CN117917477A - Flavone biosynthesis related enzyme and application thereof - Google Patents
Flavone biosynthesis related enzyme and application thereof Download PDFInfo
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- CN117917477A CN117917477A CN202310612980.XA CN202310612980A CN117917477A CN 117917477 A CN117917477 A CN 117917477A CN 202310612980 A CN202310612980 A CN 202310612980A CN 117917477 A CN117917477 A CN 117917477A
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- flavone
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- A01N35/00—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom having two bonds to hetero atoms with at the most one bond to halogen, e.g. aldehyde radical
- A01N35/04—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom having two bonds to hetero atoms with at the most one bond to halogen, e.g. aldehyde radical containing aldehyde or keto groups, or thio analogues thereof, directly attached to an aromatic ring system, e.g. acetophenone; Derivatives thereof, e.g. acetals
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- A01N43/00—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds
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- C07D311/30—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 4 with aromatic rings attached in position 2 or 3 with aromatic rings attached in position 2 only not hydrogenated in the hetero ring, e.g. flavones
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- C07D311/00—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings
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- C07D311/22—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 4
- C07D311/26—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 4 with aromatic rings attached in position 2 or 3
- C07D311/28—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 4 with aromatic rings attached in position 2 or 3 with aromatic rings attached in position 2 only
- C07D311/32—2,3-Dihydro derivatives, e.g. flavanones
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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
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Abstract
The invention discovers that the synthesis of the fungal flavone is obviously different from the synthesis of plants by analyzing the synthesis way of the flavone in a fungus capable of generating the flavone compound, and is a brand-new biosynthesis mechanism. Wherein, cfoA, cfoK and CfoJ are responsible for catalyzing the synthesis of the core skeleton of flavone. By heterologous reconstitution of these three enzymes in Aspergillus oryzae, aspergillus terreus and Saccharomyces cerevisiae, the host can produce flavonoids. Based on the guidance of enzymatic elements in the fungal flavone synthesis pathway, the construction of a novel flavone cell factory is realized.
Description
Technical Field
The invention belongs to the fields of microbial gene resources and genetic engineering, and particularly relates to a flavone biosynthesis related enzyme and application thereof.
Background
Flavonoids are a large class of natural products that are mainly found in plants, have diverse structural types and broad biological activities, and they have important physiological roles in plant growth, regulation and defense. The biosynthetic pathways of flavonoids in plants have been studied very clearly. Firstly, L-phenylalanine forms coumaroyl-CoA under the action of Phenylalanine Ammonia Lyase (PAL), cinnamic acid 4-hydroxylase (C4H) and 4-hydroxycinnamoyl-CoA ligase (4 CL), then is condensed with three molecules of malonyl CoA under the action of chalcone synthase (CHS, III PKS) to generate chalcone, and the chalcone isomerase (CHI) catalyzes the chalcone to form flavanone with specific configuration, and finally forms a flavone skeleton under the action of flavone synthase (FNS). The flavonoid compound with various structure types and biological activities is formed by modification of hydroxylation, methylation, glycosylation and the like on the basis of the framework (shown in figure 1).
There are many flavonoid-related functional foods and medicines on the market at present, such as anthocyanin and quercetin with anti-inflammatory and anti-aging effects, soybean isoflavone with phytoestrogen function, silybin with liver protection effect, and the like. The flavonoid compounds have a plurality of applications in food, health care and medicine development, and play a very important role in the health of people. Flavonoids are reported to have a market of about $2 billion per year. The sources of the flavonoids are mainly extracted from plants, and the flavonoids can not meet a great deal of market demands due to low content in the plants, complex extraction process and the like. Thus, the production of flavonoids by microorganisms using synthetic biotechnology is possible. The lack of endogenous genes for flavone synthesis in microorganisms is currently established in microorganisms based on the synthetic pathways of flavone in plants. Because of the low compatibility of microbial chassis and enzymatic elements in plants, the yield of microbial flavone cell factories is low and cannot meet the commercial requirements. In the present invention, the applicant has found a fungus capable of producing flavonoids. The synthesis route analysis shows that the synthesis of the fungal flavone is obviously different from that of plants, and the fungal flavone is a brand-new biosynthesis mechanism. Wherein, cfoA, cfoK and CfoJ are responsible for catalyzing the synthesis of the core skeleton of flavone. By heterologous reconstitution of these three enzymes in Aspergillus oryzae, aspergillus terreus and Saccharomyces cerevisiae, the host can produce flavonoids. Based on the guidance of enzymatic elements in the fungal flavone synthesis pathway, the construction of a novel flavone cell factory is realized.
Disclosure of Invention
The present invention provides genes related to flavone synthesis.
In one embodiment, the gene associated with flavone synthesis is selected from one or a combination of any of ester hydrolase CfoK, NADPH dependent FMN reductase CfoJ, hybrid non-ribosomal peptide synthase and polyketide synthase CfoA.
In one embodiment, the amino acid sequence of CfoK has at least 80%, 85%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8% or 99.9% sequence identity compared to SEQ ID No. 18; preferably, the CfoK is derived from aspergillus candidus, for example aspergillus candidus MEFC1001; more preferably, the amino acid sequence of CfoK has at least 99% sequence identity to SEQ ID No.18 and the CfoK is derived from aspergillus candidus; the aspergillus candidus comprises aspergillus candidus MEFC1001,1001. More preferably, the amino acid sequence of CfoK is shown as SEQ ID No.18, and the coding gene sequence is shown as SEQ ID No. 6.
In one embodiment, the amino acid sequence of CfoJ has at least 80%, 85%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8% or 99.9% sequence identity compared to SEQ ID No. 21; preferably, the CfoJ is derived from aspergillus candidus, for example aspergillus candidus MEFC1001; more preferably, the amino acid sequence of CfoJ has at least 99% sequence identity to SEQ ID No.21 and the CfoJ is derived from aspergillus candidus; the aspergillus candidus comprises aspergillus candidus MEFC1001,1001. More preferably, the amino acid sequence of CfoJ is shown as SEQ ID No.21, and the coding gene sequence is shown as SEQ ID No. 9.
In one embodiment, the amino acid sequence of CfoA has at least 80%, 85%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8% or 99.9% sequence identity compared to SEQ ID No. 24; preferably, the CfoA is derived from aspergillus candidus, for example aspergillus candidus MEFC1001; more preferably, the amino acid sequence of CfoA has at least 99% sequence identity to SEQ ID No.24 and the CfoA is derived from aspergillus candidus; the aspergillus candidus comprises aspergillus candidus MEFC1001,1001. More preferably, the amino acid sequence of CfoA is shown as SEQ ID No.24, and the coding gene sequence is shown as SEQ ID No. 12.
The aspergillus candidus MEFC is 1001 with a preservation number of CGMCC No.3.15294, can be obtained commercially and is a fungus preserved in the common microorganism center of China Committee for culture Collection of microorganisms.
In another aspect, the invention provides the use of the above-described gene associated with flavone synthesis in the preparation of a flavonoid compound.
On the other hand, the invention also provides application of the gene related to flavone synthesis in preparing a genetic engineering strain capable of producing the flavone compounds.
On the other hand, the invention also provides a genetic engineering strain capable of producing the flavonoid compound, wherein the genetic engineering strain is obtained by introducing the gene related to the flavonoid synthesis into a starting strain.
The "introduction" includes the step of expressing, preferably overexpressing, the above-mentioned gene of interest in the starting strain. For example, the gene of interest is constructed on an expression vector, which is transferred into a host cell to express the gene of interest, preferably over-expressed. In other embodiments, the "introducing" comprises inserting the gene of interest into the genome of the host cell; preferably, the insertion into the genome of the host cell may be by homologous recombination double crossover; in one embodiment, insertion of the gene of interest into the appropriate genomic location may be accomplished by inserting the gene of interest and the homology arms into the vector, and then transferring the vector into the host cell, using the homology arms to double-exchange homologous recombination with the host cell genome; in other embodiments, gene editing may also be employed, for example, using a CRISPR/Cas system to cleave at a desired genomic site, while inserting the gene of interest as an exogenous donor into the cleavage site.
On the other hand, the invention also provides application of the genetically engineered strain in production of flavonoid compounds.
The original strain of the genetically engineered strain is selected from aspergillus, aspergillus nidulans, yeast, escherichia coli, plants or animals; preferably, aspergillus, e.g., aspergillus candidus, aspergillus oryzae, aspergillus terreus.
Preferably, the gene related to flavone synthesis is selected from one or a combination of any of CfoA, cfoK and CfoJ.
In another aspect, the invention also provides a method for preparing flavonoid compounds, which comprises the step of fermenting the genetically engineered strain.
In one embodiment, the flavonoid is selected from one or any of compound 22, compound 24, compound 25 and compound 26.
Specifically, separate introduction CfoA may produce compound 22 and compound 25, while introduction CfoA and CfoK may produce compound 24, while introduction CfoA, cfoK, and CfoJ may produce compound 24 and compound 26.
In a preferred embodiment, the gene associated with flavone synthesis is selected from CfoA, and the flavonoid is selected from one or any of compound 22 and compound 25; the gene related to flavone synthesis is selected from CfoA and CfoK, and the flavonoid compound is selected from compound 24; the genes related to flavone synthesis are selected from CfoA, cfoK and CfoJ, and the flavonoid compounds are selected from one or more of compounds 24 and 26.
The structural formula of the compound 22 is shown as follows:
the structural formula of the compound 24 is shown as follows:
the structural formula of the compound 25 is shown as follows:
the structural formula of the compound 26 is shown as follows:
In another aspect, the invention also provides uses of CfoK and CfoJ.
In one embodiment, the invention provides the use of CfoK in the catalytic synthesis of compound 27 and/or compound 24; in other embodiments, the invention provides the use of CfoJ in the catalytic synthesis of compound 26 and/or compound 3.
Specifically, cfoK may catalyze the formation of compound 20 to form compound 27; cfoK can catalyze the formation of compound 22 to compound 24; cfoJ can catalyze compound 24 to form compound 26; cfoJ can catalyze the formation of compound 18 to compound 3.
On the other hand, the invention also provides a genetically engineered strain introduced CfoK and/or CfoJ, and the original strain of the genetically engineered strain can be yeast or escherichia coli.
On the other hand, the invention also provides application of the genetically engineered strain introduced CfoK and/or CfoJ in preparing compound 27, compound 24, compound 26 or compound 3.
In the present application, compound 27 has the structural formula shown below:
In addition, the present application found that CfoK from amino acid H at position 33 and amino acid Y at position 50 of the N-terminus are key sites for their activity, and that mutation of amino acid 33 (e.g., mutation to A or Q) or mutation of amino acid 50 (e.g., mutation to A or F) was not possible for mutated CfoK to catalyze synthesis of compound 22 to compound 24.
CfoJ amino acid P at position 112 and amino acid W at position 154 from the N-terminus are key sites for their activity, and mutation of amino acid at position 112 (e.g., mutation to a) or mutation of amino acid at position 154 (e.g., mutation to a) is not possible for mutated CfoJ to catalyze synthesis of compound 24 to compound 26.
Drawings
FIG. 1. Biosynthetic pathway of flavonoids in plants. The substrate phenylalanine generates coumaroyl CoA through phenylalanine ammonolysis, and then generates a flavone core skeleton under the sequential actions of chalcone synthase CHS, chalcone isomerase CHI and flavone synthase FNS.
FIG. 2. Metabolite analysis of Aspergillus candidus and mutant strain Delta cfoA and the corresponding flavonoids. Compared with the wild type, the metabolites 1-4 in the mutant strain delta cfoA disappear, and the metabolites are all flavonoid compounds through separation and identification, wherein the compound 1 is the final product.
FIG. 3. Gene composition in the gene cluster cfo of synthetic flavones in A.leucini. The gene cluster cfo contains 12 genes, wherein the core gene is cfoA, the self-resistance gene is cfoL, and further comprises a P450 enzyme gene (cfoH), an NADPH-dependent FMN reductase gene (cfoJ), an NTF2-like protein gene (cfoI), a cocE/NonD family hydrolase gene (cfoK), three monooxygenase genes (cfoE, cfoF and cfoG) and three methyltransferase genes (cfoB, cfoC and cfoD).
FIG. 4 analysis of metabolites of Aspergillus candidus and different mutants and their corresponding flavonoids. The metabolites of all gene deletion mutants were analyzed and 23 flavonoids, which were all intermediates in the biosynthesis of end product 1, were identified by isolation.
FIG. 5 biosynthesis pathway of flavonoids in fungi. The hybrid enzyme CfoA is used as a core enzyme, benzoic acid or parahydroxybenzoic acid is used as an initial unit to be condensed with 4 molecules of malonyl CoA to generate key intermediate chalcone, then a flavone core skeleton is formed under the actions of CfoK and CfoJ, and on the basis, the core skeleton forms a final product 1 under the catalysis of different post-modification enzymes.
Figure 6 isotopic distribution in fungal flavone and plant flavone structures. The bolded chemical bond in compound 1 represents sodium acetate from [1,2- 13C2 ] and the black dot in naringenin represents malonyl-CoA from [1,3- 14C2 ].
FIG. 7.CfoK analyzed the CHI phylogenetic tree that has been found in nature. CfoK are located on the same branch as the CocE/NonD family hydrolase, whereas plant CHI and bacterial CHI are located on two other independent branches, respectively.
FIG. 8 in vitro enzyme activity assay of CfoK and mutants and its catalytic mechanism. In vitro enzyme activity analysis of cfok and mutants thereof; the catalytic mechanism of CfoK, the N on H33 pulls out the hydrogen on the substrate C2' -OH, induces oxa-Michelal addition reaction, forms a C ring intermediate containing enol anions in a ring-closing way of 6-endo-trig, is stabilized by forming hydrogen bonds with Y50, and the hydrogen ions on the H33 imidazole ring are supplemented and enol tautomerism, thus finally forming the flavanone product 24 containing tetrahydropyranone.
FIG. 9. Analysis of CfoJ with the FNS tree currently found in nature. CfoJ are located on the same branch as the NADPH dependent FMN reductase family, while FNS I and FNS II are located on two separate branches, respectively.
In vitro enzyme activity assays of cfoj and mutants and their catalytic mechanism. In vitro enzyme activity analysis of cfoj and different cofactors, in vitro enzyme activity analysis of CfoJ mutants; the catalytic mechanism of cfoj first H 2 O strips off the hydrogen atom at the C3 position of the activated substrate and then electrons flow between C2 and C3, causing the hydrogen atom at C2 to flow in the form of a hydride carrying a pair of electrons to the N5 position of FMN, with the formation of a double bond between C2 and C3, which is reduced to FMNH 2.
In vitro enzyme activity assays of cfok and CfoJ. Separating and purifying the CfoK protein and performing in vitro enzyme activity analysis; separation and purification of CfoJ protein and in vitro enzyme activity analysis.
FIG. 12 analysis of metabolites of engineering strains and their corresponding flavonoids. (a) analysis of metabolites of Aspergillus oryzae engineering strains; (b) analysis of metabolites of the engineering strain of aspergillus terreus; (c) analysis of metabolites of the Saccharomyces cerevisiae engineering strain; (d) separating the flavonoid compound from the engineering strain.
Detailed Description
The following examples are further illustrative of the invention and are not intended to be limiting thereof.
The experimental procedures, which do not address the specific conditions in the examples below, are generally carried out under conventional conditions or standard operating conditions for filamentous fungi or under conditions recommended by the manufacturer. Percentages and parts are by weight unless otherwise indicated. Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.
Enzymolysis liquid: 0.4g of cellulase (Sigma product, catalog number: C1184), 0.4g of lyase (Sigma product, catalog number: L1412) and 0.2g of snailase (product of Biotechnology Co., ltd., shanghai, catalog number: SB 0870) were weighed out, dissolved in 50ml of 0.6M MgSO4 aqueous solution, and sterilized by filtration through a sterile filter of 0.22. Mu.m.
In the invention, the plasmid extraction adopts an OMEGA PLASMID MINI KIT I Kit (D6942-01), the DNA fragment recovery adopts an OMEGA Cycle-Pure Kit (D6492-01), and the gel recovery adopts an OMEGA Gel Extraction Kit Kit (D2500-01).
PDBS plate: 24g/L potato culture medium PDB dry powder (BD company product, catalog number: 7114771), 1.2M sorbitol, 4g/L agarose, the balance deionized water, and after autoclaving at 121℃for 20 minutes, incubation at 48 ℃.
PDA plate: 39g/L potato medium PDA dry powder (BD company product, catalog number: 633840), balance deionized water, and autoclaved at 121℃for 20 minutes, and cooled to about 60℃to prepare a flat plate.
PDAS plates: 39g/L potato medium PDA dry powder (BD company product, catalog number: 633840), 1.2M sorbitol, balance deionized water, autoclaved at 121℃for 20 minutes, and cooled to about 60℃to prepare a plate.
SGCY medium: 2% sucrose, 1% glucose, 0.5% casein hydrolysate, 0.5% yeast extract powder, 1% MgCl 2·6H2 O, and the balance deionized water, and autoclaving at 121deg.C for 20 min, cooling to about 30deg.C, and inoculating and culturing.
SYT medium: 1% of soluble starch, 0.4% of yeast extract powder, 0.2% of tryptone, 0.1% of CaCO 3,0.004％FeSO4·7H2 O,0.1% of KBr and the balance of deionized water, and sterilizing at 121 ℃ for 20 minutes, and cooling to about 30 ℃ for inoculation culture.
