CN114214354A - Double-vector expression system and construction method and application thereof - Google Patents

Abstract

The invention belongs to the field of genetic engineering and fermentation engineering, and particularly relates to a method for quickly constructing an enzyme preparation high-yield strain and a special carrier thereof. The invention constructs an auxiliary vector pUB-toxic and an integrated expression vector pUB-Exp. Cloning target gene into pUB-Exp to obtain recombinant expression plasmid pUB-Enz, transferring auxiliary vector pUB-toxic and recombinant expression plasmid pUB-Enz into host cell successively, raising culture temperature, inducing agent and high concentration antibiotic to induce auxiliary vector pUB-toxic loss and pUB-Enz to integrate into host cell genome and make gene amplification, and screening to obtain high-yield strain of target enzyme preparation. The method provided by the invention can conveniently and quickly obtain the enzyme-producing high-yield strain with good genetic stability and high enzyme-producing level, and can be applied to the high-efficiency preparation and large-scale low-cost production of corresponding enzyme preparations.

Description

Double-vector expression system and construction method and application thereof

The technical field is as follows:

the invention belongs to the field of genetic engineering and fermentation engineering, and particularly relates to a double-vector expression system and application thereof in quickly constructing a high-yield strain of an enzyme preparation.

Background art:

the availability of a particular enzyme preparation usually requires the fermentative production of the strain selected from nature or the cloning of its structural genes followed by heterologous expression in conjunction with appropriate expression elements. Higher enzyme protein yields of the producing strains require a certain number of gene copies. Heterologous protein expression generally takes two forms, one being free expression and the other being integral expression. In previous studies, it was found that appropriate and stable copy number of target genes in host cells has an effect on the yield of target enzyme proteins and even on the physiological characteristics of host bacteria (minium et al, journal of bioengineering, 2009.). Because the copy number of free plasmids in host cells cannot be regulated and the stability is poor, the target gene multicopy enzyme-producing strain constructed by cloning the target gene into a relaxed high-copy free plasmid can not meet the requirements of industrial production generally. Therefore, the way of achieving multiple gene copies and stable integration of the target gene in the host genome is of great significance in the construction of high producing strains.

The construction of multiple copies of the enzyme-encoding gene strain is usually accomplished by sequentially completing the construction of multiple individual copies. For example, waning peak and the like (Chinese patent of invention ZL201610410811.8) successfully integrate a plurality of target genes on a bacillus licheniformis genome through multiple rounds of homologous recombination by adopting a gene homologous recombination traceless integration method, thereby realizing the construction of a high-yield recombinant bacterium; or as in the Watzlawick et al research (Watzlawick H, Altenbuchner J.multiple integration of the gene ganA into the Bacillus subtilis chromosome for enhanced beta-galactosidase production using the CRISPR/Cas9 system AMB Express,2019,9(1):158.DOI:10.1186/s13568-019-0884-4), the construction of the beta-galactosidase multicopy strain is completed by sequentially integrating the coding gene of the beta-galactosidase at a plurality of sites of the Bacillus subtilis REG19 strain genome through a plurality of rounds of genetic operations based on the CRISPR/Cas9 gene editing method; similarly, gene modification methods based on Cre/loxP system, Xer/dif system and FLP/FRT system are used for multi-copy integration expression of target genes on genome. The method for integrating the genes needs to carry out multiple times of genetic operations to realize the construction of the recombinant bacteria carrying the multi-copy target genes, and has complex operation process and long time consumption. Therefore, the method for quickly realizing the integrated expression of multiple copies of the target gene and further improving the efficiency of constructing the high-yield strain has obvious significance.

The invention content is as follows:

the invention aims to establish a high-efficiency method for integrating and expressing target protein by bacillus, so that the efficiency of constructing multi-copy strains in bacillus genomes can be remarkably improved, and high-yield strains of enzyme preparations can be conveniently and quickly obtained.

In order to realize the purpose, the invention constructs a pair of new vectors by a temperature-sensitive starting vector through an in vitro recombination technology to form a double-vector expression system.

One temperature-sensitive vector pUB-toxic carries toxic protein coding gene, the expression of which is strictly controlled by an induced promoter, and a temperature-sensitive replication origin is reserved. Under the condition of raising the culture temperature, the temperature sensitive vector pUB-toxic is slowly lost from the host cell; in the presence of an inducer (such as lactose or IPTG, sucrose, xylose, etc.), toxic proteins lethal to the host cells are expressed, causing the death of host cells incapable of rapidly losing pUB-toxic; the toxic protein coding gene is a type of endonuclease gene widely existing in bacteria.

Another constructed temperature-sensitive vector is pUB-Exp, which carries an expression cassette element, a DNA sequence homologous with the host cell is used as a homology arm, a replicase gene repF in a vector framework is damaged and cannot be replicated in the Bacillus host cell, and the self-replication capability of the vector in the Bacillus host cell can be recovered by sharing the replicase carried by pUB-toxic under the existence condition of the pUB-toxic. The vectors pUB-toxic and pUB-Exp carry different antibiotic selection markers.

The expression plasmid pUB-Enz shares replicase of the auxiliary temperature-sensitive vector pUB-toxic to realize autonomous replication of pUB-Enz in bacillus, in the process, an inducer for inducing expression of toxic protein is added, cells can grow only if the auxiliary plasmid pUB-toxic is lost, at the moment, the expression plasmid pUB-Enz can only be stably integrated into host cell genome because of no replicase, and stable high-copy strains are finally obtained through pressure selection of antibiotics with certain high concentration.

One of the technical schemes provided by the invention is a temperature-sensitive vector pUB-toxic, the vector has a temperature-sensitive replication origin and carries a toxic protein coding gene, and the expression of the toxic protein is strictly controlled by an induced promoter;

furthermore, the construction of the vector pUB-toxic takes a temperature-sensitive replication type escherichia coli-bacillus shuttle plasmid as a framework, and a toxalbumin coding gene toxic expression cassette strictly controlled by an inducer is inserted into a cloning site of the vector pUB-toxic to obtain the vector pUB-toxic;

further, the temperature-sensitive replication-competent Escherichia coli-Bacillus shuttle plasmid may be any Escherichia coli-Bacillus temperature-sensitive shuttle plasmid, including but not limited to pT2^tspNZT1, pKGFP194ts, pKVM1 plasmid, etc.;

further, toxalbumin-encoding Gene toxic is mazF derived from the MazF-mazF system in Escherichia coli K12 (Gene ID:947252), ccdB derived from the CcdA-CcdB system of pEC _ L46 plasmid (Gene ID: 9538168), relE derived from the RelB-RelE system in Escherichia coli K12 (Gene ID: 947549), endoA derived from Bacillus licheniformis self EndoAI-EndoA system (Gene ID: 3097382), or the like;

preferably, the toxalbumin Gene toxic is MazF derived from the MazE-MazF system in escherichia coli K12, and the nucleotide sequence can be determined by querying Gene ID:947252, obtaining;

further, the expression of the toxalbumin gene is controlled by a strict inducible promoter;

still further, the promoters include, but are not limited to, lactose-inducible promoters, sucrose-inducible promoters, xylose-inducible promoters;

preferably, the promoter is lactose-inducible promoter P_lacAs shown in SEQ ID NO.1, the adopted inducer is IPTG or lactose;

preferably, the promoter is sucrose-inducible promoter P_sacAs shown in SEQ ID NO.2, the adopted inducer is sucrose;

preferably, the promoter is xylose inducible promoter P_xylAs shown in SEQ ID NO. 3; the adopted inducer is xylose;

further, the helper vector pUB-toxic also carries a resistance marker including, but not limited to, a kanamycin resistance marker, a tetracycline resistance marker, a gentamicin resistance marker, or an erythromycin resistance marker;

preferably, the temperature-sensitive vector pUB-toxic, in particular to the recombinant plasmid pUB-mazF, is the plasmid pT2^tsThe construction of an expression cassette inserted with a toxic protein coding gene mazF induced by lactose or IPTG is completed;

more preferably, the vector pUB-mazF has a sequence shown in a sequence table SEQ ID NO. 4.

The second technical scheme provided by the invention is a temperature-sensitive integrated expression vector pUB-Exp, wherein the pUB-Exp carries a target gene expression cassette element, a DNA sequence which is homologous with a host cell is used as a homology arm, and a replicase gene repF in a vector skeleton is damaged;

furthermore, the expression cassette element comprises a promoter, a signal peptide, a restriction enzyme site and a termination sequence, and can mediate the expression of a target gene in a host;

because the replicase gene repF in the vector skeleton is damaged, the replicase can not be replicated in the bacillus host cell, and under the existence condition of the vector pUB-toxic, the replicase carried by the pUB-toxic is shared, so that the self-replication capacity of the pUB-Exp in the bacillus host cell can be recovered;

further, the integrated expression vector pUB-Exp also carries a resistance marker;

furthermore, the vectors pUB-toxic and pUB-Exp carry different antibiotic selection markers;

further, a vector framework adopted by the expression vector pUB-Exp is a temperature-sensitive plasmid;

furthermore, the vector framework adopted by the expression vector pUB-Exp is a temperature-sensitive replication-competent Escherichia coli-Bacillus shuttle plasmid, including but not limited to pT2^ts，pNZT1，pKGFP194ts，pKVM1；

Further, the sources of the expression cassette elements (nucleotide sequences such as promoter, signal peptide, cleavage site, termination sequence, etc.) carried by the vector pUB-Exp include, but are not limited to, pHY-WZX, pHT1469(MoBiTec), pWH1520(Rygus and Hillen, 1991);

further, the vector pUB-Exp is the plasmid pT2^tsThe recombinant bacillus licheniformis amylase is obtained by taking a skeleton, destroying a coding gene (GenBank accession number: MN735463.1) of replicase RePF after reverse amplification, and inserting an expression cassette element sequence from an expression vector pHY-WZX and a sequence of about 500bp from the 3' -end of bacillus licheniformis amylase gene as an integration homology arm;

preferably, the vector pUB-Exp has a sequence shown in a sequence table SEQ ID NO. 5.

The third technical scheme of the invention is the application of the temperature-sensitive vector pUB-toxic and the expression vector pUB-Exp, in particular to the application in a bacillus expression system;

furthermore, the bacillus expression system adopts a host of bacillus licheniformis CBB 3008.

