CN109136120B - Microorganisms and uses thereof - Google Patents

Microorganisms and uses thereof Download PDF

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CN109136120B
CN109136120B CN201710464916.6A CN201710464916A CN109136120B CN 109136120 B CN109136120 B CN 109136120B CN 201710464916 A CN201710464916 A CN 201710464916A CN 109136120 B CN109136120 B CN 109136120B
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gene
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nucleotide sequence
lycopene
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CN109136120A (en
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刘天罡
叶紫玲
沈佳
刘然
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Wuhan Hesheng Technology Co ltd
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Wuhan Hesheng Technology Co ltd
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    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/14—Fungi; Culture media therefor
    • C12N1/16—Yeasts; Culture media therefor
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/37—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi
    • C07K14/39—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi from yeasts
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    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/14—Fungi; Culture media therefor
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    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/14—Fungi; Culture media therefor
    • C12N1/16—Yeasts; Culture media therefor
    • C12N1/18—Baker's yeast; Brewer's yeast
    • C12N1/185—Saccharomyces isolates
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
    • C12N15/81—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P5/00—Preparation of hydrocarbons or halogenated hydrocarbons
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    • C12P5/00—Preparation of hydrocarbons or halogenated hydrocarbons
    • C12P5/02—Preparation of hydrocarbons or halogenated hydrocarbons acyclic
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    • C12P1/00—Preparation of compounds or compositions, not provided for in groups C12P3/00 - C12P39/00, by using microorganisms or enzymes
    • C12P1/02—Preparation of compounds or compositions, not provided for in groups C12P3/00 - C12P39/00, by using microorganisms or enzymes by using fungi
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    • C12R2001/00—Microorganisms ; Processes using microorganisms
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    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/645—Fungi ; Processes using fungi
    • C12R2001/85—Saccharomyces
    • C12R2001/865—Saccharomyces cerevisiae

Abstract

The invention provides a microorganism, application thereof and a method for obtaining lycopene, wherein the microorganism overexpresses PaCrtE, PagCrtB and BtCrtI genes. By utilizing the microorganism provided by the embodiment of the invention, the yield of lycopene is obviously improved, the production period is short, and the production efficiency is high.

Description

Microorganisms and uses thereof
Technical Field
The invention relates to the field of bioengineering, in particular to a microorganism and application thereof, and more particularly to a microorganism, a method for obtaining lycopene and application of the microorganism in preparation of lycopene.
Background
The development and production of lycopene mainly include natural product extraction, chemical synthesis, microbial fermentation and other methods. The natural product extraction is mainly to obtain lycopene by extraction and purification of mature fruits, however, the production mode is influenced by a plurality of uncontrollable factors such as climate, variety, geographical position, maturity and the like, has obvious seasonality and unstable content, and has higher cost and lower content in large-area planting and breeding. In addition, high purity lycopene is very difficult in extraction and purification technology, which together make the final lycopene product very expensive. The chemical synthesis method has the advantages of easily available and cheap raw materials, mild reaction conditions, high reaction rate and easy separation of products from a reaction system, but the quality, safety and application range of the products are limited due to the difficulty in controlling the stereoselectivity of double bonds and the chemical reagent residues with different degrees. The microbial fermentation method mainly utilizes the biological metabolism of microorganisms to convert cheap raw materials such as glucose, starch, soybean cake powder and the like into lycopene, is not influenced by factors such as seasons, regions, climate and the like, has the advantages of easily obtained raw materials, short production period, simple process operation, low cost, controllable product quality, easily purified product, high safety and less environmental pollution, and not only solves the problem of occupying a large amount of cultivated land due to planting plants and the like, but also solves the defect of environmental pollution caused by chemical synthesis. Most importantly, lycopene produced by fermentation belongs to natural products, the activity of the lycopene is consistent with that of active ingredients extracted from natural plants, and the lycopene is considered to be the most promising method for producing the lycopene.
Disclosure of Invention
The present invention is directed to solving, at least to some extent, one of the technical problems in the related art.
Currently, the main study on the lycopene-producing bacteria is Blakeslea trispora. However, the Blakeslea trispora is easy to degenerate during passage, so that the yield of lycopene is reduced, and synthesis of toxic components such as aflatoxin and the like is accompanied.
Therefore, an object of the present invention is to provide a microorganism which produces lycopene with high yield, short production period and high production efficiency.
In a first aspect of the invention, a microorganism is presented. According to an embodiment of the invention, the microorganism overexpresses the PaCrtE, PagCrtB and BtCrtI genes. By utilizing the microorganism provided by the embodiment of the invention, the yield of lycopene is obviously improved, the production period is short, and the production efficiency is high.
According to an embodiment of the present invention, the microorganism may further comprise at least one of the following additional technical features:
according to an embodiment of the present invention, the microorganism further overexpresses at least one gene selected from the group consisting of tmg 1, INO2, yap1, spt15-5, taf25-3, GapN, PYC2, SMAE1, MDH2, POS5, pntAB, ADH2, ACS6, ALD6, EUTE, ERG12, IDI1, ERG10, MVD1, ERG13, ERG 8. The inventors found that the microorganism further overexpresses at least one of the above genes, and the lycopene production is further improved.
According to an embodiment of the invention, the microorganism further silences at least one gene selected from the group consisting of GAL1, GAL7, GAL10, GAL80, ROX1, VBA5, DOS2, Ypl062W, Yjl064W, Yer130C, Yer134C, Ynr063W, Exg1, Yor292C, Sfk1, Mef 1. The inventors found that the microorganism further silences at least one of the above genes, and the yield of lycopene is further increased.
According to an embodiment of the invention, the microorganism is a yeast. The growth cycle of the yeast is short, and the efficiency of producing the lycopene by using the microorganism of the embodiment of the invention is further improved.
According to an embodiment of the invention, the microorganism further comprises an ERG9 gene operably regulated. According to the embodiment of the invention, the operable regulation and control of the ERG9 gene comprises, but is not limited to, replacement of a promoter of the ERG9 gene, so that the expression of the ERG9 gene can be regulated and controlled as required, and the efficiency of producing lycopene by the microorganism is further improved.
In a second aspect of the invention, the invention proposes a method for obtaining lycopene. According to an embodiment of the invention, the method comprises: subjecting the microorganism to a fermentation treatment; and subjecting the fermentation treatment product to an extraction treatment to obtain said lycopene. By utilizing the method for obtaining the lycopene, the lycopene can be obtained with high yield and high efficiency, and the purity of the lycopene is high.
According to an embodiment of the present invention, the above method for obtaining lycopene may further comprise at least one of the following additional technical features:
according to the embodiment of the invention, the lycopene is obtained with higher yield, efficiency and purity by using the method for obtaining the lycopene according to the embodiment of the invention.
According to an embodiment of the invention, the fermentation process is carried out by: subjecting the microorganism to a basal fermentation treatment in a basal fermentation medium and a two-stage fed-batch fermentation treatment by sequentially supplementing a first feed medium and a second feed medium on the basis of the basal fermentation medium, wherein the basal fermentation medium is a YPD medium containing 24g/L peptone, 12g/L yeast extract and 12g/L glucose; the first feed medium is YPD medium containing 500g/L glucose and 15g/L yeast extract; the second feed medium was 75% ethanol or 50% glycerol. The inventors found through experiments that the fermentation treatment method has high microbial amplification rate and further improves lycopene yield.
According to an embodiment of the invention, the extraction process comprises: and carrying out ultrasonic crushing treatment and organic extraction treatment on the fermentation treatment product. The inventor discovers that in the process of extracting the fermentation product, the degradation of the lycopene is greatly reduced and the yield of the lycopene obtained after the extraction treatment is further improved when the ultrasonic crushing treatment is carried out on the fermentation product compared with the method of stewing by adopting hydrochloric acid.
In a third aspect of the invention, the invention proposes the use of a microorganism as described hereinbefore for the preparation of lycopene. As described above, the microorganisms according to the embodiments of the present invention have high lycopene production yield, short production cycle, and high production efficiency.
Drawings
FIG. 1 is a schematic structural diagram of a knockout cassette fragment 1 according to example 1 of the present invention;
FIG. 2 is a schematic structural diagram of a knockout cassette fragment 2 according to example 1 of the present invention;
FIG. 3 is a schematic structural diagram of a knockout cassette fragment 3 according to example 1 of the present invention;
FIG. 4 shows the results of shake flask fermentation of the second generation of engineering strains;
FIG. 5 is a schematic structural diagram of knockout cassette fragment 4 according to example 4 of the present invention; and
FIG. 6 is a graph showing the screening results of fermentation media according to an embodiment of the present invention.
Detailed Description
The following describes embodiments of the present invention in detail. The following examples are illustrative only and are not to be construed as limiting the invention.
Microorganisms
In a first aspect of the invention, a microorganism is presented. According to embodiments of the invention, the microorganism overexpresses the PaCrtE, PagCrtB and BtCrtI genes. The microorganism provided by the embodiment of the invention has the advantages of high yield, short production period and high production efficiency.
According to a specific embodiment of the present invention, the microorganism may further overexpress at least one gene selected from the group consisting of tmg 1, INO2, yap1, spt15-5, taf25-3, GapN, PYC2, SMAE1, MDH2, POS5, pntAB, ADH2, ACS6, ALD6, EUTE, ERG12, IDI1, ERG10, MVD1, ERG13, ERG 8.
According to yet another embodiment of the present invention, the microorganism may further silence at least one gene selected from the group consisting of GAL1, GAL7, GAL10, GAL80, ROX1, VBA5, DOS2, Ypl062W, Yjl064W, Yer130C, Yer134C, Ynr063W, Exg1, Yor292C, Sfk1, Mef 1. The inventors found that the microorganism further silences at least one of the above genes, and the yield of lycopene is further increased.
The inventor finds that the microorganism further overexpresses or silences at least one of the genes, and the yield of lycopene is further remarkably improved.
Compared with the prior art, the yield of the microorganism used for producing the lycopene is obviously improved and can reach at least 2.5 g/L.
According to a specific embodiment of the present invention, the microorganism may further comprise an ERG9 gene operably regulated. The operable regulation of ERG9 gene means that the original promoter of ERG is replaced, so that the expression of ERG9 is regulated through glucose, and the yield of lycopene is increased.
The inventors found, through experiments, that: the overexpression of PaCrtE, PagCrtB and BtCrtI genes can achieve the aim of efficiently synthesizing lycopene in a yeast body; silencing GAL1,7,10 or GAL1,7,10, 80 can achieve the purpose of producing lycopene by utilizing galactose induction or glucose concentration regulation; the over-expressed INO2, yap1, spt15-5 and taf25-3 genes are stress-resistant genes and can increase the yield of lycopene; over-expression of one or more genes of GapN, PYC2, SMAE1, MDH2, POS5 and pntAB can balance the reducing power in yeast, and can increase the yield of lycopene; overexpression of one or more genes in ADH2, ALD6 and EUTE can increase the supply of precursors for synthesizing lycopene and increase the yield of lycopene; over-expression of one or more genes selected from ERG12, IDI1, ERG10, MVD1, ERG13 and ERG8, can balance MVA pathway related to lycopene synthesis, and increase lycopene yield; silencing at least one of ROX1, VBA5, DOS2, Ypl062W, Yjl064W, Yer130C, Yer134C, Ynr063W, Exg1, Yor292C, Sfk1, Mef1 genes can regulate the yeast system as a whole, thereby increasing the yield of lycopene. With the microorganisms according to the embodiments of the present invention, the yield of lycopene is significantly improved.
Method for obtaining lycopene
In a second aspect of the invention, the invention proposes a method for obtaining lycopene. According to an embodiment of the invention, the method comprises: subjecting the microorganism to a fermentation treatment; and subjecting the fermentation treatment product to an extraction treatment to obtain said lycopene. By utilizing the method for obtaining the lycopene, the lycopene can be obtained with high yield and high efficiency, and the purity of the lycopene is high.
According to a particular embodiment of the invention, the fermentation treatment is carried out by a basic fermentation treatment and a two-stage fed-batch fermentation treatment, in particular as follows:
the fermenter was filled with a small amount of pure water and then air-extinguished at 115 ℃ for 30 minutes. Preparing fermentation liquor and feed liquor for batch culture, calibrating pH electrodes of the fermentation tank, clamping pipelines of the fermentation tank, and sterilizing at 115 ℃ for 30 minutes. The fermentation tank is taken out, connected with an air pipeline, aerated for a little (about 0.1 vvm), and simultaneously a cooling water device is opened for cooling. When the temperature is reduced to about 50 ℃, the rotating speed is opened to 100 rpm. And calibrating the dissolved oxygen electrode. The state of the unconnected circuit was set to a dissolved oxygen value of 0%, then the speed was increased to 600rpm while the ventilation was adjusted to 2vvm, the circuit of the dissolved oxygen electrode was connected, and after the dissolved oxygen stabilized, the value was set to 100%. The aeration and rotation speed were then reduced to the values required for fermentation (aeration 1.5vvm, rotation speed 300rpm-600 rpm). The pH value is naturally adjusted to 30 ℃. Inoculation was started and the initial inoculation OD adjusted to 0.5 and the required seed liquid volume was calculated according to the formula. (if the fermentation liquid volume is 2500mL, the seed liquid OD value is n, then the seed liquid volume is 2500x0.5/n mL). After the start of fermentation, the pH of the medium was controlled to 5.5 with 2M NaOH at the time of feeding, the initial aeration rate was 1.5vvm, the initial stirring rate was set at 300rpm, and the dissolved oxygen was maintained at 30% or more (correlation stirring rate, 300-600 rpm).
When the glucose concentration is reduced to about 2g/L during batch culture, glucose feeding is started, the initial feeding rate is 10mL/L fermentation liquor/h, so as to maintain the residual concentration of the glucose in the fermentation liquor to be about 2-3g/L (not to be reduced to 0g/L), an OD600 value is sampled and measured once every two hours, the glucose content is detected once, and the feeding rate is increased when the glucose concentration is lower than 1 g/L. When the OD value increases slowly (begins to enter a stationary phase), the glucose feeding is stopped, the ethanol residual quantity is monitored, when the ethanol concentration is reduced to 5-10g/L, the ethanol glycerol feeding is started, and the initial feeding rate is 2mL/L fermentation liquor/h. The ethanol and glycerol content was then checked every 4h sample while adjusting the feed rate to maintain the ethanol and glycerol in the range of 5-10 g/L. After the color changes, the product is extracted and the product change is detected, and the fermentation is finished when the lycopene concentration is not increased any more.
The inventor finds that the growth speed and the growth state of the thalli are obviously superior to those of the prior art when the thalli are cultured in the basic fermentation culture medium compared with the culture medium in the prior art, the supplemented first supplemented culture medium effectively ensures the rapid and healthy growth of the thalli, and the supplemented second supplemented culture medium effectively improves the yield of lycopene. According to yet another embodiment of the present invention, the extraction process comprises: and carrying out ultrasonic crushing treatment and organic extraction treatment on the product after the fermentation treatment. The inventor carries out optimized screening on the extraction process after fermentation treatment, and finds that the degradation rate of the lycopene is greatly reduced and the yield of the lycopene is stable by adopting an ultrasonic treatment mode compared with a hydrochloric acid cooking mode. In addition, it should be noted that the term "organic extraction treatment" as used herein refers to extraction of the ultrasonication treatment product with an organic solvent, and the manner of organic extraction is not particularly limited, and for example, according to an embodiment of the present invention, lycopene may be extracted from the ultrasonication treatment product with an acetone solvent.
Use of
In a third aspect of the invention, the invention proposes the use of a microorganism as described hereinbefore for the preparation of lycopene. The inventor proves through experiments that the microorganism has remarkable advantages in the aspect of producing lycopene, the yield of lycopene is high, the degradation rate of lycopene is low, and the production period is short.
The following describes embodiments of the present invention in detail. The following examples are illustrative only and are not to be construed as limiting the invention. The examples, where specific techniques or conditions are not indicated, are to be construed according to the techniques or conditions described in the literature in the art or according to the product specifications. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products commercially available.
EXAMPLE 1 construction of the first Generation engineered Strain J1011-C-3
In this example, the inventors described the construction of the J1011-C-3 strain in detail.
Δ LEU2 was first constructed: pGAL 1-PaCrE (Pantoea ananatis) knockout box, namely knockout box fragment 1, designing upstream and downstream primers to PCR amplify each fragment, enabling the fragments to have 60-80bp overlapped fragments, recombining all the fragments together in a homologous recombination mode, and obtaining delta LEU2 through enzyme digestion linearization: : pGAL1-PaCrtE (Pantoea ananatis) knockout cassette, as knockout cassette fragment 1 is shown in FIG. 1, and knockout cassette fragment 1 has the sequence shown in SEQ ID NO: 1. The fragments are respectively integrated on a yeast 30000B genome by utilizing a yeast homologous recombination mechanism of a yeast self through a lithium acetate method yeast transformation, the integration site is LEU2, as LEU2 in the original yeast has no activity, the integrated homologous left arm contains LEU2 complete genes, an SD-Leu solid plate (6.7 g/L of synthetic yeast nitrogen source YNB, 20g/L of glucose, 1.3g/L of leucine-deficient mixed amino acid powder and 2% agar powder) is adopted for screening after solid transformation, the yeast genome is extracted after pure culture for PCR verification, and a successfully verified strain J1011-C-1 is named as a transformant.
ATAACGAGAACACACAGGGGCGCTATCGCACAGAATCAAATTCGATGACTGGAAATTTTTTGTTAATTTCAGAGGTCGCCTGACGCATATACCTTTTTCAACTGAAAAATTGGGAGAAAAAGGAAAGGTGAGAGCGCCGGAACCGGCTTTTCATATAGAATAGAGAAGCGTTCATGACTAAATGCTTGCATCACAATACTTGAAGTTGACAATATTATTTAAGGACCTATTGTTTTTTCCAATAGGTGGTTAGCAATCGTCTTACTTTCTAACTTTTCTTACCTTTTACATTTCAGCAATATATATATATATATTTCAAGGATATACCATTCTAATGTCTGCCCCTAAGAAGATCGTCGTTTTGCCAGGTGACCACGTTGGTCAAGAAATCACAGCCGAAGCCATTAAGGTTCTTAAAGCTATTTCTGATGTTCGTTCCAATGTCAAGTTCGATTTCGAAAATCATTTAATTGGTGGTGCTGCTATCGATGCTACAGGTGTTCCACTTCCAGATGAGGCGCTGGAAGCCTCCAAGAAGGCTGATGCCGTTTTGTTAGGTGCTGTGGGTGGTCCTAAATGGGGTACCGGTAGTGTTAGACCTGAACAAGGTTTACTAAAAATCCGTAAAGAACTTCAATTGTACGCCAACTTAAGACCATGTAACTTTGCATCCGACTCTCTTTTAGACTTATCTCCAATCAAGCCACAATTTGCTAAAGGTACTGACTTCGTTGTTGTCAGAGAATTAGTGGGAGGTATTTACTTTGGTAAGAGAAAGGAAGACGATGGTGATGGTGTCGCTTGGGATAGTGAACAATACACCGTTCCAGAAGTGCAAAGAATCACAAGAATGGCCGCTTTCATGGCCCTACAACATGAGCCACCATTGCCTATTTGGTCCTTGGATAAAGCTAATGTTTTGGCCTCTTCAAGATTATGGAGAAAAACTGTGGAGGAAACCATCAAGAACGAATTCCCTACATTGAAGGTTCAACATCAATTGATTGATTCTGCCGCCATGATCCTAGTTAAGAACCCAACCCACCTAAATGGTATTATAATCACCAGCAACATGTTTGGTGATATCATCTCCGATGAAGCCTCCGTTATCCCAGGTTCCTTGGGTTTGTTGCCATCTGCGTCCTTGGCCTCTTTGCCAGACAAGAACACCGCATTTGGTTTGTACGAACCATGCCACGGTTCTGCTCCAGATTTGCCAAAGAATAAGGTCAACCCTATCGCCACTATCTTGTCTGCTGCAATGATGTTGAAATTGTCATTGAACTTGCCTGAAGAAGGTAAGGCCATTGAAGATGCAGTTAAAAAGGTTTTGGATGCAGGTATCAGAACTGGTGATTTAGGTGGTTCCAACAGTACCACCGAAGTCGGTGATGCTGTCGCCGAAGAAGTTAAGAAAATCCTTGCTTAAATTTAACTCCTTAAGTTACTTTAATGATTTAGTTTTTATTATTAATAATTCATGCTCATGACATCTCATATACACGTTTATAAAACTTAAATAGATTGAAAATGTATTAAAGATTCCTCAGGGATTCGATTTTTTTGGAAGTTTTTGTTTTTTTTTCCTTGAGATGCTGTAGTATTTGGGAACAATTATACAATCGAAAGATATATGCTTACATTCGACCGTTTTAGCCGTGATCATTATCCTATAGTAACATAACCTGAAGCATAACTGACACTACTATCATCAATACTTGTCACATGAGAACTCTGTGAATAATTAGGCCACTGAAATTTGATGCCTGAAGGACCGGCATCACGGATTTTCGATAAAGCACTTAGTATCACACTAATTGGCTTTTCGCGCAAATTAAAGCCTTCGAGCGTCCCAAAACCTTCTCAAGCAAGGTTTTCAGTATAATGTTACATGCGTACACGCGTTTGTACAGAAAAAAAAGAAAAATTTGAAATATAAATAACGTTCTTAATACTAACATAACTATAAAAAAATAAATAGGGACCTAGACTTCAGGTTGTCTAACTCCTTCCTTTTCGGTTAGAGCGGATGTGGGGGGAGGGCGTGAATGTAAGCGTGACATAACTAATTACATGATATCGACAAAGGAAAAGGGGCCTGTTTATATTGAATTTTCAAAAATTCTTACTTTTTTTTTGGATGGACGCAAAGAAGTTTAATAATCATATTACATGGCAATACCACCATATACATATCCATATCTAATCTTACTTATATGTTGTGGAAATGTAAAGAGCCCCATTATCTTAGCCTAAAAAAACCTTCTCTTTGGAACTTTCAGTAATACGCTTAACTGCTCATTGCTATATTGAAGTACGGATTAGAAGCCGCCGAGCGGGCGACAGCCCTCCGACGGAAGACTCTCCTCCGTGCGTCCTGGTCTTCACCGGTCGCGTTCCTGAAACGCAGATGTGCCTCGCGCCGCACTGCTCCGAACAATAAAGATTCTACAATACTAGCTTTTATGGTTATGAAGAGGAAAAATTGGCAGTAACCTGGCCCCACAAACCTTCAAATCAACGAATCAAATTAACAACCATAGGATAATAATGCGATTAGTTTTTTAGCCTTATTTCTGGGGTAATTAATCAGCGAAGCGATGATTTTTGATCTATTAACAGATATATAAATGCAAAAGCTGCATAACCACTTTAACTAATACTTTCAACATTTTCGGTTTGTATTACTTCTTATTCAAATGTCATAAAAGTATCAACAAAAAATTGTTAATATACCTCTATACTTTAACGTCAAGGAGAAAAAACTATAATGACTGTTTGTGCTAAGAAACATGTTCATTTGACAAGAGATGCTGCAGAACAATTGTTGGCAGATATCGATAGAAGATTGGATCAATTGTTACCAGTTGAAGGTGAAAGAGATGTTGTTGGTGCTGCAATGAGAGAAGGTGCTTTGGCACCAGGTAAAAGAATCAGACCAATGTTGTTGTTGTTGACTGCTAGAGATTTGGGTTGTGCAGTTTCTCATGATGGTTTGTTAGATTTGGCTTGTGCAGTTGAAATGGTTCATGCTGCATCATTGATCTTGGATGATATGCCATGTATGGATGATGCTAAATTGAGAAGAGGTAGACCAACTATTCATTCTCATTATGGTGAACATGTTGCTATTTTAGCTGCAGTTGCTTTGTTATCAAAAGCATTTGGTGTTATTGCTGATGCAGATGGTTTGACACCATTGGCTAAAAATAGAGCAGTTTCTGAATTGTCAAACGCTATCGGTATGCAAGGTTTGGTTCAAGGTCAATTCAAAGATTTGTCTGAAGGTGACAAACCAAGATCAGCTGAAGCAATTTTGATGACTAACCATTTCAAGACTTCTACATTATTTTGTGCTTCTATGCAAATGGCATCAATTGTTGCTAATGCATCTTCAGAAGCTAGAGATTGTTTGCATAGATTTTCATTGGATTTGGGTCAAGCATTTCAATTGTTAGATGATTTGACTGATGGTATGACTGATACTGGTAAAGATTCTAATCAAGATGCTGGTAAATCAACATTGGTTAATTTGTTAGGTCCAAGAGCTGTTGAAGAAAGATTGAGACAACATTTGCAATTAGCATCTGAACATTTGTCAGCTGCATGTCAACATGGTCATGCTACACAACATTTCATCCAAGCATGGTTCGATAAGAAATTAGCTGCAGTTTCTTAAGTCTGAAGAATGAATGATTTGATGATTTCTTTTTCCCTCCATTTTTCTTACTGAATATATCAATGATATAGACTTGTATAGTTTATTATTTCAAATTAAGTAGCTATATATAGTCAAGATAACGTTTGTTTGACACGATTACATTATTCGTCGACATCTTTTTTCAGCCTGTCGTGGTAGCAATTTGAGGAGTATTATTAATTGAATAGGTTCATTTTGCGCTCGCATAAACAGTTTTCGTCAGGGACAGTATGTTGGAATGAGTGGTAATTAATGGTGACATGACATGTTATAGCAATAACCTTGATGTTTACATCGTAGTTTAATGTACACCCCGCGAATTCGTTCAAGTAGGAGTGCACCAATTGCAAAGGGAAAAGCTGAATGGGCAGTTCGAATAAAAGATTCTCTTTTTTTATGATATTTGTACATAAACTTTATAAATGAAATTCATAATAGAAACGACACGAAATTACAAAATGGAATATGTTCATAGGGTAGACGAAACTATATACGCAATCTACATACATTTATCAAGAAGGAGAAAAAGGAGGATGTAAAGGAATACAGGTAAGCAAATTGATACTAATGGCTCAACGTGATAAGGAAAAAGAATTGCACTTTAACATTAATATTGACAAGGAGGAGGGCACCACACAAAAAGTTAGGTGTAACAGAAAATCATGAAACTATGATTCCTAATTTATATATTGGAGGATTTTCTCTAAAAAAAAAAAAATACAACAAATAAAAAACACTCAATGACCTGACCATTTGATGGAGTTTAAGTCAATACCTTCTTGAACCATTTCCCATAATGGTGAAAGTTCCCTCAAGAATTTTACTCTGTCAGAAACGGCCTTAACGACGTAGTCGACCTCCTCTTCAGTACTAAATCTACCAATACCAAATCTGATGGAAGAATGGGCTAATGCATCATCCTTACCCAGCGCATGTAAAACATAAGAAGGTTCTAGGGAAGCAGATGTACAGGCTGAACCCGAGGATAATGCGATATCCCTTAGTGCCATCAATAAAGATTCTCCTTCCACGTAGGCGAAAGAAACGTTAACACACCCTGGATAACGATGATCTGGAGATCCGTTCAACGTGGTATGTTCAGCGGATAATAGACCTTTGACTAATTTATCGGATAGTCTTTTGATGTGAGCTTGGTCGTTGTC(SEQ ID NO：1)。
Reconstruction of Δ URA 3: pGAL1-PagCrtB (Pantoea agglomerans); pGAL10-btcrti (blakeslea trispora) knockout cassette, knockout cassette fragment 2 having the sequence set forth in SEQ ID NO: 2. Designing upstream and downstream primers to amplify each fragment by PCR, enabling the fragments to have overlapping fragments of 60-80bp, recombining all the fragments together in a homologous recombination mode, and obtaining delta URA3 through enzyme digestion linearization: : pGAL1-PagCrtB (Pantoea agglomerans); pGAL10-BtCrtI (Blakeslea trispora) knockout cassette, fragment 2 of which is shown in FIG. 2. The fragments are respectively integrated on a J1011-C-1 genome of the yeast by utilizing a homologous recombination mechanism of the yeast through a lithium acetate method yeast transformation, the integration site is URA3, an SD-HIS solid plate (a synthetic yeast nitrogen source YNB6.7g/L, glucose 20g/L, mixed amino acid powder without histidine 1.3g/L and 2% agar powder) is adopted for screening after the transformation, the obtained transformant is subjected to pure culture, the yeast genome is extracted for PCR verification, and a successfully verified strain is named as J1011-C-2.
