CN111471634A - Method for genetically modifying bacillus subtilis, strain obtained by method and application of strain - Google Patents

Abstract

The invention discloses a method for genetically modifying bacillus subtilis, which comprises the following steps: starting strain MK3-MEP 123-deltadhbBConstructing; (II) overexpression of Glycerol kinase GeneglpKAnd glycerol-3-phosphate dehydrogenase geneglpD；(III) knocking out methylglyoxal synthase coding genemgsAAnd a gene encoding glycerol-1-phosphate dehydrogenasearaM。The invention is sequentially over-expressedglpK、glpDIncreasing glycerol contentThe utilization rate is used for promoting the synthesis of dihydroxyacetone phosphate and further increasing the synthesis of MK-7; knock out in sequencemgsA、araMRespectively blocking the synthesis of pyruvaldehyde and glycerol 1-phosphate, reducing the consumption of dihydroxyacetone phosphate and promoting the synthesis of MK-7, fermenting the obtained recombinant strain BSMK _4 in a 500m L shake flask for 96 hours, wherein the yield of MK-7 is 70.3 mg/L, which is compared with that of a starter strain MK3-MEP 123-deltadhbB(55.6 mg/L) by 26.4%.

Description

Method for genetically modifying bacillus subtilis, strain obtained by method and application of strain

The technical field is as follows:

the invention relates to a method for genetically modifying bacillus subtilis, belonging to the field of biological genetic engineering.

Background art:

the natural fat-soluble vitamin K comprises plant-derived vitamin K₁(also called phytoquinone, PK) and vitamin K derived from bacteria₂(also known as menadione, MK). According to the number of isoprene units in the side chain, there are 14 kinds of menaquinones, which are marked as MK-n, and MK-4, MK-7 and the like are common. In prokaryotes, MK-n is involved in electron transport in the respiratory chain. For humans and other mammals, vitamin K, because it is an important cofactor for the translational conversion of glutamate residues of specific proteins in blood and bone to gamma-carboxyglutamic acid (Gla), serves to maintain calcium homeostasis, inhibit vascular wall calcification, support endothelial integrity, promote bone mineralization, and participate in tissue turnover and cell growth control. Common vitamin K-dependent proteins include coagulation factors (II, VII, IX, X and prothrombin), protein C and protein S, osteocalcin, Matrix Gla Protein (MGP), periostin, and the like. Studies have shown that, despite the high PK content in food, PK is less bioactive than MK-n (especially MK-7); long-chain MK-n, such as MK-7, has a more durable effect on normal coagulation than PK and MK-4. Therefore, MK-7 is more popular in the food, pharmaceutical and health industries due to its long half-life and good bioavailability, and is widely used as a dietary supplement or drug for the treatment of osteoporosis [8 ]]Arterial calcification, cardiovascular disease, cancer, parkinson's disease, and the like.

The traditional MK-7 production method comprises a chemical synthesis method and a microbial fermentation method, although the process for producing MK-7 by the chemical synthesis method is continuously innovated, the defects of low yield, more byproducts, organic solvent residue, production environment danger and environmental potential are not completely solved, and the production and application of MK-7 are restricted, the large-scale and high-density continuous submerged fermentation (mainly based on fermentation of Bacillus natto) of the microorganisms produces MK-7 which is more advantageous due to the characteristics of controllable quality, safety, greenness, nature and the like, Toshiro Sato and the like separates a strain B.subtilis from Japanese food natto, screens a menaquinone resistant strain by the traditional mutagenesis, can produce 35 mg/L MK-7 after fermenting for 4 days in a 7L tank, screens the screened diphenylamine resistant strain, can be fermented for 1 day at 37 ℃, can produce 60 mg/L MK-7 after fermenting for 5 days at 45 ℃, screens a 1-hydroxy-2-naphthoic acid resistant mutant strain B.subtilis and the strain B.7, the screened by a fermentation method for producing 1-hydroxy-2-naphthoic acid resistant mutant strain B.7, the strain A.7, the strain can be produced by fermentation for 4 days at 37 ℃ and the strain A, the strain A2 strain A, the strain A3, the strain A, the strain can be produced by the fermentation process of fermentation of the strain A, the strain A, the strain A3, the strain A, the strain B-7, the strain is produced by the strain A, the strain B.7, the strain is produced by the strain A, the fermentation, the strain A, the fermentation of the strain A, the strain is produced by the strain A, the fermentation of the strain is produced by the fermentation of the strain A, the strain is produced by the strain A, the fermentation of the strain is produced by the fermentation, the fermentation of the fermentation, the fermentation of the strain A, the fermentation of the strain A, the.

Until now, the traditional mutagenesis or fermentation optimization is basically adopted for producing MK-7 by using Bacillus natto, and the methods can improve the yield of MK-7, but cannot accurately modify a target gene, have higher secondary mutation probability and longer genetic breeding period.

Disclosure of Invention

The invention aims to solve the technical problem of providing a gene modification method, and particularly provides a gene modification method which takes MK3-MEP 123-delta dhbB derived from bacillus subtilis 168 as an initial strain, surveys the influence of the initial strain on MK-7 synthesis by promoting the synthesis of dihydroacetone phosphate and reducing the consumption of the dihydroacetone phosphate, and provides a foundation for constructing an MK-7 high-yield strain by genetically modifying bacillus natto in the future.

Glycerol has been shown to be the optimal carbon source for MK-7 synthesis. In bacillus subtilis, glycerol enters cells by a mode of promoting diffusion with the help of membrane channel protein, and is sequentially subjected to phosphorylation by glycerol kinase and oxidation by glycerol-3-phosphate dehydrogenase to generate dihydroxyacetone phosphate (DHAP). The enzymatic synthesis of glyceraldehyde-3-phosphate (G3P) from DHAP by triose phosphate isomerase, and the subsequent glycolysis process provides precursors for shikimic acid pathway and methylerythritol-4-phosphate pathway, respectively, to synthesize chorismate and heptaenylpyrophosphate, which are important precursors for the synthesis of menaquinone-7. In addition, DHAP can also be catalyzed by methylglyoxal synthase to generate methylglyoxal; catalyzing by glycerol-1-phosphate dehydrogenase to generate glycerol-1-phosphate for synthesizing phosphoglycerol glycolipid.

To date, no comprehensive systematic study of the de novo MK-7 biosynthetic pathway has been discovered. Bacillus subtilis is one of the best characterized model microorganisms, as it has many studies on biochemistry, genetics and molecular biology, its growth rate is fast and it is 'generally regarded as safe' (GRAS). And the complete genome sequence of the bacillus subtilis 168 is sequenced, which is beneficial to constructing industrial production strains by means of genetic engineering. Thus, the present invention selects Bacillus subtilis 168 as a underpant cell for a comprehensive systematic study of the overall synthetic pathway for MK-7 synthesis from glycerol.

The invention takes bacillus subtilis 168 as a starting bacterium, and constructs a recombinant strain MK3-MEP 123-delta dhbB by overexpressing 1, 4-dihydroxy-2-naphthoic acid heptanenyltransferase (MenA) of a menaquinone-7 pathway, 1-deoxyxylulose-5-phosphate synthase (Dxs) of a methylerythritol-4-phosphate pathway, 1-deoxyxylulose-5-phosphate reductoisomerase (Dxr), methylerythritol 4-phosphocytidylyltransferase (YacM) and methylerythritol 2, 4-cyclodiphosphate synthase (YacN) and knocking out dhbB gene (coding isochorismate lyase). FIG. 1 shows the biosynthesis pathway of MK-7 in Bacillus subtilis, which uses constructed MK3-MEP 123-delta dhbB as an initial strain, firstly overexpresses glycerol kinase (GlpK) and glycerol-3-phosphate dehydrogenase (GlpD), promotes the synthesis of DHAP, and examines the influence of the DHAP on the synthesis of MK-7; then, the mgsA gene (coding a methylglyoxal synthetase) and the araM gene (coding a glycerol-1-phosphate dehydrogenase) are knocked out, the consumption of DHAP is reduced, and the influence of the DHAP on MK-7 synthesis is examined; finally, the synthesis of DHAP can promote the synthesis of MK-7, namely DHAP is an important intermediate metabolite influencing the synthesis of MK-7, and theoretical basis is provided for industrially constructing the high-yield MK-7 strain of Bacillus natto by a metabolic engineering means in the future.

Description of the drawings:

FIG. 1 is the biosynthetic pathway for MK-7 in Bacillus subtilis.