The fourth technical scheme provided by the invention is a method for expressing target protein in a bacillus system by adopting the temperature-sensitive vector pUB-toxic and the expression vector pUB-Exp, and comprises the following steps:

(1) constructing a temperature-sensitive vector pUB-toxic and an expression vector pUB-Exp;

(2) cloning a target protein coding gene to be expressed into pUB-Exp to obtain an expression plasmid pUB-Enz;

(3) through genetic transformation, sequentially transforming pUB-Toxic and pUB-Enz into bacillus to form a transformant containing double plasmids;

further, transforming an auxiliary vector pUB-toxic into a host, and forcing the pUB-toxic to be integrated into a genome of the bacillus licheniformis by raising the culture temperature (35-48 ℃) and selecting antibiotics to obtain a recombinant bacterium;

further, the recombinant plasmid pUB-Enz was transferred to a recombinant bacterium having pUB-toxic integrated therein, and the correct transformant was selected on an LB plate containing two antibiotics (double antibody) required for the pair of vectors at 30-35 ℃;

(4) culturing the transformant, and screening out a high-yield strain of the target protein under the conditions of raising the culture temperature and adding an inducer and the screening pressure of high-concentration antibiotics;

further, raising the temperature to 35-48 ℃;

further, the inducer is an inducer for controlling the expression of toxic proteins on pUB-toxic, and includes but is not limited to IPTG, lactose, sucrose or xylose;

further, IPTG was used in the concentration range: 0.1-1 mmol/L;

further, lactose is used in the concentration range: 0.01-3% (w/v);

further, sucrose was used in the concentration range: 0.1-3% (w/v);

further, the xylose use concentration range: 0.1-3% (w/v);

further, the antibiotic is a resistance marker carried on pUB-Exp, including but not limited to kanamycin, tetracycline, gentamicin, erythromycin;

further, kanamycin concentration range: 5-1000 mug/mL;

further, tetracycline concentration range: 3-100 mug/mL;

further, the gentamicin concentration range: 3-500 mug/mL;

furthermore, the concentration range of the erythromycin is 2-500 mug/mL;

(5) producing target enzyme protein by using the strain;

further, the protein of interest may be any protein, in particular an enzyme protein, including but not limited to alpha-amylase, pullulanase, lactase, amylosucrase, and the like;

further, the bacillus includes but is not limited to: bacillus licheniformis, Bacillus amyloliquefaciens, Bacillus subtilis, Bacillus megaterium, Bacillus stearothermophilus, Bacillus thuringiensis and the like;

preferably, the selected host bacterium is bacillus licheniformis;

more preferably, the host bacteria are Bacillus licheniformis CBB3008 and derivative strains thereof, Bacillus licheniformis BCBT0529 (Wangzhengxiang et al, Chinese invention patent, CN112574977A,2020) and the like.

Has the advantages that:

the invention provides a method for quickly constructing a high-yield strain of an enzyme preparation and a special carrier thereof. Constructing a pair of new vectors based on the temperature-sensitive vector and the toxic protein coding gene: the pUB-toxic and pUB-Exp can mediate the rapid, efficient and stable integration of exogenous genes by improving the culture temperature and adding proper antibiotics and inducers to induce the expression of toxic proteins, thereby realizing the rapid construction of enzyme proteins, in particular industrial enzyme preparations such as high-yield strains of alpha-amylase (alpha-amylase), pullulanase (pullulanase), protease (protease) and beta-galactosidase (beta-galactosidase), and improving the construction efficiency of high-yield strains of enzyme.

The problems that temperature-sensitive autonomously replicable plasmids are low in loss efficiency, integrated expression plasmids are synchronously lost from host cells, integrated expression plasmids carrying enzyme coding genes are unstable in integration, and efficient amplification cannot be achieved in genomes in the prior art are solved. The loss efficiency of the temperature-sensitive autonomously replicable plasmid can reach 100 percent, the integration efficiency of the integration expression plasmid carrying the enzyme coding gene can also reach 100 percent, and the stable integration can be realized.

The high-yield strain constructed by the invention can realize the high-efficiency preparation of the enzyme preparation, improve the production efficiency, simplify the production process of the enzyme preparation and reduce the production cost of industrial enzyme preparations.

Description of the drawings:

FIG. 1 physical map of vector

a physical map of pUB-mazF; physical map of pUB-Exp;

FIG. 2 verification results of Bacillus licheniformis CBB3008 amylase encoding gene amyL deleted strain BL-109 and amyS-containing single copy strain BS-109

M: DNA marker; 1: BL-109 strain verification results; 2: the verification result of the BS-109 strain;

FIG. 3 Rapid selection of high producing strains

The arrow indicates a new strain with a higher level of enzyme production;

FIG. 4 evaluation of stability of high-producing New strains

a, amylase activity of the new strain at first passage; b, amylase activity after passage 15 times;

FIG. 5 enzyme production process curve.

The specific implementation mode is as follows:

in order to make the objects, technical solutions and advantages of the present invention more apparent, the following detailed description of the present invention is provided in conjunction with the accompanying drawings. It should be understood that the specific embodiments described herein are merely illustrative of the present patent and are not intended to limit the present invention.

Plasmid pT2 for use in the present invention^tsFor the prior art (shown in SEQ ID NO. 6), the public is also available from the institute of biocatalysis and biotransformation, institute of chemical and materials, Tianjin science and technology university.

The plasmid pHY-WZX used in the invention is the prior art (shown in SEQ ID NO. 7), and can be obtained by the public through a biological catalysis and biological transformation laboratory of chemical and material colleges of Tianjin science and technology university.

The pKSGFP 194ts plasmid used in the present invention is disclosed in the Chinese patent ZL 201610410811.8.

The strain Bacillus licheniformis CBB3008 adopted by the invention is the prior art, and has been preserved in China center for type culture Collection (CCTCC for short), the preservation date is 2008, 11 and 25 days, and the preservation number is CCTCC NO: m208236.

The main experimental method adopted by the invention is as follows:

1. gene cloning, molecular evolution and construction of expression plasmids

Conventional Molecular Cloning procedures (plasmid DNA and genomic DNA extraction, digestion, agarose gel DNA recovery, etc.) were performed according to the literature reference methods (Sambrook et al Molecular Cloning: A Laboratory Manual, 1989).

2. Gene amplification

DNA amplification was performed in 0.2mL PCR thin wall tubes. The PCR amplification conditions were: 11(95 ℃ for 5 min); 301(94 ℃ C. for 10s, 58 ℃ C. for 30s, 72 ℃ C. for 307300 s); 11(72 ℃ C. for 10 min). The extension time of PCR amplification varies depending on the amplification length. PCR amplification uses a high fidelity DNA polymerase, such as Pfu DNA polymerase.

3. Overlapping PCR

The overlap PCR method reference (Krishnan B R, et al, direct and cross PCR amplification to failure Tn 5. sup. pF-based sequencing of lambda phase clones. nucleic Acids Research,1991,22: 6177-. The general steps are as follows: firstly, primers (P1+ P2; P3+ P4, and primers P2 and P3 are reverse complementary sequences) of a fragment F1 and a fragment F2 are used for mediating PCR amplification to respectively obtain two product fragments F1 and F2; gel recovering and purifying the amplified fragments F1 and F2; the two fragments F1 and F2 obtained by purification are diluted by proper times, mixed at a molar ratio of 1:1 and used as a template, and new PCR amplification is mediated by primers P1 and P4 to obtain a full-length sequence fused with the two fragments.

4. Genetic transformation of bacillus

The procedure was carried out with suitable modifications according to the method described in the literature reference (slow sensitivity, horse courser, king's auspicious; effect of high osmotic pressure on the electrical conversion of bacteria, proceedings of Wuxi university of light industry, 2004(04): 98-100). The method mainly comprises the following steps: inoculating fresh single colony into liquid LB culture medium, culturing overnight at 37 deg.C at 200r/min, transferring 5% bacteria liquid into new LB culture medium, and culturing until OD600 is 0.75-0.95. The culture solution is subjected to ice bath for 10min, and then centrifuged at 6000r/min for 10min at 4 ℃ to collect the thallus. The cells were washed repeatedly 4 times with pre-cooled electrotransfer wash solution (0.5mol/L sorbitol, 0.5mol/L mannitol and 10% glycerol). The cell pellet was suspended in 1mL of pre-chilled electrotransfer wash solution to complete the preparation of competent cells. Taking 1 microliter (50ng) of plasmid DNA and about 100 microliter of competent cells, uniformly mixing, immediately shocking by electricity under the electrotransformation condition of 1800v 5ms, immediately adding electrotransformation tempering liquid (LB culture medium containing 0.5mol/L sorbitol and 0.5mol/L mannitol), resuscitating and culturing at 37 ℃ and 160r/min for 3h, then coating a corresponding resistant LB plate, and culturing at a proper temperature until a single colony grows out. The correct transformant is verified by colony PCR verification, plasmid extraction and enzyme digestion, fermentation verification functions and other methods.

5. Multi-copy strain construction and screening process

1) Transforming the auxiliary vector pUB-toxic into a bacillus host, and then increasing the culture temperature (35-48 ℃) and selecting by antibiotics (resistance markers carried on the pUB-toxic) to force the pUB-toxic to be integrated into a bacillus licheniformis genome to obtain a recombinant bacterium BL-UBT;

2) cloning a coding gene of a target enzyme protein to be expressed into a restriction enzyme site of pUB-Exp to obtain a recombinant plasmid pUB-Enz;

3) transferring the recombinant plasmid pUB-Enz into the Bacillus licheniformis BL-UBT obtained in the step 1), and screening a correct transformant BL-Enz on an LB (lysogeny broth) plate which is at 30735 ℃ and contains two antibiotics (double antibodies) required by the pair of vectors;

4) subjecting BL-enz obtained in step 3) to subculture in liquid LB culture medium, passing 1 generation every 6-12h, passing 4-16 generations, and adding antibiotics with proper concentration, wherein the antibiotics are required by integrating expression vector pUB-Exp and inducer with corresponding type and concentration, and inducing expression of toxic protein coding gene, and the culture condition is 35748 ℃ and 50-300 r/min;

5) after the culture of each generation 1 is properly diluted, the culture is spread on an LB plate without resistance, colonies obtained from the plate are respectively spotted on the LB plate with proper concentration required by the corresponding two vectors pUB-toxic and pUB-Enz one by one, colonies which do not grow on the antibiotic plate required by the corresponding vector pUB-toxic but grow on the antibiotic plate required by the pUB-Enz are selected, and the stability and the enzyme production level of the screened strain are further verified. And (5) passage is carried out until strains with stable and high yield of the target protein are successfully screened out.

6. Fermentation test

The shake flask fermentation medium consists of: lactose 174%, cottonseed powder 073.5%, soybean meal 073%, ammonium sulfate 0.173%, balance water, initial pH 678.

The fermentation medium of the fermentation tank comprises the following components: 175 percent of malt syrup, 075 percent of cottonseed meal, 0 to 4 percent of corn steep liquor, 0.575 percent of bean cake meal, 0.175 percent of ammonium sulfate and the balance of water, and the pH value is 6.078.0; fermenting in 50L710 t fermenter with inoculum size of 5-10%; in the fermentation process, the fermentation temperature is 33745 ℃, the dissolved oxygen is controlled to be 0.1720 percent, the pH value is 6.077.8, 30760 percent (w/v) of maltose syrup is fed-batch, and the content of reducing sugar is maintained to be 0.1-5 percent.