ACGCAGATAATTCCAGGTATTTTGAAGCAGAACTTTTATTTATCATCGAATTGACTATTGCATTATTTCTATTTTGCAAAGAGGAGAAAGAATTAGGAAAGTTCATACTTCAAAAAGTTTTCCAACTTTCTCACACGAAAGGCCTCACGAAAAGGACTGTTCGTAGAATGCTAACATACAAAATTTTGTTAATTTCGTTATGTGCGGATCAGACGGAGTACTTGTCCAAATTAATAAACGATGAGCTGTTAAAAAAGGGGGATATTTTTACCCAAAAATTTTTTGCAACTAATCAAGGTAAGGAATTTTTGAAGAGACTATTTTCATTGACCGAATCAGAGTTTTATAGAGGATTTTTACTAGGAAATGAGAATTTTTGGAAATTTTTAAGAAAAGTTACAGCAATGAAAGAGCAGAGCGAGAGCATTTTTGAATATTTAAATGAATCGATCAAGACAGACAGCAATATTTTGACAAATGAGAACTTCATGTGGGTCCTAGGACTATTAGATGAAATTTCATCAATGGGTGCCGTTGGAAATCACTGGGAAATAGAATACAAGAAATTGACAGAAAGTGGTCATAAAATTGATAAGGAGAATCCATACAAGAAATCGATCGAATTATCATTGAAATCCATTCAACTAACATCACACTTGCTGGAAGATAATAACGATCTGCGTAAAAACGAGATATTCGCTATTATTCAAGCTTTGGCACATCAATGCATCAATCCGTGTAAGCAGATAAGTGAATTTGCAGTGGTAACGCTAGAGCAGACGCTCATCAATAAAATCGAAATTCCAACTAATGAGATGGAATCGGTAGAAGAATTAATTGAGGGCGGATTACTACCGTTGCTAAATTCGAGTGAAACACAGGAAGACCAGAAAATCCTCATTTCATCCATATTAACAATAATTTCAAATGTTTATTTGCATTATTTGAAACTAGGGAAGACAAGCAACGAAACGTTTTTGAAAATTTTGAGTATTTTCAATAAATTTGTAGAGGACTCAGATATTGAAAAAAAGCTACAGCAATTAATACTTGATAAGAAGAGTATTGAGAAGGGCAACGGTTCATCATCTCATGGATCTGCACATGAACAAACACCAGAGTCAAACGACGTTGAAATTGAGGCTACTGCGCCAATTGATGACAATACAGACGATGATAACAAACCGAAGTTATCTGATGTAGAAAAGGATTAAAGATGCTAAGAGATAGTGATGATATTTCATAAATAATGTAATTCTATATATGTTAATTACCTTTTTTGCGAGGCATATTTATGGTGAAGGATAAGTTTTGACCATCAAAGAAGGTTAATGTGGCTGTGGTTTCAGGGTCCATAAAGCTTTTCAATTCATCATTTTTTTTTTATTCTTTTTTTTGATTTCGGTTTCCTTGAAATTTTTTTGATTCGGTAATCTCCGAACAGAAGGAAGAACGAAGGAAGGAGCACAGACTTAGATTGGTATATATACGCATATGTAGTGTTGAAGAAACATGAAATTGCCCAGTATTCTTAACCCAACTGCACAGAACAAAAACCTGCAGGAAACGAAGGTACCCAATTCGCCCTATAGTGAGTCGTATTACGCGCGCTCACTGGCCGTCGTTTTACAACGTCGTGACTGGGAAAACCCTGGCGTTACCCAACTTAATCGCCTTGCAGCACATCCCCCTTTCGCCAGCTGGCGTAATAGCGAAGAGGCCCGCACCGATCGCCCTTCCCAACAGTTGCGCAGCCTGAATGGCGAATGGCGCGACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACCTCGACCCCAAAAAACTTGATTAGGGTGATGGTTCACGTAGTGGGCCATCGCCCTGATAGACGGTTTTTCGCCCTTTGACGTTGGAGTCCACGTTCTTTAATAGTGGACTCTTGTTCCAAACTGGAACAACACTCAACCCTATCTCGGTCTATTCTTTTGATTTATAAGGGATTTTGCCGATTTCGGCCTATTGGTTAAAAAATGAGCTGATTTAACAAAAATTTAACGCGAATTTTAACAAAATATTAACGTTTACAATTTCCTGATGCGGTATTTTCTCCTTACGCATCTGTGCGGTATTTCACACCGCATAGATCCGTCGAGTTCAAGAGAAAAAAAAAGAAAAAGCAAAAAGAAAAAAGGAAAGCGCGCCTCGTTCAGAATGACACGTATAGAATGATGCATTACCTTGTCATCTTCAGTATCATACTGTTCGTATACATACTTACTGACATTCATAGGTATACATATATACACATGTATATATATCGTATGCTGCAGCTTTAAATAATCGGTGTCACTACATAAGAACACCTTTGGTGGAGGGAACATCGTTGGTACCATTGGGCGAGGTGGCTTCTCTTATGGCAACCGCAAGAGCCTTGAACGCACTCTCACTACGGTGATGATCATTCTTGCCTCGCAGACAATCAACGTGGAGGGTAATTCTGCTAGCCTCTGCAAAGCTTTCAAGAAAATGCGGGATCATCTCGCAAGAGAGATCTCCTACTTTCTCCCTTTGCAAACCAAGTTCGACAACTGCGTACGGCCTGTTCGAAAGATCTACCACCGCTCTGGAAAGTGCCTCATCCAAAGGCGCAAATCCTGATCCAAACCTTTTTACTCCACGCGCCAGTAGGGCCTCTTTAAAAGCTTGACCGAGAGCAATCCCGCAGTCTTCAGTGGTGTGATGGTCGTCTATGTGTAAGTCACCAATGCACTCAACGATTAGCGACCAGCCGGAATGCTTGGCCAGAGCATGTATCATATGGTCCAGAAACCCTATACCTGTGTGGACGTTAATCACTTGCGATTGTGTGGCCTGTTCTGCTACTGCTTCTGCCTCTTTTTCTGGGAAGATCGAGTGCTCTATCGCTAGGGGACCACCCTTTAAAGAGATCGCAATCTGAATCTTGGTTTCATTTGTAATACGCTTTACTAGGGCTTTCTGCTCTGTCATCTTTGCCTTCGTTTATCTTGCCTGCTCATTTTTTAGTATATTCTTCGAAGAAATCACATTACTTTATATAATGTATAATTCATTATGTGATAATGCCAATCGCTAAGAAAAAAAAAGAGTCATCCGCTAGGGGAAAAAAAAAAATGAAAATCATTACCGAGGCATAAAAAAATATAGAGTGTACTAGAGGAGGCCAAGAGTAATAGAAAAAGAAAATTGCGGGAAAGGACTGTGTTATGACTTCCCTGACTAATGCCGTGTTCAAACGATACCTGGCAGTGACTCCTAGCGCTCACCAAGCTCTTAAAACGGGAATTTATGGTGCACTCTCAGTACAATCTGCTCTGATGCCGCATAGTTAAGCCAGCCCCGACACCCGCCAACACGCGCTGACGCGCCCTGACGGGCTTGTCTGCTCCCGGCATCCGCTTACAGACAAGCTGTGAGCAAATTAAAGCCTTCGAGCGTCCCAAAACCTTCTCAAGCAAGGTTTTCAGTATAATGTTACATGCGTACACGCGTTTGTACAGAAAAAAAAGAAAAATTTGAAATATAAATAACGTTCTTAATACTAACATAACTATAAAAAAATAAATAGGGACCTAGACTTCAGGTTGTCTAACTCCTTCCTTTTCGGTTAGAGCGGATGTGGGGGGAGGGCGTGAATGTAAGCGTGACATAACTAATTACATGATATCGACAAAGGAAAAGGGGCCTGTTTATATCCTAATATCGTTAGAGTTCTGTCCTTGGAAGACGTTTGGCAACAACTGATTAATAAAAGATGCTGGAGTAGTAGTGTCGTCCCTAGGAAAGAAATAGAAGAACAAGAAAGTGACAAAGTAGCATGCTAAGCAGTAATAAATCCAGTGTGACTCAGTCTTCCTTGTTTGCTCAGGTGCATATTTCTTCTGAGAGTCCTGTAACTTCCTAGGCAAAGGGTTTTGTCCGAATGACTTACAGACCTGGTCAGAAGTTAATTTAGATCCTGCCAAGACTATTGGGACTCCTGTACCAGGGTGAGTTGATGCACCGACGAAAAATAAATTGTCATATCTGTTAGTAGAGTCCTTTGTTGAAGGTCTGAACCACAAAACCTGAAAGACATCATGTGATAAACCTAATATTGATCCCCTCCACAAGTTGAACTTAGATTGCCAGACTGATGGGTCGTTGACCTCCTCGTGTTCTATCAAGTTAGCGAAGTTGTTGACTCCCAACCTCCTCTCTATGACCTCCAAGACCATCTTTCTAGCCCTGTTGACCAACTCTGGGTAGTTCTCCTCTGCTGAGTTACCTGTCTTAGACTTCATGTGACCAATTGGAACTAAAACTATAATTGAGTCCTTATTTGGAGGTGCTGCAGACTCGTCTATCCTTGAAGGAACGTTGACGTAGAAAGATGCTTCAGAAGGCAAACCGAAATCGTTAAAAATCTCGTCGAATGACTCCTTGTAAGCCTCAGCCAAGAAAATGTTGTGGACGTCCAATTGAGGGACCTTAGTAGACATTGACCAGTAAAAAGAAATTGATGAAGATGTTAATTTCTTAGATGCCAAAGTCTTCTTTGTCCAGTTGCAAGGTGGCAACAAGTGGTGGTAAGCGTAGACCAAGTCAGCGTTGCAGACGACAGCGTCTGCCTCAATGACTTCTCCAGACTCCAAAGTGACACCAGTGACCCTCTTGTCTTTATCGACAGTGTTAATCTTAGCGACAGGAGATTGGTACCTGAACTCAGCTCCGTACTTCTTAGAAGCTATAGACTCCAACTTTTGGACGACCATGTTGAAACCACCCCTTGGGTACCAAATTCCCTCTGCGAACTCTGTATATTGCAACAATGAGTAGACTGCAGGTGCGTCGTAAGGTGACATACCCATGTACATTGTTTGAAAAGTAAAAGCCATCCTCATCTTTTTTGTTTGGAAGTATTTTGATGCTCTGTCGTATATCTTACCGAATAAGTGCAACCTAAAAATCTCTGGGACGTACTGTAACCTTATTAAGTCCCATATAGTTTCGAAGTTTCTCTTTATAGCAATGAATGTACCCTGCTCGTAGTGGACGTGTGTCTCTTTCATGAAATCTAAGAACCTACCGAATCCTAAAGGTCCCTCAATCCTGTCCAACTCACCCTTCATCTTTGTTAAGTCTGATGACAATTGGACAGCGTCACCGTCGTCGAAATGGACTTTGTAATTGTTGTCACATCTTAATAAGTCCAAGTGGTCTCCTATCCTCTCGTCTAAGTCAGCGAAAGCGTCCTCAAACAACTTAGGCATCAAGTACAATGAAGGTCCCTGGTCGAACCTGTGACCGTCGTGGTGAATGAAAGAGCACCTTCCTCCAGAGAAGTCGTTCTTCTCGACGACAGTGACTCTGAAACCCTCCCTTGCTAACCTTGCTGCTGTTGCAGTACCTCCTATTCCAGCACCTATGACGACAATGTGCTTCTTCTGATCAGACATTTATATTGAATTTTCAAAAATTCTTACTTTTTTTTTGGATGGACGCAAAGAAGTTTAATAATCATATTACATGGCAATACCACCATATACATATCCATATCTAATCTTACTTATATGTTGTGGAAATGTAAAGAGCCCCATTATCTTAGCCTAAAAAAACCTTCTCTTTGGAACTTTCAGTAATACGCTTAACTGCTCATTGCTATATTGAAGTACGGATTAGAAGCCGCCGAGCGGGCGACAGCCCTCCGACGGAAGACTCTCCTCCGTGCGTCCTGGTCTTCACCGGTCGCGTTCCTGAAACGCAGATGTGCCTCGCGCCGCACTGCTCCGAACAATAAAGATTCTACAATACTAGCTTTTATGGTTATGAAGAGGAAAAATTGGCAGTAACCTGGCCCCACAAACCTTCAAATCAACGAATCAAATTAACAACCATAGGATAATAATGCGATTAGTTTTTTAGCCTTATTTCTGGGGTAATTAATCAGCGAAGCGATGATTTTTGATCTATTAACAGATATATAAATGCAAAAGCTGCATAACCACTTTAACTAATACTTTCAACATTTTCGGTTTGTATTACTTCTTATTCAAATGTCATAAAAGTATCAACAAAAAATTGTTAATATACCTCTATACTTTAACGTCAAGGAGAAAAAACTATAATGTCACAACCACCATTATTGGACCACGCTACACAAACTATGGCAAACGGTTCTAAATCTTTCGCTACTGCTGCTAAATTATTCGACCCAGCAACAAGAAGATCTGTATTGATGTTGTACACCTGGTGTAGACATTGCGATGACGTTATAGATGACCAAACTCACGGTTTTGCTTCAGAAGCTGCAGCCGAAGAAGAAGCTACACAAAGATTGGCAAGATTAAGAACTTTGACATTAGCTGCATTCGAAGGTGCCGAAATGCAAGATCCAGCTTTTGCCGCTTTCCAAGAAGTTGCATTAACCCATGGTATTACTCCTAGAATGGCTTTGGATCACTTAGACGGTTTTGCAATGGATGTCGCCCAAACAAGATACGTAACCTTCGAAGACACTTTAAGATATTGTTACCATGTCGCCGGTGTTGTCGGTTTGATGATGGCTAGAGTAATGGGTGTTAGAGATGAAAGAGTTTTAGATAGAGCATGTGACTTGGGTTTAGCCTTCCAATTGACAAACATAGCTAGAGATATAATAGATGACGCAGCCATAGACAGATGCTATTTGCCAGCTGAATGGTTACAAGATGCAGGTTTGACTCCTGAAAATTACGCTGCAAGAGAAAACAGAGCCGCTTTAGCCAGAGTTGCTGAAAGATTGATAGATGCAGCCGAACCATATTACATCTCTTCACAAGCTGGTTTGCATGATTTGCCACCTAGATGCGCATGGGCCATTGCTACCGCAAGATCTGTTTACAGAGAAATCGGTATTAAAGTCAAGGCTGCAGGTGGTTCCGCATGGGATAGAAGACAACACACTTCTAAAGGTGAAAAGATCGCTATGTTGATGGCCGCTCCTGGTCAAGTTATTAGAGCAAAGACCACCAGAGTCACCCCAAGACCAGCCGGTTTATGGCAAAGACCTGTTTAAATTGAATTGAATTGAAATCGATAGATCAATTTTTTTCTTTTCTCTTTCCCCATCCTTTACGCTAAAATAATAGTTTATTTTATTTTTTGAATATTTTTTATTTATATACGTATATATAGACTATTATTTATCTTTTAATGATTATTAAGATTTTTATTAAAAAAAATTACGCTCCTCTTTTAATGCCTTTATGCAGTTTTTTTTCCCATTCGATATTTCTATGTTCGGGTTCAGCGTATTTTAAGTTTAATAACTCGAAAATTCTGCGTTCGTTGTAAATGCATGTATACTAAACTCACAAATTAGAGCTTCAATTTAATTATATCAGTTATTACCCGGGAATCTCGGTCGTAATGATTTCTATAATGACGAAAAAAAAAAAATTGGAAAGAAAAAGCTTCATGGCCTTTATAAAAAGGAACTATCCAATACCTCGCCAGAACCAAGTAACAGTATTTTACGGGGCACAAATCAAGAACAATAAGACAGGACTGTAAAGATGGACGCATTGAACTCCAAAGAACAACAAGAGTTCCAAAAAGTAGTGGAACAAAAGCAAATGAAGGATTTCATGCGTTTGTACTCTAATCTGGTAGAAAGATGTTTCACAGACTGTGTCAATGACTTCACAACATCAAAGCTAACCAATAAGGAACAAACATGCATCATGAAGTGCTCAGAAAAGTTCTTGAAGCATAGCGAACGTGTAGGGCAGCGTTTCCAAGAACAAAACGCTGCCTTGGGACAAGGCTTGGGCCGATAAGGTGTACTGGCGTATATATATCTAATTATGTATCTCTGGTGTAGCCCATTTTTAGCATGTAAATATAAAGAGAAACCATATCTAATCTAACCAAATCCAAACAAAATTCAATAGTTACTATCGCTTTTTTCTTTCTGTATCGCAAATAAGTGAAAATTAAAAAAGAAAGATTAAATTGGAAGTTGGATATGGGCTGGAACAGCAGCAGTAATCGGTATCGGGTTCGCCACTAATGACGTCCTACGATTGCACTCAACAGACCTTGACGCTCACGCCGTAGCGGGCGACAAGTCAAACGGAACAACCGTTGCCGTTCCCATCGGAGTCCGACCTAGGCCGAACTCCGTGAATTTCTGATAACAACGGTCGGTAAAGACTGGTTCCCCAGTATATTTCTTCTCTCAGGAGCAGGGGCCAATGCCAAAAGCGACATTAACCCGGAGGACAAGGCTCCACTGTGTTCCACCGAATTTCCCACCTGATAATATCTGATAACCCGCCCATAGGTGGGGATCCTTCTGTAAACAGGTTTCTTAATCGTAGGAATTACCACTGTTCCACTGCCAATCGCAGCTCCCAGAGTTTCGTTCCCAGCCGCGAGCACCACAGCGTACCATGTGCGCCACGAGGCCTCAAACGTAAAACAATCGAAACGAAAAGAAACAGACTATAGGGGAGTATAGAGACAGCCGGCCAATAAGAAGAGGAAAAAGAAATACTAGCGTTTATCAATGTGGGTCGTTAACATATCCTGTTGACAATGATTACAGGTTAAAAGGTAGCGTAAGTGAATATTAACTATGGATATTCTTATACTTAGATAAGAGACATATAAAACACACGATGATTGATTGATTTTTATGACCAATATATGTAATTCGTAATTCAGATAGTTTTTATACTTTTAATGTGTGCCGAACCATATTACTAGTATATGTAACTACCATGTTATGTTCAATTGGCAGATCTTTAACTCGGCTTTAGTTATCCAAGTTACTTGCAATATTTCCTTCTGCGAGAGTACATTTGCCCTTAAACG(SEQ ID NO：2)。
And constructing a delta gal1, a delta gal7 and a delta gal10 pGAL10-tHMG1 knockout box, namely knockout box fragment 3, wherein the knockout box fragment 3 has the sequence shown in SEQ ID NO: 3. Designing upstream and downstream primers to amplify each fragment by PCR, enabling the fragments to have 60-80bp overlapped fragments, recombining all the fragments together in a homologous recombination mode, and carrying out enzyme digestion linearization to obtain delta gal1, delta gal7 and delta gal10, wherein pGAL10-tHMG1 knockout fragments and the structure of knockout box fragment 3 are shown in FIG. 3. The fragments are respectively integrated on a J1011-C-2 genome of the yeast by utilizing a homologous recombination mechanism of the yeast per se through the conversion of the yeast by a lithium acetate method, the integration sites are GAL1, GAL7 and GAL10, an SD-Trp solid plate (6.7 g/L of a synthetic yeast nitrogen source YNB, 20g/L of glucose, 1.3g/L of mixed amino acid powder lacking tryptophan and 2% of agar powder) is adopted for screening after the conversion, the obtained transformant is subjected to pure culture, the yeast genome is extracted for PCR verification, and a successfully verified strain is named as J1011-C-3.
TTCACCGATTCTGAGCGAATCACAGGTGAGAAATTTGGATTCGAAATAAACCTAAAAAAACTATCCAATAAGGCTTCCATAGGCTTCGTATTTCCCGACCATTCCAATTGGAAAAATTGAGCGCTGTCTTCCATGATCTTAGATAAAGCCTTAATACTTGGCTCATTTCCATTTGAGGTTAGCTCTAGCAACTGGTAAAGCATTTGAAGACCAGTTGGATCATCTCTATGCTGCCTATAGTAAGTGGAAAATAAAGTAGTAATAACGATACCTGTCACGTTCGAAACGAGATTATTGTTCGAATTATAAAGGCCATGTATAATGTTTGATTTGACGTATCCTAAGTCGTGGCTATTGCTCTTAATTAAATTGTTTCCCCCTAGCATTGAATTTTTCAACAGCATACCGGCGGTAGCTCTATTGTTCTGAAGATCCTGTAGGGAGTAGTGTTGCTTCAATACATCATCAGATTCGCCTTCAATTAAAATATAACACAAATAATTGAGAAATTCAGGTTGCAGCTGGAAGTTCTCCATAGCTTCCATTGCGTTATTACGAATCTCTGGATTTGGTGACATACAGTTCTGTAAAAGAGTTGCTAGTTGCAACACATAGTCTTCGGCGGGCTTCCATGTCGATGCCATCTTTATTCACTTAACTACTGCTAACAATTCTGGAAACCAAAGACTGCGGAATATTCTGATATGTATTACTACTATTCGCTGCTCTTCTGCATAATTAATACTGAAAAGTTTTTCATACTTTTAAACATAACCTTTTTTTAAGCAAAACTCTATGACCCGGATTAGAAAACTACGAAAAGAGGGTAATAACATAGGTGCAGGATTTCCATCGATAACGACGCCGACAATGAGCCTTGCTGCAACATCCAATTAGGACTAATAACTATCGTAGGAATTTCTACGTAATAAACTTCAACAGAGCCTAAAATTTGAAAATAAATAATCTAGAGGGGAAACTTAAAGAAATTCTATTCTTGTCAATAAAGTGGAAATCTGTCAGATGTCACAGTTTCTTTATTTGTGACACATATTTTCAACATAAATTCAGGCATTAGTGCTGTAAGCACAAAAAGTTGTGGCGATATGAATATTCCAGATTTTACTTACAAGCTGCATTGTAGTCTTACAATTCTTTTTTTCTTTTTTTTTTTTTATGGAAAGGACCACTCTTACATAACTAGAATAGCATTAAGAATCAGATTTACAGATAAAGATGACATTATTTTATATATATATTGTCACTCCGTTCAAGTCGACAACCAATAAAAAATTTAAAAAAAGCCAGGCAGTTAATAGAAAAAATATGATATGAATGAATATTCCACTTTCTTTTCACAGCTTGTCTGTAAGCGGATGCCGGGAGCAGACAAGCCCGTCAGGGCGCGTCAGCGGGTGTTGGCGGGTGTCGGGGCTGGCTTAACTATGCGGCATCAGAGCAGATTGTACTGAGAGTGCACCATAAACGACATTACTATATATATAATATAGGAAGCATTTAATAGACAGCATCGTAATATATGTGTACTTTGCAGTTATGACGCCAGATGGCAGTAGTGGAAGATATTCTTTATTGAAAAATAGCTTGTCACCTTACGTACAATCTTGATCCGGAGCTTTTCTTTTTTTGCCGATTAAGAATTAATTCGGTCGAAAAAAGAAAAGGAGAGGGCCAAGAGGGAGGGCATTGGTGACTATTGAGCACGTGAGTATACGTGATTAAGCACACAAAGGCAGCTTGGAGTATGTCTGTTATTAATTTCACAGGTAGTTCTGGTCCATTGGTGAAAGTTTGCGGCTTGCAGAGCACAGAGGCCGCAGAATGTGCTCTAGATTCCGATGCTGACTTGCTGGGTATTATATGTGTGCCCAATAGAAAGAGAACAATTGACCCGGTTATTGCAAGGAAAATTTCAAGTCTTGTAAAAGCATATAAAAATAGTTCAGGCACTCCGAAATACTTGGTTGGCGTGTTTCGTAATCAACCTAAGGAGGATGTTTTGGCTCTGGTCAATGATTACGGCATTGATATCGTCCAACTGCATGGAGATGAGTCGTGGCAAGAATACCAAGAGTTCCTCGGTTTGCCAGTTATTAAAAGACTCGTATTTCCAAAAGACTGCAACATACTACTCAGTGCAGCTTCACAGAAACCTCATTCGTTTATTCCCTTGTTTGATTCAGAAGCAGGTGGGACAGGTGAACTTTTGGATTGGAACTCGATTTCTGACTGGGTTGGAAGGCAAGAGAGCCCCGAAAGCTTACATTTTATGTTAGCTGGTGGACTGACGCCAGAAAATGTTGGTGATGCGCTTAGATTAAATGGCGTTATTGGTGTTGATGTAAGCGGAGGTGTGGAGACAAATGGTGTAAAAGACTCTAACAAAATAGCAAATTTCGTCAAAAATGCTAAGAAATAGGTTATTACTGAGTAGTATTTATTTAAGTATTGTTTGTGCACTTGCCTATGCGGTGTGAAATACCGCACAGATGCGTAAGGAGAAAATACCGCATCAGGAAATTGTAAACGTTAATATTTTGTTAAAATTCGCGTTAAATTTTTGTTAAATCAGCTCATTTTTTAACCAATAGGCCGAAATCGGCAAAATCCCTTATAAATCAAAAGAATAGACCGAGATAGGGTTGAGTGTTGTTCCAGTTTGGAACAAGAGTCCACTATTAAAGAACGTGGACTCCAACGTCAAAGGGCGAAAAACCGTCTATCAGGGCGATGGCCCACTACGTGAACCATCACCCTAATCAAGTTTTTTGGGGTCGAGGTGCCGTAAAGCACTAAATCGGAACCCTAAAGGGAGCCCCCGATTTAGAGCTTGACGGGGAAAGCCGGCGAACGTGGCGAGAAAGGAAGGGAAGAAAGCGAAAGGAGCGGGCGCTAGGGCGCTGGCAAGTGTAGCGGTCACGCTGCGCGTAACCACCACACCCGCCGCGCTTAATGCGCCGCTACAGGGCGCGTCGCGCCATTCGCCATTCAGGCTGCGCAACTGTTGGGAAGGGCGATCGGTGCGGGCCTCTTCGCTATTACGCCAGCTGGCGAAAGGGGGATGTGCTGCAAGGCGATTAAGTTGGGTAACGCCAGGGTTTTCCCAGTCACGACGTTGTAAAACGACGGCCAGTGAGCGCGCGTAATACGACTCACTATAGGGCGAATTGGGTACTACACGGTCCAATGGATAAACATTTTTTATCAACACTATGATATATAAATATAATAGTTTTTCGTATATATATTCCTTTTTTTGGTCAATTTTTGAAATTTTCGTAGAAAAGGGAGAGACAAAACACATTATATCAATGAAAACGTACAAAAAGTAGATAAAGTCAGTGCTTAAACACGTCTTTTCCTTAAAAATACTTTATTATTTTTATTTTATTGAGAGGGTGGTTTAAAAATAGAAATAGAGAGAGAGGTACATACATAAACATACGCGCACAAAAGCAGAGATTAGGATTTAATGCAGGTGACGGACCCATCTTTCAAACGATTTATATCAGTGGCGTCCAAATTGTTAGGTTTTGTTGGTTCAGCAGGTTTCCTGTTGTGGGTCATATGACTTTGAACCAAATGGCCGGCTGCTAGGGCAGCACATAAGGATAATTCACCTGCCAAGACGGCACAGGCAACTATTCTTGCTAATTGACGTGCGTTGGTACCAGGAGCGGTAGCATGCGGGCCTCTTACACCTAATAAGTCCAACATGGCACCTTGTGGTTCTAGAACAGTACCACCACCGATGGTACCTACTTCGATGGATGGCATGGATACGGAAATTCTCAAATCACCGTCCACTTCTTTCATCAATGTTATACAGTTGGAACTTTCAACATTTTGTGCAGGATCTTGTCCTAATGCCAAGAAAACAGCTGTCACTAAATTAGCTGCATGTGCGTTAAATCCACCAACAGACCCAGCCATTGCAGATCCAACCAAATTCTTAGCAATGTTCAACTCAACCAATGCGGAAACATCACTTTTTAACACTTTTCTGACAACATCACCAGGAATAGTAGCTTCTGCGACGACACTCTTACCACGACCTTCGATCCAGTTGATGGCAGCTGGTTTTTTGTCGGTACAGTAGTTACCAGAAACGGAGACAACCTCCATATCTTCCCAGCCATACTCTTCTACCATTTGCTTTAATGAGTATTCGACACCTTTAGAAATCATATTCATACCCATTGCGTCACCAGTAGTTGTTCTAAATCTCATGAAGAGTAAATCTCCTGCTAGACAAGTTTGAATATGTTGCAGACGTGCAAATCTTGATGTAGAGTTAAAAGCTTTTTTAATTGCGTTTTGTCCCTCTTCTGAGTCTAACCATATCTTACAGGCACCAGATCTTTTCAAAGTTGGGAAACGGACTACTGGGCCTCTTGTCATACCATCCTTAGTTAAAACAGTTGTTGCACCACCGCCAGCATTGATTGCCTTACAGCCACGCATGGCAGAAGCTACCAAACAACCCTCTGTAGTTGCCATTGGTATATGATAAGATGTACCATCGATAACCAAGGGGCCTATAACACCAACGGGCAAAGGCATGTAACCTATAACATTTTCACAACAAGCGCCAAATACGCGGTCGTAGTCATAATTTTTATATGGTAAACGATCAGATGCTAATACAGGAGCTTCTGCCAAAATTGAAAGAGCCTTCCTACGTACCGCAACCGCTCTCGTAGTATCACCTAATTTTTTCTCCAAAGCGTACAAAGGTAACTTACCGTGAATAACCAAGGCAGCGACCTCTTTGTTCTTCAATTGTTTTGTATTTCCACTACTTAATAATGCTTCTAATTCTTCTAAAGGACGTATTTTCTTATCCAAGCTTTCAATATCGCGGGAATCATCTTCCTCACTAGATGATGAAGGTCCTGATGAGCTCGATTGCGCAGATGATAAACTTTTGACTTTCGATCCAGAAATGACTGTTTTATTGGTTAAAACCATTTATATTGAATTTTCAAAAATTCTTACTTTTTTTTTGGATGGACGCAAAGAAGTTTAATAATCATATTACATGGCAATACCACCATATACATATCCATATCTAATCTTACTTATATGTTGTGGAAATGTAAAGAGCCCCATTATCTTAGCCTAAAAAAACCTTCTCTTTGGAACTTTCAGTAATACGCTTAACTGCTCATTGCTATATTGAAGTACGGATTAGAAGCCGCCGAGCGGGCGACAGCCCTCCGACGGAAGACTCTCCTCCGTGCGTCCTGGTCTTCACCGGTCGCGTTCCTGAAACGCAGATGTGCCTCGCGCCGCACTGCTCCGAACAATAAAGATTCTACAATACTAGCTTTTATGGTTATGAAGAGGAAAAATTGGCAGTAACCTGGCCCCACAAACCTTCAAATCAACGAATCAAATTAACAACCATAGGATAATAATGCGATTAGTTTTTTAGCCTTATTTCTGGGGTAATTAATCAGCGAAGCGATGATTTTTGATCTATTAACAGATATATAAATGCAAAAGCTGCATAACCACTTTAACTAATACTTTCAACATTTTCGGTTTGTATTACTTCTTATTCAAATGTCATAAAAGTATCAACAAAAAATTGTTAATATACCTCTATACTTTAACGTCAAGGAGAAAAAACTATAGCGCAATGATTGAATAGTCAAAGATTTTTTTTTTTTAATTTTTTTTTTTTCATAGAACTTTTTATTTAAATAAATCACGTCTATATATGTATCAGTATAACGTAAAAAAAAAAACACCGTCAGTTAAACAAAACATAAATAAAAAAAAAAAGAAGTGTCAAATCAAGTGTCAAATGTATACTTCTTTTTTTTACTTTGTTCAGAACAACTTCTCATTTTTTTCTACTCATAACTTTAGCATCACAAAGTACACAATAATAACGAGTAGTAACACTTTTATAGTTCATACATGCTTCAACTACTTAATAAATGATTGTATGATAATGTTTTCAATGTAAGAGATTTCGATTATCCACAAACTTTGAAACACAGGGACACAATTCTTGATATGCTTTCAACCGCTGCGTTTTGGATACCTATTCTTGACATAATATGACTACCATTTTGTTATTGTACGTGGGGCAGTTGACGTCTTATCATATGTCAAAGTCATTTGCGAAGTTCTTGGCAAGTTGCCAACTGACGAGATGCAGTAAAAAGAGATTGCCGTCTTGAAACTTTTTGTCCTTTTTTTTTTCCGGGGACTCTACGAGAACCCTTTGTCCTACTGATTAATTTTGTACTGAATTTGGACAATTCAGATTTTAGTAGACAAGCGCGAGGAGGAAAAGAAATGACAGAAAAATTCCGATGGACAAGAAGATAGGAAAAAAAAAAAGCTTTCACCGATTTCCTAGACCGGAAAAAAGTCGTATGACATCAGAATGAAAAATTTTCAAGTTAGACAAGGACAAAATCAGGACAAATTGTAAAGATATGATAAATCATTTGATTCAGCGCCAATTTGCCCTTTTCCATTTTCCATTAAATCTCTGTTCTCTCTTACTTATATGATGATTAGGTATCATCTGTATAAAACTCCTTTCTTAATTTCACTCTAAAGCATACCCCATAGAGAAGATCTTTCGGTTCGAAGACATTCCTACGCATAATAAGAATAGGAGGGAATAATGCCAGACAATCTATCATTACATTTAAGCGGCTCTTCAAAAAGATTGAACTCTCGCCAACTTATGGAATCTTCCAATGAGACCTTTGCGCCAAATAATGTGGATTTGGAAAAAGAGTATAAGTCATCTCAGAGTAATATAACTACCGAAGTTTATGAGGCATCGAGCTTTGAAGAAAAAGTAAGCTCAGAAAAACCTCAATACAGCTCATTCTGGAAGAAAATCTATTATGAATATGTGGTCGTTGACAAATCAATCTTGGGTGTTTCTATTCTGGATTCATTTATGTACAACCAGGACTTGAAGCCCGTCGAAAAAGAAAGGCGGGTTTGGTCCTGGTACAATTATTGTTACTTCTGGCTTGCTGAATGTTTCAATATCAACACTTGGCAAATTGCAGCTACAGGTCTACAACTGGGTCTAAATTGGTGGCAGTGTTGGATAACAATTTGGATTGGGTACGGTTTCGTTGGTGCTTTTGTTGTTTTGGCCTCTAGAGTTGGATCTGCTTATCATTTGTCATTCCCTATATCATCTAGAGCATCATTCGGTATTTTCTTCTCTTTATGGCCTGTTATTAATAGAGTCGTCATGGCCATCGTTTGGTATAGTGTCCAAGCTTATATTGCGGCAACTCCCGTATCATTAA(SEQ ID NO：3)。
In the following experiments, the inventors further engineered the J1011-C-3 strain as follows:
example 2 selection of promoters and terminators
In order to be able to induce galactose, it is necessary to knock out the galactose-metabolizing genes GAL1, GAL7, GAL 10. The background strain for the characteristic promoter is thus the knockout gene GAL1,7, 10. Galactose is high in price and is not suitable for industrial production, so that GAL80 is knocked out on the basis of knocking out genes GAL1,7 and 10, and regulation of gene expression is expected to be achieved through control over glucose amount. The obtained promoter information is used for constructing and controlling the expression of genes related to the synthesis of the carotenoid.
The results show that the strengths of both inducible and constitutive promoters were substantially the same in both the Δ GAL1/7/10/80 and Δ GAL1/7/10 strains during stationary phase. It can be seen that the knockout of GAL80 can induce gene expression without addition of galactose, and the induction intensity is comparable to that of galactose.
Yeast belongs to eukaryote, the proper promoter and terminator need to be selected when genes are over-expressed in the yeast, and through repeated experiments, the inventor selects P GAL1 ，P GAL7 ，P GAL10 ，P GAL1-10 ，P GAL10-1 And P HXT1 As a promoter for expressing a gene. Wherein, P GAL1 Has the sequence shown in SEQ ID NO: 4, P GAL7 Has the sequence shown in SEQ ID NO: 5, P GAL10 Has the sequence shown in SEQ ID NO: 6, P GAL1-10 Has the sequence shown in SEQ ID NO: 7, P GAL10-1 Has the sequence shown in SEQ ID NO: 8, P HXT1 Has the sequence shown in SEQ ID NO: 9, or a nucleotide sequence shown in the specification.
