CN117917478A - Novel flavonoid compound and application thereof - Google Patents
Novel flavonoid compound and application thereof Download PDFInfo
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- CN117917478A CN117917478A CN202310612994.1A CN202310612994A CN117917478A CN 117917478 A CN117917478 A CN 117917478A CN 202310612994 A CN202310612994 A CN 202310612994A CN 117917478 A CN117917478 A CN 117917478A
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- C07D311/32—2,3-Dihydro derivatives, e.g. flavanones
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Abstract
The invention provides a gene related to flavone synthesis and a novel flavonoid compound, wherein the gene related to flavone synthesis is subjected to gene manipulation in a target strain, so that the novel flavonoid compound can be obtained, and the flavonoid compound has a good antibacterial effect.
Description
Technical Field
The invention belongs to the fields of microbial gene resources and genetic engineering, and particularly relates to novel flavonoid compounds and application thereof.
Background
Flavones are a large class of natural products widely existing in plants, and have important application values in the fields of functional foods, medicines and the like, 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. At present, flavonoid compounds are mainly extracted from plants, and the flavonoid compounds can not meet the market demands all the time due to low content in the plants, complex extraction process and the like.
The use of synthetic biotechnology to modify microorganisms to make their production of flavonoids possible has been reported in many studies on the synthesis of plant-derived flavonoids in microorganisms. In the patent, the applicant discovers chlorofluoroflavonoid, namely chlorofluoromycin (chlorflavonin, CAS: 23363-64-6) and a biosynthesis gene cluster cfo thereof in aspergillus candidus, and constructs engineering strains for producing different flavones by knocking out genes in the cfo. A series of flavonoid compounds are obtained through separation, wherein the flavonoid compounds contain flavonoid compounds with novel structures, and an activity test shows that the flavonoid compounds have antibacterial activity, so that the engineering strain and the flavonoid compounds have potential application values.
Disclosure of Invention
In one aspect, the invention also provides a gene cluster associated with flavone biosynthesis or a gene associated with flavone synthesis.
In one embodiment, the nucleic acid sequence of the gene cluster is shown in SEQ ID No. 1.
In other embodiments, the gene associated with flavone synthesis is selected from one or any combination of a FAD-dependent oxidase CfoG, monooxygenase cfofa, SAM-dependent methyltransferase CfoD, cytochrome P450 enzyme CfoH, ester hydrolase CfoK, scytalone dehydratase CfoI, SAM-dependent methyltransferase CfoC, NADPH-dependent FMN reductase CfoJ, SAM-dependent methyltransferase CfoB, FAD-dependent oxidase CfoE, hybrid non-ribosomal peptide synthase, and polyketide synthase CfoA.
In one embodiment, the amino acid sequence of CfoG 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. 14; preferably, the CfoG is derived from aspergillus candidus, for example aspergillus candidus MEFC1001; more preferably, the amino acid sequence of CfoG has at least 99% sequence identity to SEQ ID No.14 and the CfoG is derived from aspergillus candidus; the aspergillus candidus comprises aspergillus candidus MEFC1001,1001. More preferably, the amino acid sequence of CfoG is shown as SEQ ID No.14, and the coding gene sequence is shown as SEQ ID No. 2.
In one embodiment, the amino acid sequence of CfoF 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. 15; preferably, the CfoF is derived from aspergillus candidus, for example aspergillus candidus MEFC1001; more preferably, the amino acid sequence of CfoF has at least 99% sequence identity to SEQ ID No.15 and the CfoF is derived from aspergillus candidus; the aspergillus candidus comprises aspergillus candidus MEFC1001,1001. More preferably, the amino acid sequence of CfoF is shown as SEQ ID No.15, and the coding gene sequence is shown as SEQ ID No. 3.
In one embodiment, the amino acid sequence of CfoD 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. 16; preferably, the CfoD is derived from aspergillus candidus, for example aspergillus candidus MEFC1001; more preferably, the amino acid sequence of CfoD has at least 99% sequence identity to SEQ ID No.16 and the CfoD is derived from aspergillus candidus; the aspergillus candidus comprises aspergillus candidus MEFC1001,1001. More preferably, the amino acid sequence of CfoD is shown as SEQ ID No.16, and the coding gene sequence is shown as SEQ ID No. 4.