Substance abbreviation: gly, glycerol; Gly-3P, glycerol-3-phosphate; DHAP, dihydroxyacetone phosphate; G3P, glyceraldehyde-3-phosphate; MG, methylglyoxal; G1P, glyceraldehyde-1-phosphate; E4P, erythrose-4-phosphate; PEP, phosphoenolpyruvate; pyr, pyruvic acid; CHA, chorismic acid; HepPP, heptaenylpyrophosphoric acid; MK-7, menaquinone-7; SA, shikimic acid; MEP, methylerythritol-4-phosphate. Enzyme: GlpK, glycerol kinase; GlpD, glycerol-3-phosphate dehydrogenase; MgsA, methylglyoxal synthase; AraM, glycerol-1-phosphate dehydrogenase.

FIG. 2 is a diagram showing the growth of the starting bacteria MK3-MEP 123-. DELTA.dhbB and the recombinant bacteria BSMK _1 and BSMK _2 (FIG. 2A) and the yield of MK-7 (FIG. 2B); relative transcript level of the gene glpK in recombinant BSMK _1 compared to the starting strain MK3-MEP 123-. DELTA.dhbB (FIG. 2C); relative transcript level of the gene glpD in recombinant strain BSMK _2 compared to control strain BSMK _1 (fig. 2D). FIG. 3 is a graph showing the growth of cells (FIG. 3A) and the MK-7 production of control bacteria BSMK _2 and knockout bacteria BSMK _3 and BSMK _4 (FIG. 3B).

The specific implementation mode is as follows:

(A) Material

Strains, plasmids and media

All strains and plasmid information referred to in this example are detailed in table 1.

L B medium (peptone 10 g/L, yeast extract 5 g/L0, sodium chloride 10 g/L) was used for general culture of B.subtilis, solid medium was supplemented with 15 g/L agar powder, neomycin 16 μ g/m L, or chloramphenicol 8 μ g/m L, fermentation medium: glycerol 30m L/L, soybean peptone 60 g/L, yeast extract 5 g/L, K₂HPO₄3g/L，MgSO₄·7H₂O0.5g/L，pH7.3。

Table 1 strains and plasmids involved in the experiment

Reagent and instrument

FastTaq enzyme, Hifi DNA polymerase and dNTP are all purchased from Beijing Quanjin Biotechnology Limited, bacterial total RNA extraction Kit TRIzol Reagent is purchased from Invitrogen, ReverTra Ace qPCR RT Kit, SYBRGreen Real-time PCR Master Mix Kit is purchased from TOYOBO, standard MK-7 is purchased from ChromaDex, and the rest biochemical reagents are imported or domestic analytical pure reagents.

Primers used for PCR are shown in Table 2.

TABLE 2PCR primer sequences

Method of producing a composite material

DNA manipulation

All DNA fragments were from PCR amplification; the DNA fragments used for transformation are spliced by adopting an overlapping PCR method. Preparation and transformation of the subtilis competent cells Using Spizisen method [23 ]]. Both gene overexpression and gene knockout on chromosomes employ label-free modification methods [24]I.e. using counter-selection boxes (P)_araNeo) and selection cassette (cat-araR). Primer synthesis and DNA sequencing were both responsible for Jinweizhi Biotechnology, Inc. (Suzhou, China).

The method comprises the following specific steps:

construction of starting Strain MK3-MEP 123-. DELTA.dhbB

Overexpression of the menA gene at the yxlA locus on the chromosome of B.subtilis. Firstly, using chromosome of B.subtilis 168 as a template, and respectively amplifying fragments U (1115bp), A (1057bp), D (1053bp) and G (806bp) by using primers yxlA-menA-U1/yxlA-menA-U2q, yxlA-menA-1q/yxlA-menA-2, yxlA-menA-D1q/yxlA-menA-D2, yxlA-menA-G1 q/yxlA-menA-G2; the plasmid pUC57-1.8k-P1 was used as a template, and the promoter-containing P was amplified with primers yxlA-menA-P1/yxlA-menA-P2_lapSFragment P of (442 bp); fragment CR (2069bp) was amplified using primer yxlA-menA-CR1q/CR2, using chromosome of BS168NUm as a template. Then, splicing the segment U, P and A into a segment UPA (2614bp) by using a primer yxlA-menA-U1/yxlA-menA-2 through an overlapping PCR method; then the fragment UPA and the fragment D are spliced into a fragment UPAD (3667bp) by using a primer yxlA-menA-U1/yxlA-menA-D2; finally, the fragment UPAD, the fragment CR and the fragment G are spliced into a fragment UPADCRG (6542bp) by using a primer yxlA-menA-U1/yxlA-menA-G2. And transforming the UPADCRG fragment into a competent cell of a recipient bacterium BS168NU, and finally obtaining a recombinant strain MK3 with menA gene integrated at a yxlA locus through two-step screening.

Overexpression of dxs Gene at the yjoB site on the B.subtilis chromosome. Firstly, using chromosome of B.subtilis 168 as a template, and respectively amplifying fragments U (1236bp), s (1947bp), D (808bp) and G (697bp) by using primers yjoB-dxs-U1/yjoB-dxs-U2q, yjoB-dxs-1q/yjoB-dxs-2, yjoB-dxs-D1q/yjoB-dxs-D2 and yjoB-dxs-G1 q/yjoB-dxs-G2; plasmid pUC57-1.8k-P2 was used as a template, and primer P was used₄₃1/P ₄₃2 amplification of a plasmid containing promoter P₄₃Fragment P of (232 bp); fragment CR (2069bp) was amplified using primer yjoB-dxs-CR1q/CR2, using chromosome of BS168NUm as a template. Then, the primer yjoB-dxs-U1/yjoB-dxs-2 is used for splicing the segment U, P and s into a segment UPs (3415bp) by an overlapping PCR method; then splicing the fragment UPs and the fragment D into a fragment UPsD (4223bp) by using a primer yjoB-dxs-U1/yjoB-dxs-D2; finally, the fragment UPsD, the fragment CR and the fragment G were spliced into a fragment UPsDCRG (6989bp) by using the primer yjoB-dxs-U1/yjoB-dxs-G2. The UPsDCRG fragment is transformed into a competent cell of receptor bacterium MK3, and the recombinant strain MK3-MEP1 with dxs gene integrated at the yjoB locus is finally obtained through two-step screening.

On the basis of the strain MK3-MEP1, dxr and yacM-yacN are sequentially overexpressed to obtain a recombinant strain MK3-MEP 123. Using chromosome of B.subtilis 168 as template, respectively amplifying fragments U (1003bp), D (815bp) and G (605bp) by primers dhbB-U1/dhbB-U2, dhbB-D1q/dhbB-D2 and dhbB-G1 q/dhbB-G2; fragment CR (2069bp) carrying the selection cassette (cat-araR) was amplified with primer dhbB-CR1q/CR2 using chromosome of BS168NUm as template. Firstly, splicing the fragments U and D into UD (1818bp) by overlap PCR by using primers dhbB-U1/dhbB-D2; the three fragments UD, GR and G were then spliced into UDCRG (4492bp) by the overlap PCR method using primers dhbB-U1/dhbB-G2. And transforming the UDCRG fragment into a competent cell of a receptor bacterium MK3-MEP123, and finally screening to obtain a recombinant strain MK3-MEP 123-delta dhbB with a gene dhbB knocked out.

Patent publication No. CN108715824A, which discloses the sequence of primers and fragments for over-expressing menA.

(II) overexpression of the Glycerol kinase Gene glpK and the Glycerol-3-phosphate dehydrogenase Gene glpD

Construction of glpK overexpression strains. Firstly, using chromosome of starting bacterium B.subtilis MK3-MEP 123-delta dhbB as template, respectively using primers pksJ-glpK-U1/pksJ-glpK-U2q, pksJ-glpK-1q/pksJ-glpK-2, pksJ-glpK-D1q/pksJ-glpK-D2 and pksJ-glpK-G1 q/pksJ-glpKK-G2 to amplify fragment U (shown as SEQ ID No.36, 1164bp), K (shown as SEQ ID No.37, 1783bp), D (shown as SEQ ID No.38, 948bp) and G (shown as SEQ ID No.39, 990 bp); amplifying a fragment CR (2069bp shown as SEQ ID No. 40) by using a primer pksJ-glpK-CR1q/CR2 by using a chromosome of BS168NUm as a template; plasmid pUC57-1.8k-P1 was used as a template, and primer P was used_lapS1/P_lapS2Amplification of a nucleic acid molecule containing promoter P_lapSFragment P of the expression cassette (shown as SEQ ID No.41, 256 bp). Then, splicing the U, P fragment and the K fragment into a UPK fragment (3203bp) by using a primer pksJ-glpK-U1/pksJ-glpK-2 through an overlapping PCR method; splicing the fragment UPK and the fragment D into a fragment UPKD (4151bp) by using a primer pksJ-glpK-U1/pksJ-glpK-D2; finally, the fragment UPKD, the fragment CR and the fragment G were spliced into a fragment UPKDCRG (7210bp) using the primers pksJ-glpK-U1/pksJ-glpK-G2. And transforming the UPKDCRG fragment into competent cells of receptor bacteria MK3-MEP 123-delta dhbB, and finally obtaining a recombinant strain BSMK _1 with glpK genes integrated at a pksJ locus through two-step screening. UPKDCRG fragment sequence (shown as SEQ ID No. 42).