AGTACGGATTAGAAGCCGCCGAGCGGGCGACAGCCCTCCGACGGAAGACTCTCCTCCGTGCGTCCTGGTCTTCACCGGTCGCGTTCCTGAAACGCAGATGTGCCTCGCGCCGCACTGCTCCGAACAATAAAGATTCTACAATACTAGCTTTTATGGTTATGAAGAGGAAAAATTGGCAGTAACCTGGCCCCACAAACCTTCAAATCAACGAATCAAATTAACAACCATAGGATAATAATGCGATTAGTTTTTTAGCCTTATTTCTGGGGTAATTAATCAGCGAAGCGATGATTTTTGATCTATTAACAGATATATAAATGCAAAAGCTGCATAACCACTTTAACTAATACTTTCAACATTTTCGGTTTGTATTACTTCTTATTCAAATGTCATAAAAGTATCAACAAAAAATTGTTAATATACCTCTATACTTTAACGTCAAGGAGAAAAAACTATA(SEQ ID NO：4)。
GACGGTAGCAACAAGAATATAGCACGAGCCGCGAAGTTCATTTCGTTACTTTTGATATCGCTCACAACTATTGCGAAGCGCTTCAGTGAAAAAATCATAAGGAAAAGTTGTAAATATTATTGGTAGTATTCGTTTGGTAAAGTAGAGGGGGTAATTTTTCCCCTTTATTTTGTTCATACATTCTTAAATTGCTTTGCCTCTCCTTTTGGAAAGCTATACTTCGGAGCACTGTTGAGCGAAGGCTCATTAGATATATTTTCTGTCATTTTCCTTAACCCAAAAATAAGGGAAAGGGTCCAAAAAGCGCTCGGACAACTGTTGACCGTGATCCGAAGGACTGGCTATACAGTGTTCACAAAATAGCCAAGCTGAAAATAATGTGTAGCTATGTTCAGTTAGTTTGGCTAGCAAAGATATAAAAGCAGGTCGGAAATATTTATGGGCATTATTATGCAGAGCATCAACATGATAAAAAAAAACAGTTGAATATTCCCTCAAAA(SEQ ID NO：5)。
TACTTCAATATAGCAATGAGCAGTTAAGCGTATTACTGAAAGTTCCAAAGAGAAGGTTTTTTTAGGCTAAGATAATGGGGCTCTTTACATTTCCACAACATATAAGTAAGATTAGATATGGATATGTATATGGTGGTATTGCCATGTAATATGATTATTAAACTTCTTTGCGTCCATCCAAAAAAAAAGTAAGAATTTTTGAAAATTCAATATAA(SEQ ID NO：6)。
TATAGTTTTTTCTCCTTGACGTTAAAGTATAGAGGTATATTAACAATTTTTTGTTGATACTTTTATGACATTTGAATAAGAAGTAATACAAACCGAAAATGTTGAAAGTATTAGTTAAAGTGGTTATGCAGCTTTTGCATTTATATATCTGTTAATAGATCAAAAATCATCGCTTCGCTGATTAATTACCCCAGAAATAAGGCTAAAAAACTAATCGCATTATTATCCTATGGTTGTTAATTTGATTCGTTGATTTGAAGGTTTGTGGGGCCAGGTTACTGCCAATTTTTCCTCTTCATAACCATAAAAGCTAGTATTGTAGAATCTTTATTGTTCGGAGCAGTGCGGCGCGAGGCACATCTGCGTTTCAGGAACGCGACCGGTGAAGACCAGGACGCACGGAGGAGAGTCTTCCGTCGGAGGGCTGTCGCCCGCTCGGCGGCTTCTAATCCGTACTTCAATATAGCAATGAGCAGTTAAGCGTATTACTGAAAGTTCCAAAGAGAAGGTTTTTTTAGGCTAAGATAATGGGGCTCTTTACATTTCCACAACATATAAGTAAGATTAGATATGGATATGTATATGGTGGTATTGCCATGTAATATGATTATTAAACTTCTTTGCGTCCATCCAAAAAAAAAGTAAGAATTTTTGAAAATTCAATATAA(SEQ ID NO：7)。
TTATATTGAATTTTCAAAAATTCTTACTTTTTTTTTGGATGGACGCAAAGAAGTTTAATAATCATATTACATGGCAATACCACCATATACATATCCATATCTAATCTTACTTATATGTTGTGGAAATGTAAAGAGCCCCATTATCTTAGCCTAAAAAAACCTTCTCTTTGGAACTTTCAGTAATACGCTTAACTGCTCATTGCTATATTGAAGTACGGATTAGAAGCCGCCGAGCGGGCGACAGCCCTCCGACGGAAGACTCTCCTCCGTGCGTCCTGGTCTTCACCGGTCGCGTTCCTGAAACGCAGATGTGCCTCGCGCCGCACTGCTCCGAACAATAAAGATTCTACAATACTAGCTTTTATGGTTATGAAGAGGAAAAATTGGCAGTAACCTGGCCCCACAAACCTTCAAATCAACGAATCAAATTAACAACCATAGGATAATAATGCGATTAGTTTTTTAGCCTTATTTCTGGGGTAATTAATCAGCGAAGCGATGATTTTTGATCTATTAACAGATATATAAATGCAAAAGCTGCATAACCACTTTAACTAATACTTTCAACATTTTCGGTTTGTATTACTTCTTATTCAAATGTCATAAAAGTATCAACAAAAAATTGTTAATATACCTCTATACTTTAACGTCAAGGAGAAAAAACTATA(SEQ ID NO：8)。
GATTTTACGTATATCAACTAGTTGACGATTATGATATCTTTATAGATTTTAAGGTGGGGAAAGAACATGAGACCCCAGATGGAATTGATTATGGGGACATTGTTGCCTTTATATATAATTTCAATATACTAATTCAAATGATTAAAAACGTGAGGGGGACACGCAACTTCGGGTGTTAAGAAATATTTTGCTACATTAGATAATGGTGGAGTTTCCTGGCTTGTCGGATAAAAGCCATCAAATGTCGCAGCAGCTCATGTTTACGTTTGCTGTCTTCTGCCCACGTCATATGAGTGGTATTCTTCTATCAGCACTTGATGAATATTCTTTTTCTCATATATCTGAAAGACAAAAGATCGGCACGGCAATGCCCTGCAGCATTTCTTCCTAGTTTTTCCGAATTTCCATTACGTATTGGATCTTGTGCGCATATTTGTCAGTCCTTCACGGAAAAAAAAAAAGAGCACTGGGTCACTTCGGAAAAACTTTTGACTCAATGCAACAGTGTCATAATCCTTTGCGCTGTCTCTTTGAAGAAAAATCAGGAGTGCAAGATATCGATTAATTCCTTGGAAGTTATGATGGTTAGTCTTAGTTTAACTCTCTTGAAGAAGGGTTTTTTCAGTTGGTCAACACTCTTTAGAGGTAAAAAAAAAAAAAAAAAAAAAAAAAAGAGAATTCTTCATGTAATTTACCATGATTCTACGTTTTTGCAAGCAAAAATGAAGATAATCCGAGCGCATGCGCAAGTAGTCCCTGCCATGCCGCTTCGGAAAAACTTTCGAAACCAATACTCCTTACATCCGCCTTGAATTTAGGACCTACAATTGTTGTCTCTTCTGCTTGAACCAATAAGCCCTAGAAACCTTACACCCTAATTTGCACAAGAAAACTACGAAAAAATTTTTTCCTCTTTTTTCCGCAGCTCCAATCTCGCCCTGCTATTTTTTTGGTTCGGAGAAAAATAGTCGGTCGATATGAATTGAAGTTTCATTGTGGCCACTCGTTTAGGAATGGAATAACCTCCGCGAATGGAAAAAAAGAACTCGGAATATCAAAAATTCCGGCTCAAAGGAAAAATTTGCTTCAGGAGGGGGGAATTATATTCCAGATGAGACCTGCA(SEQ ID NO：9)。
Selecting an arbitrary terminator (e.g. T) ADH2 ，T GAL10 ，T CYC1 ，T GPM1 And T PGK1 Etc.) as a terminator for expressing a gene, the combination and arrangement of these elements enables overexpression of a plurality of genes.
EXAMPLE 3 construction of second Generation engineered strains
In this example, the inventors adjusted the copy numbers of three genes PaCrtE (Pantoea ananatis), PagCrtB (Pantoea agglomerans) and BtCrtI (Blakeslea trispora) on the J1011-C3 strain based on the J1011-C3 strain to obtain a second generation strain.
Wherein the specific characteristics of the second generation engineering strain are shown in Table 1. Selecting proper promoters and terminators according to design, amplifying each fragment by using upstream and downstream primers through PCR, enabling the fragments to have 60-80bp overlapped fragments, recombining all the fragments together in a homologous recombination mode, obtaining each corresponding fragment through enzyme digestion linearization, converting the fragments by using a yeast homologous recombination mechanism of a yeast through a lithium acetate method, respectively integrating the fragments on a yeast J1011-C-3 genome, screening by using a screening resistant solid plate after conversion, extracting a yeast genome for PCR verification after the obtained transformant is subjected to pure culture, and obtaining successfully verified strains shown in Table 1.
Table 1: second generation engineering strain
Bacterial strains Property and knock-out box
J1011-C-5 A J1011-C-3 derived strain; over-expressing BtCrtI on the original basis.
J1011-C-6 A J1011-C-3 derived strain; overexpression of BtCrtI and PagCrtB on an as-received basis
J1011-C-7 A J1011-C-3 derived strain; overexpression of BtCrtI and PagCrtB and PaCrtE on an as-received basis
J1011-C-33 A J1011-C-3 derived strain; overexpression of PagCrtB and PaCrtE on an original basis
J1011-C-34 A J1011-C-3 derived strain; overexpression of PaCrtE on an as-received basis
J1011-C-35 A J1011-C-3 derived strain; overexpression of BtCrtI and PaCrtE on an as-received basis
J1011-C-36 A J1011-C-3 derived strain; the PagCrtB was overexpressed on an as-received basis.
Wherein BtCrtI has the sequence shown in SEQ ID NO: 10, PagCrtB has the nucleotide sequence shown in SEQ ID NO: 11, and PaCrtE has the nucleotide sequence shown in SEQ ID NO: 12.
TTATATCCTAATATCGTTAGAGTTCTGTCCTTGGAAGACGTTTGGCAACAACTGATTAATAAAAGATGCTGGAGTAGTAGTGTCGTCCCTAGGAAAGAAATAGAAGAACAAGAAAGTGACAAAGTAGCATGCTAAGCAGTAATAAATCCAGTGTGACTCAGTCTTCCTTGTTTGCTCAGGTGCATATTTCTTCTGAGAGTCCTGTAACTTCCTAGGCAAAGGGTTTTGTCCGAATGACTTACAGACCTGGTCAGAAGTTAATTTAGATCCTGCCAAGACTATTGGGACTCCTGTACCAGGGTGAGTTGATGCACCGACGAAAAATAAATTGTCATATCTGTTAGTAGAGTCCTTTGTTGAAGGTCTGAACCACAAAACCTGAAAGACATCATGTGATAAACCTAATATTGATCCCCTCCACAAGTTGAACTTAGATTGCCAGACTGATGGGTCGTTGACCTCCTCGTGTTCTATCAAGTTAGCGAAGTTGTTGACTCCCAACCTCCTCTCTATGACCTCCAAGACCATCTTTCTAGCCCTGTTGACCAACTCTGGGTAGTTCTCCTCTGCTGAGTTACCTGTCTTAGACTTCATGTGACCAATTGGAACTAAAACTATAATTGAGTCCTTATTTGGAGGTGCTGCAGACTCGTCTATCCTTGAAGGAACGTTGACGTAGAAAGATGCTTCAGAAGGCAAACCGAAATCGTTAAAAATCTCGTCGAATGACTCCTTGTAAGCCTCAGCCAAGAAAATGTTGTGGACGTCCAATTGAGGGACCTTAGTAGACATTGACCAGTAAAAAGAAATTGATGAAGATGTTAATTTCTTAGATGCCAAAGTCTTCTTTGTCCAGTTGCAAGGTGGCAACAAGTGGTGGTAAGCGTAGACCAAGTCAGCGTTGCAGACGACAGCGTCTGCCTCAATGACTTCTCCAGACTCCAAAGTGACACCAGTGACCCTCTTGTCTTTATCGACAGTGTTAATCTTAGCGACAGGAGATTGGTACCTGAACTCAGCTCCGTACTTCTTAGAAGCTATAGACTCCAACTTTTGGACGACCATGTTGAAACCACCCCTTGGGTACCAAATTCCCTCTGCGAACTCTGTATATTGCAACAATGAGTAGACTGCAGGTGCGTCGTAAGGTGACATACCCATGTACATTGTTTGAAAAGTAAAAGCCATCCTCATCTTTTTTGTTTGGAAGTATTTTGATGCTCTGTCGTATATCTTACCGAATAAGTGCAACCTAAAAATCTCTGGGACGTACTGTAACCTTATTAAGTCCCATATAGTTTCGAAGTTTCTCTTTATAGCAATGAATGTACCCTGCTCGTAGTGGACGTGTGTCTCTTTCATGAAATCTAAGAACCTACCGAATCCTAAAGGTCCCTCAATCCTGTCCAACTCACCCTTCATCTTTGTTAAGTCTGATGACAATTGGACAGCGTCACCGTCGTCGAAATGGACTTTGTAATTGTTGTCACATCTTAATAAGTCCAAGTGGTCTCCTATCCTCTCGTCTAAGTCAGCGAAAGCGTCCTCAAACAACTTAGGCATCAAGTACAATGAAGGTCCCTGGTCGAACCTGTGACCGTCGTGGTGAATGAAAGAGCACCTTCCTCCAGAGAAGTCGTTCTTCTCGACGACAGTGACTCTGAAACCCTCCCTTGCTAACCTTGCTGCTGTTGCAGTACCTCCTATTCCAGCACCTATGACGACAATGTGCTTCTTCTGATCAGACAT(SEQ ID NO：10)。
ATGTCACAACCACCATTATTGGACCACGCTACACAAACTATGGCAAACGGTTCTAAATCTTTCGCTACTGCTGCTAAATTATTCGACCCAGCAACAAGAAGATCTGTATTGATGTTGTACACCTGGTGTAGACATTGCGATGACGTTATAGATGACCAAACTCACGGTTTTGCTTCAGAAGCTGCAGCCGAAGAAGAAGCTACACAAAGATTGGCAAGATTAAGAACTTTGACATTAGCTGCATTCGAAGGTGCCGAAATGCAAGATCCAGCTTTTGCCGCTTTCCAAGAAGTTGCATTAACCCATGGTATTACTCCTAGAATGGCTTTGGATCACTTAGACGGTTTTGCAATGGATGTCGCCCAAACAAGATACGTAACCTTCGAAGACACTTTAAGATATTGTTACCATGTCGCCGGTGTTGTCGGTTTGATGATGGCTAGAGTAATGGGTGTTAGAGATGAAAGAGTTTTAGATAGAGCATGTGACTTGGGTTTAGCCTTCCAATTGACAAACATAGCTAGAGATATAATAGATGACGCAGCCATAGACAGATGCTATTTGCCAGCTGAATGGTTACAAGATGCAGGTTTGACTCCTGAAAATTACGCTGCAAGAGAAAACAGAGCCGCTTTAGCCAGAGTTGCTGAAAGATTGATAGATGCAGCCGAACCATATTACATCTCTTCACAAGCTGGTTTGCATGATTTGCCACCTAGATGCGCATGGGCCATTGCTACCGCAAGATCTGTTTACAGAGAAATCGGTATTAAAGTCAAGGCTGCAGGTGGTTCCGCATGGGATAGAAGACAACACACTTCTAAAGGTGAAAAGATCGCTATGTTGATGGCCGCTCCTGGTCAAGTTATTAGAGCAAAGACCACCAGAGTCACCCCAAGACCAGCCGGTTTATGGCAAAGACCTGTTTAA(SEQ ID NO：11)。
ATGACTGTTTGTGCTAAGAAACATGTTCATTTGACAAGAGATGCTGCAGAACAATTGTTGGCAGATATCGATAGAAGATTGGATCAATTGTTACCAGTTGAAGGTGAAAGAGATGTTGTTGGTGCTGCAATGAGAGAAGGTGCTTTGGCACCAGGTAAAAGAATCAGACCAATGTTGTTGTTGTTGACTGCTAGAGATTTGGGTTGTGCAGTTTCTCATGATGGTTTGTTAGATTTGGCTTGTGCAGTTGAAATGGTTCATGCTGCATCATTGATCTTGGATGATATGCCATGTATGGATGATGCTAAATTGAGAAGAGGTAGACCAACTATTCATTCTCATTATGGTGAACATGTTGCTATTTTAGCTGCAGTTGCTTTGTTATCAAAAGCATTTGGTGTTATTGCTGATGCAGATGGTTTGACACCATTGGCTAAAAATAGAGCAGTTTCTGAATTGTCAAACGCTATCGGTATGCAAGGTTTGGTTCAAGGTCAATTCAAAGATTTGTCTGAAGGTGACAAACCAAGATCAGCTGAAGCAATTTTGATGACTAACCATTTCAAGACTTCTACATTATTTTGTGCTTCTATGCAAATGGCATCAATTGTTGCTAATGCATCTTCAGAAGCTAGAGATTGTTTGCATAGATTTTCATTGGATTTGGGTCAAGCATTTCAATTGTTAGATGATTTGACTGATGGTATGACTGATACTGGTAAAGATTCTAATCAAGATGCTGGTAAATCAACATTGGTTAATTTGTTAGGTCCAAGAGCTGTTGAAGAAAGATTGAGACAACATTTGCAATTAGCATCTGAACATTTGTCAGCTGCATGTCAACATGGTCATGCTACACAACATTTCATCCAAGCATGGTTCGATAAGAAATTAGCTGCAGTTTCTTAA(SEQ ID NO：12)。
Furthermore, the inventor verifies through experiments that the results are shown in FIG. 5, and J1011-C-5, 6, 7 and J1011-C-33-36 have higher lycopene production performance than J1011-C-3 under the condition of shaking the bottle.
EXAMPLE 4 construction of Gene silencing engineered strains
In this example, the inventors have described in detail the experimental process of knocking out some other genes to obtain silent genetically engineered strains based on the above engineered bacteria.
In Saccharomyces cerevisiae, there are many potential genes that have a significant effect on cell accumulation of lycopene compounds after knock-out, and therefore a series of background strains were constructed for increasing lycopene production. The construction method is that hygromycin resistance gene is used as a marker to construct corresponding knockout box fragment in the gene to be inactivated, and the knockout box fragment is shown in figure 4. The fragments are respectively integrated into the engineering bacteria through the transformation of a lithium acetate yeast, and finally, a plate containing hygromycin resistance is used for screening to obtain suspected strains with resistance, and PCR verification is carried out to obtain fourth generation engineering strains shown in a table 2. Where the inactivated gene sequence in the knockout cassette fragment is downloadable from NCBI, the knockout box of FIG. 6 can be designed.
Table 2: silencing engineered Strain Properties
Figure BDA0001325723840000191
Figure BDA0001325723840000201
Furthermore, the inventor verifies through experiments that J1011-C-9,10,11,13,15,19 all have higher lycopene production performance than J1011-C-3.
EXAMPLE 5 construction of engineered strains overexpressing genes
In this example, the inventors have detailed experimental procedures related to overexpression on the above engineered strains.
Selecting proper promoters and terminators to construct expression frames of related genes in the table 3, selecting proper genes as insertion sites on the basis of the embodiment 4, designing upstream and downstream primers to amplify each segment by PCR, so that 60-80bp overlapped segments exist among the segments, recombining all the segments together in a homologous recombination mode, and obtaining gene segments capable of realizing silent genes and over-expressed genes by enzyme digestion linearization. The fragments are respectively integrated on the genome of the engineering strain by utilizing the homologous recombination mechanism of yeast per se through the transformation of the lithium acetate yeast, a screening plate is adopted for screening after the transformation, the obtained transformant is subjected to pure culture and then the yeast genome is extracted for PCR verification, and the successfully verified strains are detailed in a table 3.
Furthermore, the inventor verifies through experiments that J1011-C-4 and J1011-C-20-32 both have the performance of high-yield lycopene.
Table 3: engineered strains overexpressing genes
Figure BDA0001325723840000202
Figure BDA0001325723840000211
Wherein, the INO2 gene has the sequence shown in SEQ ID NO: 13, the gapN gene has the nucleotide sequence shown as SEQ ID NO: 14, the PYC2 gene has the nucleotide sequence shown in SEQ ID NO: 15, the SMAE1 gene has a nucleotide sequence shown in SEQ ID NO: 16, and the MDH2 gene has a nucleotide sequence shown as SEQ ID NO: 17, and the POS5 gene has a nucleotide sequence shown as SEQ ID NO: 18, the pntA gene has the nucleotide sequence shown as SEQ ID NO: 19, the pntB gene has the nucleotide sequence shown as SEQ ID NO: 20, and the ADH2 gene has a nucleotide sequence shown as SEQ ID NO: 21, and the ACS6 gene has a nucleotide sequence shown as SEQ ID NO: 22, and the ALD6 gene has a nucleotide sequence shown as SEQ ID NO: 23, the EUTE gene has a nucleotide sequence shown as SEQ ID NO: 24, the ERG12 gene has the nucleotide sequence shown in SEQ ID NO: 25, and the IDI1 gene has the nucleotide sequence shown in SEQ ID NO: 26, ERG10 gene has the nucleotide sequence shown in SEQ ID NO: 27, and the MVD1 gene has a nucleotide sequence shown as SEQ ID NO: 28, ERG13 gene has the nucleotide sequence shown in SEQ ID NO: 29, and the gene tHMG1 has the nucleotide sequence shown in SEQ ID NO: 30, ERG8 gene has the nucleotide sequence shown in SEQ ID NO: 31, yap1 gene has the nucleotide sequence shown in SEQ ID NO: 32. The spt15-5 gene has the sequence shown in SEQ ID NO: 51. taf25-3 gene has the sequence shown in SEQ ID NO: 52.
ATGTCCCAAGCAACTGGGAACGAATTACTGGGTATCCTAGATCTGGATAACGATATAGACTTTGAAACTGCTTACCAAATGCTCAGCAGTAACTTCGACGACCAAATGTCTGCGCACATACATGAAAACACGTTTAGTGCAACTTCCCCTCCTCTGTTAACACACGAGCTCGGCATAATTCCTAACGTGGCAACCGTGCAACCCTCTCACGTAGAAACTATACCTGCCGATAACCAAACTCATCATGCTCCTTTGCATACTCATGCTCACTATCTAAATCACAACCCTCATCAACCAAGCATGGGTTTTGATCAAACGCTTGGTCTCAAGTTGTCTCCTTCCAGTTCGGGGTTGTTGAGCACGAATGAATCGAATGCCATTGAACAGTTTTTAGACAATCTAATATCACAGGATATGATGTCTTCCAACGCTTCCATGAACTCCGATTCACATCTACATATAAGATCACCAAAAAAGCAGCATAGGTATACCGAATTAAATCAAAGATATCCTGAAACACATCCACACAGTAACACAGGGGAGTTACCCACAAACACAGCAGATGTGCCAACTGAGTTCACCACGAGGGAAGGACCTCATCAGCCTATCGGCAATGACCACTACAACCCGCCACCGTTTTCAGTACCTGAGATACGAATCCCAGACTCTGATATTCCAGCCAATATCGAGGACGACCCTGTGAAGGTACGGAAATGGAAACACGTTCAAATGGAGAAGATACGAAGAATAAACACCAAAGAAGCCTTTGAAAGGCTCATTAAATCAGTAAGGACCCCACCAAAGGAAAACGGGAAAAGAATTCCCAAGCATATTCTTTTAACTTGTGTAATGAACGATATCAAGTCCATTAGAAGCGCAAATGAAGCACTACAGCACATACTGGATGATTCCTGA(SEQ ID NO：13)。
ATGACTAAGCAATACAAGAACTACGTTAACGGTGAATGGAAGTTGTCTGAAAACGAAATTAAAATCTATGAACCTGCTTCAGGTGCAGAATTAGGTTCCGTTCCAGCTATGAGTACAGAAGAAGTAGATTACGTTTACGCTTCTGCTAAGAAAGCCCAACCTGCTTGGAGAGCATTGTCATATATCGAAAGAGCTGCATACTTACATAAGGTTGCTGATATATTGATGAGAGACAAGGAAAAGATTGGTGCAATCTTATCTAAAGAAGTAGCCAAGGGTTATAAATCCGCTGTTAGTGAAGTTGTCAGAACCGCTGAAATTATAAACTACGCCGCTGAAGAAGGTTTAAGAATGGAAGGTGAAGTTTTGGAAGGTGGTTCTTTCGAAGCAGCCTCTAAAAAGAAAATTGCAGTAGTTAGAAGAGAACCTGTCGGTTTAGTATTGGCCATTTCCCCTTTCAATTATCCAGTAAACTTAGCTGGTAGTAAGATCGCCCCAGCTTTGATTGCTGGTAATGTTATAGCCTTTAAACCACCTACACAAGGTTCTATTTCAGGTTTGTTATTGGCAGAAGCCTTCGCTGAAGCAGGTTTGCCTGCTGGTGTTTTTAACACAATTACCGGTAGAGGTTCCGAAATCGGTGACTACATTGTCGAACACCAAGCTGTAAACTTCATCAACTTCACTGGTTCTACAGGTATAGGTGAAAGAATCGGTAAAATGGCTGGTATGAGACCAATTATGTTAGAATTGGGTGGTAAAGATTCTGCAATAGTTTTAGAAGATGCCGACTTAGAATTGACCGCTAAGAATATCATTGCCGGTGCTTTTGGTTATTCCGGTCAAAGATGTACTGCAGTCAAAAGAGTTTTAGTCATGGAAAGTGTTGCCGATGAATTGGTCGAAAAGATAAGAGAAAAGGTTTTGGCATTGACTATCGGTAATCCTGAAGATGACGCCGACATCACTCCATTGATCGATACAAAGTCAGCTGACTACGTTGAAGGTTTGATTAATGATGCAAACGACAAGGGTGCAACCGCCTTGACTGAAATTAAAAGAGAGGGTAACTTAATCTGCCCAATCTTGTTCGATAAGGTTACTACAGACATGAGATTAGCTTGGGAAGAACCATTTGGTCCTGTCTTGCCAATAATCAGAGTTACATCTGTCGAAGAAGCTATCGAAATATCTAATAAGTCAGAATATGGTTTGCAAGCATCAATCTTTACAAACGATTTCCCTAGAGCCTTTGGTATTGCTGAACAATTGGAAGTAGGTACCGTTCATATCAACAACAAGACACAAAGAGGTACCGATAATTTTCCATTCTTGGGTGCTAAGAAATCTGGTGCAGGTATCCAAGGTGTCAAATACTCCATTGAAGCTATGACCACTGTAAAGAGTGTCGTATTTGATATTAAATAA(SEQ ID NO：14)。
ATGTATCCAAGGGTCTATGAAGATTTCCAAAAGATCAGAGAAACATACGGTGATTTATCAGTTCTACCAACCAAAAATTTCCTAGCACCAGCAGAACCTGATGAAGAAATCGAAGTCACCATCGAACAAGGTAAGACTTTGATTATCAAATTGCAAGCTGTTGGTGACTTAAATAAGAAAACTGGGCAAAGAGAAGTGTATTTTGAATTGAACGGTGAATTAAGAAAGATCAGAGTTGCAGACAAGTCACAAAACATACAATCTGTTGCTAAACCAAAGGCTGATGTCCACGATACTCACCAAATCGGTGCACCAATGGCTGGTGTTATCATAGAAGTTAAAGTACATAAAGGGTCTTTGGTGAAAAAGGGCGAATCGATTGCTGTTTTGAGTGCCATGAAAATGGAAATGGTTGTCTCTTCACCAGCAGATGGTCAAGTTAAAGATGTTTTCATTAGGGATGGTGAAAGTGTTGACGCATCAGATTTGTTGGTTGTCCTAGAAGAAGAAACCCTACCCCCATCCCAAAAAAAGTAA(SEQ ID NO：15)。
ATGTGGCCTATTCAGCAATCGCGTTTATATTCTTCTAACACTAGATCGCATAAAGCTACCACAACAAGAGAAAATACTTTCCAAAAGCCATACAGCGACGAGGAGGTCACTAAAACACCCGTCGGTTCTCGCGCCAGAAAGATCTTCGAAGCTCCTCACCCACATGCCACTCGTTTGACTGTAGAAGGTGCCATAGAATGTCCCTTGGAGAGCTTTCAACTTTTAAACTCTCCTTTATTTAACAAGGGTTCTGCATTTACACAAGAAGAAAGGGAAGCGTTTAATTTAGAAGCATTGCTACCACCACAAGTGAACACTTTGGACGAACAACTGGAAAGAAGCTACAAGCAGTTATGCTATTTGAAGACGCCCTTGGCCAAAAACGACTTCATGACGTCTTTGAGAGTACAGAACAAAGTCCTATATTTTGCATTAATAAGGAAACATATCAAGGAATTAGTTCCTATCATTTACACCCCAACCGAAGGTGATGCTATTGCTGCCTATTCCCACAGGTTCAGAAAGCCAGAAGGTGTGTTTTTAGACATTACCGAACCTGATTCCATCGAACGTAGATTGGCTACATACGGTGGAGACAAAGATGTAGACTACATCGTTGTGTCGGATTCGGAAGGTATTCTGGGAATTGGTGACCAAGGTATCGGTGGTGTACGTATTGCTATCTCCAAATTGGCATTGATGACGCTGTGCGGTGGTATTCATCCCGGCCGTGTGCTACCTGTGTGTTTGGACGTCGGTACTAACAACAAGAAACTAGCCCGTGACGAATTGTACATGGGTAACAAGTTCTCCAGAATCAGGGGTAAGCAATATGACGACTTCTTGGAAAAATTCATCAAGGCCGTTAAGAAAGTGTATCCAAGCGCCGTTCTGCATTTCGAAGATTTCGGTGTTAAGAACGCTAGAAGATTGCTAGAAAAGTACAGGTACGAATTGCCATCATTCAACGATGACATTCAGGGCACCGGTGCCGTCGTGATGGCCTCGTTGATTGCTGCTTTGAAACATACCAACAGAGACTTGAAAGACACCAGAGTGCTTATTTACGGTGCCGGGTCTGCGGGCCTCGGTATCGCAGACCAAATTGTGAATCATATGGTCACGCACGGCGTTGACAAGGAAGAAGCGCGCAAGAAAATCTTCTTGATGGACAGACGTGGGTTAATTCTACAATCTTACGAGGCTAACTCCACTCCCGCCCAACACGTATACGCTAAGAGTGATGCGGAATGGGCTGGTATCAACACCCGCTCTTTACATGATGTGGTGGAGAACGTCAAACCAACGTGTTTGGTTGGCTGCTCCACACAAGCAGGCGCATTCACTCAAGATGTCGTAGAAGAAATGCACAAGCACAATCCTAGACCGATCATTTTCCCATTATCCAACCCTACTAGACTACACGAAGCCGTTCCTGCCGATTTAATGAAGTGGACCAACAACAACGCTCTTGTAGCTACCGGATCTCCTTTCCCACCTGTTGACGGTTACCGTATCTCGGAGAACAACAATTGTTACTCTTTCCCAGGTATCGGTTTAGGTGCCGTACTATCGCGTGCCACCACTATCACAGACAAGATGATCTCCGCTGCAGTGGACCAACTAGCCGAATTGTCGCCACTAAGAGAGGGCGACTCGAGACCTGGGTTGCTACCCGGCCTGGACACCATCACCAACACCTCTGCGCGTCTAGCTACCGCTGTGATCTTGCAAGCACTCGAGGAGGGAACCGCCCGTATCGAGCAAGAACAAGTACCGGGAGGAGCTCCCGGCGAAACTGTCAAGGTTCCTCGTGACTTTGACGAATGTTTACAGTGGGTCAAAGCCCAAATGTGGGAGCCTGTGTACAGACCTATGATCAAGGTCCAACATGACCCATCGGTGCACACCAACCAATTGTAG(SEQ ID NO：16)。
