CN114134054A

CN114134054A - Construction of an Aspergillus oryzae chassis strain with high terpenoid production and an automated high-throughput mining platform for terpenoid natural products

Info

Publication number: CN114134054A
Application number: CN202111283704.0A
Authority: CN
Inventors: 刘天罡
Original assignee: Wuhan University WHU
Current assignee: Wuhan Hesheng Technology Co ltd
Priority date: 2021-11-01
Filing date: 2021-11-01
Publication date: 2022-03-04
Anticipated expiration: 2041-11-01
Also published as: CN114134054B

Abstract

The invention discloses an aspergillus oryzae chassis for high yield of terpenes and a method for automatically excavating a terpene biosynthesis gene cluster FgJ02895 in high flux, which aim at high yield of terpene products, high flux heterologous reconstruction of the terpene biosynthesis gene cluster and activity-oriented compound screening. The invention is based on CRISPR/Cas9 technology mediated gene homologous recombination, integrates over-expresses endogenous MVA pathway genes at gene high expression sites in a genome, establishes an Aspergillus oryzae chassis cell for high-yield terpenoids, and realizes 41-fold and 111-fold increase of mangicine and mangicil J yield. The high-throughput excavation strategy comprising PCR amplification, plasmid library and strain library construction established in the chassis realizes the excavation of related products of the gene cluster FgJ02895 and obtains a product library of the gene cluster. And meanwhile, the anti-inflammatory activity of the compounds produced in the strain bank can be evaluated.

Description

Aspergillus oryzae chassis strain capable of producing terpenoids at high yield and construction of automatic high-flux excavation platform for terpenoids natural products

Technical Field

The invention relates to the technical field of natural product biosynthesis, in particular to construction of a aspergillus oryzae high-yield terpene chassis and construction of an automatic high-flux terpene natural product excavating platform based on a chassis strain.

Background

Fungal natural products are one of the important sources of natural drugs, and the traditional excavation strategy has helped researchers find various active natural products from microorganisms, such as the potent antibiotics penicillin, the hypolipidemic agent lovastatin, the immunosuppressant cyclosporine and the like. However, as more and more natural products are discovered, the difficulty and challenge of being able to mine into new natural products is increasing. With the development of bioinformatics and sequencing technologies, the information of biosynthetic gene clusters of a large number of fungal natural products, particularly silent gene clusters, has been revealed to hold a great potential for synthesizing novel natural products. To fully release the synthetic potential of these natural product biosynthetic gene clusters, researchers developed a variety of strategies, such as homologous global or specific pathway regulation, exogenous activator activation, CRISPR/Cas strategy gene editing expression of silenced gene clusters, and the development of compelling heterologous reconstitution strategies, etc., unlocking the synthetic mechanisms of a series of novel natural products. In addition, the development of genome mining and artificial chromosome technology based on bioinformatics and computer network model predictive analysis technology in recent years further promotes the research progress in the field of natural product biosynthesis. Although the development of the above strategy greatly enriches natural product libraries, there are still problems to be solved in the implementation process, such as low throughput, low yield, etc.

Terpenoids are a generic term for compounds containing isoprene units. It is widely found in nature, and up to now, more than 12 tens of thousands of terpenoids are found in animals, plants and microorganisms. The compounds have a plurality of physiological activities and are widely applied to the industries of food, cosmetics and medicines. The invention takes the fungus terpene product as a research target, takes a well-known strain Aspergillus oryzae (Aspergillus oryzae) with biological safety as a heterologous expression host, creates an Aspergillus oryzae chassis for high-yield terpene, establishes a filamentous fungus terpene high-yield chassis for improving the yield of the terpene natural product, establishes a set of complete fungus terpene product genome automatic high-flux excavation research scheme, improves the flux of the research of the terpene natural product gene cluster, and solves the problem of low product yield.

Disclosure of Invention

Aiming at the defects of the prior art, the invention provides an Aspergillus oryzae chassis for high yield of terpenes and an automatic high-flux excavation platform for terpene natural products, and particularly relates to the transformation of the Aspergillus oryzae chassis for high yield of terpenes and the efficient synthesis of sesterterpene compound mangicol J with anti-inflammatory activity, and simultaneously relates to the high-flux heterologous reconstruction of a natural product biosynthesis gene cluster FgJ02895, the PCR amplification of functional elements of the natural product biosynthesis gene cluster, the construction of a plasmid library, the construction of a strain library and the screening of compounds with anti-inflammatory activity as a guide.

In order to achieve the purpose, the technical scheme of the invention is as follows:

in a first aspect of the invention, there is provided a microbial underpan cell comprising a gene that enhances the ability of the underpan cell to produce terpenoids, said gene being that of an aspergillus oryzae derived MVA pathway gene module; the genes are ERG10, ERG13, tHMG1, ERG12, ERG8, MVD1 and IDI genes from Aspergillus oryzae. Preferably, the nucleotide sequence of the ERG10 gene is shown in SEQ ID NO: 10 is shown in the figure; the nucleotide sequence of the ERG13 gene is shown as SEQ ID NO: 11 is shown in the figure; the nucleotide sequence of the tHMG1 gene is shown as SEQ ID NO: 12 is shown in the specification; the nucleotide sequence of the ERG12 gene is shown as SEQ ID NO: 13 is shown in the figure; the nucleotide sequence of the ERG8 gene is shown as SEQ ID NO: 14 is shown in the figure; the nucleotide sequence of the MVD1 gene is shown as SEQ ID NO: 15 is shown in the figure; the nucleotide sequence of IDI gene is shown as SEQ ID NO: shown at 16.

In one or more embodiments of the invention, the microbial underpan cell is a eukaryotic cell or a prokaryotic cell; preferably, the microbial underpan cell is aspergillus oryzae, escherichia coli, saccharomyces cerevisiae, pichia pastoris, bacillus, aspergillus nidulans, aspergillus niger, neurospora crassa, alternaria alternata or fusarium; more preferably, the underpan cells are aspergillus oryzae.

In one or more embodiments of the invention, the terpenoid is selected from one or more of a hemiterpene, a monoterpene, a sesquiterpene, a diterpene, a triterpenoid, a tetraterpenoid, and a polyterpene. In one or more embodiments of the present invention, the gene tmgb 1 is a rate-limiting enzyme gene, and preferably, the rate-limiting enzyme expressed by gene tmgb 1 is overexpressed in the underpan cells; more preferably, the rate-limiting enzyme expressed by said gene tmgb 1 is overexpressed in 4 copies.

In a second aspect of the invention, the invention provides the use of genes ERG10, ERG13, HMG1, ERG12, ERG8, MVD1 and IDI genes from aspergillus oryzae for increasing the ability of Chassis cells to produce terpenoids.

In a third aspect of the present invention, there is provided a method for preparing the microbial underpan cells according to the first aspect of the present invention, comprising the steps of:

step 1): establishing a microbial chassis strain promoter library: screening promoters from different sources, and characterizing the strength of the promoter suitable for aspergillus oryzae by regulating and controlling the expression of GUS genes;

step 2): selecting mevalonate pathway synthases ERG10, ERG13, tHMG1, ERG12, ERG8, MVD1 and IDI from Aspergillus oryzae original sources from a fungal genome database by sequence homology comparison analysis by taking a saccharomyces cerevisiae derived mevalonate pathway as a reference;

step 3): constructing 7 mevalonate pathway synthetases of the mevalonate pathway from the aspergillus oryzae source screened in the step 2) on different plasmids respectively under the control of promoters with different intensities characterized in the step 1), and increasing the copy number of the rate-limiting enzyme expressed by the rate-limiting enzyme gene tHMG1 to obtain the chassis cell.

In other embodiments of the present invention, the preparation method of the underpan cell according to the first aspect of the present invention comprises integrating ERG10, ERG13, tmhmg 1, ERG12, ERG8, MVD1 and IDI genes derived from aspergillus oryzae into the high expression site of aspergillus oryzae genome respectively by using CRISPR/Cas9 strategy, and increasing the copy number of the rate-limiting enzyme expressed by the rate-limiting enzyme gene tmhmg 1 to obtain the microbial underpan cell.

In the fourth aspect of the invention, the invention provides a bacterial strain with high terpene yield, wherein the Chassis cells in the third aspect of the invention are used as starting bacterial strains to respectively over-express a sesterterpene synthase gene mgcD, co-express a sesterterpene synthase gene mgcD and a cytochrome P450 enzyme gene mgcE, and the bacterial strain with high terpene yield is obtained. Preferably, the nucleotide sequence of gene mgcD is as shown in SEQ ID NO: 17 is shown; the nucleotide sequence of gene mgcE is shown as SEQ ID NO: 18, respectively.

In a fifth aspect of the present invention, the present invention provides a high throughput automated mining method for terpene biosynthetic gene cluster, comprising the following steps:

1) PCR amplification; 2) constructing a plasmid library; 3) constructing a microbial strain library; 4) activity-directed compound screening.

In one or more embodiments of the present invention, the step 2) includes the steps of:

a. respectively designing 40bp overlapping sequences at the 5 '-end and the 3' -end of each functional gene, promoter and terminator sequence in the terpene biosynthesis gene cluster, preparing a DNA polymerase system by an automatic workstation, and performing PCR amplification to obtain each fragment;

b. assembling each fragment by automated yeast (figure 2) to obtain a recombinant plasmid;

c. the recombinant plasmid was transformed into E.coli (FIG. 4), and the plasmid library was obtained by enrichment.

In one or more embodiments of the present invention, the step 3) includes the steps of: transforming the plasmid library obtained in step 2) into the microbial underpan cells according to the first aspect of the invention (fig. 6) based on an automated platform according to a rational combination of different functional elements (fig. 5) comprising terpene synthases, cytochrome P450 enzymes, acyltransferases, glycosyltransferases to obtain a strain library.

In one or more embodiments of the present invention, the step 4) includes the steps of: acting the fermentation product of the microbial strain library obtained in the step 3) on mouse macrophage RAW264.7 induced by lipopolysaccharide, and screening terpenoid compounds (figure 7) with anti-inflammatory activity, such as mangicols sesterterpene compounds, by taking the generation of Nitric Oxide (NO) as a detection index.

In a sixth aspect of the invention, the invention provides a sesquiterpene compound of novel structure, the structure of which is selected from one of the following structures (fig. 9):

17 (as characterized in fig. 10-16),

20 (as characterized in FIGS. 17-23),

28 (characterization as in figures 24-30).

In a seventh aspect, the present invention provides a use of the sesquiterpene compound of the sixth aspect of the present invention in the preparation of an anti-inflammatory agent.

In an eighth aspect of the invention, the invention provides a sesquiterpene synthesis gene cluster BGC6-FgJ02895 for synthesizing a sesquiterpene compound according to the sixth aspect of the invention. The sesquiterpene synthesis gene cluster BGC6-FgJ02895 comprises FgJ02892 gene, FgJ02895 gene, FgJ02896 gene, FgJ02897 gene, FgJ02898 gene, FgJ02899 gene, FgJ02900 gene, FgJ02901 gene and FgJ02902 gene, which respectively encode decarboxylase, terpene synthase, transcription factor, cytochrome P450 enzyme, acetyl transferase, Tf, cytochrome P450 enzyme, monooxygenase and copper ion dependent ATPase (as shown in figure 8).

Preferably, the nucleotide sequence of the FgJ02892 gene is shown in SEQ ID NO: 23 is shown; the nucleotide sequence of the FgJ02895 gene is shown as SEQ ID NO: shown at 24; the nucleotide sequence of the FgJ02896 gene is shown as SEQ ID NO: 25 is shown; the nucleotide sequence of the FgJ02897 gene is shown as SEQ ID NO: 26 is shown; the nucleotide sequence of the FgJ02898 gene is shown as SEQ ID NO: 27 is shown; the nucleotide sequence of the FgJ02899 gene is shown as SEQ ID NO: 28 is shown; the nucleotide sequence of the FgJ02900 gene is shown as SEQ ID NO: 29 is shown; the nucleotide sequence of the FgJ02901 gene is shown as SEQ ID NO: 30 is shown in the figure; the nucleotide sequence of the FgJ02902 gene is shown as SEQ ID NO: shown at 31.

In a ninth aspect of the invention, the invention provides a vector or vector mixture comprising a gene as described in the first and/or eighth aspect of the invention.

Compared with the prior art, the invention has the following advantages and beneficial effects:

1. the invention provides a microbial underpan cell which is modified to have the capacity of highly producing terpenoids.

2. The invention provides a preparation method of the microbial underpan cell, which takes aspergillus oryzae as a dominant expression host, overexpresses the whole MVA (multi-domain mosaic Virus) pathway in vivo based on a multi-step and screening marker-reusable genetic transformation strategy in the aspergillus oryzae host mediated by a CRISPR/Cas9 technology through rational design, adds 4 copies of tHMG1 gene, establishes the aspergillus oryzae underpan cell with high terpenoid yield, and provides a new solution for solving the problem of low yield in the biosynthesis process of natural products.

3. The invention provides a strain with high terpene yield, which is obtained by modifying the underpan cells.

4. The invention provides a method for producing terpenoid by using the strain with high terpenoid yield.

5. The invention provides application of ERG10, ERG13, HMG1, ERG12, ERG8, MVD1 and IDI genes derived from aspergillus oryzae in improving the high yield of terpenoids in Chassis cells.

6. In the invention, preferably, the constructed rice koji microbial chassis cells are taken as dominant expression hosts, and the terpene natural product biosynthesis gene cluster BGC-FgJ02895 is efficiently and heterologously reconstructed in vivo on the basis of the constructed automatic high-throughput platform through rational design, so that PCR amplification and plasmid library construction to strain library construction can be completed, and a solution is provided for solving the problem of low throughput in the natural product biosynthesis process.

7. The invention provides a sesquiterpene compound with a new structure, which is characterized in that: having the structure shown in

compounds

17, 20 and 28.

8. The invention provides a sesquiterpene synthesis gene cluster BGC6-FgJ02895, which is characterized in that: encoding and synthesizing the three sesquiterpene compounds with the new structure.

Additional aspects and advantages of the invention will be set forth in part in the description which follows and, in part, will be obvious from the description, or may be learned by practice of the invention.

Drawings

FIG. 1 is a schematic diagram of a terpene high-yield chassis construction process and partial experimental results according to an embodiment of the invention.

FIG. 1a is a schematic diagram of a process for preparing an Aspergillus oryzae mutant strain of the present invention; FIG. 1b is a schematic diagram showing the results of comparison of the yields of mangicriene from the starting strain AO-Y51, the mutant AO-S96 and the mutant AO-S84; FIG. 1c is a schematic diagram of the transformation of Aspergillus oryzae mutants AO-S84, AO-S94, AO-S96, AO-S98; FIG. 1d is a graph showing the comparison of the yield of Mangicol J of the starting strain AO-Y52, mutant AO-S98 and mutant AO-S94.

FIG. 2 is a schematic diagram of the process of constructing plasmid library by assembling high-throughput yeast according to an embodiment of the present invention.

FIG. 3 is a schematic diagram of a high throughput plasmid extraction process according to one embodiment of the present invention.

FIG. 4 is a schematic diagram of a high-throughput E.coli transformation procedure according to an embodiment of the present invention.

FIG. 5 is a schematic diagram showing the principle of construction of a high-throughput A.oryzae mutant strain according to an embodiment of the present invention.

FIG. 6 is a schematic diagram of the construction process of the high-throughput Aspergillus oryzae mutant library according to one embodiment of the present invention.

FIG. 7 is a schematic view of the flow chart of the anti-inflammatory activity assay in the example of the present invention.

FIG. 8a is a diagram showing the result of bioinformatics analysis of gene cluster BGC6-FgJ 02895; FIG. 8b is a schematic diagram of heterologous reconstitution of BGC6-FgJ02895 gene cluster.

FIG. 9 is a GC/MS and HR-LC/MS detection map of gene cluster BGC6-FgJ 02895.

FIG. 10 is a schematic structural diagram of Compound (17) according to one embodiment of the present invention;

FIG. 11 shows a compound of the present invention (17)¹H NMR spectrum;

FIG. 12 shows a compound of the present invention (17)¹³C NMR spectrum;

FIG. 13 shows a compound of the present invention (17)¹H-¹H COSY spectrogram;

FIG. 14 shows HSQC spectra of compound (17) of the present invention;

FIG. 15 shows a HMBC spectrum of the compound (17) of the present invention;

FIG. 16 is a ROESY spectrum of the compound (17) of the present invention.

FIG. 17 is a schematic structural diagram of Compound (20) according to one embodiment of the present invention;

FIG. 18 shows a scheme for preparing a compound (20) of the present invention¹H NMR spectrum;

FIG. 19 shows a scheme for preparing a compound (20) of the present invention¹³C NMR spectrum;

FIG. 20 shows a scheme for preparing a compound (20) of the present invention¹H-¹H COSY spectrogram;

FIG. 21 is an HSQC spectrum of compound (20) of the present invention;

FIG. 22 is an HMBC spectrum of compound (20) of the present invention;

FIG. 23 is a ROESY spectrum of the compound (20) of the present invention.

FIG. 24 is a schematic structural diagram of compound (28) according to one embodiment of the present invention;

FIG. 25 is a drawing showing a scheme for preparing a compound (28) of the present invention¹H NMR spectrum;

FIG. 26 shows a scheme for preparing a compound (28) of the present invention¹³C NMR spectrum;

FIG. 27 is a photograph of Compound (28) of the present invention¹H-¹H COSY spectrogram;

FIG. 28 is an HSQC spectrum of compound (28) of the present invention;

FIG. 29 is an HMBC spectrum of compound (28) of the present invention;

FIG. 30 is a ROESY spectrum of the compound (28) of the present invention.

Detailed Description

The scheme of the invention will be explained with reference to the examples. It will be appreciated by those skilled in the art that the following examples are illustrative of the invention only and should not be taken as limiting the scope of the invention. The examples, where specific techniques or conditions are not indicated, are to be construed according to the techniques or conditions described in the literature in the art or according to the product specifications. The methods used are conventional methods known in the art unless otherwise specified, and the consumables and reagents used are commercially available unless otherwise specified. Unless otherwise defined, technical and scientific terms used herein have the same meaning as is familiar to those skilled in the art. In addition, any methods or materials similar or equivalent to those described herein can also be used in the present invention. Wherein "-" represents gene linkage, for example hlyA (nucleotide sequence shown in SEQ ID NO: 9) -tHMG1 (nucleotide sequence shown in SEQ ID NO: 12) NOs (nucleotide sequence shown in SEQ ID NO: 22) represents a nucleotide sequence shown in SEQ ID NO: 9, the nucleotide sequence of the gene hlyA is shown as SEQ ID NO: 12, and a nucleotide sequence of the gene tHMG1 shown in SEQ ID NO: 22 are linked in sequence.

