CN107602707B - Dcas 9-omega fusion protein for specifically regulating bacillus subtilis exogenous gene expression and application thereof - Google Patents

Abstract

The invention discloses a dcas 9-omega fusion protein for specifically regulating the expression of a bacillus subtilis exogenous gene and application thereof. The amino acid sequence of the dcas 9-omega fusion protein is shown as SEQ ID NO.1 in the sequence table, the fusion protein can be guided to a specific targeting site and combined with the specific targeting site, and the RNA polymerase omega subunit recruits other subunits of RNA polymerase to assemble into RNA polymerase at the site to promote downstream gene transcription, so that the expression level of a target gene is improved, namely the fusion protein dcas 9-omega retains the DNA binding capacity of dcas9 and the transcription activation capacity of the RNA polymerase subunit, so that the target gene expression is specifically improved.

Description

Dcas 9-omega fusion protein for specifically regulating bacillus subtilis exogenous gene expression and application thereof

Technical Field

The invention belongs to the technical field of genetic engineering, and particularly relates to a dcas 9-omega fusion protein for expressing dcas9 and RNA polymerase subunits, in particular to a dcas 9-omega fusion protein for specifically regulating the expression of a bacillus subtilis exogenous gene and application thereof.

Background

Coli expression system is the most commonly used prokaryotic expression system, however, the expression system still has certain defects, such as that foreign protein is easy to form inclusion body, and endotoxin is produced. In recent years, Bacillus subtilis expression systems have been used by an increasing number of researchers to express proteins of interest. The bacillus subtilis has strong secretion capacity, can directly secrete exogenous protein into a culture medium, not only effectively prevents the protein from forming an inclusion body, but also can greatly simplify the subsequent purification steps and reduce the production cost. The bacillus subtilis does not produce endotoxin such as lipopolysaccharide, belongs to GRAS strains, and can be used for producing related proteins and metabolites such as food, medicines and the like. In addition, the existing method is mature in the genetic manipulation of bacillus subtilis and large-scale fermentation technology. Over the course of many years of effort, many prokaryotic and eukaryotic genes have been cloned and expressed in Bacillus expression systems, some of which have been used in industrial production. However, the protein expressed by using bacillus subtilis as an expression host is mainly derived from a bacillus kindred species, and the expression capability of the protein expressing non-bacillus sources is poor, so that how to improve the expression amount of foreign proteins in the bacillus subtilis is a key problem for further developing the bacillus subtilis as a cell factory and expanding the application range of the bacillus subtilis.

At present, various strategies are used for improving the expression level of foreign proteins, including gene level, transcription level, translation level, post-translational modification and the like, which can be used as modified targets, such as methods for improving gene copy number, ribosome engineering, codon optimization and the like. In prokaryotes, it is the simplest and most effective way to increase the expression level of a target gene by increasing the transcript level of the target gene, for example, by screening high-efficiency promoters through promoter engineering and the like, for increasing the expression level of the target gene. At present, reports of constructing a promoter library and screening high-efficiency promoters in escherichia coli, pichia pastoris, saccharomyces cerevisiae and the like are available. In previous researches, the inventor constructs a promoter library with GFP as a reporter gene in Bacillus subtilis. However, when the selected promoter is used for expressing a target gene, the expression level of the gene is very low, and the expression level is not proportional to the strength of the promoter, which suggests that the promoter has certain gene specificity, i.e., when a strong promoter selected by using the A gene as a reporter gene is used for expressing the B gene, it is often difficult to ensure efficient expression level. Therefore, it is important to establish a method for improving gene transcription with wider applicability.

Disclosure of Invention

In view of the defects of the prior art, the technical purpose of the invention is to establish a method for improving the transcription expression of the bacillus subtilis exogenous gene with wider applicability, and based on the technical purpose, the invention provides a dcas 9-omega fusion protein for specifically regulating the expression of the bacillus subtilis exogenous gene and application thereof.

In order to achieve the purpose of the invention, the inventor selects gram-positive bacteria bacillus subtilis as a research object, and expresses RNA polymerase and dcas9 protein through fusion, establishes a CRISPR system in the gram-positive bacteria for the first time, and realizes the specific expression of exogenous genes.

Specifically, dcas9 is a cas9 mutant (dead cas9) that lacks endonuclease activity, but still retains the ability to recognize and double-strand bind to guide RNA-targeted DNA. The bacillus subtilis RNA polymerase consists of five subunits, and the holoenzyme is alpha 2 beta' sigma omega, and has the functions of recognizing a promoter sequence and initiating and extending DNA transcription. The dCas9 and the subunit omega of the Bacillus subtilis RNA polymerase are subjected to fusion expression, guide RNA of different regions at the upstream of a target gene promoter in a Bacillus subtilis cell is targeted, a fusion protein dCas 9-omega is guided to a specific targeting site and combined with the specific targeting site, and the subunit omega of the RNA polymerase recruits other subunits of the RNA polymerase to assemble into the RNA polymerase at the site to promote downstream gene transcription, so that the expression quantity of a target gene is improved, namely the fusion protein dCas 9-omega retains the DNA binding capacity of dCas9 and the transcription activation capacity of the subunit of the RNA polymerase, and the target gene expression is specifically improved.