PPM medium: 15% sucrose, 2.5% soybean meal, 0.5% peptone, 0.1% NaNO 3 and the balance deionized water, and sterilizing at 121deg.C for 20 min, cooling to about 30deg.C, and inoculating and culturing.
YPED medium: 2% sucrose, 1% casein hydrolysate, 1% polypeptone, 1% yeast extract, and the balance deionized water, sterilizing at 121deg.C for 20 min, cooling to about 30deg.C, and inoculating and culturing.
MM medium ：0.2％ NH4Cl、0.1％(NH4)2SO4、0.05％ KCl、0.05％ NaCl、0.1％ KH2PO4、0.05％ Mg2SO4·7H2O、0.002％ FeSO4·7H2O and 2% sucrose, balance deionized water, autoclaved at 121 ℃ for 20 minutes.
CD medium: 0.2% of soluble starch, 0.1% of KH 2PO4, 0.1% of yeast extract, 0.05% of Mg 2SO4·7H2O、0.01％ FeSO4·7H2O、0.055％ CaCl2, 2% of maltose, 1% of polypeptone and the balance of deionized water, and sterilizing at 121 ℃ for 20 minutes, and cooling to about 30 ℃ for inoculation culture.
Example 1 Co-localization of self-resistance Gene cfo Gene clusters were found
1.1 First, ALS (acetolactate synthase) gene was found in 459 strain of sequenced fungal genome, and ALS gene was found to be multicopy in 49 strain of genome. 2765 secondary metabolite biosynthetic gene clusters were found to be present in 49 genomes by ANTISMASH predictions. The cfo gene cluster derived from aspergillus candidus was finally locked to contain ALS gene by co-localization analysis.
1.2 By genomic sequencing and bioinformatic analysis of a laboratory-maintained strain of Aspergillus candidus MEFC1001 (commercially available, deposited in the China general microbiological culture Collection center, accession number: CGMCC No. 3.15294), a cfo gene cluster (nucleic acid sequence shown in SEQ ID No. 1) was located which contained 12 genes (FIG. 2-FIG. 3, table 1), of which cfoA, cfoB, cfoC, cfoD, cfoE, cfoF, cfoG, cfoH, cfoI, cfoJ, cfoK total 11 genes are responsible for the synthesis of the flavonoid carbon skeleton, and cfoL is a self-resistant gene encoding the ALS protein.
TABLE 1 functional analysis of proteins encoded by the genes in Gene Cluster cfo
The sequence information is as follows:
SEQ ID No.1
ctaccagccaagaaactcatctgccagcgtaaatgaaggatcatatatcttcattgcgtctatcgcagcacctgcaacatcatacttataattatggatcatcatatcatgctggccgcctaaacacagggttagcttttgtataccaaatcaacaagcgaaaacagacaggattggtgatcatacgtggcatttggtcatcgtgaagatattctttcagatcctcaactgatacggagtgatctgcacccatgttgctaaggatctgaattacagaagcacgttttatgcgtacttcctgatagatttgcagccgcttctcgatatcatcacgcgtcgtcgccttgtgaagtatgaccccaagcgccacggcgtcttctatgccctgagcaccgccctgagctagaactaggcagggtcagcacttcgccaaggagacatgaagagtaaggaaggtatcaacgtacaaggaagcatagcatggcaagcatctccagccagagtcatacaccctttagtccattggtgaatgggcttgcggtagatcagtggccagcggcgtgcatcgggcgctttgctacaagcttgttagaacggcacttgagaagctagggcgctacatgccttaaaaccttgagcagtctggggtcgtagtccgagaatttcttgagaacatctgggattttgaagcgggcgtgccaatctgcacgggatcggtcagtgcggtccatactagacggtggtctgctcccaccttctgcacacatatttgtatcattctcctcattattaatgagaacaaaattctgtatagtattgctagatgccattagttgcagctccaaatgctcgtcggaaaagtcggtacctccggcaagggtatgacactaccgagcgccccgttacgctatcgatcatgacacgggaccagccgtcagagtcctcattccaaaatcgcgtctcaggatttgcctcgattgcactcgcaggcaccagaaagcggtagcatgtgttagcaaacctcgggcgcattgtctggaccttgtcgccgatgatggcctcgacggcaccagagcgcacgccgtcagcgcctatgacgacgtccgcagtaatgacctccccgctggccagagtgacggacggcgtcaaggggtcctagaatataacggtcaacatattgcatgatacgggatgaatagggcgtacataagccaccacattgctcatgaggtggatcgtagccggcttccccgggccttcagctcgcgtagccatccatctaagacagccatgcagatcgacgcggtgggcatagtacagctctgcaccggcaacctccttggcggtcatgccatccgacagcgtcattttcaattccagcgaggtatggtccaggaaagagaccttttgcgacttgacaaagcgccacttgtctacgtcgagaccccacggtagcaagaactggctgacattgggtggcacgtagatggcagcgccgatttcgttgtcttgtgctgacttttcgtacaggtcgacggtgtggccggcgcggcggagggacgttgctgtcgccaggccggcgatgcctgcaccgacgataattatgtgaagacccggtgaagattccatggtgggctgtgctgtatccgttgagtctgcttttggtcggcttgccagattaaatgtggcagaagctgcccttaacgctgcagactaggcgggtcattgcagtgacttggcgaaccagatttataagatatatgtaactagttgtcttttatttatagactggttatggataattacggttcaaagttatgattactagtttacaaaatgtttcttcacctcaaatgtcaaacagcctgtatcttaacacgtacaacagtgtaggaagttgaccttatatctactaccagacagtcagccgaccacgcgacccctgcgattacagtcacatacactataactggtgaatgttaataatctttggaaattctcgaacactgagtgctgagaatgcaaaatgagtatgatagtcttggccactacgccaaatcattgattagtgtctagtattgggcccaaccagactgacatcccaacataaagtgttttcattatcatgttcaggagcaacaactccactggaagcctcacaggaagactacgaatatatccgtccgttgattcataaggggggttaaaacacaccatgttagatgctttcatggtctgattcgtcaactattgctctgtcccactaaaaagtcatttgcgcctgaaccgtccgtaaactgctcacgatcaagctgcgtggtatgtgcgctggagatgcgctcatgcgacctgtcgagtgtacgaagggctctcgggacactctcacggtcgaactgcgagatggcgtcttgtatggatgacgctttacacaattgctccgctaggtcatagccgtccttgagggccacgttggcgccgttgcccgccagtggagaaaaggcgtgattactgtcgccgataaagaccacgcctctcaggtgctcgtggctaaaaggtcgcttgtccattcctgccatgataaacatggtggacgggtcggttctctccacgatggtagcaaaaggctcttcaatcacctggccgcgccggagagcctctgactttaggccctgggtatactcgatggaccaagttgatggaggcgcacgaggactctgttcccgccagctgaggccccagtgaaatccaaacttgcttgtcggcgacaaaaaacaggatacaccctggtctgtgagaaccatgccccatttgtctgtgatgtgcgctggaacggcgccgtcggggaattcggcgacaccacccatcatcatcaggcccgtgtattgcagtctatcatctggacggaagctggtccggatcttgctatttgcgccgtcggcgacgataagcagatcgcacgttcttgcttcggtgcttccaccgtcctccgtcttgcggaggcgtatgcggactcgaccgccgtcgaggcattcaggcacttcacatgatacgccccaacgaatctcaataccggcagcctccgcagcttcaatgagtatcattcgcagaatgcgtcgggggatgcgcacagcggaggtcggcatgtcgccataagctggcacattgaagctgggggaagcgatccaatctttg
tcccatatcgtgaatttgctgatggcgtctgtcatcacggcgctatctatgactttgctcaacagccccaattgcttcaagacaacgagcccggt
gtcttctttgcttcagattgcacatgtcagtggccactcttgcagtcggttcgtgtacgtatacatacatgccactcagcgtctgtgagtatccttgc
cgtctgaggtcgacttctcgcttgtcgcgatcatagacgaccaccttcggcggatggccactatctccccattgtttctgcaggttgagtgcaaa
tgcgagaccagctactcctgctccagcgacaacgatgtcctttccttccaggcagtgttctcgtgtcatgtcgagattctgtgctcttcagcgaga
ctttcgctcatgtcttaccacgctttgtagcgtttttgtgacaataaagtggaaatgtatgacactgtaaggagccccagtcagcatatactcattg
gccactacctatgatacttactccatacggcgtcggtaaggagttgtccggttacgagtcgtcatcttctggtgcaggctgccgcccccacttcg
catagcgccctagtccaggggccctgaacctgcagcgcactagcccttatagtgctgcagtagtgctatcgcgcagccagcatgatatttcac
ggagtcataccgagatcgcgcccggctcgtgtgatggacgcagccaagatgaggggaagacaatgctcgcaatctgggcgcttcgactgc
tttgcatgtaatacgcgttacgagcatagcacagccagtattggtaatactgtggttttcctagcccagttacctgaatacttcgttgcgcgctatc
gtcagtactgtcgtcagactgcaggaggaagtgtcgccgaatggtgctactagcgcacttcatgcccatgaagacaaatccggtcactctgat
cgcattgtctggggttcattggaatgtcaaccttctgaagaagtcttgaagtctacagcgaaagtgaagtccagaagcattgctattttttatcgcg
gagctggcttcttaccaaaatggctagcaagcgtctggagacactctgcaagtctctgactcgagaagtcggaagtctttcttcgctcactgga
agggacgattatgttgcccggagcaaggtcgcggacattgccagagagctcattaatgaaacgactcaccctagtgacgtcgcggcccagt
acacaataaatgtattcaccctctgctggaggacctctcattggaccgattactaacaaacatgcagatggcagaaatggcatgtctacgtatgt
ttcagaaatggaagctcctggacaagatccccgccagtggcagcatttcatatgaggatctctctgccagcatcaacgccgataaaaacctca
ttggtgcgtctgccataccaccctttcacggccatctatggaatgcaactaacctcatcaaagcccgcatgggccaaatgctcgtcgcgacag
gcaaactgcgacagccctccccatcgcacgtggcgcacactcgcctgagcacagccttcgcccatcgttcgcctccagccgtatggttctcg
atgagcttcgacgagaccctcggcccatggacccactggccgcgctacttcgccaaatacggcccgcaacagccaagcggccagaccgct
gtgcccatgacattcgcagagggggtagacggcgagttgacgtgctacgaggtcattgcgcgcggtggaccggagcgaatggctgactttg
cggatgggatgcaggggatccccgaactgatgcctgctgctgggatatatgactttgagtgggtcgggcaggcagttgcgaaaggcgaggt
cgatctggatgtgccgctgattgtagacgtgggggggaaccttggtcaggcgctgctggagattatcgcgcataccgagtcgtcgattccccc
tgacaggtgtgttttgcaggatcgggcggatgttatatctgctgcagaggggctggagaactcagtgttgaagaagatacgcaaaatgcctgtt
gattaccaccaggggcagcctcttaaaggtaagcatattcggtgtcctccagtcgaacggcttgcttatgtctggaataggagccttgatatact
atgttcgccgctgtctccatggttttacagatgaggtttctgtcaacttgcttaaacacttggccgctgctctaccggctgatccacgtgccagggt
tctcatctgcgagcaaatcacaacaacgccgccagacccgtatacgacaatgatggatatctgtatgatgaatgttggctctaaggagagaag
tgaagatgactttacgagattagtggctgctgctggcatgaaagtaatcaagttccaccgaggagagggcgttgcaacacatgtcatcgaatgt
gcacgagcttagcctgtcgcctggaaatcaatatagttactctttgtatgtgtggaagataactcgataactcgtcattctagtggccttaaccga
gatccatccccaatggcacatgtaccccttccctcctcttaatcctcaaggcaatgccagactgcctagggaacatgctgttatgtaccttccact
ccctggctggatcagccaactcaaactcaaacaccgcaatcaacgtcgccaccgtcttatagacctccatggtagcaaaattaacaccaagac
acttgcgactgcccgccccaaatgacatgtcactgctgttcatcacgcgcagtcgctctttgaaccgctccgccgactcatcttcaccctgtaac
caacgctcgggccgaaaatcctcagcgtcgtcgccccacacgtctttgttccggtgtatcacgtaggcattgaaccccaccgccacgccctc
ggggacgaaggagccgtcggggagtatcagccccccaggcggtacataccgctcctgcgcgaagcaactccccggccacagacgcatg
gattcgcggatgacagcctcaaggtacgtcacggcgcgcgcctgcgaataaggaacgggcagctctgtgaaagaggccctgagaatctgc
gcttccagcttctcccataccctccggtccttcagagcgaggtagaagatggcgcgtagtgtcgtggccgtcgtgtccaccccggcagccac
gttgacaagaatgtacgacatcaggcgcggctcgtccaccacgtcggggtactttcgcatggcgtccagataacagtctagaaagtcgggct
tggatggatcgtggtcgtcctcgccggtgcgtcgcttcgtgaggtggccaaaggcggcgttcgccaccggcacgaaactcggtgggccga
agcgaaagacggggtttttgtcgaggaatttatccagaacaggttgcattccgactgtgacgagatagtccatgactttcgcagacgtgtccac
cataccgtcaaagtcaaaggcgtgctctaggtgtcctgctcgcctgctccacgtgctctgcgcaatggtgtcccaggcgtctgttggcacagtc
gtatggggttagtcgtatataggctgtgtgggcaaattagaacttacaaaacgaagcccactcgtcaaactttagaggtttgccgtatccagaac
gctcagtaaactggtcatccagctgcttgacgaaaaacgaaagcacgctgttgacgtgcggttccatgacgctgacgcctgtggatgagaaat
atttggcaactggttttttgatccgcgcatgctcggccgggttgcactcgctgaagatgttgtataggattttgttacccagcttgacgcctgatat
gccgtgccattctgtctggtgatagcgttagtaccattcactgacgcttgatttactttagttttacgatggagtttcgtagggcttgttgggaacca
gtggaagaggacgaaacgaacctttttccaaacaccatgactatcgagacacgtactgatcaaggagggatagtcgagatcgaggtagtttg
gggacatcctcactaccggtccataccttttgtgcaaggcatggctcacgagatgcattgatccacctgcagcatggtacatgcgccagtaact
tgttaaggctgtcgaggatcagcggttaatgctctccatgtttggttgtcaaaggagtttaccaacaagcaagcaatggaccacgtcggccacg
aatcggcgaggtgaaatacggaataatgtaccagagcaagacagctgcacaaaaaaaagcgaagagggtcaagataggatgtgctgtgag
cctttccacaactctcatggagccctcggccagcgtgagcactttcgtctccatgatagacgtcattcaaaaaatcaatggctgtgatatctcca
gcatagcagtctttgagagaagaaatgtgtatgactgagatacaagaagactaggccagaacatagctagcccaaaatagaatatacaaagc
tgcagcgcacatcagctcgcaacatcatccgttttttacattcccaactcttgggattactttttttttttttctaagactgacgatagtagttgtaagtt
atatgtactagatactctacagtactttacatacacctcagtacgagcgtctacttattagacaaggtttgcgaatacacccatgcaaagcttctcc
actccctcgcatgggatgcatagtaagcactaaagctgataagcaaggaaccaagtaatgacttccaggtgcatgacatgtccggagtgcctt
acatcgtacatcaccttgatgtcaattcctcctcctaccggtccaagattttgatattggctgttcatcgagaccgtatttgttgattcgccagggca
cacgacagtgaccttaccattttaattatttcactcattgcaaacaccatgtccaacaatccaaagattattcaaatgtgtgtggcagggcagagg
aagaaagggtggtcggacgagcagttcgcccatgaatttacagtcgtgcatgcggaaatcaccaaagcaacagcccagaaaacaccagca
ctccttggctatcgacaaaccttagcaatcccaagaccgagaatatcagcgttcaacctgggccacagcaagtgggattcccaggcagtgttg
acgtggtcgagcatcgaagaactttcctccctcctcaaatccgaagggtatcgcgccaacgcaggaaaccacgtcttcaccgagccggatat
