CN110093366A - A kind of bacillus subtilis gluconate inducing expression element and construction method - Google Patents

Abstract

The invention discloses a kind of bacillus subtilis gluconate inducing expression element and construction methods, belong to field of genetic engineering.The present invention is with constitutive promoter P_lytETranscriptional regulation protein GntR is expressed, further by constitutive promoter P_thrSIn, integrate transcriptional regulation protein GntR binding sequence, realize with bacillus subtilis (Bacillus subtilis) 168 be host when, after adding gluconate, reach the induced expression level and lower leakage expression of higher-strength, its inducing expression intensity reaches 22500a.u., and leakage rate is reduced to 3.0% from 15.6%, lays a good foundation for screening-gene engineering bacteria or modulin expression.

Description

A kind of bacillus subtilis gluconate inducing expression element and construction method

Technical field

The present invention relates to a kind of bacillus subtilis gluconate inducing expression element and construction methods, belong to hereditary work Journey field.

Background technique

In bacillus subtilis metabolic engineering field, the promoter element of small-molecule substance inducing expression is responded, is answered extensively For directions such as metabolic regulations.Currently, being usually used in the evoked promoter in bacillus subtilis, there is xylose evoked promoter P_xyl With IPTG evoked promoter P_grac.But xylose evoked promoter P_xylThere is a problem of that xylose is expensive, IPTG induction is opened Mover P_grac, it is expensive that there are IPTG, and there are problems that certain toxicity to cell.Secondly, xylose and IPTG induction are opened There is higher leakage expression problem in mover.Disadvantages described above seriously limits it in bacillus subtilis metabolic engineering field Application.Gluconate is cheap as a kind of simple carbon source, and is a kind of suitable inducer to cytotoxic.It is logical Genetic engineering transformation promoter is crossed, the evoked response element for being more appropriate to bacillus subtilis can be obtained.However, how to be transformed Promoter realizes higher inducing expression intensity and lower leakage expression with response glucose hydrochlorate, is a value The problem of must furtheing investigate.Only by releasing the carbon metabolism reptation behavior of withered grass gemma born of the same parents bacillus PgntWT original promoter, It is not enough to realize the strong inducing expression response element of response glucose hydrochlorate (referring to paper " Bacillus disclosed in 2016 subtilis GntR regulation modified to devise artificial transient induction Systems "), therefore inducing expression intensity limits gluconic acid Salt treatment far below xylose and IPTG evoked promoter Application of the Expression element in bacillus subtilis metabolic engineering field.

Summary of the invention

In order to solve the above technical problems, realizing the inducing expression promoter element in response to grape hydrochlorate, and reach Higher induced expression level and lower leakage expression,

The first purpose of the invention is to provide a kind of inducing expression promoter elements；The inducing substrate of the element responds is Gluconate；The element is to contain GntR albumen, P_lytEPromoter, P_thrSWith the plasmid of GntR binding sequence；The P_lytE Promoter regulation GntR albumen；The P_thrSIt is integrated with GntR binding sequence, the nucleotide sequence such as SEQ ID NO.4 after integration It is shown；Promoter P_lytEWith promoter P_thrSTranscriptional orientation it is opposite.

In one embodiment of the invention, the P_lytEThe nucleotide sequence of promoter is as shown in SEQ ID NO.1.

In one embodiment of the invention, the transcriptional regulation protein GntR contains ammonia shown in SEQ ID NO.2 Base acid sequence.

In one embodiment of the invention, the nucleotide sequence such as SEQ ID of the transcriptional regulation protein is encoded Shown in NO.6.

In one embodiment of the invention, the plasmid is pHT01, pP43NMK or pSTOP1622 plasmid.

In one embodiment of the invention, GntR promoter is also contained in the element；Encode the GntR starting The nucleotide sequence of son is as shown in SEQ ID NO.8.

In one embodiment of the invention, the GntR albumen, P_lytEPromoter and integrate GntR binding sequence Promoter P_thrSIt is arranged on same plasmid.

In one embodiment of the invention, the nucleotide sequence of the plasmid is as shown in SEQ NO.11.

A second object of the present invention is to provide the genetic engineering bacteriums for containing the gluconate inducing expression element.

In one embodiment of the invention, the genetic engineering bacterium is host with bacillus subtilis.

Third mesh of the invention is to provide a kind of gluconate inducing expression element and is metabolized work in bacillus subtilis The application in journey field.

In one embodiment of the invention, the application is to promote the expression of destination protein.

In one embodiment of the invention, the gene of the destination protein is encoded by promoter P_lytERegulation.

In one embodiment of the invention, the application is to reduce leakage expression.

In one embodiment of the invention, the application is the genetic engineering bacterium of screening expression target gene.

In one embodiment of the invention, the activation condition of the gluconate inducing expression element are as follows: culture Contain gluconate in environment；Condition of culture is 30~37 DEG C of 16~48h of culture.

The utility model has the advantages that (1) present invention is by by transcriptional regulation protein GntR binding site sequence ATACTTGTATACAAGTATACT is integrated into stronger composition type expression promoter P_thrSIn, it is higher to obtain intensity of activation Gluconate inducing expression promoter element.

(2) present invention is relatively strong under the conditions of fermentation medium by replacement transcriptional regulation protein GntR original promoter Constitutive promoter P_lytE, transcriptional regulation protein GntR expression quantity is improved, gluconate inducing expression element is reduced and exists Without the leakage expression under gluconate existence condition.

(3) gluconate inducing expression promoter element provided by the invention activates table in bacillus subtilis Reach 22000a.u. (with strong promoter P in bacillus subtilis up to intensity₄₃Expression intensity 30000a.u. is close).Further lead to Reinforcement transcriptional regulation protein GntR expression is crossed, so that gluconate inducing expression promoter element is in no gluconate Leakage expression under existence condition is only 3% or so.Further to pass through genetic engineering, transformed bacillus bacillus grape Sugar lime inducing expression promoter is laid a good foundation.Gluconate inducing expression promoter construction method letter provided by the invention It is single, it is easy to use, there is good metabolic engineering application prospect.

