CN112143741B - Biomembrane gene island GIVal43097 and excision method thereof - Google Patents

Abstract

The invention discloses a biomembrane gene island GIVal43097 and a cutting method thereof. The nucleotide sequence of the biomembrane gene island GIVal43097 is shown in SEQ ID NO. 1. The invention utilizes molecular biology means, and identifies and directionally excises the gene island for enhancing the biofilm formation, thereby eliminating the carried gene on one hand, preventing the horizontal transmission of the gene island on the other hand, simultaneously protecting the flora balance to the maximum extent, being a novel strategy for eliminating the bacterial biofilm and achieving the purpose of weakening the pathogenic vibrio biofilm.

Description

Biomembrane gene island GIVal43097 and excision method thereof

The technical field is as follows:

the invention belongs to the fields of biochemistry and molecular biology, and particularly relates to a biomembrane gene island GIVal43097 and a excision method thereof.

Background art:

biofilms are the predominant form of bacteria found in the environment. Biofilm (bifilm), also known as biofilm, is a bacterial community formed by adhesion of bacteria to extracellular matrices such as polysaccharides, proteins, and DNA secreted from the surfaces of living or non-living organisms. Extracellular secretions in biofilms provide a barrier to the survival of microorganisms, and bacteria in the biofilm state are better resistant to environmental stresses, and thus biofilms are also referred to as semi-dormant states of bacteria. The existence form of the biofilm is various, and the biofilm comprises a solid-liquid biofilm or an adhesive biofilm formed between a solid medium and a liquid medium, a gas-liquid biofilm formed between a gas medium and a liquid medium, and a gas-solid biofilm formed between the gas medium and the solid medium and composed of a bacterial community. An article of the national institutes of health in the united states of 2014 indicated that biofilms were involved in more than 60% of microbial infection processes. Some pathogenic bacteria can regulate and control the formation of a biological membrane to enhance the pathogenic capability of the pathogenic bacteria, for example, when the human pathogenic bacteria vibrio cholerae enters the intestinal tract, the pathogenic bacteria can adapt to the acidic environment in the intestinal tract of a human well by regulating and controlling the large formation of the biological membrane, protect the pathogenic bacteria from being influenced by bile and low pH, and maintain the pathogenic capability; when the vibrio cholerae begins to infect human bodies, the expression of intracellular polysaccharide is inhibited, so that the biological membrane is promoted to be thinned, and the infection of hosts is facilitated. Coral pathogenic bacteria, namely vibrio coralyticus, quickly forms a biological membrane on the surface layer of coral mucus when infecting coral antler, and then induces the expression of virulence factors to exert pathogenicity.

Research shows that horizontal transfer elements such as gene islands and prophages participate in the formation and regulation of biological membranes. For example, the excision of the original phage CP4So in Shewanella MR-1 can enhance the biofilm of host bacteria and increase the survival rate of the host at low temperature. Meanwhile, the formation of the biological membrane can further regulate and control the cutting-off and the release of horizontal transfer elements such as prophages and the like. For example, when Escherichia coli K-12 forms a biofilm, the CP4-57 prophage on its genome is excised, further promoting the formation of a population biofilm. The Pf filamentous prophage is largely replicated and released in the process of forming the pseudomonas aeruginosa biofilm, and the pathogenicity of the bacteria is enhanced. However, the research objects of the biofilm are mainly concentrated in escherichia coli model bacteria and pathogenic bacteria of human and animals at present, and the research on the biofilm in marine bacteria is relatively less due to the pathogenic action and mechanism. Some species of Vibrio, such as Vibrio alginolyticus and Vibrio coraolyticus, cause coral diseases, and the presence of biofilm enhances the pathogenicity. Therefore, there is an urgent need to identify gene islands that regulate biofilm formation in these pathogenic bacteria, and to establish a highly efficient and targeted gene island excision method that ultimately attenuates bacterial biofilm formation.

The invention content is as follows:

the first purpose of the invention is to provide a gene island GIVal43097 for regulating and controlling the formation of bacterial biofilm in Vibrio alginolyticus.

The nucleotide sequence of the biomembrane gene island GIVal43097 is shown in SEQ ID NO. 1.

The second objective of the invention is to provide a guanylate cyclase gene, the nucleotide sequence of which is shown as 1907-2812 base of SEQ ID NO.1 or as 5342-6748 base of SEQ ID NO. 1.

The invention also provides application of the guanylate cyclase gene in promoting bacterial biofilm formation.

The third purpose of the invention is to provide the application of the dgc137 gene in the knock-out biomembrane gene island GIVal43097 in reducing the biomembrane synthesis.

The fourth object of the present invention is to provide an excision enzyme alpA, the nucleotide sequence of which is the reverse complement of the sequence shown by the 15008-15217 th base of SEQ ID NO. 1.

A fifth object of the present invention is to provide the use of an overexpressed excisionase alpA to mediate excision of the biofilm gene GIVal43097, thereby further removing the formation of bacterial biofilms in organisms, such as Vibrio alginolyticus SCSIO 43097 biofilms.

The sixth object of the present invention is to provide a method for excising the biofilm gene island GIVal43097 by overexpressing the excision enzyme alpA in an organism containing the biofilm gene island GIVal 43097.

The existing bacterial biofilm removal method is removal or inhibition by physical and chemical means, and the application in organisms is greatly limited. The invention utilizes molecular biology means, and identifies and directionally excises the gene island for enhancing the biofilm formation, thereby eliminating the carried gene on one hand, preventing the horizontal transmission of the gene island on the other hand, simultaneously protecting the flora balance to the maximum extent, being a novel strategy for eliminating the bacterial biofilm and achieving the purpose of weakening the pathogenic vibrio biofilm.

Compared with the prior art, the advantages are as follows:

1. the invention utilizes molecular biology means to remove bacterial biofilms in organisms. The current biofilm removal method mainly aims at the biofilms on medical devices or chronic wounds on body surfaces, and cannot be directly used for removing the biofilms formed by bacteria in organisms because mechanical treatment or chemical substance treatment is used.

2. The gene island is cut off by utilizing the overexpression of the excision enzyme coded by the gene island, so that the method is a specific knockout method, and compared with a chemical substance treatment method, the method cannot damage the flora balance.

3. High efficiency, the existing physical and chemical method is used for removing the biological membrane, the selected chemical reagent needs experimental test, and the removal effect cannot be estimated. The efficiency of gene island excision by the excision enzyme overexpression method is close to 100 percent.

Description of the drawings:

FIG. 1 shows the sequence analysis (A) of GIVal43097 gene island and the domain analysis and knock-out PCR detection of the biomembrane associated proteins Dgc137(B), Dgc139(C) and Dgc140 (D);

FIG. 2 is the identification of the biofilm-associated gene on Gene island GIVal 43097. Knocking out mutant strains delta dgc137, delta dgc139 and delta dgc140 from genes dgc137, dgc139 and dgc140, and carrying out phenotype detection on a gas-liquid biomembrane (A), a solid-liquid biomembrane (B) and a gas-solid biomembrane (C) of a gene island GIVal43097 excised strain delta GIVal43097 (abbreviated as delta GI, the same below); and dgc137 gene complemented gas-solid biofilm, gas-liquid biofilm (D) and solid-liquid biofilm (E), WT is wild type, WT/pBBR1Cm represents expression of empty vector in wild type, delta dgc137/pBBR1Cm represents expression of empty vector in delta dgc137, delta dgc137/pDGc137 represents expression of pBBR1Cm containing dgc137 complemented plasmid in delta dgc137, and delta GI/pBBR1Cm represents expression of empty vector in delta GI.

FIG. 3 is a schematic diagram (A) and excision efficiency (B) of excision of GIVal43097 gene island mediated by excision enzyme AlpA, and detection of solid-liquid biofilm (C), gas-solid biofilm (D) and gas-liquid biofilm (E) of gene island GIVal43097 deletion mutant strain, 43097 is wild strain containing GIVal 43097.