First, using chromosome of starting bacterium B.subtilis MK3-MEP 123-delta dhbB as template, respectively using primers pks L-glpD-U1/pks L-glpD-U2 q, pks L-glpD-1 q/pks L-glpD-2, pks L-glpD-D1 q/pks L-glpD-D2 and pks L-glpD-G1 q/pks L-glpD pUC-G2 to amplify fragment U (shown as SEQ ID No.43, 1118bp), D (shown as SEQ ID No.44, 1749bp), D (shown as SEQ ID No.45, 1004bp) and G (shown as SEQ ID No.46, SEQ ID No. 769bp), using chromosome of starting bacterium 168, pks 3-MEP 123-delta dhbB as template, using primers pks 1-glpD-1 q 11-glpD-9-pD 2 to amplify fragment U (shown as SEQ ID No.43, 1118, SEQ ID No.45, 9bp), using chromosome of primer, chromosome of chromosome 168, and PCR 3-2068, and plasmid of plasmid 1-1-369, 9, and plasmid of plasmid_lapS1/P_lapS2Amplification of a nucleic acid molecule containing promoter P_lapSFragment P of the expression cassette (256 bp as shown in SEQ ID No. 41) then fragments U, P and D were spliced into fragment UPD (3123bp) by the overlap PCR method using primers pks L-glpD-U1/pks L-glpD-2, fragment UPD and fragment D were spliced into fragment UPDD (4127bp) using primers pks L-glpD-U1/pks L-glpD-D2, fragment UPDD (4127bp) was finally spliced into fragment UPDDRG (6965bp) using primers pks L-glpD-U1/pks L-glpD-G2, fragment UPCR and fragment G were finally transformed into competent cells of recipient bacterium BSMK _1, and through two steps, recombinant strain BSMK _ 2. UPMK sequence of the DDC integrated glpD gene at the site of pks L was finally obtained as shown in SEQ ID No. 47.

The invention promotes the synthesis of dihydroxyacetone phosphate (DHAP) by regulating glpK and glpD genes, and inspects the influence of the dihydroxyacetone phosphate on MK-7 synthesis. Within B.subtilis, glycerol kinase GlpK catalyzes the phosphorylation of glycerol to glycerol-3-phosphate (Gly-3P), which is oxidized by glycerol-3-phosphate dehydrogenase GlpD to DHAP. DHAP is catalyzed by triose phosphate isomerase to synthesize glyceraldehyde-3-phosphate (G3P), and then enters glycolysis process to provide precursors for shikimic acid pathway and methylerythritol-4-phosphate pathway, which are used for synthesizing chorismate and heptaene pyrophosphoric acid respectively, and the chorismate and heptaene pyrophosphoric acid are finally synthesized into MK-7 through MK-7 pathway. An MK-7 producing strain B.subtilis MK3-MEP 123-delta dhbB has been obtained in this laboratory. As the fermentation carbon source is glycerol, in order to further improve the yield of MK-7, MK3-MEP 123-delta dhbB is used as an initiating bacterium and P is utilized_lapSpksJ and pks of the expression cassette on its chromosomeL sites respectively over-express glpK and glpD genes, improve the glycerol utilization rate and obtain recombinant bacteria BSMK _1 and BSMK _2, and the thallus growth conditions of the two bacteria and the MK-7 yield of 96h after fermentation are obtained through shake flask fermentation.

Shake flask fermentation culture and determination of thallus growth

TABLE 3 fermentation media and fermentation conditions

Parameter(s)	Range of
		Glycerol	20～80mL/L
Soybean peptone
		60～180g/L
Yeast extract
		0～20g/L
K2HPO4			1～5g/L
	MgSO4·7H2O	0.1～0.8g/L
pH			6.5～7.5
	Amount of inoculation	1％～6％
Temperature of			35～45℃
	Rotational speed	100～250r/min
Time of fermentation			72-144h

500m L shake flask fermentation, selecting newly activated plate single colony, inoculating into 250m L shake flask containing 30m L L B culture medium, shake culturing at 37 deg.C for 14h at 200r/min, transferring into 500m L conical flask containing 50m L fermentation culture medium (three parallel bottles) at 1% inoculum size, shake culturing at 37 deg.C and dark at 220r/min for 120 h.

Determination of biomass: sampling after fermenting for 6h, then sampling after 24h, taking a proper amount of fermentation liquor every 24h, centrifuging for 1min at 13000r/min to precipitate cells, washing, then re-suspending, diluting by proper times, and determining OD of the bacterial suspension₆₀₀The value is obtained.

Real-time quantitative reverse transcription PCR analysis:

taking 1m L fermentation liquor of initial (24h) growth stable period of the thallus, centrifuging at 12000r/min for 1min to collect the thallus, extracting RNA by using a total RNA extraction Kit TRIzol Reagent, adding the extracted RNA into a reaction system according to ReverTra Ace qPCR RT Kit instruction for reverse transcription to obtain cDNA, and finally adding the cDNA into the reaction system according to SYBR Green Real-time PCR Master Mix instruction for fluorescent quantitative PCR to obtain C_tValue according to formula 2^-△△CtCalculating the relative expression amount of the gene to be detected (wherein, △△ C)_t＝[(C_{t(target,test)}-C_t(ref,test))-(C_{t(target,calibrator)}-C_{t(ref,calibrator)})](ii) a target: a target gene; ref: an internal reference gene, wherein ccpA is selected as the internal reference gene in the experiment; test: a sample to be tested; calibrator: reference sample).

Extraction and HP L C detection of MK-7:

taking a 96-hour fermentation liquid of 750 mu L, adding 1m L of isopropanol and 2m L of n-hexane into the fermentation liquid, carrying out vortex oscillation for 2min, centrifuging at 5000r/min and 4 ℃ for 6min, taking a supernatant of 1m L, carrying out vacuum centrifugal concentration, finally adding 750 mu L of methanol for dissolution, filtering by a 0.25 mu m filter membrane, taking a filtrate, carrying out HP L C detection, and avoiding light as much as possible in all processes.

Chromatographic conditions of a chromatographic column, a Shim-pack GIST C18 chromatographic column (250mm × 4.6.6 mm, 5 mu m), a detector, ShimadzuSPD-20A, a mobile phase, pure methanol, a column temperature of 50 ℃, a detection wavelength of 270nm and a flow rate of 1.0m L/min, wherein a standard solution of a standard substance MK-7 is measured by using the same chromatographic conditions, a concentration-peak area standard curve is drawn, and MK-7 is quantified.

As is clear from Table 4, the growth tendency of the recombinant bacteria was almost unchanged and the growth thereof was not significantly changed as compared with the growth of the initial bacteria. As is clear from Table 5, the promoter P was used_lapSThe mRNA expression level of glpK in the recombinant strain BSMK _1 obtained after the expression cassette overexpresses glpK is 2.2 times of that of the original strain MK3-MEP 123-delta dhbB; by P_lapSThe expression level of mRNA of glpD in the recombinant strain BSMK _2 obtained after the expression of the glpD by the expression cassette is 1.5 times that of the recombinant strain BSMK _1, and the result shows that the expression level of the glpD is 1.5 times that of the recombinant strain BSMK _1_lapSAs shown in Table 6, after 96h of fermentation, the MK-7 yield of the starting strain is 55.6 mg/L, the MK-7 yield of the recombinant strain BSMK _1 is 58.9 mg/L which is 5.9 percent higher than that of the starting strain, the MK-7 yield of the recombinant strain BSMK _2 is 61.1 mg/L which is 9.9 percent higher than that of the starting strain, and the results show that the synthesis of metabolite DHAP is promoted as glpK and glpD are sequentially overexpressed, and the MK-7 yield is gradually increased.

TABLE 4 growth of the cells

Note:^*shows significant difference (P < 0.05) compared with the starting bacterium MK3-MEP 123-delta dhbB;^#shows a very significant difference (P < 0.01) compared with the starting bacterium MK3-MEP 123-delta dhbB.