In one embodiment, the amino acid sequence of CfoH 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. 17; preferably, the CfoH is derived from aspergillus candidus, for example aspergillus candidus MEFC1001; more preferably, the amino acid sequence of CfoH has at least 99% sequence identity to SEQ ID No.17 and the CfoH is derived from aspergillus candidus; the aspergillus candidus comprises aspergillus candidus MEFC1001,1001. More preferably, the amino acid sequence of CfoH is shown as SEQ ID No.17, and the coding gene sequence is shown as SEQ ID No. 5.
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 CfoI 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. 19; preferably, the CfoI is derived from aspergillus candidus, for example aspergillus candidus MEFC1001; more preferably, the amino acid sequence of CfoI has at least 99% sequence identity to SEQ ID No.19 and the CfoI is derived from aspergillus candidus; the aspergillus candidus comprises aspergillus candidus MEFC1001,1001. More preferably, the amino acid sequence of CfoI is shown as SEQ ID No.19, and the coding gene sequence is shown as SEQ ID No. 7.
In one embodiment, the amino acid sequence of CfoC 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. 20; preferably, the CfoC is derived from aspergillus candidus, for example aspergillus candidus MEFC1001; more preferably, the amino acid sequence of CfoC has at least 99% sequence identity to SEQ ID No.20 and the CfoC is derived from aspergillus candidus; the aspergillus candidus comprises aspergillus candidus MEFC1001,1001. More preferably, the amino acid sequence of CfoC is shown as SEQ ID No.20, and the coding gene sequence is shown as SEQ ID No. 8.
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 CfoB 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. 22; preferably, the CfoB is derived from aspergillus candidus, for example aspergillus candidus MEFC1001; more preferably, the amino acid sequence of CfoB has at least 99% sequence identity to SEQ ID No.22 and the CfoB is derived from aspergillus candidus; the aspergillus candidus comprises aspergillus candidus MEFC1001,1001. More preferably, the amino acid sequence of CfoB is shown as SEQ ID No.22, and the coding gene sequence is shown as SEQ ID No. 10.
In one embodiment, the amino acid sequence of CfoE 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. 23; preferably, the CfoE is derived from aspergillus candidus, for example aspergillus candidus MEFC1001; more preferably, the amino acid sequence of CfoE has at least 99% sequence identity to SEQ ID No.23 and the CfoE is derived from aspergillus candidus; the aspergillus candidus comprises aspergillus candidus MEFC1001,1001. More preferably, the amino acid sequence of CfoE is shown as SEQ ID No.23, and the coding gene sequence is shown as SEQ ID No. 11.
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.
On the other hand, the invention also provides a novel flavonoid compound, wherein the flavonoid compound is selected from one or any of the compounds 2-23.
The structural formula of the compound 2 is shown as follows:
the structural formula of the compound 3 is shown as follows:
The structural formula of the compound 4 is shown as follows:
the structural formula of the compound 5 is shown as follows:
the structural formula of the compound 6 is shown as follows:
the structural formula of the compound 7 is shown as follows:
the structural formula of the compound 8 is shown as follows:
The structural formula of the compound 9 is shown as follows:
The structural formula of the compound 10 is shown as follows:
The structural formula of the compound 11 is shown as follows:
the structural formula of the compound 12 is shown as follows:
The structural formula of the compound 13 is shown as follows:
The structural formula of the compound 14 is shown as follows:
the structural formula of the compound 15 is shown as follows:
The structural formula of the compound 16 is shown as follows:
the structural formula of the compound 17 is shown as follows:
The structural formula of the compound 18 is shown as follows:
the structural formula of the compound 19 is shown as follows:
The structural formula of the compound 20 is shown as follows:
The structural formula of the compound 21 is shown as follows:
the structural formula of the compound 22 is shown as follows:
the structural formula of the compound 23 is shown as follows:
In another aspect, the present invention also provides the use of the novel flavonoid compounds described above, including use in inhibiting the growth of microorganisms, or in the preparation of an agent for inhibiting the growth of microorganisms, or in the preparation of a medicament for the treatment of a disease caused by microorganisms; in one embodiment, the agent that inhibits microbial growth is an antibiotic; in a preferred embodiment, the microorganism is selected from one or any of Candida albicans (Candida albicans), aspergillus fumigatus (Aspergillus fumigatus), sclerotium rolfsii (Sclerotium rolfsii sacc.), botrytis cinerea (Botrytis cinerea).