agtgggatcaatttgccaagttgcgggggagttcatgtttgatccggtcggatacagcagccaggaatcgcgctttatggtctttgtttatatccc
gcgggcgatcaggagcagtcgggagctggttacagaggaagaagtggctcagagacttgatagtatcacgagaatcggtgctggaacgg
gtctcttaagatatgttatcaatcgcgacgtgagcccgtctgatccaggtgaactgtttgatggcactccgttcatcactggtgattggggggtga
tgggcgtgacggaacaatattggtttaaggatgcagatgcggcagaggccttctttgcggatgaggtacgagtggacgcgttaatgggagta
cccagctcactggacagtacgagctgcgttgctgttgctggtcaagaaacggtcctagttagcaaataatcagcttcgtagtataccttactctg
gaggtcagaagcaaatgctttactaggagatgtcttgccatttcgacaacactggtgaaatttaaatgtacctgctaggttaactttgtattttacct
ccagctatataaaggtctgcacctgggccaaggttacttcaccctctacatctctctacaacccaatgcttgtatagttacccagaacttctactcc
catcttcagcactccctcaatcatgactttcaacatatacatccccgagaatcttgcgttctccaactatattgcagtgatccaggtagcgagaac
ctgggcggatgcccaagacagaaaggtcagtcaaaatacattccccttccagaccatagaaactgatacatattgtctcccaggaccccgaa
cgattcctcgccaccgtagcaccccaagtcaccatcgactacagccttctcatccccgcatggaaaagcaaagtctacacggctgacgagttc
gtcgcggcatggctggcccccgatcgggtcggactgtccgttctggcaacgcagcacctgctaggaatgccatacatcaagagcgccacg
ccggatgagattatcgtggagtttcaagaggtggcctcgcacgggcgacgccaggatgacgacggcgcgttcggcggcaatatcggagag
acggctgatgggagggggtgggtggagcatcgttacgtcaagattgatggccagtggaagattgatctcatcaagccttctataatctatatgg
cgggggattgggagcgagttcggcgggctgagggggcggagtaatgcagcggtaggatgggctatcatgcacgaatcggggacggtctg
gctgttgttctttctgtcgcaatagtagttatggtttctattagagtatgcgcactagtgtctccttgcatgtccgtggtaaagcaatggcggttcaat
ggtctctcacccgaacagctccgtcgacaaggatacgaggtcattgagagtggcatcagttgactcaagcacgattccccgctcatgctatca
gcacttatgcatgttggcggagagataaaacatttgcaaggatgatcggccggcttatccgctgagtagaagattcttactttagcctgtttatgtt
tcgcgagatagagttttagcatatactgtagtaagattttagcatggcaggggagagttgatccctgcagtataatatatctaatctattaaaatatt
acaactttgattaccactagtaaatgtaccttgaagaaataccagacaatgattacatttacaagtgattatttttacaagtgatcacatcaatgcag
cttactgttggtcggtaactcgtagcccatttagggtagccggtggaccaccttgcagccaggtctagaagaaatatgtatagtaatacagacta
ctcctggaactagtgtggagttgcagcacgagggccgaaggaggggccagaccaagttcagggtttgtatcagtacagttcgccgtgatgtg
cgcagataggaattgctatcaaaccatatgccccccctgaagaaaacatcgcaatacattttgacctacggaccatggacccagaagttagca
aactgctcgcgacggtggcaagcctcggcgagacagcaaagaagcatgctagcaccgacaaggacagccccgcgagccgcgagtcaa
ggagagagctcctgagcgcggcgcgacaactgacaaacgagctgcagaatgagggccaaattgtagaaggatatctttacggggtagagc
cccctggcaaacaaccgcgagtcaatgctaacgggaccgccagacactcgacccgctgttgctcaagatgggcattgagctgggcattttcc
ggaatctggtggacagtactaagcccgtgacgctgggagatctagtggccgcttcgggcgccgacgaggtgctgttggctcgcattatgag
ggggctttcatcgattcatgcggtaaatgaggtcggggttgagctgtatgagcctaataaggttacccgtgcttttacgactgtcaagggcgagt
cagggctgaatgttttgtaggtgtatattgtttttgactacaggaacagaaatactgacggtaggttgccaaacagtcacaatataaaccacccc
ggctggcagtccctgcccgaatgtcttcgagcgatgggctacatgaaccccacagatcccgccaaaatgtggttcggcagacagttcaatgg
tgagcactacttcgactggctggggaaacgcccggagctcctccattcattccaccggttcatgtcaacgcagcgagacggacatgcccact
ggctcgacttttacccgttccagcaacagctgctacctgattttgacgtggaaagccccgacgccgttttcatggttgatgtgggtggtagtgta
ggtcatgagatccaggaagtcaagagacgctatccagagattccaggacgcatggtcctgcaggatgttccggccacaattgcacgagtcgt
cccagaaaatgatatggaagctatgtcgcatgatttcttcacaccgaatccagtcaagggtatgtgacaacaccattatcatctcctcccctctcg
aaagacaattttttcctactgcccacgaaggaacgtggcacaggttaacatacgtacaggcgctcgagcatactacctccgcaacgtgctgca
cgactgggacgatgaccgctgccgcgtcattctgaagcatatccgagacgccatgactccgggatattctgtgcttctgatcaatgaatttagta
tacccctcaaaggcgcctgttcgttcgccacccattctgactttatgcttatggctattaatgctgctgtggagcgaactgagcaacagtggtacc
atcttatggagtcggttggcttgcaaattaaaaagatatggacgctagagccggacactgagagtctgctggaggtcacacgtggggaatag
ggctcaaaggtggctgcattatttctttgtagaacgtgggcgttgagattatgatcttcgatgcaatgtgatttgtctgtacatagagcgagagaat
tttctgtatacagatctaaatgcacgtatatctatatcgacatggatgtatttacatcgtgactcggctgggcggcgcaacgacccagttctgacc
gagcttcacaaattcttctttaacatctgacacctcagggttttccacttgaaacccttgctcaagatcccgcaaatgcttctccagtcccgcccgc
ctgagtacagagcagctctcgccatcggcgtcagtcacaaactcgacacatccatcgtcaccgacgcccatcttgcccttcgccccacaagta
gcacagtcaacagcatcttgaccgacaaaattgaacatactcaaatggcacaccggacacatgccccgcggaccgacgtatgtagcctccc
cttccgtcgtcgcccaggcctgccgcgccaggttcctccccagttcctccgccctagcgaccgcctcgccgttgcaaagaaaactatccggg
acgcccgtgccaaacacctgcatctggtcgatgatctgggcgccgagagggaaaaacacctggtgtagtagaggaagcgtaaacggcgcc
cactcggtcgtggaagcgccgcccagcgtcaccagcgcgagaactctcggcttgaagatgcgttgatcgactaggtggcccttgcctgtgtc
gactagtcttttcgccatcgtcacgtcttggaacgggcccagcgtgctgtcttggaagcgcttgacttcccatgggacggtccgcgtcatgcat
ggagcaccgaggatgatggcgtcggcttccataatctgatcgagcataaaaggacggtcatcggggactgttcctgaggttgggctcgactg
ccccactacgggcagcggaaggtggtcatctagggcttcaaaaccgattgaaagatccttaagccggatcaaggagatggttgccggcgct
gttgccgtctcttgggctgctcgaagggcggccagaagagcttgttcggtgttgccccccggggagccaccgctgaggccaaggaatctca
tattggagagcagaattgcaggaccaaactgctctatcggtctcactgacttatgttaacgatatctgacccgaataattcaatacagatgcatac
agtattcagacattacccatcttactagtacttcagggatgagataaatgcactccgtaagccaggggtgtcgctgcaggcaaccatgtcatgtc
caatttagacccacggtgaacgcggtcgcatcaggttctacacaccaattgcccaagggagttagtggcttgtatagatgtacatttgaatacat
ccatgtgattgacaatttggctgtattgttgaaactcaatcatcgcactaacacaaggtttggccgagtggacgatccacagtattacaacagac
cgtcaggtgtgcgcgccttgtataagtgcagcgtatcatacaaccgctagccaggaaactgcttacttacctactagccacacatatccgatcc
agcatcatctccccgtgctcaatggccaagtcaaacgcaaaagatagcctcttcgcgagctacgacgccgaatactggaacacctacctgga
cgctcgtccaacatactctcccgacttctacaacctcatattcgaccaccacagccacaaaggcaataacagctggaccttagcgcacgacgt
ggggaccgggcccgggaacgtagcggccgtcctcgcggagcgttttgcccaagtcatcgccacagacaccagtcctgacaatgtcaacgc
cgcacggcaacgccaactacagacaaacaagatcaggtttgcagaatgcaacggcgaggacctagcccgcgcggcgctgtcaccccctc
gcaccgccgacctcgtcgccaacgccgaagcgatcccactcatggacgccgaggaagcgatcgggtgctttgcggagctgttggcgccc
ggtggaacgtgtgccgtgtggttctacgggcggccgacgtttgcaggacctgatgccgctgtgaatgaggcgtgtcagcgcatcttctaccg
catctcgacgcggttgctgaacaagataggcggcatgagcgggccgctttgggagcggtctacgcgcactattgcgtcgcagctggataat
gttgcgtttcctgcggagcagtggcgtcatgtggttaggtataagtggaattgtgagcagacttgtatgctcttccatgatgagagtcagtttggc
gggccggttgagcgggtgaactgtgttgggccggctgaggaggtggtttcgaagacggaccctgggttttggcagatgcagtggggcgct
gctgaggtgcggaggtggtttgaggcaaatttgccaacttggtttgaagataaggctcaggatctggagctggagagttgctatgaggagttg
gatcgtgtcatgggaggcgagtctttgccggtgacttggcctgtggttttgctcctcgcgacacgggtgtaatgtgtcgtgcatcaggtttggca
atatagtatttgaaaatccgattatacgtctaaatatattgaaagagtttttttgtttgtttactctcataacacattgatggttgagaaattggcgaaat
actgcacgaaagctacatgatatatcccggggacaagaattcagaaatacactggagtgtattatcaagtgatagatacattcacagtaaataa
atggttcgatctcaccaaagtcaaccaaaacacaatccccaacaaactaccatctggatggcgcaaataagcacgtgaacatccacaacaag
agatgcacagaaccaaacaccacgagcggtacaccggatagcacaatctgtttgaaaaacccccctcgccgcttcacaacatcccacagct
ccccggtcataacctccaccgcaccatcccacttccacggcccgcacaagcgaaacttggtattgaactcagccgcaagaagccaaacaag
cggaagccgccaccactccttacgcaaccaacccatcctcacagcgtgcagaaaagacggtgaagcattcatatcgacagccaattgattcg
cgtagtcgtcgtggctgacgccgtacttgatgcgcgcatccgaaggcatgccaagtcgatagtgcggctcgtcctggggaagaaggggctg
gagacgctcgggcgcgaggagattcaccacccaaagctgcgcctgcagttccgccagtggcgggattgcgccgaaaccgggtctcacga
atccgatgaagccaatgcttggctcgtcgcgacgccagacgtctcgtacgtctaggtcggcagtgcttgggtatgtcgcgtccaggaagtcg
aagcgttgggtatagcctgtgccgaagacgagcacgtcgggtcgtactgcctcagcttctttcatgcgtcgatactctggtcgaccgttgtccat
aaagtggagcacgcccttgctgtcgatgtgggatggccagggcgctatgtcgatgtgccggtcgtgtgtctcggggattggcacttggatgat
actggagcgcaaacgttggaatatcgtgtcttttctgtatggcttgctgatgtagggcatggctttgctagacttggtgaagatgactgtaggcatt
tatagtcagcgactgaatttgtattgttgttttgactgctacctgggataccccgtgaggaactccgaagtaacgagggagacgtcaccattcga
tgtatgccagcgtggagaaggataaccgcccacccactgatcatagccggcactcgtgccagtcatgagccacatgccctgcttgaccaaac
tgtcaaatatatttccggggaggtttccccatttgtggctagcgtgtagatatgtatctaccggcacgggaaggttcggcgactgcagatggctt
gccagggaagggaatacaacggggctcggtgttcgctgaaaccataattgttagtcaattgttgcgacaagataaaaacagccgcaggtagt
tattgagaacaaatcgaataatgattatgaacctaccttggcaacaacactgaacccgtcgcgatgacatataacgacccttttggttggcgccg
tcacggccagatgggcgatgtccatggcagtttctcccgtgcctagaacaaccaccgttttatcggagccaaactgctcgcgcgacttgaagt
cagacgagtgaaaggtcgtgggaaccttgtcgatgccgctgattgacggtatattgggtgttacatgcagacccgagcagacggcgacagc
gtcgcatatccatgattcctccttgccatctggcgtggcatagctgatgacatgggagcgacctttgcgtcgaacggcagtcaccggagtgga
cagatggatgaacggccacaggttgaacgctgtggcatactgaccgaggtactcgaggtatctcgctgcaggcaggtaatcgggatcgtctt
cacgggcccggaagtcggaaaaggctgtgagatatttggatgaaaccaacttgatatgatcagtcaatgagtctcaattgactagatgttcaac
agctgcgcagctctggagagctgatacgtacctcggcgtcttcgtacatgcgcttgagaaaggtaccgcctatagcttcttccgattcaaacag
cttcgcttcgacgggatccccgccaacaaactcgtgcgcttcaagcaaatgcttgagcgtggtcagtccggccggaccgcctccaatgactg
caactctcatggtggcaggttgctacgaagcactctctctcaggcgtgaatgggatccgttgcacagaggcaaagcttctagctgtagcgaaa
ggagaagcaattctgcagtgtcagatcgtgttcgtacttcaacagggggcctcatgtcattacacacgatgaatacactctactaattggaaagt
ccctcaacttagcttgaagtgacggtaggaaactctgtggctgtgaataacgaagtcagtcatacaaccttagcatctcttccaagtcgattcagt
gttacaaaacatcatcttacatgaagtagatcggcataccgtgcatgaaacaccacctagtaagcttacttcacatggagtatgatctagtaatttt
aatctacagggagtataacctaagtatcaccgagttagctagcactctatactatttatttcatcttgtgaatgtgctaagtctccacttacctgcgta
agtgcttacttaacaagtggggagtatagaagcatgcacacttttgttatccggtatcttaagacgccttgaggacccaatcatggtaggtaacg
cactcggcgtgatgatggccagtgctctgcttacctgcaagggatgtgcattcacagacctaatcactggtctccacagtctgggatccatggt
agcaacagtagggcttatggctatagttagtatacaaggggctgcaggaccacaggtgaacagatgcctccctgattagggctggaggctac
tgcagattgagccaaccgcggattccgcgccgaacaagtaacccatgtataacacaagatactcgtacgcgagaagattatatcacactgtat
atccgtacttaccagaggattgggtcccctgcagatgctaaagaatgacattgtataatacgaataagcctgcggaatgcggcatgcgtctgat
gagggggtggcgaaccgtgctgcaggggttggagcaggttagtgaacacgaggacaagaatgggaaaagcaaataatttctgaagacata
atatgattagctagtacacggtgtaaacaccttgggagcctcagggagaagtgtaatactagtttaagtcgttttgatattatttgattggtgggta
atatatattcttggcccctctgtgggccacggcttgcgccgaacggaaggaccccgcatggcggctttagtacagacgctaggataagataat
actattagacgatctggtggattgcttgatcgatcaattggatgagggctcaagactgtatttgagtatttcacaagttgttaacaatgtcatgcaa
gtcgttaaagaacaaatgtacacccaactctatatctcttagtagcatgcgctcttatctgggtcatctaaatcaagtgtaaaaaaccctccactttt
ccgaaattaagtttgccagggactaaaaaatagttagatttggggcggactagactatcataccgagacccgatttgatggagataatacagta
ttcaaaagccgaggtagaaagttgaaatgcaggtggggcgtctgcgtcagcctggccggtgtccttgtttctgattctgagacacaccaagatt
atagcgtcgcggagagtaaaagatctagaatttagtatggatctatgcggagcactagcaggttcctctcagggtggggctggataagcggtt
cgtgtcatggagcaaggctgtgatcacacaacgcagccctagttagaggtaggactcccggggatgaactgatgaaatctcacccccaagct
agggaatcaggttgtcgtagacaaggggtatttaagctcgtcagtggtggaagccttcgtaataacgcattgccacagactacgatcctctacc
aacctcatttcaataacacccatggagccaaagttgtcctaaactaccacgaggacatgtctcgactcgagttgataccagatattctaatccgc
catgccgacgagtctggccggaaggtcgctttcgcaggtccaggatggacaatagtatgtcttttgtgtttctagacccatgttttactccttgca