TABLE 5 relative transcript levels of glpK and glpD

Bacterial strains	Relative transcriptional level of glpK	Relative transcriptional levels of glpD
			MK3-MEP123-ΔdhbB	1.0±0.1	－－
BSMK_1	2.2#±0.3	1.0±0.1
			BSMK_2	－－	1.5±0.1

Note:^*shows significant difference (P < 0.05) compared with the starting bacterium MK3-MEP 123-delta dhbB;^#shows a very significant difference (P < 0.01) compared with the starting bacterium MK3-MEP 123-delta dhbB.

TABLE 6MK-7 production

Note:^*shows significant difference (P < 0.05) compared with the starting bacterium MK3-MEP 123-delta dhbB;^#shows a very significant difference (P < 0.01) compared with the starting bacterium MK3-MEP 123-delta dhbB.

(III) knocking out methylglyoxal synthetase coding gene mgsA and glycerol-1-phosphate dehydrogenase coding gene araM

Using chromosome of starting bacterium B.subtilis MK3-MEP 123-delta dhbB as template, respectively amplifying fragment U (1382 bp shown as SEQ ID No. 48), D (1130 bp shown as SEQ ID No. 49) and G (813 bp shown as SEQ ID No. 50) by using primers mgsA-U1/mgsA-U2, mgsA-D1q/mgsA-D2 and mgsA-G1 q/mgsA-G2; fragment CR (2069bp, shown in SEQ ID No. 40) carrying the selection cassette (cat-araR) was amplified using the primer mgsA-CR1q/CR2, using chromosome BS168NUm as a template. Firstly, splicing the fragments U and D into UD (2512bp) by overlapping PCR by using primers mgsA-U1/mgsA-D2; the three fragments UD, GR and G were then spliced into UDCRG (5394bp) by overlap PCR using primers mgsA-U1/mgsA-G2. And transforming the UDCRG fragment into a competent cell of a recipient bacterium BSMK _2, and finally screening to obtain a recombinant strain BSMK _3 with the mgsA knocked out gene. UDCRG fragment sequence (shown as SEQ ID No. 51).

Using chromosome of starting bacterium B.subtilis MK3-MEP 123-delta dhbB as template, respectively amplifying fragment U (1487 bp shown by SEQ ID No. 52), D (1026 bp shown by SEQ ID No. 53) and G (682 bp shown by SEQ ID No. 54) by using primers araM-U1/araM-U2, araM-D1q/araM-D2 and araM-G1 q/araM-G2; fragment CR (shown as SEQ ID No.40, 2069bp) was amplified using primer araM-CR1q/CR2 with chromosome of BS168NUm as a template. Firstly, splicing fragments U and D into UD (2513bp) by overlapping PCR by using primers araM-U1/araM-D2; the three fragments UD, GR and G were then spliced into UDCRG (5264bp) by overlap PCR using primers araM-U1/araM-G2. And transforming the UDCRG fragment into a competent cell of a receptor strain BSMK _3, and finally screening to obtain a recombinant strain BSMK _4 with the knocked-out gene araM. UDCRG fragment sequence (shown as SEQ ID No. 55).

As can be seen from FIG. 1, dihydroxyacetone phosphate (DHAP) can provide a precursor for MK-7 synthesis in glycolysis, and can also be catalyzed by methylglyoxal synthase (MgsA) to form Methylglyoxal (MG); it can also be used for synthesis of phosphoglycerol glycolipid by catalyzing glycerol-1-phosphate dehydrogenase (AraM) to generate glycerol-1-phosphate (G1P). Therefore, the invention takes BSMK _2 as a starting bacterium to reduce the consumption of DHAP and make the DHAP as much as possibleThe influence of (2) on MK-7 synthesis was examined. The growth of the knockout strains is shown in Table 7, the growth of the strains is slightly changed, and the maximum OD is shown₆₀₀As can be seen from Table 8, after 96h of fermentation, the bacterial strain BSMK _3 obtained by knocking out the gene mgsA encoding the methylglyoxal synthase has the MK-7 yield of 68.3 mg/L which is increased by 11.8 percent compared with the BSMK _1 of the original bacterium, and the bacterial strain BSMK _4 obtained by knocking out the gene mgsA encoding the methylglyoxal synthase and the gene araM encoding the glycerol-1-phosphate dehydrogenase sequentially has the MK-7 yield of 70.3 mg/L which is increased by 15 percent compared with the BSMK _1 of the original bacterium, which indicates that the consumption of DHAP is reduced, and the synthesis of MK-7 can be promoted, as shown in FIG. 3.

TABLE 7 growth of cells

Note:^*shows significant difference (P < 0.05) compared with BSMK _ 2;^#shows a very significant difference (P < 0.01) compared to BSMK _ 2.

TABLE 8MK-7 yields

Note:^*shows significant difference (P < 0.05) compared with BSMK _ 2;^#shows a very significant difference (P < 0.01) compared to BSMK _ 2.

The recombinant strain BSMK _4 is fermented in a 500m L shake flask for 96 hours, the yield of the MK-7 is 70.3 mg/L, and the yield of the MK-7 is improved by 26.4% compared with that of a starter MK3-MEP 123-delta dhbB (55.6 mg/L).

Sequence listing

<110> Qingdao ocean technology research institute of Tianjin university

<120> method for genetically modifying bacillus subtilis, strain obtained by method and application of strain

