjevae 26SrDNA sequence (NO.:EF595746) of announcing according to NCBI, design pair of primers: Rb26S-Rtura5-p1:5 '-AGCGGCGAGCGAAGCGGTAAG-3 ' and Rb26S-Rtura5-p2:5 '-gagttttaacagataggcggcaACGCTGCGTTCCTCAGTCCCC-3 ' (wherein capitalization partial sequence and R.babjevae 26SrDNA sequence 3 ' are held homology, and the lowercase partial sequence is complementary with the red winter spore yeast pura5 promotor 5 ' end of circle).

2.Rb26S-Rtura5gfp, the structure of Rb26S-Rtura5ble, Rb26S-Rtura5kan

Utilize respectively Pura5gfp, Pura5ble and Pura5kan carrier among embodiment 5, embodiment 7 and the embodiment 9 to be template, carry out respectively the structure of 3 amalgamation and expression boxes such as Rb26S-Rtura5gfp, Rb26S-Rtura5ble, Rb26S-Rtura5kan.PCR system (each 500 μ l): 10 * Speedbuffer, 50.0 μ l, dNTPs (10mmol/l) 10.0 μ l, upstream primer (10 μ mol/l) 20.0 μ l, downstream primer (10 μ mol/l) 20.0 μ l, SpeedSTAR ^TMHS archaeal dna polymerase 5.0 μ l, dna profiling plasmid Pura5gfp or Pura5ble or Pura5kan (all 120ng/ μ l) 15.0 μ l, ddH ₂O adds to 500 μ l.Reaction conditions: 98 ℃ of 1min, 98 ℃ of 10s, 65 ℃ of 60s, 35 circulations, 72 ℃ of 10min, 4 ℃ are finished reaction.

The PCR product utilizes PCR fragment purification test kit (available from the green skies) to carry out purifying after 1% (mass/volume concentration) agarose gel electrophoresis analysis.Rb26S-Rtura5gfp behind the purifying, Rb26S-Rtura5ble and Rb26S-Rtura5kan expression cassette concentration are respectively 310ng/ μ l, 300ng/ μ l and 270ng/ μ l, equal 45 μ l, and-20 ℃ save backup.

3.R.babjevae ATCC90942 competent cell preparation

R.babjevae ATCC90942 is available from available from the biological product of USS collecting center.At first, choose colony inoculation 10ml YEPD substratum, 28 ℃, 200rpm cultivates 24h; 1: 50 ratio of culture fresh YEPD substratum of transferring, 100ml (500ml Erlenmeyer flask, liquid amount 100ml), 28 ℃, 200rpm cultivates 7-10h, and the OD value reaches 0.6-1.2; Culture ice bath 10-30min, 4 ℃, the centrifugal 5min of 4000rpm abandons supernatant; 0 ℃ of aseptic Milli-Q washes 1 time; 0 ℃ of 1mol/l sorbyl alcohol washs 2 times; Ice bath, for subsequent use.

4.R.babjevae the electric shock of ATCC90942 transforms and expression of results is observed

Get 3 part of 100 μ l R.babjevae ATCC90942 competent cell, add respectively each 10 μ l of Rb26S-Rtura5gfp, Rb26S-Rtura5ble and Rb26S-Rtura5kan expression cassette, move in the electric shock cup that is chilled in advance 0 ℃ behind the mixing, voltage 0.8-2.0 kilovolt, resistance 200 Ω, electric capacity 25 μ F, time 4-10ms; Add immediately 1ml YEPD after the electric shock, 30 ℃ of incubation 1-2h; YEPD is dull and stereotyped for the coating of Rb26S-Rtura5gfp conversion fluid, and 10 μ l/ are dull and stereotyped; The coating of Rb26S-Rtura5ble conversion fluid contains the YEPD flat board of 20 μ g/mlZeocin (available from Invitrogen company), and 200 μ l/ are dull and stereotyped; The coating of Rb26S-Rtura5kan conversion fluid contains the YEPD flat board of 50 μ g/ml G418 (available from Beijing boat ancient cooking vessel state), and 200 μ l/ are dull and stereotyped; Cultivate 2-10d for equal 28 ℃.

R.babjevae ATCC90942/Rb26S-Rtura5gfp transformant is chosen one by one mono-clonal and is utilized fluorescent microscope to carry out microscopy, and the positive recombinant of a luciferase expression can be arranged in per 40 transformants, and the fluorescence photograph slightly; The coating of Rb26S-Rtura5ble conversion fluid contains the YEPD flat board of 20 μ g/ml Zeocin, can be observed resistance recon not of uniform size during 28 ℃ of cultivation 5d, and average 100/flat board transforms 200 recons of recombination efficiency/μ g expression cassette DNA; The coating of Rb26S-Rtura5kan conversion fluid contains the YEPD flat board of 50 μ g/ml G418, can be observed resistance recon not of uniform size during 28 ℃ of cultivation 6d, and average 100/flat board transforms 200 recons of recombination efficiency/μ g expression cassette DNA.

Each expression cassette is at the apparent conversion recombination efficiency of R.babjevae ATCC90942, than the height in R.toruloides ATCC 10788, may be because what carry out at R.babjevae ATCC90942 is the unit point homologous recombination, and what carry out at R.toruloides ATCC10788 be the reason of dibit point homologous recombination.

Above embodiment proves that the ura5 promotor can start green fluorescence protein gene, bleomycin resistant gene and the Geneticin resistant gene conformability in R.toruloides ATCC 10788 and express; Ura5gfp expression cassette, ura5ble expression cassette, ura5kan expression cassette are that the linear DNA of fundamental construction knocks out box, can knock out the ura5 target gene on the red winter spore Yeast genome of circle.Use the have dna molecular of promoter activity and the DNA with transcription terminator activity of the present invention, the expression in the red winter spore yeast of circle of foreign gene or native gene be can realize, promotor, terminator and a series of DNA construct for the red winter spore yeast of genetic engineering circle the invention provides.For the red winter spore yeast of circle has been opened a breeding new way, and therefore can provide spore yeast of red winter of the Novel circular with industrial use.And method and platform are provided for other saccharomycetic genetic manipulation of Rhodosporidium.

Embodiment 14: the structural characterization of the red winter spore yeast orotate phosphoribosyl transferase OPRTase of circle

The reference molecule cloning experimentation guide third edition (Pehanorm Brooker work, Huang Peitang etc. translate, Science Press publishes) obtain the red winter spore yeast OPRTase of circle through size-exclusion, ion-exchange purification.The OPRTase crystal at room temperature obtains by sessile drop method.Adopt the sparse matrix sampling method, grope to obtain to satisfy the parameter that high resolution structures is resolved the crystal that requires.Final crystallization condition is as follows: RtFBA protein solution (15mg/ml) and isopyknic mother liquor (0.1M Tris-HCl (pH 7.4), 0.08M magnesium acetate, 26% (w/v) polyethylene glycol 6000) mix, and 2-3 crystal occurs in week.Determine the position phase with multi-wavelength anomalous scattering method (MAD), the MAD data are collected at ADSC Quantum-4R ccd detector, and all data are unified with the DPS software package, carry out coordinate modification and processing with the CCP4 software package.Model makes up and proofreaies and correct at Silicon Graphics OCTANE with XtalView 4.0 softwares, refines with the REFMAC program.The red winter spore yeast OPRTase crystal of circle belongs to spacer P4 ₃2 ₁2, lattice parameter is

Obtain 3 d structure model according to diffraction data and see Figure 12.

The invention has the beneficial effects as follows:

For the yeast of justifying red winter spore yeast or Rhodosporidium provides promotor, terminator, selected marker's expression cassette and genetic transforming method, with effectively promotion spore yeast of red winter of circle from now on or the saccharomycetic strain improvement research of Rhodosporidium, accelerate the red winter spore yeast metabolism engineering research of circle.