FIG. 4 shows the detection of solid-liquid biomembrane (A) and gas-solid biomembrane (B) after heterologous expression of key genes dgc137, dgc139 and dgc140 on gene island GIVal43097 in Pseudomonas aeruginosa PAO1, and PAO1/pHERD20T is a control of expression empty vector in PAO 1.

The specific implementation mode is as follows:

the following examples are further illustrative of the present invention and are not intended to be limiting thereof.

Example 1:

firstly, extracting the genomic DNA of Vibrio algyrinolyticus SCSIO 43097:

the strain Vibrio alginolyticus SCSIO 43097 preserved at-80 ℃ is streaked on a 2216E culture medium plate, and is kept in an incubator at 30 ℃ for overnight; picking single colony in a 250ml triangular flask containing 25ml fresh 2216E culture medium, culturing in an incubator at 25 ℃ for 12 hours under 220rpm shaking; transferred at 1:100 into a 250ml Erlenmeyer flask containing 25ml of fresh 2216E medium, and shake-cultured at 220rpm for 5-6 hours (to log phase); centrifuging at 12,000rpm for 1min, collecting thallus, and removing supernatant; total DNA extraction was performed using a bacterial genomic DNA extraction kit (TIANGEN, BEIJING) to obtain SCSIO 43097 genomic DNA, which was used for whole genome sequencing and annotation analysis of SCSIO 43097, and by using Mauve software to perform sequence alignment with the closely related bacterium Vibrio algyrinthulius ATCC 33787, it was found that SCSIO 43097 contained a 22kb or so genetic island having the nucleotide sequence shown in SEQ ID NO.1, which encodes 1 integrase, 1 excise, 3 guanylate cyclases and 16 putative proteins (Table 1 and FIG. 1A).

TABLE 1 functional annotation of the genes contained in the Gene island GIVal43097

Identification of biomembrane related gene on GIVal43097 gene island

The guanylate cyclase is reported to be involved in the synthesis of C-di-GMP so as to regulate bacterial biomembrane, so that the gene island is supposed to regulate the formation of host bacterial biomembrane. The GIVal43097 gene island contains three genes dgc137, dgc139 and dgc140 containing GGDEF domain and encoding guanylate cyclase. Specific primers (table 2) were designed, and pMBLcas9 vector was used to knock out these three genes, respectively, to finally obtain three single-gene knock-out mutants Δ dgc, Δ 63139, and Δ dgc (fig. 1B-D). It was found that biofilm was significantly reduced after dgc137 knockdown, and that dgc139 and dgc140 knockdown had no significant effect on biofilm (fig. 2). In conclusion, dgc137 is an essential gene for SCSIO 43097 to form a large number of biofilms.

TABLE 2 primers used in the present invention

The method for gene knockout and gene complementation in Vibrio alginolyticus SCSIO 43097 comprises the following steps:

in the present study, a plasmid pMBLcas9 (the construction method is shown in Wang et al, Journal of analytical chemistry 2019,74(9), 2559-2565) depending on a CRISPR/Cas9 system is selected for gene knockout. First, gene fragments of upstream and downstream homologous arms and sgrnas were obtained by PCR amplification using primers (table 2); the homologous arms and sgRNA fragments were purified, pMBLcas9 was digested with AhdI, multi-fragment recombination was performed using ClonExpress MultiS kit (Vazyme), the recombinant fragments were introduced into E.coli WM3064 competent cells by chemical transformation, and the recombinant plasmids were verified by sequencing. The recombinant plasmid was subsequently transferred into v.algyrinolyticus SCSIO 43097 using the conjugative transfer method. Briefly, WM3064 containing recombinant plasmid is used as donor bacteria, SCSIO 43097 is used as recipient bacteria to perform conjugation transfer experiment, and the donor bacteria and the recipient bacteria are respectively cultured to OD ₆₀₀ At 1.0 rpm, the cells were centrifuged at 6000rpm for 2 minutes to collect cells, which were then cultured in a mediumEach wash was performed 2 times, the supernatants were removed and resuspended in 50. mu.L of LB, and both cells were spotted onto LB plates containing 0.3mM DAP. After overnight incubation, the lawn was streaked out using an inoculating loop to 2216E plates containing chloramphenicol (15. mu.g/mL). The zygotes obtained on the resistant plates are subjected to wF/wR primer PCR to determine correct knockout strains, so that dgc137, dgc139 and dgc140 are knocked out, and three single-gene knockout mutants, namely delta dgc137, delta dgc139 and delta dgc140, are obtained.

The gene dgc137 was complemented back in this study using plasmid pBBR1Cm (see Zeng et al, applied Microbiol Biotechnol,2015,99, 10127-. First, the coding region and promoter region of gene dgc137 were amplified by PCR using primers (dgc 137-pBBR1Cm-F and dgc137-pBBR1Cm-R in Table 2); the PCR fragment was purified and recombinantly inserted into the SalI and KpnI sites of the pBBR1Cm vector using the Clonexpress II One Step Cloning Kit (Vazyme); the plasmid is introduced into escherichia coli WM3064 competent cells by a chemical transformation method, and the plasmid is supplemented by dgc137 through sequencing verification. Subsequently, the dgc137 complementation plasmid was transferred into the single knockout mutant Δ dgc137 by the conjugative transfer method using an LB plate containing 0.3mM DAP. After overnight culture, the lawn was picked with an inoculating loop and streaked onto 2216E plates containing chloramphenicol (15. mu.g/mL) to detect the correct transformants, thereby achieving complementation of dgc137 to obtain the complemented strain Δ dgc137/pDGc 137.

Expression of the excision enzyme AlpA in v.algyrinyticus SCSIO 43097 and targeted excision of gene island GIVal 43097:

the construction of pHGECm vector for the expression of the excision enzyme AlpA in SCSIO 43097 (see Wang et al, Environmental Microbiology,2017,19(11): 4620-4637) was used. First, the excision enzyme gene alpA encoded by the gene island GIVal43097 itself was obtained by PCR amplification using primers (Table 2), the PCR fragment was ligated between pHGECm vector EcoRI and BamHI cleavage sites, introduced into Escherichia coli WM3064 competent cells by chemical transformation, and the recombinant plasmid pHGECm-alpA was verified by sequencing, and the obtained strain was WM 3064/pHGECm-alpA. Performing conjugation transfer experiment by using WM3064/pHGECm-alpA as donor bacteria and SCSIO 43097 as acceptor bacteria, and cleaning bacteria liquid with culture mediumResuspend, mix well and spot onto LB plate containing 0.3mM DAP. After overnight culture, the lawn was picked with an inoculating loop and streaked onto LB plate containing chloramphenicol (30. mu.g/mL), and the recombinant strain was obtained by confirmation. The correct recombinant strain 43097/pHGECm-alpA was activated overnight and 1/100 was transferred to 25mL fresh 2216E medium containing chloramphenicol (15. mu.g/mL) to OD ₆₀₀ When the gene island GIVal43097 is directionally excised, IPTG is added when the gene island GIVal43097 is changed to about 1.0, the strain is subjected to shake culture for 6-8 hours at 220rpm in a shaking table at 30 ℃, an inoculating loop of the strain liquid is streaked to an LB plate to obtain a single colony, and a correct gene island excised mutant strain is determined by using 43097GI-wF/-wR primers, so that a mutant strain delta GIVal43097 with the gene island GIVal43097 directionally excised is obtained.