Further, the technical scheme of the invention is summarized as follows: a dcas 9-omega fusion protein, the amino acid sequence of which is shown in SEQ ID NO.1 in the sequence table.

In another aspect, the present invention also provides a DNA fragment encoding the above-described dcas9- ω fusion protein. In a most preferred embodiment of the present invention, the nucleotide sequence of the above DNA fragment is shown as SEQ ID NO.2 of the sequence Listing.

In another aspect, the present invention may also provide a recombinant expression vector comprising the above nucleotide sequence.

In another aspect, the present invention may also provide a genetically engineered host cell comprising a recombinant expression vector as described above. Further preferably, the genetically engineered host cell of the present invention further comprises a vector targeting a target gene, the vector comprising a P-RS-sgRNA element, wherein P is a transcription promoter of the sgRNA, RS is a type IIs restriction site, and the sgRNA is a sgRNA backbone without a guide RNA, the backbone being capable of linking to a guide RNA targeting sequence. Still further preferably, the P-RS-sgRNA element is P242-BsmBI-BsmBI-sgRNA, and the nucleotide sequence of the P-RS-sgRNA element is shown as SEQ ID NO.3 in the sequence table. In a most preferred embodiment of the present invention, the above genetically engineered host cell comprises a vector targeting a target gene comprising two cleavage sites intermediate the transcription promoter and the sgRNA backbone for enzymatic ligation of a guide RNA targeting sequence.

Since the inventor establishes the CRISPR system in gram-positive bacillus subtilis for the first time and realizes the specific expression of exogenous genes, the host cell is preferably bacillus subtilis.

On the other hand, the invention also provides the application of the dcas 9-omega fusion protein in improving the expression quantity of foreign proteins in the bacillus subtilis.

In addition, the invention also provides a method for specifically regulating the expression of the exogenous gene of the bacillus subtilis based on fusion expression of dcas9 and RNA polymerase subunit, which comprises the following steps:

1) constructing a pDGT-P43-GFP vector, and integrating a T1-P43-GFP-T1T2 expression unit into a bacillus subtilis amyE site; wherein, P43 is a promoter, GFP is a reporter gene, and the integration site is bacillus subtilis amylase gene AmyE, but the invention is not limited to the above expression element, reporter gene and integration site, for example, the promoter may be other suitable promoters such as P242; the pDG-P43-GFP construction process is specifically disclosed in patent CN105296486A, and the expression unit sequence of T1-P43-GFP-T1T2 is disclosed in SEQ ID NO. 4;

2) transforming the vector obtained in the step 1) into bacillus subtilis SCK6 to obtain a recombinant strain SG;

3) constructing a pHT01-dcas 9-omega vector, wherein the vector is used for expressing a dcas 9-omega fusion protein;

4) converting the recombinant vector pHT01-dcas 9-omega obtained in the step 3) into SG to obtain a strain SG/pHT01-dcas 9-omega;

5) constructing a pHY-P242-BsmBI-BsmBI-sgRNA vector which is used for connecting a guide RNA targeting sequence;

6) constructing a guide RNA targeting vector: pHY-P242-sgR1, pHY-P242-sgR2, pHY-P242-sgR3, pHY-P242-sgR4 and pHY-P242-sgR5, wherein the vectors respectively target different sites at the upstream of the P242-GFP expression unit and are respectively named as sgR1, sgR2, sgR3, sgR4 and sgR 5;

7) respectively transforming the recombinant vector obtained in the step 6 into SG/pHT01-dcas 9-omega to obtain strains SG/pHT01-dcas 9-omega/sgR 1, SG/pHT01-dcas 9-omega/sgR 2, SG/pHT01-dcas 9-omega/sgR 3, SG/pHT01-dcas 9-omega/sgR 4 and SG/pHT01-dcas 9-omega/sgR 5;

8) culturing the recombinant strain obtained in the step 7), and adding IPTG (isopropyl-beta-thiogalactoside) to induce expression of the dcas 9-omega fusion protein.

Further preferably, the method for specifically regulating the expression of the exogenous gene of bacillus subtilis based on fusion expression of dcas9 and RNA polymerase subunit as described above, wherein the process of constructing the vector in step 5) comprises the steps of artificially synthesizing P242-BsmBI-BsmBI-sgRNA (SEQ ID NO.3), cloning the synthesized vector into a vector pHY300PLK vector, and obtaining the vector pHY-P242-BsmBI-BsmBI-sgRNA. Wherein, P242 is a transcription promoter of the sgRNA, the sgRNA is a sgRNA framework without a 20bp targeting sequence, BsmBI is a IIs type restriction enzyme cutting site, and two BsmBI restriction enzyme cutting sites are arranged between the P242 and the sgRNA framework.

Further preferably, the process for specifically regulating the expression of the exogenous gene of bacillus subtilis based on fusion expression dcas9 and RNA polymerase subunit as described above, wherein the guide RNA targeting vector constructed in step 6) comprises the steps of designing a pair of complementary primers according to the targeting sequence, mixing and annealing the two primers to obtain a DNA double-stranded fragment with a sticky end, and enzymatically ligating the DNA double-stranded fragment to a BsmBI-cleaved phyy-P242-BsmBI-sgRNA vector.