SEQ ID NO：1

TGCCGCCTAT CTGTTAAAAC TCAATGGATA CAATGCAAAG TCGGCTCGCG CCCTCTTCTC 60

CCGTTCCCCC GTACCTAGAC ATGCGTCGTG AGTGCTTCCC TCCGTCATGC AACCGTCGCA 120

AGCTCCTCGC TCCGTCGTCG CTGCTGAAGC TGCTCCTGCG CAATCCGCTG CGCGGCGGGT 180

ACCTCGCCTC GCTCCCTTTC CGCCGCCCGA ATCGCCTGTG TGAGCCTGTT CCTCCGAGAC 240

ACGACCATCG TCTTGGGATT GGCTTGTGGT ACGGGCAGAG GGCTGGTCTC TGCAATTGAG 300

CAAGCGAGCC GAGGCAGCGA TGGTCAGTTC CGGTCGCAGA CCGAGGCTCC AGGGAAGGGA 360

CGGCGGGACG CACTGATGAC GCCGGCCTGA ATGAGCAGCT TCTCAAAGCG CTTTGTCGGC 420

AGCGCACCCT GGCTGAGCCA GTACCGGACC CGCTCCAGGT TCCACTCGAC CTTCTTCTGC 480

CCGACCTTGG CGACGCCCGT GTTGGGCGTG TGCTGCCGTG GTCCCCACTC CTTCGGGTCC 540

CTGACCTGAC CGTTTGGCGA GACCGACGGC GCTGGCACGA CGACCGGTCG CGGGTTGTAC 600

GAACCGAGGA GCTCGAGGGG TTTGGCGGTC TGGCGCTGCG GCGCGCGGAT GGCGACGAGC 660

GAGTAGACGG GGTTGTTGCG CGTGCAGTGG TGGCGCGCGA GGCGCAATCG GACCGACATC 720

TCGTGCGCTG GCGGAGAGCG AGTGTGCGAG GACGAGGAAA AGGAGCGGGA GGAGGAGGGT 780

TGGGAGCGAA CCACCTCGTG CAGCTGGACT GGACGCGCTA GACGACTGCC AGACTCGCTA 840

TACTGCTCTT TACATCCGCT AGAATCCTGC ACCCGCTCGA ACCAGCTCCT CCCACCGTCA 900

GTTCGCCTCC CCCTCTCATC CTCATCTCTC AAAGGACTTC CTCGCTCGCA ACTCGCTGGA 960

GGGAGCTGCA AGACTGACAC GAGGTCGAAA ACACCGTCAG 1000

SEQ ID NO：2

ATACAAGTGC GTCTGGGCAG TTCCCCTCTT TTGCGTAGCG TACCCGCACG ATTTACTGAC 60

GCAACTCGGG CGCTACAGTC CTGTCAATCT ATCGCGATCT TCGCAGCGAC ACTCGCCGCT 120

TCCGATTCCC GACTTTGCGT CCATCTCGCT CGCTTCGATA AGCTTCTTTG GCTGCTACAG 180

ACGGCTCTCA ACTCTGCTGG AAGATGCAGC ATCAGCACTC ACGGGCTCCG CAACAGCTGC 240

TGTCACGCCT CGAGTTCAGT CGGGCAGCTC TCGCCATCTC GCAACCCGTT ATGGCGATGG 300

TCGACCCGTC TCGCGGCTTT CCCAGCGCTG CGTTTCCGCT TCAGCTGTCG CGGTACAAAC 360

TTCATCGCCC CGTAGCGAGC CGTCTCTCGC GCAACGAGGC AATCTCGCGC TTCTCTCCCT 420

AACGCTTTCT ATCGGTCCCG GCCTCTCTCC CGCGGACTCT AAGAGACGTC GACGCAGTTT 480

CGATATCGTC AACAGCGAGC GCAGACGAGC ACTGCCCGAG CAGTGAACGA CGACGAGCTC 540

TGTACGAAGC GATCGGATCC GCCGCCTTCG CTCGCGACAT GCTCGAGCTT TCCTGATCAC 600

CTTGTCTGAG TTGCTCAGGC TCTGCTAAGT CAGTCGTGCG CATCGGAGTT TGCTGAGGCG 660

CGAGAAGGAT ATTACAGCTC GTACGAAGGA CGGCGACTCT GGCTCGCTGT CCGTCACACG 720

AGACAGAGGC CAGCCTCGAC CTCGTCAGCG AGTCGGTCTG TCCATGACGG CTAACTAATG 780

AGGGTGGCTG AACAGATTGA ATGGC 805

SEQ ID NO:3 (the red winter spore yeast orotate phosphoribosyl transferase cDNA sequence of circle)

ATGAGCGCCA CGTCCTACGC CGCCTCGATC ATCGAGACCG CTCTCGCGAG CGAGCAGCCC 60

ATCCTCCGCT TCGGCACCTT CACCCTCAAG TCTGGCCGCT CCTCGCCCTA CTTCTTCAAC 120

TTTGGCCTCT TCAACACCGG CTCTCTCCTC CTCGCTCTCG CCTCGGCCTT CGCAGACGCC 180

ATCCTCGACG CCTACCCCGA GATTGGCTCC TCCTCCGCCG GTCCCGACAC GCCCAAAGTC 240

CTCTTCGGAC CCGCCTACAA GGGCATCCCC CTCGTCGCTG CTATCGCATC CGAACTCGCA 300

CGACGAGGAC GTGATATCGG CTACAGCTAC AACCGCAAGG AGAAGAAGGA CCACGGCGAG 360

GGCGGGTCGA TTGTCGGTGC GCCGCTCAAG GGACAGAAGG TCCTCATCGT CGACGACGTG 420

ATCACGGCCG GTACTGCGAT TCGCGAGGCG CACAAGATCG TCGAGTCGGA GGGCGGTCAG 480

ACGGCGGGTA TTGTCGAGGC CCTCGACCGG GAGGAGCGCG GACAGGGCGA GCTCAGTACG 540

GTCCAGGAAG TCGAGAAGGA GCTCGGCGTC AAGGTCACGA GCGTCGTCAA GATGCGCGAC 600

ATCGTTGCGT GGCTCAAAGA GAAGGGCAAG CTCGACGAGA TGAAGGCCAT GGAGGAGTAC 660

CGCGCAAAGT GGGGCGTTAA CTAG 684

SEQ ID NO：4

Met Ser Ala Thr Ser Tyr Ala Ala Ser Ile Ile Glu Thr Ala Leu

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

Ser Gly Arg Ser Ser Pro Tyr Phe Phe Asn Phe Gly Leu Phe Asn

Thr Gly Ser Leu Leu Leu Ala Leu Ala Ser Ala Phe Ala Asp Ala

Ile Leu Asp Ala Tyr Pro Glu Ile Gly Ser Ser Ser Ala Gly Pro

Asp Thr Pro Lys Val Leu Phe Gly Pro Ala Tyr Lys Gly Ile Pro

Leu Val Ala Ala Ile Ala Ser Glu Leu Ala Arg Arg Gly Arg Asp

Ile Gly Tyr Ser Tyr Asn Arg Lys Glu Lys Lys Asp His Gly Glu

Gly Gly Ser Ile Val Gly Ala Pro Leu Lys Gly Gln Lys Val Leu

Ile Val Asp Asp Val Ile Thr Ala Gly Thr Ala Ile Arg Glu Ala

His Lys Ile Val Glu Ser Glu Gly Gly Gln Thr Ala Gly Ile Val

Glu Ala Leu AsP Arg Glu Glu Arg Gly Gln Gly Glu Leu Ser Thr

Val Gln Glu Val Glu Lys Glu Leu Gly Val Lys Val Thr Ser Val

Val Lys Met Arg Asp Ile Val Ala Trp Leu Lys Glu Lys Gly Lys

Leu Asp Glu Met Lys Ala Met Glu Glu Tyr Arg Ala Lys Trp Gly

Val Asn

SEQ ID NO:5 (the red winter spore yeast whey acid phosphoric acid ribose transferase gene group dna sequence dna of circle)