For example, compound 3, compound 4 and compound 13 can inhibit the growth of candida albicans, sclerotium rolfsii and botrytis cinerea.
Compound 6 can inhibit the growth of candida albicans and aspergillus fumigatus.
Compound 7 and compound 8 can inhibit the growth of candida albicans, aspergillus fumigatus, sclerotium rolfsii and botrytis cinerea.
Compound 10 can inhibit the growth of candida albicans and sclerotium rolfsii.
Compound 14 can inhibit the growth of candida albicans, aspergillus fumigatus and sclerotium rolfsii.
Compound 19 can inhibit the growth of candida albicans and sclerotium rolfsii.
The agent and the medicine in the invention can be prepared into different dosage forms, including liquid preparation, solid preparation, semisolid preparation or gas preparation.
The agent and the drug of the present invention may contain other auxiliary agents in addition to the above-mentioned compounds as active ingredients.
On the other hand, the invention also provides a genetic engineering strain which is obtained by carrying out genetic mutation on the genes related to flavone synthesis in aspergillus candidus.
The original strain of the genetically engineered strain can also be selected from aspergillus, aspergillus nidulans, yeast, escherichia coli, plants or animals; preferably, aspergillus, e.g., aspergillus candidus, aspergillus oryzae.
In one embodiment, the gene associated with flavone synthesis is selected from one or more of FAD-dependent oxidase CfoG, monooxygenase cfofe, SAM-dependent methyltransferase CfoD, cytochrome P450 enzyme CfoH, ester hydrolase CfoK, scytalone dehydratase CfoI, SAM-dependent methyltransferase CfoC, NADPH-dependent FMN reductase CfoJ, SAM-dependent methyltransferase CfoB, FAD-dependent oxidase CfoE, hybrid non-ribosomal peptide synthase and polyketide synthase CfoA as described above.
The mutation described in the present invention includes a loss of gene function or activity by means of gene deletion, gene insertion or gene substitution.
In one embodiment, the gene mutation may be accomplished using techniques conventional in the art, for example, by homologous recombination to knock-in or knock-out resulting in loss of gene function or activity; or by mutating the gene using a gene editing means such as zinc finger endonuclease (ZFN), transcription activator-like effector nuclease (TALEN) or CRISPR technique, resulting in loss of gene function or activity.
On the other hand, the invention also provides application of the genetically engineered strain in production of flavonoid compounds.
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 the compounds 2-23.
In one embodiment, the mutated gene is CfoB, and the flavonoid compound is one or any of compound 5, compound 6 and compound 7;
in one embodiment, the mutated gene is CfoC, and the flavonoid compound is one or any of compound 8, compound 9, compound 10, compound 11 and compound 12;
In one embodiment, the mutated gene is CfoD, and the flavonoid compound is one or any of compound 3, compound 4, compound 12, compound 13 and compound 14;
In one embodiment, the mutated gene is CfoE, and the flavonoid compound is one or any of compound 2 and compound 4;
In one embodiment, the mutated gene is CfoF, and the flavonoid compound is one or any of compound 3, compound 4, compound 12 and compound 13;
In one embodiment, the mutated gene is CfoG, and the flavonoid compound is one or any of compound 12 and compound 15;
In one embodiment, the mutated gene is CfoH and the flavonoid is one or any of compound 4 and compound 12;
In one embodiment, the mutated gene is CfoI, and the flavonoid compound is one or any of compound 3, compound 4 and compound 12;
in one embodiment, the mutated gene is CfoJ, and the flavonoid compound is one or any of compound 16, compound 17, compound 18 and compound 19;
In one embodiment, the mutated gene is CfoK and the flavonoid compound is one or any of compound 20, compound 21, compound 22 and compound 23.