Compared with the prior art, the invention has the following advantages and remarkable progress: the invention obviously improves the expression quantity of the exogenous gene in the bacillus subtilis. As can be seen from the specific examples, the P43-GFP expression unit integrated on the Bacillus subtilis genome is used as a report system, and the transcription and expression of GFP are successfully and specifically improved by designing guide RNAs targeting different sites upstream of a promoter P43, wherein the expression level (relative fluorescence intensity) of GFP is improved by 1.5 times to the maximum, and the expression level of GFP is improved by 2.3 times to the maximum.

Drawings

FIG. 1: five sgRNA-targeted positions;

FIG. 2: pHT01-dcas 9-omega vector map;

FIG. 3: pHY-P242-BsmBI-BsmBI-sgRNA vector map;

FIG. 4: targeting different sites of sgrnas resulted in GFP expression profiles;

FIG. 5: targeting different sites of sgrnas resulted in varying analysis patterns of GFP transcript levels.

Detailed Description

The principles and features of this invention are described below in conjunction with the following drawings, which are set forth by way of illustration only and are not intended to limit the scope of the invention.

The first embodiment is as follows:

a vector pHT01-dcas 9-omega for fusion expression of dcas9 and RNA polymerase omega subunit is constructed, which can express dcas 9-omega fusion protein. Specifically, a vector pHT01-dcas9 for expressing dcas9 is constructed, then the omega subunit gene of RNA polymerase is amplified, and after enzyme digestion, the omega subunit gene is enzymatically linked with the vector pHT01-dcas9 after the same enzyme digestion, so that a vector pHT01-dcas 9-omega is obtained.

Step 1, constructing pHT01-dcas 9-omega vector for expressing dcas 9-omega protein, and the vector map is shown in figure 2.

1) Designing a primer for pET-dcas9-VP64-6XHis aiming at an amplification template of the dcas9 gene, and using the primer

The DNA Polymerase was used for PCR to clone the dcas9 gene into pHT01 vector to obtain pHT01-dcas 9. Primers for amplification of dcas9 were:

dcas9-F：CGCGGATCCATGGACAAGAAGTACAGCATCGGCCTGGCCA

dcas9-R：gctctagaTCCTGATGAGCCTGAGCCGTGGTGGTGGTGGTGGTGGTC

and (3) PCR reaction system: 2 × PrimeSTAR Mix, 25 μ l; forward primer (10mM), 2.5. mu.l; reverse primer (10mM), 2.5. mu.l; template, 0.5 μ l; add ddH₂O to 50. mu.l. The amplification conditions were: 10s at 98 ℃; 5s at 55 ℃; 72 ℃ for 20 s; 28 x; 72 ℃ for 5 min; 4 ℃ and infinity.

2) The dcas9 gene fragment and pHT01 vector were digested with XbaI and BamHI. The enzyme cutting system is as follows: xba I, 0.5. mu.l; BamHI, 1.5. mu.l; 10 × buffer, 5 μ l; the product was recovered by PCR in 30. mu.l. After treatment at 37 ℃ for 2h, the cells were recovered on a 1% agarose gel.

3) The dcas9 gene treated in 2) and pHT01 carrier are connected by enzyme with T4 ligase, the enzyme is: 10 XT 4DNA ligase buffer, 1. mu.l; t4DNA ligase, 0.5. mu.l; the molar ratio of the enzyme digestion fragment to the enzyme digestion vector is 1: 3; add ddH₂O to 10. mu.l, and reacted at 25 ℃ for 2 h.

4) And (3) transforming the enzyme-linked product of the step (3) into escherichia coli DH5 alpha, verifying positive clones by colony PCR, and obtaining a recombinant vector pHT01-dcas9 after sequencing verification.

Step 2, pHT01-dcas 9-omega is constructed, and the vector is used for expressing the dcas 9-omega fusion protein.

5) And (3) amplifying the omega subunit of the RNA polymerase by using the genome of the bacillus subtilis SCK6 as a template, and recovering a PCR product through agarose gel. Primers for the ω subunit were:

ω-F：GCTCTAGAATGTTAGATCCGTCAATTGATTCTTTAATGAA

ω-R：TCCCCCCGGGCTATTCGCGGTCTTCCTTTTCAAACG

and (3) PCR reaction system: 10 Xpfu buffer, 5. mu.l; dNTP (2.5mM), 2. mu.l; forward primer (10 μ M), 2 μ l; reverse primer (10. mu.M), 2. mu.l; pfu polymerase, 2. mu.l; template, 1. mu.l; add ddH₂O to 50. mu.l. The amplification conditions were: 95 ℃ for 5 min; 30 cycles of 95 ℃, 30s, 55 ℃, 30s, 72 ℃, 30 s; 72 ℃ for 5 min; 4 ℃ and infinity.

6) The fragment obtained in step 5) and the vector pHT01-dcas9 obtained in step 1 were digested with XbaI/XmaI in the following manner: xba I, 0.5. mu.l; xma i, 1.5 μ l; 10 × buffer, 5 μ l; the product was recovered by PCR in 30. mu.l. After treatment at 37 ℃ for 2h, the cells were recovered on a 1% agarose gel.

7) And (3) carrying out enzyme ligation on the vector and the fragment subjected to enzyme digestion by using T4 ligase, wherein an enzyme ligation system is shown as 3), transforming an enzyme ligation product into escherichia coli DH5 alpha, carrying out colony PCR (polymerase chain reaction) verification on positive clone, and carrying out sequencing verification to obtain a recombinant vector pHT01-dcas 9-omega.