7. Enzyme activity assay

The enzyme activities of alpha-amylase, pullulanase and the like are measured according to the related national industry standard GB 1886.174-2016.

The protease activity is determined according to the national standard method (GB/T23527) -2009).

The enzyme activity of the beta-galactosidase is determined according to the national standard GB/T33409-2016.

The general method for measuring the activity of the amylosucrase comprises the following steps: preparing 0.1mol/L sucrose solution with 50mmol/L Tris-HCl pH 7.0; preheating 1.5mL of sucrose solution in a test tube at 35 ℃ for 5min, adding 500 mu L of enzyme solution to be tested, and reacting at 35 ℃ for 30 min; adding 3mL of DNS to terminate the reaction; boiling in water bath for 10min, adding 10mL of deionized water into the test tube, and measuring the light absorption value at 550 nm; the control group is treated by reacting at 35 ℃ for 30min without enzyme solution, adding 3mL DNS and 500 μ L enzyme solution to be detected, boiling water bath for 10min, adding 10mL deionized water into the test tube, and measuring the light absorption value at 550 nm. The activity of the amylosucrase enzyme is defined as that the enzyme amount required by catalyzing and generating 1 mu moL of reducing sugar per minute by using 0.1moL/L of sucrose as a substrate under the conditions of 35 ℃ and pH 7.0 is 1U.

8. Genetic stability test

The genetic stability verification of the recombinant bacteria and the enzyme production stability verification of the recombinant bacteria are carried out according to the following methods: the culture solution of the recombinant bacteria is subjected to subculture in an anti-LB-free liquid culture medium, the subculture is carried out once every 6-12h, the culture conditions are 30-48 ℃, 100-300r/min, and 10-18 generations are carried out. And synchronously using the culture solution as seed solution to perform shake flask fermentation experiments at the same time of each passage, and verifying the change of the enzyme production level of the recombinant bacteria along with the passage times.

9. Other methods of analysis

Protein content was determined according to the literature method (Bradford. anal Chem,1976), glucose content was determined by the enzyme electrode method (SBA-90, Shandong) and cell density was determined by a spectrophotometer (UV-2000, USA) at 600 nm.

The invention will be further explained below by means of specific examples.

Example 1: preparation of mazF expression cassette carrying lactose-induced promoter control

A method for constructing and preparing an expression cassette carrying a lactose inducible promoter mazF. 1) Using a primer mazF-F/mazF-R (SEQ ID NO.8/9) to amplify from Escherichia coli K12 genomic DNA to obtain a mazF gene fragment and adding a termination sequence through the primer mazF-R; 2) amplification of the lactose promoter P from the Bacillus licheniformis CBB3008 genomic DNA template with the primer lacZ-F/lacZ-R (SEQ ID NO.10/11)_lac(SEQ ID NO. 1); using the obtained mixed solution of two DNA fragments with the molar ratio of 1:1 as a template, using primers lacZ-F and mazF-R to mediate overlapping PCR amplification, and finally obtaining the lactose-induced promoter P_lacThe sequence of the induced mazF expression cassette (SEQ ID NO.12) was carefully controlled.

Similarly, other toxalbumin gene expression cassettes under the control of promoters, such as the lactose-inducible promoter P, can be constructed in the same manner as described above_lac(SEQ ID NO.1), or a sucrose-inducible promoter P_sac(SEQ ID NO.2), or xylose-inducible promoter P_xyl(SEQ ID NO.3) with mazF of the MazE-MazF system in Escherichia coli K12 (GenBank accession: Gene ID:947252), or ccdB of the CcdA-CcdB system derived from the pEC _ L46 plasmid (Gene ID: 9538168), or from Escherichia coli K12relE (Gene ID: 947549) of the RelB-RelE system, endoA (Gene ID: 3097382) of the EndoAI-EndoA system of Bacillus licheniformis, and the like. The construction mode refers to the scheme, and corresponding primers are replaced according to actual conditions.

Example 2: construction of an auxiliary vector pUB-mazF and an integration expression vector pUB-Exp, functional evaluation 1 and construction of the vector pUB-mazF.

First the starting plasmid pT2 was replaced^tsThe kanamycin (Km) resistance marker of (1) is the tetracycline (Tet) resistance marker: removing Km resistance marker sequence by primer Tet-F and Tet-R (SEQ ID NO.13/14) mediated reverse PCR method, connecting obtained PCR product with Tet resistance marker sequence (GenBank accession number: D00946.1) obtained by amplification from plasmid pHY300PLK by primer Km-UpF/Km-DnR (SEQ ID NO.15/16) to obtain plasmid pUB-Tet; then, the lactose-inducible promoter P_lacThe sequence of the expression cassette of the regulated mazF (SEQ ID NO.12) is inserted into the SmaI site of the plasmid pUB-Tet, and finally the auxiliary plasmid pUB-mazF (shown in SEQ ID NO.4 and shown in figure 1a) which can be autonomously replicated in bacillus and carries the endonuclease gene mazF is obtained.

2. An integrated expression vector pUB-Exp was constructed.

The starting plasmid pT2 was first disrupted by PCR amplification with the primers repF-UpF/repF-DnR (SEQ ID NO.17/18)^tsThe obtained fragment is cyclized and connected by self to obtain an intermediate plasmid pUB-sint which can not be autonomously replicated in bacillus; then, a fragment amyL '(SEQ ID NO.21) with the size of about 0.5kb upstream of the 3' -end of the amylase gene amyL is amplified from the Bacillus licheniformis CBB3008 genomic DNA sequence by using a primer 3-amyL-F/3-amyL-R (SEQ ID NO.19/20), is cut by BamHI and inserted into the BamHI site of the intermediate plasmid pUB-sint to be used as an integration homology arm; meanwhile, the expression cassette of pHY-WZX is amplified by a primer pUBB-I (SEQ ID NO.22) and a primer pUBB-II (SEQ ID NO.23), digested by AvrII and inserted into SmaI/XbaI site of pUB-sint, and the construction of an integrated expression vector pUB-Exp which can not autonomously replicate in bacillus is completed (shown in SEQ ID NO.5 and shown in figure 1 b).

3. The loss efficiency of the helper vector pUB-mazF and the integration efficiency of the integrated expression vector pUB-Exp were verified.

1) And transforming the auxiliary vector pUB-mazF into the Bacillus licheniformis BL-109, and integrating the auxiliary vector pUB-mazF into a BL-109 genome in a random integration manner by the recombinant bacteria carrying the vector pUB-mazF through raising the culture temperature to 48 ℃ and adding 2 mu g/mL tetracycline into the culture medium to obtain the recombinant bacteria BL-UBM. Wherein BL-109 is a mutant strain of Bacillus licheniformis CBB3008 genome with deleted amylase gene amyL (for BL-109 construction details see example 3).

2) After the newly constructed auxiliary vector pUB-mazF is transformed into Bacillus licheniformis BL-109, subculture is carried out under two conditions of increasing the temperature (48 ℃) and adding 1mmol/L IPTG, and then the loss efficiency of the auxiliary vector pUB-mazF is verified.

The strain is subcultured in a non-anti LB medium for 4 times only by a culture mode of increasing the temperature (48 ℃), the strain liquid is diluted and then coated on a non-anti LB plate, and after the strain liquid is cultured at 30 ℃, 100 colonies are selected to be spotted on a plate with the tetracycline concentration of 2 mu g/mL, and the result is shown in Table 1, 97 colonies can grow on the tetracycline plate, which indicates that the loss of the auxiliary carrier is little.

② subculture is carried out under the culture condition that 1mmol/L IPTG is added into the culture medium while the culture temperature is increased (48 ℃), and the rest conditions are the same as the first step. The colony spotted tetracycline plate grown from the non-resistant plate almost grows in a sterile colony, which shows that the loss rate of the auxiliary carrier can reach nearly 100 percent.

And (3) transforming the integrated expression vector pUB-Exp into the recombinant Bacillus licheniformis BL-UBM containing the auxiliary vector pUB-mazF in the step 1) by electric shock, and screening on an LB plate containing kanamycin and tetracycline at 35 ℃ to obtain a correct transformant.

And (3) respectively carrying out subculture on positive transformants under two conditions of increasing the temperature (48 ℃) and adding 200 mu g/mL kanamycin into the culture medium, and combining increasing the temperature with adding 1mmol/L IPTG and 200 mu g/mL kanamycin to verify the integration efficiency of pUB-Exp. The bacterial liquid obtained by culture is firstly coated on an antibiotic-free plate, and then an LB resistance plate with tetracycline concentration of 2 mu g/mL and an LB resistance plate with kanamycin concentration of 200 mu g/mL are respectively inoculated after bacterial colonies grow. The results show (table 1):

only by raising the temperature and adding 200. mu.g/mL kanamycin, there was also a large number of colonies growing on both resistant plates, indicating that a larger proportion of helper vector was still present in the cells. When 1mmol/L IPTG and 200. mu.g/mL kanamycin were added to the medium while increasing the temperature, the colony seeding results (Table 1) showed that colonies grew aseptically on tetracycline resistant plates; the growth of 100 colonies can be seen on the kanamycin plate, which shows that the integration efficiency of the integration vector can be improved while the loss efficiency of the auxiliary vector is obviously improved under the condition, and both the integration efficiency and the loss efficiency reach 100 percent, thereby being beneficial to the successful construction of high-copy strains.

TABLE 1 loss and integration of plasmids pUB-mazF and pUB-Exp under different culture conditions

Example 3: construction of Strain BL-109 and Strain BS-109

The construction method of the single-copy strain is completed by two homologous recombinations mediated by temperature-sensitive plasmids, and is described in a specific operation reference (Vanryi. Bacillus licheniformis is modified by a genetic engineering technology to realize the high-efficiency expression of the medium-temperature alpha-amylase [ D ]. university of south Jiangnan, 2014.). Briefly described as follows:

using Bacillus licheniformis CBB3008 genome DNA as a template, and using amyL-up1(SEQ ID NO.24), amyL-up2(SEQ ID NO.25), amyL-dn1(SEQ ID NO.26) and amyL-dn2(SEQ ID NO.27) as primers, respectively amplifying the upper and lower homologous arm fragments to obtain PCR products with correct sizes, respectively purifying by using a gel recovery method, and performing overlapping PCR by using the gel recovery product DNA as a template to obtain a deletion mutation box fragment, namely delta amyL. The mutation cassette was purified, digested with XbaI, and cloned into plasmid pT2^tsIn the SmaI and XbaI sites of (4), Escherichia coli JM109 competent cells were transformed, cultured on an LB plate containing 20. mu.g/mL of kanamycin, and screened to obtain the correct recombinant plasmid pT 2-. DELTA.amyL. According to the procedure described in the method for genetic transformation of Bacillus licheniformisAnd carrying out two times of homologous recombination after transforming the plasmid pT 2-delta amyL to the Bacillus licheniformis CBB3008 to complete the deletion of the amylase gene amyL, verifying by primers amyL-F (SEQ ID NO.28) and amyL-R (SEQ ID NO.29) designed at two sides of a homologous arm to correctly construct a strain with the size of 1120bp (figure 2), and obtaining a recombinant bacterium which deletes the amylase gene amyL on the genome of the Bacillus licheniformis CBB3008 and is named as Bacillus licheniformis BL-109.