ATGCCTCACTCAGTTACACCATCCATAGAACAAGATTCGTTAAAAATTGCCATTTTAGGTGCTGCCGGTGGTATCGGGCAGTCGTTATCGCTGCTTTTGAAAGCTCAGTTGCAATACCAGTTAAAGGAGAGCAACCGGAGCGTTACCCACATTCATCTGGCTCTTTACGATGTCAACCAAGAAGCCATCAACGGTGTTACCGCCGACTTGTCTCATATAGACACCCCCATTTCCGTGTCGAGCCACTCTCCTGCAGGTGGCATTGAGAACTGTTTGCATAACGCTTCTATTGTTGTCATTCCTGCAGGTGTTCCAAGAAAACCTGGCATGACTCGTGATGACTTATTTAACGTGAATGCTGGTATCATTAGCCAGCTCGGTGATTCTATTGCAGAATGTTGTGATCTTTCCAAGGTCTTCGTTCTTGTCATTTCCAACCCTGTTAATTCTTTAGTCCCAGTGATGGTTTCTAACATTCTTAAGAACCATCCTCAGTCTAGAAATTCCGGCATTGAAAGAAGGATCATGGGTGTCACCAAGCTCGACATTGTCAGAGCGTCCACTTTTCTACGTGAGATAAACATTGAGTCAGGGCTAACTCCTCGTGTTAACTCCATGCCTGACGTCCCTGTAATTGGCGGGCATTCTGGCGAGACTATTATTCCGTTGTTTTCACAGTCAAACTTCCTATCGAGATTAAATGAGGATCAATTGAAATATTTAATACATAGAGTCCAATACGGTGGTGATGAAGTGGTCAAGGCCAAGAACGGTAAAGGTAGTGCTACCTTATCGATGGCCCATGCCGGTTATAAGTGTGTTGTCCAATTTGTTTCTTTGTTATTGGGTAACATTGAGCAGATCCATGGAACCTACTATGTGCCATTAAAAGATGCGAACAACTTCCCCATTGCTCCTGGGGCAGATCAATTATTGCCTCTGGTGGACGGTGCAGACTACTTTGCCATACCATTAACTATTACTACAAAGGGTGTTTCCTATGTGGATTATGACATCGTTAATAGGATGAACGACATGGAACGCAACCAAATGTTGCCAATTTGCGTCTCCCAGTTAAAGAAAAATATCGATAAGGGCTTGGAATTCGTTGCATCGAGATCTGCATCATCTTAA(SEQ ID NO：17)。
ATGTTTGTCAGGGTTAAATTGAATAAACCAGTAAAATGGTATAGGTTCTATAGTACGTTGGATTCACATTCCCTAAAGTTACAGAGCGGCTCGAAGTTTGTAAAAATAAAGCCAGTAAATAACTTGAGGAGTAGTTCATCAGCAGATTTCGTGTCCCCACCAAATTCCAAATTACAATCTTTAATCTGGCAGAACCCTTTACAAAATGTTTATATAACTAAAAAACCATGGACTCCATCCACAAGAGAAGCGATGGTTGAATTCATAACTCATTTACATGAGTCATACCCCGAGGTGAACGTCATTGTTCAACCCGATGTGGCAGAAGAAATTTCCCAGGATTTCAAATCTCCTTTGGAGAATGATCCCAACCGACCTCATATACTTTATACTGGTCCTGAACAAGATATCGTAAACAGAACAGACTTATTGGTGACATTGGGAGGTGATGGGACTATTTTACACGGCGTATCAATGTTCGGAAATACGCAAGTTCCTCCGGTTTTAGCATTTGCTCTGGGCACTCTGGGCTTTCTATTACCGTTTGATTTTAAGGAGCATAAAAAGGTCTTTCAGGAAGTAATCAGCTCTAGAGCCAAATGTTTGCATAGAACACGGCTAGAATGTCATTTGAAAAAAAAGGATAGCAACTCATCTATTGTGACCCATGCTATGAATGACATATTCTTACATAGGGGTAATTCCCCTCATCTCACTAACCTGGACATTTTCATTGATGGGGAATTTTTGACAAGAACGACAGCAGATGGTGTTGCATTGGCCACTCCAACGGGTTCCACAGCATATTCATTATCAGCAGGTGGATCTATTGTTTCCCCATTAGTCCCTGCTATTTTAATGACACCAATTTGTCCTCGCTCTTTGTCATTCCGACCACTGATTTTGCCTCATTCATCCCACATTAGGATAAAGATAGGTTCCAAATTGAACCAAAAACCAGTCAACAGTGTGGTAAAACTTTCTGTTGATGGTATTCCTCAACAGGATTTAGATGTTGGTGATGAAATTTATGTTATAAATGAGGTCGGCACTATATACATAGATGGTACTCAGCTTCCGACGACAAGAAAAACTGAAAATGACTTTAATAATTCAAAAAAGCCTAAAAGGTCAGGGATTTATTGTGTCGCCAAGACCGAGAATGACTGGATTAGAGGAATCAATGAACTTTTAGGATTCAATTCTAGCTTTAGGCTGACCAAGAGACAGACTGATAATGATTAA(SEQ ID NO：18)。
ATGCGAATTGGCATACCAAGAGAACGGTTAACCAATGAAACCCGTGTTGCAGCAACGCCAAAAACAGTGGAACAGCTGCTGAAACTGGGTTTTACCGTCGCGGTAGAGAGCGGCGCGGGTCAACTGGCAAGTTTTGACGATAAAGCGTTTGTGCAAGCGGGCGCTGAAATTGTAGAAGGGAATAGCGTCTGGCAGTCAGAGATCATTCTGAAGGTCAATGCGCCGTTAGATGATGAAATTGCGTTACTGAATCCTGGGACAACGCTGGTGAGTTTTATCTGGCCTGCGCAGAATCCGGAATTAATGCAAAAACTTGCGGAACGTAACGTGACCGTGATGGCGATGGACTCTGTGCCGCGTATCTCACGCGCACAATCGCTGGACGCACTAAGCTCGATGGCGAACATCGCCGGTTATCGCGCCATTGTTGAAGCGGCACATGAATTTGGGCGCTTCTTTACCGGGCAAATTACTGCGGCCGGGAAAGTGCCACCGGCAAAAGTGATGGTGATTGGTGCGGGTGTTGCAGGTCTGGCCGCCATTGGCGCAGCAAACAGTCTCGGCGCGATTGTGCGTGCATTCGACACCCGCCCGGAAGTGAAAGAACAAGTTCAAAGTATGGGCGCGGAATTCCTCGAGCTGGATTTTAAAGAGGAAGCTGGCAGCGGCGATGGCTATGCCAAAGTGATGTCGGACGCGTTCATCAAAGCGGAAATGGAACTCTTTGCCGCCCAGGCAAAAGAGGTCGATATCATTGTCACCACCGCGCTTATTCCAGGCAAACCAGCGCCGAAGCTAATTACCCGTGAAATGGTTGACTCCATGAAGGCGGGCAGTGTGATTGTCGACCTGGCAGCCCAAAACGGCGGCAACTGTGAATACACCGTGCCGGGTGAAATCTTCACTACGGAAAATGGTGTCAAAGTGATTGGTTATACCGATCTTCCGGGCCGTCTGCCGACGCAATCCTCACAGCTTTACGGCACAAACCTCGTTAATCTGCTGAAACTGTTGTGCAAAGAGAAAGACGGCAATATCACTGTTGATTTTGATGATGTGGTGATTCGCGGCGTGACCGTGATCCGTGCGGGCGAAATTACCTGGCCGGCACCGCCGATTCAGGTATCAGCTCAGCCGCAGGCGGCACAAAAAGCGGCACCGGAAGTGAAAACTGAGGAAAAATGTACCTGCTCACCGTGGCGTAAATACGCGTTGATGGCGCTGGCAATCATTCTTTTTGGCTGGATGGCAAGCGTTGCGCCGAAAGAATTCCTTGGGCACTTCACCGTTTTCGCGCTGGCCTGCGTTGTCGGTTATTACGTGGTGTGGAATGTATCGCACGCGCTGCATACACCGTTGATGTCGGTCACCAACGCGATTTCAGGGATTATTGTTGTCGGAGCACTGTTGCAGATTGGCCAGGGCGGCTGGGTTAGCTTCCTTAGTTTTATCGCGGTGCTTATAGCCAGCATTAATATTTTCGGTGGCTTCACCGTGACTCAGCGCATGCTGAAAATGTTCCGCAAAAATTAA(SEQ ID NO：19)
ATGTCTGGAGGATTAGTTACAGCTGCATACATTGTTGCCGCGATCCTGTTTATCTTCAGTCTGGCCGGTCTTTCGAAACATGAAACGTCTCGCCAGGGTAACAACTTCGGTATCGCCGGGATGGCGATTGCGTTAATCGCAACCATTTTTGGACCGGATACGGGTAATGTTGGCTGGATCTTGCTGGCGATGGTCATTGGTGGGGCAATTGGTATCCGTCTGGCGAAGAAAGTTGAAATGACCGAAATGCCAGAACTGGTGGCGATCCTGCATAGCTTCGTGGGTCTGGCGGCAGTGCTGGTTGGCTTTAACAGCTATCTGCATCATGACGCGGGAATGGCACCGATTCTGGTCAATATTCACCTGACGGAAGTGTTCCTCGGTATCTTCATCGGGGCGGTAACGTTCACGGGTTCGGTGGTGGCGTTCGGCAAACTGTGTGGCAAGATTTCGTCTAAACCATTGATGCTGCCAAACCGTCACAAAATGAACCTGGCGGCTCTGGTCGTTTCCTTCCTGCTGCTGATTGTATTTGTTCGCACGGACAGCGTCGGCCTGCAAGTGCTGGCATTGCTGATAATGACCGCAATTGCGCTGGTATTCGGCTGGCATTTAGTCGCCTCCATCGGTGGTGCAGATATGCCAGTGGTGGTGTCGATGCTGAACTCGTACTCCGGCTGGGCGGCTGCGGCTGCGGGCTTTATGCTCAGCAACGACCTGCTGATTGTGACCGGTGCGCTGGTCGGTTCTTCGGGGGCTATCCTTTCTTACATTATGTGTAAGGCGATGAACCGTTCCTTTATCAGCGTTATTGCGGGTGGTTTCGGCACCGACGGCTCTTCTACTGGCGATGATCAGGAAGTGGGTGAGCACCGCGAAATCACCGCAGAAGAGACAGCGGAACTGCTGAAAAACTCCCATTCAGTGATCATTACTCCGGGGTACGGCATGGCAGTCGCGCAGGCGCAATATCCTGTCGCTGAAATTACTGAGAAATTGCGCGCTCGTGGTATTAATGTGCGTTTCGGTATCCACCCGGTCGCGGGGCGTTTGCCTGGACATATGAACGTATTGCTGGCTGAAGCAAAAGTACCGTATGACATCGTGCTGGAAATGGACGAGATCAATGATGACTTTGCTGATACCGATACCGTACTGGTGATTGGTGCTAACGATACGGTTAACCCGGCGGCGCAGGATGATCCGAAGAGTCCGATTGCTGGTATGCCTGTGCTGGAAGTGTGGAAAGCGCAGAACGTGATTGTCTTTAAACGTTCGATGAACACTGGCTATGCTGGTGTGCAAAACCCGCTGTTCTTCAAGGAAAACACCCACATGCTGTTTGGTGACGCCAAAGCCAGCGTGGATGCAATCCTGAAAGCTCTGTAA(SEQ ID NO：20)
ATGTCTATTCCAGAAACTCAAAAAGCCATTATCTTCTACGAATCCAACGGCAAGTTGGAGCATAAGGATATCCCAGTTCCAAAGCCAAAGCCCAACGAATTGTTAATCAACGTCAAGTACTCTGGTGTCTGCCACACCGATTTGCACGCTTGGCATGGTGACTGGCCATTGCCAACTAAGTTACCATTAGTTGGTGGTCACGAAGGTGCCGGTGTCGTTGTCGGCATGGGTGAAAACGTTAAGGGCTGGAAGATCGGTGACTACGCCGGTATCAAATGGTTGAACGGTTCTTGTATGGCCTGTGAATACTGTGAATTGGGTAACGAATCCAACTGTCCTCACGCTGACTTGTCTGGTTACACCCACGACGGTTCTTTCCAAGAATACGCTACCGCTGACGCTGTTCAAGCCGCTCACATTCCTCAAGGTACTGACTTGGCTGAAGTCGCGCCAATCTTGTGTGCTGGTATCACCGTATACAAGGCTTTGAAGTCTGCCAACTTGAGAGCAGGCCACTGGGCGGCCATTTCTGGTGCTGCTGGTGGTCTAGGTTCTTTGGCTGTTCAATATGCTAAGGCGATGGGTTACAGAGTCTTAGGTATTGATGGTGGTCCAGGAAAGGAAGAATTGTTTACCTCGCTCGGTGGTGAAGTATTCATCGACTTCACCAAAGAGAAGGACATTGTTAGCGCAGTCGTTAAGGCTACCAACGGCGGTGCCCACGGTATCATCAATGTTTCCGTTTCCGAAGCCGCTATCGAAGCTTCTACCAGATACTGTAGGGCGAACGGTACTGTTGTCTTGGTTGGTTTGCCAGCCGGTGCAAAGTGCTCCTCTGATGTCTTCAACCACGTTGTCAAGTCTATCTCCATTGTCGGCTCTTACGTGGGGAACAGAGCTGATACCAGAGAAGCCTTAGATTTCTTTGCCAGAGGTCTAGTCAAGTCTCCAATAAAGGTAGTTGGCTTATCCAGTTTACCAGAAATTTACGAAAAGATGGAGAAGGGCCAAATTGCTGGTAGATACGTTGTTGACACTTCTAAATAA(SEQ ID NO：21)。
ATGTCTCAGACTCACAAGCATGCTATTCCTGCTAACATAGCTGACAGATGCTTAATAAATCCAGAACAGTATGAAACTAAATACAAGCAATCTATAAACGATCCTGACACTTTCTGGGGAGAGCAGGGTAAGATATTGGACTGGATTACACCATACCAAAAGGTCAAGAACACTTCATTTGCTCCTGGTAATGTTTCTATTAAATGGTACGAGGACGGTACATTAAACTTAGCAGCAAACTGCTTGGACAGGCACTTGCAAGAGAACGGAGACAGAACTGCAATAATTTGGGAGGGTGACGACGCATCTCAATCTAAGCACATATCATACAGAGAGTTACACAGAGACGTTTGCAGATTCGCTAACACATTGTTGGATTTGGGAATAAAGAAAGGTGACGTTGTTGCAATTTATATGCCTATGGTTCCTGAGGCTGCTGTCGCTATGTTGGCTTGCGCAAGGATAGGAGCTGTCCATTCTGTCATTTTTGGTGGTTTTTCACCTGAGGCAATAGCAGGTAGGATAATTGATTCATCTTCAAGGTTGGTCATAACTGCAGATGAGGGAGTCAGGGCAGGAAGATCTATTCCATTGAAGAAGAACGTTGACGATGCATTGAAGAACCCAAACGTTACTTCTGTCGAGCACGTCATTGTCTTAAAAAGAACTGGAAACGACATAGACTGGCAAGAGGGTAGGGACTTATGGTGGAGGGACTTGATTGAGAAGGCTTCACCTGAACACCAGCCAGAGGCAATGAATGCAGAAGACCCATTGTTTATTTTATATACATCTGGATCTACAGGAAAGCCAAAGGGAGTCTTGCATACTACAGGTGGTTACTTGGTCTATGCAGCAACAACTTTTAAGTATGTCTTCGACTACCACCCTGGTGACATTTACTGGTGCACAGCTGACGTTGGATGGGTCACAGGTCACTCTTACTTGTTGTACGGACCTTTGGCATGCGGTGCTACTACATTGATGTTCGAGGGAGTCCCTAACTGGCCTACACCTGCTAGGATGTGCCAGGTTGTCGACAAGCACCAGGTCAACATACTGTACACAGCACCAACAGCTATTAGGGCATTGATGGCAGAGGGTGACAAGGCAATTGAGGGAACAGACAGGTCTTCTTTGAGAATATTGGGATCTGTCGGTGAGCCAATAAACCCAGAGGCATGGGAGTGGTACTGGAAGAAGATAGGAAAGGAGAAATGTCCAGTCGTCGACACATGGTGGCAGACAGAGACAGGTGGATTTATGATAACTCCTTTGCCAGGAGCTATAGAGTTGAAGGCTGGTTCAGCAACAAGGCCATTCTTCGGAGTCCAGCCTGCATTGGTCGACAACGAGGGTCATCCACAGGAAGGTGCTACAGAGGGTAACTTAGTCATTACTGACTCTTGGCCTGGTCAGGCTAGGACATTGTTCGGTGACCATGAGAGGTTCGAGCAGACTTACTTTTCTACATTTAAAAATATGTACTTTTCTGGAGACGGTGCTAGAAGGGATGAAGATGGTTACTATTGGATAACTGGTAGGGTCGATGATGTCTTAAACGTCTCAGGACACAGGTTGGGAACTGCTGAAATTGAATCAGCATTGGTCGCTCACCCTAAGATAGCTGAGGCAGCTGTCGTCGGAATTCCTCACGCAATTAAGGGACAGGCAATTTACGCATACGTCACTTTGAACCACGGTGAAGAGCCATCACCAGAGTTGTACGCAGAGGTTAGGAACTGGGTCAGGAAGGAGATTGGACCTTTGGCAACTCCAGACGTCTTGCACTGGACAGACTCATTGCCTAAGACAAGGTCTGGAAAAATAATGAGGAGAATATTGAGGAAAATTGCTGCTGGAGATACTTCAAACTTGGGTGACACTTCTACATTGGCTGACCCAGGTGTTGTCGAGAAACCATTGGAGGAAAAACAGGCAATAGCTATGCCATCTTAA(SEQID NO：22)。
ATGACTAAGCTACACTTTGACACTGCTGAACCAGTCAAGATCACACTTCCAAATGGTTTGACATACGAGCAACCAACCGGTCTATTCATTAACAACAAGTTTATGAAAGCTCAAGACGGTAAGACCTATCCCGTCGAAGATCCTTCCACTGAAAACACCGTTTGTGAGGTCTCTTCTGCCACCACTGAAGATGTTGAATATGCTATCGAATGTGCCGACCGTGCTTTCCACGACACTGAATGGGCTACCCAAGACCCAAGAGAAAGAGGCCGTCTACTAAGTAAGTTGGCTGACGAATTGGAAAGCCAAATTGACTTGGTTTCTTCCATTGAAGCTTTGGACAATGGTAAAACTTTGGCCTTAGCCCGTGGGGATGTTACCATTGCAATCAACTGTCTAAGAGATGCTGCTGCCTATGCCGACAAAGTCAACGGTAGAACAATCAACACCGGTGACGGCTACATGAACTTCACCACCTTAGAGCCAATCGGTGTCTGTGGTCAAATTATTCCATGGAACTTTCCAATAATGATGTTGGCTTGGAAGATCGCCCCAGCATTGGCCATGGGTAACGTCTGTATCTTGAAACCCGCTGCTGTCACACCTTTAAATGCCCTATACTTTGCTTCTTTATGTAAGAAGGTTGGTATTCCAGCTGGTGTCGTCAACATCGTTCCAGGTCCTGGTAGAACTGTTGGTGCTGCTTTGACCAACGACCCAAGAATCAGAAAGCTGGCTTTTACCGGTTCTACAGAAGTCGGTAAGAGTGTTGCTGTCGACTCTTCTGAATCTAACTTGAAGAAAATCACTTTGGAACTAGGTGGTAAGTCCGCCCATTTGGTCTTTGACGATGCTAACATTAAGAAGACTTTACCAAATCTAGTAAACGGTATTTTCAAGAACGCTGGTCAAATTTGTTCCTCTGGTTCTAGAATTTACGTTCAAGAAGGTATTTACGACGAACTATTGGCTGCTTTCAAGGCTTACTTGGAAACCGAAATCAAAGTTGGTAATCCATTTGACAAGGCTAACTTCCAAGGTGCTATCACTAACCGTCAACAATTCGACACAATTATGAACTACATCGATATCGGTAAGAAAGAAGGCGCCAAGATCTTAACTGGTGGCGAAAAAGTTGGTGACAAGGGTTACTTCATCAGACCAACCGTTTTCTACGATGTTAATGAAGACATGAGAATTGTTAAGGAAGAAATTTTTGGACCAGTTGTCACTGTCGCAAAGTTCAAGACTTTAGAAGAAGGTGTCGAAATGGCTAACAGCTCTGAATTCGGTCTAGGTTCTGGTATCGAAACAGAATCTTTGAGCACAGGTTTGAAGGTGGCCAAGATGTTGAAGGCCGGTACCGTCTGGATCAACACATACAACGATTTTGACTCCAGAGTTCCATTCGGTGGTGTTAAGCAATCTGGTTACGGTAGAGAAATGGGTGAAGAAGTCTACCATGCATACACTGAAGTAAAAGCTGTCAGAATTAAGTTGTAA(SEQID NO：23)。
ATGAATCAACAGGATATTGAACAGGTGGTGAAAGCGGTACTGCTGAAAATGCAAAGCAGTGACACGCCGTCCGCCGCCGTTCATGAGATGGGCGTTTTCGCGTCCCTGGATGACGCCGTTGCGGCAGCCAAAGTCGCCCAGCAAGGGTTAAAAAGCGTGGCAATGCGCCAGTTAGCCATTGCTGCCATTCGTGAAGCAGGCGAAAAACACGCCAGAGATTTAGCGGAACTTGCCGTCAGTGAAACCGGCATGGGGCGCGTTGAAGATAAATTTGCAAAAAACGTCGCTCAGGCGCGCGGCACACCAGGCGTTGAGTGCCTCTCTCCGCAAGTGCTGACTGGCGACAACGGCCTGACCCTAATTGAAAACGCACCCTGGGGCGTGGTGGCTTCGGTGACGCCTTCCACTAACCCGGCGGCAACCGTAATTAACAACGCCATCAGCCTGATTGCCGCGGGCAACAGCGTCATTTTTGCCCCGCATCCGGCGGCGAAAAAAGTCTCCCAGCGGGCGATTACGCTGCTCAACCAGGCGATTGTTGCCGCAGGTGGGCCGGAAAACTTACTGGTTACTGTGGCAAATCCGGATATCGAAACCGCGCAACGCTTGTTCAAGTTTCCGGGTATCGGCCTGCTGGTGGTAACCGGCGGCGAAGCGGTAGTAGAAGCGGCGCGTAAACACACCAATAAACGTCTGATTGCCGCAGGCGCTGGCAACCCGCCGGTAGTGGTGGATGAAACCGCCGACCTCGCCCGTGCCGCTCAGTCCATCGTCAAAGGCGCTTCTTTCGATAACAACATCATTTGTGCCGACGAAAAGGTACTGATTGTTGTTGATAGCGTAGCCGATGAACTGATGCGTCTGATGGAAGGCCAGCACGCGGTGAAACTGACCGCAGAACAGGCGCAGCAGCTGCAACCGGTGTTGCTGAAAAATATCGACGAGCGCGGAAAAGGCACCGTCAGCCGTGACTGGGTTGGTCGCGACGCAGGCAAAATCGCGGCGGCAATCGGCCTTAAAGTTCCGCAAGAAACGCGCCTGCTGTTTGTGGAAACCACCGCAGAACATCCGTTTGCCGTGACTGAACTGATGATGCCGGTGTTGCCCGTCGTGCGCGTCGCCAACGTGGCGGATGCCATTGCGCTAGCGGTGAAACTGGAAGGCGGTTGCCACCACACGGCGGCAATGCACTCGCGCAACATCGAAAACATGAACCAGATGGCGAATGCTATTGATACCAGCATTTTCGTTAAGAACGGACCGTGCATTGCCGGGCTGGGGCTGGGCGGGGAAGGCTGGACCACCATGACCATCACCACGCCAACCGGTGAAGGGGTAACCAGCGCGCGTACGTTTGTCCGTCTGCGTCGCTGTGTATTAGTCGATGCGTTTCGCATTGTTTAA(SEQ ID NO：24)。
ATGTCATTACCGTTCTTAACTTCTGCACCGGGAAAGGTTATTATTTTTGGTGAACACTCTGCTGTGTACAACAAGCCTGCCGTCGCTGCTAGTGTGTCTGCGTTGAGAACCTACCTGCTAATAAGCGAGTCATCTGCACCAGATACTATTGAATTGGACTTCCCGGACATTAGCTTTAATCATAAGTGGTCCATCAATGATTTCAATGCCATCACCGAGGATCAAGTAAACTCCCAAAAATTGGCCAAGGCTCAACAAGCCACCGATGGCTTGTCTCAGGAACTCGTTAGTCTTTTGGATCCGTTGTTAGCTCAACTATCCGAATCCTTCCACTACCATGCAGCGTTTTGTTTCCTGTATATGTTTGTTTGCCTATGCCCCCATGCCAAGAATATTAAGTTTTCTTTAAAGTCTACTTTACCCATCGGTGCTGGGTTGGGCTCAAGCGCCTCTATTTCTGTATCACTGGCCTTAGCTATGGCCTACTTGGGGGGGTTAATAGGATCTAATGACTTGGAAAAGCTGTCAGAAAACGATAAGCATATAGTGAATCAATGGGCCTTCATAGGTGAAAAGTGTATTCACGGTACCCCTTCAGGAATAGATAACGCTGTGGCCACTTATGGTAATGCCCTGCTATTTGAAAAAGACTCACATAATGGAACAATAAATACAAACAATTTTAAGTTCTTAGATGATTTCCCAGCCATTCCAATGATCCTAACCTATACTAGAATTCCAAGGTCTACAAAAGATCTTGTTGCTCGCGTTCGTGTGTTGGTCACCGAGAAATTTCCTGAAGTTATGAAGCCAATTCTAGATGCCATGGGTGAATGTGCCCTACAAGGCTTAGAGATCATGACAAAGTTAAGTAAATGTAAAGGCACCGATGACGAGGCCGTAGAAACTAATAATGAACTGTATGAACAACTATTGGAATTGATAAGAATAAATCATGGACTGCTTGTCTCAATCGGTGTTTCTCATCCTGGATTAGAACTTATTAAAAATCTGAGCGATGATTTGAGAATTGGCTCCACAAAACTTACCGGTGCTGGTGGCGGCGGTTGCTCTTTGACTTTGTTACGAAGAGACATTACTCAAGAGCAAATTGACAGTTTCAAAAAGAAATTGCAAGATGATTTTAGTTACGAGACATTTGAAACAGACTTGGGTGGGACTGGCTGCTGTTTGTTAAGCGCAAAAAATTTGAATAAAGATCCTAAAATCAAATCCCTAGTATTCCAATTATTTGAAAATAAAACTACCACAAAGCAACAAATTGACGATCTATTATTGCCAGGAAACACGAATTTACCATGGACTTCATAA(SEQ ID NO：25)。
ATGACTGCCGACAACAATAGTATGCCCCATGGTGCAGTATCTAGTTACGCCAAATTAGTGCAAAACCAAACACCTGAAGACATTTTGGAAGAGTTTCCTGAAATTATTCCATTACAACAAAGACCTAATACCCGATCTAGTGAGACGTCAAATGACGAAAGCGGAGAAACATGTTTTTCTGGTCATGATGAGGAGCAAATTAAGTTAATGAATGAAAATTGTATTGTTTTGGATTGGGACGATAATGCTATTGGTGCCGGTACCAAGAAAGTTTGTCATTTAATGGAAAATATTGAAAAGGGTTTACTACATCGTGCATTCTCCGTCTTTATTTTCAATGAACAAGGTGAATTACTTTTACAACAAAGAGCCACTGAAAAAATAACTTTCCCTGATCTTTGGACTAACACATGCTGCTCTCATCCACTATGTATTGATGACGAATTAGGTTTGAAGGGTAAGCTAGACGATAAGATTAAGGGCGCTATTACTGCGGCGGTGAGAAAACTAGATCATGAATTAGGTATTCCAGAAGATGAAACTAAGACAAGGGGTAAGTTTCACTTTTTAAACAGAATCCATTACATGGCACCAAGCAATGAACCATGGGGTGAACATGAAATTGATTACATCCTATTTTATAAGATCAACGCTAAAGAAAACTTGACTGTCAACCCAAACGTCAATGAAGTTAGAGACTTCAAATGGGTTTCACCAAATGATTTGAAAACTATGTTTGCTGACCCAAGTTACAAGTTTACGCCTTGGTTTAAGATTATTTGCGAGAATTACTTATTCAACTGGTGGGAGCAATTAGATGACCTTTCTGAAGTGGAAAATGACAGGCAAATTCATAGAATGCTATAA(SEQID NO：26)。
ATGTCTCAGAACGTTTACATTGTATCGACTGCCAGAACCCCAATTGGTTCATTCCAGGGTTCTCTATCCTCCAAGACAGCAGTGGAATTGGGTGCTGTTGCTTTAAAAGGCGCCTTGGCTAAGGTTCCAGAATTGGATGCATCCAAGGATTTTGACGAAATTATTTTTGGTAACGTTCTTTCTGCCAATTTGGGCCAAGCTCCGGCCAGACAAGTTGCTTTGGCTGCCGGTTTGAGTAATCATATCGTTGCAAGCACAGTTAACAAGGTCTGTGCATCCGCTATGAAGGCAATCATTTTGGGTGCTCAATCCATCAAATGTGGTAATGCTGATGTTGTCGTAGCTGGTGGTTGTGAATCTATGACTAACGCACCATACTACATGCCAGCAGCCCGTGCGGGTGCCAAATTTGGCCAAACTGTTCTTGTTGATGGTGTCGAAAGAGATGGGTTGAACGATGCGTACGATGGTCTAGCCATGGGTGTACACGCAGAAAAGTGTGCCCGTGATTGGGATATTACTAGAGAACAACAAGACAATTTTGCCATCGAATCCTACCAAAAATCTCAAAAATCTCAAAAGGAAGGTAAATTCGACAATGAAATTGTACCTGTTACCATTAAGGGATTTAGAGGTAAGCCTGATACTCAAGTCACGAAGGACGAGGAACCTGCTAGATTACACGTTGAAAAATTGAGATCTGCAAGGACTGTTTTCCAAAAAGAAAACGGTACTGTTACTGCCGCTAACGCTTCTCCAATCAACGATGGTGCTGCAGCCGTCATCTTGGTTTCCGAAAAAGTTTTGAAGGAAAAGAATTTGAAGCCTTTGGCTATTATCAAAGGTTGGGGTGAGGCCGCTCATCAACCAGCTGATTTTACATGGGCTCCATCTCTTGCAGTTCCAAAGGCTTTGAAACATGCTGGCATCGAAGACATCAATTCTGTTGATTACTTTGAATTCAATGAAGCCTTTTCGGTTGTCGGTTTGGTGAACACTAAGATTTTGAAGCTAGACCCATCTAAGGTTAATGTATATGGTGGTGCTGTTGCTCTAGGTCACCCATTGGGTTGTTCTGGTGCTAGAGTGGTTGTTACACTGCTATCCATCTTACAGCAAGAAGGAGGTAAGATCGGTGTTGCCGCCATTTGTAATGGTGGTGGTGGTGCTTCCTCTATTGTCATTGAAAAGATATGA(SEQ ID NO：27)。
ATGACCGTTTACACAGCATCCGTTACCGCACCCGTCAACATCGCAACCCTTAAGTATTGGGGGAAAAGGGACACGAAGTTGAATCTGCCCACCAATTCGTCCATATCAGTGACTTTATCGCAAGATGACCTCAGAACGTTGACCTCTGCGGCTACTGCACCTGAGTTTGAACGCGACACTTTGTGGTTAAATGGAGAACCACACAGCATCGACAATGAAAGAACTCAAAATTGTCTGCGCGACCTACGCCAATTAAGAAAGGAAATGGAATCGAAGGACGCCTCATTGCCCACATTATCTCAATGGAAACTCCACATTGTCTCCGAAAATAACTTTCCTACAGCAGCTGGTTTAGCTTCCTCCGCTGCTGGCTTTGCTGCATTGGTCTCTGCAATTGCTAAGTTATACCAATTACCACAGTCAACTTCAGAAATATCTAGAATAGCAAGAAAGGGGTCTGGTTCAGCTTGTAGATCGTTGTTTGGCGGATACGTGGCCTGGGAAATGGGAAAAGCTGAAGATGGTCATGATTCCATGGCAGTACAAATCGCAGACAGCTCTGACTGGCCTCAGATGAAAGCTTGTGTCCTAGTTGTCAGCGATATTAAAAAGGATGTGAGTTCCACTCAGGGTATGCAATTGACCGTGGCAACCTCCGAACTATTTAAAGAAAGAATTGAACATGTCGTACCAAAGAGATTTGAAGTCATGCGTAAAGCCATTGTTGAAAAAGATTTCGCCACCTTTGCAAAGGAAACAATGATGGATTCCAACTCTTTCCATGCCACATGTTTGGACTCTTTCCCTCCAATATTCTACATGAATGACACTTCCAAGCGTATCATCAGTTGGTGCCACACCATTAATCAGTTTTACGGAGAAACAATCGTTGCATACACGTTTGATGCAGGTCCAAATGCTGTGTTGTACTACTTAGCTGAAAATGAGTCGAAACTCTTTGCATTTATCTATAAATTGTTTGGCTCTGTTCCTGGATGGGACAAGAAATTTACTACTGAGCAGCTTGAGGCTTTCAACCATCAATTTGAATCATCTAACTTTACTGCACGTGAATTGGATCTTGAGTTGCAAAAGGATGTTGCCAGAGTGATTTTAACTCAAGTCGGTTCAGGCCCACAAGAAACAAACGAATCTTTGATTGACGCAAAGACTGGTCTACCAAAGGAATAA(SEQ ID NO：28)。
ATGAAACTCTCAACTAAACTTTGTTGGTGTGGTATTAAAGGAAGACTTAGGCCGCAAAAGCAACAACAATTACACAATACAAACTTGCAAATGACTGAACTAAAAAAACAAAAGACCGCTGAACAAAAAACCAGACCTCAAAATGTCGGTATTAAAGGTATCCAAATTTACATCCCAACTCAATGTGTCAACCAATCTGAGCTAGAGAAATTTGATGGCGTTTCTCAAGGTAAATACACAATTGGTCTGGGCCAAACCAACATGTCTTTTGTCAATGACAGAGAAGATATCTACTCGATGTCCCTAACTGTTTTGTCTAAGTTGATCAAGAGTTACAACATCGACACCAACAAAATTGGTAGATTAGAAGTCGGTACTGAAACTCTGATTGACAAGTCCAAGTCTGTCAAGTCTGTCTTGATGCAATTGTTTGGTGAAAACACTGACGTCGAAGGTATTGACACGCTTAATGCCTGTTACGGTGGTACCAACGCGTTGTTCAACTCTTTGAACTGGATTGAATCTAACGCATGGGATGGTAGAGACGCCATTGTAGTTTGCGGTGATATTGCCATCTACGATAAGGGTGCCGCAAGACCAACCGGTGGTGCCGGTACTGTTGCTATGTGGATCGGTCCTGATGCTCCAATTGTATTTGACTCTGTAAGAGCTTCTTACATGGAACACGCCTACGATTTTTACAAGCCAGATTTCACCAGCGAATATCCTTACGTCGATGGTCATTTTTCATTAACTTGTTACGTCAAGGCTCTTGATCAAGTTTACAAGAGTTATTCCAAGAAGGCTATTTCTAAAGGGTTGGTTAGCGATCCCGCTGGTTCGGATGCTTTGAACGTTTTGAAATATTTCGACTACAACGTTTTCCATGTTCCAACCTGTAAATTGGTCACAAAATCATACGGTAGATTACTATATAACGATTTCAGAGCCAATCCTCAATTGTTCCCAGAAGTTGACGCCGAATTAGCTACTCGCGATTATGACGAATCTTTAACCGATAAGAACATTGAAAAAACTTTTGTTAATGTTGCTAAGCCATTCCACAAAGAGAGAGTTGCCCAATCTTTGATTGTTCCAACAAACACAGGTAACATGTACACCGCATCTGTTTATGCCGCCTTTGCATCTCTATTAAACTATGTTGGATCTGACGACTTACAAGGCAAGCGTGTTGGTTTATTTTCTTACGGTTCCGGTTTAGCTGCATCTCTATATTCTTGCAAAATTGTTGGTGACGTCCAACATATTATCAAGGAATTAGATATTACTAACAAATTAGCCAAGAGAATCACCGAAACTCCAAAGGATTACGAAGCTGCCATCGAATTGAGAGAAAATGCCCATTTGAAGAAGAACTTCAAACCTCAAGGTTCCATTGAGCATTTGCAAAGTGGTGTTTACTACTTGACCAACATCGATGACAAATTTAGAAGATCTTACGATGTTAAAAAATAA(SEQ ID NO：29)。