And fourthly, detecting the biofilm phenotype of gene island excisions (single gene knockout mutants delta dgc137, delta dgc139 and delta dgc140, and a mutant strain delta GIVal 43097):

1. solid-liquid biofilm determination:

first, a single colony of the strain was inoculated into a flask containing 25mL of 2216E medium and cultured overnight at 30 ℃. Subsequently, the overnight-cultured broth was diluted to OD ₆₀₀ When the medium was changed to 0.05, the medium was transferred to fresh 2216E medium, and 250 μ L of each diluted medium was put in a 96-well plate, and a sample to which only 2216E medium was added was used as a blank. Then placing 96-well plate in 30 deg.C constant temperature incubator for static culture (without shaking during culture), culturing for 2 hr, 4 hr, 6 hr or 8 hr respectively, and measuring OD with enzyme labeling instrument ₆₂₀ The numerical value of (c). The culture solution was decanted, the 96-well plate was gently rinsed with tap water 5 times, the free cells were washed off, and 280. mu.L of a 0.1% crystal violet solution was added for staining. Standing at room temperature for 20min, pouring out the crystal violet solution, continuously soaking and washing the 96-well plate with clean tap water gently for 5 times, washing off free crystal violet, inverting the 96-well culture plate on water-absorbing filter paper to remove residual water, naturally drying, adding 300 μ L of absolute ethyl alcohol, and standing for 5-10min to fully dissolve the crystal violet. Finally, the OD of the 96-well plate was read by a microplate reader ₅₄₀ Numerical values. Each strain was made in 3 replicates, each replicate was made in 3 replicates. The numerical calculation of the solid-liquid biofilm adopts Sample (OD540 nm) -Blank (OD540 nm)/Sample (OD620nm) -Blank (OD620 nm).

2. And (3) measuring the gas-liquid biofilm:

first, a single clone was picked and inoculated into a flask containing 25mL of 2216E medium and cultured overnight at 30 ℃. Then, the overnight-cultured broth was diluted to OD ₆₀₀ Transfer to fresh 2216E medium (0.05), aspirate 4mL of diluted inoculum into a 10mL glass tube. The cells were kept standing in an incubator at 30 ℃ and observed, recorded and photographed several times a day (the test tube was not shaken when the observation was noticed).

3. Measurement of gas-solid biofilm:

first, a single clone was picked and inoculated into a flask containing 25mL of 2216E medium and cultured overnight at 30 ℃. Sucking 10 μ L of bacterial liquid to contain FeCl ₃ The culture medium is statically cultured in an incubator at 30 ℃ on a plate of seawater LB culture medium, LB culture medium or other nutrient culture media, and the colony morphology is observed, recorded and photographed every day.

The gene dgc137 encoding the gene GIVal43097 can obviously enhance the biofilm formation of host vibrio. Meanwhile, the gene island is an important vector for gene horizontal transfer, generally has horizontal transfer capacity and can be transmitted among different species of bacteria in the same ecological niche, so that the establishment of a method for directly cutting the gene island from a genome can remove genes contained in the gene island and also eliminate the transmission capacity of the gene island. The research shows that the gene island coded excision AlpA can mediate the excision of the gene island (FIGS. 3A and B), and we use the method to obtain the gene island deletion mutant delta GIVal 43097. Subsequently, we compared the biofilm formation abilities of the genetic island deletion mutant Δ GIVal43097 and the wild strain, and the results showed that the deletion of GIVal43097 significantly reduced the host bacteria solid-liquid biofilm, gas-solid biofilm, and gas-liquid biofilm formation abilities (fig. 3C-E).

And fifthly, carrying out biomembrane phenotype detection on heterologous expression of biomembrane key genes on the gene island (dgc137, dgc139 and dgc140 are respectively expressed in pseudomonas aeruginosa PAO 1):

genes dgc137, dgc139 and dgc140 are expressed in Pseudomonas aeruginosa PAO1 and constructed by using pHERD20T vector (the construction method is shown in the literature: Li et al, Molecular Microbiology,2019,111(2), 495-. First, the coding regions of genes dgc137, dgc139 and dgc140 were amplified by PCR using primers (Table 2), the PCR fragment was ligated between the restriction sites of the vector NcoI and HindIII of pHERD20T, introduced into competent cells of Escherichia coli WM3064 by chemical transformation, and the recombinant plasmids pdgc137, pdgc139 and pdgc140 were verified by sequencing. WM3064 containing recombinant plasmids is used as donor bacteria, PAO1 is used as recipient bacteria to carry out a conjugal transfer experiment, the bacterial liquid is cleaned and resuspended by using a culture medium, the mixture is dripped on an LB plate containing DAP of 0.3mM after being mixed evenly, after being cultured for 6h at 37 ℃, lawn is picked by an inoculating loop and is streaked on the LB plate containing carbenicillin (100 mu g/mL), thereby obtaining recombinant bacteria PAO1/pdgc137, PAO1/pdgc139 and PAO1/pdgc140 of which the related genes on a gene island are expressed heterologously in pseudomonas aeruginosa. As a control, PAO1/pHERD20T was used which had been transferred into the empty vector pHERD 20T.

The correct recombinant strains were activated by overnight incubation at 37 ℃ and subsequently the overnight incubated broth was diluted to OD ₆₀₀ When the sample was changed to 0.05, the sample was transferred to a fresh LB medium containing carbenicillin (100 μ g/mL) and 10mM arabinose, and 250 μ L of each diluted culture solution was put in a 96-well plate, and a sample to which only LB medium was added was used as a blank. Then placing 96-well plate in 37 deg.C constant temperature incubator for static culture (no shaking during culture), respectively culturing for 2 hr, and measuring OD with enzyme labeling instrument ₆₂₀ The numerical value of (c). The culture solution was decanted, the 96-well plate was gently rinsed with tap water 5 times, the free cells were washed off, and 280. mu.L of a 0.1% crystal violet solution was added for staining. Standing at room temperature for 20min, pouring out the crystal violet solution, further gently washing the 96-well plate with clean tap water for 5 times, washing off free crystal violet, inverting the 96-well culture plate on water-absorbing filter paper to remove residual water, naturally air drying, adding 300 μ L of anhydrous ethanol, and standing for 5-10min to fully dissolve the crystal violet. Finally, the OD of the 96-well plate was read with a microplate reader ₅₄₀ Numerical values. 3 replicates were made for each strain, 3 replicates for each. The numerical calculation of the solid-liquid biofilm was carried out by sampling (OD540 nm) -Blank (OD540 nm)/sampling (OD620nm) -Blank (OD620 nm).

The biofilm forming capability of the recombinant bacteria is detected by the method, and dgc137 shows that the biofilm forming capability of PAO1 is remarkably enhanced; dgc140 also enhanced biofilm-forming ability of PAO1, but was less potent than dgc 137; dgc139 had no effect on the biofilm-forming ability of PAO1 and was not different from the empty vector pHERD20T (FIG. 4).

Sequence listing

<110> Nanhai ocean institute of Chinese academy of sciences

<120> biomembrane gene island GIVal43097 and excision method thereof

<160> 1

<170> SIPOSequenceListing 1.0

<210> 1

<211> 21792

<212> DNA

<213> Vibrio corallinus (Vibrio algillyliticus SCSIO 43097)