gtctttgtcttcagttgtggagactggacttccgagggtgccgacctcgaggggttttggtccgcgcagtattttcacatgtatggagataactga
cggtttgaatttgaaagacgtatggcgaccttgaacaacggactaggcgtctagcagcccatctagtccgtgccggcatagggcgaggtcaa
ttcgtggccattgtactaggcagatgtcttcagaccgtcgagtccataatcgcaatcacaagagctggtgccgtgggagtacctctggattcgc
gctcgccctcatcggagttggctaaagttttggagcatagcggcgcgcgcgtgatcatcacagatgaccgccacttggctacggtgcacact
gctgctgcggaaggaagcttaattgttttgaataccaagcttcccaatgtgaatgccaaagatgaagggcatgagcttgtgcgataccaagact
ggattgaagacgaggagtgctcgaccttggatatcaagatcgacgatctcggggaggatgaggaagcctttctacactacacttctgggacg
accagtttgcccaagggggtactgtccaaccagaagagttggctgttgaatgtgaagagcctggtgtcagcgttcgaactgacgcccgagga
ccgcttcttttggcctcttcccctgttccactgcatcggccacttgttgtgtatcatgggaactgtggtcgtcggtgccagcgcatatctccctgat
gccgatcagacaatgcttgacagcctcagagatacaaacgctcgagaaacaaccctcattgtgggcgcgcccaccaccttccacgacctgat
agatgccgcgaagcggtcagatccgacgtcgcccttgtctttaccgaggctgcgagcatgtatgtatgcgggttcctcggcatcagggtctct
aggtacccaagtcaaggagttgctcggtgttccccttctgaacaactatgggtgtaccgaaggttgcggctctatcgctgttagcaaaacgagc
cacacctatcgcgacaattccagtatctcgctactgccgcactgggagattaagctagtggacccggatgggaacccagtcaaagatggcga
gcaaggcgaggtctgcataggcggccccggtctgatgctcgagtactaccgagagacgcggacgccgtttacgccggatggctggtaccc
tactggcgacatcgcaattcgctcgagctcggcggctgatgcagaattgaccttggtgggacgtaggaaagagatcattattcgagggggcg
aaaacattcatcctcatgagttggagcacgtcttgcttcggcatcctggcgtggcggatgtcgtcgtggctggaatgccacatagactgcttgg
agaaactcctgctgcctttattgtgaagagcgctgcggacgtggactttgacctatcggccttgcttgctgcgtgtcgcaaagtcttgccagatta
taagatacccactggtaatttgcattgttctctttctctaaaatcatacacattctaacaccggttagccttttatgagatcgacaccgttcctcgaac
cgtcatcggtaaaccgaaaaggctgaccatgacatcttacacaaacaaaccactcactgcgcgatctattttgcagtcaagagattcaatcgaa
gcactagtaatggcggaaacagtcagtgcatgcactataggcgccgaacgggagggcgagtcaaacacagactggcttcgtcgaaacctt
gaccagcccttttcgtttctgggcctgagctcaatggctagtgtggtcctccgtgaccgacttgccggtctcactggtttggctgatcttcccaat
actctcgtgtttgactatgcgaccccggcagctgtgagcacgtacctgtgtagccgcttgttggagccagaatcgacacctctgccccgttcaa
caccgactacgacgtcggattgtgaagtggagcccattgctattgtatccatggcctgtcggtatcctggaggcatctcttcgccggaggactt
gtgggagctagtttcagatgaaattgacgcgaccaccgagttcccagacgacgtacgtactacttcatgttctcccaagcctcttcttgttaaata
ctaacaaaccacagcgcggctgggatatcgacgcattgtatagcactgaccccgacactcctaatacctcaaccaccaagcgcggcggtttc
ctgcccgatttcgcccactttgacgctggtctttttggcatggcgccgcgtgaggctcttgccactgatccccagcagcggctcttattggagac
gacgtgggaattggccgagcgagcgggtattgctccgttatcgcttcaaggaagccagacgggcgtgtttgtgggtactctgtatgaggatta
tgaagagaacggctttgggaacaacggtaagtatttttctttggtagctctgatttgacagcatgaacgcgtgtctatttactgacaagtctcgcca
atgatacagagttggaagcacaccttggactgggctcgtccagcagtgtcgtctctgggcgtgtgtcgtactgctttgtgagtattttgcaatcat
ctgtctatatcgccagatggaaaaaacactctgaataacccgaagctaagatactgatttggatcataaaaagggtctccacggtccatctctag
tcgtttcgacaggctgctcgtcctcactagtagcaatccacctagcggcccaatccctccgaaatagagagtgctcgctgaccatcgcgggag
gaataacagccatggccaccccacgcccgtttaccatgttcagcagacgacgaggcctttcgtcggacgggcgatgtcgggcttattcaagt
gatgcaagcggtactggctggtccgagggtgtaggcctgcttctcctcgagcgactctcggatgccaaacgcaacgggcatcagattctcgg
tttgatccgtgggtccgctgtcaactctgatggaaaatctaatggcttgacggcaccgaacgggccggcccagcagatgtgtatccagagcg
ccttggcccaggcggggatgtcgccggaaaacgtggacgtgctagaaggccatggcactgcgacgcccttgggtgatcctattgaggttca
ggctgtgatttcggcgtatgggaatggcgatagaaagaccagcgacagcggcactcgccgtttggaaccgctgttgctgggctcgatcaagt
ccaagtgagtttcacaattgtcatacgttcttcatctatggcgtggctaacgcaccgagtgacgtgttagcatcggtcacacgcaagctgccgct
gctgtcgcaggaataatcaagatggtgcaggctatgcgccacggcgttgccccgacgtcattgcatatccgtgaaccgtcaccccaaatcga
ctgggaagggagtggtgttgagctgctcagcaaggccaggcagtggccgtctgtgaatagacccagacgggctgcggtgtcttcatttggta
ttgggggcacgaatagcgtgagtcttctccacatttccggtttgagcagtacactaactttgacgttgaagcacatcattctagagcagcctgag
cctgccgaggaggaagactccaattccaagagaatatccgccaccgttccctggctcatctccggtgcaagcgaggctgcattacgcggac
aggctcatgcattactggcagcttggagacaagacgataacacatttagctccctccgtaatcaggatcctaccgacatagcgttctcacttgct
actgcgcgatccgctctcaaatacagagccacggtgacctacacgctgggagtgaacttccacgatcaggtcgaaacagcactcaaaagcc
ttgcccaaggcgaaccacaccccgacgtcgtgacagcacacaccagtactaccggtagtcagccccgtctggcgtgcttattttcaggtcag
ggcagtcggatgcccagcatagacacactcgccgagttacgcgctaccttccccgttttctccacggcattccaggcagcttgtgacgaagtg
gaccagcacttggagtgcccactcgtacatgctattggtaacagcattctagaccgcaccgaatttgcacaggccacccttttcgtctttgaggt
ggctatgttccgcctcctggagtcgttcaatatccgtccggactttgtcgctggtcattctctgggagagatcgccgcggcgcatgctgctggtg
ctctgtctcttcgtgatgctgctactatcgtcaccacgcgagcaaagcttatggcatctctaccacccaacggcggtatggtgagcatatcggct
acggaggtggaggttgccagtgagctggcacaattgggcggcagtgccacgattgcggcagtcaactcgcagaactcggtcgtggtgtctg
gcactcaagaggcgatcaaggccgtcgcggacatgttcgccagcttaggacggagagcaaccgtgctgcgaaacgtcaaacatggctttca
ctccccgttgatgaacggcatactccccgacctagagaaagcgctcccatcgtcaatggaagacgacagcccgactgccataccgctcgttt
ccactaccacaagcaagcgagccgacgcagctcagcttcgctcttcagcccattggatccgccacgttagcgagcccgtccgctttgcagac
gcgatcgacgagctcaagtcaaacgaacgcgtctctgtttttgtggagattggcccgtcggctgtcctgtctccacatgttccggaggccgcc
gctacacatggcactgttgacaagctacttagaatgctgggtcagctgtggactcggggtgtaccggtcgactggcaagctgtctttgagggt
agtggtgcgcgatttgtcgacctgcctgtctatgctttccagcggcagagatactggcttccctacacgccactgctgccagtggcatccactg
gagcagtcgcacagcaggctcaaggacagaccgtgggcgctggtggtgactcgcgactgcgccacgggatgctcttcaacgccacttctat
ccctggaaccggcactgtcatctgctctgggtgcctgtcaacgacccgacagccttggcttcgtgaccacgttatcggtggacaatcgctcgtt
ccggcctcggcatttgctgagctagccatgcgagctgcacaagaatgtgcagagtataccgaatcttactcgatggtactggatgagatggtc
cttgttgcgccgctggccgtctcttcggcagaggacgaggaacaagaagattcatctggcgaattcgagattcaagtactcatagcttggtcgc
agcgtgaggacgagggaactgagacccaaaggaccgtcgaggtgtattcacgccctagcggcgtagcaacccaacacgaatggacacac
cacgccacgggttctctcaaactcacaccacagtccaacgccgaccactcttctacaaatggcacagattctacgagaacggagtcagatgt
ggacatttccgaagcgtacaccatcttagccgatgcaggtctcacatacgggccgtctttccagctcgtacgtgccatttggcgtctacgtgac
gatgacctgctcgtgcagatcgatccacccgaggatcaggctcagatgtcgacgttcgtcctgcacccggctgtgctagatgctgcgctgcat
gcgtctacccttgcctcggcggagaaggtcgctggcggtgacattcgactgcctttctcgctccgaggcgtccaatttttgacgacggctggtg
cctctggccccattctagcacgcatcagtcaagatagcgagaacagtttctcgttgaccctcacggacaagtcatcgggtgtgctggtcgctac
agtttctgaagtccagctgcgcgcctggcagcctgctgttgctggaggtgatctctaccgcctagaatggagggagattgagtctaagcccatt
caaacaaccacgtctaccgagaccgacaagattgttcgggtccaaagcgctcgcgatattgatgcagcagcagtcagcaaagctgtgcacg
aagctgtcgccgaggtgctccacacgattcatgagtggagggctgaaaatacacatgctggcgatggtgtccgcctcgtctttgtgaccgaga
gagcgacgtcggctgacaaaagctccaatatcaatgtcgttgctgccgctgtatggggctttgtacgatcagcccaggcagagtttggcggg
gaccggattgtgctcgtcgacatggatgggacaaccgagtcggaagaggctctatcggctgcgcttgcttccagggaggaagttgttgctgt
gaatggtggaaagattacgattcctagaataagtaaacagcctccgatgccagagtccccccaagcgatggccctcgatgtcagcgggactg
tcttgatcacaggcggtacaggcgggctgggtgcaacactcagtcgagacattgtgcacactcatggagcgaaaaatctgctcctcgtcagc
cggtcaggtattgaagcgcgtggagcacgcgagttatctgaagagctgcaaagtgcaaacgcagctgtgcgcgtcgaggcgtgcgacgtc
ggtgactacacccagctggccacattgctagacaaccatacccagcatcaatacccacccatcaccgccgtcatccattgtgcaggaactgtc
agcgacgcattcctgggctcccaaaaccaagagcgagtatctagtgtgctacgtcccaaggtcgacgccgcatggaatctccacgaactcgt
ccccgagacggtccgctcgttcgtcctcttctcgtcgtacgtcagcgtcctcgggaacgaaggccaggcggcatacagtgcaggcaacgcg
ttcctggatgcactggcccgcttgcgtgtcgcacgtggactgcctgccctgtcgctggcttggggcccgtgggcgaatgaggccgggatgg
ccggcggaagcaaactggccgcgattcccccgcgcatcgccaacgctcggccgtttacggatcagcaaggactgcatctgtttcatagggc
gttgcatgtgcagacgaggaccccgccggagccggttcttctgccgctgctgctgcgcgggccgttcccgctggtgccgtctgctggtacgg
agtccaagcccaaggagaagtcgtctgcgaggggtgggtcggcatctggtgctatatggcgcaggagcatcgctgcagtgtcattggagaa
ccgtcacgacgctctgctgggcctagtgcgggatgagattgctgctgtgctgggctaccagagccaagatatgcttccggatcagcgacttg
aagacctcggttttgactcgtttacttcagtcatgctcacgaatcgactgagggttttgactgggctcggctaccttcctgttacactcgcgctgga
ttatgatactacgtcggcgctggtcgagtacctgttacctcgcattgaagccgagccacagccacagccacagccagaagtcgacatggact
caaatgcatccacgaccggcggggacacgtcagtttcccatgacagtggaaaggcggacgggttgagcccatcgtctagtgtgactacact
cgctccggaggagcacgacgacctcaaccccgagactttccgagggctagccaccatccaccgacggctgtctcagttagagcagtacac
ggcggcagcagatctcctcgcctcggccgcactggcaatgccgaccttccccaagaccgggtcgagtctgcccagctacgcagcggagc
cccagcgcctagcgaccggtccatcgggcacctcaaactcagaaccgcccctgccgctcgtcttcatcgcgcctttcttcccgcgcatcaag
gtcgagggcattggcctcagcgtgtacagcaatctcgcatcagcgatgaccggaaaacgcgacgtgttcgagcttccccacccagaagggc
aggtcgtcccaggggacctcgccacgctggccgacctgcacgtccacaccatccgcaagcacttctcggaccgctcgggcattctcctcgc
gggttactccgcggggggcaccgtcgcatacgccgtggcgtcccgactggctcacgccgcagacaggcagcctcggctggcgggcttcg
tcttggtggacacgtatctcacgatgacgggtcggggggatcccgactggctgaacgcgctgccggcggaggccctcgtttcgcgcctcca
ggttccccccagcttgggtcaccccaaaggcatgggcagtgacagtctcgtgggcgacttggacgtggcgctggcgaaggtgggtgggtat
tttaggacgctgcgggactgggatctcgagctacatccgctgcctgatgcattgtcgacgctgttcgtgcgtgccgtggatgcgtcggacaag
atgcccaaggattcggacgtttggcgtccccgatggccgcgggcggatttgacggttgatgtgccggggagtcatctggctctgctggacaa
gcggtatgcacctggtgttgccgttgagattgaacggtgggcgagggagttgaatgcttagtgggatctgaagttgatactctgttggggtact
gttagtctttggttcttgtgacaaattgaagaaatttataagatcgagggttatttaacatgatcttgagagtcgattcagacaaccatgtatgtcgtg
gtttgatagcggttggttgtatgagcgctgagctctcatacatccggatatagaacgagacatgtactttgtgtttgaaacaggcacacccagtc
ctggcttcttgtcatcctgagaaattatatctagacagtttttatgttcatgttaaatgatatgtgtttttttgaacacgctgtatgaacaccatggcgac
atgccaccaactaataggatagccacattgatcagtgacgctgctaggtttgtatccctgtagtcctattctcatacggcatcgttcctgttcatca
gtcacattctgacccctgtgggtgcaattgacagattgtacaagactcagagagctacgtgcttcttctgccgagaggatgaaccaaaggcct
gatgaaaggttccaccgtgactcacctttccttattcacaaacactttcaagctttagtctaatcaggagttgagattacatgtgaagatgcatcgt
ccgaagccggagaggatcgagtcatggcctggactatagtctactagattattacatcttatttcccctctctgaaccttgtttggccaagtaaagt
accctcaatggtggatgaaaagttaccgagtataaggcattgaaccttgagagataattccctgatagatataattcgcctcctggtaaatatact
cctgaatacatctgatcctaatagcttagatagtgctttcaaaacggaattgaattattatactccgtccgaaatcacacgatcatcctgaccggtg
ccagattcggcgcctgaatggttgaagaagagtttttcaggctgcgacccctgtctgtccagcctccggtgccggcctgccctcttagtcagca
tcccatgagtatttgacctttccctgctggtcaatcaacatctggaatctccattctttcatccgtccactggtgttgtggatattacaccctgttcgg
agaccataatttcatatggtcaagatataaaacagatagcccccgcccactccacagacaccccgattcccgacttccgattcccgaagcggc
ggtgtcgatcttccccaaggggatacatgactcatcagactcgcgccccccagcctttctatatctcgatcccaacgaactccaccatcgccca
cttctgattctgtggtatacctttttttttttaccgactcgcaccatgctccgaagtcgccaggcagcaaccgccctgagggccgtgggccagac
ccggccattgcggtcccagacaccattggccttcacgcagtcgctcaacaaggtccctgtgaatcgcaggaccgaggccacggcggccac
tgcttcttctacggcgtcgtatgtgtcttgtctggaataacatccctttgagaagagaagatgagggctgaccgtctgcttgagtggggcgatca
atagccaggtccgacccactccgagcccgacgttcaatcagtatgatagccaggtgcagccgttgacgggcgtgtcgaaaaatgtcacgga
tgagtcgtatgttcctatatcgtgatggtgttgagaatggaagctaattggtgggtggtggaattaggtttatcggcaagtccggtggcgagatct
