CN112143741A - 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

A kind of biofilm gene island GIVal43097 and its excision method

Technical field:

The invention belongs to the field of biochemistry and molecular biology, in particular to a biofilm gene island GIVal43097 and an excision method thereof.

Background technique:

Biofilms are the predominant form of bacteria in the environment. Biofilm, also translated as biofilm, is a bacterial community formed by the adhesion of bacteria on the surface of living or inanimate bodies through the adhesion of extracellular matrices such as polysaccharides, proteins, and DNA secreted by them. The extracellular secretions in the biofilm provide a barrier for the survival of microorganisms, and the bacteria in the biofilm state have better tolerance to the pressure of the environment, so the biofilm is also called the semi-dormant state of the bacteria. Biofilms exist in various forms, including solid-liquid biofilms or adhesive biofilms formed between solid and liquid media, gas-liquid biofilms formed between gas and liquid media, and gas and solid media formed by Air-solid biofilms composed of bacterial communities. A 2014 National Institutes of Health article noted that biofilms are involved in more than 60 percent of microbial infections. Some pathogenic bacteria can regulate the formation of biofilms to enhance their pathogenic ability. For example, when the human pathogenic bacterium Vibrio cholerae enters the intestinal tract, it can well adapt to the acidic environment in the human intestinal tract by regulating the formation of a large number of biofilms. , protect itself from the influence of bile and low pH, and maintain the pathogenic ability; and when Vibrio cholerae begins to infect the human body, it will promote the thinning of the biofilm by inhibiting the expression of intracellular polysaccharides, which is conducive to infecting the host. When the coral pathogen Vibrio acoralis infects staghorn coral, a biofilm is rapidly formed on the surface of coral mucus, and then the expression of virulence factors is induced to exert pathogenicity.

Studies have shown that horizontal transfer elements such as gene islands and prophages are involved in the formation and regulation of biofilms. For example, excision of the prophage CP4So in Shewanella MR-1 can enhance the biofilm of the host bacteria and improve the survival rate of the host at low temperature. At the same time, the formation of biofilms will further regulate the excision and release of horizontal transfer elements such as prophages. For example, when E. coli K-12 forms a biofilm, the CP4-57 prophage on its genome will be excised, which further promotes the formation of a colony biofilm. Pf filamentous prophages are massively replicated and released during Pseudomonas aeruginosa biofilm formation, enhancing bacterial pathogenicity. However, the current research objects of biofilms mainly focus on Escherichia coli model bacteria and human and animal pathogenic bacteria. Focusing on their pathogenic effects and mechanisms, there are relatively few studies on biofilms in marine bacteria. Some species of Vibrio, such as Vibrio alginolyticus and Vibrio alginolyticus, can cause disease in corals, and the presence of biofilms enhances their pathogenicity. Therefore, there is an urgent need to identify the gene islands that regulate biofilm formation in these pathogenic bacteria, and to establish an efficient and targeted gene island excision method that ultimately attenuates bacterial biofilm formation.

Invention content:

The first object of the present invention is to provide a gene island GIVal43097 that regulates the formation of bacterial biofilms in Vibrio alginolyticus.

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

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

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

The third object of the present invention is to provide the application of knocking out the dgc137 gene in the biofilm gene island GIVal43097 in reducing biofilm synthesis.

The fourth object of the present invention is to provide a cleavage enzyme alpA, the nucleotide sequence of which is the reverse complement of the sequence shown in bases 15008-15217 of SEQ ID NO.1.

The fifth object of the present invention is to provide the application of overexpressing the excision enzyme alpA to mediate the excision of the biofilm gene island GIVal43097, thereby further removing the formation of bacterial biofilms in organisms, such as the biofilm of Vibrioalginolyticus SCSIO 43097. form.

The sixth object of the present invention is to provide a method for excising the biofilm gene island GIVal43097, which is to overexpress the excision enzyme alpA in the organism containing the biofilm gene island GIVal43097.

The current bacterial biofilm removal methods are all removed or inhibited by physical and chemical means, and their application in vivo is greatly limited. The invention utilizes molecular biology means to identify and directionally excise the gene island that enhances the formation of biofilm, on the one hand eliminates the genes carried by it, on the other hand prevents the horizontal spread of the gene island, and at the same time protects the balance of the flora to the greatest extent , is a novel strategy to eliminate bacterial biofilms to attenuate pathogenic Vibrio biofilms.