Example 1 determination of mevalonate pathway synthase in Aspergillus oryzae

The mevalonate pathway synthases ERG10, ERG13, HMG1, ERG12, ERG8, MVD1 and IDI originally derived from Aspergillus oryzae were screened from the fungal genome database by sequence homology alignment analysis with reference to the mevalonate pathway (MVA) derived from Saccharomyces cerevisiae.

Example 2 construction of expression vector

All genes were obtained by PCR amplification using primers as shown in sequence table 1:

table 1 primer sequences used in the examples

The plasmid construction mainly adopts four methods, enzyme digestion and enzyme ligation, golden gate assembly, and Gibson method and Yeast assembly method. The specific construction method comprises the following steps:

construction of the pSC11 plasmid: using Aspergillus oryzae genome as template, wA up and wA down were amplified with primers 11-2F/11-2R, 11-7F/11-7R, respectively (DOI:10.1038/nature 04300). pGB123(DOI:10.1002/ biot.201600697) As a template, the promoter alcA (nucleotide sequence shown as SEQ ID NO: 1) are shown. GUS was amplified using pCAMBIA1301 as a template and a primer set 11-4F/11-4R. The reaction was performed with pAdeA (DOI:10.1021/ ja3116636) As a template, TamyB and AdeA were amplified with primers 11-5F/11-5R, 11-6F/11-6R, respectively. The dna fragment was expressed as pRS426(DOI:10.1016/j.biortech.2012.08.104) As a template, the vector sequence was amplified with primers 11-1F/11-1R. The 7 amplified fragments were assembled by the Yeast assembly method to obtain the plasmid pSC 11. Construction of plasmid pSC12-pSC 19: the genes identified as pGB99, pGB98, pGB93, pGB96, pGB92 and rice koji genome (DOI:10.1002/biot.201600697) As a template, the promoter agdA (nucleotide sequence shown in SEQ ID NO: 2), glaA (nucleotide sequence shown in SEQ ID NO: 3), gpdA (nucleotide sequence shown in SEQ ID NO: 4), and oliC (nucleotide sequence shown in SEQ ID NO: 5), trpC (nucleotide sequence shown in SEQ ID NO: shown at 6). Using Aspergillus oryzae genome as template, primers 17-1F/17-1R, 18-1F/18-1R, 19-1F/19-1R were used to amplify amyB (nucleotide sequence shown in SEQ ID NO: 7), enoA (nucleotide sequence shown in SEQ ID NO: 8), hlyA (nucleotide sequence shown in SEQ ID NO: 9). Using pCAMBIA1301 as a template, GUS (DOI:10.1016/j.synbio.2016.07.002). The pSC12-pSC19 plasmids were constructed by ligating together the 8 promoter fragments with the corresponding GUS fragments by overlap extension PCR (OE-PCR), cleaved with PacI/MreI, and then inserted into the PacI/MreI cleaved pSC11 plasmid.

Based on a promoter with stronger promoter capacity in a fungal host reported in the literature, the promoter strength suitable for Aspergillus oryzae is characterized by regulating the expression of a GUS reporter gene, and in order to characterize the promoter strength of a series of promoters in an Aspergillus oryzae host, the plasmid pSC11-pSC19 is transformed into Aspergillus oryzae to obtain strains AO-S11 to AO-S19 containing the corresponding promoters. Finally obtaining the promoter suitable for Aspergillus oryzae, wherein the sequence is hlyA > oliC > amyB > glaA > enoA > gpdA > agdA > trpC > alcA.

Construction of the pSC59 plasmid: using pSC11 plasmid as a template, the vector fragment was amplified with primers 44-1F/44-1R. A homologous arm fragment ku80-up and ku80-down (DOI:10.1038/nature04300) was amplified using Aspergillus oryzae genomic DNA as a template and primers 44-2F/44-2R, 44-13F/44-13R. Using Aspergillus oryzae cDNA as template, the fragments ERG10 (nucleotide sequence shown in SEQ ID NO: 10), ERG13 (nucleotide sequence shown in SEQ ID NO: 11) and tHMG1 (nucleotide sequence shown in SEQ ID NO: 12) were amplified with primers 44-4F/44-4R,44-7F/44-7R, and 44-10F/44-10R, respectively. pGB96 and Aspergillus oryzae genomic DNA were used as templates to amplify promoter fragments oliC (nucleotide sequence shown in SEQ ID NO: 5), amyB (nucleotide sequence shown in SEQ ID NO: 7) and hlyA (nucleotide sequence shown in SEQ ID NO: 9) with primers 44-3F/44-3R, 44-6F/44-6R and 44-9F/44-9R, respectively. Terminator fragment niaD (nucleotide sequence shown in SEQ ID NO: 20), amyB (nucleotide sequence shown in SEQ ID NO: 19) and NOs (nucleotide sequence shown in SEQ ID NO: 22) were amplified using A.nidulans FGSC A4, Aspergillus oryzae genome and pGB96 as templates and primers 44-5F/44-5R, 44-8F/44-8R and 44-11F/44-11R, respectively. Plasmid pAdeA (DOI:10.1021/ja3116636) As a template, the fragment AdeA was amplified with primers 44-12F/44-12R. The fragments of similar size were subjected to overlap extension PCR (OE-PCR) and finally yeast assembly to give plasmid pSC 44. Using pSC44 as a template, a fragment hlyA (nucleotide sequence shown in SEQ ID NO: 9) -tHMG1 (nucleotide sequence shown in SEQ ID NO: 12) -NOs (nucleotide sequence shown in SEQ ID NO: 22) was amplified with primers 59-1F/59-1R, digested with SacI, and inserted into the SacI-digested pSC44 plasmid to construct a pSC59 plasmid.

Construction of the pSC71 plasmid: the vector fragment was amplified with primers 71-1F/71-1R using pSC11 plasmid as a template. A homologous arm fragment ku70-up and ku70-down (DOI:10.1038/nature04300) were amplified using Aspergillus oryzae genomic DNA as a template and primers 71-2F/71-2R and 71-11F/71-11R, respectively. Using Aspergillus oryzae cDNA as template, primers 71-5F/71-5R, 71-8F/71-8R were used to amplify fragments MVD1 (nucleotide sequence shown in SEQ ID NO: 15) and IDI (nucleotide sequence shown in SEQ ID NO: 16). Using pSC44 as a template, a fragment hlyA (nucleotide sequence shown in SEQ ID NO: 9) -tHMG1 (nucleotide sequence shown in SEQ ID NO: 12) -NOs (nucleotide sequence shown in SEQ ID NO: 22) was amplified with primers 71-3F/71-3R. The promoters oliC (nucleotide sequence shown in SEQ ID NO: 5) and glaA (nucleotide sequence shown in SEQ ID NO: 3) were amplified using Aspergillus oryzae genome and pGB98 as templates and primers 71-4F/71-4R, 71-7F/71-7R. Using pYJ152 as a template, a fragment argB-PamyB-mgcD (nucleotide sequence shown in SEQ ID NO: 17) -TamyB was amplified with primers 71-10F/71-10R. Terminator agdA (nucleotide sequence shown in SEQ ID NO: 21) and niaD (nucleotide sequence shown in SEQ ID NO: 20) were amplified using Aspergillus oryzae genomic DNA and A. nidulans FGSC A4 as templates and primers 71-6F/71-6R, 71-9F/71-9R. The fragments of similar size were subjected to overlap extension PCR (OE-PCR) and finally yeast assembly to give plasmid pSC 71.

Construction of the pSC73 plasmid: using the pSC11 plasmid as a template, the vector fragment was amplified with primers 73-1F/73-1R. A homologous arm fragment ku70-up and ku70-down (DOI:10.1038/nature04300) were amplified using Aspergillus oryzae genomic DNA as a template and primers 73-2F/73-2R, 73-11F/73-11R, respectively. The Aspergillus oryzae cDNA was used as template, and primers 73-4F/73-4R, 73-7F/73-7R were used to amplify fragments ERG12 (nucleotide sequence shown in SEQ ID NO: 13) and ERG8 (nucleotide sequence shown in SEQ ID NO: 14). Using pSC44 as a template, a fragment hlyA (nucleotide sequence shown in SEQ ID NO: 9) -tHMG1 (nucleotide sequence shown in SEQ ID NO: 12) -niaD (nucleotide sequence shown in SEQ ID NO: 20) was amplified with primers 73-9F/73-9R. The promoter glaA (nucleotide sequence shown as SEQ ID NO: 3) and enoA (nucleotide sequence shown as SEQ ID NO: 8) were amplified using pGB98 and Aspergillus oryzae genomic DNA as templates and primers 73-3F/73-3R and 73-6F/73-6R. The terminator NOs (nucleotide sequence shown in SEQ ID NO: 22) and amyB (nucleotide sequence shown in SEQ ID NO: 19) were amplified using pGB96 and the Aspergillus oryzae genome as templates with primers 73-5F/73-5R and 73-8F/73-8R. sC was amplified using pUSA as template and primers 73-10F/73-10R. The fragments of similar size were subjected to overlap extension PCR (OE-PCR) and finally yeast assembly to give plasmid pSC 73.

Construction of pSC111 plasmid: the terminator NOs (nucleotide sequence shown in SEQ ID NO: 22) was amplified using the primer 111-1F/111-1R with pGB98 as a template. The vector fragment was amplified using the primer 111-3F/111-3R, using the pSC11 plasmid as a template. A homologous arm fragment koj-down and koj-up (DOI:10.1038/nature04300) were amplified using Aspergillus oryzae genomic DNA as a template and primers 111-2F/111-2R, respectively. Using Aspergillus oryzae genome DNA as a template, a ptrA (DOI:10.1007/s10529-015-2015-x). Using Aspergillus oryzae genome DNA as template, hlyA (nucleotide sequence shown in SEQ ID NO: 9) was amplified with primers 111-6F/111-6R. Using pYJ153 as a template, mgcE (nucleotide sequence shown in SEQ ID NO: 18) was amplified using primers 111-7F/111-7R. The fragments with similar sizes were subjected to overlap extension PCR (OE-PCR), and finally yeast assembly was performed to obtain plasmid pSC 111.

Construction of the pSC112 plasmid: a terminator agdA (nucleotide sequence shown in SEQ ID NO: 21) was amplified using Aspergillus oryzae genomic DNA as a template and primers 112-1F/112-1R. Using pSC111 as a template, hlyA (nucleotide sequence shown in SEQ ID NO: 9) -mgcE (nucleotide sequence shown in SEQ ID NO: 18) was amplified using primers 112-2F/112-2R. The two fragments were subjected to overlap extension PCR (OE-PCR), digested with NotI/HpaI, and inserted into NotI/HpaI-digested pSC111 plasmid to construct pSC112 plasmid.

Construction of pSC253 plasmid: vector sequences were amplified using primers 251-1F/251-1R, using pSC11 as a template. The dna fragment was identified with pAdeA-Cas9(DOI:10.1007/s10529-015-2015-x) As a template, PamyB-Cas9-TamyB was amplified with primers 251-2F/251-2R. hAMA1(DOI:10.1006/fgbi.1997.0980) was amplified with primer 251-3F/251-3R using A.nidulans FGSC A4 as template. Using pSC111 as a template, ptrA (DOI:10.1271/ bbb.64.1416). The yeast was assembled to give plasmid pSC 251. Using pSC11 as a template, the fragment CEN was amplified using primers 78-1F/78-1R. The plasmid pSC78 was constructed by XhoI/BamHI digestion and insertion into the XhoI/BamHI digested pET28 plasmid. Using Aspergillus oryzae genome DNA as template, and using primers 251-1F/251-1R, 251-2F/251-2R, 251-3F/251-3R, 251-4F/251-4R, 251-5F/251-5R, 251-6F[ 251-6 ] R amplification of P_U6-1，T_U6-1，P_U6-2，T_U6-2，P_U6-3，T_U6-3. Will P_U6-1 and T_U6-1，P_U6-2 and T_U6-2，P_U6-3 and T_U6-3 overlap extension PCR (OE-PCR) was performed and the three fragments were assembled with pSC78 by GoldeGate to construct the pSC252 plasmid. A fragment containing 3 sgrnas of pSC252 digested with NotI/PacI was inserted into pSC251 digested with NotI/PacI to construct pSC253 plasmid.

Construction of the pSC184 plasmid: argB and vector sequences were amplified using pSC71 as template and primers 87-1F/87-1R. The reaction was performed with pAdeA (DOI:10.1021/ja3116636) As a template, PamyB-Cas9-TamyB was amplified with primers 87-2F/87-2R. hAMA1 was amplified with primer 87-3F/87-3R using A.nidulans FGSC A4 as template. PyrG was amplified using A.fumigagatus as template with primers 87-4F/87-4R. Four-fragment yeast assembly to construct pSC 87. Amplification of P with 98-1F/98-1R,98-2F/98-2R, 98-3F/98-3R, 98-4F/98-4R, 98-5F/98-5R, 98-6F/98-6R_U6-1，T_U6-1，P_U6-2，T_U6-2，P_U6-3，T_U6-3. Will P_U6-1 and T_U6-1，P_U6-2 and T_U6-2，P_U6-3 and T_U6-3 overlap extension PCR (OE-PCR) was performed and the three fragments were assembled with pSC78 by GoldeGate to construct the pSC98 plasmid. The NotI/SmaI digested fragment of pSC98 containing 3 sgRNAs was inserted into NotI/SmaI digested pSC87 to construct the pSC184 plasmid.

Construction of plasmid pSC 249: a. nidulans FGSC A4 was used as a template, and primers 134-1F/134-1R, 134-2F/134-2R were used to amplify the AMA1 sequence split into two fragments. PyrG was amplified using pSC87 as a template and primers 134-3F/134-3R. Vector sequences and PamyB-Cas9-TamyB were amplified using pSC251 as template and primers 134-4F/134-4R, 134-5F/134-5R. Four-fragment USER Cloning pSC134 was constructed. P amplification with the Aspergillus oryzae genome as template and primers 249-1F/249-1R, 249-2F/249-2R_U6，T_U6The plasmid pSC249 was constructed by overlap extension PCR (OE-PCR), NotI/PacI cleavage, and insertion of NotI/PacI cleaved pSC 134.

Construction of the pSC246 plasmid: vector sequences were amplified using primers 246-1F/246-1R, using pSC11 as a template. HS401 up and HS401 down (DOI:10.1038/nature04300) were amplified using Aspergillus oryzae genome as template with primers 246-2F/246-2R, 246-5F/246-5R. Using pSC44 as a template, PhlyA-tHMG1-Tnos was amplified using primers 246-3F/246-3R. PoliC-MVD-TagdA-PamyB-IDI-TniaD was amplified using primer 246-4F/246-4R with pSC71 as template. The 5 fragments were assembled in yeast to construct the pSC246 plasmid.

Construction of plasmid pSC 247: plasmid pSC247 was constructed by amplifying Tnos-tHMG1-PhlyA, MreI/SalI digested, using pSC246 as a template, with primers 247-1F/247-1R, and inserting MreI/SalI digested pSC 246.

Construction of pSC263 plasmid: p amplification with primer 263-1F/263-1R, 263-2F/263-2R using Aspergillus oryzae genome as template_U6，T_U6The plasmid pSC263 was constructed by overlap extension PCR (OE-PCR), NotI/PacI cleavage, and insertion of NotI/PacI cleaved pSC 134.

Construction of pSC260 plasmid: vector sequences were amplified using pSC11 as a template and primers 260-1F/260-1R. Using Aspergillus oryzae genome as template, primers 260-2F/260-2R, 260-4F/260-4R were used to amplify HS201 up and HS201 down (DOI:10.1038/nature 04300). PoliC-ERG10-TniaD-PamyB-ERG13-TamyB and PhlyA-tHMG1-Tnos were amplified with primers 260-3F/260-3R using pSC44 as a template. The 4 fragments were assembled in yeast to construct the pSC260 plasmid.

Construction of the pSC248 plasmid: p amplification with the Aspergillus oryzae genome as template and the primers 248-1F/248-1R, 248-2F/248-2R_U6，T_U6The plasmid pSC248 was constructed by overlap extension PCR (OE-PCR), NotI/PacI cleavage, and insertion of NotI/PacI cleaved pSC 134.

Construction of the pSC262 plasmid: vector sequences were amplified using primers 262-1F/262-1R using pSC11 as a template. Using Aspergillus oryzae genome as template, using primers 262-2F/262-2R, 262-4F/262-4R to amplify HS601 up and HS601 down (DOI:10.1038/nature04300) and using pSC73 as template, using primers 262-3F/262-3R to amplify PglaA-ERG12-Tnos-PenoA-ERG8-TamyB and PhlyA-tHMG 1-TniaD. The 4 fragments were assembled in yeast to construct the pSC262 plasmid.

Construction of the pSC24 plasmid: using Aspergillus oryzae genome as template, and primer 24-1F/24-1R, 24-2F/24-2R amplification of P_U6，T_U6The plasmid pSC24 was constructed by overlap extension PCR (OE-PCR), NotI/PacI cleavage, and insertion of NotI/PacI cleaved pSC 134.

Construction of the pSC162 plasmid: vector sequences were amplified using primer 162-1F/162-1R with pSC11 as template. Using Aspergillus oryzae genome as template, HS801 up and HS801 down (DOI:10.1038/nature04300) were amplified using primers 162-2F/162-2R, 162-4F/162-4R. Using pSC71 as a template, PhlyA-mgcD-Tnos was amplified using primers 162-3F/162-3R. The 4 fragments were assembled in yeast to construct the pSC162 plasmid.

Construction of the pSC27 plasmid: vector sequences were amplified using primers 25-1F/25-1R using pSC11 as a template. Using Aspergillus oryzae genome as template, HS801 up and HS801 down (DOI:10.1038/nature04300) were amplified with primers 25-2F/25-2R, 25-4F/25-4R. The primer 25-3F/25-3R is used for amplifying the PamyB-mgcD-TamyB by taking pYJ152 as a template. The 4 fragments were assembled in yeast to construct the pSC25 plasmid. Using pSC111 as a template, PhlyA-mgcE-Tnos was amplified using primers 27-1F/27-1R. NotI/PacI digested was inserted into NotI/PacI digested pSC25 to construct pSC27 plasmid.