<400> 1

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cgtacaatga aaactcgtct ttccccgtac cccacgccgt acccagagcc gtacccagaa 180

atacagaggt ttttcagtca tgatcaagcc agtcgataaa attagtacga atgccacgct 240

gaaagcattg aagccacaag gcaaggaata tttaattcca gataagaaaa tcgaagggtt 300

gaatattcgc gttcgaccaa cgggcacgat gacttggatc tttcgctttc aggctggtaa 360

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tccgaaacag gaactggcac gtgaacagcg cgagcaagag gaagcaaaaa cttttgatga 540

tgcttttgcc agttttgacg agaagcgttt gtccaagcag ctacgaggag aacaagcgag 600

ggcaacctat agccgggatg tcaaaccatt tcttggtcac atcaaattag aagagttgag 660

aattcatgat cttaataaag tatttgatgc aaaggttgat gagaatggac agcgaaaagc 720

gggtgcgatt aatgcatgcc atagaattat caatcaggtg atcaaccatg cgctagcttt 780

gaatatgatt ggtgcacacc ctgcaccaca tttgaaatcc aaggatgtcg gtggtggttc 840

taaggtcaca aaacgcaatt tgagcttttc agaattgaaa acattgctaa caagcttgcc 900

ttcttggcgt acagagccct caaacgtccg tctaatgcag tttttgctcg ggtgtgggca 960

acgtattagt gctgtgcttg aaatgcgttg gagtgaggta gatttgcaag agaaggtatg 1020

gttgttgcca gccagttcaa aagaacgtca caccaagagt caggagagcc gaaaagtacc 1080

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gaaacttgtt tggccgcagc ttatggatga caaacttcaa gaacccgcag cagtacgtgc 1200