The recombinant vector pHT01-dcas 9-omega inducible expression vector induces the expression of the dcas 9-omega by IPTG.

Example two:

a vector pHY-P242-BsmBI-BsmBI-sgRNA capable of expressing sgRNA was constructed, and the vector map is shown in FIG. 3.

1) Artificially synthesizing (Shanghai Biotechnology engineering Co., Ltd.) gene sequence P242-BsmBI-BsmBI-sgRNA, wherein the gene sequence comprises a P242 promoter, two BsmBI enzyme cutting sites and a sgRNA framework (shown in SEQ ID NO. 3).

2) The synthesized gene sequence and pHY300PLK vector are subjected to enzyme digestion treatment by EcoRI and HindIII, and the enzyme digestion system is as follows: EcoRI, 0.5. mu.l; hind III, 1.5. mu.l; 10 × buffer, 5 μ l; synthesis of the sequence, 30. mu.l. After treatment at 37 ℃ for 2h, the cleaved product was recovered on a 1% agarose gel.

3) Enzyme-linking the enzyme-digested vector with the fragment, wherein the enzyme-linking system is as follows: the molar ratio of the enzyme digestion vector to the enzyme digestion fragment is 1: 3; 10 XT 4DNA ligase buffer, 1. mu.l; t4DNA ligase, 0.5. mu.l; add ddH₂O to 10 mul, reaction at 25 ℃ for 2 h.

4) And transforming the enzyme-linked product into an escherichia coli DH5 alpha strain, and carrying out PCR (polymerase chain reaction) and sequencing verification on a colony of the transformant to obtain a recombinant vector pHY-P242-BsmBI-BsmBI-sgRNA. The promoter of this vector is a constitutive promoter, and it can transcribe a downstream gene without an inducer, and since there is no ribosome binding site (RBS sequence) behind the promoter P242, this vector can transcribe only a gene downstream of the promoter P242 into RNA, but cannot translate it into a polypeptide, thereby obtaining a sgRNA sequence.

5) Designing a guide RNA sequence targeting a specific site, synthesizing two complementary primers according to the guide RNA sequence, annealing the two primers, and simultaneously using BsmB I to enzyme-cut the vector pHY-P242-BsmB I-BsmBI-sgRNA. The annealing conditions are as follows: 95 ℃ for 5 min; 95 deg.C, -1 deg.C/cycle, 40 cycles; at 55 ℃ for 30 min; 55 deg.C, -1 deg.C/cycle, 30 cycles; 4 ℃ and infinity. The enzyme cutting system is as follows: BsmBI, 0.5. mu.l; 10 × buffer, 5 μ l; pHY-P242-BsmB I-BsmBI-sgRNA plasmid, 30. mu.l, plus ddH₂O to 50. mu.l. After treatment at 55 ℃ for 2 hours, the digested vector was recovered on a 1% agarose gel.

6) And (3) connecting the annealed primer with the enzyme-digested pHY-P242-BsmBI-BsmBI-sgRNA plasmid to construct a targeting vector: pHY-P242-sgR1, pHY-P242-sgR2, pHY-P242-sgR3, pHY-P242-sgR4 and pHY-P242-sgR5, which are targeted to different sites upstream of the P43-GFP expression unit, and are named sgR1, sgR2, sgR3, sgR4 and sgR 5. The enzyme connecting system is as follows: annealed fragment (50mM), 1. mu.l; enzyme digestion vector, 1 μ l; 10 XT 4DNA ligase buffer, 1. mu.l; t4DNA ligase, 0.5. mu.l; add ddH₂O to 10. mu.l, and reacted at 25 ℃ for 2 h.

The sgrnas targeting different positions of the GFP promoter have the targeting sequences respectively (see fig. 1):

sgR 1: GATGGCCTTTTACGCGTCGA, 267bp from the Transcription Start Site (TSS) of GFP;

sgR 2: TAAGCAGAAGGCCATCCTGA, 283bp from the Transcription Start Site (TSS) of GFP;

sgR 3: CGGTGAACGCTCTCCTGAGT, 323bp from the Transcription Start Site (TSS) of GFP;

sgR 4: TCGTTTTATCTGTTGTTTGT, 343bp from the Transcription Start Site (TSS) of GFP;

sgR 5: ACTGGTCTGATCGGATCTTC, 410bp from the Transcription Start Site (TSS) of GFP.

Example three:

pHT01-dcas 9-omega and sgRs (including sgR1, sgR2, sgR3, sgR4 and sgR5) were transformed with Bacillus subtilis SG, respectively.

1) The bacillus subtilis competence is prepared by the following specific steps: SG was cultured overnight in YN medium (nutrient broth 18g/L, yeast extract 8g/L), and the overnight culture was transferred to fresh YN medium to OD₆₀₀Equal to 1, induced with 1.5% xylose for 2h, resulting in competent cells.

2) SG/pHT01-dcas 9-omega was obtained by transforming SG competent cells with pHT01-dcas 9-omega and plating them on LB medium containing chloramphenicol. SG/pHT01-dcas 9-omega competent cells were prepared according to 1), and plasmids sgRs were transformed, respectively, and plated in LB medium containing chloramphenicol and tetracycline to obtain strain SG/pHT01-dcas 9-omega/sgRs.