Construction of the pSC28 plasmid: using pSC111 as a template, PhlyA-mgcE-Tnos was amplified using primers 28-1F/28-1R. The plasmid pSC28 was constructed by PacI digestion and insertion of PacI digested pSC 27.

EXAMPLE 3 Aspergillus oryzae mutant Strain construction strategy for high yield of terpenoids

In order to highly produce target terpenoid, the invention rationally designs and constructs a series of Aspergillus oryzae mutant strains. In the construction of the strains, plasmids containing different genes are transformed into Aspergillus oryzae by protoplast transformation. After the transformants grow out, 3-4 transformants are selected to extract the genome, and PCR verification is carried out on the target gene. And (4) carrying out subculture expansion on the strains with positive PCR results for subsequent seed preservation and fermentation experiments.

In order to realize the yield increase of a sesterterpene skeleton compound mangicidine, the invention co-transforms plasmids pSC59, pSC71 and pSC73 into Aspergillus oryzae to obtain a mutant strain AO-S84. In order to achieve an increased yield of the compound mangicol J with anti-inflammatory activity, the present invention transformed plasmids pSC111 and pSC112 into the A.oryzae mutant strain AO-S84 by increasing the copy number of mgcE (2 copies) to obtain mutants AO-S93 and AO-S94. In order to obtain a free plasmid Aspergillus oryzae chassis strain with high terpene yield, the invention transforms plasmid pSC253 into Aspergillus oryzae mutant strain AO-S84 to obtain mutant strain AO-S85.

EXAMPLE 4 construction of Aspergillus oryzae mutants with high yield of terpenoids

To obtain an A.oryzae strain containing a pyrG-deficient plasmid, the present invention transformed the pSC184 plasmid into A.oryzae NSAR1 to obtain the mutant strain AO-S184. In order to obtain a high expression site integration aspergillus oryzae chassis strain with high terpene yield, the pSC249 and pSC246 plasmids are co-transformed into the strain AO-S184. The mutant strain having the gene fragment of pSC246 plasmid integrated at the HS401 site was cultured for 2-3 generations on a plate without selection pressure, and spores of the strain were collected (10)⁴Spores/5 μ L) were plated on a medium containing uracil and 5-fluoroorotic acid, and a strain in which homologous recombination had occurred and the pSC249 plasmid had been lost was selected as a starting strain for the next round of gene editing (fig. 1). Meanwhile, the plasmids used for the second and third rounds of gene editing were pSC263 and pSC260, pSC248 and pSC262, respectively, resulting in an integrated aspergillus oryzae chassis AO-S95 that over-expresses the entire MVA pathway and an additional 3 copies of thgl 1 of high-yielding terpenes. In order to realize the yield increase of a sesterterpene skeleton compound mangicidine, the invention co-transforms plasmids pSC24 and pSC162 into Aspergillus oryzae to obtain a mutant strain AO-S96. In order to achieve an increased yield of the compound mangicol J with anti-inflammatory activity, the present invention co-transformed plasmids pSC24 and pSC27, pSC24 and pSC28 into Aspergillus oryzae mutant strain AO-S95, respectively, to obtain mutants AO-S97 and AO-S98, by increasing the copy number (2 copies) of mgcE.

EXAMPLE 5 enrichment and purification of terpenoids

In order to obtain sufficient product for compound yield calibration, the present invention performs a number of fermentations of Aspergillus oryzae mutants AO-S84 and AO-S98. Placing the rice culture medium (5-10kg) with thallus into 30 deg.C incubator, and standing for 20-25 d. Crushing the cultured thallus with a stirrer, adding n-hexane or ethyl acetate with the same volume for extraction for 3-4 times, combining the extracted organic layers, and concentrating with a rotary evaporator to obtain crude extracts A and B. And (3) purifying the crude extract A by multi-time semi-preparative HPLC, eluting by using pure acetonitrile and ultrapure water as mobile phases according to the volume ratio of 75:25 and 90:10, and collecting fractions to obtain the sesterterpene compound of mangicidine. And (3) purifying the crude extract B by multi-time semi-preparative HPLC, eluting by using pure acetonitrile and ultrapure water as mobile phases according to the volume ratio of 75:25 and 90:10, and collecting fractions to obtain the sesterterpene compound of the Mangicol J.

Example 6 Synthesis and detection of terpenoids

For detecting target terpenoid, 2mL of the constructed mutant strain containing 2.5X10 was amplified on a screening plate⁷The spore suspension of (2) was inoculated into 200mL of DPY liquid medium, and 1% each of maltose and glucose was added to induce expression. Culturing at 140rpm and 30 ℃ for 7 days. Filtering cultured thallus with nylon cloth, discarding culture medium, mincing thallus with stirrer, adding equal volume of n-hexane or ethyl acetate, extracting for three times, and mixing the upper organic layers. Spin-drying with rotary evaporator, adding chromatographic grade ethyl acetate or methanol, re-dissolving the sample, high speed centrifuging, collecting supernatant, and detecting with GCMS or HR-LCMS.

The invention respectively carries out 3 parallel fermentation culture on three mutant strains of AO-S84, AO-S96 and AO-Y51. And (4) re-dissolving the fermentation product by using normal hexane, and detecting by GCMS. The results showed that mangicriene yields of 87.84mg/L and 27.38mg/L were detected for AO-S84 and AO-S96, respectively, which were 133 and 41 fold higher than the control strain AO-Y51(0.66mg/L), respectively. The invention respectively carries out 3 parallel fermentation culture on three mutant strains of AO-S93, AO-S94, AO-S97, AO-S98 and AO-Y52. And (4) after re-dissolving the fermentation product by ethyl acetate, and detecting by GCMS. The results showed that the yields of Mangicol J detected by AO-S93 and AO-S97 were 9.14mg/L and 7.26mg/L, respectively. After increasing the mgcE copy, the yields of Mangicol J detected by AO-S94 and AO-S98 were 12.09mg/L and 8.93mg/L, respectively, which were improved by 151 and 111 times, respectively, compared to the original strain AO-Y52(0.08 mg/L).

Example 7 high throughput amplification of recombinant fragments

DNA fragments required for constructing the gene cluster BGC-FgJ02895 recombinant plasmid library are obtained by PCR amplification. Phusion high fidelity DNA polymerase required for amplification was purchased from NEB (New England Biolabs, NEB) and Prime STAR GXL DNA polymerase from TaKaRa (TaKaRa Bio, Inc., Shiga, Japan). PCR primers were purchased from Kinry Biotechnology Ltd (Table 2 below). Magnetic beads for PCR purification of amplified fragments were purchased from Novozam Biotechnology Ltd (N411-01, Vazyme).

TABLE 2 PCR primers

The 5 '-end and the 3' -end of all functional genes, promoters, terminators and vector sequences all contain overlapping fragments of 40bp bases, so that the plasmid library can be obtained by utilizing the inherent recombination capability of saccharomyces cerevisiae in the following assembly. Preparation of PCR System by automated pipetting station (Biomek FX)^PLaboratory Automation Workstation, Beckman Coulter). PCR amplification adopts a 40 mu L amplification system, required primers are synthesized and dissolved and then are loaded in a 96-well plate, a DNA polymerase system (containing buffer, dNTPs and DNA polymerase) is prepared according to the required amount (589 fragment amplification amounts), then the obtained product is evenly distributed in the 96-well plate (34 mu L/hole), and a template is diluted and then loaded in the 96-well plate (25 ng/mu L). And respectively and sequentially adding 2 mu L of front primer, 2 mu L of rear primer and 2 mu L of template into the DNA polymerase system, uniformly mixing by blowing and sucking, transferring into a PCR instrument, and setting a program for PCR amplification. The amplification procedure used in this example was as follows:

example 8 construction of recombinant plasmids by Yeast Assembly

The construction of recombinant plasmids related to the gene cluster BGC-FgJ02895 mainly adopts a Yeast assembly (Yeast assembly) method. Saccharomyces cerevisiae CEN. PK2-1D, EUROSCARF) (genotype MATaurara 3-52trp1-289leu 2-3112 his3 Δ 1MAL2-8C SUC2) was used as an experimental strain for homologous recombination, and an optimized lithium acetate/polyethylene glycol (LiAc/PEG) chemical transformation method was used to construct recombinant plasmids. The experimental operations (e.g. "pipetting", "shaking", "plate transfer", "pipetting", etc.) involved in the examples were carried out by automated pipetting stations (Biomek FX)^PLaboratory Automation Workstation, Beckman Coulter), and the liquid dispensing operation is performed by an automatic liquid dispenser (Thermo Scientific)^TMMultidrop comb SMART Dispenser), the cell monoclonal picking is performed by a Qpix 460 monoclonal picking system (Molecular Device Qpix 460), "centrifugation" is performed by a centrifuge provided in an automated workstation, and "cell culture" is performed in an incubator provided in the automated workstation. Plasmid extraction beads were purchased from magenta Bio (Magpure Plasmid LQ Kit) and a Plasmid miniprep Kit was purchased from Axygen (Cat. No. AP-MN-P-250). The specific construction method is described in detail below.

The coding sequence, promoter, terminator and linear vector fragments required for construction of each recombinant plasmid related to gene cluster BGC-FgJ02895 obtained in example 7 were taken at 300ng each and mixed until use (total volume 10. mu.L). And transferring the 10 mu L of DNA mixed solution into a 96-hole deep-hole plate (100 mu L/hole) containing saccharomyces cerevisiae competent cells by using an MP 20096-Tip mechanical arm in an automatic liquid transfer workstation, sequentially adding boiled milt DNA (20 mu L/hole) and LiAc/TE/40% PEG4000 solution (700 mu L/hole), setting an "asparate" program, uniformly blowing and sucking, and incubating in an incubator at 30 ℃ for 30 min. Taking out the culture plate, adding DMSO (88 mu L/hole), blowing and sucking uniformly, transferring the culture plate to a 42 ℃ incubator for heat shock for 8min, centrifuging at 1500rpm for 5min, discarding the supernatant, adding 1mL YPD culture medium for resuspending thallus, transferring the culture plate to a 30 ℃ incubator, and incubating for 60 min. Taking out the culture plate, centrifuging at 1500rpm for 5min, removing the supernatant, adding 1mL of TE solution to wash the thallus once, centrifuging at 1500rpm for 5min, removing part of the supernatant, remaining about 100 mu L of TE solution to resuspend the thallus, sucking the bacteria liquid, adding the bacteria liquid into a 96-hole deep-hole plate containing 1mL of uracil-deficient solid culture medium (SC-Ura), transferring the culture plate to a shaking module, setting a shake program for 30s, and uniformly coating the bacteria liquid on the solid culture medium. The plates were transferred to a 30 ℃ incubator for 3 days. The specific operation flow is shown in fig. 2.

Example 9 extraction of plasmids by the paramagnetic particle method

A uracil-deficient liquid medium (SC-Ura) (200. mu.L/well) was added to the cell culture plate, and the cells were dissolved by aspiration to obtain a bacterial solution. The bacterial suspension was transferred to a 96-well deep-well plate containing 1.5mL of SC-Ura liquid medium and cultured overnight on a horizontal shaker at 30 ℃. Centrifuging at 3500rpm for 8min, discarding supernatant, and extracting yeast plasmid by optimized magnetic bead method. Lysozyme (500U/mL, Sigma-Aldrich,20210108) and plasmid miniprep buffer S1 solution were added sequentially to the well plate and the plate was transferred to a 25 ℃ incubator for 2h incubation. The plate was removed from the well, buffer S2 solution was added, and the plate was transferred to the "shake" module, 700rpm, shake for 45S. Buffer S3 was added, 700rpm, shaking for 45S. Subsequently, the mixture was centrifuged at 3500rpm for 10min, the supernatant was aspirated into a new 96-well deep-well plate, 400. mu.L of magnetic bead solution was added thereto, and the mixture was stirred and allowed to stand for 5 min. Transferring the pore plate to a magnetic frame, and adsorbing for 5 min. Discarding the supernatant, sequentially adding buffer W1 and buffer W2, washing, discarding the supernatant, standing for 5min to volatilize the solution, adding 100 μ L sterile water, transferring the well plate to a magnetic rack, standing for 5min, and dissolving plasmid DNA. The plasmid solution was pipetted into a new 96-well PCR plate to obtain a recombinant plasmid library related to the gene cluster BGC-FgJ 02895. The specific operation flow is shown in fig. 3.

Example 10 enrichment of recombinant plasmids in E.coli

Using CaCl₂Coli (Escherichia coli DH10B) competent cells were prepared chemically. Competent cells were dispensed into 96-well deep-well plates at 70. mu.L/well by an automatic aliquotter. Add 10. mu.L of plasmid DNA solution to E.coli DH10B competent cells and incubate at 4 ℃ for 30 min. Subsequently, the plate containing the cells was transferred to a preheated 42 ℃ incubator, incubated for 3min, and 800. mu.L of LB medium was added to each well, and incubated at 37 ℃ for 45min to resuscitate the cells. Subsequently, the plate was transferred to a centrifuge, centrifuged at 1500g for 8min, and the cells were collected. The partial supernatant was discarded, and about 50. mu.L of the culture medium was used to resuspend the cells, spread on Ampicillin (Ampicillin, Amp) -resistant LA solid medium, and cultured overnight at 37 ℃. The strain is picked up to LB culture medium by Qpix 460 instrument, and enlarged culture is carried out to enrich plasmid. The obtained plasmid related to the gene cluster BGC-FgJ02895 is verified by restriction enzyme digestion. The specific operation flow is shown in fig. 3.

Specifically, the method comprises the following construction of plasmids (FgJ genome, namely genome of fusarium graminearum J1-012(Fusarium graminearum J1-012)):

construction of pYJ182 plasmid: the FgJ genome and pYJ152 are respectively used as templates, a primer pair HQD02895gDNA-Tamy GR (182)/HQD02895-Pamy GF (182) and a primer pair HQD02895gDNA GR (182)/Pamy-HQD02895gDNA GF (182) are respectively used for amplifying fragments pYJ182-F1 and pYJ182-F2, and the 2 fragments are subjected to yeast assembly to obtain the plasmid pYJ 182.

Construction of pYJ183 plasmid: FgJ genome and pYJ153 are respectively used as templates, a primer pair HQD2897gDNA-Tamy GR (183)/HQD2897gDNA-Pamy GF (183) and a primer pair HQD2897gDNA GR (183)/Pamy-HQD2897gDNA GF (183) are respectively used for amplifying fragments pYJ183-F1 and pYJ183-F2, and the 2 fragments are subjected to yeast assembly to obtain the plasmid pYJ 183.

Construction of pYJ184 plasmid: fragments pYJ184-F1 and pYJ184-F2 were amplified from HQD2900g DNA-Tamy GR (184)/HQD2900g DNA-Pamy GF (184) and Tamy-HQD2900g DNA GR (184)/Pamy-HQD2900g DNA GF (184) using FgJ genome and pYJ174 as templates, respectively, and these 2 fragments were yeast-assembled to give plasmid pYJ 184.

Construction of pYJ185 plasmid: fragments pYJ185-F1 and pYJ185-F2 were amplified from HQD2901gDNA-Tamy GR (185)/HQD2901gDNA-Pamy GF (185) and Tamy-HQD2901gDNA GR (185)/Pamy-HQD2901gDNA GF (185) respectively using FgJ genome and pYJ174 as templates, respectively, and these 2 fragments were yeast-assembled to obtain plasmid pYJ 185.

Construction of pYJ199 plasmid: pGB98, FgJ genome, pGB127 and pYJ184 are respectively used as templates, and the fragments pYJ199-F1, pYJ199-F2, pYJ199-F3 and pYJ199-F4 are respectively amplified by using primer pairs PglaA-7577gDNA GF (175-) -176)/PglaA-HQD02901gDNA GR (199), HQD02901-TniaD GR (199), TniaD-HQD02901 GF (199)/TniaD-Tamy GR (175-) -176), Tamy-TniaD GF (175-) -176)/AdeA-PglaAGR (175-) -176, and the 4 fragments are respectively used for yeast assembly to obtain the pYJ 199.

Construction of pYJ200 plasmid: pGB127, FgJ genome, pYJ177, pGB98, FgJ genome, pGB127 and pYJ184 are taken as templates respectively, the plasmid was assembled by using primers TagdA-AdeAGF (177)/TagdA-HQD02898 GR (200), HQD02898-TagdA GR (200)/HQD02898-PhlyA GF (200), PhlyA-HQD02898 GR (200)/PhlyA-PglaAGF (177), PglaA-PhlyA GF (177)/PglaA-HQD02901gDNA GR (199), HQD02901-PglaA GF (199)/HQD02901-TniaD GR (199), TniaD-HQD 901 GF (199)/TniaD-Tamy GR (175-176), Tamy-TniaD (175-176)/AdeA-TagdA GR (177) to amplify pYJ 200-F0254, pYJ 200-1, pYJ200-F2, pYJ 200-3, yeast-200-pF 5, yeast-200-YF 3957, and more than 200 pYJ 46F 29.

Construction of pYJ201 plasmid: pGB98, FgJ genome, pGB127 and pYJ183 are respectively used as templates, and the plasmids pYJ201 are obtained by respectively amplifying the fragments pYJ201-F1, pYJ201-F2, pYJ201-F3 and pYJ201-F4 by using primer pairs PglaA-HQD07575GF (178)/PglaA-HQD02896 GR (201), HQD02896-PglaA GF (201)/HQD02896-TniaD GR (201), TniaD-HQD02896GF (201)/TniaD-Tamy GR (175-.

Construction of pYJ202 plasmid: pGB127, FgJ genome, pYJ177, pGB98, FgJ genome, pGB127 and pYJ183 as templates, the plasmid fragments pYJ202-F1, pYJ202-F2, pYJ202-F3, pYJ 202-F202-34, yeast 202-202, and pYJ 202-5929 were assembled by using primers such as TagdA-sC GR (180)/TagdA-HQD02899 GF (202), HQD02899 GR (202)/PhlyA-PglaA GF (177), PglaA-HQD02896 GR (201), HQD02896-PglaA GF (201)/HQD02896-TniaD GR (201), TniaD-HQD 96GF (201)/TniaD 02896-Tamy (175 GR 176), Tamy-TniaD GF (175-.