tctgatcaaa cgcaatttac cggaagattt tgagcgtttt tcgccacacg atctacgccg 1260

cacctttatc agccgttgta gcgaaatggg gctggatatc gtggcgattg aaaagacggt 1320

tggtcaccag ctaccaggta tgttgcgggt atataaccat cacgactacc ttgaagagca 1380

gttagtggta ctgcaagcat ggggagaaaa gttgcaagcg ttagatacaa gcaatgtcat 1440

tcctttagag tcgatgaagt tgcctaagca tttgtaaaat tcttagagat tattcttact 1500

agttaggggg gtgttactct ttatctgagc gaaatttaat cgtggctctt tgactatgtg 1560

ggcttcatga atggggaagt cttttggagg acgattgaga gaatacataa ctcgctgaga 1620

tttttcgtat gctcccttct cttgtccatc ggcagcatcc actatcatct ctccaactaa 1680

acgagcttag ggttaacctt catcaaaacg ttgactgtta gatagatcaa ataactactc 1740

caaaaagtcc ctcacattga cggtgcttta gtactattga gtgggatgtt taatacctct 1800

ttagtattgc taaaatcaat attttcaaat gtaatgcagt acaccaataa acataacaaa 1860

aaattatcat atacataaaa tattttgctg catgctggag gccataatgt atcttcaagt 1920

aggaaggttg ggagttaatc ttaatgatct tattgaatct cttgatcatc tcgagacata 1980

tgtgttcatc aaagatacgc aatccagata tatctatgca aataacctaa gcctgcagtt 2040

aatgagaatt gataagaaaa aactgggtca aataagagat tcggattttt taccagagga 2100

taccgttgag ttcataaagc atattgatgc taaggtcatc aagggagaaa cgactaggga 2160

agaggttttc attaatgatg caaatggtaa gcaacggatt tatcttgaga tgaaatcacc 2220

aatttatagc tccgaatcct ctaatgaggt tgtagctatc ttaggtattg cgactgatat 2280

tacaaagcaa aaggaactcg aaaagaaggc atgccagcaa gctcaaatgg atgaattaac 2340

taaagttctc aacaggcgtg gactcatcga aagcgtacaa caatacctaa aaaagaggat 2400

aagtgaaaac actttttctg cttttctatt tattgacttg aacgatttca aaaaaattaa 2460

tgatgatcat ggccatacag taggcgatca catattacgt gaagtaagtg aacgtatgaa 2520

aagtagtgtg agggcttccg gcctagtatc aaggtatggc ggagatgagt ttgtcgtatt 2580

tttagggagc gttgggagta gcaatagaga agcggaatac tacacggaag aggtgatgtc 2640

gagaattgag agtgcactcg ctggtccaat aaatatcggc aaaggtaagg tgcaggtagg 2700

tacgtcaata ggtaagtata ttattgaagg agcagacttt tccctagagt cagcgataac 2760

taaagccgat gagaatatgt actacgagaa aacgcgagta aagaagattt agactgaact 2820

aaagtgtcgc aactcatgct tgaccttaca aacccacagg ttagtacaac taggtcgtac 2880

tgcgaatgtg ctggtgtcgt aactcacaat gggacaaagc gatttgcaaa gagtatggta 2940

tctactcgaa agttttagta gtttgactga tgaactaaga tgccgtaggt gatttgagtt 3000

gtggtcagtc atatagagag aaaactagag aagttgctta aaaatgaacc tcacttgact 3060

agggctgaag cagttaagct actagcaaaa ttgaagcaga aaaaaacaaa aagcaaaagt 3120

aaacatcaag cactgaaaac cgtttcaggg ggagttgttt ctcctcgata gaacaagttg 3180

tctagcatca caccagtatt gagtaacgcg ctcaattaat gatgcttgtc gtcggcagtt 3240

cagcaatcct ccacagcatg ctgtttggga caaacgctcc ttcagttttt gggtctgcta 3300

gacaccgcaa tgaaatcaac tttattgtct tgcgaagtta tatttaattt ctcttttaat 3360

tcttcaacag gtattgggcg gcttagatag aaaccttggg cttgttcaca gcctaactct 3420

tcaagaattc gtaactggtg atcggtttct actccttccg caacgacttg aattcctaac 3480

ccttttgcaa tgtcgataat gcctgaagtg atcgctcttg aacgataatc aaccgcaata 3540

tctttgatga aacaacgatc tatcttgatt tcatcgacgt cgagtctatt gaaaattcca 3600

aatgaggtat aaccgacccc aaagtcatcg atagagaacg ttacaccaga ccttttcatt 3660

tttgttacta ggcttgcaca tttttctata tccttaagtg tggctgtctc tgtgatttca 3720

aggcataggt attttgaaag ttcagtgtct tgatttagct gatactcaat ttgatggata 3780

cccttagtgc tatttagctc agtcgctgaa atatttacag atacgctttg gatgacacgg 3840

cctaggagct gagcttcttt aagttgcttt gatgcttgct tgattactag cttcgttaaa 3900

gcgctactaa gattattttc ttctgccagc gtaacaaata ctgatggagg tatatctccg 3960

tattgagagt gggtccagcg agatagcact tcaactcccg ctattctctt gtcttttagg 4020

cgatattttg gttgaaagac tatctgtatt tgctcttctt gaatcgcatg acgcaactct 4080

ataatcatgc gttgtttttg gaaatcttct tcagcgagtt ttttgtcgtg gtatcggata 4140

gtattttttg ttttttttgc ctgagctaga gcatattttg ccgacgacat gagttgcttg 4200

acactctctc catcatacgg gtagaaggcc acaccaatag agcatgtcac atcaatttca 4260

tgattttttg catgttgatt aaactgcttt tgaacgcgag ctaagtgcag agctatttgg 4320

ttttcatcat gcattcgtac caaacaagca aacccatcag agtgtattcg tgatgagaga 4380

agaggaaaga ccgtatgaga gtcaatagat tgtgcgatat cctttagaaa ctgactactt 4440

ttcgtaaaac cgaggtgatc ggctatgggt ttaaagttat caatatcaag aagaaaaaga 4500

acgtagtgtt catcttcttt ggcactttgg attttttgtt ctaggtagcg aaaaaatacc 4560

aaagggctgt gtaaaccggt tacagagtca atgtcattcg cttgttcaag ttctcgattg 4620

ttctttttta ccagtcccca cagcaataaa gatgtgacca ctacaaataa cattcccttc 4680

agtgattgaa ccaaggtata gacttgtgta ttgctcgata ggagttctac tgctttatct 4740

gagaaccaaa cccatagcga cgcaaacaca aagtaagtaa aaacgaactt agtggaggat 4800

tttatatctc ttttcatact gaccacagta ttatttgtgt actgcccatg agtaagttta 4860

tacttaaata agtaaattat cattaaattg catgtcaatc tagaggcaca tcactaaagt 4920

gtttaaaatg ttacatcgag catcaatcac agactgttag ggctattctg gcgttttaat 4980

gtatgttggt tagtcacttc ttggagttac tctctatggg gttttgctca agataactct 5040

aagtaaggag tgatgtttca tacagagtat tcaaaggtct gattaagtct ttgagttagt 5100

atagatctgt gagtggcaat gtcttttata tctaatgaaa aaatggatga tttaacttat 5160

ggagtatatg gaggaaatgt aacagatgag ggataagtta aatctgagaa agttaattct 5220

gtgcctgagt gtgttcagtg tactagtcac tcttcttaat acttttcact ccatatatag 5280

agttcagcat gagctgctag ttaacaatac catagagtcc aaccgtgtat atgccgttaa 5340

aatggcggaa ataacggata cttttattga ttatgcttta tcccaactag agtacagcgc 5400

accgataatc agccgtagaa tgcttgagcc gcttgtattg cagcaagagg caaatcgatt 5460

gcgaactcaa accgactctt tcaactctgt cgtaattgtt aatgcggacg gcgtcatcat 5520

atcggtttca ccagaaacta ttgaagtgaa aggtgttcaa ttaactgaag aaagcgcgtt 5580

acagtctcta aatgcgaaaa ctccacttat cacgaatcca tttgtatccc ctgctggcaa 5640

ctatttgaca agcatgtcgt atcctatctt ttctgaaaac ggcgattact taggctatgt 5700

tgcgggaacc atctacctcg atcaaaaaaa tatattaagg acaattcttg ggcagcatag 5760

ttacaaagat gggtcatatt tgtatgttgt cgataagcat cagacgttga tttatcaccc 5820

tgatccgcaa cgaattggag aagttattac taataacgaa gcaataagag cggtaagtcg 5880

tggtgaggaa gggggaaaag ctatcgttaa ctcccatagc gttggcatgc tctctggata 5940

cgcgcccgta aaacatgctg gctggggggt tattgctgag aagccagaag agcgcgtttt 6000

gtctatgctt gacgatcaaa tgtggcatat gtttatagag tcactcccag ttagtgttct 6060

tatatttttg attatttggg tttcttcgat ttttatatcg aaacctttat ggcaattagc 6120

cagtgtagtc aaaagttttg aaagccataa ctctaccgag caagacttaa atgaagtgaa 6180

gccttggtat tttgaagcgt catacttgaa acgttcattt ctcagcacgt tcaatattgt 6240

atccagcact attgatcagc tgcacctaga tactttaact gatcccatga ctgggctact 6300

gaaccgcaga ggtcaaggta aggcgattga taccttgcga gcacaaaaaa ctcccttttc 6360

tgtgctagct ttggatcttg attactttaa aaaagtgaat gatagctttg gtcatgatgt 6420

cggcgatgat ttattagtca gtgtggcaag tatgatcaag gctcaggcac gagaagttga 6480