ATGGTTTTAACCAATAAAACAGTCATTTCTGGATCGAAAGTCAAAAGTTTATCATCTGCGCAATCGAGCTCATCAGGACCTTCATCATCTAGTGAGGAAGATGATTCCCGCGATATTGAAAGCTTGGATAAGAAAATACGTCCTTTAGAAGAATTAGAAGCATTATTAAGTAGTGGAAATACAAAACAATTGAAGAACAAAGAGGTCGCTGCCTTGGTTATTCACGGTAAGTTACCTTTGTACGCTTTGGAGAAAAAATTAGGTGATACTACGAGAGCGGTTGCGGTACGTAGGAAGGCTCTTTCAATTTTGGCAGAAGCTCCTGTATTAGCATCTGATCGTTTACCATATAAAAATTATGACTACGACCGCGTATTTGGCGCTTGTTGTGAAAATGTTATAGGTTACATGCCTTTGCCCGTTGGTGTTATAGGCCCCTTGGTTATCGATGGTACATCTTATCATATACCAATGGCAACTACAGAGGGTTGTTTGGTAGCTTCTGCCATGCGTGGCTGTAAGGCAATCAATGCTGGCGGTGGTGCAACAACTGTTTTAACTAAGGATGGTATGACAAGAGGCCCAGTAGTCCGTTTCCCAACTTTGAAAAGATCTGGTGCCTGTAAGATATGGTTAGACTCAGAAGAGGGACAAAACGCAATTAAAAAAGCTTTTAACTCTACATCAAGATTTGCACGTCTGCAACATATTCAAACTTGTCTAGCAGGAGATTTACTCTTCATGAGATTTAGAACAACTACTGGTGACGCAATGGGTATGAATATGATTTCTAAAGGTGTCGAATACTCATTAAAGCAAATGGTAGAAGAGTATGGCTGGGAAGATATGGAGGTTGTCTCCGTTTCTGGTAACTACTGTACCGACAAAAAACCAGCTGCCATCAACTGGATCGAAGGTCGTGGTAAGAGTGTCGTCGCAGAAGCTACTATTCCTGGTGATGTTGTCAGAAAAGTGTTAAAAAGTGATGTTTCCGCATTGGTTGAGTTGAACATTGCTAAGAATTTGGTTGGATCTGCAATGGCTGGGTCTGTTGGTGGATTTAACGCACATGCAGCTAATTTAGTGACAGCTGTTTTCTTGGCATTAGGACAAGATCCTGCACAAAATGTTGAAAGTTCCAACTGTATAACATTGATGAAAGAAGTGGACGGTGATTTGAGAATTTCCGTATCCATGCCATCCATCGAAGTAGGTACCATCGGTGGTGGTACTGTTCTAGAACCACAAGGTGCCATGTTGGACTTATTAGGTGTAAGAGGCCCGCATGCTACCGCTCCTGGTACCAACGCACGTCAATTAGCAAGAATAGTTGCCTGTGCCGTCTTGGCAGGTGAATTATCCTTATGTGCTGCCCTAGCAGCCGGCCATTTGGTTCAAAGTCATATGACCCACAACAGGAAACCTGCTGAACCAACAAAACCTAACAATTTGGACGCCACTGATATAAATCGTTTGAAAGATGGGTCCGTCACCTGCATTAAATCCTAA(SEQ ID NO：30)
ATGTCAGAGTTGAGAGCCTTCAGTGCCCCAGGGAAAGCGTTACTAGCTGGTGGATATTTAGTTTTAGATACAAAATATGAAGCATTTGTAGTCGGATTATCGGCAAGAATGCATGCTGTAGCCCATCCTTACGGTTCATTGCAAGGGTCTGATAAGTTTGAAGTGCGTGTGAAAAGTAAACAATTTAAAGATGGGGAGTGGCTGTACCATATAAGTCCTAAAAGTGGCTTCATTCCTGTTTCGATAGGCGGATCTAAGAACCCTTTCATTGAAAAAGTTATCGCTAACGTATTTAGCTACTTTAAACCTAACATGGACGACTACTGCAATAGAAACTTGTTCGTTATTGATATTTTCTCTGATGATGCCTACCATTCTCAGGAGGATAGCGTTACCGAACATCGTGGCAACAGAAGATTGAGTTTTCATTCGCACAGAATTGAAGAAGTTCCCAAAACAGGGCTGGGCTCCTCGGCAGGTTTAGTCACAGTTTTAACTACAGCTTTGGCCTCCTTTTTTGTATCGGACCTGGAAAATAATGTAGACAAATATAGAGAAGTTATTCATAATTTAGCACAAGTTGCTCATTGTCAAGCTCAGGGTAAAATTGGAAGCGGGTTTGATGTAGCGGCGGCAGCATATGGATCTATCAGATATAGAAGATTCCCACCCGCATTAATCTCTAATTTGCCAGATATTGGAAGTGCTACTTACGGCAGTAAACTGGCGCATTTGGTTGATGAAGAAGACTGGAATATTACGATTAAAAGTAACCATTTACCTTCGGGATTAACTTTATGGATGGGCGATATTAAGAATGGTTCAGAAACAGTAAAACTGGTCCAGAAGGTAAAAAATTGGTATGATTCGCATATGCCAGAAAGCTTGAAAATATATACAGAACTCGATCATGCAAATTCTAGATTTATGGATGGACTATCTAAACTAGATCGCTTACACGAGACTCATGACGATTACAGCGATCAGATATTTGAGTCTCTTGAGAGGAATGACTGTACCTGTCAAAAGTATCCTGAAATCACAGAAGTTAGAGATGCAGTTGCCACAATTAGACGTTCCTTTAGAAAAATAACTAAAGAATCTGGTGCCGATATCGAACCTCCCGTACAAACTAGCTTATTGGATGATTGCCAGACCTTAAAAGGAGTTCTTACTTGCTTAATACCTGGTGCTGGTGGTTATGACGCCATTGCAGTGATTACTAAGCAAGATGTTGATCTTAGGGCTCAAACCGCTAATGACAAAAGATTTTCTAAGGTTCAATGGCTGGATGTAACTCAGGCTGACTGGGGTGTTAGGAAAGAAAAAGATCCGGAAACTTATCTTGATAAATAG(SEQ ID NO：31)。
ATGAGTGTGTCTACCGCCAAGAGGTCGCTGGATGTCGTTTCTCCGGGTTCATTAGCGGAGTTTGAGGGTTCAAAATCTCGTCACGATGAAATAGAAAATGAACATAGACGTACTGGTACACGTGATGGCGAGGATAGCGAGCAACCGAAGAAGAAGGGTAGCAAAACTAGCAAAAAGCAAGATTTGGATCCTGAAACTAAGCAGAAGAGGACTGCCCAAAATCGGGCCGCTCAAAGAGCTTTTAGGGAACGTAAGGAGAGGAAGATGAAGGAATTGGAGAAGAAGGTACAAAGTTTAGAGAGTATTCAGCAGCAAAATGAAGTGGAAGCTACTTTTTTGAGGGACCAGTTAATCACTCTGGTGAATGAGTTAAAAAAATATAGACCAGAGACAAGAAATGACTCAAAAGTGCTGGAATATTTAGCAAGGCGAGATCCTAATTTGCATTTTTCAAAAAATAACGTTAACCACAGCAATAGCGAGCCAATTGACACACCCAATGATGACATACAAGAAAATGTTAAACAAAAGATGAATTTCACGTTTCAATATCCGCTTGATAACGACAACGACAACGACAACAGTAAAAATGTGGGGAAACAATTACCTTCACCAAATGATCCAAGTCATTCGGCTCCTATGCCTATAAATCAGACACAAAAGAAATTAAGTGACGCTACAGATTCCTCCAGCGCTACTTTGGATTCCCTTTCAAATAGTAACGATGTTCTTAATAACACACCAAACTCCTCCACTTCGATGGATTGGTTAGATAATGTAATATATACTAACAGGTTTGTGTCAGGTGATGATGGCAGCAATAGTAAAACTAAGAATTTAGACAGTAATATGTTTTCTAATGACTTTAATTTTGAAAACCAATTTGATGAACAAGTTTCGGAGTTTTGTTCGAAAATGAACCAGGTATGTGGAACAAGGCAATGTCCCATTCCCAAGAAACCCATCTCGGCTCTTGATAAAGAAGTTTTCGCGTCATCTTCTATACTAAGTTCAAATTCTCCTGCTTTAACAAATACTTGGGAATCACATTCTAATATTACAGATAATACTCCTGCTAATGTCATTGCTACTGATGCTACTAAATATGAAAATTCCTTCTCCGGTTTTGGCCGACTTGGTTTCGATATGAGTGCCAATCATTACGTCGTGAATGATAATAGCACTGGTAGCACTGATAGCACTGGTAGCACTGGCAATAAGAACAAAAAGAACAATAATAATAGCGATGATGTACTCCCATTCATATCCGAGTCACCGTTTGATATGAACCAAGTTACTAATTTTTTTAGTCCGGGATCTACCGGCATCGGCAATAATGCTGCCTCTAACACCAATCCCAGCCTACTGCAAAGCAGCAAAGAGGATATACCTTTTATCAACGCAAATCTGGCTTTCCCAGACGACAATTCAACTAATATTCAATTACAACCTTTCTCTGAATCTCAATCTCAAAATAAGTTTGACTACGACATGTTTTTTAGAGATTCATCGAAGGAAGGTAACAATTTATTTGGAGAGTTTTTAGAGGATGACGATGATGACAAAAAAGCCGCTAATATGTCAGACGATGAGTCAAGTTTAATCAAGAACCAGTTAATTAACGAAGAACCAGAGCTTCCGAAACAATATCTACAATCGGTACCAGGAAATGAAAGCGAAATCTCACAAAAAAATGGCAGTAGTTTACAGAATGCTGACAAAATCAATAATGGCAATGATAACGATAATGATAATGATGTCGTTCCATCTAAGGAAGGCTCTTTACTAAGGTGTTCGGAAATTTGGGATAGAATAACAACACATCCGAAATACTCAGATATTGATGTCGATGGTTTATGTTCCGAGCTAATGGCAAAGGCAAAATGTTCAGAAAGAGGGGTTGTCATCAATGCAGAAGACGTTCAATTAGCTTTGAATAAGCATATGAACTAA(SEQ ID NO：32)。
ATGGCCGATGAGGAACGTTTAAAGGAGTTTAAAGGGGCAAACAAGATAGTGTTTGATCCAAATACCAGACAAGTATGGGAAAACCAGAATCGAGATGGAACAAAACCAGCAACTACTTTCCAGAGTGAAGAGGTCATAAAAAGAGCTGCCCCAGAATCTGAAAAAGACACCTCCGCCACATCAAGTATTGTTCCAGAGCTATAGAACATTGTGGCAACTGTGACTTTGGGGAGCAGGTTAGATCTGAAAACAGTTGCGCTACATGCCCGTATGCAGAATATAACCCCTAGCGTTTTGCTGCTGTCATCATGCGTATAGAGAGCCAAAAACTACAGCTTTAATTTTTGCCTCAGGGAAAATGGTTGTTACCGGTGCAAAAAGTGAGGATGACTCAAAGCTGGCCAGAAGAATATATGCAGGAATTATCCAAAAAATCAGGTTTGCAGCTAAATTCACAGACTTCAAAATACAAAATATTGTCGGTTCGTGTGACGTTAATTTCCCTATACGTCTAGAAGGGTTAGCATTCAGTCATGGTACTTTCTCCTCCTATGAGCCAGAATTGTTTCCTGGTTTGATCTATAGAATGGTGAAGCCGAAAATTGTGTTGTTAATTTTTGTTTCAGGAAAGATTGTTCTTCTGGAGCAAAGCAAAGGGAGGAAATTTACCAAGCTTTTGAAGCTATATACCCAGTTCTAAGTGAATTTAGAAAAATGTGA(SEQ ID NO：51)
ATGGATTTTGAGGAAGATTACGATGCGGAGTTTGATGATAATCAAGAAGGACAATTAGAAACACCTTTTCCATCGGTTGCGGGAGCCGATGATGGGGACAATGATAATGATGACTCTGTCGCAGAAAACATGAAGAAGAAGCAAAAGAGAGAGGCTGTAGTGGATGATGGGAGTGAAAATGCATTTGGTATACCCGAATTTACAAGAAAAGATAAGACTCTGGAGGAGATTTTAGAGATGATGGACAGTACTCCTCCTATCATTCCCGATGCAGTAATAGACTACTATTTAACCAAAAACGGGTTTAACGTAGCATATGTACAAGTGAAACGACTTTTAGCACTTGCTACTCAGAAATTTGTTAGTGATATAGCTAAGGATGCCTACGAATATTCCAGGATCAGGTCTTCCGTAGCGGTATCTAATGCTAACAACAGTCAGGCGAGAGCTAGGCAGCTATTGCAAGGACAGCGACAGACTGGCGTGCAGCAGATTTCACAACAACAACATCAACAGAATGAGAAGACTACAGCAAGCATAGTTGTTCTGACGGTGAACGATCCCAGTAGCGCTGTTGCTGAATACGGGCTCAATATAGGTCGCCCAGACTTTTATCGTTAG(SEQ ID NO：52)
EXAMPLE 8 construction of a New Generation of engineered Strain
According to examples 1-7, the effective modification is combined in a superposition manner, for example, two effective knockout genes Ypl062W, Yer130C, Yer134c and Exg1 are combined in pairs for knockout, and the result shows that double knockout can obtain strains with higher yield.
On the basis, effective transformation is superposed and combined to form a new generation of engineering strains, and the strain characteristics are shown in the following table 4.
TABLE 4 characteristics of the New Generation of engineered strains
Figure BDA0001325723840000371
Figure BDA0001325723840000381
Figure BDA0001325723840000391
Note: "√" indicates that the gene was knocked out, "+" indicates that the gene was overexpressed in one copy, "+" indicates that the gene was overexpressed in at least 2 copies, "-" indicates that the gene was not manipulated (i.e., not knocked out or not overexpressed)
Furthermore, the inventor proves that the new generation of engineering strains J1011-C-37-84 have the performance of high-yield lycopene through research.
EXAMPLE 9 construction of New Generation engineered Strain
Based on the previous generation of engineering strains, 18 dominant strains are selected, and on the basis, a new generation of engineering strains are constructed to explore the influence of gapN and EUTE genes on the yield of lycopene, and the strain characteristics are shown in the following table 5.
TABLE 5 characteristics of the New Generation of engineered strains
Figure BDA0001325723840000392
Figure BDA0001325723840000401
Note: "√" indicates that the gene is overexpressed, and "-" indicates that the gene is not overexpressed
Furthermore, the inventor proves that the new generation of engineering strains J1011-C-85-138 have the performance of high-yield lycopene through research.
Example 10 engineering bacteria shake flask culture fermentation Process
In this example, the inventors have described in detail the fermentation culture process of the engineered strains obtained in examples 1 to 9.
Shake flask fermentation adopts two-stage seed culture, the recombinant strain on the plate is picked into a PA bottle containing 5mL YPD medium, the first-stage seed solution is shaken by a 30-degree shaking table, and after overnight culture (generally 14-18h), the thalli grow to logarithmic growthAt this stage (OD around 5-8), the strain was transferred to a 250mL shake flask containing 50mL YPD medium at an inoculum size of 1% and shake-cultured to obtain a secondary seed solution. After about 14-18h, the OD of the secondary seeds is measured 600 Then, the amount of the second-stage seed solution required by inoculating the cells into a 500mL shake flask containing 200mL of a fermentation medium YPDG was calculated so that the final concentration OD600 of the cells became 0.5, and then the calculated volume was taken out and centrifuged, and the supernatant was removed, and the cells were suspended in the corresponding fermentation medium and added to the corresponding 500mL shake flask, and then placed on a 30 ℃ shaking table to start shake flask fermentation. The cell concentration was measured by sampling at intervals (about 4h) and after 8h the flask was cleared of newspaper and after about 48h the sample was initially stored in a-80 ℃ freezer for determination of lycopene production.
Example 11 comparison of product extraction modes
In this example, the inventors screened the product obtained after the fermentation treatment by extraction, as follows.
The method comprises the following steps: the sample was thawed by taking out from the refrigerator, 500. mu.L of the fermentation broth was placed in a 15mL centrifuge tube (precooled on ice), centrifuged at 5000g 4 ℃ for 2min to collect the cells, the supernatant was removed and then the cells were resuspended and washed with 1mL pure water, 1mL 3N HCl was added and the cells were disrupted in a boiling water bath for 3min, centrifuged to remove HCl and washed once with water. Then adding 1mL of acetone (HPLC grade), 0.2g of glass beads and 1% of antioxidant, shaking for 5min, then centrifuging for 2min at 5000g and 4 ℃, and transferring the supernatant into a 50mL centrifuge tube; repeatedly adding the extracting solution again, shaking, and collecting the extract until the thallus has no obvious yellow color; the collected acetone extracts were mixed well, 2mL was removed, centrifuged at 12000rpm for 10 minutes, and 1.2mL to brown supernatant was collected and transferred to a HPLC flask for HPLC analysis.
The second method comprises the following steps: the sample was thawed by taking out from the refrigerator, 500. mu.L of the fermentation broth was placed in a 15mL centrifuge tube (precooled on ice), and centrifuged at 5000g 4 ℃ for 2min to collect the cells, after the supernatant was removed, the cells were resuspended in 1mL pure water and washed. Adding 4mL of acetone (HPLC grade), 0.2g of glass beads and 1% of antioxidant, shaking for 5min, performing ice bath ultrasonic treatment for 5-10min, centrifuging at 5000g and 4 ℃ for 2min, and transferring the supernatant into a 50mL centrifuge tube; repeatedly adding the extracting solution again, shaking, and collecting the extract until the thallus has no obvious yellow color; the collected acetone extracts were mixed well, 2mL was removed, centrifuged at 12000rpm for 10 minutes, and 1.2mL to brown supernatant was collected and transferred to a HPLC flask for HPLC analysis.
The detection method comprises the following steps: the detection of lycopene was carried out using a quaternary HPLC, the detector being a uv detector, the absorption wavelength of lycopene being 474nm, the columns Agilent Zorbax C18(150mm 4.6mm 5 μm), mobile phase a (acetonitrile: water ═ 9: 1) and mobile phase B (methanol: isopropanol ═ 3: 2) were analyzed under the following conditions: 0-90% B (0-15min), 90% B (15-30min), 90% -0B (30-35min), and flow rate of 1 mL/min.
The results show that: the lycopene is degraded after the product is boiled by hydrochloric acid in the method I, the yield detection is unstable, the sample obtained by the method can be detected again at intervals of a period of time, even can be degraded from 100mg/L to 10mg/L, the sample obtained by the method II is stable, and the sample obtained by the method II is detected again at intervals of a period of time, and only from 100mg/L to 90 mg/L. The extraction scheme of the second method is more stable and better than that of the first method.
Example 12 comparison of fermentation methods in fermentation tank for engineering bacteria
In this example, the inventors performed an optimized screening of the conditions of the fermentation medium, and the experimental procedures were as follows:
the strain in this example was J1011-C-3, the seed medium was YPD medium, the fermentation medium was shake-flask optimized 4 media, and the optimum medium at fermenter level was further explored, with the following media recipes:
no. 2 YPD salt-containing culture medium
2.5L batch Medium: 2% peptone, 1% yeast extract, 0.8% KH 2 PO 4 And 2% glucose;
1L glucose feed medium: 500g/L glucose, 5g/L MgSO 4 ，3.5g/L K 2 SO 4 ，0.28g/L Na 2 SO 4 10g/L yeast extract.
1L of ethanol feed medium is 100% ethanol.
② No. 5 culture medium: YPD rich medium
2.5L batch Medium: 60g peptone, 30g yeast extract and 30g glucose (more abundant than YPD);
1L feed medium 1: 500g/L glucose, 15g/L yeast extract
500mL of feed medium 2:300mL of absolute ethanol, 100mL of glycerol, and 100mL of pure water.
The method comprises the steps of adopting fed-batch culture in a fed-batch mode, feeding after the glucose in a carbon source is basically consumed, and adopting two-stage feeding modes, namely a glucose feeding stage (for synthesizing biomass) and an ethanol-glycerol feeding stage (for synthesizing products). The specific method is as follows.
The fermenter was filled with a small amount of pure water and then air-extinguished at 115 ℃ for 30 minutes. Preparing fermentation liquor and feed liquor for batch culture, calibrating pH electrodes of the fermentation tank, clamping pipelines of the fermentation tank, and sterilizing at 115 ℃ for 30 minutes. The fermentation tank is taken out, connected with an air pipeline, aerated for a little (about 0.1 vvm), and simultaneously a cooling water device is opened for cooling. When the temperature is reduced to about 50 ℃, the rotating speed is opened to 100 rpm. And calibrating the dissolved oxygen electrode. The state of the unconnected circuit was set to a dissolved oxygen value of 0%, then the speed was increased to 600rpm while the ventilation was adjusted to 2vvm, the circuit of the dissolved oxygen electrode was connected, and after the dissolved oxygen stabilized, the value was set to 100%. The aeration and rotation speed were then reduced to the values required for fermentation (aeration 1.5vvm, rotation speed 300-600 rpm). The pH value is naturally adjusted to 30 ℃. Inoculation was started and the initial inoculation OD adjusted to 0.5 and the required seed liquid volume was calculated according to the formula. (if the fermentation liquid volume is 2500mL, the seed liquid OD value is n, then the seed liquid volume is 2500x0.5/n mL). After the start of fermentation, the pH of the medium was controlled to 5.5 with 2M NaOH at the time of feeding, the initial aeration rate was 1.5vvm, the initial stirring rate was set at 300rpm, and the dissolved oxygen was maintained at 30% or more (correlation stirring rate, 300-600 rpm).
Fermentation procedure: when the glucose concentration is reduced to about 2g/L during batch culture, glucose feeding is started, the initial feeding rate is 10mL/L fermentation liquor/h, so as to maintain the residual concentration of the glucose in the fermentation liquor to be about 2-3g/L (not to be reduced to 0g/L), an OD600 value is sampled and measured once every two hours, the glucose content is detected once, and the feeding rate is increased when the glucose concentration is lower than 1 g/L. When the OD value increases slowly (begins to enter a stationary phase), the glucose feeding is stopped, the ethanol residual quantity is monitored, when the ethanol concentration is reduced to 5-10g/L, the ethanol glycerol feeding is started, and the initial feeding rate is 2mL/L fermentation liquor/h. The ethanol and glycerol content was then checked every 4h sample while adjusting the feed rate to maintain the ethanol and glycerol in the range of 5-10 g/L. After the color changes, the product is extracted and the product change is detected, and the fermentation is finished when the lycopene concentration is not increased any more.
The product was detected in the same manner as in the second method of example 9.
The result shows that the yield of the culture medium No. 5 is superior to that of the culture medium No. 2, so the culture medium is the optimal culture medium, the fermentation strategy is the optimized two-step feeding fermentation, the glucose long thalli are firstly fed, and the ethanol is fed in the stable period of the growth to improve the yield.
EXAMPLE 13 in-tank fermentation culture of the Strain
In this example, the inventors fermented some of the constructed engineered strains according to the optimal fermentation culture method obtained in example 12, and the results are shown in Table 6.
Table 6:
Figure BDA0001325723840000431
Figure BDA0001325723840000441
it can be seen that the engineered strain constructed according to the embodiment of the invention has the property of high lycopene yield
In the description herein, references to the description of the term "one embodiment," "some embodiments," "an example," "a specific example," or "some examples," etc., mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the invention. In this specification, the schematic representations of the terms used above are not necessarily intended to refer to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples. Furthermore, various embodiments or examples and features of different embodiments or examples described in this specification can be combined and combined by one skilled in the art without contradiction.
Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention, and that variations, modifications, substitutions and alterations can be made to the above embodiments by those of ordinary skill in the art within the scope of the present invention.