Example four:

the effect of different targeting sgrnas on the expression level of GFP of the targeting gene was analyzed, and the results are shown in fig. 4.

1) Bacillus subtilis SG containing sgR1/pHT01-dcas 9-omega, sgR2/pHT01-dcas 9-omega, sgR3/pHT01-dcas 9-omega, sgR4/pHT01-dcas 9-omega, sgR5/pHT01-dcas 9-omega vectors was inoculated into chloramphenicol-resistant and tetracycline-resistant 3ml LB liquid medium and cultured overnight at 37 ℃.

2) Mu.l of the overnight culture was transferred to 3ml of fresh LB medium containing chloramphenicol and tetracycline resistance, and 0.5mM IPTG was added to induce expression of dcas 9-. omega.for another 10 hours.

3) 100. mu.l of each bacterial solution was mixed with 100. mu.l of water on a 96-well black-wall transparent bottom fluorescent plate, and the cell density (OD) was measured₆₀₀) And fluorescence intensity (488nm/511nm), and the relative fluorescence intensity is obtained by dividing the fluorescence intensity by the cell density.

As can be seen from fig. 4, when sgRNA is targeted upstream of the promoter (P43) of the gene of interest (GFP), e.g., sgR1, sgR2, sgR3, sgR4, sgR5, dcas9- ω fusion protein can promote expression of GFP. The expression level (relative fluorescence intensity) of GFP is improved by 1.5 times to the maximum.

Example five:

the effect of different targeted sgrnas on the level of GFP transcription of the targeted gene was analyzed, see fig. 5.

1) Strains containing sgRs (sgR1, sgR2, sgR3, sgR4, sgR5) vector promoting GFP expression were cultured at 37 ℃ in 3ml of LB medium containing chloramphenicol and tetracycline resistance, 200. mu.l of overnight culture was transferred to 3ml of fresh LB medium containing chloramphenicol and tetracycline resistance after overnight culture, IPTG was added to induce expression of dcas 9-. omega., and further culture was carried out for 10 hours to collect cells.

2) The specific steps of RNA extraction refer to a kit: bacterial RNA Kit (Omega Co.).

3) The specific steps of carrying out DNase treatment on the sample are as follows: to an RNase-free EP tube was added 3. mu.l of RNA, 10 × Reaction buffer with MgCl₂Mu.l of DNase I (RNase-free) was added to 10. mu.l of water. Incubate at 37 ℃ for 1h, add 1. mu.l of 50mM EDTA, and incubate at 65 ℃ for 10 min.

4) See kit for specific procedures for reverse transcription: RevertAId First Strand cDNA Synthesis Kit (ThermoFisher Co., Ltd.)

5) The specific steps of the fluorescent quantitative PCR refer to a kit: iTaq Universal SYBR Green Supermix (Axygen Co., Ltd.)

The internal reference gene is as follows: rpsj

The primers are as follows: RPSJ-F: GATGGAAGCGTTCAACTAGCAGAC

RPSJ-R：GCAGATTGTGTGGACAGGTAATGG

The reaction conditions are as follows: at 95 ℃ for 3 min; 95 ℃ for 10 s; 30s at 55 ℃; 39 cycles were performed; 95 ℃ for 10 s; 65 ℃, 5s, 95 ℃, 0.5 ℃/s.

The target gene is as follows: GFP (green fluorescent protein)

The primers are as follows: GFP-F: ACAAATACAAAGATTCTCGTGAGC

GFP-R：CTAATCGCATAAGAGCATCAACAG

The reaction conditions are as follows: at 95 ℃ for 3 min; 95 ℃ for 10 s; 30s at 55 ℃; 39 cycles were performed; 95 ℃ for 10 s; 65 ℃, 5s, 95 ℃, 0.5 ℃/s.

As shown in FIG. 5, the relative fluorescence quantitative analysis showed that the GFP expression level of the strain was increased, and the maximum transcription level was increased by 2.3 times, indicating that dcas 9-omega indeed increased the GFP expression level at the transcription level, and the principle of the present invention was satisfied.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.

Sequence listing

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Phe Asp Asn Leu Thr Lys Ala Glu Arg Gly Gly Leu Ser Glu Leu Asp