<160>55

<170>SIPOSequenceListing 1.0

<210>1

<211>18

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>1

ccttgtttct ggcattgg 18

<210>2

<211>40

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>2

cttcatcatc atattcttcg gctcctccta acggttctga 40

<210>3

<211>42

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>3

tagaggggag agaggacaaa tggaaactta catcttatct tt 42

<210>4

<211>19

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>4

cttagtgcga ccttcattc 19

<210>5

<211>37

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>5

gaatgaaggt cgcactaaga ggctggattg gattgat 37

<210>6

<211>18

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>6

ctggactgct tgctgtat 18

<210>7

<211>41

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>7

gcacgaaaac tgaatgaata accaagaaca ctgatgacaa t 41

<210>8

<211>18

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>8

gtaatcaccg ctggctta 18

<210>9

<211>39

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>9

gatacagcaa gcagtccagt cttcaactaa agcacccat 39

<210>10

<211>20

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>10

ttattcattc agttttcgtg 20

<210>11

<211>20

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>11

agagtcagga gcatttaacc 20

<210>12

<211>18

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>12

ttgtcctctc tcccctct 18

<210>13

<211>19

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>13

tgcctcctgt aatctctga 19

<210>14

<211>39

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>14

ggttaaatgc tcctgactct gccatctaag tgtccgtaa 39

<210>15

<211>41

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>15

agaggggaga gaggacaaat gatgaatcac caattttcta g 41

<210>16

<211>19

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>16

gcgtctgaca cttgataac 19

<210>17

<211>40

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>17

ggttatcaag tgtcagacgc ggcagacaac attgatgaat 40

<210>18

<211>21

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>18

ttgatttggc tgactctatt g 21

<210>19

<211>39

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>19

gcacgaaaac tgaatgaata aaggctgatt acgcaatgg 39

<210>20

<211>19

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>20

cttacctgct tccgctctt 19

<210>21

<211>41

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>21

caatagagtc agccaaatca atcttcaact aaagcaccca t 41

<210>22

<211>19

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>22

ttctccaagc atcacacaa 19

<210>23

<211>20

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>23

taggcagtcg taaattgaac 20

<210>24

<211>41

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>24

ggttcaattt acgactgcct acattccgat tctactcctc t 41

<210>25

<211>18

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>25

ccgaaacatc tccgacat 18

<210>26

<211>39

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>26

gcacgaaaac tgaatgaata aatgaggcga taggtcttc 39

<210>27

<211>18

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>27

gtaggcgatg aggctttg 18

<210>28

<211>39

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>28

gatgtcggag atgtttcggt cttcaactaa agcacccat 39

<210>29

<211>18

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>29

gccgatgact tggttgtt 18

<210>30

<211>20

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>30

tcccagacaa tgacaatatg 20

<210>31

<211>39

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>31

ttttgacgaa atccagccct cccagacaat gacaatatg 39

<210>32

<211>19

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>32

cggtctgatg gctatcttc 19

<210>33

<211>39

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>33

gcacgaaaac tgaatgaata acgaaacaac caacggatg 39

<210>34

<211>19

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>34

tgataagccg attggatgg 19

<210>35

<211>39

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>35

gaagatagcc atcagaccgt attcaactaa agcacccat 39

<210>36

<211>1164

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>36

ccttgtttct ggcattggcc agcacgatag accaagggag atttgatacg cctaaacgaa 60

ttggctgttt ttcctacgga tccggctgct gttctgaatt ttacagcgga attacaactc 120

cgcaaggcca ggagcgtcag cgaacatttg gtattgagaa gcatttggat cgccggtatc 180

agctctccat ggaagagtat gagctattgt ttaaggggag cgggatggtg cggtttggga 240

cgcgcaatgt aaagctggac tttgagatga taccgggcat catgcagtcc actcaagaga 300

agccgcgctt gtttttagaa gaaatttctg agtttcatcg gaagtacagg tggatctcgt 360

gacctatcaa acgataaagg tccgctttca agcatcggtt tgctatatta cctttcaccg 420

tcctgaagcg aataatacga ttaatgacac gctgatagaa gaatgcttgc aagtgttaaa 480

ccaatgtgaa acatcaacgg tgacggttgt cgttttagag gggcttcccg aggtgttttg 540

tttcggagcg gattttcaag aaatctatca ggaaatgaaa aggggcagaa aacaggcaag 600

ctcccaagag cctctctatg atttgtggat gaaattgcag accggccctt atgtcacgat 660

ttcgcatgtc aggggaaaag tgaatgccgg cggtcttgga tttgtatctg ccacagatat 720

tgccattgct gatcagacgg cgtcattcag tctctctgag ctgctattcg gcctgtaccc 780

tgcttgtgtt ttaccgtttc tgatccgccg tatcggccgg cagaaagcgc attatatgac 840

gcttatgaca aagccgattt ccgttcagga agccagtgaa tgggggttaa tagatgcttt 900

tgatgctgaa agtgacgtgc tgctgagaaa gcatttattg cgtttgcgga ggctgaataa 960

aaaaggaatc gcacattata aacagtttat gagctcactt gatcatcaag tcagtcgtgc 1020

gaaagccacc gctttaactg caaatcaaga catgttttct gatcctcaaa accaaatggg 1080

aatcatcagg tatgttgaaa caggacaatt cccatgggag gatcagtaac cgtttaaaaa 1140

tgacaatcag aaccgttagg agga 1164

<210>37

<211>1783

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>37

atggaaacgt acattttatc cttagatcag gggacgacaa gttcaagagc gattctgttt 60

aataaagaag gcaaaattgt ccactctgct caaaaggaat ttacacaata cttcccgcat 120

cctggctggg ttgagcataa tgccaatgaa atttggggct ctgtcctcgc ggttatcgcc 180

tcagtcatct ctgaatcagg aatcagcgct tctcaaattg ccggcatcgg catcacgaac 240

cagcgcgaga cgacggttgt gtgggataaa gatacaggaa gtcctgtcta taatgcaatc 300

gtttggcagt ccagacagac gtccggcatt tgtgaggaac ttcgtgaaaa aggatataat 360

gataaattca gagaaaaaac agggctttta atcgatcctt acttctccgg cacgaaggtg 420

aagtggattt tagacaatgt ggaaggcgca agagaaaaag cggaaaaagg cgagctgctg 480

tttggaacga ttgatacgtg gctcatttgg aaaatgtcag gcggaaaagc gcatgtgacc 540

gattactcca atgcctcaag aacactgatg tttaatattt acgatttaaa atgggacgat 600

caactgctcg acattctagg cgtaccgaaa tccatgctcc ctgaagtgaa gccgtcctct 660

catgtgtatg cggagactgt tgattatcac ttcttcggaa aaaatatccc gattgctgga 720

gcggcaggcg accagcagtc cgcattgttc ggccaggcat gctttgaaga aggcatgggg 780

aaaaacactt acggcacagg atgtttcatg ctgatgaata ccggggaaaa agcaattaag 840

tccgaacatg ggcttttgac aacaatcgct tggggcattg acggaaaagt gaactatgcg 900

ttagaaggga gcatttttgt cgcaggctct gccatccagt ggcttagaga cggtttgaga 960

atgttccagg attcatcgct aagcgaatct tatgcagaaa aagtggattc aactgacggc 1020