The amyS expression cassette fragment from recombinant plasmid pUB-amyS (described in detail below) was amplified using 5' -phosphorylated primers pUPB-I and pUPB-III (SEQ ID NO.30) and cloned into the SmaI site of plasmid pT2- Δ amyL, intermediate the two homologous arm fragments. The correct plasmid was verified and was designated pT2-amyL:: amyS. amyS is transformed into a Bacillus licheniformis host cell BL-109, a recombinant bacterium for knocking out amyL and simultaneously expressing amyS is obtained after two homologous recombinations, primers amyL-F (SEQ ID NO.28) and amyL-R (SEQ ID NO.29) on both sides of a homologous arm are used for colony PCR, and the verification of a transformant is carried out, wherein the size of the correct transformant is 2919bp (figure 2). The correct strain was verified and named BS-109.

Example 4: obtaining multicopy integration of strains

The process for constructing a high-temperature resistant alpha-amylase high-producing strain based on the vectors pUB-mazF and pUB-Exp constructed in example 2 is as follows:

1) transforming the auxiliary vector pUB-mazF into Bacillus licheniformis BL-109, and integrating the auxiliary vector pUB-mazF into a BL-109 genome in a random integration manner by culturing the recombinant bacteria carrying the vector pUB-mazF in an LB liquid culture medium added with 2 mu g/mL tetracycline under the condition of raising the culture temperature to 48 ℃ and 200r/min to obtain recombinant bacteria BL-UBM; 2) inserting the gene amyS (GenBank accession number: HV536604.1) of G.stearothermophilus coding high-temperature resistant alpha-amylase into XbaI/SmaI enzyme cutting sites of a vector pUB-Exp after amplifying G.stearothermophilus genome DNA by using primers amyS-F/amyS-R (SEQ ID NO.31/32) to obtain a recombinant plasmid pUB-amyS; 3) plasmid pUB-amyS was transformed into Bacillus licheniformis BL-UBM. Transformants were selected on starch plates (LB plates containing 1% starch) containing 2. mu.g/mL tetracycline and 20. mu.g/mL kanamycin, cultured at 30 ℃ and the correct transformants were designated BL-amyS; 4) culturing BL-amyS in liquid LB culture medium, adding kanamycin and IPTG to make the final concentration 500 mug/mL and 1mmol/L respectively, carrying out passage for 1 time every 12h for 4 generations, and culturing at 48 ℃ and 200 r/min; 5) the culture is diluted by a proper time and coated on a starch plate without resistance, and bacterial colonies form hydrolysis rings with different sizes on the starch plate (figure 3); 6) 10 of the transformants were selected by selecting strains having a large starch hydrolysis loop and incapable of growing in a medium containing 2. mu.g/mL tetracycline, and named as BLi-1S, BLi-2S, BLi-3S, BLi-4S, BLi-5S, BLi-6S, BLi-7S, BLi-8S, BLi-9S, BLi-10S, respectively.

The recombinant bacteria obtained by screening are verified by a shake flask fermentation experiment. The shake flask fermentation is carried out at 37 ℃ and 220r/min, and the components of the culture medium are as follows: maltose syrup 4%, cotton seed powder 1.0%, bean cake powder 1.5%, ammonium sulfate 0.3%, and water in balance, and the initial pH is 7.0.

The control was made with a single copy of amyS, recombinant B.licheniformis BS-109, and the results showed (Table 2) that strain BLi-3S had the highest enzyme production level of 1746. + -.27U/mL, which was about 4.3 times the enzyme production level of the single copy strain (406. + -.17U/mL).

TABLE 2 recombinant bacteria shake flask fermentation enzyme production results

Selecting a new construction strain BLi-3S to verify the genetic stability: the enzyme production level in shake flask fermentation did not change much during 15 passages (passage culture conditions: culture in LB non-resistant liquid medium at 37 ℃ at 200r/min, passage culture every 12h, co-passage culture 15.) (Table 3). Wherein, the 15 th shake flask fermentation level is 1750 +/-35U/mL, which is close to the enzyme production level 1746 +/-27U/mL of the first passage strain. Meanwhile, the first passage bacterial liquid and the 15 th passage bacterial liquid are taken and coated on an LB plate containing 2% starch, and bacterial colonies obtained from the step shown in figure 4 all have starch hydrolysis rings, so that the stability of the obtained recombinant bacteria is further illustrated.

TABLE 3 enzyme production data in Shake flasks during 15 passages of BLi-3S

And selecting a newly constructed strain BLi-3S to carry out an amplification fermentation experiment to verify the enzyme production level. The fermentation was carried out in a 10t fermenter, with an inoculum size of 5%, the fermentation medium composition being: 4% of maltose syrup, 2.5% of cottonseed meal, 2% of corn steep liquor, 3.5% of bean cake meal, 0.5% of ammonium sulfate and the balance of water, wherein the pH value is 6.5; in the fermentation process, the fermentation temperature is 45 ℃, the dissolved oxygen is controlled to be more than 20 percent, the pH value is 7.0, and 60 percent (w/v) of maltose syrup is fed-batch while the content of reducing sugar is maintained at 5 percent. Sampling at fixed time to analyze the enzyme activity, and stopping fermentation when the increase speed of the enzyme activity is lower than 5U/(mL & h). A typical enzyme production process curve of the high-temperature resistant alpha-amylase is shown in figure 5, and the enzyme activity of the high-temperature resistant alpha-amylase reaches 120,828U/mL at most.

Example 5: the method is used for constructing high-yield strains for producing other enzyme preparations

Similarly, the method of the embodiment 1-4 is utilized to realize the high-efficiency expression of enzyme preparations such as mesophilic alpha-amylase, pullulanase and beta-galactosidase in the bacillus licheniformis BL-109 (host cells are bacillus licheniformis BCBT0529, king zhengxiang, Chinese invention patent CN112574977A,2020), neutral protease, alkaline protease, amylosucrase and the like in bacillus. The expression levels of several industrial enzyme preparations are shown in Table 4, and it can be seen that by using the method disclosed in the patent, a plurality of high-yield strains of important enzyme preparations can be rapidly constructed.

TABLE 4 enzyme production levels of other enzyme preparations using this method

The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the patent. It should be noted that, for those skilled in the art, various changes, combinations and improvements can be made in the above embodiments without departing from the patent concept, and all of them belong to the protection scope of the patent. Therefore, the protection scope of this patent shall be subject to the claims.

SEQUENCE LISTING

<110> Tianjin science and technology university

<120> double-vector expression system and construction method and application thereof