900 905 910

Lys Ala Gly Phe Ile Lys Arg Gln Leu Val Glu Thr Arg Gln Ile Thr

915 920 925

Lys His Val Ala Gln Ile Leu Asp Ser Arg Met Asn Thr Lys Tyr Asp

930 935 940

Glu Asn Asp Lys Leu Ile Arg Glu Val Lys Val Ile Thr Leu Lys Ser

945 950 955 960

Lys Leu Val Ser Asp Phe Arg Lys Asp Phe Gln Phe Tyr Lys Val Arg

965 970 975

Glu Ile Asn Asn Tyr His His Ala His Asp Ala Tyr Leu Asn Ala Val

980 985 990

Val Gly Thr Ala Leu Ile Lys Lys Tyr Pro Lys Leu Glu Ser Glu Phe

995 1000 1005

Val Tyr Gly Asp Tyr Lys Val Tyr Asp Val Arg Lys Met Ile Ala Lys

1010 1015 1020

Ser Glu Gln Glu Ile Gly Lys Ala Thr Ala Lys Tyr Phe Phe Tyr Ser

1025 1030 1035 1040

Asn Ile Met Asn Phe Phe Lys Thr Glu Ile Thr Leu Ala Asn Gly Glu

1045 1050 1055

Ile Arg Lys Arg Pro Leu Ile Glu Thr Asn Gly Glu Thr Gly Glu Ile

1060 1065 1070

Val Trp Asp Lys Gly Arg Asp Phe Ala Thr Val Arg Lys Val Leu Ser

1075 1080 1085

Met Pro Gln Val Asn Ile Val Lys Lys Thr Glu Val Gln Thr Gly Gly

1090 1095 1100

Phe Ser Lys Glu Ser Ile Leu Pro Lys Arg Asn Ser Asp Lys Leu Ile

1105 1110 1115 1120

Ala Arg Lys Lys Asp Trp Asp Pro Lys Lys Tyr Gly Gly Phe Asp Ser

1125 1130 1135

Pro Thr Val Ala Tyr Ser Val Leu Val Val Ala Lys Val Glu Lys Gly

1140 1145 1150

Lys Ser Lys Lys Leu Lys Ser Val Lys Glu Leu Leu Gly Ile Thr Ile

1155 1160 1165

Met Glu Arg Ser Ser Phe Glu Lys Asn Pro Ile Asp Phe Leu Glu Ala

1170 1175 1180

Lys Gly Tyr Lys Glu Val Lys Lys Asp Leu Ile Ile Lys Leu Pro Lys

1185 1190 1195 1200

Tyr Ser Leu Phe Glu Leu Glu Asn Gly Arg Lys Arg Met Leu Ala Ser

1205 1210 1215

Ala Gly Glu Leu Gln Lys Gly Asn Glu Leu Ala Leu Pro Ser Lys Tyr

1220 1225 1230

Val Asn Phe Leu Tyr Leu Ala Ser His Tyr Glu Lys Leu Lys Gly Ser

1235 1240 1245

Pro Glu Asp Asn Glu Gln Lys Gln Leu Phe Val Glu Gln His Lys His

1250 1255 1260

Tyr Leu Asp Glu Ile Ile Glu Gln Ile Ser Glu Phe Ser Lys Arg Val

1265 1270 1275 1280

Ile Leu Ala Asp Ala Asn Leu Asp Lys Val Leu Ser Ala Tyr Asn Lys

1285 1290 1295

His Arg Asp Lys Pro Ile Arg Glu Gln Ala Glu Asn Ile Ile His Leu

1300 1305 1310

Phe Thr Leu Thr Asn Leu Gly Ala Pro Ala Ala Phe Lys Tyr Phe Asp

1315 1320 1325

Thr Thr Ile Asp Arg Lys Arg Tyr Thr Ser Thr Lys Glu Val Leu Asp

1330 1335 1340

Ala Thr Leu Ile His Gln Ser Ile Thr Gly Leu Tyr Glu Thr Arg Ile

1345 1350 1355 1360

Asp Leu Ser Gln Leu Gly Gly Asp Gly Ser Ala Ala Ser Arg Met Leu

1365 1370 1375

Asp Pro Ser Ile Asp Ser Leu Met Asn Lys Leu Asp Ser Lys Tyr Thr

1380 1385 1390

Leu Val Thr Val Ser Ala Arg Arg Ala Arg Glu Met Gln Ile Lys Lys

1395 1400 1405

Asp Gln Met Ile Glu His Thr Ile Ser His Lys Tyr Val Gly Lys Ala

1410 1415 1420

Leu Glu Glu Ile Asp Ala Gly Leu Leu Ser Phe Glu Lys Glu Asp Arg

1425 1430 1435 1440

Glu

<210> 2

<211> 4326

<212> DNA

<213> Artificial sequence (artificial sequence)