SEQUENCE LISTING
<110> Wuhan Zhen Zhi Biotechnology GmbH
<120> microorganism and use thereof
<130> PIDC3172409
<160> 34
<170> PatentIn version 3.3
<210> 1
<211> 4863
<212> DNA
<213> Artificial
<220>
<223> knockout cassette fragment 1 sequence
<400> 1
ataacgagaa cacacagggg cgctatcgca cagaatcaaa ttcgatgact ggaaattttt 60
tgttaatttc agaggtcgcc tgacgcatat acctttttca actgaaaaat tgggagaaaa 120
aggaaaggtg agagcgccgg aaccggcttt tcatatagaa tagagaagcg ttcatgacta 180
aatgcttgca tcacaatact tgaagttgac aatattattt aaggacctat tgttttttcc 240
aataggtggt tagcaatcgt cttactttct aacttttctt accttttaca tttcagcaat 300
atatatatat atatttcaag gatataccat tctaatgtct gcccctaaga agatcgtcgt 360
tttgccaggt gaccacgttg gtcaagaaat cacagccgaa gccattaagg ttcttaaagc 420
tatttctgat gttcgttcca atgtcaagtt cgatttcgaa aatcatttaa ttggtggtgc 480
tgctatcgat gctacaggtg ttccacttcc agatgaggcg ctggaagcct ccaagaaggc 540
tgatgccgtt ttgttaggtg ctgtgggtgg tcctaaatgg ggtaccggta gtgttagacc 600
tgaacaaggt ttactaaaaa tccgtaaaga acttcaattg tacgccaact taagaccatg 660
taactttgca tccgactctc ttttagactt atctccaatc aagccacaat ttgctaaagg 720
tactgacttc gttgttgtca gagaattagt gggaggtatt tactttggta agagaaagga 780
agacgatggt gatggtgtcg cttgggatag tgaacaatac accgttccag aagtgcaaag 840
aatcacaaga atggccgctt tcatggccct acaacatgag ccaccattgc ctatttggtc 900
cttggataaa gctaatgttt tggcctcttc aagattatgg agaaaaactg tggaggaaac 960
catcaagaac gaattcccta cattgaaggt tcaacatcaa ttgattgatt ctgccgccat 1020
gatcctagtt aagaacccaa cccacctaaa tggtattata atcaccagca acatgtttgg 1080
tgatatcatc tccgatgaag cctccgttat cccaggttcc ttgggtttgt tgccatctgc 1140
gtccttggcc tctttgccag acaagaacac cgcatttggt ttgtacgaac catgccacgg 1200
ttctgctcca gatttgccaa agaataaggt caaccctatc gccactatct tgtctgctgc 1260
aatgatgttg aaattgtcat tgaacttgcc tgaagaaggt aaggccattg aagatgcagt 1320
taaaaaggtt ttggatgcag gtatcagaac tggtgattta ggtggttcca acagtaccac 1380
cgaagtcggt gatgctgtcg ccgaagaagt taagaaaatc cttgcttaaa tttaactcct 1440
taagttactt taatgattta gtttttatta ttaataattc atgctcatga catctcatat 1500
acacgtttat aaaacttaaa tagattgaaa atgtattaaa gattcctcag ggattcgatt 1560
tttttggaag tttttgtttt tttttccttg agatgctgta gtatttggga acaattatac 1620
aatcgaaaga tatatgctta cattcgaccg ttttagccgt gatcattatc ctatagtaac 1680
ataacctgaa gcataactga cactactatc atcaatactt gtcacatgag aactctgtga 1740
ataattaggc cactgaaatt tgatgcctga aggaccggca tcacggattt tcgataaagc 1800
acttagtatc acactaattg gcttttcgcg caaattaaag ccttcgagcg tcccaaaacc 1860
ttctcaagca aggttttcag tataatgtta catgcgtaca cgcgtttgta cagaaaaaaa 1920
agaaaaattt gaaatataaa taacgttctt aatactaaca taactataaa aaaataaata 1980
gggacctaga cttcaggttg tctaactcct tccttttcgg ttagagcgga tgtgggggga 2040
gggcgtgaat gtaagcgtga cataactaat tacatgatat cgacaaagga aaaggggcct 2100
gtttatattg aattttcaaa aattcttact ttttttttgg atggacgcaa agaagtttaa 2160
taatcatatt acatggcaat accaccatat acatatccat atctaatctt acttatatgt 2220
tgtggaaatg taaagagccc cattatctta gcctaaaaaa accttctctt tggaactttc 2280
agtaatacgc ttaactgctc attgctatat tgaagtacgg attagaagcc gccgagcggg 2340
cgacagccct ccgacggaag actctcctcc gtgcgtcctg gtcttcaccg gtcgcgttcc 2400
tgaaacgcag atgtgcctcg cgccgcactg ctccgaacaa taaagattct acaatactag 2460
cttttatggt tatgaagagg aaaaattggc agtaacctgg ccccacaaac cttcaaatca 2520
acgaatcaaa ttaacaacca taggataata atgcgattag ttttttagcc ttatttctgg 2580
ggtaattaat cagcgaagcg atgatttttg atctattaac agatatataa atgcaaaagc 2640
tgcataacca ctttaactaa tactttcaac attttcggtt tgtattactt cttattcaaa 2700
tgtcataaaa gtatcaacaa aaaattgtta atatacctct atactttaac gtcaaggaga 2760
aaaaactata atgactgttt gtgctaagaa acatgttcat ttgacaagag atgctgcaga 2820
acaattgttg gcagatatcg atagaagatt ggatcaattg ttaccagttg aaggtgaaag 2880
agatgttgtt ggtgctgcaa tgagagaagg tgctttggca ccaggtaaaa gaatcagacc 2940
aatgttgttg ttgttgactg ctagagattt gggttgtgca gtttctcatg atggtttgtt 3000
agatttggct tgtgcagttg aaatggttca tgctgcatca ttgatcttgg atgatatgcc 3060
atgtatggat gatgctaaat tgagaagagg tagaccaact attcattctc attatggtga 3120
acatgttgct attttagctg cagttgcttt gttatcaaaa gcatttggtg ttattgctga 3180
tgcagatggt ttgacaccat tggctaaaaa tagagcagtt tctgaattgt caaacgctat 3240
cggtatgcaa ggtttggttc aaggtcaatt caaagatttg tctgaaggtg acaaaccaag 3300
atcagctgaa gcaattttga tgactaacca tttcaagact tctacattat tttgtgcttc 3360
tatgcaaatg gcatcaattg ttgctaatgc atcttcagaa gctagagatt gtttgcatag 3420
attttcattg gatttgggtc aagcatttca attgttagat gatttgactg atggtatgac 3480
tgatactggt aaagattcta atcaagatgc tggtaaatca acattggtta atttgttagg 3540
tccaagagct gttgaagaaa gattgagaca acatttgcaa ttagcatctg aacatttgtc 3600
agctgcatgt caacatggtc atgctacaca acatttcatc caagcatggt tcgataagaa 3660
attagctgca gtttcttaag tctgaagaat gaatgatttg atgatttctt tttccctcca 3720
tttttcttac tgaatatatc aatgatatag acttgtatag tttattattt caaattaagt 3780
agctatatat agtcaagata acgtttgttt gacacgatta cattattcgt cgacatcttt 3840
tttcagcctg tcgtggtagc aatttgagga gtattattaa ttgaataggt tcattttgcg 3900
ctcgcataaa cagttttcgt cagggacagt atgttggaat gagtggtaat taatggtgac 3960
atgacatgtt atagcaataa ccttgatgtt tacatcgtag tttaatgtac accccgcgaa 4020
ttcgttcaag taggagtgca ccaattgcaa agggaaaagc tgaatgggca gttcgaataa 4080
aagattctct ttttttatga tatttgtaca taaactttat aaatgaaatt cataatagaa 4140
acgacacgaa attacaaaat ggaatatgtt catagggtag acgaaactat atacgcaatc 4200
tacatacatt tatcaagaag gagaaaaagg aggatgtaaa ggaatacagg taagcaaatt 4260
gatactaatg gctcaacgtg ataaggaaaa agaattgcac tttaacatta atattgacaa 4320
ggaggagggc accacacaaa aagttaggtg taacagaaaa tcatgaaact atgattccta 4380
atttatatat tggaggattt tctctaaaaa aaaaaaaata caacaaataa aaaacactca 4440
atgacctgac catttgatgg agtttaagtc aataccttct tgaaccattt cccataatgg 4500
tgaaagttcc ctcaagaatt ttactctgtc agaaacggcc ttaacgacgt agtcgacctc 4560
ctcttcagta ctaaatctac caataccaaa tctgatggaa gaatgggcta atgcatcatc 4620
cttacccagc gcatgtaaaa cataagaagg ttctagggaa gcagatgtac aggctgaacc 4680
cgaggataat gcgatatccc ttagtgccat caataaagat tctccttcca cgtaggcgaa 4740
agaaacgtta acacaccctg gataacgatg atctggagat ccgttcaacg tggtatgttc 4800
agcggataat agacctttga ctaatttatc ggatagtctt ttgatgtgag cttggtcgtt 4860
gtc 4863
<210> 2
<211> 9017
<212> DNA
<213> Artificial
<220>
<223> knockout cassette fragment 2 sequence
<400> 2
acgcagataa ttccaggtat tttgaagcag aacttttatt tatcatcgaa ttgactattg 60
cattatttct attttgcaaa gaggagaaag aattaggaaa gttcatactt caaaaagttt 120
tccaactttc tcacacgaaa ggcctcacga aaaggactgt tcgtagaatg ctaacataca 180
aaattttgtt aatttcgtta tgtgcggatc agacggagta cttgtccaaa ttaataaacg 240
atgagctgtt aaaaaagggg gatattttta cccaaaaatt ttttgcaact aatcaaggta 300
aggaattttt gaagagacta ttttcattga ccgaatcaga gttttataga ggatttttac 360
taggaaatga gaatttttgg aaatttttaa gaaaagttac agcaatgaaa gagcagagcg 420
agagcatttt tgaatattta aatgaatcga tcaagacaga cagcaatatt ttgacaaatg 480
agaacttcat gtgggtccta ggactattag atgaaatttc atcaatgggt gccgttggaa 540
atcactggga aatagaatac aagaaattga cagaaagtgg tcataaaatt gataaggaga 600
atccatacaa gaaatcgatc gaattatcat tgaaatccat tcaactaaca tcacacttgc 660
tggaagataa taacgatctg cgtaaaaacg agatattcgc tattattcaa gctttggcac 720
atcaatgcat caatccgtgt aagcagataa gtgaatttgc agtggtaacg ctagagcaga 780
cgctcatcaa taaaatcgaa attccaacta atgagatgga atcggtagaa gaattaattg 840
agggcggatt actaccgttg ctaaattcga gtgaaacaca ggaagaccag aaaatcctca 900
tttcatccat attaacaata atttcaaatg tttatttgca ttatttgaaa ctagggaaga 960
caagcaacga aacgtttttg aaaattttga gtattttcaa taaatttgta gaggactcag 1020
atattgaaaa aaagctacag caattaatac ttgataagaa gagtattgag aagggcaacg 1080
gttcatcatc tcatggatct gcacatgaac aaacaccaga gtcaaacgac gttgaaattg 1140
aggctactgc gccaattgat gacaatacag acgatgataa caaaccgaag ttatctgatg 1200
tagaaaagga ttaaagatgc taagagatag tgatgatatt tcataaataa tgtaattcta 1260
tatatgttaa ttaccttttt tgcgaggcat atttatggtg aaggataagt tttgaccatc 1320
aaagaaggtt aatgtggctg tggtttcagg gtccataaag cttttcaatt catcattttt 1380
tttttattct tttttttgat ttcggtttcc ttgaaatttt tttgattcgg taatctccga 1440
acagaaggaa gaacgaagga aggagcacag acttagattg gtatatatac gcatatgtag 1500
tgttgaagaa acatgaaatt gcccagtatt cttaacccaa ctgcacagaa caaaaacctg 1560
caggaaacga aggtacccaa ttcgccctat agtgagtcgt attacgcgcg ctcactggcc 1620
gtcgttttac aacgtcgtga ctgggaaaac cctggcgtta cccaacttaa tcgccttgca 1680
gcacatcccc ctttcgccag ctggcgtaat agcgaagagg cccgcaccga tcgcccttcc 1740
caacagttgc gcagcctgaa tggcgaatgg cgcgacgcgc cctgtagcgg cgcattaagc 1800
gcggcgggtg tggtggttac gcgcagcgtg accgctacac ttgccagcgc cctagcgccc 1860
gctcctttcg ctttcttccc ttcctttctc gccacgttcg ccggctttcc ccgtcaagct 1920
ctaaatcggg ggctcccttt agggttccga tttagtgctt tacggcacct cgaccccaaa 1980
aaacttgatt agggtgatgg ttcacgtagt gggccatcgc cctgatagac ggtttttcgc 2040
cctttgacgt tggagtccac gttctttaat agtggactct tgttccaaac tggaacaaca 2100
ctcaacccta tctcggtcta ttcttttgat ttataaggga ttttgccgat ttcggcctat 2160
tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga attttaacaa aatattaacg 2220
tttacaattt cctgatgcgg tattttctcc ttacgcatct gtgcggtatt tcacaccgca 2280
tagatccgtc gagttcaaga gaaaaaaaaa gaaaaagcaa aaagaaaaaa ggaaagcgcg 2340
cctcgttcag aatgacacgt atagaatgat gcattacctt gtcatcttca gtatcatact 2400
gttcgtatac atacttactg acattcatag gtatacatat atacacatgt atatatatcg 2460
tatgctgcag ctttaaataa tcggtgtcac tacataagaa cacctttggt ggagggaaca 2520
tcgttggtac cattgggcga ggtggcttct cttatggcaa ccgcaagagc cttgaacgca 2580
ctctcactac ggtgatgatc attcttgcct cgcagacaat caacgtggag ggtaattctg 2640
ctagcctctg caaagctttc aagaaaatgc gggatcatct cgcaagagag atctcctact 2700
ttctcccttt gcaaaccaag ttcgacaact gcgtacggcc tgttcgaaag atctaccacc 2760
gctctggaaa gtgcctcatc caaaggcgca aatcctgatc caaacctttt tactccacgc 2820
gccagtaggg cctctttaaa agcttgaccg agagcaatcc cgcagtcttc agtggtgtga 2880
tggtcgtcta tgtgtaagtc accaatgcac tcaacgatta gcgaccagcc ggaatgcttg 2940
gccagagcat gtatcatatg gtccagaaac cctatacctg tgtggacgtt aatcacttgc 3000
gattgtgtgg cctgttctgc tactgcttct gcctcttttt ctgggaagat cgagtgctct 3060
atcgctaggg gaccaccctt taaagagatc gcaatctgaa tcttggtttc atttgtaata 3120
cgctttacta gggctttctg ctctgtcatc tttgccttcg tttatcttgc ctgctcattt 3180
tttagtatat tcttcgaaga aatcacatta ctttatataa tgtataattc attatgtgat 3240
aatgccaatc gctaagaaaa aaaaagagtc atccgctagg ggaaaaaaaa aaatgaaaat 3300
cattaccgag gcataaaaaa atatagagtg tactagagga ggccaagagt aatagaaaaa 3360
gaaaattgcg ggaaaggact gtgttatgac ttccctgact aatgccgtgt tcaaacgata 3420
cctggcagtg actcctagcg ctcaccaagc tcttaaaacg ggaatttatg gtgcactctc 3480
agtacaatct gctctgatgc cgcatagtta agccagcccc gacacccgcc aacacgcgct 3540
gacgcgccct gacgggcttg tctgctcccg gcatccgctt acagacaagc tgtgagcaaa 3600
ttaaagcctt cgagcgtccc aaaaccttct caagcaaggt tttcagtata atgttacatg 3660
cgtacacgcg tttgtacaga aaaaaaagaa aaatttgaaa tataaataac gttcttaata 3720
ctaacataac tataaaaaaa taaataggga cctagacttc aggttgtcta actccttcct 3780
tttcggttag agcggatgtg gggggagggc gtgaatgtaa gcgtgacata actaattaca 3840
tgatatcgac aaaggaaaag gggcctgttt atatcctaat atcgttagag ttctgtcctt 3900
ggaagacgtt tggcaacaac tgattaataa aagatgctgg agtagtagtg tcgtccctag 3960
gaaagaaata gaagaacaag aaagtgacaa agtagcatgc taagcagtaa taaatccagt 4020
gtgactcagt cttccttgtt tgctcaggtg catatttctt ctgagagtcc tgtaacttcc 4080
taggcaaagg gttttgtccg aatgacttac agacctggtc agaagttaat ttagatcctg 4140
ccaagactat tgggactcct gtaccagggt gagttgatgc accgacgaaa aataaattgt 4200
catatctgtt agtagagtcc tttgttgaag gtctgaacca caaaacctga aagacatcat 4260
gtgataaacc taatattgat cccctccaca agttgaactt agattgccag actgatgggt 4320
cgttgacctc ctcgtgttct atcaagttag cgaagttgtt gactcccaac ctcctctcta 4380
tgacctccaa gaccatcttt ctagccctgt tgaccaactc tgggtagttc tcctctgctg 4440
agttacctgt cttagacttc atgtgaccaa ttggaactaa aactataatt gagtccttat 4500
ttggaggtgc tgcagactcg tctatccttg aaggaacgtt gacgtagaaa gatgcttcag 4560
aaggcaaacc gaaatcgtta aaaatctcgt cgaatgactc cttgtaagcc tcagccaaga 4620
aaatgttgtg gacgtccaat tgagggacct tagtagacat tgaccagtaa aaagaaattg 4680
atgaagatgt taatttctta gatgccaaag tcttctttgt ccagttgcaa ggtggcaaca 4740
agtggtggta agcgtagacc aagtcagcgt tgcagacgac agcgtctgcc tcaatgactt 4800
ctccagactc caaagtgaca ccagtgaccc tcttgtcttt atcgacagtg ttaatcttag 4860
cgacaggaga ttggtacctg aactcagctc cgtacttctt agaagctata gactccaact 4920
tttggacgac catgttgaaa ccaccccttg ggtaccaaat tccctctgcg aactctgtat 4980
attgcaacaa tgagtagact gcaggtgcgt cgtaaggtga catacccatg tacattgttt 5040
gaaaagtaaa agccatcctc atcttttttg tttggaagta ttttgatgct ctgtcgtata 5100
tcttaccgaa taagtgcaac ctaaaaatct ctgggacgta ctgtaacctt attaagtccc 5160
atatagtttc gaagtttctc tttatagcaa tgaatgtacc ctgctcgtag tggacgtgtg 5220
tctctttcat gaaatctaag aacctaccga atcctaaagg tccctcaatc ctgtccaact 5280
cacccttcat ctttgttaag tctgatgaca attggacagc gtcaccgtcg tcgaaatgga 5340
ctttgtaatt gttgtcacat cttaataagt ccaagtggtc tcctatcctc tcgtctaagt 5400
cagcgaaagc gtcctcaaac aacttaggca tcaagtacaa tgaaggtccc tggtcgaacc 5460
tgtgaccgtc gtggtgaatg aaagagcacc ttcctccaga gaagtcgttc ttctcgacga 5520
cagtgactct gaaaccctcc cttgctaacc ttgctgctgt tgcagtacct cctattccag 5580
cacctatgac gacaatgtgc ttcttctgat cagacattta tattgaattt tcaaaaattc 5640
ttactttttt tttggatgga cgcaaagaag tttaataatc atattacatg gcaataccac 5700
catatacata tccatatcta atcttactta tatgttgtgg aaatgtaaag agccccatta 5760
tcttagccta aaaaaacctt ctctttggaa ctttcagtaa tacgcttaac tgctcattgc 5820
tatattgaag tacggattag aagccgccga gcgggcgaca gccctccgac ggaagactct 5880
cctccgtgcg tcctggtctt caccggtcgc gttcctgaaa cgcagatgtg cctcgcgccg 5940
cactgctccg aacaataaag attctacaat actagctttt atggttatga agaggaaaaa 6000
ttggcagtaa cctggcccca caaaccttca aatcaacgaa tcaaattaac aaccatagga 6060
taataatgcg attagttttt tagccttatt tctggggtaa ttaatcagcg aagcgatgat 6120
ttttgatcta ttaacagata tataaatgca aaagctgcat aaccacttta actaatactt 6180
tcaacatttt cggtttgtat tacttcttat tcaaatgtca taaaagtatc aacaaaaaat 6240
tgttaatata cctctatact ttaacgtcaa ggagaaaaaa ctataatgtc acaaccacca 6300
ttattggacc acgctacaca aactatggca aacggttcta aatctttcgc tactgctgct 6360
aaattattcg acccagcaac aagaagatct gtattgatgt tgtacacctg gtgtagacat 6420
tgcgatgacg ttatagatga ccaaactcac ggttttgctt cagaagctgc agccgaagaa 6480
gaagctacac aaagattggc aagattaaga actttgacat tagctgcatt cgaaggtgcc 6540
gaaatgcaag atccagcttt tgccgctttc caagaagttg cattaaccca tggtattact 6600
cctagaatgg ctttggatca cttagacggt tttgcaatgg atgtcgccca aacaagatac 6660
gtaaccttcg aagacacttt aagatattgt taccatgtcg ccggtgttgt cggtttgatg 6720
atggctagag taatgggtgt tagagatgaa agagttttag atagagcatg tgacttgggt 6780
ttagccttcc aattgacaaa catagctaga gatataatag atgacgcagc catagacaga 6840
tgctatttgc cagctgaatg gttacaagat gcaggtttga ctcctgaaaa ttacgctgca 6900
agagaaaaca gagccgcttt agccagagtt gctgaaagat tgatagatgc agccgaacca 6960
tattacatct cttcacaagc tggtttgcat gatttgccac ctagatgcgc atgggccatt 7020
gctaccgcaa gatctgttta cagagaaatc ggtattaaag tcaaggctgc aggtggttcc 7080
gcatgggata gaagacaaca cacttctaaa ggtgaaaaga tcgctatgtt gatggccgct 7140
cctggtcaag ttattagagc aaagaccacc agagtcaccc caagaccagc cggtttatgg 7200
caaagacctg tttaaattga attgaattga aatcgataga tcaatttttt tcttttctct 7260
ttccccatcc tttacgctaa aataatagtt tattttattt tttgaatatt ttttatttat 7320
atacgtatat atagactatt atttatcttt taatgattat taagattttt attaaaaaaa 7380
attacgctcc tcttttaatg cctttatgca gttttttttc ccattcgata tttctatgtt 7440
cgggttcagc gtattttaag tttaataact cgaaaattct gcgttcgttg taaatgcatg 7500
tatactaaac tcacaaatta gagcttcaat ttaattatat cagttattac ccgggaatct 7560
cggtcgtaat gatttctata atgacgaaaa aaaaaaaatt ggaaagaaaa agcttcatgg 7620
cctttataaa aaggaactat ccaatacctc gccagaacca agtaacagta ttttacgggg 7680
cacaaatcaa gaacaataag acaggactgt aaagatggac gcattgaact ccaaagaaca 7740
acaagagttc caaaaagtag tggaacaaaa gcaaatgaag gatttcatgc gtttgtactc 7800
taatctggta gaaagatgtt tcacagactg tgtcaatgac ttcacaacat caaagctaac 7860
caataaggaa caaacatgca tcatgaagtg ctcagaaaag ttcttgaagc atagcgaacg 7920
tgtagggcag cgtttccaag aacaaaacgc tgccttggga caaggcttgg gccgataagg 7980
tgtactggcg tatatatatc taattatgta tctctggtgt agcccatttt tagcatgtaa 8040
atataaagag aaaccatatc taatctaacc aaatccaaac aaaattcaat agttactatc 8100
gcttttttct ttctgtatcg caaataagtg aaaattaaaa aagaaagatt aaattggaag 8160
ttggatatgg gctggaacag cagcagtaat cggtatcggg ttcgccacta atgacgtcct 8220
acgattgcac tcaacagacc ttgacgctca cgccgtagcg ggcgacaagt caaacggaac 8280
aaccgttgcc gttcccatcg gagtccgacc taggccgaac tccgtgaatt tctgataaca 8340
acggtcggta aagactggtt ccccagtata tttcttctct caggagcagg ggccaatgcc 8400
aaaagcgaca ttaacccgga ggacaaggct ccactgtgtt ccaccgaatt tcccacctga 8460
taatatctga taacccgccc ataggtgggg atccttctgt aaacaggttt cttaatcgta 8520
ggaattacca ctgttccact gccaatcgca gctcccagag tttcgttccc agccgcgagc 8580
accacagcgt accatgtgcg ccacgaggcc tcaaacgtaa aacaatcgaa acgaaaagaa 8640
acagactata ggggagtata gagacagccg gccaataaga agaggaaaaa gaaatactag 8700
cgtttatcaa tgtgggtcgt taacatatcc tgttgacaat gattacaggt taaaaggtag 8760
cgtaagtgaa tattaactat ggatattctt atacttagat aagagacata taaaacacac 8820
gatgattgat tgatttttat gaccaatata tgtaattcgt aattcagata gtttttatac 8880
ttttaatgtg tgccgaacca tattactagt atatgtaact accatgttat gttcaattgg 8940
cagatcttta actcggcttt agttatccaa gttacttgca atatttcctt ctgcgagagt 9000
acatttgccc ttaaacg 9017
<210> 3
<211> 7319
<212> DNA
<213> Artificial
<220>
<223> knockout cassette fragment 3 sequence
<400> 3
ttcaccgatt ctgagcgaat cacaggtgag aaatttggat tcgaaataaa cctaaaaaaa 60
ctatccaata aggcttccat aggcttcgta tttcccgacc attccaattg gaaaaattga 120
gcgctgtctt ccatgatctt agataaagcc ttaatacttg gctcatttcc atttgaggtt 180
agctctagca actggtaaag catttgaaga ccagttggat catctctatg ctgcctatag 240
taagtggaaa ataaagtagt aataacgata cctgtcacgt tcgaaacgag attattgttc 300
gaattataaa ggccatgtat aatgtttgat ttgacgtatc ctaagtcgtg gctattgctc 360
ttaattaaat tgtttccccc tagcattgaa tttttcaaca gcataccggc ggtagctcta 420
ttgttctgaa gatcctgtag ggagtagtgt tgcttcaata catcatcaga ttcgccttca 480
attaaaatat aacacaaata attgagaaat tcaggttgca gctggaagtt ctccatagct 540
tccattgcgt tattacgaat ctctggattt ggtgacatac agttctgtaa aagagttgct 600
agttgcaaca catagtcttc ggcgggcttc catgtcgatg ccatctttat tcacttaact 660
actgctaaca attctggaaa ccaaagactg cggaatattc tgatatgtat tactactatt 720
cgctgctctt ctgcataatt aatactgaaa agtttttcat acttttaaac ataacctttt 780
tttaagcaaa actctatgac ccggattaga aaactacgaa aagagggtaa taacataggt 840
gcaggatttc catcgataac gacgccgaca atgagccttg ctgcaacatc caattaggac 900
taataactat cgtaggaatt tctacgtaat aaacttcaac agagcctaaa atttgaaaat 960
aaataatcta gaggggaaac ttaaagaaat tctattcttg tcaataaagt ggaaatctgt 1020
cagatgtcac agtttcttta tttgtgacac atattttcaa cataaattca ggcattagtg 1080
ctgtaagcac aaaaagttgt ggcgatatga atattccaga ttttacttac aagctgcatt 1140
gtagtcttac aattcttttt ttcttttttt ttttttatgg aaaggaccac tcttacataa 1200
ctagaatagc attaagaatc agatttacag ataaagatga cattatttta tatatatatt 1260
gtcactccgt tcaagtcgac aaccaataaa aaatttaaaa aaagccaggc agttaataga 1320
aaaaatatga tatgaatgaa tattccactt tcttttcaca gcttgtctgt aagcggatgc 1380
cgggagcaga caagcccgtc agggcgcgtc agcgggtgtt ggcgggtgtc ggggctggct 1440
taactatgcg gcatcagagc agattgtact gagagtgcac cataaacgac attactatat 1500
atataatata ggaagcattt aatagacagc atcgtaatat atgtgtactt tgcagttatg 1560
acgccagatg gcagtagtgg aagatattct ttattgaaaa atagcttgtc accttacgta 1620
caatcttgat ccggagcttt tctttttttg ccgattaaga attaattcgg tcgaaaaaag 1680
aaaaggagag ggccaagagg gagggcattg gtgactattg agcacgtgag tatacgtgat 1740
taagcacaca aaggcagctt ggagtatgtc tgttattaat ttcacaggta gttctggtcc 1800
attggtgaaa gtttgcggct tgcagagcac agaggccgca gaatgtgctc tagattccga 1860
tgctgacttg ctgggtatta tatgtgtgcc caatagaaag agaacaattg acccggttat 1920
tgcaaggaaa atttcaagtc ttgtaaaagc atataaaaat agttcaggca ctccgaaata 1980
cttggttggc gtgtttcgta atcaacctaa ggaggatgtt ttggctctgg tcaatgatta 2040
cggcattgat atcgtccaac tgcatggaga tgagtcgtgg caagaatacc aagagttcct 2100
cggtttgcca gttattaaaa gactcgtatt tccaaaagac tgcaacatac tactcagtgc 2160
agcttcacag aaacctcatt cgtttattcc cttgtttgat tcagaagcag gtgggacagg 2220
tgaacttttg gattggaact cgatttctga ctgggttgga aggcaagaga gccccgaaag 2280
cttacatttt atgttagctg gtggactgac gccagaaaat gttggtgatg cgcttagatt 2340
aaatggcgtt attggtgttg atgtaagcgg aggtgtggag acaaatggtg taaaagactc 2400
taacaaaata gcaaatttcg tcaaaaatgc taagaaatag gttattactg agtagtattt 2460
atttaagtat tgtttgtgca cttgcctatg cggtgtgaaa taccgcacag atgcgtaagg 2520
agaaaatacc gcatcaggaa attgtaaacg ttaatatttt gttaaaattc gcgttaaatt 2580
tttgttaaat cagctcattt tttaaccaat aggccgaaat cggcaaaatc ccttataaat 2640
caaaagaata gaccgagata gggttgagtg ttgttccagt ttggaacaag agtccactat 2700
taaagaacgt ggactccaac gtcaaagggc gaaaaaccgt ctatcagggc gatggcccac 2760
tacgtgaacc atcaccctaa tcaagttttt tggggtcgag gtgccgtaaa gcactaaatc 2820
ggaaccctaa agggagcccc cgatttagag cttgacgggg aaagccggcg aacgtggcga 2880
gaaaggaagg gaagaaagcg aaaggagcgg gcgctagggc gctggcaagt gtagcggtca 2940
cgctgcgcgt aaccaccaca cccgccgcgc ttaatgcgcc gctacagggc gcgtcgcgcc 3000
attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat 3060
tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt 3120
tttcccagtc acgacgttgt aaaacgacgg ccagtgagcg cgcgtaatac gactcactat 3180
agggcgaatt gggtactaca cggtccaatg gataaacatt ttttatcaac actatgatat 3240
ataaatataa tagtttttcg tatatatatt cctttttttg gtcaattttt gaaattttcg 3300
tagaaaaggg agagacaaaa cacattatat caatgaaaac gtacaaaaag tagataaagt 3360
cagtgcttaa acacgtcttt tccttaaaaa tactttatta tttttatttt attgagaggg 3420
tggtttaaaa atagaaatag agagagaggt acatacataa acatacgcgc acaaaagcag 3480
agattaggat ttaatgcagg tgacggaccc atctttcaaa cgatttatat cagtggcgtc 3540
caaattgtta ggttttgttg gttcagcagg tttcctgttg tgggtcatat gactttgaac 3600
caaatggccg gctgctaggg cagcacataa ggataattca cctgccaaga cggcacaggc 3660
aactattctt gctaattgac gtgcgttggt accaggagcg gtagcatgcg ggcctcttac 3720
acctaataag tccaacatgg caccttgtgg ttctagaaca gtaccaccac cgatggtacc 3780
tacttcgatg gatggcatgg atacggaaat tctcaaatca ccgtccactt ctttcatcaa 3840
tgttatacag ttggaacttt caacattttg tgcaggatct tgtcctaatg ccaagaaaac 3900
agctgtcact aaattagctg catgtgcgtt aaatccacca acagacccag ccattgcaga 3960
tccaaccaaa ttcttagcaa tgttcaactc aaccaatgcg gaaacatcac tttttaacac 4020
ttttctgaca acatcaccag gaatagtagc ttctgcgacg acactcttac cacgaccttc 4080
gatccagttg atggcagctg gttttttgtc ggtacagtag ttaccagaaa cggagacaac 4140
ctccatatct tcccagccat actcttctac catttgcttt aatgagtatt cgacaccttt 4200
agaaatcata ttcataccca ttgcgtcacc agtagttgtt ctaaatctca tgaagagtaa 4260
atctcctgct agacaagttt gaatatgttg cagacgtgca aatcttgatg tagagttaaa 4320
agctttttta attgcgtttt gtccctcttc tgagtctaac catatcttac aggcaccaga 4380
tcttttcaaa gttgggaaac ggactactgg gcctcttgtc ataccatcct tagttaaaac 4440
agttgttgca ccaccgccag cattgattgc cttacagcca cgcatggcag aagctaccaa 4500
acaaccctct gtagttgcca ttggtatatg ataagatgta ccatcgataa ccaaggggcc 4560
tataacacca acgggcaaag gcatgtaacc tataacattt tcacaacaag cgccaaatac 4620
gcggtcgtag tcataatttt tatatggtaa acgatcagat gctaatacag gagcttctgc 4680
caaaattgaa agagccttcc tacgtaccgc aaccgctctc gtagtatcac ctaatttttt 4740
ctccaaagcg tacaaaggta acttaccgtg aataaccaag gcagcgacct ctttgttctt 4800
caattgtttt gtatttccac tacttaataa tgcttctaat tcttctaaag gacgtatttt 4860
cttatccaag ctttcaatat cgcgggaatc atcttcctca ctagatgatg aaggtcctga 4920
tgagctcgat tgcgcagatg ataaactttt gactttcgat ccagaaatga ctgttttatt 4980
ggttaaaacc atttatattg aattttcaaa aattcttact ttttttttgg atggacgcaa 5040
agaagtttaa taatcatatt acatggcaat accaccatat acatatccat atctaatctt 5100
acttatatgt tgtggaaatg taaagagccc cattatctta gcctaaaaaa accttctctt 5160
tggaactttc agtaatacgc ttaactgctc attgctatat tgaagtacgg attagaagcc 5220
gccgagcggg cgacagccct ccgacggaag actctcctcc gtgcgtcctg gtcttcaccg 5280
gtcgcgttcc tgaaacgcag atgtgcctcg cgccgcactg ctccgaacaa taaagattct 5340
acaatactag cttttatggt tatgaagagg aaaaattggc agtaacctgg ccccacaaac 5400
cttcaaatca acgaatcaaa ttaacaacca taggataata atgcgattag ttttttagcc 5460
ttatttctgg ggtaattaat cagcgaagcg atgatttttg atctattaac agatatataa 5520
atgcaaaagc tgcataacca ctttaactaa tactttcaac attttcggtt tgtattactt 5580
cttattcaaa tgtcataaaa gtatcaacaa aaaattgtta atatacctct atactttaac 5640
gtcaaggaga aaaaactata gcgcaatgat tgaatagtca aagatttttt ttttttaatt 5700
tttttttttt catagaactt tttatttaaa taaatcacgt ctatatatgt atcagtataa 5760
cgtaaaaaaa aaaacaccgt cagttaaaca aaacataaat aaaaaaaaaa agaagtgtca 5820
aatcaagtgt caaatgtata cttctttttt ttactttgtt cagaacaact tctcattttt 5880
ttctactcat aactttagca tcacaaagta cacaataata acgagtagta acacttttat 5940
agttcataca tgcttcaact acttaataaa tgattgtatg ataatgtttt caatgtaaga 6000
gatttcgatt atccacaaac tttgaaacac agggacacaa ttcttgatat gctttcaacc 6060
gctgcgtttt ggatacctat tcttgacata atatgactac cattttgtta ttgtacgtgg 6120
ggcagttgac gtcttatcat atgtcaaagt catttgcgaa gttcttggca agttgccaac 6180
tgacgagatg cagtaaaaag agattgccgt cttgaaactt tttgtccttt tttttttccg 6240
gggactctac gagaaccctt tgtcctactg attaattttg tactgaattt ggacaattca 6300
gattttagta gacaagcgcg aggaggaaaa gaaatgacag aaaaattccg atggacaaga 6360
agataggaaa aaaaaaaagc tttcaccgat ttcctagacc ggaaaaaagt cgtatgacat 6420
cagaatgaaa aattttcaag ttagacaagg acaaaatcag gacaaattgt aaagatatga 6480
taaatcattt gattcagcgc caatttgccc ttttccattt tccattaaat ctctgttctc 6540
tcttacttat atgatgatta ggtatcatct gtataaaact cctttcttaa tttcactcta 6600
aagcataccc catagagaag atctttcggt tcgaagacat tcctacgcat aataagaata 6660
ggagggaata atgccagaca atctatcatt acatttaagc ggctcttcaa aaagattgaa 6720