Specific embodiment

Sequence involved in specific embodiment is as follows:

Composing type P_lytEPromoter nucleotide sequence is as shown in SEQ ID NO.1；

The amino acid sequence of transcriptional regulation protein GntR is as shown in SEQ ID NO.2；

Constitutive promoter P_thrSNucleotide sequence as shown in SEQ ID NO.3；

Constitutive promoter P_thrSNucleotide sequence after integrating GntR binding sequence is as shown in SEQ ID NO.4；

The original base sequence of transcriptional regulation protein GntR is as shown in SEQ ID NO.5；

120 bit bases of transcriptional regulation protein GntR are changed to the nucleotide sequence after T as C as shown in SEQ ID NO.6；

The nucleotide sequence of GntR original promoter is encoded as shown in SEQ ID NO.7；

Transcriptional regulation protein GntR promoter sequence after code optimization is as shown in SEQ ID NO.8；

The amino acid sequence of green fluorescent protein GFP is as shown in SEQ NO.9；

Gluconate inducing expression element plasmid pHT-BaL-optimal base sequence after optimizing original GntR promoter Column are as shown in SEQ NO.10；

GntR promoter is replaced with into composing type P_lytEGluconate inducing expression element plasmid pHT- after promoter BaL-lytE base sequence is as shown in SEQ NO.11.

The bacillus subtilis condition of culture of the promoter element of inducing expression containing gluconate:

Fermentation medium (g/L): sodium gluconate 60, tryptone 6, yeast powder 12, ammonium sulfate 6, dipotassium hydrogen phosphate 12.5, potassium dihydrogen phosphate 2.5, magnesium sulfate 3.

Condition of culture: 37 DEG C, 200rpm, under the conditions of cultivate 48h.

Green fluorescent protein and cell concentration detection method:

Green fluorescent protein is detected using Tecan microplate reader, excitation wavelength 490nm, launch wavelength 530nm, gain 60. Cell concentration Detection wavelength is 600nm.

Reveal expression quantity calculation formula (%): x=(a-b)/c

Wherein, a is gluconate inducing expression element, and the fluorescence intensity when being not added with gluconate, b is withered grass Fluorescence intensity of 168 bacterial strain of bacillus as control strain, c are gluconate inducing expression element in addition gluconic acid Fluorescence intensity when salt.

Embodiment 1 constructs gluconic acid response element tool plasmid pHT-BaL-optimal

Design primer pHT-F:

5 '-ACACATGGCATGGATGAACTATACAAATAATTCACGTCACGCGTCCATGGAGA-3 ' and pHT-R: 5 '-GAGCTCGAATTCACTGGCCGTCGTTTTA-3 ' expand pHT01 plasmid backbone using pHT01 plasmid as template；Design is drawn Object GntR-F:

5 '-ACGACGGCCAGTGAATTCGAGCTCCTAGTCATTGTTGTATTCAGCTCCTTTTGCCA GCA-3 ' and GntR-R:5 '-ATGCTAGACTCCAAAGACCTGTTGTATCCCG-3 ', by GntR sequence (SEQ original in bacillus subtilis Shown in ID NO.5) nucleotide sequence the 120th T is become from C, glucose catabolism reptation behavior is released, after replacing base GntR sequence be SEQ ID NO.6, coding amino acid sequence have not been changed.Original transcriptional control egg in bacillus subtilis White GntR expression promoter sequence is SEQ ID NO.7, by the 127th, 128 and 129 nucleotide of original GntR promoter by CAT It is changed to ACA, releases glucose catabolism reptation behavior, the GntR promoter sequence after replacing nucleotide is SEQ ID NO.8.Design primer PGntR-F:

5 '-GATACAACAGGTCTTTGGAGTCTAGCATACACTCACCTTCCTCACTCAAGGAGTAT ACT-3 ' and PGntR-R:

5 '-GCAATATGGTAAAAATTTAAATAAAAATTAGAAATGAAAGTGTTTGA-3 ', after codon optimization GntR gene chemical synthesis after using it as template, expand optimization gene sequence after GntR promoter sequence SEQ ID NO.8；Design Primer PthrS-F:5 '-

AATTTTTATTTAAATTTTTACCATATTGCTATGTATATTGATTCTCATTTGCTCGC GCC-3 ' and PthrS-R:5 '-

GTGTACATTTCACCTCCTTTAAGGAGTATACTTGTATACAAGTATTATAAATTGTTCAATCCAAAAAA TCAACACGAT-3 ' expands the P of the binding sequence containing GntR using 168 genome of bacillus subtilis as template_thrSPromoter sequence It arranges (SEQ ID NO.4)；Design primer 5 '-

ACTCCTTAAAGGAGGTGAAATGTACACATGGGTAAGGGAGAAGAACTTTTCACTGG A-3 ' and 5 '-TTA TTTGTATAGTTCATCCATGCCATGTGTAATC-3 ' (has been disclosed in paper with plasmid pBSX-GFP Characterization and application of endogenous phase-dependent promoters in Bacillus subtilis.Appl.Microbiol.Biotechnol.101, in 4151-61.) it is template, expand GFP gene Sequence SEQ ID NO.9.

By GntR aporepressor base shown in GntR promoter gene segment, SEQ ID NO.6 shown in SEQ ID NO.8 Because of P shown in segment, SEQ ID NO.4_thrSFluorescin GFP gene shown in promoter gene segment and SEQ ID NO.9 Segment and pHT01 plasmid fragments, above 5 DNA fragmentations by NEB Gibson Assembly Master Mix Kit into Row assembling, constructs gluconic acid response element tool plasmid pHT-BaL-optimal plasmid.

Embodiment 2 constructs gluconate inducing expression element bacillus subtilis BS-pHT-BaL-optimal

PHT-BaL-optimal plasmid conversion bacillus subtilis (Bacillus subtilis) that embodiment 1 is constructed 168, recombined bacillus subtilis engineering bacteria is obtained, BS-pHT-BaL-optimal is named as.

Embodiment 3 constructs gluconic acid response element tool plasmid pHT-BaL-lytE

Design primer pHT-lytE-F:5 '-TATGTATATTGATTCTCATTTGCTCGCGCCGCTGA-3 ' and pHT- LytE-R:5 '-AAAGGAGGTGAAATGTACACATGCTAGACTCCAAAGACCTGTTGTATCCCGCA-3 ' is with embodiment 1 The pHT-BaL-optimal plasmid of method building is template amplification plasmid backbone segment；5 '-GTGTACATTTCAC of design primer - the AATGAGAATCAAT of CTCCTTTCCCAAATGTTAACTCTATATATATGTATCTCTTTTTTTAAATTAATCT- 3 ' and 5 ' ATACATACTCCTCGAATATACTTTATCACTCATTTTTCCGATATAT-3 ' expands P_lytEPromoter sequence.