ATGAGCGCCA CGTCCTACGC CGCCTCGATC ATCGAGACCG CTCTCGCGAG CGAGCAGCCC 60

ATCCTCCGCT TCGGCACCTT CACCCTCAAG TCTGGCCGCT CCTCGCCCTA CTTCTTCAAC 120

TTTGGCCTCT TCAACACCGG CTCTCTCCTC CTCGCTCTCG CCTCGGCCTT CGCAGACGCC 180

ATCCTCGACG CCTACCCCGA GATTGGCTCC TCCTCCGCCG GTCCCGACAC GCCCAAAGTC 240

CTCTTCGGAC CCGCCTACAA GGGCATCCCC CTCGTCGCTG CTATCGCATC CGAACTCGCA 300

CGACGAGGAC GTGATATCGG CTACAGCTAC AACCGCAAGG AGAAGAAGGA CCACGGCGAG 360

GGCGGGTCGA TTGTCGGTGC GCCGCTCAAG GGACAGAAGG TCCTCATCGT CGACGACGTG 420

ATCACGGCCG GTACTGCGAT TCGCGAGGCG CACAAGATCG TCGAGTCGGA GGGCGGTCAG 480

ACGGCGGGTA TTGTCGAGGC CCTCGACCGG GAGGAGCGCG GACAGGGCGA GCTCAGTACG 540

GTCCAGGAAG TCGAGAAGGA GCTCGGCGTC AAGGTCACGA GCGTCGTCAA GATGCGCGAC 600

ATCGTTGCGT GGCTCAAAGA GAAGGGCAAG CTCGACGAGA TGAAGGCCAT GGAGGAGTAC 660

CGCGCAAAGT GGGGCGTTAA CTAG 684

SEQ ID NO：6

TGCCGCCTAT CTGTTAAAAC TCAATGGATA CAATGCAAAG TCGGCTCGCG CCCTCTTCTC 60

CCGTTCCCCC GTACCTAGAC ATGCGTCGTG AGTGCTTCCC TCCGTCATGC AACCGTCGCA 120

AGCTCCTCGC TCCGTCGTCG CTGCTGAAGC TGCTCCTGCG CAATCCGCTG CGCGGCGGGT 180

ACCTCGCCTC GCTCCCTTTC CGCCGCCCGA ATCGCCTGTG TGAGCCTGTT CCTCCGAGAC 240

ACGACCATCG TCTTGGGATT GGCTTGTGGT ACGGGCAGAG GGCTGGTCTC TGCAATTGAG 300

CAAGCGAGCC GAGGCAGCGA TGGTCAGTTC CGGTCGCAGA CCGAGGCTCC AGGGAAGGGA 360

CGGCGGGACG CACTGATGAC GCCGGCCTGA ATGAGCAGCT TCTCAAAGCG CTTTGTCGGC 420

AGCGCACCCT GGCTGAGCCA GTACCGGACC CGCTCCAGGT TCCACTCGAC CTTCTTCTGC 480

CCGACCTTGG CGACGCCCGT GTTGGGCGTG TGCTGCCGTG GTCCCCACTC CTTCGGGTCC 540

CTGACCTGAC CGTTTGGCGA GACCGACGGC GCTGGCACGA CGACCGGTCG CGGGTTGTAC 600

GAACCGAGGA GCTCGAGGGG TTTGGCGGTC TGGCGCTGCG GCGCGCGGAT GGCGACGAGC 660

GAGTAGACGG GGTTGTTGCG CGTGCAGTGG TGGCGCGCGA GGCGCAATCG GACCGACATC 720

TCGTGCGCTG GCGGAGAGCG AGTGTGCGAG GACGAGGAAA AGGAGCGGGA GGAGGAGGGT 780

TGGGAGCGAA CCACCTCGTG CAGCTGGACT GGACGCGCTA GACGACTGCC AGACTCGCTA 840

TACTGCTCTT TACATCCGCT AGAATCCTGC ACCCGCTCGA ACCAGCTCCT CCCACCGTCA 900

GTTCGCCTCC CCCTCTCATC CTCATCTCTC AAAGGACTTC CTCGCTCGCA ACTCGCTGGA 960

GGGAGCTGCA AGACTGACAC GAGGTCGAAA ACACCGTCAG ATGAGCGCCA CGTCCTACGC 1020

CGCCTCGATC ATCGAGACCG CTCTCGCGAG CGAGCAGCCC ATCCTCCGCT TCGGCACCTT 1080

CACCCTCAAG TCTGGCCGCT CCTCGCCCTA CTTCTTCAAC TTTGGCCTCT TCAACACCGG 1140

CTCTCTCCTC CTCGCTCTCG CCTCGGCCTT CGCAGACGCC ATCCTCGACG CCTACCCCGA 1200

GATTGGCTCC TCCTCCGCCG GTCCCGACAC GCCCAAAGTC CTCTTCGGAC CCGCCTACAA 1260

GGGCATCCCC CTCGTCGCTG CTATCGCATC CGAACTCGCA CGACGAGGAC GTGATATCGG 1320

CTACAGCTAC AACCGCAAGG AGAAGAAGGA CCACGGCGAG GGCGGGTCGA TTGTCGGTGC 1380

GCCGCTCAAG GGACAGAAGG TCCTCATCGT CGACGACGTG ATCACGGCCG GTACTGCGAT 1440

TCGCGAGGCG CACAAGATCG TCGAGTCGGA GGGCGGTCAG ACGGCGGGTA TTGTCGAGGC 1500

CCTCGACCGG GAGGAGCGCG GACAGGGCGA GCTCAGTACG GTCCAGGAAG TCGAGAAGGA 1560

GCTCGGCGTC AAGGTCACGA GCGTCGTCAA GATGCGCGAC ATCGTTGCGT GGCTCAAAGA 1620

GAAGGGCAAG CTCGACGAGA TGAAGGCCAT GGAGGAGTAC CGCGCAAAGT GGGGCGTTAA 1680

CTAGATACAA GTGCGTCTGG GCAGTTCCCC TCTTTTGCGT AGCGTACCCG CACGATTTAC 1740

TGACGCAACT CGGGCGCTAC AGTCCTGTCA ATCTATCGCG ATCTTCGCAG CGACACTCGC 1800

CGCTTCCGAT TCCCGACTTT GCGTCCATCT CGCTCGCTTC GATAAGCTTC TTTGGCTGCT 1860

ACAGACGGCT CTCAACTCTG CTGGAAGATG CAGCATCAGC ACTCACGGGC TCCGCAACAG 1920

CTGCTGTCAC GCCTCGAGTT CAGTCGGGCA GCTCTCGCCA TCTCGCAACC CGTTATGGCG 1980

ATGGTCGACC CGTCTCGCGG CTTTCCCAGC GCTGCGTTTC CGCTTCAGCT GTCGCGGTAC 2040

AAACTTCATC GCCCCGTAGC GAGCCGTCTC TCGCGCAACG AGGCAATCTC GCGCTTCTCT 2100

CCCTAACGCT TTCTATCGGT CCCGGCCTCT CTCCCGCGGA CTCTAAGAGA CGTCGACGCA 2160

GTTTCGATAT CGTCAACAGC GAGCGCAGAC GAGCACTGCC CGAGCAGTGA ACGACGACGA 2220

GCTCTGTACG AAGCGATCGG ATCCGCCGCC TTCGCTCGCG ACATGCTCGA GCTTTCCTGA 2280

TCACCTTGTC TGAGTTGCTC AGGCTCTGCT AAGTCAGTCG TGCGCATCGG AGTTTGCTGA 2340

GGCGCGAGAA GGATATTACA GCTCGTACGA AGGACGGCGA CTCTGGCTCG CTGTCCGTCA 2400

CACGAGACAG AGGCCAGCCT CGACCTCGTC AGCGAGTCGG TCTGTCCATG ACGGCTAACT 2460

AATGAGGGTG GCTGAACAGA TTGAATGGC 2489

SEQ ID NO:7 (green fluorescent protein encoding gene)

ATGAGTAAAG GAGAAGAACT TTTCACTGGA GTTGTCCCAA TTCTTGTTGA ATTAGATGGT 60

GATGTTAATG GGCACAAATT TTCTGTCAGT GGAGAGGGTG AAGGTGATGC AACATACGGA 120

AAACTTACCC TTAAATTTAT TTGCACTACT GGAAAACTAC CTGTTCCATG GCCAACACTT 180

GTCACTACTT TCTCTTATGG TGTTCAATGC TTTTCCCGTT ATCCGGATCA TATGAAACGG 240

CATGACTTTT TCAAGAGTGC CATGCCCGAA GGTTATGTAC AGGAACGCAC TATATCTTTC 300

AAAGATGACG GGAACTACAA GACGCGTGCT GAAGTCAAGT TTGAAGGTGA TACCCTTGTT 360

AATCGTATCG AGTTAAAAGG TATTGATTTT AAAGAAGATG GAAACATTCT CGGACACAAA 420

CTCGAGTACA ACTATAACTC ACACAATGTA TACATCACGG CAGACAAACA AAAGAATGGA 480

ATCAAAGCTA ACTTCAAAAT TCGCCACAAC ATTGAAGATG GATCCGTTCA ACTAGCAGAC 540

CATTATCAAC AAAATACTCC AATTGGCGAT GGCCCTGTCC TTTTACCAGA CAACCATTAC 600

CTGTCGACAC AATCTGCCCT TTCGAAAGAT CCCAACGAAA AGCGTGACCA CATGGTCCTT 660

CTTGAGTTTG TAACTGCTGC TGGGATTACA CATGGCATGG ATGAGCTCTA CAAATAA 717

SEQ ID NO:8 (green fluorescent protein encoding gene)

TGCCGCCTAT CTGTTAAAAC TCAATGGATA CAATGCAAAG TCGGCTCGCG CCCTCTTCTC 60

CCGTTCCCCC GTACCTAGAC ATGCGTCGTG AGTGCTTCCC TCCGTCATGC AACCGTCGCA 120

AGCTCCTCGC TCCGTCGTCG CTGCTGAAGC TGCTCCTGCG CAATCCGCTG CGCGGCGGGT 180

ACCTCGCCTC GCTCCCTTTC CGCCGCCCGA ATCGCCTGTG TGAGCCTGTT CCTCCGAGAC 240

ACGACCATCG TCTTGGGATT GGCTTGTGGT ACGGGCAGAG GGCTGGTCTC TGCAATTGAG 300

CAAGCGAGCC GAGGCAGCGA TGGTCAGTTC CGGTCGCAGA CCGAGGCTCC AGGGAAGGGA 360

CGGCGGGACG CACTGATGAC GCCGGCCTGA ATGAGCAGCT TCTCAAAGCG CTTTGTCGGC 420

AGCGCACCCT GGCTGAGCCA GTACCGGACC CGCTCCAGGT TCCACTCGAC CTTCTTCTGC 480

CCGACCTTGG CGACGCCCGT GTTGGGCGTG TGCTGCCGTG GTCCCCACTC CTTCGGGTCC 540

CTGACCTGAC CGTTTGGCGA GACCGACGGC GCTGGCACGA CGACCGGTCG CGGGTTGTAC 600

GAACCGAGGA GCTCGAGGGG TTTGGCGGTC TGGCGCTGCG GCGCGCGGAT GGCGACGAGC 660

GAGTAGACGG GGTTGTTGCG CGTGCAGTGG TGGCGCGCGA GGCGCAATCG GACCGACATC 720

TCGTGCGCTG GCGGAGAGCG AGTGTGCGAG GACGAGGAAA AGGAGCGGGA GGAGGAGGGT 780

TGGGAGCGAA CCACCTCGTG CAGCTGGACT GGACGCGCTA GACGACTGCC AGACTCGCTA 840

TACTGCTCTT TACATCCGCT AGAATCCTGC ACCCGCTCGA ACCAGCTCCT CCCACCGTCA 900

GTTCGCCTCC CCCTCTCATC CTCATCTCTC AAAGGACTTC CTCGCTCGCA ACTCGCTGGA 960

GGGAGCTGCA AGACTGACAC GAGGTCGAAA ACACCGTCAG ATGAGTAAAG GAGAAGAACT 1020

TTTCACTGGA GTTGTCCCAA TTCTTGTTGA ATTAGATGGT GATGTTAATG GGCACAAATT 1080

TTCTGTCAGT GGAGAGGGTG AAGGTGATGC AACATACGGA AAACTTACCC TTAAATTTAT 1140

TTGCACTACT GGAAAACTAC CTGTTCCATG GCCAACACTT GTCACTACTT TCTCTTATGG 1200

TGTTCAATGC TTTTCCCGTT ATCCGGATCA TATGAAACGG CATGACTTTT TCAAGAGTGC 1260

CATGCCCGAA GGTTATGTAC AGGAACGCAC TATATCTTTC AAAGATGACG GGAACTACAA 1320

GACGCGTGCT GAAGTCAAGT TTGAAGGTGA TACCCTTGTT AATCGTATCG AGTTAAAAGG 1380

TATTGATTTT AAAGAAGATG GAAACATTCT CGGACACAAA CTCGAGTACA ACTATAACTC 1440

ACACAATGTA TACATCACGG CAGACAAACA AAAGAATGGA ATCAAAGCTA ACTTCAAAAT 1500

TCGCCACAAC ATTGAAGATG GATCCGTTCA ACTAGCAGAC CATTATCAAC AAAATACTCC 1560

AATTGGCGAT GGCCCTGTCC TTTTACCAGA CAACCATTAC CTGTCGACAC AATCTGCCCT 1620

TTCGAAAGAT CCCAACGAAA AGCGTGACCA CATGGTCCTT CTTGAGTTTG TAACTGCTGC 1680

TGGGATTACA CATGGCATGG ATGAGCTCTA CAAATAGATA CAAGTGCGTC TGGGCAGTTC 1740

CCCTCTTTTG CGTAGCGTAC CCGCACGATT TACTGACGCA ACTCGGGCGC TACAGTCCTG 1800

TCAATCTATC GCGATCTTCG CAGCGACACT CGCCGCTTCC GATTCCCGAC TTTGCGTCCA 1860

TCTCGCTCGC TTCGATAAGC TTCTTTGGCT GCTACAGACG GCTCTCAACT CTGCTGGAAG 1920

ATGCAGCATC AGCACTCACG GGCTCCGCAA CAGCTGCTGT CACGCCTCGA GTTCAGTCGG 1980

GCAGCTCTCG CCATCTCGCA ACCCGTTATG GCGATGGTCG ACCCGTCTCG CGGCTTTCCC 2040

AGCGCTGCGT TTCCGCTTCA GCTGTCGCGG TACAAACTTC ATCGCCCCGT AGCGAGCCGT 2100

CTCTCGCGCA ACGAGGCAAT CTCGCGCTTC TCTCCCTAAC GCTTTCTATC GGTCCCGGCC 2160

TCTCTCCCGC GGACTCTAAG AGACGTCGAC GCAGTTTCGA TATCGTCAAC AGCGAGCGCA 2220

GACGAGCACT GCCCGAGCAG TGAACGACGA CGAGCTCTGT ACGAAGCGAT CGGATCCGCC 2280

GCCTTCGCTC GCGACATGCT CGAGCTTTCC TGATCACCTT GTCTGAGTTG CTCAGGCTCT 2340

GCTAAGTCAG TCGTGCGCAT CGGAGTTTGC TGAGGCGCGA GAAGGATATT ACAGCTCGTA 2400

CGAAGGACGG CGACTCTGGC TCGCTGTCCG TCACACGAGA CAGAGGCCAG CCTCGACCTC 2460

GTCAGCGAGT CGGTCTGTCC ATGACGGCTA ACTAATGAGG GTGGCTGAAC AGATTGAATG 2520

GC 2522

SEQ ID NO:9 (bleomycin resistant gene ble)