Drawings
FIG. 1 HPLC analysis of Aspergillus candidus MEFC1001 metabolite and corresponding compound structure.
FIG. 2 HPLC analysis of cfo gene clusters and mutants thereof, delta cfoA metabolites in A.leucini.
FIG. 3 HPLC analysis of metabolites of different Aspergillus candidus engineering strains.
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 aqueous MgSO 4 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.
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.
SM medium is prepared from 10% glucose, 1% sucrose, 0.5% yeast extract, 0.5% peptone, 0.1% Na 2CO3, and deionized water by autoclaving at 121deg.C for 20 min, and inoculating and culturing at 30deg.C.
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 (Table 1), of which cfoA, cfoB, cfoC, cfoD, cfoE, cfoF, cfoG, cfoH, cfoI, cfoJ, cfoK total 11 genes are genes responsible for the synthesis of the carbon skeleton of flavonoids, and cfoL is a self-resistant gene, encoding 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
NFVLINNEENDTNMCAEDWHARFKIPDVLKKFSDYDPRLLKVLSKAPDARRWPLIYRKPIHQWTKGCMTLAGDACHAMLPFLAQGGAQGIEDAVALGVILHKATTRDDIEKRLQIYQEVRIKRASVIQILSNMGADHSVSVEDLKEYLHDDQMPRGQHDMMIHNYKYDVAGAAIDAMKIYDPSFTLADEFLGW*
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
MLRSRQAATALRAVGQTRPLRSQTPLAFTQSLNKVPVNRRTEATAATASSTASQVRPTPSP
TFNQYDSQVQPLTGVSKNVTDESFIGKSGGEIFHDMMLRQGVKHIFGYPGGAILPVFDAIY
NSPHFDFILPRHEQGAGHMAEGYARASGKPGVVLVTSGPGATNIVTPMQDALLDGTPMV
VFCGQVPTTSIGSDAFQEADICGISRPCTKWNVMVKNVAELPRRINEAFQIATTGRPGPVL
VDLPKDVTAGILRRAIPTESAIPSLPSAAIQDAMDLNHKQLEASVARVAKLVNMAKQPVIY
AGQGVVQSELGPELLKQLSDLASIPVTTTLQGLGGFDELDYKSLHMLGMHGSGYANMA
MQEADLIIALGGRFDDRVTLNVSKFAPGARAAAAENRGGIVQFEIMPKNINKVVEATEAI
VGDVGTNLRLLLPHVEPRSLDDRQAWYTKIDAWKKRWPLSDYQKTERHGLIKPQTLIEEL
SNLCADRKDKTYITTGVGQHQMWTAQHFRWRHPRTMITSGGLGTMGYGLPAAIGAKVA
QPDALVVDIDGDASFNMTLTELSTAAQFNIGVKVIVLNNEEQGMVTQWQNLFYEDRYAH
THQANPDFIQLATAMGIQGQRVADPTKVKESLQWLIDTDGPALLEVITDKKVPVLPMVPGGCGLHEFIAFNPEDEKTRRGLMRERTCGLHG*
example 2 isolation and identification of flavonoids from Aspergillus candidus MEFC1001
2.1 Placing Aspergillus candidus MEFC strain 1001 (collection number: CGMCC No. 3.15294) on a PDA plate for static culture, washing spores with sterile water, inoculating to SM culture medium, culturing seed liquid at 28 ℃ and 220rpm, inoculating to PPM culture medium, fermenting at 28 ℃ and 220rpm for 14 days to obtain Aspergillus candidus MEFC strain 1001 fermentation broth.