Construction of pYJ203 plasmid: the plasmid pYJ203-F1, pYJ203-F2, pYJ203-F3 and pYJ203-F4 were respectively amplified from pGB98, FgJ genome, pGB127 and pYJ182 as templates and PglaA-Pamy GF (203)/PglaA GF (203)/HQD02902-TniaD GR (203), TniaD-HQD02902GF (203)/TniaD-ArgB GR (203) and ArgB-TniaD GF (203)/Pamy-PglaA GR (203), respectively, by using primer pairs PglaA-Pamy GF (203)/PglaA GF-HQD 02902 GR (203)/TglaD-203/HQD-203, and pYJ203-F4 as templates, and these 4 fragments were yeast-assembled to obtain pYJ 203.

Construction of pYJ204 plasmid: pGB98, FgJ genome, pGB127, FgJ genome, pYJ177 and pYJ182 are respectively used as templates, the fragments PglaA-Pamy GF (203)/PglaA-HQD02902 GR (203), HQD02902-PglaA GF (203)/HQD02902-TniaD GR (203), TniaD-HQD02902GF (203)/TniaD-TagdA GR (204), TagdA-TniaD GR (204)/TagdA-Hqd02892 GF (204), HQD02892-TagdA GR (204)/HQD02892-PhlyA GF (204), PhlyA-HQD02892 GR (204)/PhlyA-ArgB GF (204), ArgB-PhlyAGF (204)/Pamy-PglaA GR (203) were amplified with primer pairs to obtain pYJ204-F1, pYJ204-F2, pYJ 204-3, pYJ 204-PyF 39204-39204, pYJ-PyF 5, yeast 3957, and yeast strain No. 204-PyJ-593, and more than PyJ 3975.

EXAMPLE 11 Aspergillus oryzae mutant library construction for the Synthesis of terpenoids

Through bioinformatics analysis, the terpene biosynthetic gene cluster can be divided into 3 synthesis modules, which are respectively: 1) upstream terpene synthase module: contains Terpene Synthase (TS), and catalytic precursor (such as FPP, GGPP, GFPP) for synthesizing terpene skeleton compound; 2) a mid-stream oxidation module: contains cytochrome P450 enzyme (CYP 450) which catalyzes a core skeleton to synthesize double bonds, carbonyl or hydroxyl functional groups; 3) downstream post-modification module: contains optional post-modification catalytic enzymes such as Acyltransferase (ACT) and Glycosylase (GE) to catalyze the intermediate compound synthesized in the step 2), thereby synthesizing terpenoids with diverse structures and potential activity. Therefore, in order to reconstruct the terpenoid gene cluster in high flux, obtain intermediates and final terpenoid and analyze the biosynthesis pathway of the terpenoid gene cluster, the invention rationally designs the combined transformation of recombinant plasmids according to the inherent sequential catalytic properties of the terpenoid gene cluster synthesis, and constructs an aspergillus oryzae mutant strain screening library related to the gene cluster BGC-FgJ02895 (as shown in figure 5). The strain construction adopts a protoplast transformation method, and plasmids containing different genes are transformed into Aspergillus oryzae based on an automatic liquid transfer workstation. The method comprises the following specific steps:

before the experiment, the automatic liquid transfer workstation was subjected to UV sterilization. Culturing Aspergillus oryzae on DPY solid plate for about 3 days, scraping spore and mycelium, transferring to 100mL DPY liquid culture medium, culturing at 30 deg.C for 2 days, and collecting thallus. An optimized enzyme cracking method is adopted to prepare the aspergillus oryzae protoplast. The protoplast solution was dispensed into 96-well deep-well plates in an amount of 100. mu.L/well by an automatic dispenser. The plasmid DNA mixture to be transformed was added to the protoplast solution, the plate was transferred to an incubator and incubated at 30 ℃ for 1 hour. Subsequently, 1.25ml of PEG6000 solution was added and incubated at room temperature for 30 min. The plate was transferred to a centrifuge, centrifuged at 420g for 25min and a portion of the solution was discarded. To the remaining 200. mu.L of the solution, 800. mu.L of STC solution was added, and 420g was centrifuged for 25min, and part of the solution was discarded. Approximately 100. mu.L of the solution was retained to resuspend the cells, and the cells were transferred from a 96-well deep-well plate to a plate containing three defects (sC) using a flexible 8-channel robotic arm^-,ΔargB,adeA^-) Solid screening media in 24-well deep-well plates. The 24-well deep-well plate was then transferred to a shaking module, and after shaking to coat the cells, the plate was transferred to an incubator and incubated at 30 ℃ for 2-5 days (as shown in FIG. 6). After the transformant grows out, extracting a genome and carrying out PCR verification on a target gene. In the invention, BGC-FgJ02895 is taken as an example, and a strain library containing 11 mutant strains is finally constructed.

Specifically, the construction of the following Aspergillus oryzae mutant strains is included:

AO-Y23 co-transformed plasmid pGB366/pUSA/pAdeA, containing terpene synthase FgJ 02895; AO-Y24 co-transformed plasmid pGB366/pYJ183/pAdeA, containing terpene synthase FgJ02895, cytochrome P450 enzyme FgJ 02897; AO-Y25 co-transformed plasmid pGB366/pUSA/pYJ184, containing terpene synthase FgJ02895, cytochrome P450 enzyme FgJ 02900; AO-Y26 co-transformed plasmid pGB366/pUSA/pYJ185, containing terpene synthase FgJ02895, monooxygenase FgJ 02901; AO-Y27 co-transformed plasmid pGB366/pYJ183/pYJ184 containing terpene synthase FgJ02895, cytochrome P450 enzyme FgJ02897, cytochrome P450 enzyme FgJ 02900; AO-Y28 co-transformed plasmid pGB366/pYJ183/pYJ199 comprising terpene synthase FgJ02895, cytochrome P450 enzyme FgJ02897, cytochrome P450 enzyme FgJ02900, monooxygenase FgJ 02901; AO-Y29 co-transformed plasmid pGB366/pYJ183/pYJ200, comprising terpene synthase FgJ02895, cytochrome P450 enzyme FgJ02897, cytochrome P450 enzyme FgJ02900, monooxygenase FgJ02901, acetyltransferase FgJ 02898; AO-Y30 co-transformed plasmid pGB366/pYJ201/pYJ200, comprising terpene synthase FgJ02895, cytochrome P450 enzyme FgJ02897, transcription factor FgJ02896, cytochrome P450 enzyme FgJ02900, monooxygenase FgJ02901, acetyltransferase FgJ 02898; AO-Y31 co-transformed plasmid pGB366/pYJ202/pYJ200, comprising terpene synthase FgJ02895, cytochrome P450 enzyme FgJ02897, transcription factor FgJ02896, TfgJ 02899, cytochrome P450 enzyme FgJ02900, monooxygenase FgJ02901, acetyltransferase FgJ 02898; AO-Y32 co-transformed plasmid pYJ203/pYJ202/pYJ200 comprising terpene synthase FgJ02895, Cu2+ -ATPaseFgJ02902, cytochrome P450 enzyme FgJ02897, transcription factor FgJ02896, TfgJ 02899, cytochrome P450 enzyme FgJ02900, monooxygenase FgJ02901, acetyltransferase FgJ 02898; AO-Y33 co-transformed plasmid pYJ204/pYJ202/pYJ200, comprising terpene synthase FgJ02895, Cu2+ -ATPaseFgJ02902, decarboxylase FgJ02892 (nucleotide sequence shown in SEQ ID NO: 25), cytochrome P450 enzyme FgJ02897 (nucleotide sequence shown in SEQ ID NO: 30), transcription factor FgJ02896 (nucleotide sequence shown in SEQ ID NO: 29), Tf FgJ02899 (nucleotide sequence shown in SEQ ID NO: 32), cytochrome P450 enzyme FgJ02900 (nucleotide sequence shown in SEQ ID NO: 33), monooxygenase FgJ02901 (nucleotide sequence shown in SEQ ID NO: 34), acetyltransferase FgJ02898 (nucleotide sequence shown in SEQ ID NO: 31).

Example 12 Synthesis and detection of terpenoids

And transferring the Aspergillus oryzae strain with positive PCR (polymerase chain reaction) verification result to a 24-hole deep-hole plate containing a rice solid culture medium for culture, transferring 6 multiple holes (each hole contains 1g of rice and 1.5mL of deionized water), and performing fermentation culture for 2 weeks at 30 ℃. Acetone (5 mL/well) was added to the cultured 24-well plates and soaked at room temperature for 2 h. The supernatant was transferred to a new 24-well deep-well plate, and ethyl acetate (5 mL/well) was added to the cells, followed by immersion at room temperature for 2 hours. The acetone and ethyl acetate extract phases were combined, the 24-well plate was transferred to a fume hood and allowed to evaporate naturally to obtain a crude extract. The generation of terpenoid products in the mutant was detected by GC/MS and HR-LC/MS.

In the invention, 3 terpenoids (17, 20 and 28) with new structures are separated and purified, and the structures of the terpenoids are analyzed by nuclear magnetic resonance spectroscopy, and the results are as follows:

compound 17: C₁₅H₂₄，¹H-NMR and¹³C-NMR data are shown in tables 3 and 4 and FIGS. 10 to 16; HR-ESI-MS [ M + H ] in positive ion mode]⁺m/z 205.1948(calculated value 205.1951)。

Compound 20: C₁₅H₂₄O，

-80.5(c 0.1,MeOH),UV(MeOH)λ_max(logε):196(3.81)；IR(KBr)v_max3362,2955,2921,1710,1637,1460,1378,1270,1114,1064,907cm^–1；¹H-NMR and¹³C-NMR data are shown in tables 1 and 2 and FIGS. 17 to 23; HR-ESI-MS [ M + H ] in positive ion mode]⁺m/z 221.1908(calculated value 221.1900)。

Compound 28: C₁₇H₂₈O₃，

-38.5(c 0.12,MeOH),UV(MeOH)λ_max(logε):196(3.45)；IR(KBr)v_max3432,2963,2928,1737,1632,1454,1384,1243,1045cm^–1；¹H-NMR and¹³C-NMR data are shown in tables 1 and 2 and FIGS. 24 to 30; HR-ESI-MS [ M + H ] in positive ion mode]⁺m/z 281.2117(calculated value 281.2111)。

TABLE 3 preparation of

compounds

17, 20 and 28¹And attributing H-NMR data.

^a Recorded at 500MHz,and the assignments were based on DEPT,HSQC,COSY,HMBC,and ROESY experiments.

TABLE 4 preparation of

compounds

17, 20 and 28¹³C-NMR data attribution.

Example 13 high throughput screening of terpenoid anti-inflammatory Activity

1) In vitro cytotoxicity assessment

To exclude the effect of compound-induced cytotoxicity on anti-inflammatory activity screening, the toxic effect of the compound to be screened on the cells was first assessed prior to the activity screening assay. In this example, mouse macrophage RAW264.7 was used as a study subject. Cells were plated at 2.0X 10⁴Cells/well were seeded in 24-well plates at 37 ℃ with 5% CO₂After 24h of incubation in an incubator, the crude extract to be screened (final concentration 500. mu.g/mL) was added at 37 ℃ with 5% CO₂Incubate for 24 h. To each well was added 100. mu.L of CCK-8 diluted solution (10. mu.L of CCK-8 solution added to 90. mu.L of medium), 37 ℃ 5% CO₂After incubation in the incubator for 1h, the plates were transferred to a microplate reader and absorbance was measured at a wavelength of 450nm (as shown in FIG. 7).

2) In vitro anti-inflammatory Activity screening

In this example, Lipopolysaccharide (LPS) -induced mouse macrophage RAW264.7 was used as an in vitro inflammatory cell model. RAW264.7 mouse macrophages are scaled up to 3.0X 10⁵Cells/well were seeded in 24-well plates at 37 ℃ with 5% CO₂After 24 hours of culture in an incubator, LPS (1. mu.g/mL) was allowed to act on macrophages of RAW264.7 mice, and molding was performed to obtain inflammatory cells. And (3) respectively acting the crude extract (with the final concentration of 500 mu g/mL) on model cells, taking supernatant after 16h, detecting the level of NO in the supernatant by a Griess reagent color development method, and evaluating the anti-inflammatory activity of the compound by taking the capability of the tested drug for inhibiting NO release as a screening index. After the cells are treated by the drug, a flexible 8-channel machine is adoptedThe arm transfers the supernatant from the 24-well plate to a 96-well plate, followed by sequential addition of Griess I and Griess II reagents. The plate was transferred to a microplate reader and the UV absorbance was measured at 540nm wavelength (as shown in FIG. 7).

Although the embodiments of the present invention have been shown and described, it is understood that the above embodiments are illustrative and not restrictive, and that those skilled in the art may change, modify, replace and modify the above embodiments within the scope of the present invention and that they should be included in the protection scope of the present invention.

Sequence listing

<110> Wuhan university

<120> construction of Aspergillus oryzae chassis strain for high yield of terpenoid and automatic high-flux excavation platform for terpenoid natural products