ATGGCCAAGT TGACCAGTGC CGTTCCGGTG CTCACCGCGC GCGACGTCGC CGGAGCGGTC 60

GAGTTCTGGA CCGACCGGCT CGGGTTCTCC CGGGACTTCG TGGAGGACGA CTTCGCCGGT 120

GTGGTCCGGG ACGACGTGAC CCTGTTCATC AGCGCGGTCC AGGACCAGGT GGTGCCGGAC 180

AACACCCTGG CCTGGGTGTG GGTGCGCGGC CTGGACGAGC TGTACGCCGA GTGGTCGGAG 240

GTCGTGTCCA CGAACTTCCG GGACGCCTCC GGGCCGGCCA TGACCGAGAT CGGCGAGCAG 300

CCGTGGGGGC GGGAGTTCGC CCTGCGCGAC CCGGCCGGCA ACTGCGTGCA CTTCGTGGCC 360

GAGGAGCAGG ACTGA 375

SEQ ID NO:10 (bleomycin resistant gene ble)

TGCCGCCTAT CTGTTAAAAC TCAATGGATA CAATGCAAAG TCGGCTCGCG CCCTCTTCTC 60

CCGTTCCCCC GTACCTAGAC ATGCGTCGTG AGTGCTTCCC TCCGTCATGC AACCGTCGCA 120

AGCTCCTCGC TCCGTCGTCG CTGCTGAAGC TGCTCCTGCG CAATCCGCTG CGCGGCGGGT 180

ACCTCGCCTC GCTCCCTTTC CGCCGCCCGA ATCGCCTGTG TGAGCCTGTT CCTCCGAGAC 240

ACGACCATCG TCTTGGGATT GGCTTGTGGT ACGGGCAGAG GGCTGGTCTC TGCAATTGAG 300

CAAGCGAGCC GAGGCAGCGA TGGTCAGTTC CGGTCGCAGA CCGAGGCTCC AGGGAAGGGA 360

CGGCGGGACG CACTGATGAC GCCGGCCTGA ATGAGCAGCT TCTCAAAGCG CTTTGTCGGC 420

AGCGCACCCT GGCTGAGCCA GTACCGGACC CGCTCCAGGT TCCACTCGAC CTTCTTCTGC 480

CCGACCTTGG CGACGCCCGT GTTGGGCGTG TGCTGCCGTG GTCCCCACTC CTTCGGGTCC 540

CTGACCTGAC CGTTTGGCGA GACCGACGGC GCTGGCACGA CGACCGGTCG CGGGTTGTAC 600

GAACCGAGGA GCTCGAGGGG TTTGGCGGTC TGGCGCTGCG GCGCGCGGAT GGCGACGAGC 660

GAGTAGACGG GGTTGTTGCG CGTGCAGTGG TGGCGCGCGA GGCGCAATCG GACCGACATC 720

TCGTGCGCTG GCGGAGAGCG AGTGTGCGAG GACGAGGAAA AGGAGCGGGA GGAGGAGGGT 780

TGGGAGCGAA CCACCTCGTG CAGCTGGACT GGACGCGCTA GACGACTGCC AGACTCGCTA 840

TACTGCTCTT TACATCCGCT AGAATCCTGC ACCCGCTCGA ACCAGCTCCT CCCACCGTCA 900

GTTCGCCTCC CCCTCTCATC CTCATCTCTC AAAGGACTTC CTCGCTCGCA ACTCGCTGGA 960

GGGAGCTGCA AGACTGACAC GAGGTCGAAA ACACCGTCAG ATGGCCAAGT TGACCAGTGC 1020

CGTTCCGGTG CTCACCGCGC GCGACGTCGC CGGAGCGGTC GAGTTCTGGA CCGACCGGCT 1080

CGGGTTCTCC CGGGACTTCG TGGAGGACGA CTTCGCCGGT GTGGTCCGGG ACGACGTGAC 1140

CCTGTTCATC AGCGCGGTCC AGGACCAGGT GGTGCCGGAC AACACCCTGG CCTGGGTGTG 1200

GGTGCGCGGC CTGGACGAGC TGTACGCCGA GTGGTCGGAG GTCGTGTCCA CGAACTTCCG 1260

GGACGCCTCC GGGCCGGCCA TGACCGAGAT CGGCGAGCAG CCGTGGGGGC GGGAGTTCGC 1320

CCTGCGCGAC CCGGCCGGCA ACTGCGTGCA CTTCGTGGCC GAGGAGCAGG ACTGAATACA 1380

AGTGCGTCTG GGCAGTTCCC CTCTTTTGCG TAGCGTACCC GCACGATTTA CTGACGCAAC 1440

TCGGGCGCTA CAGTCCTGTC AATCTATCGC GATCTTCGCA GCGACACTCG CCGCTTCCGA 1500

TTCCCGACTT TGCGTCCATC TCGCTCGCTT CGATAAGCTT CTTTGGCTGC TACAGACGGC 1560

TCTCAACTCT GCTGGAAGAT GCAGCATCAG CACTCACGGG CTCCGCAACA GCTGCTGTCA 1620

CGCCTCGAGT TCAGTCGGGC AGCTCTCGCC ATCTCGCAAC CCGTTATGGC GATGGTCGAC 1680

CCGTCTCGCG GCTTTCCCAG CGCTGCGTTT CCGCTTCAGC TGTCGCGGTA CAAACTTCAT 1740

CGCCCCGTAG CGAGCCGTCT CTCGCGCAAC GAGGCAATCT CGCGCTTCTC TCCCTAACGC 1800

TTTCTATCGG TCCCGGCCTC TCTCCCGCGG ACTCTAAGAG ACGTCGACGC AGTTTCGATA 1860

TCGTCAACAG CGAGCGCAGA CGAGCACTGC CCGAGCAGTG AACGACGACG AGCTCTGTAC 1920

GAAGCGATCG GATCCGCCGC CTTCGCTCGC GACATGCTCG AGCTTTCCTG ATCACCTTGT 1980

CTGAGTTGCT CAGGCTCTGC TAAGTCAGTC GTGCGCATCG GAGTTTGCTG AGGCGCGAGA 2040

AGGATATTAC AGCTCGTACG AAGGACGGCG ACTCTGGCTC GCTGTCCGTC ACACGAGACA 2100

GAGGCCAGCC TCGACCTCGT CAGCGAGTCG GTCTGTCCAT GACGGCTAAC TAATGAGGGT 2160

GGCTGAACAG ATTGAATGGC 2180

SEQ ID NO:11 (Geneticin resistant gene kanmx4)

ATGGGTAAGG AAAAGACTCA CGTTTCGAGG CCGCGATTAA ATTCCAACAT GGATGCTGAT 60

TTATATGGGT ATAAATGGGC TCGCGATAAT GTCGGGCAAT CAGGTGCGAC AATCTATCGA 120

TTGTATGGGA AGCCCGATGC GCCAGAGTTG TTTCTGAAAC ATGGCAAAGG TAGCGTTGCC 180

AATGATGTTA CAGATGAGAT GGTCAGACTA AACTGGCTGA CGGAATTTAT GCCTCTTCCG 240

ACCATCAAGC ATTTTATCCG TACTCCTGAT GATGCATGGT TACTCACCAC TGCGATCCCC 300

GGCAAAACAG CATTCCAGGT ATTAGAAGAA TATCCTGATT CAGGTGAAAA TATTGTTGAT 360

GCGCTGGCAG TGTTCCTGCG CCGGTTGCAT TCGATTCCTG TTTGTAATTG TCCTTTTAAC 420

AGCGATCGCG TATTTCGTCT CGCTCAGGCG CAATCACGAA TGAATAACGG TTTGGTTGAT 480

GCGAGTGATT TTGATGACGA GCGTAATGGC TGGCCTGTTG AACAAGTCTG GAAAGAAATG 540

CATAAGCTTT TGCCATTCTC ACCGGATTCA GTCGTCACTC ATGGTGATTT CTCACTTGAT 600

AACCTTATTT TTGACGAGGG GAAATTAATA GGTTGTATTG ATGTTGGACG AGTCGGAATC 660

GCAGACCGAT ACCAGGATCT TGCCATCCTA TGGAACTGCC TCGGTGAGTT TTCTCCTTCA 720

TTACAGAAAC GGCTTTTTCA AAAATATGGT ATTGATAATC CTGATATGAA TAAATTGCAG 780

TTTCATTTGA TGCTCGATGA GTTTTTCTAA 810

SEQ ID NO:12 (Geneticin resistant gene ble)