<130> 1

<160> 32

<170> PatentIn version 3.5

<210> 1

<211> 280

<212> DNA

<213> Bacillus licheniformis ATCC14580

<400> 1

ctcaaaacct ctctccctgt aaatcgttgc tttaatcaat tataataaat tagttgattt 60

agtcaagtgt atggaaataa agttaaaaat gttaatgata gaatatattt tacaaataaa 120

gaaagagaaa ttcaatcgta caggaaaatt catccacctt ggcaggaaag agggaagaaa 180

acagagtata taatctaaca acctgttttt gaaagcgttt ttagtgtcat gtccgtctcc 240

gcgtcatatg aaaacgagga agaaaggatg aaactgtagg 280

<210> 2

<211> 349

<212> DNA

<213> Bacillus licheniformis ATCC14580

<400> 2

cgattcccgc ttatacagac tatagattca tataaaaaag gtgtctttcc gcttaaaatc 60

ggtgtcattt gcataaaaat gtataggaaa agaggaactt agaccagttg agtcccagag 120

atgagtatat tagaatgatg gtaattcaat atcgtcggga ttgttactgt ctaagcaggc 180

aagacctaaa atgtgtgaag ggcgaaatct atcttttgcc tatatgaact tgcacatttt 240

aggtcttttt tctgttcttc aggacaatgc cgcagttagg agggatgatt gggaacgttc 300

atgttgtcag aagcaatcta ttacatattg aaaaagggag gaaatattg 349

<210> 3

<211> 191

<212> DNA

<213> Bacillus licheniformis ATCC14580

<400> 3

tgtcaatcac tccattgctt tgaagctgtg aatttattat agtataacaa attttgaaaa 60

cttatttttt cctctatttc cattgaaagc gattaattga tcctgtaaaa tacatacaag 120

gaagttagtt taatggttaa acaaacattg ttttttaacg tttgcaagga aaagtgaagg 180

gggagatcgg a 191

<210> 4

<211> 5676

<212> DNA

<213> Artificial sequence

<400> 4

ggtaccgggc cccccctcga ggtcgacggt atcgatacaa attcctcgta ggcgctcggg 60

acccctatct agcgaacttt tagaaaagat ataaaacatc agagtatgga cagttgcgga 120

tgtacttcag aaaagattag atgtctaaaa agcttgtagt taaagctttt tagacatcta 180

aatctaggta aaggatcaat tttgaactct ctcccaaagt tgatccctta acgatttaga 240

aatccctttg agaatgttta tatacattca aggtaaccag ccaactaatg acaatgattc 300

ctgaaaaaag taataacaaa ttactataca gataagttga ctgatcaact tccataggta 360

acaacctttg atcaagtaag ggtatggata ataaaccacc tacaattgca atacctgttc 420

cctctgataa aaagctggta aagttaagca aactcattcc agcaccagct tcctgctgtt 480

tcaagctact tgaaacaatt gttgatataa ctgttttggt gaacgaaagc ccacctaaaa 540

caaatacgat tataattgtc atgaaccatg atgttgtttc taaaagaaag gaagcagtta 600

aaaagctaac agaaagaaat gtaactccga tgtttaacac gtataaagga cctcttctat 660

caacaagtat cccaccaatg tagccgaaaa taatgacact cattgttcca gggaaaataa 720

ttacacttcc gatttcggca gtacttagct ggtgaacatc tttcatcata taaggaacca 780

tagagacaaa ccctgctact gttccaaata taattccccc acaaagaact ccaatcataa 840

aaggtatatt tttccctaat ccgggatcaa caaaaggatc tgttactttc ctgatatgtt 900

ttacaaatat caggaatgac agcacgctaa cgataagaaa agaaatgcta tatgatgttg 960

taaacaacat aaaaaataca atgcctacag acattagtat aattcctttg atatcaaaat 1020

gaccttttat ccttacttct ttctttaata atttcataag aaacggaaca gtgataattg 1080

ttatcatagg aatgagtaga agataggacc aatgaatata atgggctatc attccaccaa 1140

tcgctggacc gactccttct cccatggcta ctatcgatcc aataagacca aatgctttac 1200

ccctattttc ctttggaata tagcgcgcaa ctacaaccat tacgagtgct ggaaatgcag 1260

ctgcaccagc cccttgaata aaacgagcca taataagtaa ggaaaagaaa gaatggccaa 1320

caaacccaat taccgacccg aaacaattta ttataattcc aaataggagt aaccttttga 1380

tgcctaattg atcagatagc tttccatata cagctgttcc aatggaaaag gttaacataa 1440

aggctgtgtt cacccagttt gtactcgcag gtggtttatt aaaatcattt gcaatatcag 1500

gtaatgagac gttcaaaacc atttcattta atacgctaaa aaaagataaa atgcaaagcc 1560

aaattaaaat ttggttgtgt cgtaaattcg attgtgaata ggatgtattc acatttcacc 1620

ctccaataat gagggcagac gtagtttata gggttaatga tacgcttccc tcttttaatt 1680

gaaccctgtt acattcatta ttcattacac ttcataatta attcctccta aacttgatta 1740

aaacatttta ccacatataa actaagtttt aaattcagta tttcatctta ttatttcctt 1800

cctcttttct acagtattta aagatacccc aagaagctaa ttataacaag acgaactcca 1860

attcactgtt ccttgcattc taaaacctta aataccagaa aacagctttt tcaaagttgt 1920

tttcaaagtt ggcgtataac atagtatcga cggagccgat tttgaaacca caattatgat 1980

agaatttaca agctataagg ttattgtcct gggtttcaag cattagtcca tgcaagtttt 2040

tatgctttgc ccattctata gatatattga taagcgcgct gcctatgcct tgccccctga 2100

aatccttaca tacggcgata tcttctatat aaaagatata ttatcttatc agtattgtca 2160

atatattcaa ggcaatctgc ctcctcatcc tcttcatcct cttcgtcttg gtagcttttt 2220

aaatatggcg cttcatagag taattctgta aaggtccaat tctcgttttc atacctcggt 2280

ataatcttac ctatcacctc aaatggttcg ctgggtttat cgataagctt gatatcgaat 2340

tcgatcagta caagaaagat actgtatttc ataaacagga actgcaagaa gttaaggatg 2400

agttacagaa ggcaaataag cagttacaga gtggaataga gcatatgagg tctacgaaac 2460

cctttgatta tgaaaatgag cgtacaggtt tgttctctgg acgtgaagag actggtagaa 2520

agatattaac tgctgatgaa tttgaacgcc tgcaagaaac aatctcttct gcagaacgga 2580

ttgttgatga ttacgaaaat attaagagca cagactatta cacagaaaat caagaattaa 2640

aaaaacgtag agagagtttg aaagaagtag tgaatacatg gaaagagggg tatcacgaaa 2700

aaagtaaaga ggttaataaa ttaaagcgag agaatgatag tttgaatgag cagttgaatg 2760

tatcagagaa atttcaagat agtacagtga ctttatatcg tgctgcgagg gcgaatttcc 2820

ctgggtttga gaaagggttt aataggctta aagagaaatt ctttaatgat tccaaattcg 2880

agcgtgtggg acagtttatg gatgttgtac aggataatgt ccagaaggtc gatagaaagc 2940

gtgagaaaca gcgtacagac gatttagaga tgtagaggta cttttatgcc gagaaaactt 3000

tttgcgtgtg acagtcctta aaatatactt agagcgtaag cgaaagtagt agcgacagct 3060

attaactttc ggttgcaaag ctctaggatt tttaatggac gcagcgcatc acacgcaaaa 3120

aggaaattgg aataaatgcg aaatttgaga tgttaattaa agaccttttt gaggtctttt 3180

tttcttagat ttttggggtt atttagggga gaaaacatag gggggtacta cgacctcccc 3240

cctaggtgtc cattgtccat tgtccaaaca aataaataaa tattgggttt ttaatgttaa 3300

aaggttgttt tttatgttaa agtgaaaaaa acagatgttg ggaggtacag tgatggttgt 3360

agatagaaaa gaagagaaaa aagttgctgt tactttaaga cttacaacag aagaaaatga 3420

gatattaaat agaatcaaag aaaaatataa tattagcaaa tcagatgcaa ccggtattct 3480

aataaaaaaa tatgcaaagg aggaatacgg tgcattttaa acaaaaaaag atagacagca 3540

ctggcatgct gcctatctat gactaaattt tgttaaatgt attagcaccg ttattatatc 3600

atgagcgaaa atgtaataaa agaaactgaa aacaagaaaa attcaagagg acgtaattgg 3660

acatttgttt tatatccaga atcagcaaaa gccgagtggt tagagtattt aaaagagtta 3720

cacattcaat ttgtagtgtc tccattacat gatagggata ctgatacaga agataggatg 3780

aaaaaagagc attatcatat tctagtgatg tatgagggta ataaatctta tgaacagata 3840

aaaataatta cagaagaatt gaatgcgact attccgcaga ttgcaggaag tgtgaaaggt 3900

cttgtgagat atatgcttca catggacgat cctaataaat ttaaatatca aaaagaagat 3960

atgatagttt atggcggtgt agatgttgat gaattattaa agaaaacaac aacagataga 4020

tataaattaa ttaaagaaat gattgagttt attgatgaac aaggaatcgt agaatttaag 4080

agtttaatgg attatgcaat gaagtttaag tttgatgatt ggttcccgct tttatgtgat 4140

aactcggcgt atgttattca agaatatata aaatcaaatc ggtataaatc tgaccgatag 4200

attttgaatt taagagtgtc acaagacact cttttttcgc accaacgaaa actggtttaa 4260

gccgactgcg caaaagacat aatcgattca caaaaaatag gcacacgaaa aacaagttaa 4320

gggatgcagt ttatgcatcc cttaacggat cccctcaaaa cctctctccc tgtaaatcgt 4380

tgctttaatc aattataata aattagttga tttagtcaag tgtatggaaa taaagttaaa 4440

aatgttaatg atagaatata ttttacaaat aaagaaagag aaattcaatc gtacaggaaa 4500

attcatccac cttggcagga aagagggaag aaaacagagt atataatcta acaacctgtt 4560

tttgaaagcg tttttagtgt catgtccgtc tccgcgtcat atgaaaacga ggaagaaagg 4620

atgaaactgt aggatggtaa gccgatacgt acccgatatg ggcgatctga tttgggttga 4680

ttttgacccg acaaaaggta gcgagcaagc tggacatcgt ccagctgttg tcctgagtcc 4740

tttcatgtac aacaacaaaa caggtatgtg tctgtgtgtt ccttgtacaa cgcaatcaaa 4800

aggatatccg ttcgaagttg ttttatccgg tcaggaacgt gatggcgtag cgttagctga 4860

tcaggtaaaa agtatcgcct ggcgggcaag aggagcaacg aagaaaggaa cagttgcccc 4920

agaggaatta caactcatta aagccaaaat taacgtactg attgggtagg ggactagttc 4980

tagagcggcc gccaccgcgg tggagctcgt agaaaagatc aaaggatctt cttgagatcc 5040

tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt 5100

ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc 5160

gcagatacca aatactgtcc ttctagtgta gccgtagtta ggccaccact tcaagaactc 5220

tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg 5280

cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg 5340

gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga 5400

actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc 5460

ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg 5520

gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg 5580

atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt 5640

tttacggttc ctggcctttt gctggccttt tgctca 5676

<210> 5

<211> 4792

<212> DNA

<213> Artificial sequence

<400> 5

ggtaccgggc cccccctcga ggtcgacggt atcgatacaa attcctcgta ggcgctcggg 60

acccctatct agcgaacttt tagaaaagat ataaaacatc agagtatgga cagttgcgga 120

tgtacttcag aaaagattag atgtctaaaa agcttgtagt taaagctttt tagacatcta 180

aatctaggta ctaaaacaat tcatccagta aaatataata ttttattttc tcccaatcag 240