<160> 31

<170> SIPOSequenceListing 1.0

<210> 1

<211> 246

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 1

cgggatagtt ccgacctagg attggatgca tgcggaaccg cacgagggcg gggcggaaat 60

tgacacacca ctcctctcca cgcaccgttc aagaggtacg cgtatagagc cgtatagagc 120

agagacggag cactttctgg tactgtccgc acgggatgtc cgcacggaga gccacaaacg 180

agcggggccc cgtacgtgct ctcctacccc aggatcgcat ccccgcatag ctgaacatct 240

atataa 246

<210> 2

<211> 720

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 2

gccatcggat gctcccgtca tggcaccact agagatggcg tggaaaaccc tcaccggcac 60

accggagggg tttaggcacc ttggaatatg aggtggggaa cgatgtattt gccagtattg 120

actctggtga atggatctct cgagaaatac taccttttca gggctcaagc gtcgtgtcgg 180

gcatttatcg ggggatggac caatcagcgt agggatatca gatgatcgcc agcattggtc 240

aggaacgttt ccaatttccg gacacggaag tactgtaact gctcccaaga atcaacacac 300

tcttttccgg tctcgtcctt tgctcggcag agattcatct cccatcgtcg gcttaaccgg 360

tactctttcg tcacgttcca aaaggcttga tcatgctgtc cccactccgt gcgggtgaag 420

ccacctcatt gctgcgtagg acctataccc ttcaactagc gtgacttctt cccctctcat 480

ggtcgagaga ttgcaggcaa tgcccctcgg acgtttgacg gggaatgttt tgccttcacg 540

gcaggtagca caaatcgatg ggaacgggac gggccatcaa ttgtgaggga tttcccgtgg 600

acacctggtt cgtcaagaca tatacatcta gctacaattc cggttcggag acggcagagg 660

ggtccgtttc ttaaaagaac aactacaaca cggtccggaa tcaacttggc ggaccacgac 720

<210> 3

<211> 745

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

ggatccgaac tccaaccggg gggagtatat tgagtggccg cagtggaagg aatcgcggca 60

gttgatgaat ttcggagcga acgacgccag tctccttacg gatgatttcc gcaacgggac 120

atatgagttc atcctgcaga ataccgcggc gttccacatc tgatgccatt ggcggagggg 180

tccggacggt caggaactta gccttatgag attaatgatg gacgtgtctg gcctcggaaa 240

aggatatatg gggatcataa tagtactagc catattaatg aagggtatat accacgcgtt 300

ggacctgcgt tatagcttcc cgttagttat agtaccatcg ttataccagc caatcaagtc 360

accacgcacg accggggacg gcgaatcccc gggaattgaa agaaattgca tcccaggcca 420

gtgaggccag cgattggcca catctccaag gcacagggcc attctgcagc gctggtggat 480

tcatcgcaat ttcccccggc ccggcccgac accgctatag gctggttctc ccacaccatc 540

ggagattcgt cgcctaatgt ctcgtccgtt cacaagctga agagcttgaa gtggcgagat 600

gcctctgcag gaattcaagc tagatgctaa gcgatattgc atggcaattt gtgttgatgc 660

atgtgcttct tccttcagct tcccctcgtg cagatgaggt ttggctataa attgaagtgg 720

ttggtcgggg ttccgtgagg ggctg 745

<210> 4

<211> 708

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

acagtgaccg gtgactcttt ctggcatgcg gagagacgga cggacgcaga gagaagggct 60

gagtaataag cgccactgcg ccagacagct ctggcggctc tgaggtgcag tggatgatta 120

ttaatccggg accggccgcc cctccgcccc gaagtggaaa ggctggtgtg cccctcgttg 180

accaagaatc tattgcatca tcggagaata tggagcttca tcgaatcacc ggcagtaagc 240

gaaggagaat gtgaagccag gggtgtatag ccgtcggcga aatagcatgc cattaaccta 300

ggtacagaag tccaattgct tccgatctgg taaaagattc acgagatagt accttctccg 360

aagtaggtag agcgagtacc cggcgcgtaa gctccctaat tggcccatcc ggcatctgta 420

gggcgtccaa atatcgtgcc tctcctgctt tgcccggtgt atgaaaccgg aaaggccgct 480

caggagctgg ccagcggcgc agaccgggaa cacaagctgg cagtcgaccc atccggtgct 540

ctgcactcga cctgctgagg tccctcagtc cctggtaggc agctttgccc cgtctgtccg 600

cccggtgtgt cggcggggtt gacaaggtcg ttgcgtcagt ccaacatttg ttgccatatt 660

ttcctgctct ccccaccagc tgctcttttc ttttctcttt cttttccc 708

<210> 5

<211> 844

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

ctgcagctgt ggagccgcat tcccgattcg ggccggattg gtcaagattt gcgtccgagg 60

tgccgtctat cattctagct tgcggtcctg ggcttgtgac tggtcgcgag ctgccactaa 120

gtggggcagt accattttat cggacccatc cagctatggg acccactcgc aaatttttac 180

atcattttct ttttgctcag taacggccac cttttgtaaa gcgtaaccag caaacaaatt 240

gcaattggcc cgtagcaagg tagtcagggc ttatcgtgat ggaggagaag gctatatcag 300

cctcaaaaat atgttgccag ctggcggaag cccggaaggt aagtggattc ttcgccgtgg 360

ctggagcaac cggtggattc cagcgtctcc gacttggact gagcaattca gcgtcacgga 420

ttcacgatag acagctcaga ccgctccacg gctggcggca ttattggtta acccggaaac 480

tcagtctcct tggccccgtc ccgaagggac ccgacttacc aggctgggaa agccagggat 540

agaatacact gtacgggctt cgtacgggag gttcggcgta gggttgttcc caagttttac 600

acacccccca agacagctag cgcacgaaag acgcggaggg tttggtgaaa aaagggcgaa 660

aattaagcgg gagacgtatt taggtgctag ggccggtttc ctccccattt ttcttcggtt 720

ccctttctct cctggaagac tttctctctc tctcttcttc tcttcttcca tcctcagtcc 780

atcttccttt cccatcatcc atctcctcac ctccatctca actccatcac atcacaatcg 840

atcc 844

<210> 6

<211> 359

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

cagaagatga tattgaagga gcactttttg ggcttggctg gagctagtgg aggtcaacaa 60

tgaatgccta ttttggttta gtcgtccagg cggtgagcac aaaatttgtg tcgtttgaca 120

agatggttca tttaggcaac tggtcagatc agccccactt gtagcagtag cggcggcgct 180

cgaagtgtga ctcttattag cagacaggaa cgaggacatt attatcatct gctgcttggt 240

gcacgataac ttggtgcgtt tgtcaagcaa ggtaagtgaa cgacccggtc ataccttctt 300

aagttcgccc ttcctccctt tatttcagat tcaatctgac ttacctattc tacccaagc 359

<210> 7

<211> 604

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

tcatggtgtt ttgatcattt taaattttta tatggcgggt ggtgggcaac tcgcttgcgc 60

gggcaactcg cttaccgatt acgttagggc tgatatttac gtaaaaatcg tcaagggatg 120

caagaccaaa gtagtaaaac cccggagtca acagcatcca agcccaagtc cttcacggag 180

aaaccccagc gtccacatca cgagcgaagg accacytcta ggcatcggac gcaccatcca 240

attagaagca gcaaagcgaa acagcccaag aaaaaggtcg gcccgtcggc cttttctgca 300

acgctgatca cgggcagcga tccaaccaac accctccaga gtgactaggg gcggaaattt 360

aaagggatta atttccactc aaccacaaat cacagtcgtc cccggtattg tcctgcagaa 420

tgcaatttaa actcttctgc gaatcgcttg gattccccgc ccctggccgt agagcttaaa 480

gtatgtccct tgtcgatgcg atgtatcaca acatataaat actagcaagg gatgccatgc 540

ttggaggata gcaaccgaca acatcacatc aagctctccc ttctctgaac aataaacccc 600

acag 604

<210> 8

<211> 737

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

cagccagcga gagtcacaga agactgatga gccccaccat ttcattggaa agattcggga 60

ggacgaggtc gagagctttt gccggggtag aggacgagga tggtacaaga actagacctt 120

tccaacttta attgttgaca cctatttaat tctctccttc ttctttattt tatttttcat 180

ttctccaacg acgactgtct cattactagt ctactagtaa ctctgtctta tcgtcatctc 240

ccataggtga gtttggttgt tttgtttcca ctgagatcat gacctcctcc taccccacca 300

tcccactatt tttgttacgg tagccatgac ccctccatgg caaagagaga ggaggacgag 360

gacgatcagg aaactgtgtc tcgccgtcat accacaatcg tgttatcctg attgacatct 420

tcttaaatat cgttgtaact gttcctgact ctcggtcaac tgaaattgga tctccccacc 480

actgcctcta ccttgtactc cgtgactgaa ccatccgatc attctttttg ggtcgtcggt 540

gaacacaacc cccgctgcta gtctccttcc aacaccgatc cagaattgtt ttgattttcc 600

attcccttcg tttatatctg tcgtctctcc tccctttccg tctcttttct tccgtcctcc 660

aagttagtcg actgaccaat tccgcagctc gtcaaaatgc ctatcaccaa gatccacgcc 720

cgctctgttt acgactc 737

<210> 9

<211> 1499

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 9

tgagtgaagt tccggtcgga gcaatcaggg tcggttggag tatatcagaa atgtatatct 60

ttggaaaggg tagttataat atgtcgaata tgaatttagg taagagtatt tgaaacaaca 120

agtatgagga tggttcactg gatggcttgc cctaacatcc acaccatcgc atccggccac 180

ctcaggaaca atcactcctc ctcacaaggt atatcccgcc attgaccaac gacaatccag 240

agtatgagat acttgcagaa cgccgagtgg cgttatcaac cgcgtgacct attcagtacc 300

tttgcctcac gccaagcaaa tttccccaca caaaagcaac cataaatgtc gttggccacc 360

gaagctcaaa ctttcggagt cgctctcagt ggttacttta ctgctggtgc ccacgggatc 420

gtaaatggac cttgaccaat tcgagtagtt ccgcagacga tcgctcatcg ctcgatttgg 480

tcgcgtgtcc agagtgttgc tacagcatgg tctggattcc aatccacgca gcagatatct 540

cttttacgca gtaactattc gatagccatt tccgttcaga tcagccgtcg gatccgagga 600

gcgacgacat caatgcgtgt tattagtcaa attttggagg ggttgcgtgc ccactgcagg 660

cagatgtagc cgtggcacca caacaccggc cagccctgga ttgggtggtg gaaccaagat 720

atgagaacca gtatctattg gcacggaggc gtttccggag cctgccgcgt gtgatacgtg 780

cagagtcaat tcctacggac tgtctggggt aggatcacaa ctaatcagtt gcagcgatgg 840

tttgacaaca ggggtcgatc aaagtttccg aagaatagga agcgaggaca gcaccaaggc 900

cgctgaacca caggaaacaa aggaaggaaa aacacaacaa aaccaaagac agacacatag 960

gaaatgacat acttaccaga gataagatga aaagcaccat ggggagggag ggtatggatg 1020

gggaagttga tcaacagtct gaaacccgcc cgaaatgaat gcatgacgcg acgattccat 1080

ctccacctaa gcttcatccc gtcaacctct aacaacgcgc tcggaggaga aaaagagggg 1140

gatcaacagc aatctaggtt gttgttcccc caggatttct ccgcaaagaa tgtttagggt 1200

tccgtggatc gggtgaccga atcggccaac cgcattgtct gatcgtctcg tcatatgatc 1260

cagtgcatga cgtcttccat caatccatca ccccagactg aatcctcaca ttaatttaac 1320

aattgtcgct cggggaaatc aataaatacc cgctcgtctc ctccctccct cgtgctggtt 1380

cagttgattg ttcactcatc gactttatca atcttccatt gaccattcca ggcttgtcgc 1440

ccactcatat aatcttcttt ccccggtctc acatcaatca cccttcacac cacaacacc 1499

<210> 10

<211> 1200

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 10

atgtcttctc ttccgccggt ctatattgtt tcctctgccc gcaccccagt cggctctttc 60

ttggggtcgc tctcaagtct cactgccccg cagttaggct ctcatgctat taaagctgcg 120

ctcagcaaag cggatggaat caagccgtct gatatccagg aggtcttctt tggcaatgtc 180

atctccgcaa acgttggaca aaatcctgct agacagtgtg ctctcggcgc tggtctcaat 240

gaatcaactg tctgtactac ggttaataag gtgtgcgcgt ctggcttgaa agcggttatt 300

ctcggtgcac agaccatcat gactggcaat gcggatattg tcgtagcagg cggtgctgaa 360

tccatgtcta acgcccctca ttaccttcca aaccttcgcg tcggtgcgaa atacggcaac 420

cagagtctgg tggacggtat tatgaaggat ggcttgacag acgcaggaaa gcaggaactc 480

atgggcttgc aagccgagga gtgtgctcag gatcatggct ttagcaggga acaacaggat 540

gattatgcca ttcgcactta cgaaaaagca caggcggctc aaaaggctgg cctttttgac 600

gaagaaattg cgcctattga acttcctggc tttaggggca agccaggtgt gactgtgtca 660

caagacgaag aaccaaagaa tcttaacccg gataagcttc gagctatcaa gcctgcattt 720

atccccggat ccggcacggt cacagccccg aattcctcac ctcttaacga cggtgctgct 780

gctgttatcc tcgtctcaga agctaaactg aaagagctta acctaaagcc tgttgcaaag 840

attcttggct ggggagatgc cgcccagcag ccaagcaaat tcacaactgc cccagctcta 900

gcaattccca aggccctcag ccatgcaggt gtggctcagg atgctgttga tgcgttcgag 960

attaacgaag cgttcagcgt agttgctctg gccaatatga aactcctggg gttggctgaa 1020

gataaagtca acatccatgg tggtgcagtg gctatcggtc atcctatcgg cgccagcggt 1080

gctcgtatct tgactacatt gctcggtgta ttgaaagcga gaaagggtaa gattggttgt 1140

gccgggattt gtaatggagg aggtggtgct agcgctattg ttgtcgaatc tctcgtctga 1200

<210> 11

<211> 1383

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 11

atgtctgctc gtcctcagaa cattggtgtc aaggccattg aggtctattt tcctaagcaa 60

tgtgtcgaac aaaccgagct ggagaagttc gacggtgtca gtgagggcaa gtacacaatc 120

ggtctgggac agacaaaaat gagcttctgt gatgaccgtg aggatatcta ctctgtcgcc 180

ctgaccactc tctcctccct ctttcgcaaa tacaacgtcg accccaagtc cgttggtcgt 240

ctcgaagtcg gtactgagac tctcctggac aaatccaagt ccgtcaagtc cgttctgatg 300

cagctctttg ccgagagcgg aaacttcaac gttgagggtg ttgataacgt caacgcttgc 360

tatggaggta ccaacgctgt cttcaacagc atcaactggc ttgagtcttc cgcctgggat 420

ggaagagatg ccgttgttgt ctgcggtgac attgctctgt atgccgaggg acctgctcgc 480

cctactggtg gtgctggctg tgttgccctc ctcattggtc ctgatgcccc tattgtcttt 540

gagcccggtc ttcgtggctc ttacgtcacc cacacctacg atttctacaa gcctgatctc 600

accagcgaat accccgttgt tgacggtcag cactcccttc agtgctacac tgaggctgtt 660

gatgcttgct acaaggccta cgccgctcgc gagaagacgc tgaaggaaaa gactcagaac 720

ggaaccaacg gtgtggccca tgatgaatcc aagactcctt tggaccgctt tgactatatc 780

cttttccact cccctacctg caagttggtc cagaagtcgt acggccgtat gctttacaac 840

gatttcctcg agaaccccac ccaccccgct ttcgctgaag tcgctcctga gctgcgcgat 900

ctggactaca gcaagtctct cactgacaag aacgtcgaga agactttcat gggtctgacc 960

aagaagcgct tcgctgagcg tgtgaagccc agccttgatg ttgccactct ctgtggtaac 1020

atgtacaccg ccaccgtcta cgccggcctg gccagcttgc tcagcaacgt caccttcgac 1080

cccagccagc ctaagcgcat tggccttttc tcctacggca gtggtctcgc tgcttccatg 1140

ttcagcgcga agattgttgg tgacgtgtct tacatggctg agaagcttga tcttcacaac 1200

cgcctcaatg ctagggatgt cttggccccc caggcctatg ttgagatgtg tgctctgcgt 1260

aagcaagctc acttgaagaa gaacttcaag ccctccggta acacggagac gcttttcccc 1320

aacacctact acctcactga ggtggacgac atgttccgcc gcaagtacga ggtcaaggca 1380

tga 1383

<210> 12

<211> 2497

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 12

atggaaaagc cgattatcct tactcgggct gtgctcaatg cttcggctga caacaagcgc 60

cgaggtggtg ctggtccttc cagccaatcg agcccgacca ctgcgaagtc catccaagac 120

tcgatccaga ccgcaatcag ggaacagggc tttgagatcg tccgggatta ctgtattgag 180

atcgctatcc ttgttgctgg cgcggcttcg ggggtgcaag gcggcctgaa acagttctgt 240

ttcctggctg cttggattct gttcttcgac tgcctcctgc tatttacctt ctacacgact 300

attctttgca tcaagcttga aatcactcgc attaagcgtc acattgcgct ccgtaaagct 360

ttggaagagg atggtattac gcaccgtgtc gccgaaaatg tggcctcaaa caatgattgg 420

cctcaggatg gctcggaaaa cagtgacacc agcatcttcg gaaggaaaat caagtcaagc 480

aatgtgcgtc gtttcaagat tctcatggtt ggaggtttcg tcttgattaa tgtggtgaac 540

ttgtctgcca ttcctttccg gaattccgct ctgggccctg ttccgttact ctcccgggta 600

tccaatgtgc tcgcgcctac tccaattgac cctttcaagg tcgctgaaaa cggtcttgat 660

tcgatctacg tgactgcgaa gagccagatg accgaaactg ttgtgactgt cattccacca 720

atcaagtaca agctcgagta tccttcagtc cattatgctg cgccaggaga tagccaatcc 780

tttgacattg aatacactga tcaactcttg gatgctgttg gtggacgcgt gattgaaagt 840

ttgctgaaga gcgtcgagga cccggtcatc agcaaatgga ttattgctgc gctcaccctg 900

agcatcgtat tgaatggtta ccttttcaac gcggcacggt ggagtatcaa ggaacccgag 960

gctgcccctg cacccaaggc cgtcgagccg aaggtttacc ccaaggtgga tttgaatgca 1020

gacagctcga agagaagtgc agaggaatgt gaagtattcc tgaaggaaaa gcgggcgccc 1080

tatttgtcgg acgaggatct gattgagctt tgcttgcgag gcaagattcc agggtatgct 1140

ttggagaaga ccatggaaaa tgaagacctt atgagccgcg ttgatgcctt cacgagggca 1200

gtcaagatca gaagggctgt ggtatctagg accaaggcta cgtctgccgt tacaagctct 1260

ttggaggcct cgaaactccc ttacaaggac tacaactata cgttggttca cggtgcatgc 1320

tgtgagaacg ttattggata tttgcctctg ccccttggag ttgctggacc tcttactatc 1380

gatggccaaa gctactttat tcccatggct accactgagg gtgtattggt agcaagtgcc 1440

agccgtggtg ccaaggctat caatgcaggt ggtggtgcag tgactgtcct caccggcgat 1500

ggtatgactc gtggtccctg tgttggtttc cctaccctgg cacgcgctgc cgctgcaaaa 1560

gtttggattg actctgagga gggtcagagt atcatgaagg ccgcgttcaa ctctaccagc 1620

cgctttgctc gtctccagac tatgaaaacc gctcttgctg gtacatacct gtatattcgt 1680

ttcaagacaa cgaccggcga tgctatgggt atgaacatga tctccaaggg cgttgagaag 1740

gcgcttcatg tgatgtctac agaatgtgga tttgatgaca tggccaccat caccatctct 1800

ggtaatttct gtacagacaa gaagtctgct gctcttaact ggatcgatgg acgtggcaag 1860

tcagttgtag cagaggccat cattcctggt gatgttgtca agagtgtgct caagagtaac 1920

gttgacgcgc tggtcgaatt gaacaccagc aagaacttga tcggaagtgc aatggctgga 1980