Compared with the existing technology, the advantages are as follows:

1. The present invention utilizes molecular biology means to remove bacterial biofilms in organisms. The current biofilm removal methods are mainly aimed at biofilms on some medical devices or chronic wounds on the body surface, because they are all mechanical treatment or chemical treatment methods, which cannot be directly used for the removal of biofilms formed by bacteria in vivo. .

2. The excision of the gene island is mediated by over-expression of the excision enzyme encoded by the gene island itself.

3. High efficiency, using existing physical and chemical methods to remove biofilms, the selected chemical reagents need to be tested experimentally, and the removal effect cannot be estimated. However, the efficiency of excising gene islands by overexpression of Dicerase is close to 100%.

Description of drawings:

Figure 1 shows the sequence analysis of gene island GIVal43097 (A) and the domain analysis and knockout PCR detection of biofilm-related proteins Dgc137 (B), Dgc139 (C) and Dgc140 (D);

Figure 2 is the identification of biofilm-related genes on the gene island GIVal43097. Gas-liquid biofilms (A), solid-liquid biofilms (B) and gas-solid biofilms of gene dgc137, dgc139 and dgc140 knockout mutants Δdgc137, Δdgc139 and Δdgc140, and gene island GIVal43097 excised strain ΔGIVal43097 (abbreviated as ΔGI, the same below) Membrane (C) phenotype assay; and gas-solid biofilms, gas-liquid biofilms (D) and solid-liquid biofilms (E) complemented by the dgc137 gene, WT is wild type, where wt/pBBR1Cm represents expression in wild type Empty vector, Adgc137/pBBR1Cm represents the expression of empty vector in Adgc137, Adgc137/pDgc137 represents the expression of pBBR1Cm containing dgc137 complement plasmid in Adgc137, ΔGI/pBBR1Cm represents the expression of empty vector in ΔGI.

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

Figure 4 shows the detection of solid-liquid biofilm (A) and gas-solid biofilm (B) after heterologous expression of key genes dgc137, dgc139 and dgc140 on gene island GIVal43097 in Pseudomonas aeruginosa PAO1, PAO1/pHERD20T is expressed in PAO1 Empty vector control.

Detailed ways:

The following examples are further illustrations of the present invention, rather than limitations of the present invention.

Example 1:

1. Extraction of genomic DNA from Vibrio alginolyticus SCSIO 43097:

Strain Vibrio alginolyticus SCSIO 43097 stored at -80°C, streaked 2216E medium plate, left at 30°C incubator overnight; pick a single colony into a 250ml Erlenmeyer flask containing 25ml fresh 2216E medium, 25°C incubator, shake at 220rpm Culture for 12 hours; transfer to a 250ml Erlenmeyer flask containing 25ml of fresh 2216E medium at 1:100, shake at 220rpm for 5-6 hours (to logarithmic phase); centrifuge at 12,000rpm for 1min, collect the cells, and remove the supernatant ; Use bacterial genomic DNA extraction kit (TIANGEN, BEIJING) for total DNA extraction, thereby obtaining SCSIO 43097 genomic DNA, which can be used for whole genome sequencing and annotation analysis of SCSIO 43097, using Mauve software and closely related bacteria Vibrioalginolyticus ATCC 33787 for sequence By comparison, it was found that SCSIO 43097 contains a gene island of about 22kb, and its nucleotide sequence is shown in SEQ ID NO. enzymes and 16 hypothetical proteins (Table 1 and Figure 1A).

Table 1 Functional annotation of genes contained in gene island GIVal43097

2. Identification of biofilm-related genes on gene island GIVal43097

According to literature reports, guanylate cyclase is involved in the synthesis of C-di-GMP, which in turn regulates bacterial biofilms. Therefore, it is speculated that this gene island may regulate the formation of host bacterial biofilms. The GIVal43097 gene island contains three GGDEF domain-encoding guanylate cyclase-encoding genes dgc137, dgc139 and dgc140. Specific primers were designed (Table 2), and the three genes were knocked out by using the pMBLcas9 vector, and finally three single-gene knockout mutants, Adgc137, Adgc139 and Adgc140, were obtained (Fig. 1B-D). The experiment found that the biofilm was significantly reduced after dgc137 knockout, and the knockout of dgc139 and dgc140 had no obvious effect on the biofilm (Figure 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 methods of gene knockout and gene complementation in Vibrio alginolyticus SCSIO 43097 are as follows:

In this study, the plasmid pMBLcas9 dependent on the CRISPR/Cas9 system (for the construction method, refer to the literature: Wang et al., Journal of Antimicrobial Chemotherapy 2019, 74(9), 2559–2565) was selected for gene knockout. First, the upstream and downstream homology arms and sgRNA gene fragments were obtained by PCR amplification with primers (Table 2); the homology arms and sgRNA fragments were purified, pMBLcas9 was digested with AhdI, and the ClonExpress MultiS kit (Vazyme) was used for digestion. For multi-fragment recombination, 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 then transferred into V. alginolyticus SCSIO43097 using the conjugative transfer method. Briefly, the WM3064 containing the recombinant plasmid was used as the donor bacteria, and the SCSIO 43097 was used as the recipient bacteria for conjugation transfer experiments. The donor bacteria and the recipient bacteria were cultured to an OD ₆₀₀ of 1.0, and the bacteria were collected by centrifugation at 6000 rpm for 2 minutes. The cells were washed twice with culture medium, the supernatant was removed, and each was resuspended with 50 μL of LB. The two cells were mixed and spotted onto LB plates containing 0.3 mM DAP. After overnight culture, the bacterial lawn was picked with an inoculating loop and streaked onto a 2216E plate containing chloramphenicol (15 μg/mL). Use wF/wR primer PCR to determine the correct knockout strain on the conjugants obtained on the resistance plate, thereby realizing the knockout of dgc137, dgc139 and dgc140, and obtaining three single-gene knockout mutants Δdgc137, Δdgc139 and Δdgc140 .

In this study, the gene dgc137 was complemented by the plasmid pBBR1Cm (see the literature for the construction method: Zeng et al., Appl Microbiol Biotechnol, 2015, 99, 10127–10139). First, the coding region and promoter region of the gene dgc137 were amplified by PCR using primers (dgc137-pBBR1Cm-F and dgc137-pBBR1Cm-R in Table 2); the PCR fragment was purified using the ClonExpress II One Step Cloning Kit (Vazyme) was recombined into the SalI and KpnI sites of the pBBR1Cm vector; it was introduced into E. coli WM3064 competent cells by chemical transformation, and the dgc137 complement plasmid was verified by sequencing. Subsequently, the dgc137 complementation plasmid was transferred into the single-gene knockout mutant Δdgc137 by conjugative transfer method using LB plates containing 0.3 mM DAP. After overnight culture, pick the bacterial lawn with an inoculation loop and streak it to a 2216E plate containing chloramphenicol (15 μg/mL), and detect the correct transformants, thereby realizing the complementation of dgc137, and obtaining the complementing strain Δdgc137/pDgc137.

3. Expression of cleavage enzyme AlpA in V.alginolyticus SCSIO 43097 and directional excision of gene island GIVal43097:

The expression of cleavage enzyme AlpA in SCSIO 43097 was constructed using pHGECm vector (for the construction method, please refer to the literature: Wang et al., Environmental Microbiology, 2017, 19(11): 4620-4637). First, the cleavage enzyme gene alpA encoded by the gene island GIVal43097 was obtained by PCR amplification using primers (Table 2), and the PCR fragment was connected between the pHGECm vector EcoRI and BamHI restriction sites, and was introduced into Escherichia coli WM3064 by chemical transformation. In competent cells, the recombinant plasmid pHGECm-alpA was verified by sequencing, and the obtained strain was WM3064/pHGECm-alpA. Using WM3064/pHGECm-alpA as the donor bacteria and SCSIO 43097 as the recipient bacteria, the conjugation transfer experiment was carried out, and the bacteria liquid was washed and resuspended with the medium, and after mixing, it was placed on the LB plate containing 0.3mM DAP. After overnight culture, the bacterial lawn was picked with an inoculation loop and streaked onto an LB plate containing chloramphenicol (30 μg/mL) to verify that the recombinant bacteria were obtained. The correct recombinant strain 43097/pHGECm-alpA was activated overnight, 1/100 was transferred to 25 ml of fresh 2216E medium containing chloramphenicol (15 μg/mL), and IPTG was added to OD ₆₀₀ = about 1.0, at 30 In a ℃ shaker, shake at 220 rpm for 6-8 hours, inoculate the bacterial solution and streak it to the LB plate to obtain a single colony, and use the 43097GI-wF/-wR primer to determine the correct gene island excision mutant strain, thereby obtaining the gene Islet GIVal43097-directed excision mutant ΔGIVal43097.