gtgtatgttg ttccagcatt tgtcggactg ggaacgcctt actgggacag cgatgtgcgc 1080

ggttcggttt tcggcctgac aagagggaca acaaaagagc actttatccg tgcgacactg 1140

gagtcattgg cttatcagac caaagatgtg cttgacgcaa tggaagcaga ttcaaacatt 1200

tcattaaaga cgctccgtgt agacggagga gctgtaaaaa acaatttcct aatgcagttc 1260

caaggagacc tgttgaatgt tcctgtggag cgcccggaaa ttaatgaaac gactgcactt 1320

ggcgcggctt atttggcggg tatcgctgtg ggattctgga aggaccgttc tgaaatcgcg 1380

aaccagtgga atctggataa acggtttgag cctgaattgg aagaagaaaa acgaaatgag 1440

ctgtataaag gctggcaaaa agccgtgaaa gcagctatgg cttttaaata aagtaatact 1500

atggtataat ggttacaagt taataagaac ggtcctgaga tgaggagaga ccacagcacc 1560

aaagtgtaag catgcacttt ggctgttgtg gtctcttttt ctatttaccg tgacaacaag 1620

gaggaaacgt aatgatgaat catcaatttt caagtcttga aagagatcgc atgctgacag 1680

acatgacgaa aaaaacatat gacctattta ttatcggagg aggaattaca ggagccggaa 1740

cagctcttga cgcggcatca aggggaatga aggtcgcact aag 1783

<210>38

<211>948

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>38

aggctggatt ggattgatca ttaccgagct tctgttacgt gggcgccgaa ttttgcgttt 60

ggtctggtta ctgattttgc agaagaaata aaagataaga aatgggatct gtcttcaatg 120

cgatacatgc tcaatggcgg tgaagcaatg gtagccaagg tagggcgtag aatacttgag 180

cttttagaac cacatggact gcctgctgac gccatccgac ctgcctgggg aatgtccgaa 240

acttcatcgg gcgttatttt ttctcatgaa tttacccggg ctggaacaag tgatgatgat 300

cattttgttg aaataggctc tccaatcccc ggcttctcta tgagaattgt gaatgaccat 360

aatgaattgg ttgaagaggg tgagatcggc cgttttcaag tctcgggtct ttctgttact 420

agcggttatt accagcggcc tgatttgaat gagagtgtgt ttacagaaga cggctggttt 480

gaaaccggag atcttggctt tttgcgaaac gggcgcttaa ccataacagg ccgtacgaaa 540

gatgcaatta tcattaacgg cattaattat tacagtcatg caattgagtc tgcagttgaa 600

gaattacctg aaattgaaac ttcttatact gctgcttgcg ctgtccgtct gggccagaat 660

tcaacagatc agctggctat cttctttgtt acttccgcaa aattaaatga tgagcagatg 720

tctcagcttc ttagaaacat acaatcccat gtctcacagg tgatcggagt caccccggaa 780

tatttgctgc ctgtacaaaa agaagagatt ccaaaaacgg ctatcggaaa gatccagcgc 840

acccagctaa aaacttcatt tgaaaatgga gagttcgacc acttgctaca taaacccaat 900

cggatgaacg atgcagtcca ggatgagggg atacagcaag cagtccag 948

<210>39

<211>990

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>39

ccaagaacac tgatgacaat ctttattctc tggcgctgga ttgcgagatt ccagttattg 60

cggcaatgca gggccatgga attggaggcg gctttgtcat ggggctcttt gctgacattg 120

tcattctgag cagagaaagc gtgtatacgg ctaattttat gaaatacgga tttacgcctg 180

gaatgggcgc tacctttatc gttcctaaaa aactgggttt cagcctggcc caggaaatat 240

tattgaatgc gggcagctac cgcggagctg atcttgaaaa aagaggggtt ccttttaagg 300

tgctgccccg tgcagaagtg ttagattatg aagtggagct ggcgcaagaa ctggcggaaa 360

agccgagaaa ctcactggtt acactgaaag atcatttggt tgccccgctt cgcgatcagc 420

tgccgcgcgt cattgaacag gaattgatga tgcatgagaa aacatttcat catgaagaag 480

tcaaaagtag aatcaaaggt ttatatggta attaactgaa aatatatata aattttgcag 540

gtgaaatgga ggcagcgtga tgagaaataa tgataatatc cgcatattaa ctaatccttc 600

tgtcagtcat ggggagcctt tacatatatc tgaaaagcag ccggcaacaa tacctgaagt 660

tttatacaga acggcgacgg agcttgggga tacaaaggga atcatttatt tgcagccgga 720

tggaactgaa gtttaccaat catacagacg attatgggat gatggattgc gcattgcgaa 780

ggggcttcgc caatcaggcc tgaaagcaaa acaaagctgg attttgcagc ttggtgacaa 840

ttcacagctt ctccctgcgt tctgggggtg tgtgttaaca ggggttgttc cggctccatt 900

agccgttccg ccaacgtatg ctgaatcgag cagcggcact caaaagctga aagacgcatg 960

gacgcttctt gataagccag cggtgattac 990

<210>40

<211>2069

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>40

tcttcaacta aagcacccat tagttcaaca aacgaaaatt ggataaagtg ggatattttt 60

aaaatatata tttatgttac agtaatattg acttttaaaa aaggattgat tctaatgaag 120

aaagcagaca agtaagcctc ctaaattcac tttagataaa aatttaggag gcatatcaaa 180

tgaactttaa taaaattgat ttagacaatt ggaagagaaa agagatattt aatcattatt 240

tgaaccaaca aacgactttt agtataacca cagaaattga tattagtgtt ttataccgaa 300

acataaaaca agaaggatat aaattttacc ctgcatttat tttcttagtg acaagggtga 360

taaactcaaa tacagctttt agaactggtt acaatagcga cggagagtta ggttattggg 420

ataagttaga gccactttat acaatttttg atggtgtatc taaaacattc tctggtattt 480

ggactcctgt aaagaatgac ttcaaagagt tttatgattt atacctttct gatgtagaga 540

aatataatgg ttcggggaaa ttgtttccca aaacacctat acctgaaaat gctttttctc 600

tttctattat tccatggact tcatttactg ggtttaactt aaatatcaat aataatagta 660

attaccttct acccattatt acagcaggaa aattcattaa taaaggtaat tcaatatatt 720

taccgctatc tttacaggta catcattctg tttgtgatgg ttatcatgca ggattgttta 780

tgaactctat tcaggaattg tcagataggc ctaatgactg gcttttataa tatgagataa 840

tgccgactgt actttttaca gtcggttttc taatgtcact aacctgcccc gttagttgaa 900

ggcattttct gtcaatgttt tcttacaaag aacgctgtga tatactgaaa tttgtccgta 960

tacattttgg aggaatggat atgttaccaa aatacgcgca agtaaaagaa gaaatcagtt 1020

cttggattaa tcaaggcaaa atactgcccg atcaaaaaat ccctaccgaa aacgaattaa 1080

tgcagcaatt cggcgtcagc cggcatacca tccgcaaagc gatcggagac ctcgtatcac 1140

aaggtctgct gtacagcgtg caaggcggag gcacctttgt cgcttcacgc tctgctaagt 1200

cagcgctgca ttccaataaa acgatcggtg ttttgacaac ttacatatca gactatattt 1260

tcccgagcat catcagagga atcgagtcct atttaagcga gcaggggtat tctatgcttt 1320

tgacaagcac aaacaacaac ccggacaatg aaagaagagg cttagaaaac ctgctgtccc 1380

agcatattga cggactcatc gtagaaccga caaaaagcgc ccttcaaacc ccaaacatcg 1440

gctattatct gaacttggag aaaaacggca ttccttttgc gatgattaac gcgtcatatg 1500

ccgagcttgc cgcgccaagt tttaccttgg atgatgtgaa aggcgggatg atggcggcgg 1560

agcatttgct ttctctcggc cacacgcata tgatgggtat ttttaaagct gatgacacac 1620

aaggcgtgaa acggatgaac ggatttatac aggcgcaccg ggagcgtgag ttgtttcctt 1680

ctccggatat gatcgtgaca tttacaacgg aagaaaaaga atcaaaactt ctggagaaag 1740

taaaagccac actggagaaa aacagcaagc acatgccgac agccattctt tgttataacg 1800

atgaaattgc gctgaaggtg attgatatgc tgagggagat ggatcttaaa gtgccggagg 1860

atatgtctat tgtcgggtac gatgattcac atttcgccca aatctcagaa gtgaaactaa 1920

cctctgtcaa acatccgaaa tcagtgcttg gaaaagcagc cgccaaatat gtcattgact 1980

gcttagagca taaaaagccg aagcaagagg atgtcatatt tgagcctgag ttgatcattc 2040

gccagtccgc acgaaaactg aatgaataa 2069

<210>41

<211>256

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>41

agagtcagga gcatttaacc taaaaaagca tgacatttca gcataatgaa catttactca 60

tgtctatttt cgttcttttc tgtatgaaaa tagttatttc gagtctctac ggaaatagcg 120

agagatgata caagaacgtc ctgatcttat tataatataa gcaaaaaact cataaaaagg 180

aaaagcattg acctgaaaac ttatcggtaa agtatgatat aatacaaaaa gaccgattag 240

aggggagaga ggacaa 256

<210>42

<211>7210

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>42

ccttgtttct ggcattggcc agcacgatag accaagggag atttgatacg cctaaacgaa 60

ttggctgttt ttcctacgga tccggctgct gttctgaatt ttacagcgga attacaactc 120

cgcaaggcca ggagcgtcag cgaacatttg gtattgagaa gcatttggat cgccggtatc 180

agctctccat ggaagagtat gagctattgt ttaaggggag cgggatggtg cggtttggga 240