gcttgatccc cagtaagtca aaaaatagct cgacatactg ttcttccccg atatcctccc 300

tgatcgaccg gacgcagaag gcaatgtcat accacttgtc cgccctgccg cttctcccaa 360

gatcaataaa gccacttact ttgccatctt tcacaaagat gttgctgtct cccaggtcgc 420

cgtgggaaaa gacaagttcc tcttcgggct tttccgtctt taaaaaatca tacagctcgc 480

gcggatcttt aaatggagtg tcttcttccc agttttcgca atccacatcg gccagatcgt 540

tattcagtaa gtaatccaat tcggctaagc ggctgtctaa gctattcgta tagggacaat 600

ccgatatgtc gatggagtga aagagcctga tgcactccgc atacagctcg ataatctttt 660

cagggctttg ttcatcttca tactcttccg agcaaaggac gccatcggcc tcactcatga 720

gcagattgct ccagccatca tgccgttcaa agtgcaggac ctttggaaca ggcagctttc 780

cttccagcca tagcatcatg tccttttccc gttccacatc ataggtggtc cctttatacc 840

ggctgtccgt catttttaaa tataggtttt cattttctcc caccagctta tataccttag 900

caggagacat tccttccgta tcttttacgc agcggtattt ttcgatcagt tttttcaatt 960

ccggtgatat tctcatttta gccatttatt atttccttcc tcttttctac agtatttaaa 1020

gataccccaa gaagctaatt ataacaagac gaactccaat tcactgttcc ttgcattcta 1080

aaaccttaaa taccagaaaa cagctttttc aaagttgttt tcaaagttgg cgtataacat 1140

agtatcgacg gagccgattt tgaaaccaca attatgatag aatttacaag ctataaggtt 1200

attgtcctgg gtttcaagca ttagtccatg caagttttta tgctttgccc attctataga 1260

tatattgata agcgcgctgc ctatgccttg ccccctgaaa tccttacata cggcgatatc 1320

ttctatataa aagatatatt atcttatcag tattgtcaat atattcaagg caatctgcct 1380

cctcatcctc ttcatcctct tcgtcttggt agctttttaa atatggcgct tcatagagta 1440

attctgtaaa ggtccaattc tcgttttcat acctcggtat aatcttacct atcacctcaa 1500

atggttcgct gggtttatcg ataagcttga tatcgaattc gatcagtaca agaaagatac 1560

tgtatttcat aaacaggaac tgcaagaagt taaggatgag ttacagaagg caaataagca 1620

gttacagagt ggaatagagc atatgaggtc tacgaaaccc tttgattatg aaaatgagcg 1680

tacaggtttg ttctctggac gtgaagagac tggtagaaag atattaactg ctgatgaatt 1740

tgaacgcctg caagaaacaa tctcttctgc agaacggatt gttgatgatt acgaaaatat 1800

taagagcaca gactattaca cagaaaatca agaattaaaa aaacgtagag agagtttgaa 1860

agaagtagtg aatacatgga aagaggggta tcacgaaaaa agtaaagagg ttaataaatt 1920

aaagcgagag aatgatagtt tgaatgagca gttgaatgta tcagagaaat ttcaagatag 1980

tacagtgact ttatatcgtg ctgcgagggc gaatttccct gggtttgaga aagggtttaa 2040

taggcttaaa gagaaattct ttaatgattc caaattcgag cgtgtgggac agtttatgga 2100

tgttgtacag gataatgtcc agaaggtcga tagaaagcgt gagaaacagc gtacagacga 2160

tttagagatg tagaggtact tttatgccga gaaaactttt tgcgtgtgac agtccttaaa 2220

atatacttag agcgtaagcg aaagtagtag cgacagctat taactttcgg ttgcaaagct 2280

ctaggatttt taatggacgc agcgcatcac acgcaaaaag gaaattggaa taaatgcgaa 2340

atttgagatg ttaattaaag acctttttga ggtctttttt tcttagattt ttggggttat 2400

ttaggggaga aaacataggg gggtactacg acctcccccc taggtgtcca ttgtccattg 2460

tccaaacaaa taaataaata ttgggttttt aatgttaaaa ggttgttttt tatgttaaag 2520

tgaaaaaaac agatgttggg aggtacagtg atggttgtag atagaaaaga agagaaaaaa 2580

gttgctgtta ctttaagact tacaacagaa gaaaatgaga tattaaatag aatcaaagaa 2640

aaatataata ttagcaaatc agatgcaacc ggtattctaa taaaaaaata tgcaaaggag 2700

gaatacggtg cattttaaac aaaaaaagat agacagcact ggcatgctgc ctatctatga 2760

ctaaattttg ttaaatgtat tagcaccgtt attatatcat gagcgaaaat gtaataaaag 2820

aaactgaaaa caagaaaaat tcaagaggac gtaattggac atttgtttta tatccagaat 2880

cagcaaaagc cgagtggtta gagtatttaa aagagttaca cattcaattt gtagtgtctc 2940

cattacatga tagggatact gatacagaag ataggagtga taactcggcg tatgttattc 3000

aagaatatat aaaatcaaat cggtataaat ctgaccgata gattttgaat ttaagagtgt 3060

cacaagacac tcttttttcg caccaacgaa aactggttta agccgactgc gcaaaagaca 3120

taatcgattc acaaaaaata ggcacacgaa aaacaagtta agggatgcag tttatgcatc 3180

ccttaacgga tccgagtttc acgtaaacgg cgggtcggtt tcaatttatg ttcaaagata 3240

gaagagcaga gaggacggat ttcctgaagg aaatccgttt ttttattttg cccgtcttat 3300

aaatttcttt gattacattt tataattaat tttaacaaag tgtcatcagc cctcaggaag 3360

gacttgctga cagtttgaat cgcataggta aggcggggat gaaatggcaa cgttatctga 3420

tgtagcaaag aaagcaaatg tgtcgaaaat gacggtatcg cgggtgatca atcatcctga 3480

gactgtgacg gatgaattga aaaagcttgt tcattccgca atgaaggagc tcaattatat 3540

accgaactat gcagcaagag cgctcgttca aaacagaaca caggtcgtca agctgctcat 3600

actggaagaa atggatacaa cagaacctta ttatatgaat ctgttaacgg gaatcagccg 3660

cgagctggac cgtcatcatt atgctttgcg gatccctcca ttggtaactg tatctcagct 3720

tgaagaagtg aagaagcaga gaggctattg aataaatgag tagaaagcgc catatcggcg 3780

cttttctttt ggaagaaaat atagggaaaa tggtacttgt taaaaattcg gaatatttat 3840

acaatatcat atgtttcaca ttgaaagggg aggagaatca tgaaacaaca aaaacggctt 3900

tacgcccgat tgctgacgct gttatttgcg ctcatcttct tgctgcctca ttctgcagca 3960

gcggcggcaa atctagaatt cgagctcccg ggtaccatgg catgctaatt agatagagca 4020

gagaggacgg atttcctgaa ggaaatccgt ttttttattt tgcccgtctt ataagacaag 4080

ggaaaacgca agcgcctaga gcggccgcca ccgcggtgga gctcgtagaa aagatcaaag 4140

gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac 4200

cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa 4260

ctggcttcag cagagcgcag ataccaaata ctgtccttct agtgtagccg tagttaggcc 4320

accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag 4380

tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac 4440

cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc 4500

gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc gccacgcttc 4560

ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca 4620

cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg tttcgccacc 4680

tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg 4740

ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct ca 4792

<210> 6

<211> 4258

<212> DNA

<213> Artificial sequence

<400> 6

ggtaccgggc cccccctcga ggtcgacggt atcgatacaa attcctcgta ggcgctcggg 60

acccctatct agcgaacttt tagaaaagat ataaaacatc agagtatgga cagttgcgga 120

tgtacttcag aaaagattag atgtctaaaa agcttgtagt taaagctttt tagacatcta 180

aatctaggta ctaaaacaat tcatccagta aaatataata ttttattttc tcccaatcag 240

gcttgatccc cagtaagtca aaaaatagct cgacatactg ttcttccccg atatcctccc 300

tgatcgaccg gacgcagaag gcaatgtcat accacttgtc cgccctgccg cttctcccaa 360

gatcaataaa gccacttact ttgccatctt tcacaaagat gttgctgtct cccaggtcgc 420

cgtgggaaaa gacaagttcc tcttcgggct tttccgtctt taaaaaatca tacagctcgc 480

gcggatcttt aaatggagtg tcttcttccc agttttcgca atccacatcg gccagatcgt 540

tattcagtaa gtaatccaat tcggctaagc ggctgtctaa gctattcgta tagggacaat 600

ccgatatgtc gatggagtga aagagcctga tgcactccgc atacagctcg ataatctttt 660

cagggctttg ttcatcttca tactcttccg agcaaaggac gccatcggcc tcactcatga 720

gcagattgct ccagccatca tgccgttcaa agtgcaggac ctttggaaca ggcagctttc 780

cttccagcca tagcatcatg tccttttccc gttccacatc ataggtggtc cctttatacc 840

ggctgtccgt catttttaaa tataggtttt cattttctcc caccagctta tataccttag 900

caggagacat tccttccgta tcttttacgc agcggtattt ttcgatcagt tttttcaatt 960

ccggtgatat tctcatttta gccatttatt atttccttcc tcttttctac agtatttaaa 1020

gataccccaa gaagctaatt ataacaagac gaactccaat tcactgttcc ttgcattcta 1080

aaaccttaaa taccagaaaa cagctttttc aaagttgttt tcaaagttgg cgtataacat 1140

agtatcgacg gagccgattt tgaaaccaca attatgatag aatttacaag ctataaggtt 1200

attgtcctgg gtttcaagca ttagtccatg caagttttta tgctttgccc attctataga 1260

tatattgata agcgcgctgc ctatgccttg ccccctgaaa tccttacata cggcgatatc 1320

ttctatataa aagatatatt atcttatcag tattgtcaat atattcaagg caatctgcct 1380

cctcatcctc ttcatcctct tcgtcttggt agctttttaa atatggcgct tcatagagta 1440

attctgtaaa ggtccaattc tcgttttcat acctcggtat aatcttacct atcacctcaa 1500

atggttcgct gggtttatcg ataagcttga tatcgaattc gatcagtaca agaaagatac 1560

tgtatttcat aaacaggaac tgcaagaagt taaggatgag ttacagaagg caaataagca 1620

gttacagagt ggaatagagc atatgaggtc tacgaaaccc tttgattatg aaaatgagcg 1680

tacaggtttg ttctctggac gtgaagagac tggtagaaag atattaactg ctgatgaatt 1740

tgaacgcctg caagaaacaa tctcttctgc agaacggatt gttgatgatt acgaaaatat 1800

taagagcaca gactattaca cagaaaatca agaattaaaa aaacgtagag agagtttgaa 1860

agaagtagtg aatacatgga aagaggggta tcacgaaaaa agtaaagagg ttaataaatt 1920

aaagcgagag aatgatagtt tgaatgagca gttgaatgta tcagagaaat ttcaagatag 1980

tacagtgact ttatatcgtg ctgcgagggc gaatttccct gggtttgaga aagggtttaa 2040

taggcttaaa gagaaattct ttaatgattc caaattcgag cgtgtgggac agtttatgga 2100

tgttgtacag gataatgtcc agaaggtcga tagaaagcgt gagaaacagc gtacagacga 2160

tttagagatg tagaggtact tttatgccga gaaaactttt tgcgtgtgac agtccttaaa 2220

atatacttag agcgtaagcg aaagtagtag cgacagctat taactttcgg ttgcaaagct 2280

ctaggatttt taatggacgc agcgcatcac acgcaaaaag gaaattggaa taaatgcgaa 2340

atttgagatg ttaattaaag acctttttga ggtctttttt tcttagattt ttggggttat 2400

ttaggggaga aaacataggg gggtactacg acctcccccc taggtgtcca ttgtccattg 2460

tccaaacaaa taaataaata ttgggttttt aatgttaaaa ggttgttttt tatgttaaag 2520