tattgtttgc cgctctggtg gcgaagagtt tatcgttttc ttagtcaaaa cggacataca 6540

tcaggctaaa ctcgttgccg agagaattcg taaatatatt gcattaaaca agtttgatgc 6600

agtagggcat gttacgattt ctatcggagt gtctcattgg tcagcagaag atgagccgat 6660

tagcgcgact cttaaacgtg ccgatgatgc tctttatcaa gctaaacata atggtaggaa 6720

tcgcgccgaa atcgctctct tcaagtgaga ggcctgaaat taatgatata gccccattat 6780

gcgcagtgct taagtattag gaaggtcatt gctccttcct aatacctgat ttcaagtatt 6840

ttgaagaaca gaagtcgggg cgaattagtt ttgataggtg gctagaggta gttatttcac 6900

gagacttctg cttcgtttct ttaccatctg ccctcagcat cgtcagccga gtgatatgtt 6960

gagttattcc cttcaaattt cagccgacca aaactttcac ataatacatc aagagtgatg 7020

tcgctttcta agatggtttc tacaggtact gcgagagttt catcgtctga gaaatcgtta 7080

atatccagtt gatagcgctc aaagctgtgt tgttcttcgt cagttaaaga tgacttaagt 7140

ttgtgctcaa atactcgacc atataaccag catttgatat ttcctagtga aaattcaata 7200

agcggtgctt caccataaat cccatccatt cgattgttgg cttttagcat agagtgaacc 7260

gttaatatta ttgccacact gtatgatctt cctgcaacct gtagttcagg acgcgcaagc 7320

tcacaccagt gatccgaaga agatagctcg tacagtttat tgcttctaat cgttgctgcg 7380

tcaatgacct cttcgcgtaa gtagggatga gaatgagccg ttccaatgta ttgataatcc 7440

tcgccgaaca ctttagcgat atcttttttt attgaaagag agagtgggtt ccattcaact 7500

gaagagcgtt ttcttttagc tgatgtttca acagaaacat gatttatcgt acatttaaat 7560

gggtgggctt tattcgcata tccccaaagg tgggccgctg tctctatgga gactgatttt 7620

ctgccctcat gctttatcgc ataagcctct aaaccagcca tgattagctc aaatatcgca 7680

cttttactga ctgaaattgt cgcggtcatt ttctcttggt tcctattatt gacggttatt 7740

ttcgtagttt gatgagaggt aagacacaag agttattggg cgaactctac atgttctttc 7800

agtttgtttt tatagcgctc ataaagcgaa tcccatacat cttcacttac catgactagt 7860

ggtaagtatt cttcaaaaat gacaccactg tcgtcagggt aggcaagctc ggtataagca 7920

taacgtgggc catctacata aagcgcttcc caacattcga tgcattcatc tttaaaccac 7980

tcttttgtga ttggctcagg gccgttaccc agaagatgaa ttaagttctt agaatcgaag 8040

aaatgatgtt cttcactttt gatatcaaac aggtagctag aaaatagagt gatggctctt 8100

ctgcagaaat accataatcg caacattgaa actgcgcagg tgattgaaga gctgatacag 8160

tgggcgaaag agatgcaagc tgacgctgag atgatggaaa aactggggct ttccactgat 8220

gaaattgcgt tttaccgtgc attggtcacc aacgaggcat ctgtgcgcac cttaggcgat 8280

gacaacttgc gccaactcgc tattgaactc acgcaccaac tgcgtaaatc ggctactgta 8340

tattggcaga aacgagaaag cgtgcgtgct cggatgcgca accttgtacg ccgtttattg 8400

cgtcgctaga aatatccacc tgatgccgca gaagaggcaa ttaagctggt tctggatcaa 8460

gctgaagtgc tggctgacgg gtggtatgcg gcatgattaa atgaaaggca aggtctgata 8520

gccttgcctt tctattagtg ctgacattgg tcctgtctta tcatcccact ttctgctgaa 8580

tattatttaa agtgtgtttg tatttagaga atgatccctg tagttttctt ttcctcttta 8640

ggcttaacga cttttggctt cgcatgctct gtatgctttc gttttgactt cgtataagcg 8700

gacttacgtg caatactatg attttcgatt tctgcaatat ttccttttac tacttttgca 8760

taggcaaact taggcaatac tttggtcact tcaatggtcg caactttgat ttcatcgtgg 8820

cctaaactct cttttgtcac aggatcgata aatttttcac ccaaattgaa gacatcaaag 8880

tattgtcctt gcttaactgt attgccgccc gagttcaaaa ctaaagagcc attatcgcca 8940

actaaaacca ctaacggata aatgttatct tttagttggt taaatatttt tttcgaagca 9000

atatcaacca gaagattcaa attgtttcta cttgaagtgg ttggaatagt tgctgaccat 9060

ttcacttgat tattgacaac atcgagaatt tgaaatttta ccaaaacttt agagctatag 9120

taggttttca catctttaat gctttcccct gtgagctcta cggtacgtga gttatcgacg 9180

acactttcgt tagtgttagc agtaacaacc tgaccagtta caaaatactg taacgttggg 9240

tcgagtttgt catatagtac tttgaacttt cggtcttgca ccaaaagttc ttcgactttt 9300

ttgccgataa tagtgccttt tgggccagaa aaactaccaa tagcgattgt tcttcttttg 9360

gcattttgat cttttatttt ctctctaggg tcaacctcaa ccttaacctc taactttacc 9420

tcacagccat aacttgaaca cttttcatgt agcactttgt acttaacgtt ccctcgcgag 9480

tatgttgcag tgccatctgc catagagtcg tttaactgtt ttgtctttcc gtcgttaatt 9540

gatgtaagga gtgaagtgcg ggcactttca atagatacac ctgagacctg ttctaccgca 9600

cgccttaaac tgacatcaat agcctgatct aatgtttctc cttgcccatt tgttataact 9660

ttctcataac ttgcagcact agataaacat gagaataagc ttgctgcgca aagtagtata 9720

agccttttat tcatgttaaa aatccgccac atcaaacata tcttccgatt cagccgagtt 9780

aatgttatca gagagagttt ctgctcccag ggcttcttgc tccagctttt cagcactcat 9840

tccacttgcc atattttttg cactcacaat cttcttagga tgccaaacga gtaccgtccc 9900

caccatttca tgccccatca cagggtgctt acgacgccat tcaaactcag tacttacacc 9960

agtcatattt tgtacacttg atgtcattga agcactttgt tgaacggatt tgattaattc 10020

attagttaag cctggggact ttttgcgaac agcacctgtt atcaaatcga actgctcagc 10080

tttactgaca cggttttctt ctgaagttga ctcttggaaa tcgccattca gattataggt 10140

ttgtgccaag ctcgcccaag cgttgttcgt tgcaaagctc ttagctgctt tacgttcaat 10200

ttttctttcc gttggtgagg atgtataagt aacgcctgct tgaccgtatg aaatcaacat 10260

gggatagccg tcggagtcat agctaacttg agttcctacc ttcagatata gactgtcttt 10320

ttggttatag aacatatcgt agatagatgt cattttaggg ttagctttat ctttctgtgg 10380

tgtaatttga ccgccggaat cgatcattgc acgaacttga tctcgtttat ttgcagacaa 10440

agcaagaaca accccaactg tcccttgacc agaatctctg atttcttcat aggttttgat 10500

aatcatcatt ccagccaaat caccgtatga agaccttata gtctgcttga tcactgactc 10560

tttaaataga tcgcgcttga tcgatggtgg ggctgcttca aactcttttg gatcaatccc 10620

catttcttgt aattctgcat caagtttacc ttctagcacc gcaaccactt tatctaagaa 10680

gctcgccatc tgagagtcac tcttaaaatc ctcttcagtc ggggtcggca agccgctatt 10740

ttttgtcatg gagtaaagca tgttattttc gacactggta tttagtgttt gtaagtattc 10800

tttttgcgcc tctaatacgg ccttatctaa tgctgctact cgaaactctg cccatcgagg 10860

gtcattgcga tctgccatga tttcagcaga accagaataa ataaatacac tatgttgctt 10920

accctgttta tataaggtgc gttttacgtt tgacacataa gcatcaagct tttcatcaat 10980

ggcatcagtg gtatcaacat cgttttgaac ttggaccaca tctttatcgg tttgtttgcc 11040

cgcagcctct tctgttattt ctgctaccgt tgttgtatct actaaattac cttcttgaga 11100

tgcatgtgtt ggtaatgaca agccagcgat taccattgct gtaaataatt ggtttaattt 11160

catttttaat cacctaaccc tctaactaaa agtaatgggc agctttcact gcccatcatt 11220

tgacgctacc taccagctca cacttgagcc atccgctacc ttgccgacta ctcgctctcc 11280

ttcccaatac gctaaaccat tttctatatt ggttagtgac atttggaaat agtactcaat 11340

tcgtgtatct ccgttatcta gagttgagtc tcgctgaagg attttgcctg ataggctaaa 11400

gtctggggct agtacttggt tatctccttt gacacttgcc tgtttgacca ttttggaatt 11460

ttttaaatca cgcatttttt ttgtggcatc gtcctcaccg aatgcagttg ttactaagaa 11520

ttttcctgat tgaagtaact ttactcttaa agatttggtt agctgatcag tgtctaagcg 11580

ctgcatagtg tcattctcaa tgccacttac ataaataatg tatcgaccac caacatctgc 11640

ttgagggtga gtcatcaatt tattagaaat aatgtcatca gccatttcat ttgctgcttt 11700

cgagaaatca gcgtaagaga gggctgctac cacatttgtc gttgtatcag cagcatcaat 11760