The above pHT-BaL-optimal plasmid is template amplification plasmid backbone segment and P_lytEPromoter fragment totally 2 DNA Segment is assembled by NEB Gibson Assembly Master Mix Kit, constructs gluconic acid response element tool Plasmid pHT-BaL-lytE plasmid.

Embodiment 4 constructs gluconate inducing expression element bacillus subtilis BS-pHT-BaL-lytE

PHT-BaL-lytE plasmid conversion bacillus subtilis (Bacillus subtilis) prepared by embodiment 3 168, recombined bacillus subtilis engineering bacteria is obtained, BS-pHT-BaL-lytE is named as.

5 bacillus subtilis BS-pHT-BaL-optimal of embodiment adds sodium gluconate induction fermentation

By the bacterial strain BS-pHT-BaL-optimal prepared according to the method for embodiment 2 in 37 DEG C, 200rpm, containing 48h is cultivated in the fermentation medium of 60g/L sodium gluconate.Finally measurement fermentation liquid green fluorescent protein intensity is 22500a.u. successfully obtains the gluconate inducing expression element of strong inducing expression intensity.When not added in fermentation medium When sodium gluconate, ferment 48h, and fermentation liquid green fluorescent protein intensity is 3500a.u., and leakage expression quantity is 15.6%.

6 bacillus subtilis BS-pHT-BaL-lytE of embodiment adds sodium gluconate induction fermentation

By the bacterial strain BS-pHT-BaL-lytE prepared according to the method for embodiment 4 under 37 DEG C, 200rpm, containing 48h is cultivated in the fermentation medium of 60g/L sodium gluconate.Finally measurement fermentation liquid green fluorescent protein intensity is 22000a.u., compared to control strain BS-pHT-BaL-optimal, inducing expression intensity is not decreased significantly.But when When not adding sodium gluconate in fermentation medium, ferment 48h, and fermentation liquid green fluorescent protein intensity is only 660a.u., leakage Expression quantity is reduced to 3.0% from 15.6%.

Influence of 1 different promoters of comparative example to inducing expression element regulating effect

Using the construction of strategy inducing expression element of embodiment 1, difference is, respectively by P_thrSThe replacement of promoter promoter For P_yvyDPromoter, P_phrKPromoter and P_odhAPromoter, using method validation regulating effect described in embodiment 5 or 6, as a result Show P_phrKThe inducing expression original part of promoter building, induced activation intensity only has 3000a.u., and P_yvyDPromoter and P_odhAIt opens The inducing expression original part of mover building, cannot be activated expression.

Although the present invention has been described by way of example and in terms of the preferred embodiments, it is not intended to limit the invention, any to be familiar with this skill The people of art can do various change and modification, therefore protection model of the invention without departing from the spirit and scope of the present invention Enclosing subject to the definition of the claims.

SEQUENCE LISTING

<110>Southern Yangtze University

<120>a kind of bacillus subtilis gluconate inducing expression element and construction method

<160> 11

<170> PatentIn version 3.3

<210> 1

<211> 300

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ctcctcgaat atactttatc actcattttt ccgatatatg agccgacccc gaaagttttt 60

catttatttc ctttatgttg aaaaacatcc cataaaacat gacaaaagca ctcgtttttt 120

gtcacctttg caatgaaaat gaaatattta atacccttaa aaactttttt ttagaacgaa 180

taattaagaa atttgtcaca tgaagtcaag actatttctg atgggaatct atccttataa 240

tagaaatcaa taagattaat ttaaaaaaag agatacatat atatagagtt aacatttggg 300

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Met Leu Asp Ser Lys Asp Leu Leu Tyr Pro Ala Lys Trp Leu Ser Lys

1 5 10 15

Ala Ser Thr Gly Val Arg Val Ala Tyr Glu Leu Arg Met Arg Ile Val

20 25 30

Ser Gly Leu Ile Glu Ser Gly Thr Ile Leu Ser Glu Asn Thr Ile Ala

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Ala Glu Phe Ser Val Ser Arg Ser Pro Val Arg Glu Ala Leu Lys Ile

50 55 60

Leu Ala Ser Glu Lys Ile Ile Arg Leu Glu Arg Met Gly Ala Val Val

65 70 75 80

Ile Gly Leu Thr Glu Lys Lys Ile Ala Glu Ile Tyr Asp Val Arg Leu

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Leu Leu Glu Thr Phe Val Phe Glu Arg Leu Val Lys Ile Asp Ile Glu

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Pro Leu Val Lys Asp Leu Ser Lys Ile Leu Glu Met Met Lys Val Ser

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

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His Glu Thr Ile Ile Arg Ala Ile Asp His Ser Tyr Ile Gln Met Ile

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Trp Asn Asn Leu Lys Pro Val Met Glu Ser Phe Ile Leu Leu Ser Met

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

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Asn His Glu Leu Tyr Ile Gln Ala Ile Lys Thr Lys Asp Arg Ala Leu

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Met Ile Gln Ser Leu His Gln Asn Phe Asp Asp Val Gln Asp Lys Val

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Glu Asp Leu Trp Leu Ser Gln Gln Met Leu Ala Lys Gly Ala Glu Tyr

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Asn Asn Asp

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tatgtatatt gattctcatt tgctcgcgcc gctgatttcc attgcgcctg atgaagtcgt 60

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gagagtggaa atgctcccgc tgcatgcttt tacagatgca gaaataccgg tgaagcactc 180

ggaaggatga gggccggcag cctgtctatt taaggctgtc ggtttaaaaa aaaggaaacg 240

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tatgtatatt gattctcatt tgctcgcgcc gctgatttcc attgcgcctg atgaagtcgt 60

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gagagtggaa atgctcccgc tgcatgcttt tacagatgca gaaataccgg tgaagcactc 180

ggaaggatga gggccggcag cctgtctatt taaggctgtc ggtttaaaaa aaaggaaacg 240

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atgctagact ccaaagacct gttgtatccc gcaaaatggc tctcaaaagc gtcaaccgga 60