TGCCGCCTAT CTGTTAAAAC TCAATGGATA CAATGCAAAG TCGGCTCGCG CCCTCTTCTC 60

CCGTTCCCCC GTACCTAGAC ATGCGTCGTG AGTGCTTCCC TCCGTCATGC AACCGTCGCA 120

AGCTCCTCGC TCCGTCGTCG CTGCTGAAGC TGCTCCTGCG CAATCCGCTG CGCGGCGGGT 180

ACCTCGCCTC GCTCCCTTTC CGCCGCCCGA ATCGCCTGTG TGAGCCTGTT CCTCCGAGAC 240

ACGACCATCG TCTTGGGATT GGCTTGTGGT ACGGGCAGAG GGCTGGTCTC TGCAATTGAG 300

CAAGCGAGCC GAGGCAGCGA TGGTCAGTTC CGGTCGCAGA CCGAGGCTCC AGGGAAGGGA 360

CGGCGGGACG CACTGATGAC GCCGGCCTGA ATGAGCAGCT TCTCAAAGCG CTTTGTCGGC 420

AGCGCACCCT GGCTGAGCCA GTACCGGACC CGCTCCAGGT TCCACTCGAC CTTCTTCTGC 480

CCGACCTTGG CGACGCCCGT GTTGGGCGTG TGCTGCCGTG GTCCCCACTC CTTCGGGTCC 540

CTGACCTGAC CGTTTGGCGA GACCGACGGC GCTGGCACGA CGACCGGTCG CGGGTTGTAC 600

GAACCGAGGA GCTCGAGGGG TTTGGCGGTC TGGCGCTGCG GCGCGCGGAT GGCGACGAGC 660

GAGTAGACGG GGTTGTTGCG CGTGCAGTGG TGGCGCGCGA GGCGCAATCG GACCGACATC 720

TCGTGCGCTG GCGGAGAGCG AGTGTGCGAG GACGAGGAAA AGGAGCGGGA GGAGGAGGGT 780

TGGGAGCGAA CCACCTCGTG CAGCTGGACT GGACGCGCTA GACGACTGCC AGACTCGCTA 840

TACTGCTCTT TACATCCGCT AGAATCCTGC ACCCGCTCGA ACCAGCTCCT CCCACCGTCA 900

GTTCGCCTCC CCCTCTCATC CTCATCTCTC AAAGGACTTC CTCGCTCGCA ACTCGCTGGA 960

GGGAGCTGCA AGACTGACAC GAGGTCGAAA ACACCGTCAG ATGGGTAAGG AAAAGACTCA 1020

CGTTTCGAGG CCGCGATTAA ATTCCAACAT GGATGCTGAT TTATATGGGT ATAAATGGGC 1080

TCGCGATAAT GTCGGGCAAT CAGGTGCGAC AATCTATCGA TTGTATGGGA AGCCCGATGC 1140

GCCAGAGTTG TTTCTGAAAC ATGGCAAAGG TAGCGTTGCC AATGATGTTA CAGATGAGAT 1200

GGTCAGACTA AACTGGCTGA CGGAATTTAT GCCTCTTCCG ACCATCAAGC ATTTTATCCG 1260

TACTCCTGAT GATGCATGGT TACTCACCAC TGCGATCCCC GGCAAAACAG CATTCCAGGT 1320

ATTAGAAGAA TATCCTGATT CAGGTGAAAA TATTGTTGAT GCGCTGGCAG TGTTCCTGCG 1380

CCGGTTGCAT TCGATTCCTG TTTGTAATTG TCCTTTTAAC AGCGATCGCG TATTTCGTCT 1440

CGCTCAGGCG CAATCACGAA TGAATAACGG TTTGGTTGAT GCGAGTGATT TTGATGACGA 1500

GCGTAATGGC TGGCCTGTTG AACAAGTCTG GAAAGAAATG CATAAGCTTT TGCCATTCTC 1560

ACCGGATTCA GTCGTCACTC ATGGTGATTT CTCACTTGAT AACCTTATTT TTGACGAGGG 1620

GAAATTAATA GGTTGTATTG ATGTTGGACG AGTCGGAATC GCAGACCGAT ACCAGGATCT 1680

TGCCATCCTA TGGAACTGCC TCGGTGAGTT TTCTCCTTCA TTACAGAAAC GGCTTTTTCA 1740

AAAATATGGT ATTGATAATC CTGATATGAA TAAATTGCAG TTTCATTTGA TGCTCGATGA 1800

GTTTTTCTAA ATACAAGTGC GTCTGGGCAG TTCCCCTCTT TTGCGTAGCG TACCCGCACG 1860

ATTTACTGAC GCAACTCGGG CGCTACAGTC CTGTCAATCT ATCGCGATCT TCGCAGCGAC 1920

ACTCGCCGCT TCCGATTCCC GACTTTGCGT CCATCTCGCT CGCTTCGATA AGCTTCTTTG 1980

GCTGCTACAG ACGGCTCTCA ACTCTGCTGG AAGATGCAGC ATCAGCACTC ACGGGCTCCG 2040

CAACAGCTGC TGTCACGCCT CGAGTTCAGT CGGGCAGCTC TCGCCATCTC GCAACCCGTT 2100

ATGGCGATGG TCGACCCGTC TCGCGGCTTT CCCAGCGCTG CGTTTCCGCT TCAGCTGTCG 2160

CGGTACAAAC TTCATCGCCC CGTAGCGAGC CGTCTCTCGC GCAACGAGGC AATCTCGCGC 2220

TTCTCTCCCT AACGCTTTCT ATCGGTCCCG GCCTCTCTCC CGCGGACTCT AAGAGACGTC 2280

GACGCAGTTT CGATATCGTC AACAGCGAGC GCAGACGAGC ACTGCCCGAG CAGTGAACGA 2340

CGACGAGCTC TGTACGAAGC GATCGGATCC GCCGCCTTCG CTCGCGACAT GCTCGAGCTT 2400

TCCTGATCAC CTTGTCTGAG TTGCTCAGGC TCTGCTAAGT CAGTCGTGCG CATCGGAGTT 2460

TGCTGAGGCG CGAGAAGGAT ATTACAGCTC GTACGAAGGA CGGCGACTCT GGCTCGCTGT 2520

CCGTCACACG AGACAGAGGC CAGCCTCGAC CTCGTCAGCG AGTCGGTCTG TCCATGACGG 2580

CTAACTAATG AGGGTGGCTG AACAGATTGA ATGGC 2615

Whey .ST25

SEQUENCE LISTING

＜110〉Dalian Inst of Chemicophysics, Chinese Academy of Sciences

＜120〉orotate phosphoribosyl transferase promotor and application and construct and carrier

<130>

<160>12

<170>PatentIn version 3.1

<210>1

<211>1000

<212>DNA

＜213〉justify red winter spore yeast (Rhodosporidium toruloides)

<220>

<221>promoter

<222>(201)..(1000)

<223>

<400>1

tgccgcctat ctgttaaaac tcaatggata caatgcaaag tcggctcgcg ccctcttctc 60

ccgttccccc gtacctagac atgcgtcgtg agtgcttccc tccgtcatgc aaccgtcgca 120

agctcctcgc tccgtcgtcg ctgctgaagc tgctcctgcg caatccgctg cgcggcgggt 180

acctcgcctc gctccctttc cgccgcccga atcgcctgtg tgagcctgtt cctccgagac 240

acgaccatcg tcttgggatt ggcttgtggt acgggcagag ggctggtctc tgcaattgag 300

caagcgagcc gaggcagcga tggtcagttc cggtcgcaga ccgaggctcc agggaaggga 360

cggcgggacg cactgatgac gccggcctga atgagcagct tctcaaagcg ctttgtcggc 420

agcgcaccct ggctgagcca gtaccggacc cgctccaggt tccactcgac cttcttctgc 480

ccgaccttgg cgacgcccgt gttgggcgtg tgctgccgtg gtccccactc cttcgggtcc 540

ctgacctgac cgtttggcga gaccgacggc gctggcacga cgaccggtcg cgggttgtac 600

gaaccgagga gctcgagggg tttggcggtc tggcgctgcg gcgcgcggat ggcgacgagc 660

gagtagacgg ggttgttgcg cgtgcagtgg tggcgcgcga ggcgcaatcg gaccgacatc 720

tcgtgcgctg gcggagagcg agtgtgcgag gacgaggaaa aggagcggga ggaggagggt 780

tgggagcgaa ccacctcgtg cagctggact ggacgcgcta gacgactgcc agactcgcta 840

tactgctctt tacatccgct agaatcctgc acccgctcga accagctcct cccaccgtca 900

gttcgcctcc ccctctcatc ctcatctctc aaaggacttc ctcgctcgca actcgctgga 960

gggagctgca agactgacac gaggtcgaaa acaccgtcag 1000

<210>2

<211>805

<212>DNA

＜213〉justify red winter spore yeast (Rhodosporidium toruloides)

<220>

<221>terminator

<222>(1)..(600)

<223>

<400>2

atacaagtgc gtctgggcag ttcccctctt ttgcgtagcg tacccgcacg atttactgac 60

gcaactcggg cgctacagtc ctgtcaatct atcgcgatct tcgcagcgac actcgccgct 120

tccgattccc gactttgcgt ccatctcgct cgcttcgata agcttctttg gctgctacag 180

acggctctca actctgctgg aagatgcagc atcagcactc acgggctccg caacagctgc 240

tgtcacgcct cgagttcagt cgggcagctc tcgccatctc gcaacccgtt atggcgatgg 300

tcgacccgtc tcgcggcttt cccagcgctg cgtttccgct tcagctgtcg cggtacaaac 360

ttcatcgccc cgtagcgagc cgtctctcgc gcaacgaggc aatctcgcgc ttctctccct 420

aacgctttct atcggtcccg gcctctctcc cgcggactct aagagacgtc gacgcagttt 480

cgatatcgtc aacagcgagc gcagacgagc actgcccgag cagtgaacga cgacgagctc 540

tgtacgaagc gatcggatcc gccgccttcg ctcgcgacat gctcgagctt tcctgatcac 600

cttgtctgag ttgctcaggc tctgctaagt cagtcgtgcg catcggagtt tgctgaggcg 660

cgagaaggat attacagctc gtacgaagga cggcgactct ggctcgctgt ccgtcacacg 720

agacagaggc cagcctcgac ctcgtcagcg agtcggtctg tccatgacgg ctaactaatg 780

agggtggctg aacagattga atggc 805

<210>3

<211>684

<212>DNA

＜213〉justify red winter spore yeast (Rhodosporidium toruloides)

<220>

<221>CDS

<222>(1)..(684)