ataagatgtg gttgttttac aaccaaccaa gctcatcgca gctacaacag ctgcaattgt 11820

caattttttc atgtttatcc ttaaatattt acgactttta cggatggttc tattaatgga 11880

gagaccgctt tcacgtagat gatgtgtttg ttagattctg tgctaagctc aactggaata 11940

gtgaaagatc ctgcctttat ttcaactgta ttgtgtttgg ctttgattcg agttgcttga 12000

tattcactcg gtagggctga gaaagttcga atatcagcac cagtagtcac catttgtaaa 12060

atggctaatg catcaccaag taaagcgttt tcatctttaa cttgtttttg agcgatggtt 12120

tttaatgtta ctcgggtgat ttcacgagct aaaatatatg gaaactcctg atcgaattcg 12180

gcactaatta tcttatccat atctgcaata gtgctggtct ttgaaccatt aattgtaatg 12240

ctgtcaaatg ttgtaccgcg ttttttcaag gtaggaattg cgataccagc ataagataca 12300

ttgttagaca gtaagaaaat tggcagatca attcgacgct cttctttcac tggcgatttg 12360

ccattttcaa aaatcaccca aacttgatct tttagcctag attggcttgg atttttaacc 12420

atactacgtg ccaattgata gtccgctttg gcagcaggtg aaccagtaag agagtgtaca 12480

cgcttgaaag agttagcagc tttttcaaag tctgacttag atttacctgt gatgagtaaa 12540

tgaagaccgt aagagtatgt agtaaaaggg tttacgtagc cttcataagg tttccattgg 12600

ccttgctcaa tacctgcttt ttttagtgta ttttgtgtca aatccgattc tatggtctgc 12660

tttaccaaag cgcttgactc accagcttca gactccagtt cttcgcttcg ctccttaatg 12720

atatctgcaa aatgttgagc tgctctacgc tgtcgctctt ctgctctatt tagttcaacg 12780

cgagcatttt gatagtcatg aagtgatata aaattccatg ctttaatggt atttgtcatg 12840

atgccgtcgt aatgcgcttg ttcgtaagcc aacatggcgt catttccaac cattgaacca 12900

ataagctcaa ctccctcaga agcagtactt tccgtatctt cttctttcat cattatttct 12960

gtttcatcaa gcagcttggt ggataattcg tattcacctg catagttcaa gcttgcacca 13020

gcttgcaatg tccatagtag gtcgcttgtt tcacctgaat cgttatcgta gccagcatgt 13080

tctaaggcta cttgagatgc tcccgaaaag tcaccagaat caagctttga attgaagcta 13140

tcgaagtcgt ttctctgcat catacttgta cagcctgaca acataaccat actgatcgca 13200

ctgctaatta gttttatatt cattgacttt attgttatct aaatgattga atttttgaac 13260

tagaatcacc ttatactcac aaatagcact gcaatcaagc tcttaatgtg tggttaatta 13320

acacttttag tattatccga ttattgaatg ctatttattt gatttatgtc attaggctca 13380

catgcgtctc ttgtctttga aagttgagtg tatctactaa acgtaagcga atatctattg 13440

ttatcagtag ggaagctctt cttatctcaa ttggaccatt tcattatgtg tagtagtaat 13500

gaaaggaatt tcagatagat attatcttcg tgtatatccc aacggtttca tttttaggag 13560

ctttttatgc acgaaaacaa ctactatttt gatggtcctg tcacagcaaa taaggtgctc 13620

gacgcggctg ctgaaatcct tgctacacgg gctctccgag gcgatcagta ttgcaatccc 13680

gaagcaacaa aaacctattt atcttgcaaa ttaaaacacc atgaaagaga ggtgtttgcg 13740

gtgatgtttc tagataatca gcatcgtctt attgcttttg aaaagctctt ctttggcaca 13800

atagattcgg caagtgtcta tcccagagaa gtcctcaaag cggctttaaa agtcaatgcc 13860

gccgcactca tatttgctca caatcaccct tctggtgatg ccacaccttc ccaagccgat 13920

aaacaaatta cgcaaaggct caaagaggct ttagcgctgg tggacattcg agttcttgat 13980

catatcgtgg ttggcgatag tgccatctct tttgcagaga gggggctact atgatccaga 14040

aaacatcggg tgaactggaa ttgcaggata gctttcatcg agcattgctt ttggtaatcc 14100

aacgttattc gattcctcca caagctcaac gtatggtcct gaactgtcga caaagacgct 14160

attaccagac agggcagggg cttcatccca ttgaaattca atttcgtcgt acatcggtca 14220

gtgaagattg gcaggttgtt tgcttagcga gttttcttat tcggaaaata aaccgagtga 14280

gatggagcct gaactctact ttcatctctt caatctttgg tgttatcaac cagatagcgg 14340

tgctgcaagt cttgcagacc cgcaagtgaa agcgctgtta gatacttggc tgaacgcgct 14400

ctctcgacac cttactcagt ctctctttga tgacattcag ttatcgatga ttcaaacctt 14460

cgatgaacac cattcatgat cacattttaa cctcggtcac ttttaggaga tctaaccatg 14520

tctatactgc attttacgcc gtcagctctt gctgtttcta ataagctttt gacgttatta 14580

aaccaacatt gtcatgcctc gatgaaatca acacagtcca ccagccatgc cctctctatc 14640

cattttcgag acaaaagcta tagcgcggaa gaggggggat atcatccggt tgagattggc 14700

ctcaacattg tgaatggtaa aacggttgat attctctaca ttaccgattt tgcgtatatg 14760

gggaactatt atccggagct agagcgatgc attgattttg attttggcaa tcaagtcgct 14820

tttaccgtcg ccactggatg gcaaagcatt cgctctccag gtatcgatga gctgtatcaa 14880

atgtgggagc aaaactttct tcagtacgtc gaatgggagt gttacgacga cattcagatt 14940

tctcattaac ttttatcaat ctagctaagc tttaacctgc tgctttatat ctccaccaaa 15000

gaatcaatca aacttctgat tgtggtaatc ccaactgttc atctcgctct gtcactcttt 15060

ctaatatcca gtcttggact tcgctttcta cccaagccac gctgcgaaag cccagtgata 15120

ccgatttagg gaacttaccc tcagccatga gcttgtacat gtacgcacgg ctcaaacctg 15180

tattatccag cacttctttt agtcgaatta gtctcatttt tctgtcctca tgatttgttc 15240

gtcataagga ggtagctagt tgtaattttt ccggacgcta actaaggagc gcctatgtcc 15300

gaacagcacg atcttttcac tacgcttgaa agcgagatac gccaggcatc cctcaatgaa 15360

cgtactcgca ccaagttact cgataacctc aaacaaatga gagccaccga gctcaacatt 15420

ctcattacag gagccaccgg agcaggaaaa agctcaacga ttaacgcact ctttgacatg 15480

agtgttgcag aggtgggaat cagttgtgag ccgcacactc agggtgttac gcaatatcgt 15540

ctgaacaatc ttattctttg ggatagtcct ggtctggggg atggtatcga agaagataag 15600

cgccatgccc gcttattgaa aaatacgctg agagcgagag atgagcagaa ccgtttcgtg 15660

attgacttgg tacttgtgat tcttgatggt agctcacgcg atttaggtac acctatcacc 15720

ttgattagcg atgtgcttat tccggtatta ggcgaagacg cacagcaccg tttaatcatc 15780

gccattaatc aagccgataa tgcgttaaaa ggaaatcaag cctgggacta cgaaagcaat 15840

acaccgacac ccgcatcaaa agcttatctt gaaaccatgg tgcgctcggt tcatcgacgc 15900

gtattgcgtg caacaggggt ttatctaaag cccgtctact acgttgcggg gcactgtgat 15960

ggattggcca agcaacggcc ctataacctt tccaagttgc tgtatttgat tgttgaacgt 16020

ttaccaaaaa ataaacggct cttactggcg aatcgcacgc tttcccctcg acatgagaat 16080

tggaaagaca acgatgccag tgattacaac aaaaaaacca gtcttagttt gtgggaggca 16140

atcttcgaca ctacgactga aggtgcgcaa tacggcgaag aaataggaag tgtgtttggc 16200

actacgggca agcacctagg caaaattata ggcggtgcgc taggggcatg cttaggcggt 16260

ttacgctatc tctttggctg gtagataagc gccgctacgg ttacatctct caaagccaat 16320

ctctcttcga cataagttgt cgcaaccacc ttttaatatc aactcattga ataatatata 16380

aaggatatct ctcatgaaac ctattgtaat taccgcagca ctaacactgc tcatgacctt 16440

tactgcgtct gctgagggct acaaagtcga atacaaagac atcaacaccc gatggggacc 16500

aaagacgaaa atatatgtaa cgtccctctt tgactactcg actgtcttca ggatcgaagt 16560

taaccgaggt aactgtgaag tcgctagacc agaaacacca aagattaccg tcaacaagaa 16620

taagtacagt caatctgaag agttactcaa aaacatgagc aaccctgatt acaaaaagcc 16680

ctccagtaac aacaagtcta gtggtgtgaa atggcttcct tattccgaag tggatttgaa 16740

gtacggagag tctactggtt atgtcactac aatgccctgt agagtcctac aaattgacat 16800

tcactctagt gaagggagtt accgctttaa tgcggattaa acaactgaaa ggccacaagg 16860

gcctttcttt tttgtcttct cacttacaac tgctgtggaa gttttttcct ttatccccaa 16920

aaggcttact ttcaagtttt gctaaatagc tgacacggta aaaagcctga ttgtattggt 16980