ctctcgccaa cttatggaat cttccaatga gacctttgcg ccaaataatg tggatttgga 6780
aaaagagtat aagtcatctc agagtaatat aactaccgaa gtttatgagg catcgagctt 6840
tgaagaaaaa gtaagctcag aaaaacctca atacagctca ttctggaaga aaatctatta 6900
tgaatatgtg gtcgttgaca aatcaatctt gggtgtttct attctggatt catttatgta 6960
caaccaggac ttgaagcccg tcgaaaaaga aaggcgggtt tggtcctggt acaattattg 7020
ttacttctgg cttgctgaat gtttcaatat caacacttgg caaattgcag ctacaggtct 7080
acaactgggt ctaaattggt ggcagtgttg gataacaatt tggattgggt acggtttcgt 7140
tggtgctttt gttgttttgg cctctagagt tggatctgct tatcatttgt cattccctat 7200
atcatctaga gcatcattcg gtattttctt ctctttatgg cctgttatta atagagtcgt 7260
catggccatc gtttggtata gtgtccaagc ttatattgcg gcaactcccg tatcattaa 7319
<210> 4
<211> 457
<212> DNA
<213> Artificial
<220>
<223> nucleotide sequence possessed by PGAL1
<400> 4
agtacggatt agaagccgcc gagcgggcga cagccctccg acggaagact ctcctccgtg 60
cgtcctggtc ttcaccggtc gcgttcctga aacgcagatg tgcctcgcgc cgcactgctc 120
cgaacaataa agattctaca atactagctt ttatggttat gaagaggaaa aattggcagt 180
aacctggccc cacaaacctt caaatcaacg aatcaaatta acaaccatag gataataatg 240
cgattagttt tttagcctta tttctggggt aattaatcag cgaagcgatg atttttgatc 300
tattaacaga tatataaatg caaaagctgc ataaccactt taactaatac tttcaacatt 360
ttcggtttgt attacttctt attcaaatgt cataaaagta tcaacaaaaa attgttaata 420
tacctctata ctttaacgtc aaggagaaaa aactata 457
<210> 5
<211> 500
<212> DNA
<213> Artificial
<220>
<223> nucleotide sequence possessed by PGAL7
<400> 5
gacggtagca acaagaatat agcacgagcc gcgaagttca tttcgttact tttgatatcg 60
ctcacaacta ttgcgaagcg cttcagtgaa aaaatcataa ggaaaagttg taaatattat 120
tggtagtatt cgtttggtaa agtagagggg gtaatttttc ccctttattt tgttcataca 180
ttcttaaatt gctttgcctc tccttttgga aagctatact tcggagcact gttgagcgaa 240
ggctcattag atatattttc tgtcattttc cttaacccaa aaataaggga aagggtccaa 300
aaagcgctcg gacaactgtt gaccgtgatc cgaaggactg gctatacagt gttcacaaaa 360
tagccaagct gaaaataatg tgtagctatg ttcagttagt ttggctagca aagatataaa 420
agcaggtcgg aaatatttat gggcattatt atgcagagca tcaacatgat aaaaaaaaac 480
agttgaatat tccctcaaaa 500
<210> 6
<211> 215
<212> DNA
<213> Artificial
<220>
<223> nucleotide sequence possessed by PGAL10
<400> 6
tacttcaata tagcaatgag cagttaagcg tattactgaa agttccaaag agaaggtttt 60
tttaggctaa gataatgggg ctctttacat ttccacaaca tataagtaag attagatatg 120
gatatgtata tggtggtatt gccatgtaat atgattatta aacttctttg cgtccatcca 180
aaaaaaaagt aagaattttt gaaaattcaa tataa 215
<210> 7
<211> 668
<212> DNA
<213> Artificial
<220>
<223> nucleotide sequence possessed by PGAL1-10
<400> 7
tatagttttt tctccttgac gttaaagtat agaggtatat taacaatttt ttgttgatac 60
ttttatgaca tttgaataag aagtaataca aaccgaaaat gttgaaagta ttagttaaag 120
tggttatgca gcttttgcat ttatatatct gttaatagat caaaaatcat cgcttcgctg 180
attaattacc ccagaaataa ggctaaaaaa ctaatcgcat tattatccta tggttgttaa 240
tttgattcgt tgatttgaag gtttgtgggg ccaggttact gccaattttt cctcttcata 300
accataaaag ctagtattgt agaatcttta ttgttcggag cagtgcggcg cgaggcacat 360
ctgcgtttca ggaacgcgac cggtgaagac caggacgcac ggaggagagt cttccgtcgg 420
agggctgtcg cccgctcggc ggcttctaat ccgtacttca atatagcaat gagcagttaa 480
gcgtattact gaaagttcca aagagaaggt ttttttaggc taagataatg gggctcttta 540
catttccaca acatataagt aagattagat atggatatgt atatggtggt attgccatgt 600
aatatgatta ttaaacttct ttgcgtccat ccaaaaaaaa agtaagaatt tttgaaaatt 660
caatataa 668
<210> 8
<211> 668
<212> DNA
<213> Artificial
<220>
<223> nucleotide sequence possessed by PGAL10-1
<400> 8
ttatattgaa ttttcaaaaa ttcttacttt ttttttggat ggacgcaaag aagtttaata 60
atcatattac atggcaatac caccatatac atatccatat ctaatcttac ttatatgttg 120
tggaaatgta aagagcccca ttatcttagc ctaaaaaaac cttctctttg gaactttcag 180
taatacgctt aactgctcat tgctatattg aagtacggat tagaagccgc cgagcgggcg 240
acagccctcc gacggaagac tctcctccgt gcgtcctggt cttcaccggt cgcgttcctg 300
aaacgcagat gtgcctcgcg ccgcactgct ccgaacaata aagattctac aatactagct 360
tttatggtta tgaagaggaa aaattggcag taacctggcc ccacaaacct tcaaatcaac 420
gaatcaaatt aacaaccata ggataataat gcgattagtt ttttagcctt atttctgggg 480
taattaatca gcgaagcgat gatttttgat ctattaacag atatataaat gcaaaagctg 540
cataaccact ttaactaata ctttcaacat tttcggtttg tattacttct tattcaaatg 600
tcataaaagt atcaacaaaa aattgttaat atacctctat actttaacgt caaggagaaa 660
aaactata 668
<210> 9
<211> 1123
<212> DNA
<213> Artificial
<220>
<223> nucleotide sequence possessed by P HXT1
<400> 9
gattttacgt atatcaacta gttgacgatt atgatatctt tatagatttt aaggtgggga 60
aagaacatga gaccccagat ggaattgatt atggggacat tgttgccttt atatataatt 120
tcaatatact aattcaaatg attaaaaacg tgagggggac acgcaacttc gggtgttaag 180
aaatattttg ctacattaga taatggtgga gtttcctggc ttgtcggata aaagccatca 240
aatgtcgcag cagctcatgt ttacgtttgc tgtcttctgc ccacgtcata tgagtggtat 300
tcttctatca gcacttgatg aatattcttt ttctcatata tctgaaagac aaaagatcgg 360
cacggcaatg ccctgcagca tttcttccta gtttttccga atttccatta cgtattggat 420
cttgtgcgca tatttgtcag tccttcacgg aaaaaaaaaa agagcactgg gtcacttcgg 480
aaaaactttt gactcaatgc aacagtgtca taatcctttg cgctgtctct ttgaagaaaa 540
atcaggagtg caagatatcg attaattcct tggaagttat gatggttagt cttagtttaa 600
ctctcttgaa gaagggtttt ttcagttggt caacactctt tagaggtaaa aaaaaaaaaa 660
aaaaaaaaaa aaagagaatt cttcatgtaa tttaccatga ttctacgttt ttgcaagcaa 720
aaatgaagat aatccgagcg catgcgcaag tagtccctgc catgccgctt cggaaaaact 780
ttcgaaacca atactcctta catccgcctt gaatttagga cctacaattg ttgtctcttc 840
tgcttgaacc aataagccct agaaacctta caccctaatt tgcacaagaa aactacgaaa 900
aaattttttc ctcttttttc cgcagctcca atctcgccct gctatttttt tggttcggag 960
aaaaatagtc ggtcgatatg aattgaagtt tcattgtggc cactcgttta ggaatggaat 1020
aacctccgcg aatggaaaaa aagaactcgg aatatcaaaa attccggctc aaaggaaaaa 1080
tttgcttcag gaggggggaa ttatattcca gatgagacct gca 1123
<210> 10
<211> 1749
<212> DNA
<213> Artificial
<220>
<223> nucleotide sequence possessed by BtCrtI
<400> 10
ttatatccta atatcgttag agttctgtcc ttggaagacg tttggcaaca actgattaat 60
aaaagatgct ggagtagtag tgtcgtccct aggaaagaaa tagaagaaca agaaagtgac 120
aaagtagcat gctaagcagt aataaatcca gtgtgactca gtcttccttg tttgctcagg 180
tgcatatttc ttctgagagt cctgtaactt cctaggcaaa gggttttgtc cgaatgactt 240
acagacctgg tcagaagtta atttagatcc tgccaagact attgggactc ctgtaccagg 300
gtgagttgat gcaccgacga aaaataaatt gtcatatctg ttagtagagt cctttgttga 360
aggtctgaac cacaaaacct gaaagacatc atgtgataaa cctaatattg atcccctcca 420
caagttgaac ttagattgcc agactgatgg gtcgttgacc tcctcgtgtt ctatcaagtt 480
agcgaagttg ttgactccca acctcctctc tatgacctcc aagaccatct ttctagccct 540
gttgaccaac tctgggtagt tctcctctgc tgagttacct gtcttagact tcatgtgacc 600
aattggaact aaaactataa ttgagtcctt atttggaggt gctgcagact cgtctatcct 660
tgaaggaacg ttgacgtaga aagatgcttc agaaggcaaa ccgaaatcgt taaaaatctc 720
gtcgaatgac tccttgtaag cctcagccaa gaaaatgttg tggacgtcca attgagggac 780
cttagtagac attgaccagt aaaaagaaat tgatgaagat gttaatttct tagatgccaa 840
agtcttcttt gtccagttgc aaggtggcaa caagtggtgg taagcgtaga ccaagtcagc 900
gttgcagacg acagcgtctg cctcaatgac ttctccagac tccaaagtga caccagtgac 960
cctcttgtct ttatcgacag tgttaatctt agcgacagga gattggtacc tgaactcagc 1020
tccgtacttc ttagaagcta tagactccaa cttttggacg accatgttga aaccacccct 1080
tgggtaccaa attccctctg cgaactctgt atattgcaac aatgagtaga ctgcaggtgc 1140
gtcgtaaggt gacataccca tgtacattgt ttgaaaagta aaagccatcc tcatcttttt 1200
tgtttggaag tattttgatg ctctgtcgta tatcttaccg aataagtgca acctaaaaat 1260
ctctgggacg tactgtaacc ttattaagtc ccatatagtt tcgaagtttc tctttatagc 1320
aatgaatgta ccctgctcgt agtggacgtg tgtctctttc atgaaatcta agaacctacc 1380
gaatcctaaa ggtccctcaa tcctgtccaa ctcacccttc atctttgtta agtctgatga 1440
caattggaca gcgtcaccgt cgtcgaaatg gactttgtaa ttgttgtcac atcttaataa 1500
gtccaagtgg tctcctatcc tctcgtctaa gtcagcgaaa gcgtcctcaa acaacttagg 1560
catcaagtac aatgaaggtc cctggtcgaa cctgtgaccg tcgtggtgaa tgaaagagca 1620
ccttcctcca gagaagtcgt tcttctcgac gacagtgact ctgaaaccct cccttgctaa 1680
ccttgctgct gttgcagtac ctcctattcc agcacctatg acgacaatgt gcttcttctg 1740
atcagacat 1749
<210> 11
<211> 930
<212> DNA
<213> Artificial
<220>
<223> nucleotide sequence possessed by PagCrtB
<400> 11
atgtcacaac caccattatt ggaccacgct acacaaacta tggcaaacgg ttctaaatct 60
ttcgctactg ctgctaaatt attcgaccca gcaacaagaa gatctgtatt gatgttgtac 120
acctggtgta gacattgcga tgacgttata gatgaccaaa ctcacggttt tgcttcagaa 180
gctgcagccg aagaagaagc tacacaaaga ttggcaagat taagaacttt gacattagct 240
gcattcgaag gtgccgaaat gcaagatcca gcttttgccg ctttccaaga agttgcatta 300
acccatggta ttactcctag aatggctttg gatcacttag acggttttgc aatggatgtc 360
gcccaaacaa gatacgtaac cttcgaagac actttaagat attgttacca tgtcgccggt 420
gttgtcggtt tgatgatggc tagagtaatg ggtgttagag atgaaagagt tttagataga 480
gcatgtgact tgggtttagc cttccaattg acaaacatag ctagagatat aatagatgac 540
gcagccatag acagatgcta tttgccagct gaatggttac aagatgcagg tttgactcct 600
gaaaattacg ctgcaagaga aaacagagcc gctttagcca gagttgctga aagattgata 660
gatgcagccg aaccatatta catctcttca caagctggtt tgcatgattt gccacctaga 720
tgcgcatggg ccattgctac cgcaagatct gtttacagag aaatcggtat taaagtcaag 780
gctgcaggtg gttccgcatg ggatagaaga caacacactt ctaaaggtga aaagatcgct 840
atgttgatgg ccgctcctgg tcaagttatt agagcaaaga ccaccagagt caccccaaga 900
ccagccggtt tatggcaaag acctgtttaa 930
<210> 12
<211> 909
<212> DNA
<213> Artificial
<220>
<223> nucleotide sequence possessed by PaCrtE
<400> 12
atgactgttt gtgctaagaa acatgttcat ttgacaagag atgctgcaga acaattgttg 60
gcagatatcg atagaagatt ggatcaattg ttaccagttg aaggtgaaag agatgttgtt 120
ggtgctgcaa tgagagaagg tgctttggca ccaggtaaaa gaatcagacc aatgttgttg 180
ttgttgactg ctagagattt gggttgtgca gtttctcatg atggtttgtt agatttggct 240
tgtgcagttg aaatggttca tgctgcatca ttgatcttgg atgatatgcc atgtatggat 300
gatgctaaat tgagaagagg tagaccaact attcattctc attatggtga acatgttgct 360
attttagctg cagttgcttt gttatcaaaa gcatttggtg ttattgctga tgcagatggt 420
ttgacaccat tggctaaaaa tagagcagtt tctgaattgt caaacgctat cggtatgcaa 480
ggtttggttc aaggtcaatt caaagatttg tctgaaggtg acaaaccaag atcagctgaa 540
gcaattttga tgactaacca tttcaagact tctacattat tttgtgcttc tatgcaaatg 600
gcatcaattg ttgctaatgc atcttcagaa gctagagatt gtttgcatag attttcattg 660
gatttgggtc aagcatttca attgttagat gatttgactg atggtatgac tgatactggt 720
aaagattcta atcaagatgc tggtaaatca acattggtta atttgttagg tccaagagct 780
gttgaagaaa gattgagaca acatttgcaa ttagcatctg aacatttgtc agctgcatgt 840
caacatggtc atgctacaca acatttcatc caagcatggt tcgataagaa attagctgca 900
gtttcttaa 909
<210> 13
<211> 915
<212> DNA
<213> Artificial
<220>
<223> nucleotide sequence possessed by INO2 gene
<400> 13
atgtcccaag caactgggaa cgaattactg ggtatcctag atctggataa cgatatagac 60
tttgaaactg cttaccaaat gctcagcagt aacttcgacg accaaatgtc tgcgcacata 120
catgaaaaca cgtttagtgc aacttcccct cctctgttaa cacacgagct cggcataatt 180
cctaacgtgg caaccgtgca accctctcac gtagaaacta tacctgccga taaccaaact 240
catcatgctc ctttgcatac tcatgctcac tatctaaatc acaaccctca tcaaccaagc 300
atgggttttg atcaaacgct tggtctcaag ttgtctcctt ccagttcggg gttgttgagc 360
acgaatgaat cgaatgccat tgaacagttt ttagacaatc taatatcaca ggatatgatg 420
tcttccaacg cttccatgaa ctccgattca catctacata taagatcacc aaaaaagcag 480
cataggtata ccgaattaaa tcaaagatat cctgaaacac atccacacag taacacaggg 540
gagttaccca caaacacagc agatgtgcca actgagttca ccacgaggga aggacctcat 600
cagcctatcg gcaatgacca ctacaacccg ccaccgtttt cagtacctga gatacgaatc 660
ccagactctg atattccagc caatatcgag gacgaccctg tgaaggtacg gaaatggaaa 720
cacgttcaaa tggagaagat acgaagaata aacaccaaag aagcctttga aaggctcatt 780
aaatcagtaa ggaccccacc aaaggaaaac gggaaaagaa ttcccaagca tattctttta 840
acttgtgtaa tgaacgatat caagtccatt agaagcgcaa atgaagcact acagcacata 900
ctggatgatt cctga 915
<210> 14
<211> 1428
<212> DNA
<213> Artificial
<220>
<223> nucleotide sequence possessed by gapN Gene
<400> 14
atgactaagc aatacaagaa ctacgttaac ggtgaatgga agttgtctga aaacgaaatt 60
aaaatctatg aacctgcttc aggtgcagaa ttaggttccg ttccagctat gagtacagaa 120
gaagtagatt acgtttacgc ttctgctaag aaagcccaac ctgcttggag agcattgtca 180
tatatcgaaa gagctgcata cttacataag gttgctgata tattgatgag agacaaggaa 240
aagattggtg caatcttatc taaagaagta gccaagggtt ataaatccgc tgttagtgaa 300
gttgtcagaa ccgctgaaat tataaactac gccgctgaag aaggtttaag aatggaaggt 360
gaagttttgg aaggtggttc tttcgaagca gcctctaaaa agaaaattgc agtagttaga 420
agagaacctg tcggtttagt attggccatt tcccctttca attatccagt aaacttagct 480
ggtagtaaga tcgccccagc tttgattgct ggtaatgtta tagcctttaa accacctaca 540
caaggttcta tttcaggttt gttattggca gaagccttcg ctgaagcagg tttgcctgct 600
ggtgttttta acacaattac cggtagaggt tccgaaatcg gtgactacat tgtcgaacac 660
caagctgtaa acttcatcaa cttcactggt tctacaggta taggtgaaag aatcggtaaa 720
atggctggta tgagaccaat tatgttagaa ttgggtggta aagattctgc aatagtttta 780
gaagatgccg acttagaatt gaccgctaag aatatcattg ccggtgcttt tggttattcc 840
ggtcaaagat gtactgcagt caaaagagtt ttagtcatgg aaagtgttgc cgatgaattg 900
gtcgaaaaga taagagaaaa ggttttggca ttgactatcg gtaatcctga agatgacgcc 960
gacatcactc cattgatcga tacaaagtca gctgactacg ttgaaggttt gattaatgat 1020
gcaaacgaca agggtgcaac cgccttgact gaaattaaaa gagagggtaa cttaatctgc 1080
ccaatcttgt tcgataaggt tactacagac atgagattag cttgggaaga accatttggt 1140
cctgtcttgc caataatcag agttacatct gtcgaagaag ctatcgaaat atctaataag 1200
tcagaatatg gtttgcaagc atcaatcttt acaaacgatt tccctagagc ctttggtatt 1260
gctgaacaat tggaagtagg taccgttcat atcaacaaca agacacaaag aggtaccgat 1320
aattttccat tcttgggtgc taagaaatct ggtgcaggta tccaaggtgt caaatactcc 1380
attgaagcta tgaccactgt aaagagtgtc gtatttgata ttaaataa 1428
<210> 15
<211> 537
<212> DNA
<213> Artificial
<220>
<223> nucleotide sequence possessed by PYC2 gene
<400> 15
atgtatccaa gggtctatga agatttccaa aagatcagag aaacatacgg tgatttatca 60
gttctaccaa ccaaaaattt cctagcacca gcagaacctg atgaagaaat cgaagtcacc 120
atcgaacaag gtaagacttt gattatcaaa ttgcaagctg ttggtgactt aaataagaaa 180
actgggcaaa gagaagtgta ttttgaattg aacggtgaat taagaaagat cagagttgca 240
gacaagtcac aaaacataca atctgttgct aaaccaaagg ctgatgtcca cgatactcac 300
caaatcggtg caccaatggc tggtgttatc atagaagtta aagtacataa agggtctttg 360
gtgaaaaagg gcgaatcgat tgctgttttg agtgccatga aaatggaaat ggttgtctct 420
tcaccagcag atggtcaagt taaagatgtt ttcattaggg atggtgaaag tgttgacgca 480
tcagatttgt tggttgtcct agaagaagaa accctacccc catcccaaaa aaagtaa 537
<210> 16
<211> 1923
<212> DNA
<213> Artificial
<220>
<223> nucleotide sequence possessed by SMAE1 gene
<400> 16
atgtggccta ttcagcaatc gcgtttatat tcttctaaca ctagatcgca taaagctacc 60
acaacaagag aaaatacttt ccaaaagcca tacagcgacg aggaggtcac taaaacaccc 120
gtcggttctc gcgccagaaa gatcttcgaa gctcctcacc cacatgccac tcgtttgact 180
gtagaaggtg ccatagaatg tcccttggag agctttcaac ttttaaactc tcctttattt 240
aacaagggtt ctgcatttac acaagaagaa agggaagcgt ttaatttaga agcattgcta 300
ccaccacaag tgaacacttt ggacgaacaa ctggaaagaa gctacaagca gttatgctat 360
ttgaagacgc ccttggccaa aaacgacttc atgacgtctt tgagagtaca gaacaaagtc 420
ctatattttg cattaataag gaaacatatc aaggaattag ttcctatcat ttacacccca 480
accgaaggtg atgctattgc tgcctattcc cacaggttca gaaagccaga aggtgtgttt 540
ttagacatta ccgaacctga ttccatcgaa cgtagattgg ctacatacgg tggagacaaa 600
gatgtagact acatcgttgt gtcggattcg gaaggtattc tgggaattgg tgaccaaggt 660
atcggtggtg tacgtattgc tatctccaaa ttggcattga tgacgctgtg cggtggtatt 720
catcccggcc gtgtgctacc tgtgtgtttg gacgtcggta ctaacaacaa gaaactagcc 780
cgtgacgaat tgtacatggg taacaagttc tccagaatca ggggtaagca atatgacgac 840
ttcttggaaa aattcatcaa ggccgttaag aaagtgtatc caagcgccgt tctgcatttc 900
gaagatttcg gtgttaagaa cgctagaaga ttgctagaaa agtacaggta cgaattgcca 960
tcattcaacg atgacattca gggcaccggt gccgtcgtga tggcctcgtt gattgctgct 1020
ttgaaacata ccaacagaga cttgaaagac accagagtgc ttatttacgg tgccgggtct 1080
gcgggcctcg gtatcgcaga ccaaattgtg aatcatatgg tcacgcacgg cgttgacaag 1140
gaagaagcgc gcaagaaaat cttcttgatg gacagacgtg ggttaattct acaatcttac 1200
gaggctaact ccactcccgc ccaacacgta tacgctaaga gtgatgcgga atgggctggt 1260
atcaacaccc gctctttaca tgatgtggtg gagaacgtca aaccaacgtg tttggttggc 1320
tgctccacac aagcaggcgc attcactcaa gatgtcgtag aagaaatgca caagcacaat 1380
cctagaccga tcattttccc attatccaac cctactagac tacacgaagc cgttcctgcc 1440
gatttaatga agtggaccaa caacaacgct cttgtagcta ccggatctcc tttcccacct 1500
gttgacggtt accgtatctc ggagaacaac aattgttact ctttcccagg tatcggttta 1560
ggtgccgtac tatcgcgtgc caccactatc acagacaaga tgatctccgc tgcagtggac 1620
caactagccg aattgtcgcc actaagagag ggcgactcga gacctgggtt gctacccggc 1680
ctggacacca tcaccaacac ctctgcgcgt ctagctaccg ctgtgatctt gcaagcactc 1740
gaggagggaa ccgcccgtat cgagcaagaa caagtaccgg gaggagctcc cggcgaaact 1800
gtcaaggttc ctcgtgactt tgacgaatgt ttacagtggg tcaaagccca aatgtgggag 1860
cctgtgtaca gacctatgat caaggtccaa catgacccat cggtgcacac caaccaattg 1920
tag 1923
<210> 17
<211> 1134
<212> DNA
<213> Artificial
<220>
<223> nucleotide sequence possessed by MDH2 gene
<400> 17
atgcctcact cagttacacc atccatagaa caagattcgt taaaaattgc cattttaggt 60
gctgccggtg gtatcgggca gtcgttatcg ctgcttttga aagctcagtt gcaataccag 120
ttaaaggaga gcaaccggag cgttacccac attcatctgg ctctttacga tgtcaaccaa 180
gaagccatca acggtgttac cgccgacttg tctcatatag acacccccat ttccgtgtcg 240
agccactctc ctgcaggtgg cattgagaac tgtttgcata acgcttctat tgttgtcatt 300
cctgcaggtg ttccaagaaa acctggcatg actcgtgatg acttatttaa cgtgaatgct 360
ggtatcatta gccagctcgg tgattctatt gcagaatgtt gtgatctttc caaggtcttc 420
gttcttgtca tttccaaccc tgttaattct ttagtcccag tgatggtttc taacattctt 480
aagaaccatc ctcagtctag aaattccggc attgaaagaa ggatcatggg tgtcaccaag 540
ctcgacattg tcagagcgtc cacttttcta cgtgagataa acattgagtc agggctaact 600
cctcgtgtta actccatgcc tgacgtccct gtaattggcg ggcattctgg cgagactatt 660
attccgttgt tttcacagtc aaacttccta tcgagattaa atgaggatca attgaaatat 720
ttaatacata gagtccaata cggtggtgat gaagtggtca aggccaagaa cggtaaaggt 780
agtgctacct tatcgatggc ccatgccggt tataagtgtg ttgtccaatt tgtttctttg 840
ttattgggta acattgagca gatccatgga acctactatg tgccattaaa agatgcgaac 900
aacttcccca ttgctcctgg ggcagatcaa ttattgcctc tggtggacgg tgcagactac 960
tttgccatac cattaactat tactacaaag ggtgtttcct atgtggatta tgacatcgtt 1020
aataggatga acgacatgga acgcaaccaa atgttgccaa tttgcgtctc ccagttaaag 1080
aaaaatatcg ataagggctt ggaattcgtt gcatcgagat ctgcatcatc ttaa 1134
<210> 18
<211> 1245
<212> DNA
<213> Artificial
<220>
<223> nucleotide sequence possessed by POS5 gene
<400> 18
atgtttgtca gggttaaatt gaataaacca gtaaaatggt ataggttcta tagtacgttg 60
gattcacatt ccctaaagtt acagagcggc tcgaagtttg taaaaataaa gccagtaaat 120
aacttgagga gtagttcatc agcagatttc gtgtccccac caaattccaa attacaatct 180
ttaatctggc agaacccttt acaaaatgtt tatataacta aaaaaccatg gactccatcc 240
acaagagaag cgatggttga attcataact catttacatg agtcataccc cgaggtgaac 300
gtcattgttc aacccgatgt ggcagaagaa atttcccagg atttcaaatc tcctttggag 360
aatgatccca accgacctca tatactttat actggtcctg aacaagatat cgtaaacaga 420
acagacttat tggtgacatt gggaggtgat gggactattt tacacggcgt atcaatgttc 480
ggaaatacgc aagttcctcc ggttttagca tttgctctgg gcactctggg ctttctatta 540
ccgtttgatt ttaaggagca taaaaaggtc tttcaggaag taatcagctc tagagccaaa 600
tgtttgcata gaacacggct agaatgtcat ttgaaaaaaa aggatagcaa ctcatctatt 660
gtgacccatg ctatgaatga catattctta cataggggta attcccctca tctcactaac 720
ctggacattt tcattgatgg ggaatttttg acaagaacga cagcagatgg tgttgcattg 780
gccactccaa cgggttccac agcatattca ttatcagcag gtggatctat tgtttcccca 840
ttagtccctg ctattttaat gacaccaatt tgtcctcgct ctttgtcatt ccgaccactg 900
attttgcctc attcatccca cattaggata aagataggtt ccaaattgaa ccaaaaacca 960
gtcaacagtg tggtaaaact ttctgttgat ggtattcctc aacaggattt agatgttggt 1020
gatgaaattt atgttataaa tgaggtcggc actatataca tagatggtac tcagcttccg 1080
acgacaagaa aaactgaaaa tgactttaat aattcaaaaa agcctaaaag gtcagggatt 1140
tattgtgtcg ccaagaccga gaatgactgg attagaggaa tcaatgaact tttaggattc 1200
aattctagct ttaggctgac caagagacag actgataatg attaa 1245
<210> 19
<211> 1533
<212> DNA
<213> Artificial
<220>
<223> nucleotide sequence possessed by pntA gene
<400> 19
atgcgaattg gcataccaag agaacggtta accaatgaaa cccgtgttgc agcaacgcca 60
aaaacagtgg aacagctgct gaaactgggt tttaccgtcg cggtagagag cggcgcgggt 120
caactggcaa gttttgacga taaagcgttt gtgcaagcgg gcgctgaaat tgtagaaggg 180
aatagcgtct ggcagtcaga gatcattctg aaggtcaatg cgccgttaga tgatgaaatt 240
gcgttactga atcctgggac aacgctggtg agttttatct ggcctgcgca gaatccggaa 300
ttaatgcaaa aacttgcgga acgtaacgtg accgtgatgg cgatggactc tgtgccgcgt 360
atctcacgcg cacaatcgct ggacgcacta agctcgatgg cgaacatcgc cggttatcgc 420
gccattgttg aagcggcaca tgaatttggg cgcttcttta ccgggcaaat tactgcggcc 480
gggaaagtgc caccggcaaa agtgatggtg attggtgcgg gtgttgcagg tctggccgcc 540
attggcgcag caaacagtct cggcgcgatt gtgcgtgcat tcgacacccg cccggaagtg 600
aaagaacaag ttcaaagtat gggcgcggaa ttcctcgagc tggattttaa agaggaagct 660
ggcagcggcg atggctatgc caaagtgatg tcggacgcgt tcatcaaagc ggaaatggaa 720
ctctttgccg cccaggcaaa agaggtcgat atcattgtca ccaccgcgct tattccaggc 780
aaaccagcgc cgaagctaat tacccgtgaa atggttgact ccatgaaggc gggcagtgtg 840
attgtcgacc tggcagccca aaacggcggc aactgtgaat acaccgtgcc gggtgaaatc 900
ttcactacgg aaaatggtgt caaagtgatt ggttataccg atcttccggg ccgtctgccg 960
acgcaatcct cacagcttta cggcacaaac ctcgttaatc tgctgaaact gttgtgcaaa 1020
gagaaagacg gcaatatcac tgttgatttt gatgatgtgg tgattcgcgg cgtgaccgtg 1080
atccgtgcgg gcgaaattac ctggccggca ccgccgattc aggtatcagc tcagccgcag 1140
gcggcacaaa aagcggcacc ggaagtgaaa actgaggaaa aatgtacctg ctcaccgtgg 1200
cgtaaatacg cgttgatggc gctggcaatc attctttttg gctggatggc aagcgttgcg 1260
ccgaaagaat tccttgggca cttcaccgtt ttcgcgctgg cctgcgttgt cggttattac 1320
gtggtgtgga atgtatcgca cgcgctgcat acaccgttga tgtcggtcac caacgcgatt 1380
tcagggatta ttgttgtcgg agcactgttg cagattggcc agggcggctg ggttagcttc 1440
cttagtttta tcgcggtgct tatagccagc attaatattt tcggtggctt caccgtgact 1500
cagcgcatgc tgaaaatgtt ccgcaaaaat taa 1533
<210> 20
<211> 1389
<212> DNA
<213> Artificial
<220>
<223> nucleotide sequence possessed by pntB gene
<400> 20
atgtctggag gattagttac agctgcatac attgttgccg cgatcctgtt tatcttcagt 60
ctggccggtc tttcgaaaca tgaaacgtct cgccagggta acaacttcgg tatcgccggg 120
atggcgattg cgttaatcgc aaccattttt ggaccggata cgggtaatgt tggctggatc 180
ttgctggcga tggtcattgg tggggcaatt ggtatccgtc tggcgaagaa agttgaaatg 240
accgaaatgc cagaactggt ggcgatcctg catagcttcg tgggtctggc ggcagtgctg 300
gttggcttta acagctatct gcatcatgac gcgggaatgg caccgattct ggtcaatatt 360
cacctgacgg aagtgttcct cggtatcttc atcggggcgg taacgttcac gggttcggtg 420
gtggcgttcg gcaaactgtg tggcaagatt tcgtctaaac cattgatgct gccaaaccgt 480
cacaaaatga acctggcggc tctggtcgtt tccttcctgc tgctgattgt atttgttcgc 540
acggacagcg tcggcctgca agtgctggca ttgctgataa tgaccgcaat tgcgctggta 600
ttcggctggc atttagtcgc ctccatcggt ggtgcagata tgccagtggt ggtgtcgatg 660
ctgaactcgt actccggctg ggcggctgcg gctgcgggct ttatgctcag caacgacctg 720
ctgattgtga ccggtgcgct ggtcggttct tcgggggcta tcctttctta cattatgtgt 780
aaggcgatga accgttcctt tatcagcgtt attgcgggtg gtttcggcac cgacggctct 840
tctactggcg atgatcagga agtgggtgag caccgcgaaa tcaccgcaga agagacagcg 900
gaactgctga aaaactccca ttcagtgatc attactccgg ggtacggcat ggcagtcgcg 960
caggcgcaat atcctgtcgc tgaaattact gagaaattgc gcgctcgtgg tattaatgtg 1020
cgtttcggta tccacccggt cgcggggcgt ttgcctggac atatgaacgt attgctggct 1080
gaagcaaaag taccgtatga catcgtgctg gaaatggacg agatcaatga tgactttgct 1140
gataccgata ccgtactggt gattggtgct aacgatacgg ttaacccggc ggcgcaggat 1200
gatccgaaga gtccgattgc tggtatgcct gtgctggaag tgtggaaagc gcagaacgtg 1260
attgtcttta aacgttcgat gaacactggc tatgctggtg tgcaaaaccc gctgttcttc 1320
aaggaaaaca cccacatgct gtttggtgac gccaaagcca gcgtggatgc aatcctgaaa 1380
gctctgtaa 1389
<210> 21
<211> 1047
<212> DNA
<213> Artificial
<220>
<223> nucleotide sequence possessed by ADH2 Gene
<400> 21
atgtctattc cagaaactca aaaagccatt atcttctacg aatccaacgg caagttggag 60
cataaggata tcccagttcc aaagccaaag cccaacgaat tgttaatcaa cgtcaagtac 120
tctggtgtct gccacaccga tttgcacgct tggcatggtg actggccatt gccaactaag 180
ttaccattag ttggtggtca cgaaggtgcc ggtgtcgttg tcggcatggg tgaaaacgtt 240
aagggctgga agatcggtga ctacgccggt atcaaatggt tgaacggttc ttgtatggcc 300
tgtgaatact gtgaattggg taacgaatcc aactgtcctc acgctgactt gtctggttac 360
acccacgacg gttctttcca agaatacgct accgctgacg ctgttcaagc cgctcacatt 420
cctcaaggta ctgacttggc tgaagtcgcg ccaatcttgt gtgctggtat caccgtatac 480
aaggctttga agtctgccaa cttgagagca ggccactggg cggccatttc tggtgctgct 540
ggtggtctag gttctttggc tgttcaatat gctaaggcga tgggttacag agtcttaggt 600
attgatggtg gtccaggaaa ggaagaattg tttacctcgc tcggtggtga agtattcatc 660
gacttcacca aagagaagga cattgttagc gcagtcgtta aggctaccaa cggcggtgcc 720
cacggtatca tcaatgtttc cgtttccgaa gccgctatcg aagcttctac cagatactgt 780
agggcgaacg gtactgttgt cttggttggt ttgccagccg gtgcaaagtg ctcctctgat 840
gtcttcaacc acgttgtcaa gtctatctcc attgtcggct cttacgtggg gaacagagct 900
gataccagag aagccttaga tttctttgcc agaggtctag tcaagtctcc aataaaggta 960
gttggcttat ccagtttacc agaaatttac gaaaagatgg agaagggcca aattgctggt 1020
agatacgttg ttgacacttc taaataa 1047
<210> 22
<211> 1959
<212> DNA
<213> Artificial
<220>
<223> nucleotide sequence possessed by ACS6 gene
<400> 22
atgtctcaga ctcacaagca tgctattcct gctaacatag ctgacagatg cttaataaat 60
ccagaacagt atgaaactaa atacaagcaa tctataaacg atcctgacac tttctgggga 120
gagcagggta agatattgga ctggattaca ccataccaaa aggtcaagaa cacttcattt 180
gctcctggta atgtttctat taaatggtac gaggacggta cattaaactt agcagcaaac 240
tgcttggaca ggcacttgca agagaacgga gacagaactg caataatttg ggagggtgac 300
gacgcatctc aatctaagca catatcatac agagagttac acagagacgt ttgcagattc 360
gctaacacat tgttggattt gggaataaag aaaggtgacg ttgttgcaat ttatatgcct 420
atggttcctg aggctgctgt cgctatgttg gcttgcgcaa ggataggagc tgtccattct 480