<223>

<400>3

atg agc gcc acg tcc tac gcc gcc tcg atc atc gag acc gct ctc gcg 48

Met Ser Ala Thr Ser Tyr Ala Ala Ser Ile Ile Glu Thr Ala Leu Ala

1 5 10 15

agc gag cag ccc atc ctc cgc ttc ggc acc ttc acc ctc aag tct ggc 96

Ser Glu Gln Pro Ile Leu Arg Phe Gly Thr Phe Thr Leu Lys Ser Gly

20 25 30

cgc tcc tcg ccc tac ttc ttc aac ttt ggc ctc ttc aac acc ggc tct 144

Arg Ser Ser Pro Tyr Phe Phe Asn Phe Gly Leu Phe Asn Thr Gly Ser

35 40 45

ctc ctc ctc gct ctc gcc tcg gcc ttc gca gac gcc atc ctc gac gcc 192

Leu Leu Leu Ala Leu Ala Ser Ala Phe Ala Asp Ala Ile Leu Asp Ala

50 55 60

tac ccc gag att ggc tcc tcc tcc gcc ggt ccc gac acg ccc aaa gtc 240

Tyr Pro Glu Ile Gly Ser Ser Ser Ala Gly Pro Asp Thr Pro Lys Val

65 70 75 80

ctc ttc gga ccc gcc tac aag ggc atc ccc ctc gtc gct gct atc gca 288

Leu Phe Gly Pro Ala Tyr Lys Gly Ile Pro Leu Val Ala Ala Ile Ala

85 90 95

tcc gaa ctc gca cga cga gga cgt gat atc ggc tac agc tac aac cgc 336

Ser Glu Leu Ala Arg Arg Gly Arg Asp Ile Gly Tyr Ser Tyr Asn Arg

100 105 110

aag gag aag aag gac cac ggc gag ggc ggg tcg att gtc ggt gcg ccg 384

Lys Glu Lys Lys Asp His Gly Glu Gly Gly Ser Ile Val Gly Ala Pro

115 120 125

ctc aag gga cag aag gtc ctc atc gtc gac gac gtg atc acg gcc ggt 432

Leu Lys Gly Gln Lys Val Leu Ile Val Asp Asp Val Ile Thr Ala Gly

130 135 140

act gcg att cgc gag gcg cac aag atc gtc gag tcg gag ggc ggt cag 480

Thr Ala Ile Arg Glu Ala His Lys Ile Val Glu Ser Glu Gly Gly Gln

145 150 155 160

acg gcg ggt att gtc gag gcc ctc gac cgg gag gag cgc gga cag ggc 528

Thr Ala Gly Ile Val Glu Ala Leu Asp Arg Glu Glu Arg Gly Gln Gly

165 170 175

gag ctc agt acg gtc cag gaa gtc gag aag gag ctc ggc gtc aag gtc 576

Glu Leu Ser Thr Val Gln Glu Val Glu Lys Glu Leu Gly Val Lys Val

180 185 190

acg agc gtc gtc aag atg cgc gac atc gtt gcg tgg ctc aaa gag aag 624

Thr Ser Val Val Lys Met Arg Asp Ile Val Ala Trp Leu Lys Glu Lys

195 200 205

ggc aag ctc gac gag atg aag gcc atg gag gag tac cgc gca aag tgg 672

Gly Lys Leu Asp Glu Met Lys Ala Met Glu Glu Tyr Arg Ala Lys Trp

210 215 220

ggc gtt aac tag 684

Gly Val Asn

225

<210>4

<211>227

<212>PRT

＜213〉justify red winter spore yeast (Rhodosporidium toruloides)

<400>4

Met Ser Ala Thr Ser Tyr Ala Ala Ser Ile Ile Glu Thr Ala Leu Ala

1 5 10 15

Ser Glu Gln Pro Ile Leu Arg Phe Gly Thr Phe Thr Leu Lys Ser Gly

20 25 30

Arg Ser Ser Pro Tyr Phe Phe Asn Phe Gly Leu Phe Asn Thr Gly Ser

35 40 45

Leu Leu Leu Ala Leu Ala Ser Ala Phe Ala Asp Ala Ile Leu Asp Ala

50 55 60

Tyr Pro Glu Ile Gly Ser Ser Ser Ala Gly Pro Asp Thr Pro Lys Val

65 70 75 80

Leu Phe Gly Pro Ala Tyr Lys Gly Ile Pro Leu Val Ala Ala Ile Ala

85 90 95

Ser Glu Leu Ala Arg Arg Gly Arg Asp Ile Gly Tyr Ser Tyr Asn Arg

100 105 110

Lys Glu Lys Lys Asp His Gly Glu Gly Gly Ser Ile Val Gly Ala Pro

115 120 125

Leu Lys Gly Gln Lys Val Leu Ile Val Asp Asp Val Ile Thr Ala Gly

130 135 140

Thr Ala Ile Arg Glu Ala His Lys Ile Val Glu Ser Glu Gly Gly Gln

145 150 155 160

Thr Ala Gly Ile Val Glu Ala Leu Asp Arg Glu Glu Arg Gly Gln Gly

165 170 175

Glu Leu Ser Thr Val Gln Glu Val Glu Lys Glu Leu Gly Val Lys Val

180 185 190

Thr Ser Val Val Lys Met Arg Asp Ile Val Ala Trp Leu Lys Glu Lys

195 200 205

Gly Lys Leu Asp Glu Met Lys Ala Met Glu Glu Tyr Arg Ala Lys Trp

210 215 220

Gly Val Asn

225

<210>5

<211>684

<212>DNA

＜213〉justify red winter spore yeast (Rhodosporidium toruloides)

<220>

<221>gene

<222>(1)..(684)

<223>

<400>5

atgagcgcca cgtcctacgc cgcctcgatc atcgagaccg ctctcgcgag cgagcagccc 60

atcctccgct tcggcacctt caccctcaag tctggccgct cctcgcccta cttcttcaac 120

tttggcctct tcaacaccgg ctctctcctc ctcgctctcg cctcggcctt cgcagacgcc 180

atcctcgacg cctaccccga gattggctcc tcctccgccg gtcccgacac gcccaaagtc 240

ctcttcggac ccgcctacaa gggcatcccc ctcgtcgctg ctatcgcatc cgaactcgca 300

cgacgaggac gtgatatcgg ctacagctac aaccgcaagg agaagaagga ccacggcgag 360

ggcgggtcga ttgtcggtgc gccgctcaag ggacagaagg tcctcatcgt cgacgacgtg 420

atcacggccg gtactgcgat tcgcgaggcg cacaagatcg tcgagtcgga gggcggtcag 480

acggcgggta ttgtcgaggc cctcgaccgg gaggagcgcg gacagggcga gctcagtacg 540

gtccaggaag tcgagaagga gctcggcgtc aaggtcacga gcgtcgtcaa gatgcgcgac 600

atcgttgcgt ggctcaaaga gaagggcaag ctcgacgaga tgaaggccat ggaggagtac 660

cgcgcaaagt ggggcgttaa ctag 684

<210>6

<211>2489

<212>DNA

＜213〉justify red winter spore yeast (Rhodosporidium toruloides)

<220>

<221>promoter

<222>(201)..(1000)

<223>

<220>

<221>terminator

<222>(1685)..(2489)

<223>

<400>6

tgccgcctat ctgttaaaac tcaatggata caatgcaaag tcggctcgcg ccctcttctc 60

ccgttccccc gtacctagac atgcgtcgtg agtgcttccc tccgtcatgc aaccgtcgca 120

agctcctcgc tccgtcgtcg ctgctgaagc tgctcctgcg caatccgctg cgcggcgggt 180

acctcgcctc gctccctttc cgccgcccga atcgcctgtg tgagcctgtt cctccgagac 240

acgaccatcg tcttgggatt ggcttgtggt acgggcagag ggctggtctc tgcaattgag 300

caagcgagcc gaggcagcga tggtcagttc cggtcgcaga ccgaggctcc agggaaggga 360

cggcgggacg cactgatgac gccggcctga atgagcagct tctcaaagcg ctttgtcggc 420

agcgcaccct ggctgagcca gtaccggacc cgctccaggt tccactcgac cttcttctgc 480

ccgaccttgg cgacgcccgt gttgggcgtg tgctgccgtg gtccccactc cttcgggtcc 540

ctgacctgac cgtttggcga gaccgacggc gctggcacga cgaccggtcg cgggttgtac 600

gaaccgagga gctcgagggg tttggcggtc tggcgctgcg gcgcgcggat ggcgacgagc 660

gagtagacgg ggttgttgcg cgtgcagtgg tggcgcgcga ggcgcaatcg gaccgacatc 720

tcgtgcgctg gcggagagcg agtgtgcgag gacgaggaaa aggagcggga ggaggagggt 780

tgggagcgaa ccacctcgtg cagctggact ggacgcgcta gacgactgcc agactcgcta 840

tactgctctt tacatccgct agaatcctgc acccgctcga accagctcct cccaccgtca 900

gttcgcctcc ccctctcatc ctcatctctc aaaggacttc ctcgctcgca actcgctgga 960

gggagctgca agactgacac gaggtcgaaa acaccgtcag atgagcgcca cgtcctacgc 1020

cgcctcgatc atcgagaccg ctctcgcgag cgagcagccc atcctccgct tcggcacctt 1080

caccctcaag tctggccgct cctcgcccta cttcttcaac tttggcctct tcaacaccgg 1140

ctctctcctc ctcgctctcg cctcggcctt cgcagacgcc atcctcgacg cctaccccga 1200

gattggctcc tcctccgccg gtcccgacac gcccaaagtc ctcttcggac ccgcctacaa 1260

gggcatcccc ctcgtcgctg ctatcgcatc cgaactcgca cgacgaggac gtgatatcgg 1320

ctacagctac aaccgcaagg agaagaagga ccacggcgag ggcgggtcga ttgtcggtgc 1380

gccgctcaag ggacagaagg tcctcatcgt cgacgacgtg atcacggccg gtactgcgat 1440

tcgcgaggcg cacaagatcg tcgagtcgga gggcggtcag acggcgggta ttgtcgaggc 1500

cctcgaccgg gaggagcgcg gacagggcga gctcagtacg gtccaggaag tcgagaagga 1560

gctcggcgtc aaggtcacga gcgtcgtcaa gatgcgcgac atcgttgcgt ggctcaaaga 1620

gaagggcaag ctcgacgaga tgaaggccat ggaggagtac cgcgcaaagt ggggcgttaa 1680

ctagatacaa gtgcgtctgg gcagttcccc tcttttgcgt agcgtacccg cacgatttac 1740

tgacgcaact cgggcgctac agtcctgtca atctatcgcg atcttcgcag cgacactcgc 1800

cgcttccgat tcccgacttt gcgtccatct cgctcgcttc gataagcttc tttggctgct 1860

acagacggct ctcaactctg ctggaagatg cagcatcagc actcacgggc tccgcaacag 1920

ctgctgtcac gcctcgagtt cagtcgggca gctctcgcca tctcgcaacc cgttatggcg 1980

atggtcgacc cgtctcgcgg ctttcccagc gctgcgtttc cgcttcagct gtcgcggtac 2040

aaacttcatc gccccgtagc gagccgtctc tcgcgcaacg aggcaatctc gcgcttctct 2100

ccctaacgct ttctatcggt cccggcctct ctcccgcgga ctctaagaga cgtcgacgca 2160

gtttcgatat cgtcaacagc gagcgcagac gagcactgcc cgagcagtga acgacgacga 2220

gctctgtacg aagcgatcgg atccgccgcc ttcgctcgcg acatgctcga gctttcctga 2280

tcaccttgtc tgagttgctc aggctctgct aagtcagtcg tgcgcatcgg agtttgctga 2340

ggcgcgagaa ggatattaca gctcgtacga aggacggcga ctctggctcg ctgtccgtca 2400

cacgagacag aggccagcct cgacctcgtc agcgagtcgg tctgtccatg acggctaact 2460

aatgagggtg gctgaacaga ttgaatggc 2489

<210>7

<211>717

<212>DNA

＜213〉artificial sequence

<220>

<221>gene

<222>(1)..(717)