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attttatcag aaaatacaat cgccgccgag ttttcagtaa gccgttcgcc ggttcgcgaa 180

gcgctaaaaa tactcgcatc cgaaaaaatc atccgcttag aacgaatggg agcggtcgta 240

attggtttaa ctgagaagaa aatcgcggaa atttatgatg tgcggttact attagaaaca 300

tttgtctttg aacggcttgt caaaatagac attgagcctt tagttaagga tctcagcaaa 360

attcttgaaa tgatgaaagt ctcaatcaaa tatgaggatg ctgacgaatt ttcatttcaa 420

gatgtgctgt tccatgaaac gattatccga gcgattgatc attcatacat tcagatgatc 480

tggaacaatc taaaacccgt catggaaagc tttattcttt tatcgatgcg ggtacggtta 540

aaggaaaagt atgaagactt cacaaggatt ttagataacc acgagcttta tattcaagcc 600

atcaaaacaa aggatagggc gctgatgatt cagtctcttc accaaaactt tgatgatgtg 660

caggataagg tagaagacct atggctctca caacaaatgc tggcaaaagg agctgaatac 720

aacaatgact ag 732

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atgctagact ccaaagacct gttgtatccc gcaaaatggc tctcaaaagc gtcaaccgga 60

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attttatcag aaaatacaat cgccgccgag ttttcagtaa gccgttcgcc ggttcgcgaa 180

gcgctaaaaa tactcgcatc cgaaaaaatc atccgcttag aacgaatggg agcggtcgta 240

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tttgtctttg aacggcttgt caaaatagac attgagcctt tagttaagga tctcagcaaa 360

attcttgaaa tgatgaaagt ctcaatcaaa tatgaggatg ctgacgaatt ttcatttcaa 420

gatgtgctgt tccatgaaac gattatccga gcgattgatc attcatacat tcagatgatc 480

tggaacaatc taaaacccgt catggaaagc tttattcttt tatcgatgcg ggtacggtta 540

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atcaaaacaa aggatagggc gctgatgatt cagtctcttc accaaaactt tgatgatgtg 660

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gcaatatggt aaaaatttaa ataaaaatta gaaatgaaag tgtttgcata aaagaaatat 60

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gcaatatggt aaaaatttaa ataaaaatta gaaatgaaag tgtttgacaa aaagaaatat 60

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Met Gly Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu Val

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Glu Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Ser Gly Glu

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Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Phe Ile Cys

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Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr Leu

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Thr Tyr Gly Val Gln Cys Phe Ser Arg Tyr Pro Asp His Met Lys Arg

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His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu Arg

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Thr Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu Val

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Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr Asn

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Val Leu Leu Pro Asp Asn His Tyr Leu Ser Thr Gln Ser Ala Leu Ser

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Thr Ala Ala Gly Ile Thr His Gly Met Asp Glu Leu Tyr Lys

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tattggtatg actggtttta agcgcaaaaa aagttgcttt ttcgtaccta ttaatgtatc 60