ttcacgacatgatgctacgacagggtgtcaagcatatctgtacgtgtcgagaatggaaaaaaaaagtgcacggcatgacacagggctaccat
agctaatcatggatgaatccatcatgcagttggataccccggcggcgccattctccccgtcttcgatgcaatctacaactccccgcacttcgact
tcatcctccccaggcacgagcaaggcgccggccacatggccgaaggctacgcccgggcatctggaaagccgggcgtcgtcctcgtgacg
tccggccccggcgccaccaacatcgtgacccccatgcaggacgcgctcctcgacgggacgccgatggtcgtcttctgcggccaggtcccg
accaccagtatcggcagcgacgccttccaagaggccgacatctgcggcatctcgcggccctgcaccaagtggaacgtcatggtcaagaac
gtcgccgagcttccgcgccgcatcaacgaggcgttccagatcgccaccaccggccggcccggtcccgtcctcgtcgacctgcccaaggac
gtgacggccggcatcctgcgccgggcgattccgacggagagtgcgatcccctcgctgccgagcgccgcgatccaagacgccatggacct
caaccacaaacagctcgaggcctccgttgcgcgtgtcgctaagctcgtcaacatggccaagcagccggtcatctacgccggccagggcgt
cgtccagtccgaactcgggcccgagctgctcaagcagctctccgacctcgcgtccatccccgtgaccaccacgctgcagggtctcggcgg
cttcgacgagctcgactacaagtccctgcacatgctcggcatgcacggatccggctacgccaacatggccatgcaggaagcggacctcatc
atcgccctcggcggacgcttcgacgaccgcgtgaccctcaacgtcagcaagttcgccccgggggcccgcgccgcggccgccgagaacc
gcggcggcatcgtgcagttcgagatcatgcccaagaacatcaacaaggtggtggaggccaccgaggcgatcgtcggcgacgtcggcacc
aacctgcgcctgctcctgccgcacgtcgagccccgctcgctggacgaccgccaagcgtggtacaccaagatcgacgcctggaagaagag
gtggccgctgtcggactaccagaagaccgagcgccacgggctcatcaagccgcagacgctcatcgaggagctgagcaacctctgcgccg
accgcaaggacaagacgtacatcacgaccggcgtcgggcagcaccagatgtggaccgcgcagcacttccgctggcgacatccccgcac
gatgatcacctccggcgggctcggcacgatggggtacggcctgcccgcggcgatcggggccaaggtcgcccagcccgacgccctcgtc
gtcgacatcgacggcgacgcctcgttcaacatgaccctgacggagctgtccaccgccgcgcagttcaacatcggcgtcaaggtcatcgtcct
caacaacgaggagcaggggatggtcacgcagtggcagaacctcttctacgaggaccggtacgcccatacccaccaggcgaacccggact
tcatccagcttgccaccgccatgggcatccagggccagagagtggccgatccgaccaaggtcaaggagagcctccagtggctcatcgaca
cggacgggcctgccctgctggaggtgatcacggataagaaggtgcccgtgttgccgatggtgccgggtggatgtggtctgcatgagtttatc
gcctttaatcctggtacgcactgattcccttattttgctggttgtattgttggttgtatatgaggctaacgattttacagaagatgaaaagacgcgtc
gtgggctgatgcgcgagcggacgtgcgggcttcatgggtaa
SEQ ID No.2
atggaatcttcaccgggtcttcacataattatcgtcggtgcaggcatcgccggcctggcgacagcaacgtccctccgccgcgccggccacac
cgtcgacctgtacgaaaagtcagcacaagacaacgaaatcggcgctgccatctacgtgccacccaatgtcagccagttcttgctaccgtggg
gtctcgacgtagacaagtggcgctttgtcaagtcgcaaaaggtctctttcctggaccatacctcgctggaattgaaaatgacgctgtcggatgg
catgaccgccaaggaggttgccggtgcagagctgtactatgcccaccgcgtcgatctgcatggctgtcttagatggatggctacgcgagctg
aaggcccggggaagccggctacgatccacctcatgagcaatgtggtggcttatgaccccttgacgccgtccgtcactctggccagcgggga
ggtcattactgcggacgtcgtcataggcgctgacggcgtgcgctctggtgccgtcgaggccatcatcggcgacaaggtccagacaatgcgc
ccgaggtttgctaacacatgctaccgctttctggtgcctgcgagtgcaatcgaggcaaatcctgagacgcgattttggaatgaggactctgacg
gctggtcccgtgtcatgatcgatagcgtaacggggcgctcggtagtgtcatacccttgccggagcaatactatacagaattttgttctcattaata
atgaggagaatgatacaaatatgtgtgcagaagattggcacgcccgcttcaaaatcccagatgttctcaagaaattctcggactacgacccca
gactgctcaaggttttaagcaaagcgcccgatgcacgccgctggccactgatctaccgcaagcccattcaccaatggactaaagggtgtatg
actctggctggagatgcttgccatgctatgcttccttttctagctcagggcggtgctcagggcatagaagacgccgtggcgcttggggtcatac
ttcacaaggcgacgacgcgtgatgatatcgagaagcggctgcaaatctatcaggaagtacgcataaaacgtgcttctgtaattcagatccttag
caacatgggtgcagatcactccgtatcagttgaggatctgaaagaatatcttcacgatgaccaaatgccacgcggccagcatgatatgatgat
ccataattataagtatgatgttgcaggtgctgcgatagacgcaatgaagatatatgatccttcatttacgctggcagatgagtttcttggctggtagSEQ ID No.3
atggatgtcatacatttccactttattgtcacaaaaacgctacaaagcgtgaatctcgacatgacacgagaacactgcctggaaggaaaggac
atcgttgtcgctggagcaggagtagctggtctcgcatttgcactcaacctgcagaaacaatggggagatagtggccatccgccgaaggtggt
cgtctatgatcgcgacaagcgagaagtcgacctcagacggcaaggatactcacagacgctgagtggcatcaaagaagacaccgggctcgt
tgtcttgaagcaattggggctgttgagcaaagtcatagatagcgccgtgatgacagacgccatcagcaaattcacgatatgggacaaagattg
gatcgcttcccccagcttcaatgtgccagcttatggcgacatgccgacctccgctgtgcgcatcccccgacgcattctgcgaatgatactcatt
gaagctgcggaggctgccggtattgagattcgttggggcgtatcatgtgaagtgcctgaatgcctcgacggcggtcgagtccgcatacgcct
ccgcaagacggaggacggtggaagcaccgaagcaagaacgtgcgatctgcttatcgtcgccgacggcgcaaatagcaagatccggacca
gcttccgtccagatgatagactgcaatacacgggcctgatgatgatgggtggtgtcgccgaattccccgacggcgccgttccagcgcacatc
acagacaaatggggcatggttctcacagaccagggtgtatcctgttttttgtcgccgacaagcaagtttggatttcactggggcctcagctggc
gggaacagagtcctcgtgcgcctccatcaacttggtccatcgagtatacccagggcctaaagtcagaggctctccggcgcggccaggtgatt
gaagagccttttgctaccatcgtggagagaaccgacccgtccaccatgtttatcatggcaggaatggacaagcgaccttttagccacgagcac
ctgagaggcgtggtctttatcggcgacagtaatcacgccttttctccactggcgggcaacggcgccaacgtggccctcaaggacggctatga
cctagcggagcaattgtgtaaagcgtcatccatacaagacgccatctcgcagttcgaccgtgagagtgtcccgagagcccttcgtacactcga
caggtcgcatgagcgcatctccagcgcacataccacgcagcttgatcgtgagcagtttacggacggttcaggcgcaaatgactttttagtggg
acagagcaatagttga
SEQ ID No.4
atgcagatggcagaaatggcatgtctacgtatgtttcagaaatggaagctcctggacaagatccccgccagtggcagcatttcatatgaggat
ctctctgccagcatcaacgccgataaaaacctcattgcccgcatgggccaaatgctcgtcgcgacaggcaaactgcgacagccctccccatc
gcacgtggcgcacactcgcctgagcacagccttcgcccatcgttcgcctccagccgtatggttctcgatgagcttcgacgagaccctcggcc
catggacccactggccgcgctacttcgccaaatacggcccgcaacagccaagcggccagaccgctgtgcccatgacattcgcagagggg
gtagacggcgagttgacgtgctacgaggtcattgcgcgcggtggaccggagcgaatggctgactttgcggatgggatgcaggggatcccc
gaactgatgcctgctgctgggatatatgactttgagtgggtcgggcaggcagttgcgaaaggcgaggtcgatctggatgtgccgctgattgta
gacgtgggggggaaccttggtcaggcgctgctggagattatcgcgcataccgagtcgtcgattccccctgacaggggcagcctcttaaagg
agccttga
SEQ ID No.5
atgacgtctatcatggagacgaaagtgctcacgctggccgagggctccatgagagttgtggaaaggctcacagcacatcctatcttgaccctc
ttcgcttttttttgtgcagctgtcttgctctggtacattattccgtatttcacctcgccgattcgtggccgacgtggtccattgcttgcttccttaacaag
ttactggcgcatgtaccatgctgcaggtggatcaatgcatctcgtgagccatgccttgcacaaaaggtatggaccggtagtgaggatgtcccc
aaactacctcgatctcgactatccctccttgatcagtacgtgtctcgatagtcatggtgtttggaaaaagacagaatggcacggcatatcaggcg
tcaagctgggtaacaaaatcctatacaacatcttcagcgagtgcaacccggccgagcatgcgcggatcaaaaaaccagttgccaaatatttct
catccacaggcgtcagcgtcatggaaccgcacgtcaacagcgtgctttcgtttttcgtcaagcagctggatgaccagtttactgagcgttctgg
atacggcaaacctctaaagtttgacgagtgggcttcgttttacgcctgggacaccattgcgcagagcacgtggagcaggcgagcaggacac
ctagagcacgcctttgactttgacggtatggtggacacgtctgcgaaagtcatggactatctcgtcacagtcggaatgcaacctgttctggataa
attcctcgacaaaaaccccgtctttcgcttcggcccaccgagtttcgtgccggtggcgaacgccgcctttggccacctcacgaagcgacgca
ccggcgaggacgaccacgatccatccaagcccgactttctagactgttatctggacgccatgcgaaagtaccccgacgtggtggacgagcc
gcgcctgatgtcgtacattcttgtcaacgtggctgccggggtggacacgacggccacgacactacgcgccatcttctacctcgctctgaagga
ccggagggtatgggagaagctggaagcgcagattctcagggcctctttcacagagctgcccgttccttattcgcaggcgcgcgccgtgacgt
accttgaggctgtcatccgcgaatccatgcgtctgtggccggggagttgcttcgcgcaggagcggtatgtaccgcctggggggctgatactc
cccgacggctccttcgtccccgagggcgtggcggtggggttcaatgcctacgtgatacaccggaacaaagacgtgtggggcgacgacgct
gaggattttcggcccgagcgttggttacagggtgaagatgagtcggcggagcggttcaaagagcgactgcgcgtgatgaacagcagtgac
atgtcatttggggcgggcagtcgcaagtgtcttggtgttaattttgctaccatggaggtctataagacggtggcgacgttgattgcggtgtttgag
tttgagttggctgatccagccagggagtggaaggtacataacagcatgttccctaggcagtctggcattgccttgaggattaagaggagggaa
ggggtacatgtgccattggggatggatctcggttaa
SEQ ID No.6
atgtccaacaatccaaagattattcaaatgtgtgtggcagggcagaggaagaaagggtggtcggacgagcagttcgcccatgaatttacagt
cgtgcatgcggaaatcaccaaagcaacagcccagaaaacaccagcactccttggctatcgacaaaccttagcaatcccaagaccgagaata
tcagcgttcaacctgggccacagcaagtgggattcccaggcagtgttgacgtggtcgagcatcgaagaactttcctccctcctcaaatccgaa
gggtatcgcgccaacgcaggaaaccacgtcttcaccgagccggatatagtgggatcaatttgccaagttgcgggggagttcatgtttgatccg
gtcggatacagcagccaggaatcgcgctttatggtctttgtttatatcccgcgggcgatcaggagcagtcgggagctggttacagaggaaga
agtggctcagagacttgatagtatcacgagaatcggtgctggaacgggtctcttaagatatgttatcaatcgcgacgtgagcccgtctgatcca
ggtgaactgtttgatggcactccgttcatcactggtgattggggggtgatgggcgtgacggaacaatattggtttaaggatgcagatgcggca
gaggccttctttgcggatgaggtacgagtggacgcgttaatgggagtacccagctcactggacagtacgagctgcgttgctgttgctggtcaa
gaaacggtcctagttagcaaataa
SEQ ID No.7
Atgactttcaacatatacatccccgagaatcttgcgttctccaactatattgcagtgatccaggtagcgagaacctgggcggatgcccaagac
agaaaggaccccgaacgattcctcgccaccgtagcaccccaagtcaccatcgactacagccttctcatccccgcatggaaaagcaaagtcta
cacggctgacgagttcgtcgcggcatggctggcccccgatcgggtcggactgtccgttctggcaacgcagcacctgctaggaatgccatac
atcaagagcgccacgccggatgagattatcgtggagtttcaagaggtggcctcgcacgggcgacgccaggatgacgacggcgcgttcgg
cggcaatatcggagagacggctgatgggagggggtgggtggagcatcgttacgtcaagattgatggccagtggaagattgatctcatcaag
ccttctataatctatatggcgggggattgggagcgagttcggcgggctgagggggcggagtaa
SEQ ID No.8
atggacccagaagttagcaaactgctcgcgacggtggcaagcctcggcgagacagcaaagaagcatgctagcaccgacaaggacagcc
ccgcgagccgcgagtcaaggagagagctcctgagcgcggcgcgacaactgacaaacgagctgcagaatgagggccaaattgtagaagg
atatctttacgggacactcgacccgctgttgctcaagatgggcattgagctgggcattttccggaatctggtggacagtactaagcccgtgacg
ctgggagatctagtggccgcttcgggcgccgacgaggtgctgttggctcgcattatgagggggctttcatcgattcatgcggtaaatgaggtc
ggggttgagctgtatgagcctaataaggttacccgtgcttttacgactgtcaagggcgagtcagggctgaatgtttttcacaatataaaccaccc
cggctggcagtccctgcccgaatgtcttcgagcgatgggctacatgaaccccacagatcccgccaaaatgtggttcggcagacagttcaatg
gtgagcactacttcgactggctggggaaacgcccggagctcctccattcattccaccggttcatgtcaacgcagcgagacggacatgcccac
tggctcgacttttacccgttccagcaacagctgctacctgattttgacgtggaaagccccgacgccgttttcatggttgatgtgggtggtagtgta
ggtcatgagatccaggaagtcaagagacgctatccagagattccaggacgcatggtcctgcaggatgttccggccacaattgcacgagtcgt
cccagaaaatgatatggaagctatgtcgcatgatttcttcacaccgaatccagtcaagggcgctcgagcatactacctccgcaacgtgctgca
cgactgggacgatgaccgctgccgcgtcattctgaagcatatccgagacgccatgactccgggatattctgtgcttctgatcaatgaatttagta
tacccctcaaaggcgcctgttcgttcgccacccattctgactttatgcttatggctattaatgctgctgtggagcgaactgagcaacagtggtacc
atcttatggagtcggttggcttgcaaattaaaaagatatggacgctagagccggacactgagagtctgctggaggtcacacgtggggaatagSEQ ID No.9
Atgagattccttggcctcagcggtggctccccggggggcaacaccgaacaagctcttctggccgcccttcgagcagcccaagagacggca
acagcgccggcaaccatctccttgatccggcttaaggatctttcaatcggttttgaagccctagatgaccaccttccgctgcccgtagtggggc
agtcgagcccaacctcaggaacagtccccgatgaccgtccttttatgctcgatcagattatggaagccgacgccatcatcctcggtgctccatg
catgacgcggaccgtcccatgggaagtcaagcgcttccaagacagcacgctgggcccgttccaagacgtgacgatggcgaaaagactagt
cgacacaggcaagggccacctagtcgatcaacgcatcttcaagccgagagttctcgcgctggtgacgctgggcggcgcttccacgaccga
gtgggcgccgtttacgcttcctctactacaccaggtgtttttccctctcggcgcccagatcatcgaccagatgcaggtgtttggcacgggcgtc
ccggatagttttctttgcaacggcgaggcggtcgctagggcggaggaactggggaggaacctggcgcggcaggcctgggcgacgacgg
aaggggaggctacatacgtcggtccgcggggcatgtgtccggtgtgccatttgagtatgttcaattttgtcggtcaagatgctgttgactgtgct
acttgtggggcgaagggcaagatgggcgtcggtgacgatggatgtgtcgagtttgtgactgacgccgatggcgagagctgctctgtactcag
gcgggcgggactggagaagcatttgcgggatcttgagcaagggtttcaagtggaaaaccctgaggtgtcagatgttaaagaagaatttgtga
agctcggtcagaactgggtcgttgcgccgcccagccgagtcacgatgtaa
SEQ ID No.10
Atggccaagtcaaacgcaaaagatagcctcttcgcgagctacgacgccgaatactggaacacctacctggacgctcgtccaacatactctc