agcttgggtg gcttcaacgc gcacgcatcg aacattgtta ctgccatttt cttggcaact 2040

ggtcaggacc ctgcgcagaa tgtggaaagc agtagctgca tcactactat gagaaagtaa 2100

gttacaggtt gccaatgttc acgattcaca tcagtgtact aattgtcatg ctcccagtct 2160

caacggtgac cttcagatct ccgtgtcaat gccctcaatc gaggtcggaa ccattggtgg 2220

tggtacgatt cttgaaggac agtccgctat gcttgaccta ctcggtgtgc gcggttccca 2280

ccccaccaac cctggcgaca acgcccgtca acttgcacgg attgttgccg ccgctacgct 2340

tgcaggcgaa ctgagcttgt gctctgcact tgctgccggc catcttgtcc gtgcgcacat 2400

ggcgcacaat cgcagtagcg ctcctacacg gtcatcaact cccgtctcgg ccgctgttgg 2460

cgctgctcgg ggattgacta tgacgagttc gaaatga 2497

<210> 13

<211> 1605

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 13

atgcacaacg gccgtggacg ccgaaagaac ggcagtgtta aagctccaaa gaatcgtcag 60

cgccctacta tgtcacactt aatctcggaa caatcccttc ccacccgctc aaaatcctcg 120

gtcgctagcg acgatagcgt agacacctcc gacgacacct cagccgctcc ctccttcagc 180

agtagtccgc cgagcacgaa gtctatcacc aacggtgttc acagaccagc tatggctcga 240

aaggcttctt ctccaatggc acccgccttc atggtgtccg ccccgggcaa ggtcattgtc 300

tttggtgagc atgccgtcgt gcatggtaaa gccgccatgg cggcagccat ctccctacga 360

tcctacctcc ttgttacaac cttgaccaaa tcgcaacgca ccatcacctt aaacttcaga 420

gatattggat tgaatcacac ctggagcatc gacgagctgc cgtgggactt gtttcaccaa 480

ccgaccaaga agaagtacta ttacgacctg gtcacctcga ttgaccccga acttctggac 540

gcgatcttgc ctctcgtgga gcgcatctcc ccagacctac ccgaagacaa gcgaaaacat 600

cagcgtggcg ctgcgactgc gttcctctat cttttctgtg cgttgggttc cccgcaacac 660

cctggagcga tctataccct tcggtcgacg atcccaactg gcgcggggtt gggcagcagt 720

gctagtatat gcgtttgtat cagtgctgca ctccttcttc agattcgtac tctagctgga 780

ccgcaccccg accaaccgcc cgacgaggcg gaggtgcaga tcgagcgcat caaccgatgg 840

gcattcgttg gtgagatgtg cattcacgga aatcccagcg gagtggataa cacggtcgcc 900

gcaggcggca aggccgtgat tttcagaaga ggcgattatt ccaaaccacc ggccgttagc 960

tcactcccca atttccccga gctacctcta ctgctcgtgg acactcgaca atcccgttcc 1020

acggcggttg aagtagcaaa ggtcggtcag ctgaaagaag aacaaccact agtgacggag 1080

gcgatccttg ataccatcga gaaggtgaat gcttccgctc aggagattat acgggaaacg 1140

gattcgtcag gtatttccaa ggatacgctc gagcgcattg gagcgcttat ccgcatcaac 1200

cacggcttgt tggtctcgct gggagtctct caccctcgac tcgagcgcat tcgtgagctt 1260

gtagattttg cgaacattgg ttggacgaaa ctcaccggcg ctggtggagg aggatgcgcg 1320

atcaccctcc tacgtccgga tgccgacccg agtgctatcc gccaattgga ggaaaagctg 1380

gacgaagaag gattcgcgaa gtacgagact actctgggag gagatggtgt cggtgtcctg 1440

tggcccgccg tggtccgcaa tggaaccgac gaagaaggtg gtgaggagat tgaccagcag 1500

aagttcgaaa atgcagatgg ccccgaaggc atcgagcgtc tcgtcggtgt gggcacacag 1560

gaaaagagag agggctggaa gttttggaaa cgagcaatgc attaa 1605

<210> 14

<211> 1452

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 14

atgtcttatc cgccatccgg gagcaccgcc ttgtccgccc cgggcaaggt ccttcttacc 60

ggtggttacc tcgttttaga ccgcaattac acggggacag tgatcgcact cgatgctagg 120

atccacgtta tcgttcaaca attgaaaaga ggccatcgac gtggtgcatc attcagctca 180

gtgaaggggg gtcccgatac ggagacagtc gaggacggaa gtgctgtgga cgacaaggaa 240

aaagaagacg tcgtggttgt acgctcgccg caatttgtca atgcaatttg ggagtacggt 300

atacagcgtt gtgagaatgg aggtggaatc aaagtgattc aaaggaacga cgggcgttcc 360

aatccgttcg tcgaaacttc cctcaactac gctctcacct atatcagcta tgtggccgac 420

tcgaaggact ttgggtccct ctccgtgacc atcctggccg acactgatta ctactctgag 480

actgccttct ctagggtttc tgagtcccct ggaagattcg tgaacttcgg tgttcctctt 540

cacgaggccc acaagacagg actaggttcc tctgcggctc tagtaactgc cctagtatca 600

tccctcgtta ttcaccgtac cctgcagcct gacgaccttg gagcttctcg tgacaagctt 660

cataacttgg cacaggctgc ccactgtgct gctcaaggta aagtgggatc cgggtttgat 720

gtggctgctg ctatctacgg ctcttgccta tatcgccgat tctccccaag cattttggaa 780

tccgtcggcg acgccggttc acctgggttt gaagagcggc tgtttgcagt cgtggaggac 840

gccgacccta agcatccgtg ggatacagag tgcttggatt tcggcatgcg acttcctcga 900

ggcatgcaga tggtcctgtg cgatgttgag tgcggctcaa attccccttc gatggtcaag 960

aaagtgctcg aatggcgaaa acaaaaccag caggaagccg atcttctttg ggctgccctc 1020

cagtcaaaca atgaaaggct ctgtcttcag ctcaagcagc tagcccagag ccctgaccaa 1080

gaatcgcccg aagatttcaa tgatgtccgc aacctcatcc agcgctcacg caatcacctt 1140

cgcagcatga cccgcaaggc gggagtcccc attgagccgc gggtacagac agaactgctt 1200

gatgccgtgt cagccgttga cggagtgatt ggtggtgtgg ttcctggtgc gggagggtat 1260

gatgcgattg ctgtcttgat ccgcgatgac caggaggtgc ttaaaaagtt aactgagctc 1320

tttaagaact gggaaagtaa ggtggaggac gatttcggtg gcaagatcgg aactgtccgg 1380

ctcctcggtg tgcgtcacgg ctcagatgga gtcaaaaatg aggttctcga ccaatatgcc 1440

ggttggcttt aa 1452

<210> 15

<211> 1215

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 15

atggctgctc cttctgacag tacggtcttt cgggccacca ccacagcccc agtcaatatc 60

gctgttatca aatactgggg taaaagggac gccactctga acttgcctac gaactcctcc 120

ctctctgtaa ccctctcgca gcgttctttg cgcaccctca ccactgcctc atgctctgcc 180

aagtacccga ctgccgacga gcttatcctc aatggcaaac ctcaagatat ccagtcctcc 240

aagcgcaccc ttgcttgcct ttcgaatctg cgctccctcc gccaggaact tgaagctgcc 300

gactcttctc tgccgagact gtctaccctt cccctacgga tcgtttccga gaacaacttc 360

cccaccgccg ctggcctcgc cagttccgcc gccggtttcg cagcgctcgt ccgtgccgtc 420

gccgaccttt accagcttcc ccagtccccc cgagacctca gccgcatcgc tcgtcaggga 480

tctggttccg cttgtcggtc tctgatgggc ggatatgtgg cctggcgcgc cggaaatctt 540

gccgatggta gcgacagctt ggctgaggag gttgctcccg agtcacactg gcctgagatg 600

cgtgcgctca tcctggtcgt cagcgctgaa aagaaggatg tgcccagtac ggagggtatg 660

caaaccaccg ttgctacttc caacctcttc gcgacccgcg cggaatctgt tgtacccgag 720

cggatggcgg ctattgagac tgccattcag aaccgagatt tccctgcctt cgccgagatt 780

accatgcggg actccaatgg cttccacgcc acctgtctcg actcctggcc tcccatcttc 840

tatatgaacg atgtctctcg cgccgctgtt aggctcgtcc acgatattaa ccgtgccgtc 900

ggtcgtacgg tttgcgctta cacttttgat gctggtccga acgcggtcat ctactacctt 960

gagaaggatt ctgaactcgt tgccggtacc gtcaaggcta tcctgggcgc cagcagtgag 1020

ggctgggacg gtccgttcta cgaacctctt aagagcttca ccgctccggg tgtggcattg 1080

gataaggtgg actctagggc tgttgatgtg ctcaaggatg gtgtcagtcg tgtgatctta 1140

accggcgtcg gtgagggtcc tgtcagtgtc aacgaccacc tcgtcagtga gacaggtgac 1200

attctctcca actaa 1215

<210> 16

<211> 819

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 16

atgagcgtaa ctactacaac caccgagccc cctagaatca cggcggagaa tgtcgccact 60

ctcttcccag aggtcgatac ttctttggct cgtgaagtcc tcccaaaagc cgatggcaat 120

ccctccgcag ctagcagcaa cgagctggcg ggttatgacg acgaacaggt ccgtctaatg 180

gatgaagtat gcatcgttct ggatgatgat gacaagccta ttggaagtgc cagcaagaag 240

acctgccacc tcatgaccaa cattgaccgc ggccttctcc accgtgcctt ttccgtgttc 300

ctcttcgatt ccaacaagcg cttgcttctc caacagcgcg ccactgagaa gattacattc 360

ccagatatgt ggacaaacac ttgctgctct caccctcttg gaattgctgg cgagaccggt 420

tctgagctgg atgccgctat cttgggcgtg aagcgggctg cgcagcggaa gttggaacat 480

gagcttggaa ttaagccgga gcaagtaccc ctggataagt tcgatttctt cacgagaata 540

cattacaagg ctcctagtga tgggaagtgg ggagagcatg agatcgacta tattctcttc 600

atccaggcag atgtagagct gaagcctagc ccgaatgagg ttcgagacac gaagtacgtc 660

tcggctgacg aattgaagac gatgtttgag cagccggggt tgaaattcac gccttggttc 720

aaacttatct gcaattcgat gttgttcgaa tggtggagcc atctcggctc tccaaccctg 780

gagaagtaca agggcgagaa aggtatccgg cgtatgtga 819

<210> 17

<211> 2268

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 17

atggacttca cataccgcta ttcgttcgag cctacggact atgacactga cggtctctgt 60

gatggtgttc cggtccgtat gcacaagggt gcagacttgg acgaggttgc catcttcaaa 120

gctcagtatg actgggagaa gcatgttggt cctaagctgc cctttcgggg tgcattgggg 180

ccaagacaca acttcatctg tcttactctg ccagagtgct tgcctgagag actagagatt 240

gtgtcttacg ccaatgagtt tgccttcctt cacgatgata ttactgatgt cgagtcagct 300

gagacggttg ccgctgagaa cgatgagttc cttgatgccc ttcaacaagg tgttagagaa 360

ggtgacatcc agagccgtga gtccggaaag cgtcatctcc aggcttggat cttcaagtcc 420

atggtggcca ttgaccgtga tagagctgtg gccgctatga acgcttgggc cacctttatc 480

aacacaggtg caggatgcgc tcacgataca aacttcaagt cacttgatga gtatcttcac 540

tacagggcta cagatgtcgg ctacatgttc tggcacgctc ttatcatctt cggatgcgcc 600

atcaccattc ctgaacatga gattgagcta tgccatcaac tcgctcttcc agccatcatg 660

tccgtgactt tgacaaacga catctggtca tatggcaaag aagcagaggc agctgagaaa 720

tccggcaagc ccggagattt tgtcaacgct ctcgttgttc tgatgagaga gcacaactgc 780

tccattgaag aagccgagcg tctctgcaga gcgcgaaaca aaatcgaggt agccaagtgt 840

cttcaagtca caaaagagac acgagagcga aaagatgttt cacaagatct caaagattac 900

ctctaccata tgctgtttgg tgtcagtgga aatgcgatct ggagcactca gtgccgaaga 960

tatgacatga cagcgcctta caacgaaaga cagcaggcca gactcaagca gaccaagggt 1020

gagcttactt ccacatatga tcctgttcag gctgccaagg aggccatgat ggagtctact 1080

cgtcctgaga tccacagact gcctactccc gatagtccca ggaaggagag ctttgctgtt 1140

cgtcctttgg tgaatggcag tggacaatac aatggcaaca atcacatcaa tggagtctcc 1200

aatgaagttg acgtgcgtcc ttctattgag agacatgcct caaccaagcg agctacttca 1260

gctgatgaca tcgactggac ggcacataag aaggttgata gtggggctga ccacaagaag 1320

accctgtccg atatcatgct gcaagagttg cctcctatgg aagacgatgt cgtcatggaa 1380

ccataccgat atctgtgttc tcttccctca aagggagtta gaaacaagac tattgacgct 1440

cttaacttct ggctcaaggt tcctattgaa aatgcaaaca ccatcaaggc catcactgaa 1500

agccttcacg gatcatcact catgcttgat gatatcgagg accattcaca actgcgacgt 1560

ggcaagcctt cggcccacgc tgtttttggt gaggcacaga ccatcaactc tgcaacattc 1620

cagtacattc agtctgttag cctgattagc cagcttagaa gccctaaggc tttgaacatc 1680

tttgttgatg agattcgaca acttttcatc ggtcaggctt acgagctcca gtggacctct 1740

aacatgattt gcccaccttt ggaggagtat ttgcgaatgg ttgacggaaa aactggcggg 1800

ttattccgcc ttctcactcg tctcatggct gctgagtcca ctactgaggt agatgttgac 1860

tttagccgtc tgtgccagct ttttggtcgc tacttccaga tccgagacga ttacgccaac 1920

ctcaagctcg cagactacac cgaacaaaag ggtttctgtg aagacctcga cgagggcaag 1980

ttctcactcc ctctcatcat tgccttcaac gagaacaaca aggcccccaa agccgtagct 2040

caactgcgcg gcctcatgat gcagcgctgt gtcaacggcg gcctcacctt tgaacagaag 2100

gtgctagcac tgaatctcat tgaggaggct ggtggaattt cgggcacgga gaaggtgctg 2160

cactcacttt atggtgagat ggaggctgag ctggaaaggt tggctggtgt ctttggggcg 2220

gagaatcatc agcttgagct tattctggag atgctgcgta tagattag 2268

<210> 18

<211> 1858

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 18

atggaaaggc tcaagatgga tagcctcaac atcaacatca atgactggtt cgagaaagac 60

ctgccagcca ctgcagactg gaagctcttt gctctagcca gtgttgtctt tgttgttcta 120

cgatttactt gcattgtcat ctatcgtatc tacttttctc ccctttccaa gttccctggt 180

cccaagctcg ctgctgcgac gcatctttat gagtcttact atgacttttg gaagaaaggg 240

cagtactaca aagtgattca gcgtatgcac gaggtctacg gaccgcttgt tcgtgtcacg 300

cctgatgaac tttcgatcaa cgaccctgac tattacgaca ctgtctatgt caacggtaat 360

gttcgacgta ctgagtcctt cggccattcc tttggtggcg gacttggtat tgaagacacc 420

ttcttcgcct ctcaggacca tgacctccac cgcaagagaa gaaaacccat cgagccttac 480

ttctctcgca atggtgtctt gaagctcgag aatcttatcg gtgaacgtgt tgagaaactg 540

ttccacaagt tccacgagct gtctggtact ggtgttgttg cccgtcttga ctatgccttt 600

gaggccttca ctggcgatgt catgcagcat atttgcattg agaagcctga atcactactc 660

aacagcgatg acttttcttc tgagtggttt gagatgcttc gcaatgtctc cttgtccgta 720

cctcttatgg gaatgatccc ttggcttgtc cacgtactga agttcatccc cgagagtgtc 780

atcatgtggc tcgcgccctc agctgcccac ttccagacct tccgtgttca agctggtcgt 840

cagattgagc aagccaagca cgagaaagtg gagaatgatc gcaaaggtat cactactgtc 900

ggcggcaagc ccaccctctt ccgcttcctt gtccacgaga gtggtctcgc accggaagac 960

ctgagcaccg agagactcca gaaggaggca atggttctac ttggcggtgg cactacaact 1020

actgcgcgta ctgcgaccat gacttgcttc tggatgctca gcatgcctga gaagggccaa 1080

cgtcttcgcg acgagctcaa ggacatcatg gccgagtacc ccaagaagaa gccttctttg 1140

accgagcttg agaagctgcc ttatttggga gctgtgatcc aagagagttt gagaatggcg 1200

tacggatcca tgcgtcgact tcctcgcact tctcccgatg tagctctgca gttcaaagat 1260

tgggtgatcc ctcctgggac tcctgttggc atgaatgctt attatcttca cactgatcct 1320

aatgcgttcc ccgagccatt tgagtacaag cccgaacgat ggcttggaaa tgtcacaccc 1380

gcgatgaagc gtagttttgt gcccttttcg cgcggttcgc gccgatgccc cggatctagc 1440

ttggctcttg ccgatctcca tttcgttctt gcagcattgt tcggaccaac tggacccaaa 1500

tttgagttgt tcgaatcgga caggtctgac gtggatgcca ttcatgacta cctgatgccg 1560

ctgcctcggt tggattccaa gggtgttcgc gtcactgtca agtaggatcg ttcaaacatt 1620

tggcaataaa gtttcttaag attgaatcct gttgccggtc ttgcgatgat tatcatataa 1680

tttctgttga attacgttaa gcatgtaata attaacatgt aatgcatgac gttatttatg 1740

agatgggttt ttatgattag agtcccgcaa ttatacattt aatacgcgat agaaaacaaa 1800

atatagcgcg caaactagga taaattatcg cgcgcggtgt catctatgtt actagatc 1858

<210> 19

<211> 182

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 19

gatctgtagt agctcgtgaa gggtggagag tatatgatgg tactgctatt caatctggca 60

ttggacagtg agtttgagtt tgatgtacag ttggagtcgt tactgctgtc atccccttat 120

actcttcgat tgtttttcga accctaacgc caagcacgct agtctattat aggaaaggat 180

cc 182

<210> 20

<211> 269

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 20

actagacggg ttcgcatagg tttggggttg tatcttggcg ttgggacgga ctgggtatgg 60

tgtttctttt ggatatatga catgatatgt acacggccgt gaatctttaa ctttatatca 120

ttatagaaat gcacttgcac atttcaacac gctgcgagca gaatctcgaa gattgttccg 180

caagtattag atcatgagag cattttcatt tcctttcagg cagtgggagt aggccatcct 240

gaaaacaagg cggccactgt agactagag 269

<210> 21

<211> 567

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 21

ttgtattggt attaccagaa cgtcacgaga ccgcctgccg caatgttctc atcctttctt 60

tgctactagc aagttttttc gcaatttctc gaagctttag atggtctttt cttttccagt 120

cgaaagctac tagccggtct tttctgtcct attcagctta ggcagttata ggatatcttc 180

aagattcagg tactctttga ctacacacaa tgccctatga tatgaagggt aaatgtgtgg 240

gtatcattca ttggatctta agtaaggcac agcccgcgga gcagaaatga agctccttgt 300

atgacatcac caaatggtca cttaatgcaa tttcgaaccc tttccatccg agctcaagtt 360

cgagagctca gttcccattt tactcatctt ttttacttag aagagggata taatctaata 420

acaaactgat ttaaatgaac acagagctat tactttcaaa tttggcttaa tgtccacttc 480

tcgccaccca acggtcctat ggggtagtgg ttatcccacc ggattttgat gaacaatata 540

catgtcctgt atatgtgtag gaagatc 567

<210> 22

<211> 253

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 22

gatcgttcaa acatttggca ataaagtttc ttaagattga atcctgttgc cggtcttgcg 60

atgattatca tataatttct gttgaattac gttaagcatg taataattaa catgtaatgc 120

atgacgttat ttatgagatg ggtttttatg attagagtcc cgcaattata catttaatac 180

gcgatagaaa acaaaatata gcgcgcaaac taggataaat tatcgcgcgc ggtgtcatct 240

atgttactag atc 253

<210> 23

<211> 741

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 23