gttttagaaa accgactgta aaaagtacag tcggcattat ctcatattat aaaagccagt 120

cattaggcct atctgacaat tcctgaatag agttcataaa caatcctgca tgataaccat 180

cacaaacaga atgatgtacc tgtaaagata gcggtaaata tattgaatta cctttattaa 240

tgaattttcc tgctgtaata atgggtagaa ggtaattact attattattg atatttaagt 300

taaacccagt aaatgaagtc catggaataa tagaaagaga aaaagcattt tcaggtatag 360

gtgttttggg aaacaatttc cccgaaccat tatatttctc tacatcagaa aggtataaat 420

cataaaactc tttgaagtca ttctttacag gagtccaaat accagagaat gttttagata 480

caccatcaaa aattgtataa agtggctcta acttatccca ataacctaac tctccgtcgc 540

tattgtaacc agttctaaaa gctgtatttg agtttatcac ccttgtcact aagaaaataa 600

atgcagggta aaatttatat ccttcttgtt ttatgtttcg gtataaaaca ctaatatcaa 660

tttctgtggt tatactaaaa gtcgtttgtt ggttcaaata atgattaaat atctcttttc 720

tcttccaatt gtctaaatca attttattaa agttcatttg atatgcctcc taaattttta 780

tctaaagtga atttaggagg cttacttgtc tgctttcttc attagaatca atcctttttt 840

aaaagtcaat attactgtaa cataaatata tattttaaaa atatcccact ttatccaatt 900

ttcgtttgtt gaactaatgg gtgctttagt tgaagaataa aagaccacat taaaaaatgt 960

ggtcttttgt gtttttttaa aggatttgag cgtagcgaaa aatccttttc tttcttatct 1020

tgataataag ggtaactatt gccgatcgtc cattccgaca gcatcgccag tcactatggc 1080

gtgctgctag cgccattcgc cattcaggct gcgcaactgt tgggaagggc gatcggtgcg 1140

ggcctcttcg ctattacgcc agctggcgaa agggggatgt gctgcaaggc gattaagttg 1200

ggtaacgcca gggttttccc agtcacgacg ttgtaaaacg acggccagtg aattcgagct 1260

cctagtcatt gttgtattca gctccttttg ccagcatttg ttgtgagagc cataggtctt 1320

ctaccttatc ctgcacatca tcaaagtttt ggtgaagaga ctgaatcatc agcgccctat 1380

cctttgtttt gatggcttga atataaagct cgtggttatc taaaatcctt gtgaagtctt 1440

catacttttc ctttaaccgt acccgcatcg ataaaagaat aaagctttcc atgacgggtt 1500

ttagattgtt ccagatcatc tgaatgtatg aatgatcaat cgctcggata atcgtttcat 1560

ggaacagcac atcttgaaat gaaaattcgt cagcatcctc atatttgatt gagactttca 1620

tcatttcaag aattttgctg agatccttaa ctaaaggctc aatgtctatt ttgacaagcc 1680

gttcaaagac aaatgtttct aatagtaacc gcacatcata aatttccgcg attttcttct 1740

cagttaaacc aattacgacc gctcccattc gttctaagcg gatgattttt tcggatgcga 1800

gtatttttag cgcttcgcga accggcgaac ggcttactga aaactcggcg gcgattgtat 1860

tttctgataa aatagtaccg ctttcaatca gacctgaaac gatccgcatt ctcagctcgt 1920

atgcgacacg aactccggtt gacgcttttg agagccattt tgcgggatac aacaggtctt 1980

tggagtctag catacactca ccttcctcac tcaaggagta tacttgtata caagtatgat 2040

aacgtgaata tttctttttg tcaaacactt tcatttctaa tttttattta aatttttacc 2100

atattgctat gtatattgat tctcatttgc tcgcgccgct gatttccatt gcgcctgatg 2160

aagtcgtgct ttataccgac cagcccgagc acatgatggc aaggaccatt caaaacgtat 2220

ttcaagagag agtggaaatg ctcccgctgc atgcttttac agatgcagaa ataccggtga 2280

agcactcgga aggatgaggg ccggcagcct gtctatttaa ggctgtcggt ttaaaaaaaa 2340

ggaaacgcga tcgtgttgat tttttggatt gaacaattta taatacttgt atacaagtat 2400

actccttaaa ggaggtgaaa tgtacacatg ggtaagggag aagaactttt cactggagtt 2460

gtcccaattc ttgttgaatt agatggtgat gttaatgggc acaaattttc tgtcagtgga 2520

gagggtgaag gtgatgcaac atacggaaaa cttaccctta aatttatttg cactactgga 2580

aagcttcctg ttccttggcc aacacttgtc actactctta cttatggtgt tcaatgcttt 2640

tcaagatacc cagatcatat gaagcggcac gacttcttca agagcgccat gcctgaggga 2700

tacgtgcagg agaggaccat cttcttcaag gacgacggga actacaagac acgtgctgaa 2760

gtcaagtttg agggagacac cctcgtcaac agaatcgagc ttaagggaat cgatttcaag 2820

gaggacggaa acatcctcgg ccacaagttg gaatacaact acaactccca caacgtatac 2880

atcatggcag acaaacaaaa gaatggaatc aaagttaact tcaaaattag acacaacatt 2940

gaagatggaa gcgttcaact agcagaccat tatcaacaaa atactccaat tggcgatggc 3000

cctgtccttt taccagacaa ccattacctg tccacacaat ctgccctttc gaaagatccc 3060

aacgaaaaga gagaccacat ggtccttctt gagtttgtaa cagctgctgg gattacacat 3120

ggcatggatg aactatacaa ataattcacg tcacgcgtcc atggagatct ttgtctgcaa 3180

ctgaaaagtt tataccttac ctggaacaaa tggttgaaac atacgaggct aatatcggct 3240

tattaggaat agtccctgta ctaataaaat caggtggatc agttgatcag tatattttgg 3300

acgaagctcg gaaagaattt ggagatgact tgcttaattc cacaattaaa ttaagggaaa 3360

gaataaagcg atttgatgtt caaggaatca cggaagaaga tactcatgat aaagaagctc 3420

taaaactatt caataacctt acaatggaat tgatcgaaag ggtggaaggt taatggtacg 3480

aaaattaggg gatctaccta gaaagccaca aggcgatagg tcaagcttaa agaaccctta 3540

catggatctt acagattctg aaagtaaaga aacaacagag gttaaacaaa cagaaccaaa 3600

aagaaaaaaa gcattgttga aaacaatgaa agttgatgtt tcaatccata ataagattaa 3660

atcgctgcac gaaattctgg cagcatccga agggaattca tattacttag aggatactat 3720

tgagagagct attgataaga tggttgagac attacctgag agccaaaaaa ctttttatga 3780

atatgaatta aaaaaaagaa ccaacaaagg ctgagacaga ctccaaacga gtctgttttt 3840

ttaaaaaaaa tattaggagc attgaatata tattagagaa ttaagaaaga catgggaata 3900

aaaatatttt aaatccagta aaaatatgat aagattattt cagaatatga agaactctgt 3960

ttgtttttga tgaaaaaaca aacaaaaaaa atccacctaa cggaatctca atttaactaa 4020

cagcggccaa actgagaagt taaatttgag aaggggaaaa ggcggattta tacttgtatt 4080

taactatctc cattttaaca ttttattaaa ccccatacaa gtgaaaatcc tcttttacac 4140