4. Biofilm phenotype detection of gene island excised strains (single gene knockout mutants Δdgc137, Δdgc139 and Δdgc140, and mutant ΔGIVal43097):

1. Solid-liquid biofilm determination:

First, pick a monoclonal strain and inoculate it into a Erlenmeyer flask containing 25 mL of 2216E medium, and culture it overnight at 30°C. Subsequently, the bacteria cultured overnight was diluted to OD ₆₀₀ =0.05 and transferred to fresh 2216E medium, and 250 μL of the diluted culture medium was taken into a 96-well plate, and the sample with only 2216E medium was used as a blank control. Then the 96-well plate was placed in a 30°C constant temperature incubator for static culture (do not shake during the culture), and the OD ₆₂₀ value was measured with a microplate reader after culturing for 2h, 4h, 6h or 8h. The culture solution was poured out, and the 96-well plate was gently dipped 5 times with tap water to wash away the free cells, and 280 μL of 0.1% crystal violet solution was added for staining. After standing at room temperature for 20 min, pour out the crystal violet solution, continue to gently soak the 96-well plate with clean tap water for 5 times to wash away the free crystal violet, and then invert the 96-well culture plate on absorbent filter paper to remove the residual water. , After natural air-drying, add 300 μL of absolute ethanol and let stand for 5-10min to fully dissolve the crystal violet. Finally, read the OD ₅₄₀ value of the 96-well plate with a microplate reader. Do 3 replicates for each strain and do 3 parallels for each replicate. The numerical calculation of solid-liquid biofilms was carried out using Sample(OD540 nm)-Blank(OD540 nm)/Sample(OD620 nm)-Blank(OD620 nm).

2. Determination of gas-liquid biofilm:

First, pick a single clone and inoculate it in a Erlenmeyer flask containing 25 mL of 2216E medium, and culture it overnight at 30°C. Next, the overnight cultured bacterial solution was diluted to OD ₆₀₀ =0.05 and transferred to fresh 2216E medium, and 4 mL of the diluted bacterial solution was drawn into a 10 mL glass test tube. Incubate in a 30°C incubator, observe and record and take pictures several times a day (be careful not to shake the test tube during observation).

3. Determination of gas-solid biofilm:

First, pick a single clone and inoculate it in a Erlenmeyer flask containing 25 mL of 2216E medium, and culture it overnight at 30°C. Pipette 10 μL of bacterial solution onto a plate containing FeCl ₃ in seawater LB medium, LB medium or other nutrient medium, and place it in a 30°C incubator for static culture. Observe and record and photograph the colony morphology every day.

The encoding gene dgc137 of the gene island GIVal43097 can significantly enhance the biofilm formation of the host Vibrio. At the same time, gene island is an important carrier of gene horizontal transfer. It generally has the ability of horizontal transfer and can be spread among different species of bacteria in the same ecological niche. Therefore, the establishment of a method of direct excision of gene island from the genome can not only remove the gene island Contains genes that also eliminate its ability to spread. It was found that the cleavage enzyme AlpA encoded by the gene island can mediate the excision of the gene island (Figure 3A and B). We obtained the gene island deletion mutant ΔGIVal43097 by this method. Subsequently, we compared the biofilm formation ability of the gene island deletion mutant ΔGIVal43097 and the wild strain, and the results showed that the deletion of GIVal43097 significantly reduced the ability of the host bacteria to form solid-liquid biofilms, gas-solid biofilms, and gas-liquid biofilms (Fig. 3C-E). ).

V. Biofilm phenotype detection of heterologous expression of key biofilm genes on the gene island (dgc137, dgc139 and dgc140 were respectively expressed in Pseudomonas aeruginosa PAO1):

The genes dgc137, dgc139 and dgc140 were expressed in Pseudomonas aeruginosa PAO1 and constructed using the pHERD20T vector (for the construction method, please refer to the literature: Li et al., Molecular Microbiology, 2019, 111(2), 495-513). First, the coding regions of the genes dgc137, dgc139 and dgc140 were obtained by PCR amplification with primers (Table 2) respectively, and the PCR fragments were connected between the NcoI and HindIII restriction sites of the pHERD20T vector, and were introduced into Escherichia coli WM3064 by chemical transformation. The recombinant plasmids pdgc137, pdgc139, and pdgc140 were verified by sequencing. Using WM3064 containing the recombinant plasmid as the donor bacteria and PAO1 as the recipient bacteria, the conjugation transfer experiment was carried out. The bacteria liquid was washed and resuspended with the medium, and then placed on the LB plate containing 0.3 mM DAP after mixing, at 37°C. After culturing for 6 hours, pick the bacterial lawn with an inoculation loop and streak it onto an LB plate containing carbenicillin (100 μg/mL), thereby obtaining a recombinant strain PAO1 with heterologous expression of related genes on the gene island in Pseudomonas aeruginosa. /pdgc137, PAO1/pdgc139 and PAO1/pdgc140. The control-PAO1/pHERD20T transformed into the empty vector pHERD20T was used.