2.2 Extracting the fermentation liquor with ethyl acetate with the same volume for three times, and concentrating to obtain a crude extract. 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. The components are concentrated, dissolved in methanol, filtered by a 0.22 mu m filter membrane and the semi-prepared liquid phase is purified. The purification method comprises the following steps: mobile phase a (100% water+0.05% formic acid), mobile phase B (100% acetonitrile+0.05% formic acid), column Waters X-bridge C18 (100 mm X10 mm,5 μm), flow rate 2mL/min, detection wavelength 345nm, adjusting the ratio of mobile phases a and B according to the polarity of the compound, collecting the compound in each component at the corresponding retention time. Co-isolation and purification gave 4 compounds, which were both flavonoids identified by HRESI and NMR, with chloroflavin (compound 1) as the final product and compounds 2-4 as intermediates in the biosynthetic pathway of compound 1 (shown in FIG. 1).
Example 3 construction of engineering strains producing different flavones in Aspergillus candidus and analysis of the products
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).
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.
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.
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.
3.1 Determination of the flavonoid biosynthetic Gene Cluster in Aspergillus candidus. Based on transcriptome analysis and bioinformatic prediction of aspergillus candidus in PPM medium, it was found that cfo gene clusters in aspergillus candidus might be responsible for biosynthesis of flavonoids. Therefore, according to the homologous recombination double exchange technology, the core gene cfoA in the cfo gene cluster is knocked out, and the metabolite analysis finds that the flavonoid compound disappears compared with the wild type mutant strain delta cfoA, so that the gene cluster is proved to be responsible for biosynthesis of the flavonoid compound, and the synthesized end product is the chlorothalonil (figure 2).
3.2 Construction of Aspergillus candidus engineering strains. In order to construct engineering strains for producing different flavonoid compounds in aspergillus candidus, genes (CfoG, cfoF, cfoD, cfoH, cfoK, cfoI, cfoC, cfoJ, cfoB, cfoE in table 1) in cfo are knocked out respectively to obtain different gene deletion mutant strains.
Specifically, according to the nucleotide sequence of each gene in the cfo gene cluster, a pair of external primers are respectively designed at the upstream and downstream of the cfo gene cluster. 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.L of PSTC (40% PEG4000,1.2M sorbitol, 50mM Tris-HCl-pH 8.0, 50mM CaCl 2), gently mixed, and ice-incubated 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 are selected from the screening plate and transferred to a PDAH plate (PDA+50 mg/L hygromycin B) for 5 days, and then part of hyphae on single colonies are selected, genome is extracted, and the genome is used as a template for PCR amplification by using an external primer on a homology arm. 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.
3.3 Metabolite analysis of Aspergillus candidus engineering strains. Aspergillus candidus engineering strain and wild-type MEFC strain 1001 strain constructed in 2.2 are respectively inoculated in PPM culture medium, and the spore number of the Aspergillus candidus engineering strain and the wild-type MEFC strain in the culture medium is 10 7/50 mL. The culture was carried out at 220rpm and 30℃for 7 days to obtain a fermentation broth of each strain. 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 C18RRHD 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,0.58-4.05min80％-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. Analysis showed that flavonoids metabolites different from wild type were produced in the engineering strain (fig. 3).
Example 4 separation and purification of flavonoid Compounds in engineering Strain
Based on the results of analysis of the products of the mutants in example 2.3, the scale of fermentation was increased for mutants having a change in the metabolite, and the medium and fermentation conditions were the same as in example 2.2. After the fermentation is finished, the fermentation liquid is extracted for three times by adopting 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. A small amount of HPLC analysis is taken, the analysis method is the same as in example 2.3, and the optimal preparation condition is determined according to the polarity and the retention time of 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. The corresponding compounds were collected at the corresponding retention times. Through HRESI and NMR analysis, 22 flavonoid compounds, namely compounds 2-23, are finally determined in engineering strains, wherein engineering strain delta cfoB can produce compounds 5-7, engineering strain delta cfoC can produce compounds 8-12, engineering strain delta cfoD can produce compounds 3-4 and 12-14, engineering strain delta cfoE can produce compounds 2 and 4, engineering strain delta cfoF can produce compounds 3-4 and 12-13, engineering strain delta cfoG can produce compounds 12 and 15, engineering strain delta cfoH can produce compounds 4 and 12, engineering strain delta cfoI can produce compounds 3-4 and 12, engineering strain delta cfoJ can produce compounds 16-19, and engineering strain delta cfoK can produce compounds 20-23. The structures of the different strains and the corresponding compounds are shown in Table 2, wherein compounds 6-8, 10 and 19 are novel compounds.