tgaaaaaaac agatgttggg aggtacagtg atggttgtag atagaaaaga agagaaaaaa 2580

gttgctgtta ctttaagact tacaacagaa gaaaatgaga tattaaatag aatcaaagaa 2640

aaatataata ttagcaaatc agatgcaacc ggtattctaa taaaaaaata tgcaaaggag 2700

gaatacggtg cattttaaac aaaaaaagat agacagcact ggcatgctgc ctatctatga 2760

ctaaattttg ttaaatgtat tagcaccgtt attatatcat gagcgaaaat gtaataaaag 2820

aaactgaaaa caagaaaaat tcaagaggac gtaattggac atttgtttta tatccagaat 2880

cagcaaaagc cgagtggtta gagtatttaa aagagttaca cattcaattt gtagtgtctc 2940

cattacatga tagggatact gatacagaag ataggatgaa aaaagagcat tatcatattc 3000

tagtgatgta tgagggtaat aaatcttatg aacagataaa aataattaca gaagaattga 3060

atgcgactat tccgcagatt gcaggaagtg tgaaaggtct tgtgagatat atgcttcaca 3120

tggacgatcc taataaattt aaatatcaaa aagaagatat gatagtttat ggcggtgtag 3180

atgttgatga attattaaag aaaacaacaa cagatagata taaattaatt aaagaaatga 3240

ttgagtttat tgatgaacaa ggaatcgtag aatttaagag tttaatggat tatgcaatga 3300

agtttaagtt tgatgattgg ttcccgcttt tatgtgataa ctcggcgtat gttattcaag 3360

aatatataaa atcaaatcgg tataaatctg accgatagat tttgaattta agagtgtcac 3420

aagacactct tttttcgcac caacgaaaac tggtttaagc cgactgcgca aaagacataa 3480

tcgattcaca aaaaataggc acacgaaaaa caagttaagg gatgcagttt atgcatccct 3540

taacggatcc cgggactagt tctagagcgg ccgccaccgc ggtggagctc gtagaaaaga 3600

tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa 3660

aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga 3720

aggtaactgg cttcagcaga gcgcagatac caaatactgt ccttctagtg tagccgtagt 3780

taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt 3840

taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat 3900

agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct 3960

tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca 4020

cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag 4080

agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc 4140

gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga 4200

aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctca 4258

<210> 7

<211> 6702

<212> DNA

<213> Artificial sequence

<400> 7

aggatccatt ggtaactgta tctcagcttg aagaagtgaa gaagcagaga ggctattgaa 60

taaatgagta gaaagcgcca tatcggcgct tttcttttgg aagaaaatat agggaaaatg 120

gtacttgtta aaaattcgga atatttatac aatatcatat gtttcacatt gaaaggggag 180

gagaatcatg aaacaacaaa aacggcttta cgcccgattg ctgacgctgt tatttgcgct 240

catcttcttg ctgcctcatt ctgcagcagc ggcggcaaat ctagaattcg agctcccggg 300

taccatggca tgctaattag atagagcaga gaggacggat ttcctgaagg aaatccgttt 360

ttttattttg cccgtcttat aagacaaggg aaaacgcaag cgcaaagaga aagcaggtag 420

cttgcagtgg gcttacatgg cgatagctag actgggcggt tttatggaca gcaagcgaac 480

cggaattgcc agctggggcg ccctctggta aggttgggaa gccctgcaaa gtaaactgga 540

tggctttctt gccgccaagg atctgatggc gcaggggatc aagatctgat caagagacag 600

gatgaggatc gtttcgcatg attgaacaag atggattgca cgcaggttct ccggccgctt 660

gggtggagag gctattcggc tatgactggg cacaacagac aatcggctgc tctgatgccg 720

ccgtgttccg gctgtcagcg caggggcgcc cggttctttt tgtcaagacc gacctgtccg 780

gtgccctgaa tgaactgcag gacgaggcag cgcggctatc gtggctggcc acgacgggcg 840

ttccttgcgc agctgtgctc gacgttgtca ctgaagcggg aagggactgg ctgctattgg 900

gcgaagtgcc ggggcaggat ctcctgtcat ctcaccttgc tcctgccgag aaagtatcca 960

tcatggctga tgcaatgcgg cggctgcata cgcttgatcc ggctacctgc ccattcgacc 1020

accaagcgaa acatcgcatc gagcgagcac gtactcggat ggaagccggt cttgtcgatc 1080

aggatgatct ggacgaagag catcaggggc tcgcgccagc cgaactgttc gccaggctca 1140

aggcgcgcat gcccgacggc gaggatctcg tcgtgaccca tggcgatgcc tgcttgccga 1200

atatcatggt ggaaaatggc cgcttttctg gattcatcga ctgtggccgg ctgggtgtgg 1260

cggaccgcta tcaggacata gcgttggcta cccgtgatat tgctgaagag cttggcggcg 1320

aatgggctga ccgcttcctc gtgctttacg gtatcgccgc tcccgattcg cagcgcatcg 1380

ccttctatcg ccttcttgac gagttcttct aataagggga tcttgaagtt cctattccga 1440

agttcctatt ctctagaaag tataggaact tcgaagcagc tccagcctac actggtctta 1500

tgacttgggc gcgctggaaa actatttgaa caaaacaaat tttaatcatt cagtgtttga 1560

cgtgccgctt cattatcagt tccatgctgc atcgacacag ggaggcggct atgatatgag 1620

gaaattgctg aacggtacgg tcgtttccaa gcatccgttg aaatcggtta catttgtcga 1680

taaccatgat acacagccgg ggcaatcgct tgagtcgact gtccaaacat ggtttaagcc 1740

gcttgcttac gcttttattc tcacaaggga atctggatac cctcaggttt tctacgggga 1800

tatgtacggg acgaaaggag actcccagcg cgaaattcct gccttgaaac acaaaattga 1860

acagatctca attcctgtta taaaaaaagg atcaattttg aactctctcc caaagttgat 1920

cccttaacga tttagaaatc cctttgagaa tgtttatata cattcaaggt aaccagccaa 1980

ctaatgacaa tgattcctga aaaaagtaat aacaaattac tatacagata agttgactga 2040

tcaacttcca taggtaacaa cctttgatca agtaagggta tggataataa accacctaca 2100

attgcaatac ctgttccctc tgataaaaag ctggtaaagt taagcaaact cattccagca 2160

ccagcttcct gctgtttcaa gctacttgaa acaattgttg atataactgt tttggtgaac 2220

gaaagcccac ctaaaacaaa tacgattata attgtcatga accatgatgt tgtttctaaa 2280

agaaaggaag cagttaaaaa gctaacagaa agaaatgtaa ctccgatgtt taacacgtat 2340

aaaggacctc ttctatcaac aagtatccca ccaatgtagc cgaaaataat gacactcatt 2400

gttccaggga aaataattac acttccgatt tcggcagtac ttagctggtg aacatctttc 2460

atcatataag gaaccataga gacaaaccct gctactgttc caaatataat tcccccacaa 2520

agaactccaa tcataaaagg tatatttttc cctaatccgg gatcaacaaa aggatctgtt 2580

actttcctga tatgttttac aaatatcagg aatgacagca cgctaacgat aagaaaagaa 2640

atgctatatg atgttgtaaa caacataaaa aatacaatgc ctacagacat tagtataatt 2700

cctttgatat caaaatgacc ttttatcctt acttctttct ttaataattt cataagaaac 2760

ggaacagtga taattgttat cataggaatg agtagaagat aggaccaatg aatataatgg 2820

gctatcattc caccaatcgc tggaccgact ccttctccca tggctactat cgatccaata 2880

agaccaaatg ctttacccct attttccttt ggaatatagc gcgcaactac aaccattacg 2940

agtgctggaa atgcagctgc accagcccct tgaataaaac gagccataat aagtaaggaa 3000

aagaaagaat ggccaacaaa cccaattacc gacccgaaac aatttattat aattccaaat 3060

aggagtaacc ttttgatgcc taattgatca gatagctttc catatacagc tgttccaatg 3120

gaaaaggtta acataaaggc tgtgttcacc cagtttgtac tcgcaggtgg tttattaaaa 3180

tcatttgcaa tatcaggtaa tgagacgttc aaaaccattt catttaatac gctaaaaaaa 3240

gataaaatgc aaagccaaat taaaatttgg ttgtgtcgta aattcgattg tgaataggat 3300

gtattcacat ttcaccctcc aataatgagg gcagacgtag tttatagggt taatgatacg 3360

cttccctctt ttaattgaac cctgttacat tcattattca ttacacttca taattaattc 3420

ctcctaaact tgattaaaac attttaccac atataaacta agttttaaat tcagtatttc 3480

atcacttata caacaatatg gcccgtttgt tgaactactc tttaataaaa taatttttcc 3540

gttcccaatt ccacattgca ataatagaaa atccatcttc atcggctttt tcgtcatcat 3600

ctgtatgaat caaatcgcct tcttctgtgt catcaaggtt taatttttta tgtatttctt 3660

ttaacaaacc accataggag attaaccttt tacggtgtaa accttcctcc aaatcagaca 3720

aacgtttcaa attcttttct tcatcatcgg tcataaaatc cgtatccttt acaggatatt 3780

ttgcagtttc gtcaattgcc gattgtatat ccgatttata tttatttttc ggtcgaatca 3840

tttgaacttt tacatttgga tcatagtcta atttcattgc ctttttccaa aattgaatcc 3900

attgtttttg attcacgtag ttttctgtat tcttaaaata agttggttcc acacatacca 3960

atacatgcat gtgctgatta taagaattat ctttattatt tattgtcact tccgttgcac 4020

gcataaaacc aacaagattt ttattaattt ttttatattg catcattcgg cgaaatcctt 4080

gagccatatc tgacaaactc ttatttaatt cttcgccatc ataaacattt ttaactgtta 4140

atgtgagaaa caaccaacga actgttggct tttgtttaat aacttcagca acaacctttt 4200

gtgactgaat gccatgtttc attgctctcc tccagttgca cattggacaa agcctggatt 4260

tacaaaacca cactcgatac aactttcttt cgcctgtttc acgattttgt ttatactcta 4320

atatttcagc acaatctttt actctttcag cctttttaaa ttcaagaata tgcagaagtt 4380

caaagtaatc aacattagcg attttctttt ctctccatgg tctcactttt ccactttttg 4440

tcttgtccac taaaaccctt gatttttcat ctgaataaat gctactatta ggacacataa 4500

tattaaaaga aacccccatc tatttagtta tttgtttggt cacttataac tttaacagat 4560

ggggtttttc tgtgcaacca attttaaggg ttttccaata ctttaaaaca catacatacc 4620

aacacttcaa cgcacctttc agcaactaaa ataaaaatga cgttatttct atatgtatca 4680

agataagaaa gaacaagttc aaaaccatca aaaaaagaca ccttttcagg tgcttttttt 4740

attttataaa ctcattccct gatctcgact tcgttctttt tttacctctc ggttatgagt 4800

tagttcaaat tcgttctttt taggttctaa atcgtgtttt tcttggaatt gtgctgtttt 4860

atcctttacc ttgtctacaa accccttaaa aacgttttta aaggctttta agcgtctgta 4920

cgttccttaa ggaattattc cttagtgctt tctaggttaa tgtcatgata ataatggttt 4980

cttagacgtc aggtggcact tttcggggaa atgtccgcgg aacccctatt tgtttatttt 5040

tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat 5100

aatattgaaa aaggaagagt atgagtattc aacatttccg tgtcgccctt attccctttt 5160

ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg 5220

ctgaagatca gttgggtgca cgagtgggtt acatcgaact ggatctcaac agcggtaaga 5280