<400> 2

atggataaga aatactcaat aggcttagct atcggcacaa atagcgtcgg atgggcggtg 60

atcactgatg aatataaggt tccgtctaaa aagttcaagg ttctgggaaa tacagaccgc 120

cacagtatca aaaaaaatct tataggggct cttttatttg acagtggaga gacagcggaa 180

gcgactcgtc tcaaacggac agctcgtaga aggtatacac gtcggaagaa tcgtatttgt 240

tatctacagg agattttttc aaatgagatg gcgaaagtag atgatagttt ctttcatcga 300

cttgaagagt cttttttggt ggaagaagac aagaagcatg aacgtcatcc tatttttgga 360

aatatagtag atgaagttgc ttatcatgag aaatatccaa ctatctatca tctgcgaaaa 420

aaattggtag attctactga taaagcggat ttgcgcttaa tctatttggc cttagcgcat 480

atgattaagt ttcgtggtca ttttttgatt gagggagatt taaatcctga taatagtgat 540

gtggacaaac tatttatcca gttggtacaa acctacaatc aattatttga agaaaaccct 600

attaacgcaa gtggagtaga tgctaaagcg attctttctg cacgattgag taaatcaaga 660

cgattagaaa atctcattgc tcagctcccc ggtgagaaga aaaatggctt atttgggaat 720

ctcattgctt tgtcattggg tttgacccct aattttaaat caaattttga tttggcagaa 780

gatgctaaat tacagctttc aaaagatact tacgatgatg atttagataa tttattggcg 840

caaattggag atcaatatgc tgatttgttt ttggcagcta agaatttatc agatgctatt 900

ttactttcag atatcctaag agtaaatact gaaataacta aggctcccct atcagcttca 960

atgattaaac gctacgatga acatcatcaa gacttgactc ttttaaaagc tttagttcga 1020

caacaacttc cagaaaagta taaagaaatc ttttttgatc aatcaaaaaa cggatatgca 1080

ggttatattg atgggggagc tagccaagaa gaattttata aatttatcaa accaatttta 1140

gaaaaaatgg atggtactga ggaattattg gtgaaactaa atcgtgaaga tttgctgcgc 1200

aagcaacgga cctttgacaa cggctctatt ccccatcaaa ttcacttggg tgagctgcat 1260

gctattttga gaagacaaga agacttttat ccatttttaa aagacaatcg tgagaagatt 1320

gaaaaaatct tgacttttcg aattccttat tatgttggtc cattggcgcg tggcaatagt 1380

cgttttgcat ggatgactcg gaagtctgaa gaaacaatta ccccatggaa ttttgaagaa 1440

gttgtcgata aaggtgcttc agctcaatca tttattgaac gcatgacaaa ctttgataaa 1500

aatcttccaa atgaaaaagt actaccaaaa catagtttgc tttatgagta ttttacggtt 1560

tataacgaat tgacaaaggt caaatatgtt actgaaggaa tgcgaaaacc agcatttctt 1620

tcaggtgaac agaagaaagc cattgttgat ttactcttca aaacaaatcg aaaagtaacc 1680

gttaagcaat taaaagaaga ttatttcaaa aaaatagaat gttttgatag tgttgaaatt 1740

tcaggagttg aagatagatt taatgcttca ttaggtacct accatgattt gctaaaaatt 1800

attaaagata aagatttttt ggataatgaa gaaaatgaag atatcttaga ggatattgtt 1860

ttaacattga ccttatttga agatagggag atgattgagg aaagacttaa aacatatgct 1920

cacctctttg atgataaggt gatgaaacag cttaaacgtc gccgttatac tggttgggga 1980

cgtttgtctc gaaaattgat taatggtatt agggataagc aatctggcaa aacaatatta 2040

gattttttga aatcagatgg ttttgccaat cgcaatttta tgcagctgat ccatgatgat 2100

agtttgacat ttaaagaaga cattcaaaaa gcacaagtgt ctggacaagg cgatagttta 2160

catgaacata ttgcaaattt agctggtagc cctgctatta aaaaaggtat tttacagact 2220

gtaaaagttg ttgatgaatt ggtcaaagta atggggcggc ataagccaga aaatatcgtt 2280

attgaaatgg cacgtgaaaa tcagacaact caaaagggcc agaaaaattc gcgagagcgt 2340

atgaaacgaa tcgaagaagg tatcaaagaa ttaggaagtc agattcttaa agagcatcct 2400

gttgaaaata ctcaattgca aaatgaaaag ctctatctct attatctcca aaatggaaga 2460

gacatgtatg tggaccaaga attagatatt aatcgtttaa gtgattatga tgtcgatgcc 2520

attgttccac aaagtttcct taaagacgat tcaatagaca ataaggtctt aacgcgttct 2580

gataaaaatc gtggtaaatc ggataacgtt ccaagtgaag aagtagtcaa aaagatgaaa 2640

aactattgga gacaacttct aaacgccaag ttaatcactc aacgtaagtt tgataattta 2700

acgaaagctg aacgtggagg tttgagtgaa cttgataaag ctggttttat caaacgccaa 2760

ttggttgaaa ctcgccaaat cactaagcat gtggcacaaa ttttggatag tcgcatgaat 2820

actaaatacg atgaaaatga taaacttatt cgagaggtta aagtgattac cttaaaatct 2880

aaattagttt ctgacttccg aaaagatttc caattctata aagtacgtga gattaacaat 2940

taccatcatg cccatgatgc gtatctaaat gccgtcgttg gaactgcttt gattaagaaa 3000

tatccaaaac ttgaatcgga gtttgtctat ggtgattata aagtttatga tgttcgtaaa 3060

atgattgcta agtctgagca agaaataggc aaagcaaccg caaaatattt cttttactct 3120

aatatcatga acttcttcaa aacagaaatt acacttgcaa atggagagat tcgcaaacgc 3180

cctctaatcg aaactaatgg ggaaactgga gaaattgtct gggataaagg gcgagatttt 3240

gccacagtgc gcaaagtatt gtccatgccc caagtcaata ttgtcaagaa aacagaagta 3300

cagacaggcg gattctccaa ggagtcaatt ttaccaaaaa gaaattcgga caagcttatt 3360