tgtaaaaagt ctccgtatct agtcgcttat taatttcata agtaccgtta ttagggaaaa 17040

aaatcgcact acttgcttgc ccgaccgtaa taccgattgc acttgcccaa gccttagcaa 17100

ccatccctga aacgtattca cccgtaagat catcataagg ccctattcct tgaaaaacat 17160

caccattgag caatatggct acgtgatagt gaggatacac tgaagtgact atctctctac 17220

accagataaa tcgaggttga gagtggcggt tgtgcttctt acgttttaga taagcacaca 17280

tgatagcttt gaaggactct tggaacttag taatctgttt gccagttacc ccgttaaccg 17340

gataaccatc cgggtaatgt aaatcaaatc gcattagccg tgttcgaggg tattggctta 17400

acgcataatc gaaagtcttc ttgatttttc gtaagtactt tggttcaagg gcccctttgt 17460

tggtattgaa actcaatggc ataccgttga tgtgtaactt acatgcattg ttgatgttat 17520

tcatttctat cattcctatt ggttagtgtt ctcaataggt agataggttg ttgtaattaa 17580

ttagataagg ttcatataaa tactattatt accgacagtt tttcactccc ttcctccagc 17640

cacgcatctt tgctcgacaa ggcgtcctgt agcttcaaaa cttgcaaaag ttactctgat 17700

aaaaagttag gccccccgtt tgagaaggag tctctgattt ttctcattga taccaatcgc 17760

aaattgcttt ccatcaacaa catcaagggc tttgattgta cgtgctaggt ccaatgcacg 17820

ataagcaaga aattcgatta acttattgac tttctctttc tcaagcttaa gtgttgacca 17880

ggcaaaatgt tcacgtataa cacgctcata ccactcaatg tctgtacaaa cctttccttc 17940

gacattgatg aacttaatga tttcactttc aataaagtaa tctatgtgtt ctgcaaactc 18000

tttattgcat tttacgtcag caactttttg aaataataaa ggtattgtga ggttgtaaac 18060

gggattcagc gcaccagacg attttaaatg ggcgataaag ctgtagtagc ttttttgatc 18120

gtacatggat aagttcagaa aaattattga ctttcttttc tttagaacag agtggctgag 18180

acctttgata tatcccctga tataaccaag tcttctaagc ttgctgagca ttgcttgagc 18240

tttacgttca gtgcatccca taaatctgca taaatctcta aggcttatac tatcaacaat 18300

cccagattcc gtagcctcac ttaaaagatg ggtaagcatt aagcgatttg acagcgtcaa 18360

ctctttagca tgctgattac tataccaaag atgattcatc cacctcctca tatagatggg 18420

gcgttgttca aactgactac tcaactcatc aaaatcaatg cctgtaagtt gatagcgtgt 18480

gactggtctt cctcgttttc taggtacttc tttctcaggt atcaagagaa taacttttgc 18540

ctctatcaat aagcgtagac agcgccttat aaggcgttct gaggcttggt attttcgggc 18600

catatataga acaggttttc tgtaaaaatt gtctgattcg gcatccaaat acataagcag 18660

cagtagcatt ctagcttcag cgtttagttt gtcattctct tggaaagtcc agtttacaat 18720

cataaaatca catttatatt tatttatagg gcgttttggt atgtatatta cttggttttt 18780

ctttctatgt ccttggacta cgtccaaaga catagttata gtgtgaagga tgccaactat 18840

attaaaaacg ctggaagcta gagtgatatg agtgagaaaa aaatacgtcc attagaaggt 18900

tttagtttca acaatgaatc tcatgtgaaa tggttaaata cgtatctagc aaaagccaaa 18960

actaatccaa ttgtcgaaaa gcattacttt gggttatttc agttatcagc atcacctaaa 19020

aaacgacttc aaaatttttt tatagattgt aaaaattcag ctgatggtag agagatctgg 19080

ttacttatgc atggagcttg ggggaaaagg tcgagaagag aaaagcaaaa gcggaagggg 19140

aaaaaacagt ataattttga actcacgata gaatttggtc aaaatcttca agctttagcc 19200

acaaagaggg gacagactca gcagaaggtt cttgaagagc ttgtaaatgg ttcattaaca 19260

tcaatgctcg aagttcgaaa gttacaagaa actgatagaa ttctatcact agaagctaca 19320

gccaatacca cgttagaaaa ggagttaggc tctgctcgaa aaaaaatttt agagttggag 19380

tcaaaaattg cgactcttaa aaaagagaac cttcacaagg cctctgaaag cgacaatgtt 19440

tctactaacc atcatgcaga tgaaccgtta caactgactc tagaaagaaa aggtcgttca 19500

attattcaca tgcagccaaa ggacagtgac caagactaag catctttaca ctataaatac 19560

cactagtaga gctggtggct aggcgcttgg tgtgagtatt aacatgaagg agatcgggtt 19620

attgagtgat cagcttgggt gaaaatacac acttggtgtg cttaatgtaa ggaaattgtc 19680

tgcttactgc atgtgagcat ataacgcgct gtcagcgctg actatccaca tgaacatgtt 19740

gcttattttt gcgaactagc atctaacagt taataaaatc actgtcttta aatacagcga 19800

tggtttacac tttttgcgtt atatcaaccg gattatgtaa actatttgga cgaaattgga 19860

ataaaaaatg aaagcatatc acacatacag tgctgtagac gttgcatgga gcttgctcaa 19920

gcatgcgaaa gagcaaggta aatgcttctc caatttgcag ctacaaaaac ttacctatgt 19980

ttgtcatggt ttatcattgg cacacttcca gcgtcctctc gtcgtagatg atgtatttgc 20040

ttggaagttt ggtccagtgg tgccatctgt ttactttcgc tttaaatctt acggtagtag 20100

tgtgatcact gaacaatgtg atgtagcact agaccaagaa agtgagtcta ttgttaaaga 20160

tgtagtctct aagctgggcc atttgacggg gcctcaactt gtagatttaa ctcaccgcga 20220

aggaagtcca tggcatcagg tatgggatgg cacacaccaa aaagtgattc cggaccattt 20280

gattcaagcc cattacactc aaatcaaaca atccggacga acagctagcc tgtgagtaac 20340

aaagaaaata gtgtcgtcag tttttacatt gacgacactc ccacagctga acaagcttca 20400

aaaaccttcc aagacgacta cgaactggac tttcaagatt ttatagaaag aacggtaaat 20460

gaaagtgctg ctgaagactt ctcgtactca aataatggag taaagaaagc tggaagacgg 20520

cttcgcaaga gagagggcga cctcaagtca gctactgcta caatccaaca gtttcgagcc 20580

gcacacgaaa agccattaaa cactatcgca tatcttgttg gtcgctgttg ccgtgagctg 20640

ggtatagatg ttaaaccagt tacacggtta aagcgcttag aaacaatcgt agataagttg 20700

cagcgtaaat ctcttgatgg tgaaacggca aatgctactt gtgtaaccaa tatgaatgat 20760

attggcggct gcagggcgat ttttccggat ttaatttcat tggagaaggt gaaatctcaa 20820

ctgcaactta cagttgagaa ggtaggccga gttagaatta aagatattga tgactatatt 20880

actagtccta aacctaatga ttgtggttac cgaagcttgc atgtgattta tcgctatgac 20940

catgctagcg gtaaaagttt taacattgag gctcagctac gtactcgact tcaacatctt 21000

tgggctacta cagtggaaat tgtcgacatc ctagaaggta ccaagattaa gactcactct 21060

cacagttcaa atgaaggcaa gagcagttta caggttcagt gggaagaatt gctttcgata 21120

atgagccaat gcattgctga tgctgaaggg gctatagagt taagtgatat cgataaagcc 21180

gatttttcaa agcgtttggt cgtcttggac cgcgaaatca atgcgctaaa tcggttgagt 21240

agttttaaaa ttttatctga aaaggttaat tcttactgtg atagttccat tgagcacgta 21300

ttgctagtta ttaacgaaac ttcacgagaa ttgatcttta gtgaagagtt tgaaaattac 21360

agccaagcaa tttcagttta taacgcagct gagaaagtca ctcgtagcta ctcacgagca 21420

acctcgagac tcaataccct tctcatatcc acaaaaaata tgggccaact ttcggaagcc 21480

taccctaact atctaggcga ttgtgcctca ttcatcactc tgcttgaaga cacaatgctg 21540

tctggtgaaa gttaaataca aacctttctt gttgttattg acccttgtat ctaatcctgc 21600

ttgttaaaac gtgcccaatc tgtcatcgaa tttggtatgt gtaaaatatg gcttcctgtg 21660

atggcttgag ggttagactt tattgtcccc gtaccccaag ccgtacccag ggtgtttttt 21720

gctaaagata aaagccgttc atgtccttat tttatgcgct ctagaagcac tccagactcc 21780

atatggtggg ta 21792

Claims (2)

1. The application of the guanylate cyclase gene in promoting the formation of bacterial biofilms is characterized in that the nucleotide sequence of the guanylate cyclase gene is shown as 1907-2812-bit base of SEQ ID NO.1 or is shown as 5342-6748-bit base of SEQ ID NO. 1.

2. Knockout of biomembrane gene island GIVal43097 todgc137Use of genes for reducing biofilm synthesis, saiddgc137The nucleotide sequence of the gene is shown as 1907-2812 base of SEQ ID NO. 1.

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