cgcgcaatgt aaagctggac tttgagatga taccgggcat catgcagtcc actcaagaga 300

agccgcgctt gtttttagaa gaaatttctg agtttcatcg gaagtacagg tggatctcgt 360

gacctatcaa acgataaagg tccgctttca agcatcggtt tgctatatta cctttcaccg 420

tcctgaagcg aataatacga ttaatgacac gctgatagaa gaatgcttgc aagtgttaaa 480

ccaatgtgaa acatcaacgg tgacggttgt cgttttagag gggcttcccg aggtgttttg 540

tttcggagcg gattttcaag aaatctatca ggaaatgaaa aggggcagaa aacaggcaag 600

ctcccaagag cctctctatg atttgtggat gaaattgcag accggccctt atgtcacgat 660

ttcgcatgtc aggggaaaag tgaatgccgg cggtcttgga tttgtatctg ccacagatat 720

tgccattgct gatcagacgg cgtcattcag tctctctgag ctgctattcg gcctgtaccc 780

tgcttgtgtt ttaccgtttc tgatccgccg tatcggccgg cagaaagcgc attatatgac 840

gcttatgaca aagccgattt ccgttcagga agccagtgaa tgggggttaa tagatgcttt 900

tgatgctgaa agtgacgtgc tgctgagaaa gcatttattg cgtttgcgga ggctgaataa 960

aaaaggaatc gcacattata aacagtttat gagctcactt gatcatcaag tcagtcgtgc 1020

gaaagccacc gctttaactg caaatcaaga catgttttct gatcctcaaa accaaatggg 1080

aatcatcagg tatgttgaaa caggacaatt cccatgggag gatcagtaac cgtttaaaaa 1140

tgacaatcag aaccgttagg aggaagagtc aggagcattt aacctaaaaa agcatgacat 1200

ttcagcataa tgaacattta ctcatgtcta ttttcgttct tttctgtatg aaaatagtta 1260

tttcgagtct ctacggaaat agcgagagat gatacaagaa cgtcctgatc ttattataat 1320

ataagcaaaa aactcataaa aaggaaaagc attgacctga aaacttatcg gtaaagtatg 1380

atataataca aaaagaccga ttagagggga gagaggacaa atggaaacgt acattttatc 1440

cttagatcag gggacgacaa gttcaagagc gattctgttt aataaagaag gcaaaattgt 1500

ccactctgct caaaaggaat ttacacaata cttcccgcat cctggctggg ttgagcataa 1560

tgccaatgaa atttggggct ctgtcctcgc ggttatcgcc tcagtcatct ctgaatcagg 1620

aatcagcgct tctcaaattg ccggcatcgg catcacgaac cagcgcgaga cgacggttgt 1680

gtgggataaa gatacaggaa gtcctgtcta taatgcaatc gtttggcagt ccagacagac 1740

gtccggcatt tgtgaggaac ttcgtgaaaa aggatataat gataaattca gagaaaaaac 1800

agggctttta atcgatcctt acttctccgg cacgaaggtg aagtggattt tagacaatgt 1860

ggaaggcgca agagaaaaag cggaaaaagg cgagctgctg tttggaacga ttgatacgtg 1920

gctcatttgg aaaatgtcag gcggaaaagc gcatgtgacc gattactcca atgcctcaag 1980

aacactgatg tttaatattt acgatttaaa atgggacgat caactgctcg acattctagg 2040

cgtaccgaaa tccatgctcc ctgaagtgaa gccgtcctct catgtgtatg cggagactgt 2100

tgattatcac ttcttcggaa aaaatatccc gattgctgga gcggcaggcg accagcagtc 2160

cgcattgttc ggccaggcat gctttgaaga aggcatgggg aaaaacactt acggcacagg 2220

atgtttcatg ctgatgaata ccggggaaaa agcaattaag tccgaacatg ggcttttgac 2280

aacaatcgct tggggcattg acggaaaagt gaactatgcg ttagaaggga gcatttttgt 2340

cgcaggctct gccatccagt ggcttagaga cggtttgaga atgttccagg attcatcgct 2400

aagcgaatct tatgcagaaa aagtggattc aactgacggc gtgtatgttg ttccagcatt 2460

tgtcggactg ggaacgcctt actgggacag cgatgtgcgc ggttcggttt tcggcctgac 2520

aagagggaca acaaaagagc actttatccg tgcgacactg gagtcattgg cttatcagac 2580

caaagatgtg cttgacgcaa tggaagcaga ttcaaacatt tcattaaaga cgctccgtgt 2640

agacggagga gctgtaaaaa acaatttcct aatgcagttc caaggagacc tgttgaatgt 2700

tcctgtggag cgcccggaaa ttaatgaaac gactgcactt ggcgcggctt atttggcggg 2760

tatcgctgtg ggattctgga aggaccgttc tgaaatcgcg aaccagtgga atctggataa 2820

acggtttgag cctgaattgg aagaagaaaa acgaaatgag ctgtataaag gctggcaaaa 2880

agccgtgaaa gcagctatgg cttttaaata aagtaatact atggtataat ggttacaagt 2940

taataagaac ggtcctgaga tgaggagaga ccacagcacc aaagtgtaag catgcacttt 3000

ggctgttgtg gtctcttttt ctatttaccg tgacaacaag gaggaaacgt aatgatgaat 3060

catcaatttt caagtcttga aagagatcgc atgctgacag acatgacgaa aaaaacatat 3120

gacctattta ttatcggagg aggaattaca ggagccggaa cagctcttga cgcggcatca 3180

aggggaatga aggtcgcact aagaggctgg attggattga tcattaccga gcttctgtta 3240

cgtgggcgcc gaattttgcg tttggtctgg ttactgattt tgcagaagaa ataaaagata 3300

agaaatggga tctgtcttca atgcgataca tgctcaatgg cggtgaagca atggtagcca 3360

aggtagggcg tagaatactt gagcttttag aaccacatgg actgcctgct gacgccatcc 3420

gacctgcctg gggaatgtcc gaaacttcat cgggcgttattttttctcat gaatttaccc 3480

gggctggaac aagtgatgat gatcattttg ttgaaatagg ctctccaatc cccggcttct 3540

ctatgagaat tgtgaatgac cataatgaat tggttgaaga gggtgagatc ggccgttttc 3600

aagtctcggg tctttctgtt actagcggtt attaccagcg gcctgatttg aatgagagtg 3660

tgtttacaga agacggctgg tttgaaaccg gagatcttgg ctttttgcga aacgggcgct 3720

taaccataac aggccgtacg aaagatgcaa ttatcattaa cggcattaat tattacagtc 3780

atgcaattga gtctgcagtt gaagaattac ctgaaattga aacttcttat actgctgctt 3840

gcgctgtccg tctgggccag aattcaacag atcagctggc tatcttcttt gttacttccg 3900

caaaattaaa tgatgagcag atgtctcagc ttcttagaaa catacaatcc catgtctcac 3960

aggtgatcgg agtcaccccg gaatatttgc tgcctgtaca aaaagaagag attccaaaaa 4020

cggctatcgg aaagatccag cgcacccagc taaaaacttc atttgaaaat ggagagttcg 4080

accacttgct acataaaccc aatcggatga acgatgcagt ccaggatgag gggatacagc 4140

aagcagtcca gtcttcaact aaagcaccca ttagttcaac aaacgaaaat tggataaagt 4200

gggatatttt taaaatatat atttatgtta cagtaatatt gacttttaaa aaaggattga 4260

ttctaatgaa gaaagcagac aagtaagcct cctaaattca ctttagataa aaatttagga 4320

ggcatatcaa atgaacttta ataaaattga tttagacaat tggaagagaa aagagatatt 4380

taatcattat ttgaaccaac aaacgacttt tagtataacc acagaaattg atattagtgt 4440

tttataccga aacataaaac aagaaggata taaattttac cctgcattta ttttcttagt 4500

gacaagggtg ataaactcaa atacagcttt tagaactggt tacaatagcg acggagagtt 4560

aggttattgg gataagttag agccacttta tacaattttt gatggtgtat ctaaaacatt 4620

ctctggtatt tggactcctg taaagaatga cttcaaagag ttttatgatt tatacctttc 4680

tgatgtagag aaatataatg gttcggggaa attgtttccc aaaacaccta tacctgaaaa 4740

tgctttttct ctttctatta ttccatggac ttcatttact gggtttaact taaatatcaa 4800

taataatagt aattaccttc tacccattat tacagcagga aaattcatta ataaaggtaa 4860

ttcaatatat ttaccgctat ctttacaggt acatcattct gtttgtgatg gttatcatgc 4920

aggattgttt atgaactcta ttcaggaatt gtcagatagg cctaatgact ggcttttata 4980

atatgagata atgccgactg tactttttac agtcggtttt ctaatgtcac taacctgccc 5040

cgttagttga aggcattttc tgtcaatgtt ttcttacaaa gaacgctgtg atatactgaa 5100

atttgtccgt atacattttg gaggaatgga tatgttacca aaatacgcgc aagtaaaaga 5160

agaaatcagt tcttggatta atcaaggcaa aatactgccc gatcaaaaaa tccctaccga 5220

aaacgaatta atgcagcaat tcggcgtcag ccggcatacc atccgcaaag cgatcggaga 5280

cctcgtatca caaggtctgc tgtacagcgt gcaaggcgga ggcacctttg tcgcttcacg 5340

ctctgctaag tcagcgctgc attccaataa aacgatcggt gttttgacaa cttacatatc 5400

agactatatt ttcccgagca tcatcagagg aatcgagtcc tatttaagcg agcaggggta 5460

ttctatgctt ttgacaagca caaacaacaa cccggacaat gaaagaagag gcttagaaaa 5520

cctgctgtcc cagcatattg acggactcat cgtagaaccg acaaaaagcg cccttcaaac 5580

cccaaacatc ggctattatc tgaacttgga gaaaaacggc attccttttg cgatgattaa 5640

cgcgtcatat gccgagcttg ccgcgccaag ttttaccttg gatgatgtga aaggcgggat 5700

gatggcggcg gagcatttgc tttctctcgg ccacacgcat atgatgggta tttttaaagc 5760