tgttccttta ggtgatcgcg gagggacatt atgagtgaag taaacctaaa aggaaataca 4200

gatgaattag tgtattatcg acagcaaacc actggaaata aaatcgccag gaagagaatc 4260

aaaaaaggga aagaagaagt ttattatgtt gctgaaacgg aagagaagat atggacagaa 4320

gagcaaataa aaaacttttc tttagacaaa tttggtacgc atatacctta catagaaggt 4380

cattatacaa tcttaaataa ttacttcttt gatttttggg gctatttttt aggtgctgaa 4440

ggaattgcgc tctatgctca cctaactcgt tatgcatacg gcagcaaaga cttttgcttt 4500

cctagtctac aaacaatcgc taaaaaaatg gacaagactc ctgttacagt tagaggctac 4560

ttgaaactgc ttgaaaggta cggttttatt tggaaggtaa acgtccgtaa taaaaccaag 4620

gataacacag aggaatcccc gatttttaag attagacgta aggttccttt gctttcagaa 4680

gaacttttaa atggaaaccc taatattgaa attccagatg acgaggaagc acatgtaaag 4740

aaggctttaa aaaaggaaaa agagggtctt ccaaaggttt tgaaaaaaga gcacgatgaa 4800

tttgttaaaa aaatgatgga tgagtcagaa acaattaata ttccagaggc cttacaatat 4860

gacacaatgt atgaagatat actcagtaaa ggagaaattc gaaaagaaat caaaaaacaa 4920

atacctaatc ctacaacatc ttttgagagt atatcaatga caactgaaga ggaaaaagtc 4980

gacagtactt taaaaagcga aatgcaaaat cgtgtctcta agccttcttt tgatacctgg 5040

tttaaaaaca ctaagatcaa aattgaaaat aaaaattgtt tattacttgt accgagtgaa 5100

tttgcatttg aatggattaa gaaaagatat ttagaaacaa ttaaaacagt ccttgaagaa 5160

gctggatatg ttttcgaaaa aatcgaacta agaaaagtgc aataaactgc tgaagtattt 5220

cagcagtttt ttttatttag aaatagtgaa aaaaatataa tcagggaggt atcaatattt 5280

aatgagtact gatttaaatt tatttagact ggaattaata attaacacgt agactaatta 5340

aaatttaatg agggataaag aggatacaaa aatattaatt tcaatcccta ttaaatttta 5400

acaagggggg gattaaaatt taattagagg tttatccaca agaaaagacc ctaataaaat 5460

ttttactagg gttataacac tgattaattt cttaatgggg gagggattaa aatttaatga 5520

caaagaaaac aatcttttaa gaaaagcttt taaaagataa taataaaaag agctttgcga 5580

ttaagcaaaa ctctttactt tttcattgac attatcaaat tcatcgattt caaattgttg 5640

ttgtatcata aagttaattc tgttttgcac aaccttttca ggaatataaa acacatctga 5700

ggcttgtttt ataaactcag ggtcgctaaa gtcaatgtaa cgtagcatat gatatggtat 5760

agcttccacc caagttagcc tttctgcttc ttctgaatgt ttttcatata cttccatggg 5820

tatctctaaa tgattttcct catgtagcaa ggtatgagca aaaagtttat ggaattgata 5880

gttcctctct ttttcttcaa cttttttatc taaaacaaac actttaacat ctgagtcaat 5940

gtaagcataa gatgtttttc cagtcataat ttcaatccca aatcttttag acagaaattc 6000

tggacgtaaa tcttttggtg aaagaatttt tttatgtagc aatatatccg atacagcacc 6060

ttctaaaagc gttggtgaat agggcatttt acctatctcc tctcattttg tggaataaaa 6120

atagtcatat tcgtccatct acctatccta ttatcgaaca gttgaacttt ttaatcaagg 6180

atcagtcctt tttttcatta ttcttaaact gtgctcttaa ctttaacaac tcgatttgtt 6240

tttccagatc tcgagggtaa ctagcctcgc cgatcccgca agaggcccgg cagtcaggtg 6300

gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt atttttctaa atacattcaa 6360

atatgtatcc gctcatgaga caataaccct gataaatgct tcaataatat tgaaaaagga 6420

agagtatgag tattcaacat ttccgtgtcg cccttattcc cttttttgcg gcattttgcc 6480

ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg 6540

gtgcacgagt gggttacatc gaactggatc tcaacagcgg taagatcctt gagagttttc 6600

gccccgaaga acgttttcca atgatgagca cttttaaagt tctgctatgt ggcgcggtat 6660

tatcccgtat tgacgccggg caagagcaac tcggtcgccg catacactat tctcagaatg 6720

acttggttga gtactcacca gtcacagaaa agcatcttac ggatggcatg acagtaagag 6780

aattatgcag tgctgccata accatgagtg ataacactgc ggccaactta cttctgacaa 6840

cgatcggagg accgaaggag ctaaccgctt ttttgcacaa catgggggat catgtaactc 6900

gccttgatcg ttgggaaccg gagctgaatg aagccatacc aaacgacgag cgtgacacca 6960

cgatgcctgt agcaatggca acaacgttgc gcaaactatt aactggcgaa ctacttactc 7020

tagcttcccg gcaacaatta atagactgga tggaggcgga taaagttgca ggaccacttc 7080

tgcgctcggc ccttccggct ggctggttta ttgctgataa atctggagcc ggtgagcgtg 7140

ggtctcgcgg tatcattgca gcactggggc cagatggtaa gccctcccgt atcgtagtta 7200

tctacacgac ggggagtcag gcaactatgg atgaacgaaa tagacagatc gctgagatag 7260

gtgcctcact gattaagcat tggtaactgt cagaccaagt ttactcatat atactttaga 7320

ttgatttaaa acttcatttt taatttaaaa ggatctaggt gaagatcctt tttgataatc 7380

tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa 7440

agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa 7500

aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc 7560

cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgtccttcta gtgtagccgt 7620

agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc 7680

tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac 7740

gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca 7800

gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg 7860

ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag 7920

gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt 7980

ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat 8040

ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc 8100

acatgttctt tcctgcgtta tcccctgatt ctgtggataa ccgtattacc gcctttgagt 8160

gagctgatac cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg agcgaggaag 8220