The correct recombinant strain was cultured at 37°C for overnight activation, and then, the overnight culture was diluted to OD ₆₀₀ = 0.05 and transferred to fresh LB medium containing carbenicillin (100 μg/mL) and 10 mM arabinose, And take 250 μL of the diluted culture medium in 96-well plate respectively, and take the sample with only LB medium added as blank control. Then, the 96-well plate was placed in a 37°C constant temperature incubator for static culture (do not shake during the culture), and the OD ₆₂₀ value was measured with a microplate reader after culturing for 2 hours. The culture solution was poured out, and the 96-well plate was gently dipped 5 times with tap water to wash away the free cells, and 280 μL of 0.1% crystal violet solution was added for staining. After standing at room temperature for 20 min, pour out the crystal violet solution, continue to gently soak the 96-well plate with clean tap water for 5 times to wash away the free crystal violet, and then invert the 96-well culture plate on absorbent filter paper to remove the residual water. , After natural air-drying, add 300 μL of absolute ethanol and let stand for 5-10min to fully dissolve the crystal violet. Finally, read the OD ₅₄₀ value of the 96-well plate with a microplate reader. Do 3 replicates for each strain and do 3 parallels for each replicate. The numerical calculation of solid-liquid biofilms was carried out using Sample(OD540 nm)-Blank(OD540 nm)/Sample(OD620 nm)-Blank(OD620 nm).

The biofilm formation ability of the recombinant bacteria was detected by the above method, and it was found that dgc137 significantly enhanced the biofilm formation ability of PAO1; dgc140 could also enhance the biofilm formation ability of PAO1, but the ability was weaker than that of dgc137; dgc139 significantly enhanced the biofilm formation ability of PAO1 There was no effect on capacity, no difference from the empty vector pHERD20T (Figure 4).

sequence listing

<110> South China Sea Institute of Oceanography, Chinese Academy of Sciences

<120> A kind of biofilm gene island GIVal43097 and its excision method

<160> 1

<170> SIPOSequenceListing 1.0

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<211> 21792

<212> DNA

<213> Vibrio alginolyticus SCSIO 43097

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aggcataggt attttgaaag ttcagtgtct tgatttagct gatactcaat ttgatggata 3780

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gcgctactaa gattattttc ttctgccagc gtaacaaata ctgatggagg tatatctccg 3960

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ataatcatgc gttgtttttg gaaatcttct tcagcgagtt ttttgtcgtg gtatcggata 4140

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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

ttttatctc 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

tattatttttg 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

ttttttgcgcc 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 cgcattttttt 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 ttttttaggag 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 ttttttacatt 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 ggtgttttttt 21720

gctaaagata aaagccgttc atgtccttat tttatgcgct ctagaagcac tccagactcc 21780

atatggtggg ta 21792

Claims (9)

1. A biomembrane gene island GIVal43097, characterized in that the nucleotide sequence is shown in SEQ ID NO. 1.

2. Guanylate cyclase gene, characterized in that the nucleotide sequence is shown as 1907-2812 base of SEQ ID NO.1 or as 5342-6748 base of SEQ ID NO. 1.

3. Use of the guanylate cyclase gene of claim 2 to promote bacterial biofilm formation.

4. Use of a knock-out dgc137 gene in the biomembrane gene island GIVal43097 to reduce biofilm synthesis.

5. An excisionase alpA, characterized in that the nucleotide sequence is the reverse complement of the sequence shown by the 15008-15217 th base of SEQ ID NO. 1.

6. Use of an overexpression of the excision enzyme alpA according to claim 5 for mediating the excision of the island of the biofilm gene GIVal 43097.

7. Use according to claim 6, characterized in that the use of the overexpressed excisionase alpA for removing the formation of bacterial biofilms in organisms.

8. Use according to claim 7, characterized in that the use of the overexpressed excisionase alpA for removing the biofilm formation of Vibrio alginolyticus SCSIO 43097.

9. A method for excising an island GIVal43097 of a biofilm gene, comprising overexpressing the excisionase alpA of claim 5 in an organism containing an island GIVal43097 of a biofilm gene.

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