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 has very low similarity to the amino acid sequence of CHI found in nature at present, and the phylogenetic tree analysis CfoK is located on a different branch than the plant CHI and bacterial CHI; cfoK is presumed to be 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 phylogenetic tree analysis CfoJ is located on a different branch than FNS I and FNS II; cfoJ is presumed to be a novel flavonoid synthase.
TABLE 2 corresponding flavonoids in different mutants
EXAMPLE 5 evaluation of the Activity of flavonoid Compounds against pathogenic fungi
5.1 Two strains of the human pathogenic fungi Candida albicans (Candida albicans), aspergillus fumigatus (Aspergillus fumigatus), 4 strains of the plant pathogenic fungi Rhizoctonia cerealis (Sclerotium rolfsii sacc.), botrytis cinerea (Botrytis cinerea), fusarium oxysporum (Fusarium oxysporum f.sp.cucumerinum, FOC) and Malus pumila (Colletotrichum gloeosporioides) were selected as test strains. Candida albicans is inoculated in PDB culture medium, cultured for 12 hours at 28 ℃ and 220rpm, and then diluted to 5 multiplied by 10 5/mL by the sterile PDB culture medium, and bacterial suspension is obtained for standby. Other strains were inoculated on PDA plates and incubated at 28℃for 5-7 days until the plates were full of hyphae or spores. The mycelia were washed with a sterile 0.85% NaCl solution (containing 0.25% Tween 20) and scraped off, 50mL of sterile PDB medium was added to obtain a mother liquor of the bacterial suspension, and the mother liquor was further diluted with sterile PDB medium to obtain a bacterial suspension of 5X 10 5/mL concentration for use.
5.2 Compound (3-23) obtained from Aspergillus candidus engineering strain and positive control (amphotericin B) were dissolved in 100. Mu.L DMSO to prepare 10mg/mL solution. After thoroughly mixing, 50. Mu.L of the sample solution was pipetted into another centrifuge tube, followed by 50. Mu.L of DMSO to obtain a halved concentration of the sample solution. According to this method, 15 groups of sample solutions with sequentially halved concentrations were obtained. Under aseptic conditions, 95 μl of the bacterial suspension to be tested is sequentially added into a 96-well plate, 5 μl of diluted sample to be tested is sequentially added into the 96-well plate containing the bacterial suspension, and the final concentration of the compound to be tested is sequentially 500, 250, 125, 62.5, 31.25, 15.63, 7.81, 3.91, 1.95, 0.98, 0.49, 0.24, 0.12, 0.06 and 0.03 μg/mL. After gentle shaking and mixing, 96-well plates were sealed and incubated at 28℃for 72 hours. And measuring the absorbance value of each hole (or observing whether the solution in the hole is turbid or not at a bright place by naked eyes) at the wavelength of 600nm by using an enzyme-labeled instrument, wherein the Minimum Inhibitory Concentration (MIC) of the compound is the Minimum Inhibitory Concentration (MIC) of the compound, which can completely inhibit the growth of indicator bacteria in the hole. The above-mentioned tests were performed three times in parallel, and the test results are shown in Table 3.