cggaagagcg cccaatacgc atgcttaagt 8250

<210> 11

<211> 8436

<212> DNA

<213>artificial sequence

<400> 11

tattggtatg actggtttta agcgcaaaaa aagttgcttt ttcgtaccta ttaatgtatc 60

gttttagaaa accgactgta aaaagtacag tcggcattat ctcatattat aaaagccagt 120

cattaggcct atctgacaat tcctgaatag agttcataaa caatcctgca tgataaccat 180

cacaaacaga atgatgtacc tgtaaagata gcggtaaata tattgaatta cctttattaa 240

tgaattttcc tgctgtaata atgggtagaa ggtaattact attattattg atatttaagt 300

taaacccagt aaatgaagtc catggaataa tagaaagaga aaaagcattt tcaggtatag 360

gtgttttggg aaacaatttc cccgaaccat tatatttctc tacatcagaa aggtataaat 420

cataaaactc tttgaagtca ttctttacag gagtccaaat accagagaat gttttagata 480

caccatcaaa aattgtataa agtggctcta acttatccca ataacctaac tctccgtcgc 540

tattgtaacc agttctaaaa gctgtatttg agtttatcac ccttgtcact aagaaaataa 600

atgcagggta aaatttatat ccttcttgtt ttatgtttcg gtataaaaca ctaatatcaa 660

tttctgtggt tatactaaaa gtcgtttgtt ggttcaaata atgattaaat atctcttttc 720

tcttccaatt gtctaaatca attttattaa agttcatttg atatgcctcc taaattttta 780

tctaaagtga atttaggagg cttacttgtc tgctttcttc attagaatca atcctttttt 840

aaaagtcaat attactgtaa cataaatata tattttaaaa atatcccact ttatccaatt 900

ttcgtttgtt gaactaatgg gtgctttagt tgaagaataa aagaccacat taaaaaatgt 960

ggtcttttgt gtttttttaa aggatttgag cgtagcgaaa aatccttttc tttcttatct 1020

tgataataag ggtaactatt gccgatcgtc cattccgaca gcatcgccag tcactatggc 1080

gtgctgctag cgccattcgc cattcaggct gcgcaactgt tgggaagggc gatcggtgcg 1140

ggcctcttcg ctattacgcc agctggcgaa agggggatgt gctgcaaggc gattaagttg 1200

ggtaacgcca gggttttccc agtcacgacg ttgtaaaacg acggccagtg aattcgagct 1260

cctagtcatt gttgtattca gctccttttg ccagcatttg ttgtgagagc cataggtctt 1320

ctaccttatc ctgcacatca tcaaagtttt ggtgaagaga ctgaatcatc agcgccctat 1380

cctttgtttt gatggcttga atataaagct cgtggttatc taaaatcctt gtgaagtctt 1440

catacttttc ctttaaccgt acccgcatcg ataaaagaat aaagctttcc atgacgggtt 1500

ttagattgtt ccagatcatc tgaatgtatg aatgatcaat cgctcggata atcgtttcat 1560

ggaacagcac atcttgaaat gaaaattcgt cagcatcctc atatttgatt gagactttca 1620

tcatttcaag aattttgctg agatccttaa ctaaaggctc aatgtctatt ttgacaagcc 1680

gttcaaagac aaatgtttct aatagtaacc gcacatcata aatttccgcg attttcttct 1740

cagttaaacc aattacgacc gctcccattc gttctaagcg gatgattttt tcggatgcga 1800

gtatttttag cgcttcgcga accggcgaac ggcttactga aaactcggcg gcgattgtat 1860

tttctgataa aatagtaccg ctttcaatca gacctgaaac gatccgcatt ctcagctcgt 1920

atgcgacacg aactccggtt gacgcttttg agagccattt tgcgggatac aacaggtctt 1980

tggagtctag catcccaaat gttaactcta tatatatgta tctctttttt taaattaatc 2040

ttattgattt ctattataag gatagattcc catcagaaat agtcttgact tcatgtgaca 2100

aatttcttaa ttattcgttc taaaaaaaag tttttaaggg tattaaatat ttcattttca 2160

ttgcaaaggt gacaaaaaac gagtgctttt gtcatgtttt atgggatgtt tttcaacata 2220

aaggaaataa atgaaaaact ttcggggtcg gctcatatat cggaaaaatg agtgataaag 2280

tatattcgag gagtatgtat attgattctc atttgctcgc gccgctgatt tccattgcgc 2340

ctgatgaagt cgtgctttat accgaccagc ccgagcacat gatggcaagg accattcaaa 2400

acgtatttca agagagagtg gaaatgctcc cgctgcatgc ttttacagat gcagaaatac 2460

cggtgaagca ctcggaagga tgagggccgg cagcctgtct atttaaggct gtcggtttaa 2520

aaaaaaggaa acgcgatcgt gttgattttt tggattgaac aatttataat acttgtatac 2580

aagtatactc cttaaaggag gtgaaatgta cacatgggta agggagaaga acttttcact 2640

ggagttgtcc caattcttgt tgaattagat ggtgatgtta atgggcacaa attttctgtc 2700

agtggagagg gtgaaggtga tgcaacatac ggaaaactta cccttaaatt tatttgcact 2760

actggaaagc ttcctgttcc ttggccaaca cttgtcacta ctcttactta tggtgttcaa 2820

tgcttttcaa gatacccaga tcatatgaag cggcacgact tcttcaagag cgccatgcct 2880

gagggatacg tgcaggagag gaccatcttc ttcaaggacg acgggaacta caagacacgt 2940

gctgaagtca agtttgaggg agacaccctc gtcaacagaa tcgagcttaa gggaatcgat 3000

ttcaaggagg acggaaacat cctcggccac aagttggaat acaactacaa ctcccacaac 3060

gtatacatca tggcagacaa acaaaagaat ggaatcaaag ttaacttcaa aattagacac 3120

aacattgaag atggaagcgt tcaactagca gaccattatc aacaaaatac tccaattggc 3180

gatggccctg tccttttacc agacaaccat tacctgtcca cacaatctgc cctttcgaaa 3240

gatcccaacg aaaagagaga ccacatggtc cttcttgagt ttgtaacagc tgctgggatt 3300

acacatggca tggatgaact atacaaataa ttcacgtcac gcgtccatgg agatctttgt 3360

ctgcaactga aaagtttata ccttacctgg aacaaatggt tgaaacatac gaggctaata 3420

tcggcttatt aggaatagtc cctgtactaa taaaatcagg tggatcagtt gatcagtata 3480

ttttggacga agctcggaaa gaatttggag atgacttgct taattccaca attaaattaa 3540

gggaaagaat aaagcgattt gatgttcaag gaatcacgga agaagatact catgataaag 3600

aagctctaaa actattcaat aaccttacaa tggaattgat cgaaagggtg gaaggttaat 3660

ggtacgaaaa ttaggggatc tacctagaaa gccacaaggc gataggtcaa gcttaaagaa 3720

cccttacatg gatcttacag attctgaaag taaagaaaca acagaggtta aacaaacaga 3780

accaaaaaga aaaaaagcat tgttgaaaac aatgaaagtt gatgtttcaa tccataataa 3840

gattaaatcg ctgcacgaaa ttctggcagc atccgaaggg aattcatatt acttagagga 3900

tactattgag agagctattg ataagatggt tgagacatta cctgagagcc aaaaaacttt 3960

ttatgaatat gaattaaaaa aaagaaccaa caaaggctga gacagactcc aaacgagtct 4020

gtttttttaa aaaaaatatt aggagcattg aatatatatt agagaattaa gaaagacatg 4080

ggaataaaaa tattttaaat ccagtaaaaa tatgataaga ttatttcaga atatgaagaa 4140

ctctgtttgt ttttgatgaa aaaacaaaca aaaaaaatcc acctaacgga atctcaattt 4200

aactaacagc ggccaaactg agaagttaaa tttgagaagg ggaaaaggcg gatttatact 4260

tgtatttaac tatctccatt ttaacatttt attaaacccc atacaagtga aaatcctctt 4320

ttacactgtt cctttaggtg atcgcggagg gacattatga gtgaagtaaa cctaaaagga 4380

aatacagatg aattagtgta ttatcgacag caaaccactg gaaataaaat cgccaggaag 4440

agaatcaaaa aagggaaaga agaagtttat tatgttgctg aaacggaaga gaagatatgg 4500

acagaagagc aaataaaaaa cttttcttta gacaaatttg gtacgcatat accttacata 4560

gaaggtcatt atacaatctt aaataattac ttctttgatt tttggggcta ttttttaggt 4620

gctgaaggaa ttgcgctcta tgctcaccta actcgttatg catacggcag caaagacttt 4680

tgctttccta gtctacaaac aatcgctaaa aaaatggaca agactcctgt tacagttaga 4740

ggctacttga aactgcttga aaggtacggt tttatttgga aggtaaacgt ccgtaataaa 4800

accaaggata acacagagga atccccgatt tttaagatta gacgtaaggt tcctttgctt 4860

tcagaagaac ttttaaatgg aaaccctaat attgaaattc cagatgacga ggaagcacat 4920

gtaaagaagg ctttaaaaaa ggaaaaagag ggtcttccaa aggttttgaa aaaagagcac 4980