atggttaacg gaaccttctc catccaagac gacaaggccc ttcctgtgtt tgcaccccca 60

tacacttccc aggagccctt ccactttagc ggcatgactt ctgtctctat catgtaccgc 120

gtccgtgcct cccagatcca gcaccttgtg ccgcgtgaac tagaaatgga ggacgaaccg 180

atcatgactt ccttcttcgt tcattacggc acctctaccg tcggcgagta caacgaatac 240

ggcaacgctg tccaggtcaa gtataatggc aagacgttcg actattactt ggttcttgtc 300

ctcgacaacg acggcgccat ctttgctggt agagaaatat ttggatatcc caaaatcttc 360

ggcaaaacca acttccaccc gtcagctggg tccaaggtca tgacgggaaa cgtggagcgt 420

cccgcaggac gatctctgat cgagtttgag ttcgctccca aggcgcctct tgaattgtcg 480

gcagaaccca cgatttccaa ggcgctgaac ttgcgtgtta ttcccagtac ggacccgaca 540

aagcctgcca ttaaggagtt tgtcgctgta gatatgcaag tcgaattcag cgagaaatgg 600

agcggagaag gaagtctcaa gttcaacaaa ggattcgcat cggttccatg ggccaacata 660

gacgtagtct cgtacgtcgg gtctttcatg ggaaagagca aggccattct caccaatgag 720

gtagagcgtt tccctctcta a 741

<210> 24

<211> 1273

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 24

tcacagcgcc gccaaactct ttggtatcaa caactctcgc gttttcctaa cccgagcacc 60

atcctcctta ctcttgaaat accgacccgt cttgtagctc cagagcagac taccaagagc 120

atggttcttt tcaacttcaa gaaagccaag gagtgcacgg tcagcctctt caccgaagca 180

tgggagcttc gatagtgctc tgtaatagcg tctatagcag ttgttgatca taactccgac 240

ctcgtttatg gcctgctggg ttgagtgccc cttggctttg agaagtcgga ccatgttgtg 300

gtctaccccg agacgaaggt ctttctcgta agacaggatg tcgttgacta ataggatctg 360

gtcgctgatg atgaccatga tttcgaatac ggaaggatga tcagctacct cctctggcaa 420

gtcaattccg taacaccact cgttattaac aagggctgga taagcaccga gagaacctcg 480

acgcatagcc atgtattcct ctgggcgacg tgtgtaggac cggccctcca cattagtccg 540

aacctggtcg accagtccct gccagtaaag ctcatgtgcc cacatccagc gcttatagaa 600

acgatctgat gatggctttc caatgaagaa accctctggg ctttgcttta tgcgatcaca 660

caatgtctgg aacacatagc gaatggggtg ttctgagtcg gcggtatacc gaggggcggt 720

accctccatg atctcgcgcg tcctggcaat ctcgctgatg gcgccttcta gatcattgca 780

caaatggccc tcgtcaaact ggtcatcgaa gagaaaggcc cacgagttcc aatcagctga 840

cgtacgtaag gcaaaggcac tgcagtcagg tgcccagatg ttggcaagat aggtaaagtc 900

aacccgcttg tttctgttgg tccagtctgc atctgctttg atcacgcttt ataagttgtt 960

agtagacgat gattcttttg gtctgtgtca tacgtagggg gttaactcac gaggaaatcc 1020

aatcatctgc ttctgctttg acgctagcat agtttgggtt ctcacgggct gggaccgaca 1080

tgagtgagct gaagaggtca ggaagtatga gaacctccat ctcatcacgg ccctggaact 1140

gatcatgttt gggttcgttg tagactccgt taccgttctt catcttgagt ttgacctcat 1200

gtttggtgtc gatatagacc tcatccccgg ttgtcatctc tgactcggaa ctactgctgt 1260

cgaatttgac cat 1273

<210> 25

<211> 1692

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 25

atgtttagta ctctacggca aacgtctggc gatggagacc gtctcctcag cgtggagatt 60

gctccaccct ttgacaagga caggatgtca atgcggaccg cgtgtgagag atgccgcata 120

cagaaggtag gtagtatcga agccattcac attgtgtcac gtcggagagt ctttctagat 180

aaaccctaga cgtcgttcct attctcactt gataattatg aaactaattg actgcattag 240

tccaagtgtg tcagtggcga gaatggctgt atgctgtgtg ttgccaagaa ccagaaatgc 300

gagtacctgg ttgtgtcgag gccacgacgg cgcaagtcca acattgctgg cggtggccca 360

aagtcccgcg acgataacga tgatgaggac ggcgacaaca cagtcgtcaa tcagcaaaag 420

cagtcaaatc aggctaggat gcaaaaacga tggtctcgaa tgcagacaat acgacagtca 480

tcgccattat cagggccatc atctccaccc actgatcaag tgagggtttc agcgaagccc 540

tcgattcaag atgacaacag taacataata gggccagacc tgggcctaga agatgcacat 600

caaataatat tcaacggtgg tctcttcccg gactcccttg gacaaatccc ggctctcttt 660

ccacacctcg agcccgcagc cattaaagac ttctttagca atatgcctat agctgcggag 720

aatacccatg caggccgtgg tgttaccgca gaagcattga ctgctcgagg agacagtggc 780

gggctaaaca gtcacacctc tgtatactca acctcatcag cagatagcct gtttgacttt 840

ggagttgatc agatggactt tctgatggac gagtcacaca tcactgctat agctcccaga 900

agtaccagta gcagcaataa agttgcaaga gacgttcgtg ctcctgccac tcattcttca 960

acaccatcag aatcaaactc tacatccatt tctacatctt cttcttgcag ttgtatgatg 1020

acagctgtgg gtatctatga ggctttgcaa gttgagctga actggggcga tccaattgct 1080

ggcccatcgg ctacatcaag tcccaaatcc tcatcggcgg gctcttcata ctcgtctggg 1140

ccgccatcgt ggtcagactc aggttcgata acaaaccatt caacaacact gatgacccag 1200

caaacgatct tgaaacgcca aaagacggtg ctactccgtt gtgactccct cacccggtgt 1260

ggcacctgct ggtcccgccc ggactttgtt atgctaatca ttaccatatg tgatcgcata 1320

ttgactagtc tggaggcggt tgagcgtttt gtctgtaaca agaaagacga tgacataaat 1380

agaatcagtt ccaacggctc taccactgcc gtagatatcc aggccgcacg cacggagcta 1440

gactcatccc tgtccaccac ttcccaggga ctacaatctg gggttggtgc atggcagatt 1500

gatgacgagg atgagcagga aatggttatt agcttgatca agtcccgcgt tacaagactc 1560

ggcaatctga tcaacatagc tgaggggaca attagcgcaa acgggtggcc ttggcatgag 1620

aggctggctc aggctctacg gaggaggtct aacaagcttg ccatatcctt gtcatttcgc 1680

ggttttccgt aa 1692

<210> 26

<211> 1934

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 26

atggcatatc ctatcatcct catcggcgtt attggccttc ccattacaat cctgctccgc 60

tggctcctga catacacgcg caccgtcctc aaggtccgcc gtaatggcca tcctgcaatt 120

cacagtggtg tcatgatctt tgagtccgtc gtgcgttcat ggtatcctcg aatccccttt 180

ctggtaccta tggagaagtt caccctgaag gatcccttca agaagttcgc cgatgcgcgc 240

tctgacatga tcgttataac tgaggctgta agctacccta ccctttttgc aaaaagaatg 300

accactgacg tgtgagaagt cgtcacctaa tggtatcgcc tacctcttcg gatcaccaaa 360

gatcttccgt gaaattggcc gcaacggcga catcttcctc aagccgcttg agaagattcg 420

ctatcgcatg ctcaatacct ttggactcca gctcgcctca acacagaatg gcacccagca 480

tgagagacac aagcgcgttg tcaaagcagt attcaataat gagcttatgg agaacgggtg 540

gcaaaacatg cgcaacatgt ggcgaagcct cctccgcgaa gagggcgtct atccaacagc 600

tgccaactca gacgctgctc ccattgtacg agacatgaag tcgacgatgc tgaaagtcac 660

acttggtgcc attggtgcct cgtggtttga cattgacatt ccctggaatc cggctaagga 720

gacgcaacgc caaaatgacg agttgatgcc attcgccgag acactgaaag ttgtttggga 780

ttcaccattt gtgcagacta ttctaccact gtggttcatg gaatggagcc cttcgttgca 840

tcttcgtcgc gctgcttggg cacagcgatc ccttgttacc cacatcaaga atgcgcaggc 900

tgagacaaga cgaagaatcg aggatagcaa ggataaagct caggttcaag gccggatgcg 960

aaagtacagg aacctcatcg atgcgctggt ggattctcag aatgacgtgg agatggctga 1020

gaaggccgag aagggatatt tggcccccaa tgtcggcctg tcagacaaag aagtccaagg 1080

caacattttc tctttcatgg tgtaagttca aaggcacttg atgttgaact acagaatact 1140

aataaatggt taatttagaa ctggtcatga aacatcctca cacaccctga cttgggtctt 1200

gtcactgctc gccaagaaca ccgactggca agaaaggctc tatgctgaag tcagcaaggt 1260

caacactctc cccttgaacg aggccgaaag cgcgaacggc gcaaagccac tgaagtgcct 1320

tggttatgaa gaaatggcaa acttccctct gatccttgcg gctactgtcg aaacactccg 1380

catgcgtgat ttggctatgc agatgacgcg tgtggcctct cgcaacacca cgctcagtta 1440

cacaacatgg gatggtgacg caactaaccc atccgaggcc aaggttcagc aacacacgat 1500

tacaattcct gctggcacac gtgtgcacct cgatacagcg gcctttgggg ttaacccgtt 1560

caagtgggaa gacccagaga catataaccc agaacgtcac cttcgcgaga ctgaggatat 1620

gaatggcaac aaaaaggtga cggtacgttt atctcatctt tctatttaaa ggaataaccc 1680

tgtactaaca ttcgatacag atctcatatg aggatttcat cggctactca tccggttccc 1740

gtcagtgcat tggtaaacgt tttgccgagg tgacaatggt gtgtttcctg gcacatatga 1800

ttctgaacta tcgatgggag gtcgtgcctg aggcgggcga gacacaggag caagctaaag 1860

tgagggcgtc tactggttcg gaacagttca tgttgacacc accggcgtac gatctgcgct 1920

tcatcagacg ctaa 1934

<210> 27

<211> 3949

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 27

atggctatcg gtagaaccaa gcgaggaaag atcggaaaca ggttgattcc acagattttg 60

gacgatctcg ccgctacgga gcctgatcgc atcatttact cctgggccaa gtcctcggac 120

ttgtcacagg gtttccgcca tgtatctgct cgtgccttta cgagagcagt cgataagacg 180

gcgtggttac ttcagcgaga actaggagag acctcagaga tccgcgctgt gggttatatt 240

gggcctcgtt agttaagctg tccaagagcg tcgcactcgg gccatttgac taacgacacg 300

gcatctagac gatcttcgac agatcttgtt gacattcgcc tgtatcaaag ccaactacac 360

cgtgagtttc tccttaacct caacgtgtac ctaacgactt gactctgttt gtttaacctc 420

tttttacaca ggctttattc ctctctccaa agaacagtat tgagggagct ttagccgttc 480

tcgatgcggc agattgtaac atctgggtca accccgctgg cgagaggcca gcaccactcg 540

tggatgactt cgtgcagcaa cgtgccatgc acgtgatgca cctgccgctg ttgagcgagc 600

ttcttcctga agacgagagc gagatggagg acgtgaagcc gtttccttac acaaagtgct 660

gggaagatgc gataaacgac acattctgca ttcttcatac ctcgggctcg actggcctgt 720

caaagcccat caagtggaca cacgggttga ttggtactgt ggatgctgtc cgactgcttc 780

cccctcatga aggtatggag ccctgggcga aagggtggga tgatggtgat acgctgtact 840

cgacattccc gatgagccat gtaagcattc cagtcccagt ctcaatgaaa aaatcagcat 900

gatgctaacc atgtatcaag ggagcgggga tcttgatgga cgtcgttata gcgccactat 960

ttggcctgca ctgcgtcctt gggcctcgag acgtgatacc caatttggaa ctcatatcgt 1020

ctctggcaga ccacatcgaa attgacatct ggagcatgat tccttcgctc agtgacgaac 1080

ttggcgaagc acctgacatc ttgccaaaac ttagccggtc gaaatttatt tgtgcttcag 1140

gaggtaagac agttgtcctc atgccttcca ctcccagacg tttgctgata tatataatta 1200

ccttacaggg cccgtgagca gtgttttggg ctccaaggtg aacgagttca ttcgtgtctt 1260

gaatctgacc ggcacatcag aaggtctatt catcggcaac ctgtgggtgg acagaaaaga 1320

ctggcactgg tttgccttcc acccttggtc tggctttgac tttaagatgg tcgagcctgg 1380

tctttatgaa cagtggatcc atcgtaacga acatgcagac cttttccagg gtctctttca 1440

aacctttcaa gatgtggaga gtttcaactt caaggacctg tatgttccgc acccgactaa 1500

gccgggcctc tgggcatctc acggtcgcag cgatgatgtt gtggtccttt cgaacggata 1560

caagatctcg cctcttgata cagaggccct tgtcgcatct cacccggccg ttgatgggtg 1620

tttgatggta agtccagaat ccctcaagag gttgaagaag aatgctgatt gaagtgtacc 1680

acagatcgga tcaggtaagc cacaagctgg tctactcatc gagctgaaag atccaacaat 1740

aaagaaggac gacgacaacg ctgaagcgct cttcaacagc atctgggccg tggttgagag 1800

ggccaactcc ctgtctctgc acaagaacca gctgcaccgg gactatgtcg ccttttctga 1860

agcggacaag ccatttatcc gtaccgataa gcgcaccatc aaacgtcggg ctactatggc 1920

actgtatgaa gattacatac agcgcttcta ccagtcaaga acagaggatg atagcggaga 1980

tggagctgcg gctatcgggt tcatcacagt cgacacatca tccttggact cgactactcg 2040

cgcagttcga catgtgctgg cttcgattgt gcctgtggta aaagattccc ccgcggacgc 2100

tgatatgttt actcttgggt tcgactcact tctcgtcttc cgcgctacca agacgattgc 2160

ggcagttaca gatcttggtg ggaaattctc accgaggaac ttctatgctg gcccgacgat 2220

tgaagcgatc gtcgcaactg tcatgcgact agcttctgag cgcagagcca tgataataga 2280

tggcaccgtt gcctcatcgc cgaccgaaca gcatcaacaa gacccaaagg aagtaatgat 2340

gagtacactc ataaaacgac acaaggctgt tctatcctcc aagctgggcc caatggacct 2400

ttttggaggg aacatgtacg agggtattaa cgtcttcata cctctctgtc cagatgtgcc 2460

gtttaagcag gcgtataaag tgctccagcg aggtcttgtt cgtgcgatgg agatcgtgcc 2520

ggatctcgca ggtaaagtta taccctgttc ggagcacgag attggataca agaagggaga 2580

tcttcgtctc agtcttcctc cactgccctc tactgtcttg gggatgactg ccccggagga 2640

gccacgacaa ctgcgcttca atgacctgtc gtccgtcctg ccatcctacg ctgagcagaa 2700

agtttctgga ttcttgacgt cagcttaccc cgacgagctt ctgactacgt gtccggcttt 2760

tccttcactg cctgcggacg tgtgtaatat ccaggccaat ttcatcgagg gtggttgtgt 2820

gctggccttc aatgttcatc atcacgctct tgatggtgtc ggattgttga tagcacttac 2880

ggtttgggca gaatgctgtc gattcgtcca gggtgatcag tctgcgactt gcacgtggct 2940

gcacccagag agcctcaatc gtgatatgct atctgtcttg tacgagttgg agggcttcgc 3000

gaagccggca agtgaaatcg accccaaggt ttggggtttc ctaccatacg ccgatccggc 3060

gctgaacgcc aaggacgcaa ctgcagccaa cggacatggg actgagccaa gaaccgagaa 3120

ggctcctttg tctcgcaatc tgcctgaacc gcctcgtcta cctccatgcg agcactggcc 3180

acccaaagct cgattggatg gtcgcacact ggcagcgtcg accttcctta tctcggccga 3240

gaagctgaag agactccaag agagtgtaga gcttgctgaa gctactgatc cagaacgtca 3300

aagcctcagc aacgagtcag tgtcactatc cctcggcgat gtactccaag ccttcttctg 3360

gcgcgccgcc gtccgggctc gtcgtcgccc tgagaacacc tcggccgatg acacatccat 3420

catcgagatg cccaccgacg tccgacccta cttcagcgcg cacctaccgc caacgtacat 3480

ggccaacagc gtcatcatga atcgacagca cgtgtccgtc tcgaagctct gctcatccga 3540

gacaaccatt tacgaaattg ctcaaatctg tcgcgaggcg cgcactcgaa ttgatcaaga 3600

gcttgtccac gatgcatttg gtctgcttca tacgatccaa gacaacagtc caggaaacca 3660

caccacagct ttcctgggcc agggtatcca ggacggtcca cactcgctct tcaacaacat 3720

gatgctattc cacgcaaagg atattggggc atttggcgga aacatctttg atgcgcctga 3780

tgctgtgagg gtccaaatgg attggctaaa caaagccttc aggagtctgt ttattctgcc 3840

gataagagat gatggcggtg tcgagttgct tctcgggacg tttcctgagg aactggatgc 3900

tatgagaaat gatgaggagt ttatgcagtt tgccgagttt ctgggatag 3949

<210> 28

<211> 1495

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 28

atggatatcg tggacgacgt caagataatg accgacacta tcgttctaac ctaccccgaa 60

cctactacca tcatgaacaa tacctcaaca gaccatggca tcgtctttga tgacggagtc 120

tcgaagctca agacaccggc tatcaccata gcttcatcag acgtctcgtt tgacgacaca 180

atgtcaacca gatcctccaa ctccagtctc ggtactgaga tgacgggtcc tccagccaca 240

gatgacaaaa tggaagtctc caacgaaacc aaagtctctg agatatccga gagaatttta 300

gacgtcattc tgaaatactc tctcaacaag ttcgaaagca cctccgagct ccacagcaaa 360

ggcagaccca agtttcttgc cgtcatctca cgctttgtcc aagagcaaca gaaagtggtt 420

atgtgtctgc cagcattccc attcaagtct gcgaacaagg ttgaaaaggt gctgggcagt 480

cttcccgaca aggccgagga agtatcactc gccagattaa actccatgtg taccaccatc 540

ggacagtttt atgagccggg agctcagttg accatcatct cggacggtct ggtttacaat 600

ggttcgttca cctcttttat ttctgcctca aattaatgaa gtatttcatg gctgctggca 660

catcacttac atttcttctt cgtcatatag acttgctagg catttcagac ctcgagacct 720

ggcgttacgg ctccgcgcta cgagccatgg ccgagcgcaa ggcctttact aacctatcat 780

tttcccgtct tcaagacttg gtcgcagcca agggcttgcc caatgacctc aatgagctga 840

catatgttgc caacgccacc aacttccgtc gaaccctgtt caacaagtac ggacgggacg 900

acgatctcga cattgatcac gaaatcgcca caaacgcaga cacactcggg acgtataagg 960

ggtactgccg tttcctcaag tcagatctgc aacacatcta cggcccagcc aagagttctg 1020

ccaagtacag gaaggacgtc aagtatcttg ctaagcaact gctgatccgg ggatatgtaa 1080

gtttcgagct cactagtcca accagttatt gaatactgta tcgactcctt acagctaacg 1140

caatctcaag gcctttgctg gagctgtcaa agcgcgcttc ccagaccatt tgcgtctcag 1200

tatccaccaa tcgaccgggg agcacaagat ttcaattagc cttctcaaca ccaaaagcgg 1260

cttcacaacg ccatggcatt gcagcgtggc attgatggaa gatggcgaat ggcttagcgg 1320

ccttacgatc gacttcaaag ccgatcggtt gctggaactt gtccaagagg agggacggcc 1380

gagttacttc aaagaggtcg cccgtcagcg gccatacctg acggagagcg ctaagccacg 1440

cgttgttgtt aagcaggaac cacaggccca tagacctagg atacgcgcat cttaa 1495

<210> 29

<211> 1875

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 29

tcaagatatt ttgatgcgag tcgaaatatg tcccttcttc gtctgtagca caaccagatc 60

gtggtcatac gtgaggtctt caatcgtagt ctcgaagagt tccgatttag gaatgactcg 120

aaacgccatc agggcagcca tgatgtagat ctcacacagc gcaagactac aagatacaac 180

tgttagaatg aaaacttcaa cttgaacatg taccgtagtg gacagtagga tgtaagtaag 240

actcaccttt caccaacgca aactctagat ccctttgaaa acgagataag gaatttctga 300