ccgacttctacaacctcatattcgaccaccacagccacaaaggcaataacagctggaccttagcgcacgacgtggggaccgggcccggga
acgtagcggccgtcctcgcggagcgttttgcccaagtcatcgccacagacaccagtcctgacaatgtcaacgccgcacggcaacgccaact
acagacaaacaagatcaggtttgcagaatgcaacggcgaggacctagcccgcgcggcgctgtcaccccctcgcaccgccgacctcgtcg
ccaacgccgaagcgatcccactcatggacgccgaggaagcgatcgggtgctttgcggagctgttggcgcccggtggaacgtgtgccgtgt
ggttctacgggcggccgacgtttgcaggacctgatgccgctgtgaatgaggcgtgtcagcgcatcttctaccgcatctcgacgcggttgctga
acaagataggcggcatgagcgggccgctttgggagcggtctacgcgcactattgcgtcgcagctggataatgttgcgtttcctgcggagcag
tggcgtcatgtggttaggtataagtggaattgtgagcagacttgtatgctcttccatgatgagagtcagtttggcgggccggttgagcgggtga
actgtgttgggccggctgaggaggtggtttcgaagacggaccctgggttttggcagatgcagtggggcgctgctgaggtgcggaggtggttt
gaggcaaatttgccaacttggtttgaagataaggctcaggatctggagctggagagttgctatgaggagttggatcgtgtcatgggaggcgag
tctttgccggtgacttggcctgtggttttgctcctcgcgacacgggtgtaa
SEQ ID No.11
atgagagttgcagtcattggaggcggtccggccggactgaccacgctcaagcatttgcttgaagcgcacgagtttgttggcggggatcccgt
cgaagcgaagctgtttgaatcggaagaagctataggcggtacctttctcaagcgcatgtacgaagacgccgagttggtttcatccaaatatctc
acagccttttccgacttccgggcccgtgaagacgatcccgattacctgcctgcagcgagatacctcgagtacctcggtcagtatgccacagcg
ttcaacctgtggccgttcatccatctgtccactccggtgactgccgttcgacgcaaaggtcgctcccatgtcatcagctatgccacgccagatg
gcaaggaggaatcatggatatgcgacgctgtcgccgtctgctcgggtctgcatgtaacacccaatataccgtcaatcagcggcatcgacaag
gttcccacgacctttcactcgtctgacttcaagtcgcgcgagcagtttggctccgataaaacggtggttgttctaggcacgggagaaactgcca
tggacatcgcccatctggccgtgacggcgccaaccaaaagggtcgttatatgtcatcgcgacgggttcagtgttgttgccaagcgaacaccg
agccccgttgtattcccttccctggcaagccatctgcagtcgccgaaccttcccgtgccggtagatacatatctacacgctagccacaaatggg
gaaacctccccggaaatatatttgacagtttggtcaagcagggcatgtggctcatgactggcacgagtgccggctatgatcagtgggtgggc
ggttatccttctccacgctggcatacatcgaatgtcatcttcaccaagtctagcaaagccatgccctacatcagcaagccatacagaaaagaca
cgatattccaacgtttgcgctccagtatcatccaagtgccaatccccgagacacacgaccggcacatcgacatagcgccctggccatcccac
atcgacagcaagggcgtgctccactttatggacaacggtcgaccagagtatcgacgcatgaaagaagctgaggcagtacgacccgacgtg
ctcgtcttcggcacaggctatacccaacgcttcgacttcctggacgcgacatacccaagcactgccgacctagacgtacgagacgtctggcg
tcgcgacgagccaagcattggcttcatcggattcgtgagacccggtttcggcgcaatcccgccactggcggaactgcaggcgcagctttgg
gtggtgaatctcctcgcgcccgagcgtctccagccccttcttccccaggacgagccgcactatcgacttggcatgccttcggatgcgcgcatc
aagtacggcgtcagccacgacgactacgcgaatcaattggctgtcgatatgaatgcttcaccgtcttttctgcacgctgtgaggatgggttggtt
gcgtaaggagtggtggcggcttccgcttgtttggcttcttgcggctgagttcaataccaagtttcgcttgtgcgggccgtggaagtgggatggt
gcggtggaggttatgaccggggagctgtgggatgttgtgaagcggcgaggggggtttttcaaacagattgtgctatccggtgtaccgctcgt
ggtgtttggttctgtgcatctcttgttgtggatgttcacgtgcttatttgcgccatccagatggtag
SEQ ID No.12
atgaaatctcacccccaagctagggaatcaggttgtcgtagacaaggggtatttaagctcgtcagtggtggaagccttcgtaataacgcattgc
cacagactacgatcctctaccaacctcatttcaataacacccatggagccaaagttgtcctaaactaccacgaggacatgtctcgactcgagtt
gataccagatattctaatccgccatgccgacgagtctggccggaaggtcgctttcgcaggtccaggatggacaataacgtatggcgaccttga
acaacggactaggcgtctagcagcccatctagtccgtgccggcatagggcgaggtcaattcgtggccattgtactaggcagatgtcttcaga
ccgtcgagtccataatcgcaatcacaagagctggtgccgtgggagtacctctggattcgcgctcgccctcatcggagttggctaaagttttgga
gcatagcggcgcgcgcgtgatcatcacagatgaccgccacttggctacggtgcacactgctgctgcggaaggaagcttaattgttttgaatac
caagcttcccaatgtgaatgccaaagatgaagggcatgagcttgtgcgataccaagactggattgaagacgaggagtgctcgaccttggata
tcaagatcgacgatctcggggaggatgaggaagcctttctacactacacttctgggacgaccagtttgcccaagggggtactgtccaaccag
aagagttggctgttgaatgtgaagagcctggtgtcagcgttcgaactgacgcccgaggaccgcttcttttggcctcttcccctgttccactgcat
cggccacttgttgtgtatcatgggaactgtggtcgtcggtgccagcgcatatctccctgatgccgatcagacaatgcttgacagcctcagagat
acaaacgctcgagaaacaaccctcattgtgggcgcgcccaccaccttccacgacctgatagatgccgcgaagcggtcagatccgacgtcg
cccttgtctttaccgaggctgcgagcatgtatgtatgcgggttcctcggcatcagggtctctaggtacccaagtcaaggagttgctcggtgttcc
ccttctgaacaactatgggtgtaccgaaggttgcggctctatcgctgttagcaaaacgagccacacctatcgcgacaattccagtatctcgcta
ctgccgcactgggagattaagctagtggacccggatgggaacccagtcaaagatggcgagcaaggcgaggtctgcataggcggccccgg
tctgatgctcgagtactaccgagagacgcggacgccgtttacgccggatggctggtaccctactggcgacatcgcaattcgctcgagctcgg
cggctgatgcagaattgaccttggtgggacgtaggaaagagatcattattcgagggggcgaaaacattcatcctcatgagttggagcacgtct
tgcttcggcatcctggcgtggcggatgtcgtcgtggctggaatgccacatagactgcttggagaaactcctgctgcctttattgtgaagagcgc
tgcggacgtggactttgacctatcggccttgcttgctgcgtgtcgcaaagtcttgccagattataagatacccactgccttttatgagatcgacac
cgttcctcgaaccgtcatcggtaaaccgaaaaggctgaccatgacatcttacacaaacaaaccactcactgcgcgatctattttgcagtcaaga
gattcaatcgaagcactagtaatggcggaaacagtcagtgcatgcactataggcgccgaacgggagggcgagtcaaacacagactggcttc
gtcgaaaccttgaccagcccttttcgtttctgggcctgagctcaatggctagtgtggtcctccgtgaccgacttgccggtctcactggtttggctg
atcttcccaatactctcgtgtttgactatgcgaccccggcagctgtgagcacgtacctgtgtagccgcttgttggagccagaatcgacacctctg
ccccgttcaacaccgactacgacgtcggattgtgaagtggagcccattgctattgtatccatggcctgtcggtatcctggaggcatctcttcgcc
ggaggacttgtgggagctagtttcagatgaaattgacgcgaccaccgagttcccagacgaccgcggctgggatatcgacgcattgtatagca
ctgaccccgacactcctaatacctcaaccaccaagcgcggcggtttcctgcccgatttcgcccactttgacgctggtctttttggcatggcgcc
gcgtgaggctcttgccactgatccccagcagcggctcttattggagacgacgtgggaattggccgagcgagcgggtattgctccgttatcgct
tcaaggaagccagacgggcgtgtttgtgggtactctgtatgaggattatgaagagaacggctttgggaacaacgagttggaagcacaccttg
gactgggctcgtccagcagtgtcgtctctgggcgtgtgtcgtactgctttggtctccacggtccatctctagtcgtttcgacaggctgctcgtcct
cactagtagcaatccacctagcggcccaatccctccgaaatagagagtgctcgctgaccatcgcgggaggaataacagccatggccacccc
acgcccgtttaccatgttcagcagacgacgaggcctttcgtcggacgggcgatgtcgggcttattcaagtgatgcaagcggtactggctggtc
cgagggtgtaggcctgcttctcctcgagcgactctcggatgccaaacgcaacgggcatcagattctcggtttgatccgtgggtccgctgtcaa
ctctgatggaaaatctaatggcttgacggcaccgaacgggccggcccagcagatgtgtatccagagcgccttggcccaggcggggatgtc
gccggaaaacgtggacgtgctagaaggccatggcactgcgacgcccttgggtgatcctattgaggttcaggctgtgatttcggcgtatggga
atggcgatagaaagaccagcgacagcggcactcgccgtttggaaccgctgttgctgggctcgatcaagtccaacatcggtcacacgcaagc
tgccgctgctgtcgcaggaataatcaagatggtgcaggctatgcgccacggcgttgccccgacgtcattgcatatccgtgaaccgtcacccc
aaatcgactgggaagggagtggtgttgagctgctcagcaaggccaggcagtggccgtctgtgaatagacccagacgggctgcggtgtcttc
atttggtattgggggcacgaatagccacatcattctagagcagcctgagcctgccgaggaggaagactccaattccaagagaatatccgcca
ccgttccctggctcatctccggtgcaagcgaggctgcattacgcggacaggctcatgcattactggcagcttggagacaagacgataacaca
tttagctccctccgtaatcaggatcctaccgacatagcgttctcacttgctactgcgcgatccgctctcaaatacagagccacggtgacctacac
gctgggagtgaacttccacgatcaggtcgaaacagcactcaaaagccttgcccaaggcgaaccacaccccgacgtcgtgacagcacacac
cagtactaccggtagtcagccccgtctggcgtgcttattttcaggtcagggcagtcggatgcccagcatagacacactcgccgagttacgcgc
taccttccccgttttctccacggcattccaggcagcttgtgacgaagtggaccagcacttggagtgcccactcgtacatgctattggtaacagca
ttctagaccgcaccgaatttgcacaggccacccttttcgtctttgaggtggctatgttccgcctcctggagtcgttcaatatccgtccggactttgt
cgctggtcattctctgggagagatcgccgcggcgcatgctgctggtgctctgtctcttcgtgatgctgctactatcgtcaccacgcgagcaaag
cttatggcatctctaccacccaacggcggtatggtgagcatatcggctacggaggtggaggttgccagtgagctggcacaattgggcggca
gtgccacgattgcggcagtcaactcgcagaactcggtcgtggtgtctggcactcaagaggcgatcaaggccgtcgcggacatgttcgccag
cttaggacggagagcaaccgtgctgcgaaacgtcaaacatggctttcactccccgttgatgaacggcatactccccgacctagagaaagcg
ctcccatcgtcaatggaagacgacagcccgactgccataccgctcgtttccactaccacaagcaagcgagccgacgcagctcagcttcgctc
ttcagcccattggatccgccacgttagcgagcccgtccgctttgcagacgcgatcgacgagctcaagtcaaacgaacgcgtctctgtttttgtg
gagattggcccgtcggctgtcctgtctccacatgttccggaggccgccgctacacatggcactgttgacaagctacttagaatgctgggtcag
ctgtggactcggggtgtaccggtcgactggcaagctgtctttgagggtagtggtgcgcgatttgtcgacctgcctgtctatgctttccagcggc
agagatactggcttccctacacgccactgctgccagtggcatccactggagcagtcgcacagcaggctcaaggacagaccgtgggcgctg
gtggtgactcgcgactgcgccacgggatgctcttcaacgccacttctatccctggaaccggcactgtcatctgctctgggtgcctgtcaacga
cccgacagccttggcttcgtgaccacgttatcggtggacaatcgctcgttccggcctcggcatttgctgagctagccatgcgagctgcacaag
aatgtgcagagtataccgaatcttactcgatggtactggatgagatggtccttgttgcgccgctggccgtctcttcggcagaggacgaggaac
aagaagattcatctggcgaattcgagattcaagtactcatagcttggtcgcagcgtgaggacgagggaactgagacccaaaggaccgtcga
ggtgtattcacgccctagcggcgtagcaacccaacacgaatggacacaccacgccacgggttctctcaaactcacaccacagtccaacgcc
gaccactcttctacaaatggcacagattctacgagaacggagtcagatgtggacatttccgaagcgtacaccatcttagccgatgcaggtctca
catacgggccgtctttccagctcgtacgtgccatttggcgtctacgtgacgatgacctgctcgtgcagatcgatccacccgaggatcaggctc
agatgtcgacgttcgtcctgcacccggctgtgctagatgctgcgctgcatgcgtctacccttgcctcggcggagaaggtcgctggcggtgac
attcgactgcctttctcgctccgaggcgtccaatttttgacgacggctggtgcctctggccccattctagcacgcatcagtcaagatagcgaga
acagtttctcgttgaccctcacggacaagtcatcgggtgtgctggtcgctacagtttctgaagtccagctgcgcgcctggcagcctgctgttgct
ggaggtgatctctaccgcctagaatggagggagattgagtctaagcccattcaaacaaccacgtctaccgagaccgacaagattgttcgggt
ccaaagcgctcgcgatattgatgcagcagcagtcagcaaagctgtgcacgaagctgtcgccgaggtgctccacacgattcatgagtggagg
gctgaaaatacacatgctggcgatggtgtccgcctcgtctttgtgaccgagagagcgacgtcggctgacaaaagctccaatatcaatgtcgtt
gctgccgctgtatggggctttgtacgatcagcccaggcagagtttggcggggaccggattgtgctcgtcgacatggatgggacaaccgagtc
ggaagaggctctatcggctgcgcttgcttccagggaggaagttgttgctgtgaatggtggaaagattacgattcctagaataagtaaacagcct
ccgatgccagagtccccccaagcgatggccctcgatgtcagcgggactgtcttgatcacaggcggtacaggcgggctgggtgcaacactc
agtcgagacattgtgcacactcatggagcgaaaaatctgctcctcgtcagccggtcaggtattgaagcgcgtggagcacgcgagttatctga
agagctgcaaagtgcaaacgcagctgtgcgcgtcgaggcgtgcgacgtcggtgactacacccagctggccacattgctagacaaccatac
ccagcatcaatacccacccatcaccgccgtcatccattgtgcaggaactgtcagcgacgcattcctgggctcccaaaaccaagagcgagtat
ctagtgtgctacgtcccaaggtcgacgccgcatggaatctccacgaactcgtccccgagacggtccgctcgttcgtcctcttctcgtcgtacgt
cagcgtcctcgggaacgaaggccaggcggcatacagtgcaggcaacgcgttcctggatgcactggcccgcttgcgtgtcgcacgtggact
gcctgccctgtcgctggcttggggcccgtgggcgaatgaggccgggatggccggcggaagcaaactggccgcgattcccccgcgcatcg
ccaacgctcggccgtttacggatcagcaaggactgcatctgtttcatagggcgttgcatgtgcagacgaggaccccgccggagccggttctt
ctgccgctgctgctgcgcgggccgttcccgctggtgccgtctgctggtacggagtccaagcccaaggagaagtcgtctgcgaggggtgggt
cggcatctggtgctatatggcgcaggagcatcgctgcagtgtcattggagaaccgtcacgacgctctgctgggcctagtgcgggatgagatt
gctgctgtgctgggctaccagagccaagatatgcttccggatcagcgacttgaagacctcggttttgactcgtttacttcagtcatgctcacgaa
tcgactgagggttttgactgggctcggctaccttcctgttacactcgcgctggattatgatactacgtcggcgctggtcgagtacctgttacctcg
cattgaagccgagccacagccacagccacagccagaagtcgacatggactcaaatgcatccacgaccggcggggacacgtcagtttccca
tgacagtggaaaggcggacgggttgagcccatcgtctagtgtgactacactcgctccggaggagcacgacgacctcaaccccgagactttc
cgagggctagccaccatccaccgacggctgtctcagttagagcagtacacggcggcagcagatctcctcgcctcggccgcactggcaatg
ccgaccttccccaagaccgggtcgagtctgcccagctacgcagcggagccccagcgcctagcgaccggtccatcgggcacctcaaactca
gaaccgcccctgccgctcgtcttcatcgcgcctttcttcccgcgcatcaaggtcgagggcattggcctcagcgtgtacagcaatctcgcatca