<223>

<400>7

atgagtaaag gagaagaact tttcactgga gttgtcccaa ttcttgttga attagatggt 60

gatgttaatg ggcacaaatt ttctgtcagt ggagagggtg aaggtgatgc aacatacgga 120

aaacttaccc ttaaatttat ttgcactact ggaaaactac ctgttccatg gccaacactt 180

gtcactactt tctcttatgg tgttcaatgc ttttcccgtt atccggatca tatgaaacgg 240

catgactttt tcaagagtgc catgcccgaa ggttatgtac aggaacgcac tatatctttc 300

aaagatgacg ggaactacaa gacgcgtgct gaagtcaagt ttgaaggtga tacccttgtt 360

aatcgtatcg agttaaaagg tattgatttt aaagaagatg gaaacattct cggacacaaa 420

ctcgagtaca actataactc acacaatgta tacatcacgg cagacaaaca aaagaatgga 480

atcaaagcta acttcaaaat tcgccacaac attgaagatg gatccgttca actagcagac 540

cattatcaac aaaatactcc aattggcgat ggccctgtcc ttttaccaga caaccattac 600

ctgtcgacac aatctgccct ttcgaaagat cccaacgaaa agcgtgacca catggtcctt 660

cttgagtttg taactgctgc tgggattaca catggcatgg atgagctcta caaataa 717

<210>8

<211>2522

<212>DNA

＜213〉artificial sequence

<220>

<221>promoter

<222>(201)..(1000)

<223>

<220>

<221>terminator

<222>(1718)..(2522)

<223>

<220>

<221>gene

<222>(1001)..(1717)

<223>

<400>8

tgccgcctat ctgttaaaac tcaatggata caatgcaaag tcggctcgcg ccctcttctc 60

ccgttccccc gtacctagac atgcgtcgtg agtgcttccc tccgtcatgc aaccgtcgca 120

agctcctcgc tccgtcgtcg ctgctgaagc tgctcctgcg caatccgctg cgcggcgggt 180

acctcgcctc gctccctttc cgccgcccga atcgcctgtg tgagcctgtt cctccgagac 240

acgaccatcg tcttgggatt ggcttgtggt acgggcagag ggctggtctc tgcaattgag 300

caagcgagcc gaggcagcga tggtcagttc cggtcgcaga ccgaggctcc agggaaggga 360

cggcgggacg cactgatgac gccggcctga atgagcagct tctcaaagcg ctttgtcggc 420

agcgcaccct ggctgagcca gtaccggacc cgctccaggt tccactcgac cttcttctgc 480

ccgaccttgg cgacgcccgt gttgggcgtg tgctgccgtg gtccccactc cttcgggtcc 540

ctgacctgac cgtttggcga gaccgacggc gctggcacga cgaccggtcg cgggttgtac 600

gaaccgagga gctcgagggg tttggcggtc tggcgctgcg gcgcgcggat ggcgacgagc 660

gagtagacgg ggttgttgcg cgtgcagtgg tggcgcgcga ggcgcaatcg gaccgacatc 720

tcgtgcgctg gcggagagcg agtgtgcgag gacgaggaaa aggagcggga ggaggagggt 780

tgggagcgaa ccacctcgtg cagctggact ggacgcgcta gacgactgcc agactcgcta 840

tactgctctt tacatccgct agaatcctgc acccgctcga accagctcct cccaccgtca 900

gttcgcctcc ccctctcatc ctcatctctc aaaggacttc ctcgctcgca actcgctgga 960

gggagctgca agactgacac gaggtcgaaa acaccgtcag atgagtaaag gagaagaact 1020

tttcactgga gttgtcccaa ttcttgttga attagatggt gatgttaatg ggcacaaatt 1080

ttctgtcagt ggagagggtg aaggtgatgc aacatacgga aaacttaccc ttaaatttat 1140

ttgcactact ggaaaactac ctgttccatg gccaacactt gtcactactt tctcttatgg 1200

tgttcaatgc ttttcccgtt atccggatca tatgaaacgg catgactttt tcaagagtgc 1260

catgcccgaa ggttatgtac aggaacgcac tatatctttc aaagatgacg ggaactacaa 1320

gacgcgtgct gaagtcaagt ttgaaggtga tacccttgtt aatcgtatcg agttaaaagg 1380

tattgatttt aaagaagatg gaaacattct cggacacaaa ctcgagtaca actataactc 1440

acacaatgta tacatcacgg cagacaaaca aaagaatgga atcaaagcta acttcaaaat 1500

tcgccacaac attgaagatg gatccgttca actagcagac cattatcaac aaaatactcc 1560

aattggcgat ggccctgtcc ttttaccaga caaccattac ctgtcgacac aatctgccct 1620

ttcgaaagat cccaacgaaa agcgtgacca catggtcctt cttgagtttg taactgctgc 1680

tgggattaca catggcatgg atgagctcta caaatagata caagtgcgtc tgggcagttc 1740

ccctcttttg cgtagcgtac ccgcacgatt tactgacgca actcgggcgc tacagtcctg 1800

tcaatctatc gcgatcttcg cagcgacact cgccgcttcc gattcccgac tttgcgtcca 1860

tctcgctcgc ttcgataagc ttctttggct gctacagacg gctctcaact ctgctggaag 1920

atgcagcatc agcactcacg ggctccgcaa cagctgctgt cacgcctcga gttcagtcgg 1980

gcagctctcg ccatctcgca acccgttatg gcgatggtcg acccgtctcg cggctttccc 2040

agcgctgcgt ttccgcttca gctgtcgcgg tacaaacttc atcgccccgt agcgagccgt 2100

ctctcgcgca acgaggcaat ctcgcgcttc tctccctaac gctttctatc ggtcccggcc 2160

tctctcccgc ggactctaag agacgtcgac gcagtttcga tatcgtcaac agcgagcgca 2220

gacgagcact gcccgagcag tgaacgacga cgagctctgt acgaagcgat cggatccgcc 2280

gccttcgctc gcgacatgct cgagctttcc tgatcacctt gtctgagttg ctcaggctct 2340

gctaagtcag tcgtgcgcat cggagtttgc tgaggcgcga gaaggatatt acagctcgta 2400

cgaaggacgg cgactctggc tcgctgtccg tcacacgaga cagaggccag cctcgacctc 2460

gtcagcgagt cggtctgtcc atgacggcta actaatgagg gtggctgaac agattgaatg 2520

gc 2522

<210>9

<211>375

<212>DNA

＜213〉artificial sequence

<220>

<221>gene

<222>(1)..(375)

<223>

<400>9

atggccaagt tgaccagtgc cgttccggtg ctcaccgcgc gcgacgtcgc cggagcggtc 60

gagttctgga ccgaccggct cgggttctcc cgggacttcg tggaggacga cttcgccggt 120

gtggtccggg acgacgtgac cctgttcatc agcgcggtcc aggaccaggt ggtgccggac 180

aacaccctgg cctgggtgtg ggtgcgcggc ctggacgagc tgtacgccga gtggtcggag 240

gtcgtgtcca cgaacttccg ggacgcctcc gggccggcca tgaccgagat cggcgagcag 300

ccgtgggggc gggagttcgc cctgcgcgac ccggccggca actgcgtgca cttcgtggcc 360

gaggagcagg actga 375

<210>10

<211>2180

<212>DNA

＜213〉artificial sequence

<220>

<221>promoter

<222>(201)..(1000)

<223>

<220>

<221>terminator

<222>(1376)..(2180)

<223>

<220>

<221>gene

<222>(1001)..(1375)