gtcatttttg gtggtttttc acctgaggca atagcaggta ggataattga ttcatcttca 540
aggttggtca taactgcaga tgagggagtc agggcaggaa gatctattcc attgaagaag 600
aacgttgacg atgcattgaa gaacccaaac gttacttctg tcgagcacgt cattgtctta 660
aaaagaactg gaaacgacat agactggcaa gagggtaggg acttatggtg gagggacttg 720
attgagaagg cttcacctga acaccagcca gaggcaatga atgcagaaga cccattgttt 780
attttatata catctggatc tacaggaaag ccaaagggag tcttgcatac tacaggtggt 840
tacttggtct atgcagcaac aacttttaag tatgtcttcg actaccaccc tggtgacatt 900
tactggtgca cagctgacgt tggatgggtc acaggtcact cttacttgtt gtacggacct 960
ttggcatgcg gtgctactac attgatgttc gagggagtcc ctaactggcc tacacctgct 1020
aggatgtgcc aggttgtcga caagcaccag gtcaacatac tgtacacagc accaacagct 1080
attagggcat tgatggcaga gggtgacaag gcaattgagg gaacagacag gtcttctttg 1140
agaatattgg gatctgtcgg tgagccaata aacccagagg catgggagtg gtactggaag 1200
aagataggaa aggagaaatg tccagtcgtc gacacatggt ggcagacaga gacaggtgga 1260
tttatgataa ctcctttgcc aggagctata gagttgaagg ctggttcagc aacaaggcca 1320
ttcttcggag tccagcctgc attggtcgac aacgagggtc atccacagga aggtgctaca 1380
gagggtaact tagtcattac tgactcttgg cctggtcagg ctaggacatt gttcggtgac 1440
catgagaggt tcgagcagac ttacttttct acatttaaaa atatgtactt ttctggagac 1500
ggtgctagaa gggatgaaga tggttactat tggataactg gtagggtcga tgatgtctta 1560
aacgtctcag gacacaggtt gggaactgct gaaattgaat cagcattggt cgctcaccct 1620
aagatagctg aggcagctgt cgtcggaatt cctcacgcaa ttaagggaca ggcaatttac 1680
gcatacgtca ctttgaacca cggtgaagag ccatcaccag agttgtacgc agaggttagg 1740
aactgggtca ggaaggagat tggacctttg gcaactccag acgtcttgca ctggacagac 1800
tcattgccta agacaaggtc tggaaaaata atgaggagaa tattgaggaa aattgctgct 1860
ggagatactt caaacttggg tgacacttct acattggctg acccaggtgt tgtcgagaaa 1920
ccattggagg aaaaacaggc aatagctatg ccatcttaa 1959
<210> 23
<211> 1503
<212> DNA
<213> Artificial
<220>
<223> nucleotide sequence possessed by ALD6 gene
<400> 23
atgactaagc tacactttga cactgctgaa ccagtcaaga tcacacttcc aaatggtttg 60
acatacgagc aaccaaccgg tctattcatt aacaacaagt ttatgaaagc tcaagacggt 120
aagacctatc ccgtcgaaga tccttccact gaaaacaccg tttgtgaggt ctcttctgcc 180
accactgaag atgttgaata tgctatcgaa tgtgccgacc gtgctttcca cgacactgaa 240
tgggctaccc aagacccaag agaaagaggc cgtctactaa gtaagttggc tgacgaattg 300
gaaagccaaa ttgacttggt ttcttccatt gaagctttgg acaatggtaa aactttggcc 360
ttagcccgtg gggatgttac cattgcaatc aactgtctaa gagatgctgc tgcctatgcc 420
gacaaagtca acggtagaac aatcaacacc ggtgacggct acatgaactt caccacctta 480
gagccaatcg gtgtctgtgg tcaaattatt ccatggaact ttccaataat gatgttggct 540
tggaagatcg ccccagcatt ggccatgggt aacgtctgta tcttgaaacc cgctgctgtc 600
acacctttaa atgccctata ctttgcttct ttatgtaaga aggttggtat tccagctggt 660
gtcgtcaaca tcgttccagg tcctggtaga actgttggtg ctgctttgac caacgaccca 720
agaatcagaa agctggcttt taccggttct acagaagtcg gtaagagtgt tgctgtcgac 780
tcttctgaat ctaacttgaa gaaaatcact ttggaactag gtggtaagtc cgcccatttg 840
gtctttgacg atgctaacat taagaagact ttaccaaatc tagtaaacgg tattttcaag 900
aacgctggtc aaatttgttc ctctggttct agaatttacg ttcaagaagg tatttacgac 960
gaactattgg ctgctttcaa ggcttacttg gaaaccgaaa tcaaagttgg taatccattt 1020
gacaaggcta acttccaagg tgctatcact aaccgtcaac aattcgacac aattatgaac 1080
tacatcgata tcggtaagaa agaaggcgcc aagatcttaa ctggtggcga aaaagttggt 1140
gacaagggtt acttcatcag accaaccgtt ttctacgatg ttaatgaaga catgagaatt 1200
gttaaggaag aaatttttgg accagttgtc actgtcgcaa agttcaagac tttagaagaa 1260
ggtgtcgaaa tggctaacag ctctgaattc ggtctaggtt ctggtatcga aacagaatct 1320
ttgagcacag gtttgaaggt ggccaagatg ttgaaggccg gtaccgtctg gatcaacaca 1380
tacaacgatt ttgactccag agttccattc ggtggtgtta agcaatctgg ttacggtaga 1440
gaaatgggtg aagaagtcta ccatgcatac actgaagtaa aagctgtcag aattaagttg 1500
taa 1503
<210> 24
<211> 1404
<212> DNA
<213> Artificial
<220>
<223> nucleotide sequence possessed by EUTE gene
<400> 24
atgaatcaac aggatattga acaggtggtg aaagcggtac tgctgaaaat gcaaagcagt 60
gacacgccgt ccgccgccgt tcatgagatg ggcgttttcg cgtccctgga tgacgccgtt 120
gcggcagcca aagtcgccca gcaagggtta aaaagcgtgg caatgcgcca gttagccatt 180
gctgccattc gtgaagcagg cgaaaaacac gccagagatt tagcggaact tgccgtcagt 240
gaaaccggca tggggcgcgt tgaagataaa tttgcaaaaa acgtcgctca ggcgcgcggc 300
acaccaggcg ttgagtgcct ctctccgcaa gtgctgactg gcgacaacgg cctgacccta 360
attgaaaacg caccctgggg cgtggtggct tcggtgacgc cttccactaa cccggcggca 420
accgtaatta acaacgccat cagcctgatt gccgcgggca acagcgtcat ttttgccccg 480
catccggcgg cgaaaaaagt ctcccagcgg gcgattacgc tgctcaacca ggcgattgtt 540
gccgcaggtg ggccggaaaa cttactggtt actgtggcaa atccggatat cgaaaccgcg 600
caacgcttgt tcaagtttcc gggtatcggc ctgctggtgg taaccggcgg cgaagcggta 660
gtagaagcgg cgcgtaaaca caccaataaa cgtctgattg ccgcaggcgc tggcaacccg 720
ccggtagtgg tggatgaaac cgccgacctc gcccgtgccg ctcagtccat cgtcaaaggc 780
gcttctttcg ataacaacat catttgtgcc gacgaaaagg tactgattgt tgttgatagc 840
gtagccgatg aactgatgcg tctgatggaa ggccagcacg cggtgaaact gaccgcagaa 900
caggcgcagc agctgcaacc ggtgttgctg aaaaatatcg acgagcgcgg aaaaggcacc 960
gtcagccgtg actgggttgg tcgcgacgca ggcaaaatcg cggcggcaat cggccttaaa 1020
gttccgcaag aaacgcgcct gctgtttgtg gaaaccaccg cagaacatcc gtttgccgtg 1080
actgaactga tgatgccggt gttgcccgtc gtgcgcgtcg ccaacgtggc ggatgccatt 1140
gcgctagcgg tgaaactgga aggcggttgc caccacacgg cggcaatgca ctcgcgcaac 1200
atcgaaaaca tgaaccagat ggcgaatgct attgatacca gcattttcgt taagaacgga 1260
ccgtgcattg ccgggctggg gctgggcggg gaaggctgga ccaccatgac catcaccacg 1320
ccaaccggtg aaggggtaac cagcgcgcgt acgtttgtcc gtctgcgtcg ctgtgtatta 1380
gtcgatgcgt ttcgcattgt ttaa 1404
<210> 25
<211> 1332
<212> DNA
<213> Artificial
<220>
<223> nucleotide sequence possessed by ERG12 gene
<400> 25
atgtcattac cgttcttaac ttctgcaccg ggaaaggtta ttatttttgg tgaacactct 60
gctgtgtaca acaagcctgc cgtcgctgct agtgtgtctg cgttgagaac ctacctgcta 120
ataagcgagt catctgcacc agatactatt gaattggact tcccggacat tagctttaat 180
cataagtggt ccatcaatga tttcaatgcc atcaccgagg atcaagtaaa ctcccaaaaa 240
ttggccaagg ctcaacaagc caccgatggc ttgtctcagg aactcgttag tcttttggat 300
ccgttgttag ctcaactatc cgaatccttc cactaccatg cagcgttttg tttcctgtat 360
atgtttgttt gcctatgccc ccatgccaag aatattaagt tttctttaaa gtctacttta 420
cccatcggtg ctgggttggg ctcaagcgcc tctatttctg tatcactggc cttagctatg 480
gcctacttgg gggggttaat aggatctaat gacttggaaa agctgtcaga aaacgataag 540
catatagtga atcaatgggc cttcataggt gaaaagtgta ttcacggtac cccttcagga 600
atagataacg ctgtggccac ttatggtaat gccctgctat ttgaaaaaga ctcacataat 660
ggaacaataa atacaaacaa ttttaagttc ttagatgatt tcccagccat tccaatgatc 720
ctaacctata ctagaattcc aaggtctaca aaagatcttg ttgctcgcgt tcgtgtgttg 780
gtcaccgaga aatttcctga agttatgaag ccaattctag atgccatggg tgaatgtgcc 840
ctacaaggct tagagatcat gacaaagtta agtaaatgta aaggcaccga tgacgaggcc 900
gtagaaacta ataatgaact gtatgaacaa ctattggaat tgataagaat aaatcatgga 960
ctgcttgtct caatcggtgt ttctcatcct ggattagaac ttattaaaaa tctgagcgat 1020
gatttgagaa ttggctccac aaaacttacc ggtgctggtg gcggcggttg ctctttgact 1080
ttgttacgaa gagacattac tcaagagcaa attgacagtt tcaaaaagaa attgcaagat 1140
gattttagtt acgagacatt tgaaacagac ttgggtggga ctggctgctg tttgttaagc 1200
gcaaaaaatt tgaataaaga tcctaaaatc aaatccctag tattccaatt atttgaaaat 1260
aaaactacca caaagcaaca aattgacgat ctattattgc caggaaacac gaatttacca 1320
tggacttcat aa 1332
<210> 26
<211> 867
<212> DNA
<213> Artificial
<220>
<223> IDI1 Gene sequence
<400> 26
atgactgccg acaacaatag tatgccccat ggtgcagtat ctagttacgc caaattagtg 60
caaaaccaaa cacctgaaga cattttggaa gagtttcctg aaattattcc attacaacaa 120
agacctaata cccgatctag tgagacgtca aatgacgaaa gcggagaaac atgtttttct 180
ggtcatgatg aggagcaaat taagttaatg aatgaaaatt gtattgtttt ggattgggac 240
gataatgcta ttggtgccgg taccaagaaa gtttgtcatt taatggaaaa tattgaaaag 300
ggtttactac atcgtgcatt ctccgtcttt attttcaatg aacaaggtga attactttta 360
caacaaagag ccactgaaaa aataactttc cctgatcttt ggactaacac atgctgctct 420
catccactat gtattgatga cgaattaggt ttgaagggta agctagacga taagattaag 480
ggcgctatta ctgcggcggt gagaaaacta gatcatgaat taggtattcc agaagatgaa 540
actaagacaa ggggtaagtt tcacttttta aacagaatcc attacatggc accaagcaat 600
gaaccatggg gtgaacatga aattgattac atcctatttt ataagatcaa cgctaaagaa 660
aacttgactg tcaacccaaa cgtcaatgaa gttagagact tcaaatgggt ttcaccaaat 720
gatttgaaaa ctatgtttgc tgacccaagt tacaagttta cgccttggtt taagattatt 780
tgcgagaatt acttattcaa ctggtgggag caattagatg acctttctga agtggaaaat 840
gacaggcaaa ttcatagaat gctataa 867
<210> 27
<211> 1197
<212> DNA
<213> Artificial
<220>
<223> nucleotide sequence of ERG10 gene
<400> 27
atgtctcaga acgtttacat tgtatcgact gccagaaccc caattggttc attccagggt 60
tctctatcct ccaagacagc agtggaattg ggtgctgttg ctttaaaagg cgccttggct 120
aaggttccag aattggatgc atccaaggat tttgacgaaa ttatttttgg taacgttctt 180
tctgccaatt tgggccaagc tccggccaga caagttgctt tggctgccgg tttgagtaat 240
catatcgttg caagcacagt taacaaggtc tgtgcatccg ctatgaaggc aatcattttg 300
ggtgctcaat ccatcaaatg tggtaatgct gatgttgtcg tagctggtgg ttgtgaatct 360
atgactaacg caccatacta catgccagca gcccgtgcgg gtgccaaatt tggccaaact 420
gttcttgttg atggtgtcga aagagatggg ttgaacgatg cgtacgatgg tctagccatg 480
ggtgtacacg cagaaaagtg tgcccgtgat tgggatatta ctagagaaca acaagacaat 540
tttgccatcg aatcctacca aaaatctcaa aaatctcaaa aggaaggtaa attcgacaat 600
gaaattgtac ctgttaccat taagggattt agaggtaagc ctgatactca agtcacgaag 660
gacgaggaac ctgctagatt acacgttgaa aaattgagat ctgcaaggac tgttttccaa 720
aaagaaaacg gtactgttac tgccgctaac gcttctccaa tcaacgatgg tgctgcagcc 780
gtcatcttgg tttccgaaaa agttttgaag gaaaagaatt tgaagccttt ggctattatc 840
aaaggttggg gtgaggccgc tcatcaacca gctgatttta catgggctcc atctcttgca 900
gttccaaagg ctttgaaaca tgctggcatc gaagacatca attctgttga ttactttgaa 960
ttcaatgaag ccttttcggt tgtcggtttg gtgaacacta agattttgaa gctagaccca 1020
tctaaggtta atgtatatgg tggtgctgtt gctctaggtc acccattggg ttgttctggt 1080
gctagagtgg ttgttacact gctatccatc ttacagcaag aaggaggtaa gatcggtgtt 1140
gccgccattt gtaatggtgg tggtggtgct tcctctattg tcattgaaaa gatatga 1197
<210> 28
<211> 1191
<212> DNA
<213> Artificial
<220>
<223> nucleotide sequence of MVD1 gene
<400> 28
atgaccgttt acacagcatc cgttaccgca cccgtcaaca tcgcaaccct taagtattgg 60
gggaaaaggg acacgaagtt gaatctgccc accaattcgt ccatatcagt gactttatcg 120
caagatgacc tcagaacgtt gacctctgcg gctactgcac ctgagtttga acgcgacact 180
ttgtggttaa atggagaacc acacagcatc gacaatgaaa gaactcaaaa ttgtctgcgc 240
gacctacgcc aattaagaaa ggaaatggaa tcgaaggacg cctcattgcc cacattatct 300
caatggaaac tccacattgt ctccgaaaat aactttccta cagcagctgg tttagcttcc 360
tccgctgctg gctttgctgc attggtctct gcaattgcta agttatacca attaccacag 420
tcaacttcag aaatatctag aatagcaaga aaggggtctg gttcagcttg tagatcgttg 480
tttggcggat acgtggcctg ggaaatggga aaagctgaag atggtcatga ttccatggca 540
gtacaaatcg cagacagctc tgactggcct cagatgaaag cttgtgtcct agttgtcagc 600
gatattaaaa aggatgtgag ttccactcag ggtatgcaat tgaccgtggc aacctccgaa 660
ctatttaaag aaagaattga acatgtcgta ccaaagagat ttgaagtcat gcgtaaagcc 720
attgttgaaa aagatttcgc cacctttgca aaggaaacaa tgatggattc caactctttc 780
catgccacat gtttggactc tttccctcca atattctaca tgaatgacac ttccaagcgt 840
atcatcagtt ggtgccacac cattaatcag ttttacggag aaacaatcgt tgcatacacg 900
tttgatgcag gtccaaatgc tgtgttgtac tacttagctg aaaatgagtc gaaactcttt 960
gcatttatct ataaattgtt tggctctgtt cctggatggg acaagaaatt tactactgag 1020
cagcttgagg ctttcaacca tcaatttgaa tcatctaact ttactgcacg tgaattggat 1080
cttgagttgc aaaaggatgt tgccagagtg attttaactc aagtcggttc aggcccacaa 1140
gaaacaaacg aatctttgat tgacgcaaag actggtctac caaaggaata a 1191
<210> 29
<211> 1476
<212> DNA
<213> Artificial
<220>
<223> nucleotide sequence of ERG13 gene
<400> 29
atgaaactct caactaaact ttgttggtgt ggtattaaag gaagacttag gccgcaaaag 60
caacaacaat tacacaatac aaacttgcaa atgactgaac taaaaaaaca aaagaccgct 120
gaacaaaaaa ccagacctca aaatgtcggt attaaaggta tccaaattta catcccaact 180
caatgtgtca accaatctga gctagagaaa tttgatggcg tttctcaagg taaatacaca 240
attggtctgg gccaaaccaa catgtctttt gtcaatgaca gagaagatat ctactcgatg 300
tccctaactg ttttgtctaa gttgatcaag agttacaaca tcgacaccaa caaaattggt 360
agattagaag tcggtactga aactctgatt gacaagtcca agtctgtcaa gtctgtcttg 420
atgcaattgt ttggtgaaaa cactgacgtc gaaggtattg acacgcttaa tgcctgttac 480
ggtggtacca acgcgttgtt caactctttg aactggattg aatctaacgc atgggatggt 540
agagacgcca ttgtagtttg cggtgatatt gccatctacg ataagggtgc cgcaagacca 600
accggtggtg ccggtactgt tgctatgtgg atcggtcctg atgctccaat tgtatttgac 660
tctgtaagag cttcttacat ggaacacgcc tacgattttt acaagccaga tttcaccagc 720
gaatatcctt acgtcgatgg tcatttttca ttaacttgtt acgtcaaggc tcttgatcaa 780
gtttacaaga gttattccaa gaaggctatt tctaaagggt tggttagcga tcccgctggt 840
tcggatgctt tgaacgtttt gaaatatttc gactacaacg ttttccatgt tccaacctgt 900
aaattggtca caaaatcata cggtagatta ctatataacg atttcagagc caatcctcaa 960
ttgttcccag aagttgacgc cgaattagct actcgcgatt atgacgaatc tttaaccgat 1020
aagaacattg aaaaaacttt tgttaatgtt gctaagccat tccacaaaga gagagttgcc 1080
caatctttga ttgttccaac aaacacaggt aacatgtaca ccgcatctgt ttatgccgcc 1140
tttgcatctc tattaaacta tgttggatct gacgacttac aaggcaagcg tgttggttta 1200
ttttcttacg gttccggttt agctgcatct ctatattctt gcaaaattgt tggtgacgtc 1260
caacatatta tcaaggaatt agatattact aacaaattag ccaagagaat caccgaaact 1320
ccaaaggatt acgaagctgc catcgaattg agagaaaatg cccatttgaa gaagaacttc 1380
aaacctcaag gttccattga gcatttgcaa agtggtgttt actacttgac caacatcgat 1440
gacaaattta gaagatctta cgatgttaaa aaataa 1476
<210> 30
<211> 1509
<212> DNA
<213> Artificial
<220>
<223> nucleotide sequence of tHMG1 gene
<400> 30
atggttttaa ccaataaaac agtcatttct ggatcgaaag tcaaaagttt atcatctgcg 60
caatcgagct catcaggacc ttcatcatct agtgaggaag atgattcccg cgatattgaa 120
agcttggata agaaaatacg tcctttagaa gaattagaag cattattaag tagtggaaat 180
acaaaacaat tgaagaacaa agaggtcgct gccttggtta ttcacggtaa gttacctttg 240
tacgctttgg agaaaaaatt aggtgatact acgagagcgg ttgcggtacg taggaaggct 300
ctttcaattt tggcagaagc tcctgtatta gcatctgatc gtttaccata taaaaattat 360
gactacgacc gcgtatttgg cgcttgttgt gaaaatgtta taggttacat gcctttgccc 420
gttggtgtta taggcccctt ggttatcgat ggtacatctt atcatatacc aatggcaact 480
acagagggtt gtttggtagc ttctgccatg cgtggctgta aggcaatcaa tgctggcggt 540
ggtgcaacaa ctgttttaac taaggatggt atgacaagag gcccagtagt ccgtttccca 600
actttgaaaa gatctggtgc ctgtaagata tggttagact cagaagaggg acaaaacgca 660
attaaaaaag cttttaactc tacatcaaga tttgcacgtc tgcaacatat tcaaacttgt 720
ctagcaggag atttactctt catgagattt agaacaacta ctggtgacgc aatgggtatg 780
aatatgattt ctaaaggtgt cgaatactca ttaaagcaaa tggtagaaga gtatggctgg 840
gaagatatgg aggttgtctc cgtttctggt aactactgta ccgacaaaaa accagctgcc 900
atcaactgga tcgaaggtcg tggtaagagt gtcgtcgcag aagctactat tcctggtgat 960
gttgtcagaa aagtgttaaa aagtgatgtt tccgcattgg ttgagttgaa cattgctaag 1020
aatttggttg gatctgcaat ggctgggtct gttggtggat ttaacgcaca tgcagctaat 1080
ttagtgacag ctgttttctt ggcattagga caagatcctg cacaaaatgt tgaaagttcc 1140
aactgtataa cattgatgaa agaagtggac ggtgatttga gaatttccgt atccatgcca 1200
tccatcgaag taggtaccat cggtggtggt actgttctag aaccacaagg tgccatgttg 1260
gacttattag gtgtaagagg cccgcatgct accgctcctg gtaccaacgc acgtcaatta 1320
gcaagaatag ttgcctgtgc cgtcttggca ggtgaattat ccttatgtgc tgccctagca 1380
gccggccatt tggttcaaag tcatatgacc cacaacagga aacctgctga accaacaaaa 1440
cctaacaatt tggacgccac tgatataaat cgtttgaaag atgggtccgt cacctgcatt 1500
aaatcctaa 1509
<210> 31
<211> 1356
<212> DNA
<213> Artificial
<220>
<223> nucleotide sequence of ERG8 gene
<400> 31
atgtcagagt tgagagcctt cagtgcccca gggaaagcgt tactagctgg tggatattta 60
gttttagata caaaatatga agcatttgta gtcggattat cggcaagaat gcatgctgta 120
gcccatcctt acggttcatt gcaagggtct gataagtttg aagtgcgtgt gaaaagtaaa 180
caatttaaag atggggagtg gctgtaccat ataagtccta aaagtggctt cattcctgtt 240
tcgataggcg gatctaagaa ccctttcatt gaaaaagtta tcgctaacgt atttagctac 300
tttaaaccta acatggacga ctactgcaat agaaacttgt tcgttattga tattttctct 360
gatgatgcct accattctca ggaggatagc gttaccgaac atcgtggcaa cagaagattg 420
agttttcatt cgcacagaat tgaagaagtt cccaaaacag ggctgggctc ctcggcaggt 480
ttagtcacag ttttaactac agctttggcc tccttttttg tatcggacct ggaaaataat 540
gtagacaaat atagagaagt tattcataat ttagcacaag ttgctcattg tcaagctcag 600
ggtaaaattg gaagcgggtt tgatgtagcg gcggcagcat atggatctat cagatataga 660
agattcccac ccgcattaat ctctaatttg ccagatattg gaagtgctac ttacggcagt 720
aaactggcgc atttggttga tgaagaagac tggaatatta cgattaaaag taaccattta 780
ccttcgggat taactttatg gatgggcgat attaagaatg gttcagaaac agtaaaactg 840
gtccagaagg taaaaaattg gtatgattcg catatgccag aaagcttgaa aatatataca 900
gaactcgatc atgcaaattc tagatttatg gatggactat ctaaactaga tcgcttacac 960
gagactcatg acgattacag cgatcagata tttgagtctc ttgagaggaa tgactgtacc 1020
tgtcaaaagt atcctgaaat cacagaagtt agagatgcag ttgccacaat tagacgttcc 1080
tttagaaaaa taactaaaga atctggtgcc gatatcgaac ctcccgtaca aactagctta 1140
ttggatgatt gccagacctt aaaaggagtt cttacttgct taatacctgg tgctggtggt 1200
tatgacgcca ttgcagtgat tactaagcaa gatgttgatc ttagggctca aaccgctaat 1260
gacaaaagat tttctaaggt tcaatggctg gatgtaactc aggctgactg gggtgttagg 1320
aaagaaaaag atccggaaac ttatcttgat aaatag 1356
<210> 32
<211> 1953
<212> DNA
<213> Artificial
<220>
<223> nucleotide sequence of yap1 gene
<400> 32
atgagtgtgt ctaccgccaa gaggtcgctg gatgtcgttt ctccgggttc attagcggag 60
tttgagggtt caaaatctcg tcacgatgaa atagaaaatg aacatagacg tactggtaca 120
cgtgatggcg aggatagcga gcaaccgaag aagaagggta gcaaaactag caaaaagcaa 180
gatttggatc ctgaaactaa gcagaagagg actgcccaaa atcgggccgc tcaaagagct 240
tttagggaac gtaaggagag gaagatgaag gaattggaga agaaggtaca aagtttagag 300
agtattcagc agcaaaatga agtggaagct acttttttga gggaccagtt aatcactctg 360
gtgaatgagt taaaaaaata tagaccagag acaagaaatg actcaaaagt gctggaatat 420
ttagcaaggc gagatcctaa tttgcatttt tcaaaaaata acgttaacca cagcaatagc 480
gagccaattg acacacccaa tgatgacata caagaaaatg ttaaacaaaa gatgaatttc 540
acgtttcaat atccgcttga taacgacaac gacaacgaca acagtaaaaa tgtggggaaa 600
caattacctt caccaaatga tccaagtcat tcggctccta tgcctataaa tcagacacaa 660
aagaaattaa gtgacgctac agattcctcc agcgctactt tggattccct ttcaaatagt 720
aacgatgttc ttaataacac accaaactcc tccacttcga tggattggtt agataatgta 780
atatatacta acaggtttgt gtcaggtgat gatggcagca atagtaaaac taagaattta 840
gacagtaata tgttttctaa tgactttaat tttgaaaacc aatttgatga acaagtttcg 900
gagttttgtt cgaaaatgaa ccaggtatgt ggaacaaggc aatgtcccat tcccaagaaa 960
cccatctcgg ctcttgataa agaagttttc gcgtcatctt ctatactaag ttcaaattct 1020
cctgctttaa caaatacttg ggaatcacat tctaatatta cagataatac tcctgctaat 1080
gtcattgcta ctgatgctac taaatatgaa aattccttct ccggttttgg ccgacttggt 1140
ttcgatatga gtgccaatca ttacgtcgtg aatgataata gcactggtag cactgatagc 1200
actggtagca ctggcaataa gaacaaaaag aacaataata atagcgatga tgtactccca 1260
ttcatatccg agtcaccgtt tgatatgaac caagttacta atttttttag tccgggatct 1320
accggcatcg gcaataatgc tgcctctaac accaatccca gcctactgca aagcagcaaa 1380
gaggatatac cttttatcaa cgcaaatctg gctttcccag acgacaattc aactaatatt 1440
caattacaac ctttctctga atctcaatct caaaataagt ttgactacga catgtttttt 1500
agagattcat cgaaggaagg taacaattta tttggagagt ttttagagga tgacgatgat 1560
gacaaaaaag ccgctaatat gtcagacgat gagtcaagtt taatcaagaa ccagttaatt 1620
aacgaagaac cagagcttcc gaaacaatat ctacaatcgg taccaggaaa tgaaagcgaa 1680
atctcacaaa aaaatggcag tagtttacag aatgctgaca aaatcaataa tggcaatgat 1740
aacgataatg ataatgatgt cgttccatct aaggaaggct ctttactaag gtgttcggaa 1800
atttgggata gaataacaac acatccgaaa tactcagata ttgatgtcga tggtttatgt 1860
tccgagctaa tggcaaaggc aaaatgttca gaaagagggg ttgtcatcaa tgcagaagac 1920
gttcaattag ctttgaataa gcatatgaac taa 1953
<210> 51
<211> 720
<212> DNA
<213> Artificial
<220>
<223> nucleotide sequence of spt15-5 Gene
<400> 51
atggccgatg aggaacgttt aaaggagttt aaaggggcaa acaagatagt gtttgatcca 60
aataccagac aagtatggga aaaccagaat cgagatggaa caaaaccagc aactactttc 120
cagagtgaag aggtcataaa aagagctgcc ccagaatctg aaaaagacac ctccgccaca 180
tcaagtattg ttccagagct atagaacatt gtggcaactg tgactttggg gagcaggtta 240
gatctgaaaa cagttgcgct acatgcccgt atgcagaata taacccctag cgttttgctg 300
ctgtcatcat gcgtatagag agccaaaaac tacagcttta atttttgcct cagggaaaat 360
ggttgttacc ggtgcaaaaa gtgaggatga ctcaaagctg gccagaagaa tatatgcagg 420
aattatccaa aaaatcaggt ttgcagctaa attcacagac ttcaaaatac aaaatattgt 480
cggttcgtgt gacgttaatt tccctatacg tctagaaggg ttagcattca gtcatggtac 540
tttctcctcc tatgagccag aattgtttcc tggtttgatc tatagaatgg tgaagccgaa 600
aattgtgttg ttaatttttg tttcaggaaa gattgttctt ctggagcaaa gcaaagggag 660
gaaatttacc aagcttttga agctatatac ccagttctaa gtgaatttag aaaaatgtga 720
<210> 52
<211> 621
<212> DNA
<213> Artificial
<220>
<223> taf25-3 Gene nucleotide sequence
<400> 52
atggattttg aggaagatta cgatgcggag tttgatgata atcaagaagg acaattagaa 60
acaccttttc catcggttgc gggagccgat gatggggaca atgataatga tgactctgtc 120
gcagaaaaca tgaagaagaa gcaaaagaga gaggctgtag tggatgatgg gagtgaaaat 180
gcatttggta tacccgaatt tacaagaaaa gataagactc tggaggagat tttagagatg 240
atggacagta ctcctcctat cattcccgat gcagtaatag actactattt aaccaaaaac 300
gggtttaacg tagcatatgt acaagtgaaa cgacttttag cacttgctac tcagaaattt 360
gttagtgata tagctaagga tgcctacgaa tattccagga tcaggtcttc cgtagcggta 420
tctaatgcta acaacagtca ggcgagagct aggcagctat tgcaaggaca gcgacagact 480
ggcgtgcagc agatttcaca acaacaacat caacagaatg agaagactac agcaagcata 540
gttgttctga cggtgaacga tcccagtagc gctgttgctg aatacgggct caatataggt 600
cgcccagact tttatcgtta g 621

Claims (8)

1. A microorganism, comprising:
over-expressiontHMG1、PagCrtB、BtCrtIAnd is derived fromPantoea ananatisIs/are as followsCrtEA gene;
wherein the microorganism is yeast, and is derived from yeastPantoea ananatisIs/are as followsCrtEThe integration site of the gene is LEU2,PagCrtBandBtCrtIthe integration site of the gene is URA3,tHMG1the integration sites of the genes are GAL1, GAL7 and GAL 10;
the gene tHMG1 has the sequence shown in SEQ ID NO: 30, or a nucleotide sequence shown in the specification,BtCrtIthe gene has the sequence shown in SEQ ID NO: 10, or a nucleotide sequence shown in the specification,PagCrtBthe gene has the sequence shown in SEQ ID NO: 11, derived fromPantoea ananatisIs/are as followsCrtEThe gene has the sequence shown in SEQ ID NO: 12.
2. The microorganism of claim 1, wherein further silencing comprises a silencing selected from the group consisting ofGAL80，ROX1，VBA5， DOS2，Ypl062W，Yjl064W，Yer130C，Yer134C，Ynr063W，Exg1，Yor292C，Sfk1，Mef1At least one of the genes.
3. The microorganism of claim 2, further over-expressing comprising a promoter selected from the group consisting ofINO2，yap1， spt15-5，taf25-3，GapN，PYC2，SMAE1，MDH2，POS5，pntAB，ADH2，ACS6，ALD6，EUTE，ERG12， IDI1，ERG10，MVD1，ERG13，ERG8At least one of a gene;
wherein, the INO2 gene has the sequence shown in SEQ ID NO: 13, or a nucleotide sequence represented by SEQ ID NO,GapNthe gene has the sequence shown in SEQ ID NO: 14, the PYC2 gene has a nucleotide sequence shown in SEQ ID NO: 15, the SMAE1 gene has the nucleotide sequence shown in SEQ ID NO: 16, and the MDH2 gene has a nucleotide sequence shown in SEQ ID NO: 17, and the POS5 gene has a nucleotide sequence shown in SEQ ID NO: 18, the pntA gene has the nucleotide sequence shown in SEQ ID NO: 19, the pntB gene has the nucleotide sequence shown in SEQ ID NO: 20, the ADH2 gene has a nucleotide sequence shown in SEQ ID NO: 21, the ACS6 gene has the nucleotide sequence shown in SEQ ID NO: 22, and the ALD6 gene has a nucleotide sequence shown in SEQ ID NO: 23, the EUTE gene has a nucleotide sequence shown in SEQ ID NO: 24, the ERG12 gene has the nucleotide sequence shown in SEQ ID NO: 25, and the IDI1 gene has the nucleotide sequence shown in SEQ ID NO: 26, ERG10 gene has the nucleotide sequence shown in SEQ ID NO: 27, and the MVD1 gene has the nucleotide sequence shown in SEQ ID NO: 28, ERG13 gene has the nucleotide sequence shown in SEQ ID NO: 29, ERG8 gene has the nucleotide sequence shown in SEQ ID NO: 31, yap1 gene has the nucleotide sequence shown in SEQ ID NO: 32, and the spt15-5 gene has the nucleotide sequence shown in SEQ ID NO: 51, taf25-3 gene has the nucleotide sequence shown in SEQ ID NO: 52.
4. The microorganism according to claim 1, wherein the microorganism is selected from the group consisting ofFurther comprising the operational regulationERG9A gene.
5. A method for obtaining lycopene, comprising:
subjecting the microorganism according to any one of claims 1 to 4 to a fermentation treatment; and
subjecting the fermentation treatment product to an extraction treatment to obtain said lycopene.
6. The method according to claim 5, wherein the fermentation treatment is carried out by:
subjecting the microorganism to a basic fermentation treatment in a basic fermentation medium and a two-stage fed-batch fermentation treatment by sequentially supplementing a first fed-batch medium and a second fed-batch medium on the basis of the basic fermentation medium,
wherein the basic fermentation medium is a YPD medium containing 24g/L peptone, 12g/L yeast extract and 12g/L glucose;
the first feed medium is YPD medium containing 500g/L glucose and 15g/L yeast extract;
the second feed medium is 75% ethanol and/or 50% glycerol.
7. The method of claim 5, wherein the extraction process comprises:
and (3) carrying out homogenate crushing or enzymolysis crushing treatment and organic extraction treatment on the fermentation treatment product.
8. Use of a microorganism according to any one of claims 1 to 4 in the preparation of lycopene.
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