tccttgagag ttttcgcccc gaagaacgtt ttccaatgat gagcactttt aaagttctgc 5340

tatgtggcgc ggtattatcc cgtgttgacg ccgggcaaga gcaactcggt cgccgcatac 5400

actattctca gaatgacttg gttgagtact caccagtcac agaaaagcat cttacggatg 5460

gcatgacagt aagagaatta tgcagtgctg ccataaccat gagtgataac actgcggcca 5520

acttacttct gacaacgatc ggaggaccga aggagctaac cgcttttttg cacaacatgg 5580

gggatcatgt aactcgcctt gatcgttggg aaccggagct gaatgaagcc ataccaaacg 5640

acgagcgtga caccacgatg cctgcagcaa tggcaacaac gttgcgcaaa ctattaactg 5700

gcgaactact tactctagct tcccggcaac aattaataga ctggatggag gcggataaag 5760

ttgcaggacc acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatctg 5820

gagccggtga gcgtgggtct cgcggtatca ttgcagcact ggggccagat ggtaagccct 5880

cccgtatcgt agttatctac acgacgggga gtcaggcaac tatggatgaa cgaaatagac 5940

agatcgctga gataggtgcc tcactgatta agcattggta actgtcagac caagtttact 6000

catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga 6060

tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt 6120

cagacccctt aataagatga tcttcttgag atcgttttgg tctgcgcgta atctcttgct 6180

ctgaaaacga aaaaaccgcc ttgcagggag gtttttcgaa ggttctctga gctaccaact 6240

ctttgaaccg aggtaactgg cttgcaggag cgcagtcacc aaaacttgtc ctttcagttt 6300

agccttaacc ggcgcatgac ttcaagacta actcctctaa atcaattacc agtggctgct 6360

gccagtggtg cttttgcatg tctttccggg ttggactcaa gacgatagtt accggataag 6420

gcgcagcggt cggactgaac ggggggttcg tgcatacagt ccagcttgga gcgaactgcc 6480

tacccggaac tgagtgtcag gcgtggaatg agacaaacgc ggccataaca gcggaatgac 6540

accggtaaac cgaaaggcag gaacaggaga gcgcacgagg gagccgccag ggggaaacgc 6600

ctggtatctt tatagtcctg tcgggtttcg ccaccactga tttgagcgtc agatttcgtg 6660

atgcttgtca gggggcggag cctatggaaa aacgctttgc cc 6702

<210> 8

<211> 49

<212> DNA

<213> Artificial sequence

<400> 8

gaagaaagga tgaaactgta ggatggtaag ccgatacgta cccgatatg 49

<210> 9

<211> 83

<212> DNA

<213> Artificial sequence

<400> 9

gccgacaacg ccatccggag aagccgaatg acgttgtcgg agtaagcgct ggctacccta 60

cccaatcagt acgttaattt tgg 83

<210> 10

<211> 25

<212> DNA

<213> Artificial sequence

<400> 10

ctcaaaacct ctctccctgt aaatc 25

<210> 11

<211> 49

<212> DNA

<213> Artificial sequence

<400> 11

catatcgggt acgtatcggc ttaccatcct acagtttcat cctttcttc 49

<210> 12

<211> 678

<212> DNA

<213> Artificial sequence

<400> 12

ctcaaaacct ctctccctgt aaatcgttgc tttaatcaat tataataaat tagttgattt 60

agtcaagtgt atggaaataa agttaaaaat gttaatgata gaatatattt tacaaataaa 120

gaaagagaaa ttcaatcgta caggaaaatt catccacctt ggcaggaaag agggaagaaa 180

acagagtata taatctaaca acctgttttt gaaagcgttt ttagtgtcat gtccgtctcc 240

gcgtcatatg aaaacgagga agaaaggatg aaactgtagg atggtaagcc gatacgtacc 300

cgatatgggc gatctgattt gggttgattt tgacccgaca aaaggtagcg agcaagctgg 360

acatcgtcca gctgttgtcc tgagtccttt catgtacaac aacaaaacag gtatgtgtct 420

gtgtgttcct tgtacaacgc aatcaaaagg atatccgttc gaagttgttt tatccggtca 480

ggaacgtgat ggcgtagcgt tagctgatca ggtaaaaagt atcgcctggc gggcaagagg 540

agcaacgaag aaaggaacag ttgccccaga ggaattacaa ctcattaaag ccaaaattaa 600

cgtactgatt gggtagggta gggtagccag cgcttactcc gacaacgtca ttcggcttct 660

ccggatggcg ttgtcggc 678

<210> 13

<211> 29

<212> DNA

<213> Artificial sequence

<400> 13

attggatcca ttggtaactg tatctcagc 29

<210> 14

<211> 26

<212> DNA

<213> Artificial sequence

<400> 14

aaccctaggg ttcaattttg tgtttc 26

<210> 15

<211> 36

<212> DNA

<213> Artificial sequence

<400> 15

gatgaaatac tgaatttaaa acttagttta tatgtg 36

<210> 16

<211> 27

<212> DNA

<213> Artificial sequence

<400> 16

aaggatcaat tttgaactct ctcccaa 27

<210> 17

<211> 22

<212> DNA

<213> Artificial sequence

<400> 17

atgtgataac tcggcgtatg tt 22

<210> 18

<211> 26

<212> DNA

<213> Artificial sequence

<400> 18

tcctatcttc tgtatcagta tcccta 26

<210> 19

<211> 27

<212> DNA

<213> Artificial sequence

<400> 19

ccaggatccg agtttcacgt aaacggc 27

<210> 20

<211> 34

<212> DNA

<213> Artificial sequence

<400> 20

tgcggatccg caaagcataa tgatgacggt ccag 34

<210> 21

<211> 496

<212> DNA

<213> Bacillus licheniformis CBB3008

<400> 21

gagtttcacg taaacggcgg gtcggtttca atttatgttc aaagatagaa gagcagagag 60

gacggatttc ctgaaggaaa tccgtttttt tattttgccc gtcttataaa tttctttgat 120

tacattttat aattaatttt aacaaagtgt catcagccct caggaaggac ttgctgacag 180

tttgaatcgc ataggtaagg cggggatgaa atggcaacgt tatctgatgt agcaaagaaa 240

gcaaatgtgt cgaaaatgac ggtatcgcgg gtgatcaatc atcctgagac tgtgacggat 300

gaattgaaaa agcttgttca ttccgcaatg aaggagctca attatatacc gaactatgca 360

gcaagagcgc tcgttcaaaa cagaacacag gtcgtcaagc tgctcatact ggaagaaatg 420

gatacaacag aaccttatta tatgaatctg ttaacgggaa tcagccgcga gctggaccgt 480

catcattatg ctttgc 496

<210> 22

<211> 26

<212> DNA

<213> Artificial Synthesis

<400> 22

tccattggta actgtatctc agcttg 26

<210> 23

<211> 44

<212> DNA

<213> Artificial Synthesis

<400> 23

aggcctaggc cacctaggga agatcctttg atcttttcta cgag 44

<210> 24

<211> 20

<212> DNA

<213> Artificial sequence

<400> 24

ccgaaacgat aaaacggatg 20

<210> 25

<211> 46

<212> DNA

<213> Artificial sequence

<400> 25

ccgtttacgt gaaactctcc acccgggcca ttttccctat attttc 46

<210> 26

<211> 46

<212> DNA

<213> Artificial sequence

<400> 26

gaaaatatag ggaaaatggc ccgggtggag agtttcacgt aaacgg 46

<210> 27

<211> 33

<212> DNA

<213> Artificial sequence

<400> 27

gctctagagc aaagcataat gatgacggtc cag 33

<210> 28

<211> 20

<212> DNA

<213> Artificial sequence

<400> 28

ctttgtgctt ggaagcagag 20

<210> 29

<211> 20

<212> DNA

<213> Artificial sequence

<400> 29

gatgagccct tcaaaatcgg 20

<210> 30

<211> 26

<212> DNA

<213> Artificial sequence

<400> 30

gaagatcctt tgatcttttc tacgag 26

<210> 31

<211> 33

<212> DNA

<213> Artificial sequence

<400> 31

ccatctagag ccgcaccgtt taacggtacc atg 33

<210> 32

<211> 25

<212> DNA

<213> Artificial sequence

<400> 32

aggccatgcc accaaccgtg gttcg 25

Claims (10)

1. A dual-vector expression system is characterized by comprising two temperature-sensitive vectors: pUB-toxic and pUB-Enz;

the pUB-toxic has a temperature-sensitive replication origin and carries a toxic protein coding gene, and the expression of the toxic protein is strictly controlled by an induction promoter;

the pUB-Exp, carrying the gene expression cassette element of interest, a DNA sequence homologous to the host cell and the replicase gene repF in the vector backbone is disrupted.

2. The two-vector expression system of claim 1, wherein the pUB-toxic and pUB-Enz are backbone plasmids that are temperature-sensitive replication-competent escherichia coli-bacillus shuttle plasmids.

3. The dual vector expression system of claim 2, wherein the backbone plasmid of pUB-toxic is pT2^ts，pNZT1, pKGFP194ts or pKVM1 plasmids; the backbone plasmid of pUB-Exp is pT2^tspNZT1, pKGFP194ts or pKVM1 plasmids.

4. The two-vector expression system of claim 1, wherein the toxalbumin-encoding gene is mazF, ccdB, relE or endoA.

5. The two-vector expression system of claim 1, wherein the inducible promoter is a lactose-inducible promoter, a sucrose-inducible promoter, or a xylose-inducible promoter.

6. The two-vector expression system of claim 1, wherein the pUB-toxic and pUB-Exp carry different resistance markers.

7. A two-vector expression system according to claim 1, wherein the pUB-Exp carries expression cassette elements from plasmids pHY-WZX, pHT1469 or pWH 1520.

8. The two-vector expression system of claim 1, wherein the sequence of pUB-toxic is shown as SEQ ID No.4, and the sequence of pUB-Exp is shown as SEQ ID No. 5.

9. Use of a two-vector expression system according to any one of claims 1 to 8 in a bacillus expression system.

10. A method for expressing a target protein in Bacillus by using the dual-vector expression system of any one of claims 1 to 8, which is characterized by comprising the following steps:

(1) constructing a temperature-sensitive vector pUB-toxic and an expression vector pUB-Exp;

(2) cloning a target protein coding gene to be expressed into pUB-Exp to obtain an expression plasmid pUB-Enz;

(3) through genetic transformation, sequentially transforming pUB-Toxic and pUB-Enz into bacillus to form a transformant containing double plasmids;

(4) culturing the transformant, and screening out a high-yield strain of the target protein under the conditions of raising the culture temperature and adding an inducer and the screening pressure of high-concentration antibiotics;

(5) the strain is adopted to produce the target protein.

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