<223>

<400>10

tgccgcctat ctgttaaaac tcaatggata caatgcaaag tcggctcgcg ccctcttctc 60

ccgttccccc gtacctagac atgcgtcgtg agtgcttccc tccgtcatgc aaccgtcgca 120

agctcctcgc tccgtcgtcg ctgctgaagc tgctcctgcg caatccgctg cgcggcgggt 180

acctcgcctc gctccctttc cgccgcccga atcgcctgtg tgagcctgtt cctccgagac 240

acgaccatcg tcttgggatt ggcttgtggt acgggcagag ggctggtctc tgcaattgag 300

caagcgagcc gaggcagcga tggtcagttc cggtcgcaga ccgaggctcc agggaaggga 360

cggcgggacg cactgatgac gccggcctga atgagcagct tctcaaagcg ctttgtcggc 420

agcgcaccct ggctgagcca gtaccggacc cgctccaggt tccactcgac cttcttctgc 480

ccgaccttgg cgacgcccgt gttgggcgtg tgctgccgtg gtccccactc cttcgggtcc 540

ctgacctgac cgtttggcga gaccgacggc gctggcacga cgaccggtcg cgggttgtac 600

gaaccgagga gctcgagggg tttggcggtc tggcgctgcg gcgcgcggat ggcgacgagc 660

gagtagacgg ggttgttgcg cgtgcagtgg tggcgcgcga ggcgcaatcg gaccgacatc 720

tcgtgcgctg gcggagagcg agtgtgcgag gacgaggaaa aggagcggga ggaggagggt 780

tgggagcgaa ccacctcgtg cagctggact ggacgcgcta gacgactgcc agactcgcta 840

tactgctctt tacatccgct agaatcctgc acccgctcga accagctcct cccaccgtca 900

gttcgcctcc ccctctcatc ctcatctctc aaaggacttc ctcgctcgca actcgctgga 960

gggagctgca agactgacac gaggtcgaaa acaccgtcag atggccaagt tgaccagtgc 1020

cgttccggtg ctcaccgcgc gcgacgtcgc cggagcggtc gagttctgga ccgaccggct 1080

cgggttctcc cgggacttcg tggaggacga cttcgccggt gtggtccggg acgacgtgac 1140

cctgttcatc agcgcggtcc aggaccaggt ggtgccggac aacaccctgg cctgggtgtg 1200

ggtgcgcggc ctggacgagc tgtacgccga gtggtcggag gtcgtgtcca cgaacttccg 1260

ggacgcctcc gggccggcca tgaccgagat cggcgagcag ccgtgggggc gggagttcgc 1320

cctgcgcgac ccggccggca actgcgtgca cttcgtggcc gaggagcagg actgaataca 1380

agtgcgtctg ggcagttccc ctcttttgcg tagcgtaccc gcacgattta ctgacgcaac 1440

tcgggcgcta cagtcctgtc aatctatcgc gatcttcgca gcgacactcg ccgcttccga 1500

ttcccgactt tgcgtccatc tcgctcgctt cgataagctt ctttggctgc tacagacggc 1560

tctcaactct gctggaagat gcagcatcag cactcacggg ctccgcaaca gctgctgtca 1620

cgcctcgagt tcagtcgggc agctctcgcc atctcgcaac ccgttatggc gatggtcgac 1680

ccgtctcgcg gctttcccag cgctgcgttt ccgcttcagc tgtcgcggta caaacttcat 1740

cgccccgtag cgagccgtct ctcgcgcaac gaggcaatct cgcgcttctc tccctaacgc 1800

tttctatcgg tcccggcctc tctcccgcgg actctaagag acgtcgacgc agtttcgata 1860

tcgtcaacag cgagcgcaga cgagcactgc ccgagcagtg aacgacgacg agctctgtac 1920

gaagcgatcg gatccgccgc cttcgctcgc gacatgctcg agctttcctg atcaccttgt 1980

ctgagttgct caggctctgc taagtcagtc gtgcgcatcg gagtttgctg aggcgcgaga 2040

aggatattac agctcgtacg aaggacggcg actctggctc gctgtccgtc acacgagaca 2100

gaggccagcc tcgacctcgt cagcgagtcg gtctgtccat gacggctaac taatgagggt 2160

ggctgaacag attgaatggc 2180

<210>11

<211>810

<212>DNA

＜213〉artificial sequence

<220>

<221>gene

<222>(1)..(810)

<223>

<400>11

atgggtaagg aaaagactca cgtttcgagg ccgcgattaa attccaacat ggatgctgat 60

ttatatgggt ataaatgggc tcgcgataat gtcgggcaat caggtgcgac aatctatcga 120

ttgtatggga agcccgatgc gccagagttg tttctgaaac atggcaaagg tagcgttgcc 180

aatgatgtta cagatgagat ggtcagacta aactggctga cggaatttat gcctcttccg 240

accatcaagc attttatccg tactcctgat gatgcatggt tactcaccac tgcgatcccc 300

ggcaaaacag cattccaggt attagaagaa tatcctgatt caggtgaaaa tattgttgat 360

gcgctggcag tgttcctgcg ccggttgcat tcgattcctg tttgtaattg tccttttaac 420

agcgatcgcg tatttcgtct cgctcaggcg caatcacgaa tgaataacgg tttggttgat 480

gcgagtgatt ttgatgacga gcgtaatggc tggcctgttg aacaagtctg gaaagaaatg 540

cataagcttt tgccattctc accggattca gtcgtcactc atggtgattt ctcacttgat 600

aaccttattt ttgacgaggg gaaattaata ggttgtattg atgttggacg agtcggaatc 660

gcagaccgat accaggatct tgccatccta tggaactgcc tcggtgagtt ttctccttca 720

ttacagaaac ggctttttca aaaatatggt attgataatc ctgatatgaa taaattgcag 780

tttcatttga tgctcgatga gtttttctaa 810

<210>12

<211>2615

<212>DNA

＜213〉artificial sequence

<220>

<221>promoter

<222>(201)..(1000)

<223>

<220>

<221>terminator

<222>(1811)..(2615)

<223>

<220>

<221>gene

<222>(1001)..(1810)

<223>

<400>12

tgccgcctat ctgttaaaac tcaatggata caatgcaaag tcggctcgcg ccctcttctc 60

ccgttccccc gtacctagac atgcgtcgtg agtgcttccc tccgtcatgc aaccgtcgca 120

agctcctcgc tccgtcgtcg ctgctgaagc tgctcctgcg caatccgctg cgcggcgggt 180

acctcgcctc gctccctttc cgccgcccga atcgcctgtg tgagcctgtt cctccgagac 240

acgaccatcg tcttgggatt ggcttgtggt acgggcagag ggctggtctc tgcaattgag 300

caagcgagcc gaggcagcga tggtcagttc cggtcgcaga ccgaggctcc agggaaggga 360

cggcgggacg cactgatgac gccggcctga atgagcagct tctcaaagcg ctttgtcggc 420

agcgcaccct ggctgagcca gtaccggacc cgctccaggt tccactcgac cttcttctgc 480

ccgaccttgg cgacgcccgt gttgggcgtg tgctgccgtg gtccccactc cttcgggtcc 540

ctgacctgac cgtttggcga gaccgacggc gctggcacga cgaccggtcg cgggttgtac 600

gaaccgagga gctcgagggg tttggcggtc tggcgctgcg gcgcgcggat ggcgacgagc 660

gagtagacgg ggttgttgcg cgtgcagtgg tggcgcgcga ggcgcaatcg gaccgacatc 720

tcgtgcgctg gcggagagcg agtgtgcgag gacgaggaaa aggagcggga ggaggagggt 780

tgggagcgaa ccacctcgtg cagctggact ggacgcgcta gacgactgcc agactcgcta 840

tactgctctt tacatccgct agaatcctgc acccgctcga accagctcct cccaccgtca 900

gttcgcctcc ccctctcatc ctcatctctc aaaggacttc ctcgctcgca actcgctgga 960

gggagctgca agactgacac gaggtcgaaa acaccgtcag atgggtaagg aaaagactca 1020

cgtttcgagg ccgcgattaa attccaacat ggatgctgat ttatatgggt ataaatgggc 1080

tcgcgataat gtcgggcaat caggtgcgac aatctatcga ttgtatggga agcccgatgc 1140

gccagagttg tttctgaaac atggcaaagg tagcgttgcc aatgatgtta cagatgagat 1200

ggtcagacta aactggctga cggaatttat gcctcttccg accatcaagc attttatccg 1260

tactcctgat gatgcatggt tactcaccac tgcgatcccc ggcaaaacag cattccaggt 1320

attagaagaa tatcctgatt caggtgaaaa tattgttgat gcgctggcag tgttcctgcg 1380

ccggttgcat tcgattcctg tttgtaattg tccttttaac agcgatcgcg tatttcgtct 1440

cgctcaggcg caatcacgaa tgaataacgg tttggttgat gcgagtgatt ttgatgacga 1500

gcgtaatggc tggcctgttg aacaagtctg gaaagaaatg cataagcttt tgccattctc 1560

accggattca gtcgtcactc atggtgattt ctcacttgat aaccttattt ttgacgaggg 1620

gaaattaata ggttgtattg atgttggacg agtcggaatc gcagaccgat accaggatct 1680

tgccatccta tggaactgcc tcggtgagtt ttctccttca ttacagaaac ggctttttca 1740

aaaatatggt attgataatc ctgatatgaa taaattgcag tttcatttga tgctcgatga 1800

gtttttctaa atacaagtgc gtctgggcag ttcccctctt ttgcgtagcg tacccgcacg 1860

atttactgac gcaactcggg cgctacagtc ctgtcaatct atcgcgatct tcgcagcgac 1920

actcgccgct tccgattccc gactttgcgt ccatctcgct cgcttcgata agcttctttg 1980

gctgctacag acggctctca actctgctgg aagatgcagc atcagcactc acgggctccg 2040

caacagctgc tgtcacgcct cgagttcagt cgggcagctc tcgccatctc gcaacccgtt 2100

atggcgatgg tcgacccgtc tcgcggcttt cccagcgctg cgtttccgct tcagctgtcg 2160

cggtacaaac ttcatcgccc cgtagcgagc cgtctctcgc gcaacgaggc aatctcgcgc 2220

ttctctccct aacgctttct atcggtcccg gcctctctcc cgcggactct aagagacgtc 2280

gacgcagttt cgatatcgtc aacagcgagc gcagacgagc actgcccgag cagtgaacga 2340

cgacgagctc tgtacgaagc gatcggatcc gccgccttcg ctcgcgacat gctcgagctt 2400

tcctgatcac cttgtctgag ttgctcaggc tctgctaagt cagtcgtgcg catcggagtt 2460

tgctgaggcg cgagaaggat attacagctc gtacgaagga cggcgactct ggctcgctgt 2520

ccgtcacacg agacagaggc cagcctcgac ctcgtcagcg agtcggtctg tccatgacgg 2580

ctaactaatg agggtggctg aacagattga atggc 2615

Claims (7)

1. the orotate phosphoribosyl transferase promotor is abbreviated as pRtura5, and its nucleotide sequence consists of dna sequence dna shown in SEQ ID NO:1.

2. the application of the described orotate phosphoribosyl transferase promotor of claim 1, it is characterized in that: the deoxynucleoside acid sequence shown in the SEQ ID NO:1 can be used as promotor and is used for making up saccharomyces neoformans genetic operating system and new recombinant strain, and resulting engineering strain carries corresponding pRtura5 sequence;

Described engineering strain is Rhodosporidium (Rhodosporidium) engineering strain, and described saccharomyces neoformans genetic operating system is the red winter spore yeast genetic operating system of circle.

3. DNA construct, contain the deoxynucleoside acid sequence shown in the described SEQ ID of claim 1 NO:1, or contain simultaneously the deoxynucleoside acid sequence shown in the described SEQ ID of claim 1 NO:1 and the deoxynucleoside acid sequence shown in SEQ ID NO:2, and sequence is positioned at the upstream of sequence shown in the SEQ ID NO:2 shown in the SEQ ID NO:1, is the open reading frame of an encoding gene between SEQ ID NO:1 and the SEQ ID NO:2.

4. DNA construct as claimed in claim 3, it is characterized in that: sequence shown in the described SEQ ID NO:2 is a kind of orotate phosphoribosyl transferase terminator Rtura5t.

5. DNA construct as claimed in claim 3, it is characterized in that: described open reading frame is the open reading frame of the orotate phosphoribosyl transferase gene between SEQ ID NO:1 and SEQ ID NO:2, its cDNA sequence is the deoxynucleoside acid sequence shown in SEQ ID NO:3, its genomic dna is the deoxynucleoside acid sequence shown in the SEQ ID NO:5, and its aminoacid sequence is shown in SEQ ID NO:4.

6. carrier that carries promotor pRtura5 as claimed in claim 1 or DNA construct claimed in claim 4.

7. carrier as claimed in claim 6, it is characterized in that: described carrier is plasmid vector.

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