gcgatgaccggaaaacgcgacgtgttcgagcttccccacccagaagggcaggtcgtcccaggggacctcgccacgctggccgacctgca
cgtccacaccatccgcaagcacttctcggaccgctcgggcattctcctcgcgggttactccgcggggggcaccgtcgcatacgccgtggcg
tcccgactggctcacgccgcagacaggcagcctcggctggcgggcttcgtcttggtggacacgtatctcacgatgacgggtcggggggatc
ccgactggctgaacgcgctgccggcggaggccctcgtttcgcgcctccaggttccccccagcttgggtcaccccaaaggcatgggcagtg
acagtctcgtgggcgacttggacgtggcgctggcgaaggtgggtgggtattttaggacgctgcgggactgggatctcgagctacatccgctg
cctgatgcattgtcgacgctgttcgtgcgtgccgtggatgcgtcggacaagatgcccaaggattcggacgtttggcgtccccgatggccgcg
ggcggatttgacggttgatgtgccggggagtcatctggctctgctggacaagcggtatgcacctggtgttgccgttgagattgaacggtgggc
gagggagttgaatgcttag
SEQ ID No.13
atgctccgaagtcgccaggcagcaaccgccctgagggccgtgggccagacccggccattgcggtcccagacaccattggccttcacgca
gtcgctcaacaaggtccctgtgaatcgcaggaccgaggccacggcggccactgcttcttctacggcgtcccaggtccgacccactccgagc
ccgacgttcaatcagtatgatagccaggtgcagccgttgacgggcgtgtcgaaaaatgtcacggatgagtcgtttatcggcaagtccggtgg
cgagatctttcacgacatgatgctacgacagggtgtcaagcatatctttggataccccggcggcgccattctccccgtcttcgatgcaatctaca
actccccgcacttcgacttcatcctccccaggcacgagcaaggcgccggccacatggccgaaggctacgcccgggcatctggaaagccg
ggcgtcgtcctcgtgacgtccggccccggcgccaccaacatcgtgacccccatgcaggacgcgctcctcgacgggacgccgatggtcgtc
ttctgcggccaggtcccgaccaccagtatcggcagcgacgccttccaagaggccgacatctgcggcatctcgcggccctgcaccaagtgg
aacgtcatggtcaagaacgtcgccgagcttccgcgccgcatcaacgaggcgttccagatcgccaccaccggccggcccggtcccgtcctc
gtcgacctgcccaaggacgtgacggccggcatcctgcgccgggcgattccgacggagagtgcgatcccctcgctgccgagcgccgcgat
ccaagacgccatggacctcaaccacaaacagctcgaggcctccgttgcgcgtgtcgctaagctcgtcaacatggccaagcagccggtcatc
tacgccggccagggcgtcgtccagtccgaactcgggcccgagctgctcaagcagctctccgacctcgcgtccatccccgtgaccaccacg
ctgcagggtctcggcggcttcgacgagctcgactacaagtccctgcacatgctcggcatgcacggatccggctacgccaacatggccatgc
aggaagcggacctcatcatcgccctcggcggacgcttcgacgaccgcgtgaccctcaacgtcagcaagttcgccccgggggcccgcgcc
gcggccgccgagaaccgcggcggcatcgtgcagttcgagatcatgcccaagaacatcaacaaggtggtggaggccaccgaggcgatcgt
cggcgacgtcggcaccaacctgcgcctgctcctgccgcacgtcgagccccgctcgctggacgaccgccaagcgtggtacaccaagatcg
acgcctggaagaagaggtggccgctgtcggactaccagaagaccgagcgccacgggctcatcaagccgcagacgctcatcgaggagct
gagcaacctctgcgccgaccgcaaggacaagacgtacatcacgaccggcgtcgggcagcaccagatgtggaccgcgcagcacttccgct
ggcgacatccccgcacgatgatcacctccggcgggctcggcacgatggggtacggcctgcccgcggcgatcggggccaaggtcgccca
gcccgacgccctcgtcgtcgacatcgacggcgacgcctcgttcaacatgaccctgacggagctgtccaccgccgcgcagttcaacatcggc
gtcaaggtcatcgtcctcaacaacgaggagcaggggatggtcacgcagtggcagaacctcttctacgaggaccggtacgcccatacccac
caggcgaacccggacttcatccagcttgccaccgccatgggcatccagggccagagagtggccgatccgaccaaggtcaaggagagcct
ccagtggctcatcgacacggacgggcctgccctgctggaggtgatcacggataagaaggtgcccgtgttgccgatggtgccgggtggatgt
ggtctgcatgagtttatcgcctttaatcctgaagatgaaaagacgcgtcgtgggctgatgcgcgagcggacgtgcgggcttcatgggtaaSEQ ID No.14
MESSPGLHIIIVGAGIAGLATATSLRRAGHTVDLYEKSAQDNEIGAAIYVPPNVSQFLLPWG
LDVDKWRFVKSQKVSFLDHTSLELKMTLSDGMTAKEVAGAELYYAHRVDLHGCLRWMA
TRAEGPGKPATIHLMSNVVAYDPLTPSVTLASGEVITADVVIGADGVRSGAVEAIIGDKVQ
TMRPRFANTCYRFLVPASAIEANPETRFWNEDSDGWSRVMIDSVTGRSVVSYPCRSNTIQ
NFVLINNEENDTNMCAEDWHARFKIPDVLKKFSDYDPRLLKVLSKAPDARRWPLIYRKPI
HQWTKGCMTLAGDACHAMLPFLAQGGAQGIEDAVALGVILHKATTRDDIEKRLQIYQEVRIKRASVIQILSNMGADHSVSVEDLKEYLHDDQMPRGQHDMMIHNYKYDVAGAAIDAMKIYDPSFTLADEFLGW*
SEQ ID No.15
MDVIHFHFIVTKTLQSVNLDMTREHCLEGKDIVVAGAGVAGLAFALNLQKQWGDSGHPPKVVVYDRDKREVDLRRQGYSQTLSGIKEDTGLVVLKQLGLLSKVIDSAVMTDAISKFTIWDKDWIASPSFNVPAYGDMPTSAVRIPRRILRMILIEAAEAAGIEIRWGVSCEVPECLDGGRVRIRLRKTEDGGSTEARTCDLLIVADGANSKIRTSFRPDDRLQYTGLMMMGGVAEFPDGAVPAHITDKWGMVLTDQGVSCFLSPTSKFGFHWGLSWREQSPRAPPSTWSIEYTQGLKSEALRRGQVIEEPFATIVERTDPSTMFIMAGMDKRPFSHEHLRGVVFIGDSNHAFSPLAGNGANVALKDGYDLAEQLCKASSIQDAISQFDRESVPRALRTLDRSHERISSAHTTQLDREQFTDGSGANDFLVGQSNS*
SEQ ID No.16
MQMAEMACLRMFQKWKLLDKIPASGSISYEDLSASINADKNLIARMGQMLVATGKLRQPSPSHVAHTRLSTAFAHRSPPAVWFSMSFDETLGPWTHWPRYFAKYGPQQPSGQTAVPMTFAEGVDGELTCYEVIARGGPERMADFADGMQGIPELMPAAGIYDFEWVGQAVAKGEVDLDVPLIVDVGGNLGQALLEIIAHTESSIPPDRGSLLKEP*
SEQ ID No.17
MTSIMETKVLTLAEGSMRVVERLTAHPILTLFAFFCAAVLLWYIIPYFTSPIRGRRGPLLASLTSYWRMYHAAGGSMHLVSHALHKRYGPVVRMSPNYLDLDYPSLISTCLDSHGVWKKTEWHGISGVKLGNKILYNIFSECNPAEHARIKKPVAKYFSSTGVSVMEPHVNSVLSFFVKQLDDQFTERSGYGKPLKFDEWASFYAWDTIAQSTWSRRAGHLEHAFDFDGMVDTSAKVMDYLVTVGMQPVLDKFLDKNPVFRFGPPSFVPVANAAFGHLTKRRTGEDDHDPSKPDFLDCYLDAMRKYPDVVDEPRLMSYILVNVAAGVDTTATTLRAIFYLALKDRRVWEKLEAQILRASFTELPVPYSQARAVTYLEAVIRESMRLWPGSCFAQERYVPPGGLILPDGSFVPEGVAVGFNAYVIHRNKDVWGDDAEDFRPERWLQGEDESAERFKERLRVMNSSDMSFGAGSRKCLGVNFATMEVYKTVATLIAVFEFELADPAREWKVHNSMFPRQSGIALRIKRREGVHVPLGMDLG*
SEQ ID No.18
MSNNPKIIQMCVAGQRKKGWSDEQFAHEFTVVHAEITKATAQKTPALLGYRQTLAIPRPRISAFNLGHSKWDSQAVLTWSSIEELSSLLKSEGYRANAGNHVFTEPDIVGSICQVAGEFMFDPVGYSSQESRFMVFVYIPRAIRSSRELVTEEEVAQRLDSITRIGAGTGLLRYVINRDVSPSDPGELFDGTPFITGDWGVMGVTEQYWFKDADAAEAFFADEVRVDALMGVPSSLDSTSCVAVAGQETVLVSK*
SEQ ID No.19
MTFNIYIPENLAFSNYIAVIQVARTWADAQDRKDPERFLATVAPQVTIDYSLLIPAWKSKVYTADEFVAAWLAPDRVGLSVLATQHLLGMPYIKSATPDEIIVEFQEVASHGRRQDDDGAFGGNIGETADGRGWVEHRYVKIDGQWKIDLIKPSIIYMAGDWERVRRAEGAE*
SEQ ID No.20
MDPEVSKLLATVASLGETAKKHASTDKDSPASRESRRELLSAARQLTNELQNEGQIVEGYLYGTLDPLLLKMGIELGIFRNLVDSTKPVTLGDLVAASGADEVLLARIMRGLSSIHAVNEVGVELYEPNKVTRAFTTVKGESGLNVFHNINHPGWQSLPECLRAMGYMNPTDPAKMWFGRQFNGEHYFDWLGKRPELLHSFHRFMSTQRDGHAHWLDFYPFQQQLLPDFDVESPDAVFMVDVGGSVGHEIQEVKRRYPEIPGRMVLQDVPATIARVVPENDMEAMSHDFFTPNPVKGARAYYLRNVLHDWDDDRCRVILKHIRDAMTPGYSVLLINEFSIPLKGACSFATHSDFMLMAINAAVERTEQQWYHLMESVGLQIKKIWTLEPDTESLLEVTRGE*
SEQ ID No.21
MRFLGLSGGSPGGNTEQALLAALRAAQETATAPATISLIRLKDLSIGFEALDDHLPLPVVG
QSSPTSGTVPDDRPFMLDQIMEADAIILGAPCMTRTVPWEVKRFQDSTLGPFQDVTMAKR
LVDTGKGHLVDQRIFKPRVLALVTLGGASTTEWAPFTLPLLHQVFFPLGAQIIDQMQVFGT
GVPDSFLCNGEAVARAEELGRNLARQAWATTEGEATYVGPRGMCPVCHLSMFNFVGQDA
VDCATCGAKGKMGVGDDGCVEFVTDADGESCSVLRRAGLEKHLRDLEQGFQVENPEVSDVKEEFVKLGQNWVVAPPSRVTM*
SEQ ID No.22
MAKSNAKDSLFASYDAEYWNTYLDARPTYSPDFYNLIFDHHSHKGNNSWTLAHDVGTG
PGNVAAVLAERFAQVIATDTSPDNVNAARQRQLQTNKIRFAECNGEDLARAALSPPRTAD
LVANAEAIPLMDAEEAIGCFAELLAPGGTCAVWFYGRPTFAGPDAAVNEACQRIFYRISTR
LLNKIGGMSGPLWERSTRTIASQLDNVAFPAEQWRHVVRYKWNCEQTCMLFHDESQFGG
PVERVNCVGPAEEVVSKTDPGFWQMQWGAAEVRRWFEANLPTWFEDKAQDLELESCYEELDRVMGGESLPVTWPVVLLLATRV*
SEQ ID No.23
MRVAVIGGGPAGLTTLKHLLEAHEFVGGDPVEAKLFESEEAIGGTFLKRMYEDAELVSSK
YLTAFSDFRAREDDPDYLPAARYLEYLGQYATAFNLWPFIHLSTPVTAVRRKGRSHVISYAT
PDGKEESWICDAVAVCSGLHVTPNIPSISGIDKVPTTFHSSDFKSREQFGSDKTVVVLGTGE
TAMDIAHLAVTAPTKRVVICHRDGFSVVAKRTPSPVVFPSLASHLQSPNLPVPVDTYLHAS
HKWGNLPGNIFDSLVKQGMWLMTGTSAGYDQWVGGYPSPRWHTSNVIFTKSSKAMPYI
SKPYRKDTIFQRLRSSIIQVPIPETHDRHIDIAPWPSHIDSKGVLHFMDNGRPEYRRMKEAE
AVRPDVLVFGTGYTQRFDFLDATYPSTADLDVRDVWRRDEPSIGFIGFVRPGFGAIPPLAE
LQAQLWVVNLLAPERLQPLLPQDEPHYRLGMPSDARIKYGVSHDDYANQLAVDMNASPS
FLHAVRMGWLRKEWWRLPLVWLLAAEFNTKFRLCGPWKWDGAVEVMTGELWDVVKRRGGFFKQIVLSGVPLVVFGSVHLLLWMFTCLFAPSRW*
SEQ ID No.24
MKSHPQARESGCRRQGVFKLVSGGSLRNNALPQTTILYQPHFNNTHGAKVVLNYHEDMS
RLELIPDILIRHADESGRKVAFAGPGWTITYGDLEQRTRRLAAHLVRAGIGRGQFVAIVLGR
CLQTVESIIAITRAGAVGVPLDSRSPSSELAKVLEHSGARVIITDDRHLATVHTAAAEGSLIV
LNTKLPNVNAKDEGHELVRYQDWIEDEECSTLDIKIDDLGEDEEAFLHYTSGTTSLPKGV
LSNQKSWLLNVKSLVSAFELTPEDRFFWPLPLFHCIGHLLCIMGTVVVGASAYLPDADQT
MLDSLRDTNARETTLIVGAPTTFHDLIDAAKRSDPTSPLSLPRLRACMYAGSSASGSLGTQ
VKELLGVPLLNNYGCTEGCGSIAVSKTSHTYRDNSSISLLPHWEIKLVDPDGNPVKDGEQ
GEVCIGGPGLMLEYYRETRTPFTPDGWYPTGDIAIRSSSAADAELTLVGRRKEIIIRGGENI
HPHELEHVLLRHPGVADVVVAGMPHRLLGETPAAFIVKSAADVDFDLSALLAACRKVLP
DYKIPTAFYEIDTVPRTVIGKPKRLTMTSYTNKPLTARSILQSRDSIEALVMAETVSACTIGA
EREGESNTDWLRRNLDQPFSFLGLSSMASVVLRDRLAGLTGLADLPNTLVFDYATPAAVS
TYLCSRLLEPESTPLPRSTPTTTSDCEVEPIAIVSMACRYPGGISSPEDLWELVSDEIDATTEF
PDDRGWDIDALYSTDPDTPNTSTTKRGGFLPDFAHFDAGLFGMAPREALATDPQQRLLLE
TTWELAERAGIAPLSLQGSQTGVFVGTLYEDYEENGFGNNELEAHLGLGSSSSVVSGRVS
YCFGLHGPSLVVSTGCSSSLVAIHLAAQSLRNRECSLTIAGGITAMATPRPFTMFSRRRGLS
SDGRCRAYSSDASGTGWSEGVGLLLLERLSDAKRNGHQILGLIRGSAVNSDGKSNGLTAP
NGPAQQMCIQSALAQAGMSPENVDVLEGHGTATPLGDPIEVQAVISAYGNGDRKTSDSGT
RRLEPLLLGSIKSNIGHTQAAAAVAGIIKMVQAMRHGVAPTSLHIREPSPQIDWEGSGVELL
SKARQWPSVNRPRRAAVSSFGIGGTNSHIILEQPEPAEEEDSNSKRISATVPWLISGASEAA
LRGQAHALLAAWRQDDNTFSSLRNQDPTDIAFSLATARSALKYRATVTYTLGVNFHDQV
ETALKSLAQGEPHPDVVTAHTSTTGSQPRLACLFSGQGSRMPSIDTLAELRATFPVFSTAFQ
AACDEVDQHLECPLVHAIGNSILDRTEFAQATLFVFEVAMFRLLESFNIRPDFVAGHSLGEI
AAAHAAGALSLRDAATIVTTRAKLMASLPPNGGMVSISATEVEVASELAQLGGSATIAAV
NSQNSVVVSGTQEAIKAVADMFASLGRRATVLRNVKHGFHSPLMNGILPDLEKALPSSME
DDSPTAIPLVSTTTSKRADAAQLRSSAHWIRHVSEPVRFADAIDELKSNERVSVFVEIGPSA
VLSPHVPEAAATHGTVDKLLRMLGQLWTRGVPVDWQAVFEGSGARFVDLPVYAFQRQR
YWLPYTPLLPVASTGAVAQQAQGQTVGAGGDSRLRHGMLFNATSIPGTGTVICSGCLSTT
RQPWLRDHVIGGQSLVPASAFAELAMRAAQECAEYTESYSMVLDEMVLVAPLAVSSAED
EEQEDSSGEFEIQVLIAWSQREDEGTETQRTVEVYSRPSGVATQHEWTHHATGSLKLTPQS
NADHSSTNGTDSTRTESDVDISEAYTILADAGLTYGPSFQLVRAIWRLRDDDLLVQIDPPE
DQAQMSTFVLHPAVLDAALHASTLASAEKVAGGDIRLPFSLRGVQFLTTAGASGPILARIS
QDSENSFSLTLTDKSSGVLVATVSEVQLRAWQPAVAGGDLYRLEWREIESKPIQTTTSTETD
KIVRVQSARDIDAAAVSKAVHEAVAEVLHTIHEWRAENTHAGDGVRLVFVTERATSADKS
SNINVVAAAVWGFVRSAQAEFGGDRIVLVDMDGTTESEEALSAALASREEVVAVNGGKIT
IPRISKQPPMPESPQAMALDVSGTVLITGGTGGLGATLSRDIVHTHGAKNLLLVSRSGIEAR
GARELSEELQSANAAVRVEACDVGDYTQLATLLDNHTQHQYPPITAVIHCAGTVSDAFLG
SQNQERVSSVLRPKVDAAWNLHELVPETVRSFVLFSSYVSVLGNEGQAAYSAGNAFLDA
LARLRVARGLPALSLAWGPWANEAGMAGGSKLAAIPPRIANARPFTDQQGLHLFHRALH
VQTRTPPEPVLLPLLLRGPFPLVPSAGTESKPKEKSSARGGSASGAIWRRSIAAVSLENRHD
ALLGLVRDEIAAVLGYQSQDMLPDQRLEDLGFDSFTSVMLTNRLRVLTGLGYLPVTLALD
YDTTSALVEYLLPRIEAEPQPQPQPEVDMDSNASTTGGDTSVSHDSGKADGLSPSSSVTTL
APEEHDDLNPETFRGLATIHRRLSQLEQYTAAADLLASAALAMPTFPKTGSSLPSYAAEPQ
RLATGPSGTSNSEPPLPLVFIAPFFPRIKVEGIGLSVYSNLASAMTGKRDVFELPHPEGQVV
PGDLATLADLHVHTIRKHFSDRSGILLAGYSAGGTVAYAVASRLAHAADRQPRLAGFVLV
DTYLTMTGRGDPDWLNALPAEALVSRLQVPPSLGHPKGMGSDSLVGDLDVALAKVGGYF
RTLRDWDLELHPLPDALSTLFVRAVDASDKMPKDSDVWRPRWPRADLTVDVPGSHLALLDKRYAPGVAVEIERWARELNA*
SEQ ID No.25
MLRSRQAATALRAVGQTRPLRSQTPLAFTQSLNKVPVNRRTEATAATASSTASQVRPTPSPTFNQYDSQVQPLTGVSKNVTDESFIGKSGGEIFHDMMLRQGVKHIFGYPGGAILPVFDAIYNSPHFDFILPRHEQGAGHMAEGYARASGKPGVVLVTSGPGATNIVTPMQDALLDGTPMVVFCGQVPTTSIGSDAFQEADICGISRPCTKWNVMVKNVAELPRRINEAFQIATTGRPGPVLVDLPKDVTAGILRRAIPTESAIPSLPSAAIQDAMDLNHKQLEASVARVAKLVNMAKQPVIYAGQGVVQSELGPELLKQLSDLASIPVTTTLQGLGGFDELDYKSLHMLGMHGSGYANMAMQEADLIIALGGRFDDRVTLNVSKFAPGARAAAAENRGGIVQFEIMPKNINKVVEATEAIVGDVGTNLRLLLPHVEPRSLDDRQAWYTKIDAWKKRWPLSDYQKTERHGLIKPQTLIEELSNLCADRKDKTYITTGVGQHQMWTAQHFRWRHPRTMITSGGLGTMGYGLPAAIGAKVAQPDALVVDIDGDASFNMTLTELSTAAQFNIGVKVIVLNNEEQGMVTQWQNLFYEDRYAHTHQANPDFIQLATAMGIQGQRVADPTKVKESLQWLIDTDGPALLEVITDKKVPVLPMVPGGCGLHEFIAFNPEDEKTRRGLMRERTCGLHG*
example 2 analysis of fungal flavone biosynthetic pathway
2.1 Construction of Gene deletion mutants in Gene Cluster cfo
According to the homologous recombination double-exchange technology, 12 genes contained in the cfo gene cluster in aspergillus candidus MEFC1001 are knocked out, and mutant strains with different gene deletions are constructed.