agagtgtaat caggctttcc gtcgggtagc agccatcgat cagggttaaa ggaatcaggg 360

tcgggaaaca gccgcgtgtc ccagtggtta atcatggctg tcatgctcac aggcgtgcct 420

cgcgggatca tgaattgtgt cttgccatct tgactcttat agaagagatc ttcctcacgg 480

gcgatacgag cagatcgacc ggctgcgcca ggttggtgcc gcagagactc ttgaacaagg 540

gcccagaggt agggtctctg ttcgagttgc gcccatttga gatttttagg atcaattcct 600

tcaaggtctt tcataagacg agcgtagacc ttgggttgat gaagcatttg gaacgtcatg 660

acagtaagga tagcctgtgc cgggttagct gtgaaaatta taattgtctg tcatggatcg 720

cgaactaaca gcagtggttt ccgttccagc gaggagaaag acaaatccct cacccgaaag 780

acgaaacatg gttttctcct cttcaggtag gacgttggag tttagaatct cgttaaagac 840

acggccgccg tccgggttgg ccaaggctgt cttgatgtag gctggaatga catgattcat 900

ctggtgcatg atcctcttga catcctcccc catatagtca gcgaagatgg gtaggatatc 960

agctagccgc ctagccaaag cattgtggcg cactggttgc tatccgttag caaaattgtc 1020

aagtcaaagt atttgaaagt tcaatgcaaa aacgtactca tgtaagtggt tttcaagaag 1080

gatgacgtcc aggtgccaaa gttgggctcc cagccctctt gctcaataaa gcccatgggc 1140

tctccaaatg catactgcga gaagacgtcg gcagtgtagc aattgaaggc gcccttgact 1200

tcaaaggctt cttttccagt ccagcgcagc attttctgga tgaagagctc ggcgaatttt 1260

agtacttcat cttcgagctt aagaacttgt ccacgtgaga agaagcgtgc atgggcagcc 1320

ctgcgcttgc gatgcaactc atggggccca gctgtgccgg ttgcctgtga ggctgggcca 1380

gcacctgcta tcttgaggtg gtgctgccat ttgtcgcgga ctagaagatg atagacgtga 1440

gcgacagagt cccatcgaga caataatgtt gcgactaaat aaacttactt cgacctggct 1500

ttcctgtgta gatctcgtcg gcaaagtagg gatcgctgca gtggagttca tcagggctca 1560

cgcgcacgat gggaccttga aaggctgtta gtggtcactg acgatccatc cggaatcatt 1620

caagcactta ccatactgct catgcatctt ttctatcctc ctgccatagc ggccttgaag 1680

gatccagtca tagtaagcct catagacata cgaggcagcg gcaatcttcg gtccagggaa 1740

cttggagagt gggtggaaag gagagatgtt gtaaagcgcg agagcgacac ggtaaccaag 1800

ccaaagacca gcgagtccaa gcagcgctga tggactcaca aaacgcttga gggcgatgat 1860

attgaagtca tccat 1875

<210> 30

<211> 1771

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 30

ctatgacctg cgcttgacct ccaattggac tcccggatcg gcaaacagct caaaaccaaa 60

cggcatcagc ttgggtgtat aaccctcagc caaccgcaca tcgtaatcga gcaagatggt 120

agcaatagct gtcttcacct ctgcgacagc gaagaatctt cctgggcaga taggtttgcc 180

aataccaaat acaaaatgat cctcacttgt agaagtagca gaactagcgt acgtccactt 240

gccgccctga gcacgaagct tcgcatatcg atatgcgtca aaggtgtctg ggttctcgta 300

cacctcaggg tttcggagac gagacatgta aattgagaga gcggttcctt tggggagata 360

ctggccgttg ggaagggtga cgggtgatag gacctggcgg tcgagattga ctttaatcta 420

ttagtacatt gttgtataat gatgaagtaa tcgtcttatt gacttaccaa taacagcgga 480

attcatcctc tgtgtctctt tgatgacgct atcgagcaat cgcatatttg taagcgcagc 540

gggtgaccat ccatgctcgt gaaccacggc ttcaatctct tctcggagag gtgtgatcag 600

atcatgcgag caaatctcaa tgaccgtctg cttcagcaac tccgaagtag tcacaacagc 660

agccatagac attccaagct gagcggcgac aggatcatag ctcttgcccg ccgcaatttc 720

actgaaccaa gtgatagagt catcgtgctc tttgccttca gccgctctct tggccaaatt 780

cttttccaag acttcgcgtg cacgcttaac ttctgctctg caactcttac aagcggggag 840

gaaccattga ataagaggtc tcagccatcg cggccattgt cgcaacgcgc gagccgcgag 900

gaacacagtc attccgtagt tggtactgac ttgttgccat gtctcgtcgt gtgacagctc 960

ctcgccaaca aagacggatg cggacattcg gctgatgagg ttaagaccgt ccttggccca 1020

gtcgactgct ttccaagcta ttctgggtgt cagtcaatac tcacgatggg agaatgctca 1080

atacgtaccg ccacatcctt ccggccaagc tgcaaggatg tctctctgca catttttgtg 1140

cagtttgctg acatctggaa agaaactagt cagtgggatg acgccaaggg tatggtggcg 1200

gtggacttac gagggttttg aacaagcttc ttcttgataa cattggccaa cagtttggaa 1260

gggtcagaga tggctgccgt gccattgaaa ccagggtagc ccgcaaagaa atcctaaaga 1320

gacaatttag taagttattg ataggactat ctagaaagca agggttttac cttgcgaaca 1380

agcggctggt gatcaagttg aggaccatga cgcctgatcc attcaaaatg gtcttcaggg 1440

aggatcaatc tatctcccac caatgtgatc atgcggaaag ggttttggaa ctgatttagt 1500

tattagtgtc tagttgtctg gataactatc attcgcgtac cttggcaaac ccctgtttga 1560

tgaggccatc ggcattagtg acgaaggcta tttgggcctt gatggagaac caatcattgg 1620

gatatttgtt gatgactggg attttttctt ttggcgagac aagtactaga agtgataaaa 1680

cgaggatgat ggcaccggca gcgacgaggc cagcaccctc gggagtatag tcccccacac 1740

ctagcgtagt ggtgttttga ctgaaatcca t 1771

<210> 31

<211> 3320

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 31

atgcgcctgc cagaagatgg cgatggtaca gctggcagac tttccgtgtc tggtagcgca 60

catcttgcaa caaccacgct gcaggtcggc ggcatgacgt gagtcaccac ctgttaacat 120

tttgctcttt tagtactcat aactaacgtt gtagcaataa tagatgcggt gcttgcacat 180

cagccgttga gtccggtttc aaaggcgttg acggtatcgg aacagtctct atcagtctcg 240

taatggagcg cgccgtagtc acacatgacc cacggatcat acccgccgag aagatacatg 300

agatcatcga ggatcgcggg tttgatgcag aggttctctc gacagatatt cccaacgctg 360

gggctactcg gaccaacaac cacttcaacg agagcactgc catcaatggc gaaactacaa 420

ccactgcgac aacaactttc gccatcgagg gaatgacatg tggtgcctgc acatcagcag 480

ttgagggcag ctttaagggc gtggacagta tcctcaagtt taacattagc cttttggctg 540

agcgcgccgt tattacgtac gatgaaacca agatttcccc cgaggagata gccgagatta 600

tcgaggatcg cggattcgat gctaccattc tatccacaca acgcgacatg gcctgccagg 660

gacgagacac cacatctgcc caattcaaag tctttgggtg caaagatgca acgacggccc 720

aagctctgga ggaaggcctc attgcgatcc aaggcatcca atcagtatct ctcagcctca 780

gtacggatcg tcttacagtt gtttatcaac ccatgaccat aggacttcgt ggcattgtcg 840

aagccataga ggcgcagggc ttgaatgccc tagttgcaag cggagaagat aacaacgcgc 900

aacttgaatc gttggcaaaa acgcgtgaaa tcactgagtg gaggagagca ttcaagatct 960

cactcgcctt tgcgattcct gtccttctaa ttggaatgat cattcctatg gccttccctg 1020

cgatagacat tgggagtttc gaactcattc ctggtctatt cttgggtgac attgtgtgtc 1080

tcattatcac attacctgtc cagttcggca ttggcaagag attctatatt tctggttaca 1140

agtctctcaa gcatggatca ccgacaatgg atgttctggt cgttcttggc acaacgtgtg 1200

cttttctctt tagcgtcttc tcgatgctgg tctcagttct tcttgagccg cattccaaac 1260

cttccaccat cttcgataca agtactatgc tcattacttt cataacacta tctcgatggc 1320

tcgaaaatcg cgccaagggc aagacctcca aggcattatc tcgccttatg tccctagctc 1380

cgtcaacagc agccatctat gctgatccga tcgccgtgga aaaagcagcc gagaactggg 1440

caaagtcttt tgacgagccg tcaacgccaa ggacacctgg taaccaaact ggcggatccg 1500

cttgggagga aaaggtcatc ccaacagagc tacttgaggt tgacgatatc gtggtcatcc 1560

ggccaggtga caagattcca gcagatggtg tcctggtccg gggtacaaca ttcgttgatg 1620

aaagcatggt tacaggagaa gctatgcctg tccacaagcg tataggtgat aacatgatcg 1680

ccggtactat caatggtgac ggacgtgttg atcttcgtgt tactcgagct ggccatgcta 1740

cccaattaag ccaaatcgtc aagttggttc aagatgcgca aacggcccgc gccccaatcc 1800

aggagctcgc tgataagctg gccggctact ttgttcccat gattctcatt cttggtctta 1860

gcacattcct tgtatggatg gtcctttgtc acgttttatc tcaccctcct gagatcttcc 1920

ttgaagacaa cagcggtggt aaaatcgtgg tatgtgtcaa gttgtgcatt tccgtcatcg 1980

tctttgcctg tccatgtgcc cttgggcttg ctacgcccac ggcagtcatg gtcggtacag 2040

gagttggggc cgagaacgga attctcatca aaggaggcgc tgccttggag cgtataacca 2100

aggtcacgca tatcatcctt gataaaactg gcacaattac gtacggaaaa atgagcgttg 2160

ccagcacaga tctcatctcg cagtgggcca gaagcgatgt caacaaacga ctgtggtggt 2220

ccatcgtggg tctggccgag atggggagcg aacaccccgt tggcaaggct atcctgggcg 2280

ctgcaaagga agaactgggc atggatcccg agggaaccat tgatggcact gtcggtgact 2340

tcaaagctgt tgtaggcaag ggtgtcagtg tgactgtgga gccagctacc tcgagccgta 2400

cacgatacct cgttcaagtt ggaaatctcg tctttttgca agataatggt gttgatgtcc 2460

ccgaggatgc tgtccaggct gcagagaaga tcaacttgtc ggctgacgta ggtaaatcga 2520

cggtcaagag cagcggcgct ggaaccacca acatctttgt ggccatcgat ggcgtttaca 2580

caggctatgt atgtctgtct gataagatca aggaggacgc tgctgcggct atctcggttc 2640

tgcaccgcat gggcatcaaa acctcgatag taacaggcga tcaacggtct accgcactcg 2700

ctgtcgcttc tgtcgtgggt attgatgccg ataatgtcta cgctggcgtt agtcccgatc 2760

aaaagcaagc catcgtacaa gagatccaac agtctggtga agttgtcggc atggttggtg 2820

acggcattaa tgactctcca gcccttgcga cagcagacgt tggcatcgcc atggcaagcg 2880

gcacggatgt agcgatggaa gcagcagatg tcgttcttat gagaccgaca gagctcatga 2940

ttatacctgc tgctttgact cttacacaca ctattttccg tcgaatcaag ttaaaccttg 3000

gatgggcttg tctatataac gccattggtc tcccgatcgc aatgggcttt tttcttccgc 3060

tgggtctgag cgtacaccct atcatggcga gtcttgcgat ggcgtttagc agtgtcacgg 3120

tggtggttag tagtctcatg cttaactcat ggaaaaggcc tacttggatg aatgaaatag 3180

ctatgaacga tgacaagacg cccaaggcgg agaggtgggc atttggaagg ggcatcgttg 3240

gctgggtgag ggaaatgatg ggacgtaggg gaaaggtgga ggaaattggg tatttgccgt 3300

tacagaacat ggagggctga 3320

Claims

1. a microorganism chassis cell, is characterized in that, described chassis cell contains the gene that improves described chassis cell production terpenoid ability, and described gene is the gene of the MVA approach gene module of aspergillus oryzae origin; Described gene is ERG10, ERG13, tHMG1, ERG12, ERG8, MVD1 and IDI genes from Aspergillus oryzae.

2. microorganism chassis cell according to claim 1, is characterized in that, described microorganism is eukaryotic cell or prokaryotic cell; Preferably, described microorganism is Aspergillus oryzae, Escherichia coli, Saccharomyces cerevisiae, Pichia pastoris, spore Bacillus, Aspergillus nidulans, Aspergillus niger, Neurospora crassa, Alternaria or Fusarium; more preferably, the microorganism is Aspergillus oryzae.

3. microbial chassis cell according to claim 1, is characterized in that, described terpenoid is selected from semiterpenoid, monoterpenoid, sesquiterpenoid, diterpenoid, triterpenoid, One or more of tetraterpenoids and polyterpenoids.

4. the preparation method of the microbial chassis cell according to claim 1, is characterized in that, comprises the steps:

Step 1): establish a microbial chassis strain promoter library: screen promoters from different sources, and characterize the strength of the promoter suitable for Aspergillus oryzae by regulating the expression of the GUS gene;

Step 2): with the mevalonate pathway derived from Saccharomyces cerevisiae as a reference, through sequence homology comparison analysis, the mevalonate pathway synthases ERG10, ERG13, tHMG1, ERG12, ERG8, MVD1 and IDI;

Step 3): The 7 mevalonate pathway synthases of the mevalonate pathway derived from Aspergillus oryzae screened in step 2) are respectively constructed on different plasmids under the control of promoters with different strengths characterized in step 1), The chassis cells were obtained by increasing the copy number of the rate-limiting enzyme expressed by the rate-limiting enzyme gene tHMG1.

5. the preparation method of the described microbial chassis cell of claim 1 or 2, is characterized in that, comprises adopting CRISPR/Cas9 strategy, will be derived from the ERG10, ERG13, tHMG1, ERG12, ERG8, MVD1 and IDI genes of aspergillus oryzae , respectively integrated into the high expression site of the genome of Aspergillus oryzae to increase the number of copies of the rate-limiting enzyme expressed by the rate-limiting enzyme gene tHMG1 to obtain the microbial chassis cells.

6. a high-yielding terpene microorganism bacterial strain is characterized in that, with the microorganism chassis cell described in claim 1 or 2 as a starting bacterial strain, respectively overexpressing the disesquiterpene synthase gene mgcD and co-expressing the disesquiterpene synthase. The enzyme gene mgcD and the cytochrome P450 enzyme gene mgcE are obtained to obtain the microorganism strain with high terpenoid production.

7. a terpenoid biosynthesis gene cluster high-throughput automated mining method, is characterized in that: comprise the steps:

1) PCR amplification; 2) plasmid library construction; 3) microbial strain library construction; 4) activity-oriented compound screening;

Preferably, the step 2) includes the following steps:

a. Design 40bp overlapping sequences at the 5'-end and 3'-end of each gene, promoter and terminator sequence, prepare a DNA polymerase system on an automated workstation, and obtain each fragment by PCR amplification;

b. Automated yeast assembly of each fragment to obtain a recombinant plasmid;

c. The recombinant plasmid was transformed into Escherichia coli, and enriched to obtain a plasmid library;

Optionally, the step 3) comprises the steps:

The plasmid library obtained in step 2) is transformed into the microbial chassis cell according to claim 1 or 2 according to the rational combination of different functional elements contained, based on an automated platform, to obtain a strain library, and the functional elements include terpenes. synthases, cytochrome P450 enzymes, acyltransferases, glycosyltransferases;

Preferably, the step 4) includes the following steps:

The fermentation product of the microbial strain library obtained in step 3) was acted on the mouse macrophage RAW264.7 induced by lipopolysaccharide, and the generation of nitric oxide was used as the detection index to screen out terpenoids with anti-inflammatory activity.

8. a sesquiterpene compound, is characterized in that, the structure of described sesquiterpene compound is selected from a kind of in following structure:

9. The application of the sesquiterpene compound of claim 8 in the preparation of anti-inflammatory drugs.

10. The application of a sesquiterpene synthesis gene cluster BGC6-FgJ02895 in synthesizing the sesquiterpene compound described in claim 8, wherein the sesquiterpene synthesis gene cluster BGC6-FgJ02895 comprises FgJ02892 gene, FgJ02895 Gene, FgJ02896 gene, FgJ02897 gene, FgJ02898 gene, FgJ02899 gene, FgJ02900 gene, FgJ02901 gene, FgJ02902 gene, the genes encoding decarboxylase, terpenoid synthase, transcription factor, cytochrome P450 enzyme, acetyltransferase, Tf, Cytochrome P450 enzymes, monooxygenases, copper ion-dependent ATPases;

Preferably, the nucleotide sequence of the FgJ02892 gene is shown in SEQ ID NO: 23; the nucleotide sequence of the FgJ02895 gene is shown in SEQ ID NO: 24; the nucleotide sequence of the FgJ02896 gene is shown in SEQ ID NO: 24 ID NO: 25; the nucleotide sequence of the FgJ02897 gene is shown in SEQ ID NO: 26; the nucleotide sequence of the FgJ02898 gene is shown in SEQ ID NO: 27; the nucleotide sequence of the FgJ02899 gene The acid sequence is shown in SEQ ID NO: 28; the nucleotide sequence of the FgJ02900 gene is shown in SEQ ID NO: 29; the nucleotide sequence of the FgJ02901 gene is shown in SEQ ID NO: 30; the FgJ02902 The nucleotide sequence of the gene is shown in SEQ ID NO:31.