gctcgtaaaa aagactggga tccaaaaaaa tatggtggtt ttgatagtcc aacggtagct 3420

tattcagtcc tagtggttgc taaggtggaa aaagggaaat cgaagaagtt aaaatccgtt 3480

aaagagttac tagggatcac aattatggaa agaagttcct ttgaaaaaaa tccgattgac 3540

tttttagaag ctaaaggata taaggaagtt aaaaaagact taatcattaa actacctaaa 3600

tatagtcttt ttgagttaga aaacggtcgt aaacggatgc tggctagtgc cggagaatta 3660

caaaaaggaa atgagctggc tctgccaagc aaatatgtga attttttata tttagctagt 3720

cattatgaaa agttgaaggg tagtccagaa gataacgaac aaaaacaatt gtttgtggag 3780

cagcataagc attatttaga tgagattatt gagcaaatca gtgaattttc taagcgtgtt 3840

attttagcag atgccaattt agataaagtt cttagtgcat ataacaaaca tagagacaaa 3900

ccaatacgtg aacaagcaga aaatattatt catttattta cgttgacgaa tcttggagct 3960

cccgctgctt ttaaatattt tgatacaaca attgatcgta aacgatatac gtctacaaaa 4020

gaagttttag atgccactct tatccatcaa tccatcactg gtctttatga aacacgcatt 4080

gatttgagtc agctaggagg tgacggttct gcagcttcta gaatgttaga tccgtcaatt 4140

gattctttaa tgaataaatt agattcaaaa tatacgctgg tgactgtttc tgcgagacgt 4200

gcccgtgaaa tgcaaatcaa aaaagaccaa atgattgaac atacgatttc acacaaatat 4260

gtaggcaaag ctttagaaga aattgatgca ggcctgcttt cgtttgaaaa ggaagaccgc 4320

gaatag 4326

<210> 3

<211> 404

<212> DNA

<213> Artificial sequence (artificial sequence)

<400> 3

aagcttgctc tagatatttt tttgccaaag ctgtaattga ggaatcatag aatttttaat 60

ttaaatttta tttgaatctt tacaatccta ttgatataat aaatgtagtg aggtggagga 120

gacgcgcgtc tccgttttag agctagaaat agcaagttaa aataaggcta gtccgttatc 180

aacttgaaaa agtggcaccg agtcggtgct tttttcttca gcacaattcc aagaaaaaca 240

cgatttagaa cctaaaaaga acgaatttga actaactcat aaccgagagg taaaaaaaga 300

acgaagtcga gatcagggaa tgagtttata aaataaaaaa agcacctgaa aaggtgtctt 360

tttttgatgg ttttgaactt ggactagtcc gggatcccga attc 404

<210> 4

<211> 1323

<212> DNA

<213> Artificial sequence (artificial sequence)

<400> 4

ggaaccacaa attaaaactg gtctgatcgg atcttcaggc atcaaataaa acgaaaggct 60

cagtcgaaag actgggcctt tcgttttatc tgttgtttgt cggtgaacgc tctcctgagt 120

aggacaaatc cgccgctcta gctaagcaga aggccatcct gacggatggc cttttacgcg 180

tcgacgggat cctgataggt ggtatgtttt cgcttgaact tttaaataca gccattgaac 240

atacggttga tttaataact gacaaacatc accctcttgc taaagcggcc aaggacgctg 300

ccgccggggc tgtttgcgtt tttgccgtga tttcgtgtat cattggttta cttatttttt 360

tgccaaagct gtaatggctg aaaattctta catttatttt acatttttag aaatgggcgt 420

gaaaaaaagc gcgcgattat gtaaaatata aagtgatagc ggtaccatta tagaaaggag 480

gtgataaaaa tgcgtaaagg agaagaactt ttcactggag ttgtcccaat tcttgttgaa 540

ttagatggtg atgttaatgg gcacaaattt tctgtcagtg gagagggtga aggtgatgca 600

acatacggaa aacttaccct taaatttatt tgcactactg gaaaactacc tgttccatgg 660

ccaacacttg tcactacttt cgggtatggt gttcaatgct ttgcgagata cccagatcat 720

atgaaacggc atgacttttt caagagtgcc atgcccgaag gttatgtaca ggaaagaact 780

atatttttca aagatgacgg gaactacaag acacgtgctg aagtcaagtt tgaaggtgat 840

acccttgtta atagaatcga gttaaaaggt attgatttta aagaagatgg aaacattctt 900

ggacacaaat tggaatacaa ctataactca cacaatgtat acatcatggc agacaaacaa 960

aagaatggaa tcaaagttaa cttcaaaatt agacacaaca ttgaagatgg aagcgttcaa 1020

ctagcagacc attatcaaca aaatactcca attggcgatg gccctgtcct tttaccagac 1080

aaccattacc tgtccacaca atctgccctt tcgaaagatc ccaacgaaaa gagagaccac 1140

atggtccttc ttgagtttgt aacagctgct gggattacac atggcatgga tgaactatac 1200

aaataaaagc ttgggcttaa ttaattaaga ctcctgttga tagatccagt aatgacctca 1260

gaactccatc tggatttgtt cagaacgctc ggttgccgcc gggcgttttt tattggtgag 1320

aat 1323

Claims (5)

1. A dcas 9-omega fusion protein for specifically regulating the expression of a bacillus subtilis exogenous gene has an amino acid sequence shown in SEQ ID NO.1 in a sequence table.

2. A DNA fragment encoding the dcas9- ω fusion protein according to claim 1.

3. The DNA fragment of claim 2, wherein the nucleotide sequence is represented by SEQ ID NO.2 of the sequence Listing.

4. A recombinant expression vector comprising the nucleotide sequence of claim 3.

5. A genetically engineered host cell comprising the recombinant expression vector of claim 4, wherein said host cell is Bacillus subtilis.

Images