gatgaatttg ttaaaaaaat gatggatgag tcagaaacaa ttaatattcc agaggcctta 5040

caatatgaca caatgtatga agatatactc agtaaaggag aaattcgaaa agaaatcaaa 5100

aaacaaatac ctaatcctac aacatctttt gagagtatat caatgacaac tgaagaggaa 5160

aaagtcgaca gtactttaaa aagcgaaatg caaaatcgtg tctctaagcc ttcttttgat 5220

acctggttta aaaacactaa gatcaaaatt gaaaataaaa attgtttatt acttgtaccg 5280

agtgaatttg catttgaatg gattaagaaa agatatttag aaacaattaa aacagtcctt 5340

gaagaagctg gatatgtttt cgaaaaaatc gaactaagaa aagtgcaata aactgctgaa 5400

gtatttcagc agtttttttt atttagaaat agtgaaaaaa atataatcag ggaggtatca 5460

atatttaatg agtactgatt taaatttatt tagactggaa ttaataatta acacgtagac 5520

taattaaaat ttaatgaggg ataaagagga tacaaaaata ttaatttcaa tccctattaa 5580

attttaacaa gggggggatt aaaatttaat tagaggttta tccacaagaa aagaccctaa 5640

taaaattttt actagggtta taacactgat taatttctta atgggggagg gattaaaatt 5700

taatgacaaa gaaaacaatc ttttaagaaa agcttttaaa agataataat aaaaagagct 5760

ttgcgattaa gcaaaactct ttactttttc attgacatta tcaaattcat cgatttcaaa 5820

ttgttgttgt atcataaagt taattctgtt ttgcacaacc ttttcaggaa tataaaacac 5880

atctgaggct tgttttataa actcagggtc gctaaagtca atgtaacgta gcatatgata 5940

tggtatagct tccacccaag ttagcctttc tgcttcttct gaatgttttt catatacttc 6000

catgggtatc tctaaatgat tttcctcatg tagcaaggta tgagcaaaaa gtttatggaa 6060

ttgatagttc ctctcttttt cttcaacttt tttatctaaa acaaacactt taacatctga 6120

gtcaatgtaa gcataagatg tttttccagt cataatttca atcccaaatc ttttagacag 6180

aaattctgga cgtaaatctt ttggtgaaag aattttttta tgtagcaata tatccgatac 6240

agcaccttct aaaagcgttg gtgaataggg cattttacct atctcctctc attttgtgga 6300

ataaaaatag tcatattcgt ccatctacct atcctattat cgaacagttg aactttttaa 6360

tcaaggatca gtcctttttt tcattattct taaactgtgc tcttaacttt aacaactcga 6420

tttgtttttc cagatctcga gggtaactag cctcgccgat cccgcaagag gcccggcagt 6480

caggtggcac ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac 6540

attcaaatat gtatccgctc atgagacaat aaccctgata aatgcttcaa taatattgaa 6600

aaaggaagag tatgagtatt caacatttcc gtgtcgccct tattcccttt tttgcggcat 6660

tttgccttcc tgtttttgct cacccagaaa cgctggtgaa agtaaaagat gctgaagatc 6720

agttgggtgc acgagtgggt tacatcgaac tggatctcaa cagcggtaag atccttgaga 6780

gttttcgccc cgaagaacgt tttccaatga tgagcacttt taaagttctg ctatgtggcg 6840

cggtattatc ccgtattgac gccgggcaag agcaactcgg tcgccgcata cactattctc 6900

agaatgactt ggttgagtac tcaccagtca cagaaaagca tcttacggat ggcatgacag 6960

taagagaatt atgcagtgct gccataacca tgagtgataa cactgcggcc aacttacttc 7020

tgacaacgat cggaggaccg aaggagctaa ccgctttttt gcacaacatg ggggatcatg 7080

taactcgcct tgatcgttgg gaaccggagc tgaatgaagc cataccaaac gacgagcgtg 7140

acaccacgat gcctgtagca atggcaacaa cgttgcgcaa actattaact ggcgaactac 7200

ttactctagc ttcccggcaa caattaatag actggatgga ggcggataaa gttgcaggac 7260

cacttctgcg ctcggccctt ccggctggct ggtttattgc tgataaatct ggagccggtg 7320

agcgtgggtc tcgcggtatc attgcagcac tggggccaga tggtaagccc tcccgtatcg 7380

tagttatcta cacgacgggg agtcaggcaa ctatggatga acgaaataga cagatcgctg 7440

agataggtgc ctcactgatt aagcattggt aactgtcaga ccaagtttac tcatatatac 7500

tttagattga tttaaaactt catttttaat ttaaaaggat ctaggtgaag atcctttttg 7560

ataatctcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg 7620

tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc 7680

aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc 7740

tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtc cttctagtgt 7800

agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc 7860

taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact 7920

caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac 7980

agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag 8040

aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg 8100

gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg 8160

tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga 8220

gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt 8280

ttgctcacat gttctttcct gcgttatccc ctgattctgt ggataaccgt attaccgcct 8340

ttgagtgagc tgataccgct cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg 8400

aggaagcgga agagcgccca atacgcatgc ttaagt 8436

Claims (10)

1. a kind of inducing expression element, which is characterized in that the inducing substrate of the element responds is gluconate；The element Contain GntR albumen, P_lytEPromoter, the promoter P for integrating GntR binding sequence_thrS；The P_lytEPromoter regulation GntR egg It is white；The promoter P for integrating GntR binding sequence_thrSContain the nucleotide sequence as shown in SEQ ID NO.4；Promoter P_lytEWith promoter P_thrSTranscriptional orientation it is opposite.

2. inducing expression element according to claim 1, which is characterized in that encode the P_lytEThe nucleotides sequence of promoter Column are as shown in SEQ ID NO.1.

3. inducing expression element according to claim 1, which is characterized in that the transcriptional regulation protein GntR contains SEQ Amino acid sequence shown in ID NO.2, or, the gene for encoding the transcriptional regulation protein GntR contains such as SEQ ID NO.6 institute The nucleotide sequence shown.

4. any inducing expression element according to claim 1~3, which is characterized in that the GntR albumen, P_lytEStarting Son and the promoter P for integrating GntR binding sequence_thrSIt is arranged on same plasmid.

5. inducing expression element according to claim 4, which is characterized in that the plasmid include pHT01, pP43NMK or pSTOP1622。

6. the genetic engineering bacterium containing any inducing expression element of Claims 1 to 5.

7. genetic engineering bacterium according to claim 6, which is characterized in that with bacillus subtilis be host.

8. any inducing expression element of Claims 1 to 5 is promoting destination protein expression or is reducing albumen leakage expression side The application in face.

9. application of any inducing expression element of Claims 1 to 5 in the microorganism of screening expression target gene.

10. the genetic engineering bacterium of claim 6 or 7 answering in terms of promoting destination protein expression or reducing albumen leakage expression With.