Specifically, according to the nucleotide sequence of the cfo gene cluster, a pair of external primers are respectively designed at the upstream and downstream of each gene. The method comprises the steps of obtaining left and right homologous exchange arms for homologous recombination through PCR amplification, respectively obtaining the lengths of about 1500 base pairs, fusing the homologous arms with hygromycin resistance tags hph through fusion PCR, purifying and concentrating a PCR reaction product to obtain a targeting element, knocking the targeting element into MEFC1001 strain, and screening to obtain a gene deletion mutant strain.
Specifically, MEFC1001 strain is cultured in SGCY culture medium at 28 ℃ and 220rpm for 2 days, mycelium is collected by a sterile filter cloth of 100 meshes, and cell walls are digested by an enzymolysis liquid with the weight of 10 times of the mycelium and the enzymolysis condition is that the enzymolysis liquid is digested at 30 ℃ and 130rpm for 2 hours. Filtering the enzymolysis solution by using 500-mesh sterile filter cloth, centrifuging and collecting the filtrate at 4000rpm to obtain protoplast, washing the protoplast once by using precooled 1.0M sorbitol solution, washing the protoplast once by using precooled STC (1.0M sorbitol, 50mM Tris-HCl-pH8.0, 50mM CaCl 2), and finally re-suspending the protoplast in the precooled STC, wherein the concentration is adjusted to be 5 multiplied by 10 7 per mL, thus obtaining protoplast suspension. To the suspension, 10. Mu.L of each gene targeting element (about 3. Mu.g) was added, followed by 50. Mu. LPSTC (40% PEG4000,1.2M sorbitol, 50mM Tris-HCl-pH 8.0, 50mM CaCl 2), gently mixed, and ice-bathed for 30min. 1mL of PSTC was added, the mixture was left at room temperature for 20min after mixing, and the mixture was poured into 5 PDAS screening plates (containing 50mg/L hygromycin B) after mixing with 15mL PDBS, and incubated for 5 days at 30℃in the dark.
Transformants with hygromycin resistance were selected from the screening plate and transferred to PDAH plates (PDA+50 mg/L hygromycin B), after 5 days of culture, part of mycelia on single colonies were picked up, the genome was extracted, and PCR amplification was performed using the outer primers on the homology arms as templates. If the random insertion PCR product is about 3.5kb in size, if homologous recombination occurs, the PCR product is about 5.0kb in size, indicating that the gene of interest is knocked out.
2.2 Analysis of the metabolites of knockout mutants
The constructed deletion mutant strain of each gene and the wild-type MEFC strain of each gene are respectively inoculated in PPM culture medium, and the spore number of each deletion mutant strain of each gene and the wild-type MEFC strain of each gene is 10 7/50 mL in the culture medium. The culture was carried out at 220rpm and 30℃for 7 days to obtain fermentation broths of the respective strains. Extracting the fermentation liquor by using ethyl acetate with the same volume, and concentrating to obtain a crude extract. Organic membrane filtration at 0.22. Mu.M after methanol dissolution, waters ACQUITY UPLC analysis, eclipse Plus C18 RRHD column (50 mm. Times.2.1 mm,1.8 μm), analytical method: mobile phase a (95% water +5% acetonitrile +0.05% formic acid), mobile phase B (100% acetonitrile +0.05% formic acid), gradient elution 0-0.58min 100％-80％ A,0.58-4.05min 80％-40％ A,4.05-5.79min 40％ A,5.79-6.37min 40％-0％ A,and 6.95-7.53min 100％ A, flow rate 0.6mL/min, detection wavelength 345nm.
According to the analysis result, the fermentation scale of the mutant strain is enlarged, the fermentation conditions are the same as above, the fermentation liquid is extracted three times by adopting the ethyl acetate with the same volume, and the crude extract is obtained by concentration. And (3) carrying out column chromatography on the crude extract by adopting a dry method column packing method, wherein the packing is octadecylsilane chemically bonded silica packing, and methanol water is subjected to gradient elution (10-100% of methanol volume), and each gradient elution comprises 10 column volumes. And (3) analyzing a small amount of HPLC, wherein the analysis method is the same as above, and determining the optimal preparation condition according to the polarity and retention time of the flavonoid compounds in each component. Purification conditions: mobile phase a (100% water+0.05% formic acid), mobile phase B (100% acetonitrile+0.05% formic acid), elution at a flow rate of 2mL/min, detection wavelength 345nm, column Waters X-bridge C18 (100 mm X10 mm,5 μm) by adjusting the ratio of mobile phase according to the polarity of the target compound. Collecting and obtaining the corresponding flavonoid compounds at the corresponding retention time. The metabolites in the mutant and wild type were isolated and identified (FIG. 4).
The mutant fermentation products from which three methyltransferases were knocked out were first analyzed. Compounds 5-7 accumulated in Δ cfoB, whose C7 position was changed from the original methoxy to hydroxy compared to 1, indicating that CfoB was responsible for catalyzing the methylation of C7-OH. Compounds 8-12, wherein 8, 9 and 11C 6-OH and 10C 8-OH lack methyl groups as compared to end product 1, accumulate in Δ cfoC, indicating CfoC is responsible for methylation of C6-OH or C8-OH. Compound 14 accumulated in mutant Δ cfoD, changing from methoxy to hydroxy at position C3 in 14, and thus CfoD was responsible for the methylation of C3-OH.
Mutant fermentation products knocked out of three monooxygenases and P450 enzymes were analyzed. In mutant Δ cfoE, compound 1 disappeared and 2 accumulated, so CfoE functions as a halogenation enzyme responsible for adding a chlorine atom to the C3' position of 2 to form end product 1. In mutant Δ cfoF, compounds 3,4, 12 and 13, all of which had no substituent at the C3 position, were accumulated, indicating that CfoF was responsible for hydroxylation at the C3 position. Compounds 12 and 15, which lack hydroxyl groups at both C6 and C8 compared to other intermediates, were produced in the cfoG knockdown mutant, indicating that CfoG is responsible for hydroxylation of flavonoids at either C6 or C8. The C2 'positions of compounds 4 and 12 in the P450 gene cfoH knocked-out mutant were not oxidized, indicating that CfoH was responsible for the hydroxylation of the C2' position. Thus far, based on structural information of the gene knockout experiments and intermediates, the functions of three methyltransferases, three oxidases and one P450 enzyme in the cfo gene cluster were determined (fig. 5).
2.3 Analysis of Assembly mechanism of Kelvin skeleton, a key intermediate of fungal flavone
The knockdown cfoK mutant metabolites accumulate compounds 20-23, wherein 20 and 22 are chalcones, indicating that key intermediates for flavone synthesis in fungi are similar to plants, both chalcones, and also indicating that the non-ribosomal peptide-polyketide hybrid enzyme (NRPS-PKS) encoded by core gene cfoA in gene cluster cfo can synthesize chalcones with the function of chalcone synthase (CHS), which is a type III PKS in plants. Further labeling with sodium [1,2- 13C2 ] acetate, it was found that chalcone in fungi was formed by CfoA catalyzed condensation of benzoic acid or parahydroxybenzoic acid with 4 molecules of malonyl-coa, which is significantly different from the pattern of assembly of type III PKS catalyzed p-coumaroyl-coa with 3 molecules of malonyl-coa in plants (fig. 6).
2.4 In vitro enzyme Activity assay for fungi CHI and FNS
Chalcone 20 and 22 accumulated in mutant strain Δ cfoK, presumably CfoK has a similar function to chalcone isomerase (CHI) in plants, and can catalyze an intramolecular oxa-Michael addition reaction to convert chalcone to a tricyclic flavanone. CfoK have very low amino acid sequence similarity to CHI that has been found in nature today, and the phylogenetic tree analysis CfoK is located on a different branch than plant CHI and bacterial CHI (fig. 7). To further verify the function of cfoK gene, CDS sequence of cfoK was obtained by 5'-race and 3' -race techniques, the codon was optimized and expressed in E.coli to obtain soluble protein, cfoK was obtained by separation and purification, in vitro enzyme activity analysis and point mutation experiments showed that CfoK indeed had CHI activity, and the catalytic mechanism was histidine residue mediated oxa-Michael addition reaction, which was completely different from H 2 O mediated reaction mechanism in plant CHI (FIG. 8). In general, cfoK is a novel chalcone isomerase.
Flavanones 16-19 accumulate in mutant strain Δ cfoJ, presumably CfoJ, and function similarly to the plant flavone synthase (FNS), and catalyze desaturation to form double bonds at C2 and C3, converting flavanones to flavones. Two classes of FNS are currently found in nature, FNS I (α -KG dependent dioxygenase) and FNS II (cytochrome P450), respectively. Whereas CfoJ is annotated as NADPH dependent FMN reductase, the treelet analysis CfoJ is located on a different branch than FNS I and FNS II (fig. 9). To further verify the function of cfoJ gene, CDS sequence of cfoJ was obtained by 5'-race and 3' -race techniques, the soluble protein was obtained by induced expression in E.coli after codon optimization, cfoJ was obtained by separation and purification, in vitro enzyme activity analysis and point mutation experiments showed that CfoJ indeed had the activity of FNS, and the catalytic mechanism was dependent on FMN-mediated dehydrogenation at C2-C3, which was completely different from the two types of FNS free radical catalytic mechanisms in nature (FIG. 10). In summary, cfoJ is a novel flavonoid synthase.
To further verify the function of cfoK and cfoJ genes, we performed in vitro enzyme activity experiments of the respective encoded proteins, first using 5'-race and 3' -race techniques to obtain the CDS sequences of cfoK and cfoJ, codon optimization followed by inducible expression in the large intestine to obtain soluble proteins, and isolation and purification to obtain CfoK and CfoJ (fig. 11). And constructing an enzyme activity reaction system by taking the corresponding intermediate as a substrate. The results show CfoK can catalyze the production of naringenin (27) and pinocembrin (24) from chalcones 20 and 22 (fig. 11 a), and CfoJ can catalyze the production of pinocembrin (24) and chrysin (26) and 3 from compound 18 (fig. 11 b). The function of CfoK and CfoJ in flavone biosynthesis was further verified.
So far, through experiments such as gene knockout, isotope labeling, in vitro enzyme activity, mutant activity analysis and the like, the complete biosynthesis pathway of flavonoid compounds in fungi is analyzed, and as shown in figure 5, the flavonoid compounds in fungi are completely different from the biosynthesis of plant flavonoid compounds.
Example 3 heterologous expression of a key Gene for flavone Synthesis
In order to verify the feasibility of constructing a flavone cell factory by using a fungal biosynthesis pathway, the applicant constructs flavone-producing chassis cells in aspergillus oryzae (Aspergillus oryzae), aspergillus terreus (Aspergillus terreus) and saccharomyces cerevisiae (Saccharomyces cerevisiae) respectively by using three key enzymes CfoA (CHS), cfak (CHI) and cfoJ (FNS) in the fungal flavone synthesis pathway as enzymatic elements, and lays a foundation for constructing plant-derived flavone compounds by synthetic biology. The method comprises the following steps:
3.1 construction of heterologous expression plasmids containing Critical genes
The nucleotide sequences of genes cfoA, cfoK and cfoJ were amplified by PCR using the genome of MEFC1001 as template with specific primers. The pTAex vector for heterologous expression was digested with restriction enzyme KpnI, and the linearized pTAex vector was recovered. Plasmids pTAex3-cfoA, pTAex3-cfoK and pTAex3-cfoJ were obtained by ligating the cfoA, cfoK and cfoJ genes, respectively, with a linearized pTAex3 vector using Seamless Assembly Cloning (Clone Smarter) kit. The restriction enzyme XbaI cleaves pAdeA vector for heterologous expression and linearized pAdeA vector is recovered. The cfoK gene containing amyB promoter and terminator on the pTAex-cfoK plasmid was amplified and ligated to linearized pAdeA vector by Seamless Assembly Cloning kit to construct pAdeA-cfoK plasmid. The restriction enzyme HindIII cleaves pBARI vector for heterologous expression and linearized pBARI vector is recovered. The cfoJ gene containing amyB promoter and terminator on the pTAex-cfoJ plasmid was amplified and ligated to linearized pBARI vector by Seamless Assembly Cloning kit to construct pBARI-cfoJ plasmid.
3.2 Construction of heterologous expression Strain and metabolite analysis thereof
Aspergillus oryzae was inoculated into YPED liquid medium at spore inoculum size of 1X 10 7/50 mL and cultured at 28℃for 12 hours at 220 rpm. Collection of mycelium, enzymolysis of protoplast, and plasmid transformation experiments by a PEG-CaCl 2 -mediated protoplast transformation method. Wherein the plasmid pTAex-cfoA was transformed, protoplast regeneration and selection were cultured as MMAS (MM+0.2% agar+1.2M sorbitol+0.15% methionine+0.01% > adenine). The transformant was verified by PCR to obtain a mutant strain Ao-cfoA expressing cfoA genes. The pAdeA-cfoK plasmid was transformed on the basis of the mutant strain Ao-cfoA, protoplast regeneration and selection were performed to MMAS (MM+0.2% agar+1.2M sorbitol+0.15% methionine). The transformant was verified by PCR to obtain a mutant strain Ao-cfoA-cfoK expressing cfoA and cfoK genes simultaneously. The pBARI-cfoJ plasmid was transformed on the basis of the mutant strain Ao-cfoA-cfoK, protoplast regeneration and selection were grown to MMAS (mm+0.2% agar+1.2M sorbitol+0.15% methionine+500 μg/mL glufosinate). The transformant was verified by PCR to obtain mutant strains Ao-cfoA-cfoK-cfoJ expressing cfoA, cfoK and cfoJ genes simultaneously. The construction method is similar to that of Aspergillus oryzae engineering strains, mutant strains At-cfoA, at-cfoA-cfoK and At-cfoA-cfoK-cfoJ are obtained by transformation in Aspergillus terreus, and mutant strains Sc-cfoA, sc-cfoA-cfoK and Sc-cfoA-cfoK-cfoJ are obtained by transformation in Saccharomyces cerevisiae.
The wild strains and engineering strains of Aspergillus oryzae, aspergillus terreus and Saccharomyces cerevisiae were inoculated into CD liquid medium, respectively, and cultured at 28℃and 220rpm for 3 days, and the fermentation broth treatment method, crude extract analysis method and compound separation and purification method after the completion of the culture were the same as those of example 2.2, and as a result, it was revealed that flavonoid compounds could be produced in the heterologously expressed engineering strains (FIG. 12). Specifically, expression cfoA alone may produce compound 22 and compound 25, while expression cfoA and cfoK may produce compound 24, while expression cfoA, cfoK, and cfoJ may produce compound 24 and compound 26.
The heterologous expression experiment of the key genes further verifies the biosynthesis way of the fungal flavone, and simultaneously proves that the way can provide an enzymatic element for the construction of a flavone cell factory and lays a foundation for producing the plant-derived flavone compounds in microorganisms through synthetic biology.
While the invention has been described in terms of preferred embodiments, it is not intended to limit the invention, but rather, it will be apparent to those skilled in the art that various changes and modifications can be made herein without departing from the spirit and scope of the invention as defined by the appended claims.
Claims (10)
1.A gene related to flavone synthesis, characterized in that,
The gene related to flavone synthesis is selected from one or a combination of more than one of ester hydrolase CfoK, NADPH dependent FMN reductase CfoJ, heterozygous non-ribosomal peptide synthase and polyketide synthase CfoA;
The amino acid sequence of CfoK has at least 80% sequence identity to SEQ ID No. 18;
the amino acid sequence of CfoJ has at least 80% sequence identity to SEQ ID No. 21;
The amino acid sequence of CfoA has at least 80% sequence identity to SEQ ID No. 24;
preferably, the gene associated with flavone synthesis is derived from aspergillus candidus.
2. The use of the gene related to flavone synthesis according to claim 1 for preparing a flavonoid compound.
3. The use of the gene related to flavone synthesis according to claim 1 for preparing a genetically engineered strain capable of producing a flavone compound.
4. A genetically engineered strain capable of producing a flavonoid compound, which is obtained by introducing the gene related to flavonoid synthesis of claim 1 into a starting strain.
5. The genetically engineered strain of claim 4, wherein the starting strain is selected from one or more of aspergillus, yeast, and escherichia coli.
6. The use of the genetically engineered strain of claim 4 or 5 for the production of flavonoids.
7. The use according to any one of claims 2-3 or 6, wherein said flavonoid is selected from one or more of compound 22, compound 24, compound 25 and compound 26;
the structural formula of the compound 22 is shown as follows:
the structural formula of the compound 24 is shown as follows:
the structural formula of the compound 25 is shown as follows:
the structural formula of the compound 26 is shown as follows:
8. a method for producing a flavonoid compound, comprising the step of fermenting the genetically engineered strain of claim 4 or 5.
9. Use of CfoK in claim 1 for the catalytic synthesis of compound 27 and/or compound 24;
the structural formula of the compound 24 is shown as follows:
The structural formula of the compound 27 is shown as follows:
。
10. Use of CfoJ in claim 1 for the catalytic synthesis of compound 26 and/or compound 3;
the structural formula of the compound 26 is shown as follows:
the structural formula of the compound 3 is shown as follows:
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