TABLE 3 inhibition Activity of flavonoids against pathogenic fungi
a Amphotericin B; and NI: has no inhibitory activity
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 FAD dependent oxidase CfoG, monooxygenase CfoF, SAM dependent methyltransferase CfoD, cytochrome P450 enzyme CfoH, ester hydrolase CfoK, scytalone dehydratase CfoI, SAM dependent methyltransferase CfoC, NADPH dependent FMN reductase CfoJ, SAM dependent methyltransferase CfoB, FAD dependent oxidase CfoE, heterozygous non-ribosomal peptide synthase and polyketide synthase CfoA;
The amino acid sequence of CfoG has at least 80% sequence identity with SEQ ID No.14, the amino acid sequence of CfoF has at least 80% sequence identity with SEQ ID No.15, the amino acid sequence of CfoD has at least 80% sequence identity with SEQ ID No.16, the amino acid sequence of CfoH has at least 80% sequence identity with SEQ ID No.17, the amino acid sequence of CfoK has at least 80% sequence identity with SEQ ID No.18, the amino acid sequence of CfoI has at least 80% sequence identity with SEQ ID No.19, the amino acid sequence of CfoC has at least 80% sequence identity with SEQ ID No.20, the amino acid sequence of CfoJ has at least 80% sequence identity with SEQ ID No.21, the amino acid sequence of CfoB has at least 80% sequence identity with SEQ ID No.22, the amino acid sequence of 24 has at least 80% sequence identity with SEQ ID No. CfoE, and the amino acid sequence of CfoE has at least 80% sequence identity with SEQ ID No. CfoA;
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. Use according to claim 2 or 3, wherein the flavonoid is selected from one or any of the compounds 2-23.
5. A novel flavonoid compound selected from one or more of a compound 6, a compound 7, a compound 8, a compound 10, a compound 19, a compound 2-compound 5, a compound 9, a compound 11-compound 18 and a compound 20-compound 23; preferably, the flavonoid compound is selected from one or more of compound 6, compound 7, compound 8, compound 10 or compound 19.
6. Use of a flavonoid compound according to claim 5, including use in inhibiting the growth of a microorganism, or in the preparation of an agent that inhibits the growth of a microorganism, or in the preparation of a medicament for the treatment of a disease caused by a microorganism; preferably, the microorganism is selected from one or more of Candida albicans (Candida albicans), aspergillus fumigatus (Aspergillus fumigatus), sclerotium rolfsii (Sclerotium rolfsii sacc.) and Botrytis cinerea (Botrytis cinerea).
7. A genetic engineering strain obtained by mutating genes related to flavone synthesis in aspergillus candidus; the gene related to flavone synthesis is selected from one or more of FAD dependent oxidase CfoG, monooxygenase CfoF, SAM dependent methyltransferase CfoD, cytochrome P450 enzyme CfoH, ester hydrolase CfoK, scytalone dehydratase CfoI, SAM dependent methyltransferase CfoC, NADPH dependent FMN reductase CfoJ, SAM dependent methyltransferase CfoB, FAD dependent oxidase CfoE, hybrid non-ribosomal peptide synthase and polyketide synthase CfoA in claim 1.
8. The use of the genetically engineered strain of claim 7 for the production of flavonoids; preferably, the flavonoid compound is selected from one or any of the compounds 2-23.
9. A method for producing a flavonoid compound, comprising the step of fermenting the genetically engineered strain of claim 7; preferably, the flavonoid compound is selected from one or any of the compounds 2-23.
10. The flavonoid according to claim 5, or the use according to claim 8, or the method according to claim 9,
The structural formula of the compound 2 is shown as follows:
the structural formula of the compound 3 is shown as follows:
The structural formula of the compound 4 is shown as follows:
the structural formula of the compound 5 is shown as follows:
the structural formula of the compound 6 is shown as follows:
the structural formula of the compound 7 is shown as follows:
the structural formula of the compound 8 is shown as follows:
The structural formula of the compound 9 is shown as follows:
The structural formula of the compound 10 is shown as follows:
The structural formula of the compound 11 is shown as follows:
the structural formula of the compound 12 is shown as follows:
The structural formula of the compound 13 is shown as follows:
The structural formula of the compound 14 is shown as follows:
the structural formula of the compound 15 is shown as follows:
The structural formula of the compound 16 is shown as follows:
the structural formula of the compound 17 is shown as follows:
The structural formula of the compound 18 is shown as follows:
the structural formula of the compound 19 is shown as follows:
The structural formula of the compound 20 is shown as follows:
The structural formula of the compound 21 is shown as follows:
the structural formula of the compound 22 is shown as follows:
the structural formula of the compound 23 is shown as follows:
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