tgatgacaca caaggcgtga aacggatgaa cggatttata caggcgcacc gggagcgtga 5820

gttgtttcct tctccggata tgatcgtgac atttacaacg gaagaaaaag aatcaaaact 5880

tctggagaaa gtaaaagcca cactggagaa aaacagcaag cacatgccga cagccattct 5940

ttgttataac gatgaaattg cgctgaaggt gattgatatg ctgagggaga tggatcttaa 6000

agtgccggag gatatgtcta ttgtcgggta cgatgattca catttcgccc aaatctcaga 6060

agtgaaacta acctctgtca aacatccgaa atcagtgctt ggaaaagcag ccgccaaata 6120

tgtcattgac tgcttagagc ataaaaagcc gaagcaagag gatgtcatat ttgagcctga 6180

gttgatcatt cgccagtccg cacgaaaact gaatgaataa ccaagaacac tgatgacaat 6240

ctttattctc tggcgctgga ttgcgagatt ccagttattg cggcaatgca gggccatgga 6300

attggaggcg gctttgtcat ggggctcttt gctgacattg tcattctgag cagagaaagc 6360

gtgtatacgg ctaattttat gaaatacgga tttacgcctg gaatgggcgc tacctttatc 6420

gttcctaaaa aactgggttt cagcctggcc caggaaatat tattgaatgc gggcagctac 6480

cgcggagctg atcttgaaaa aagaggggtt ccttttaagg tgctgccccg tgcagaagtg 6540

ttagattatg aagtggagct ggcgcaagaa ctggcggaaa agccgagaaa ctcactggtt 6600

acactgaaag atcatttggt tgccccgctt cgcgatcagc tgccgcgcgt cattgaacag 6660

gaattgatga tgcatgagaa aacatttcat catgaagaag tcaaaagtag aatcaaaggt 6720

ttatatggta attaactgaa aatatatata aattttgcag gtgaaatgga ggcagcgtga 6780

tgagaaataa tgataatatc cgcatattaa ctaatccttc tgtcagtcat ggggagcctt 6840

tacatatatc tgaaaagcag ccggcaacaa tacctgaagt tttatacaga acggcgacgg 6900

agcttgggga tacaaaggga atcatttatt tgcagccgga tggaactgaa gtttaccaat 6960

catacagacg attatgggat gatggattgc gcattgcgaa ggggcttcgc caatcaggcc 7020

tgaaagcaaa acaaagctgg attttgcagc ttggtgacaa ttcacagctt ctccctgcgt 7080

tctgggggtg tgtgttaaca ggggttgttc cggctccatt agccgttccg ccaacgtatg 7140

ctgaatcgag cagcggcact caaaagctga aagacgcatg gacgcttctt gataagccag 7200

cggtgattac 7210

<210>43

<211>6458

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>43

ccttgtttct ggcattggcc agcacgatag accaagggag atttgatacg cctaaacgaa 60

ttggctgttt ttcctacgga tccggctgct gttctgaatt ttacagcgga attacaactc 120

cgcaaggcca ggagcgtcag cgaacatttg gtattgagaa gcatttggat cgccggtatc 180

agctctccat ggaagagtat gagctattgt ttaaggggag cgggatggtg cggtttggga 240

cgcgcaatgt aaagctggac tttgagatga taccgggcat catgcagtcc actcaagaga 300

agccgcgctt gtttttagaa gaaatttctg agtttcatcg gaagtacagg tggatctcgt 360

gacctatcaa acgataaagg tccgctttca agcatcggtt tgctatatta cctttcaccg 420

tcctgaagcg aataatacga ttaatgacac gctgatagaa gaatgcttgc aagtgttaaa 480

ccaatgtgaa acatcaacgg tgacggttgt cgttttagag gggcttcccg aggtgttttg 540

tttcggagcg gattttcaag aaatctatca ggaaatgaaa aggggcagaa aacaggcaag 600

ctcccaagag cctctctatg atttgtggat gaaattgcag accggccctt atgtcacgat 660

ttcgcatgtc aggggaaaag tgaatgccgg cggtcttgga tttgtatctg ccacagatat 720

tgccattgct gatcagacgg cgtcattcag tctctctgag ctgctattcg gcctgtaccc 780

tgcttgtgtt ttaccgtttc tgatccgccg tatcggccgg cagaaagcgc attatatgac 840

gcttatgaca aagccgattt ccgttcagga agccagtgaa tgggggttaa tagatgcttt 900

tgatgctgaa agtgacgtgc tgctgagaaa gcatttattg cgtttgcgga ggctgaataa 960

aaaaggaatc gcacattata aacagtttat gagctcactt gatcatcaag tcagtcgtgc 1020

gaaagccacc gctttaactg caaatcaaga catgttttct gatcctcaaa accaaatggg 1080

aatcatcagg tatgttgaaa caggacaatt cccatgggag gatcagtaac cgtttaaaaa 1140

tgacaatcag aaccgttagg aggaagagtc aggagcattt aacctaaaaa agcatgacat 1200

ttcagcataa tgaacattta ctcatgtcta ttttcgttct tttctgtatg aaaatagtta 1260

tttcgagtct ctacggaaat agcgagagat gatacaagaa cgtcctgatc ttattataat 1320

ataagcaaaa aactcataaa aaggaaaagc attgacctga aaacttatcg gtaaagtatg 1380

atataataca aaaagaccga ttagagggga gagaggacaa atggaaacgt acattttatc 1440

cttagatcag gggacgacaa gttcaagagc gattctgttt aataaagaag gcaaaattgt 1500

ccactctgct caaaaggaat ttacacaata cttcccgcat cctggctggg ttgagcataa 1560

tgccaatgaa atttggggct ctgtcctcgc ggttatcgcc tcagtcatct ctgaatcagg 1620

aatcagcgct tctcaaattg ccggcatcgg catcacgaac cagcgcgaga cgacggttgt 1680

gtgggataaa gatacaggaa gtcctgtcta taatgcaatc gtttggcagt ccagacagac 1740

gtccggcatt tgtgaggaac ttcgtgaaaa aggatataat gataaattca gagaaaaaac 1800

agggctttta atcgatcctt acttctccgg cacgaaggtg aagtggattt tagacaatgt 1860

ggaaggcgca agagaaaaag cggaaaaagg cgagctgctg tttggaacga ttgatacgtg 1920

gctcatttgg aaaatgtcag gcggaaaagc gcatgtgacc gattactcca atgcctcaag 1980

aacactgatg tttaatattt acgatttaaa atgggacgat caactgctcg acattctagg 2040

cgtaccgaaa tccatgctcc ctgaagtgaa gccgtcctct catgtgtatg cggagactgt 2100

tgattatcac ttcttcggaa aaaatatccc gattgctgga gcggcaggcg accagcagtc 2160

cgcattgttc ggccaggcat gctttgaaga aggcatgggg aaaaacactt acggcacagg 2220

atgtttcatg ctgatgaata ccggggaaaa agcaattaag tccgaacatg ggcttttgac 2280

aacaatcgct tggggcattg acggaaaagt gaactatgcg ttagaaggga gcatttttgt 2340

cgcaggctct gccatccagt ggcttagaga cggtttgaga atgttccagg attcatcgct 2400

aagcgaatct tatgcagaaa aagtggattc aactgacggc gtgtatgttg ttccagcatt 2460

tgtcggactg ggaacgcctt actgggacag cgatgtgcgc ggttcggttt tcggcctgac 2520

aagagggaca acaaaagagc actttatccg tgcgacactg gagtcattgg cttatcagac 2580

caaagatgtg cttgacgcaa tggaagcaga ttcaaacatt tcattaaaga cgctccgtgt 2640

agacggagga gctgtaaaaa acaatttcct aatgcagttc caaggagacc tgttgaatgt 2700

tcctgtggag cgcccggaaa ttaatgaaac gactgcactt ggcgcggctt atttggcggg 2760

tatcgctgtg ggattctgga aggaccgttc tgaaatcgcg aaccagtgga atctggataa 2820

acggtttgag cctgaattgg aagaagaaaa acgaaatgag ctgtataaag gctggcaaaa 2880

agccgtgaaa gcagctatgg cttttaaata aagtaatact atggtataat ggttacaagt 2940

taataagaac ggtcctgaga tgaggagaga ccacagcacc aaagtgtaag catgcacttt 3000

ggctgttgtg gtctcttttt ctatttaccg tgacaacaag gaggaaacgt aatgatgaat 3060

catcaatttt caagtcttga aagagatcgc atgctgacag acatgacgaa aaaaacatat 3120

gacctattta ttatcggagg aggaattaca ggagccggaa cagctcttga cgcggcatca 3180

aggggaatga aggtcgcact aagaggctgg attggattga tcattaccga gcttctgtta 3240

cgtgggcgcc gaattttgcg tttggtctgg ttactgattt tgcagaagaa ataaaagata 3300

agaaatggga tctgtcttca atgcgataca tgctcaatgg cggtgaagca atggtagcca 3360

aggtagggcg tagaatactt gagcttttag aaccacatgg actgcctgct gacgccatcc 3420

gacctgcctg gggaatgtcc gaaacttcat cgggcgttat tttttctcat gaatttaccc 3480

gggctggaac aagtgatgat gatcattttg ttgaaatagg ctctccaatc cccggcttct 3540

ctatgagaat tgtgaatgac cataatgaat tggttgaaga gggtgagatc ggccgttttc 3600

aagtctcggg tctttctgtt actagcggtt attaccagcg gcctgatttg aatgagagtg 3660

tgtttacaga agacggctgg tttgaaaccg gagatcttgg ctttttgcga aacgggcgct 3720

taaccataac aggccgtacg aaagatgcaa ttatcattaa cggcattaat tattacagtc 3780

atgcaattga gtctgcagtt gaagaattac ctgaaattga aacttcttat actgctgctt 3840

gcgctgtccg tctgggccag aattcaacag atcagctggc tatcttcttt gttacttccg 3900

caaaattaaa tgatgagcag atgtctcagc ttcttagaaa catacaatcc catgtctcac 3960

aggtgatcgg agtcaccccg gaatatttgc tgcctgtaca aaaagaagag attccaaaaa 4020

cggctatcgg aaagatccag cgcacccagc taaaaacttc atttgaaaat ggagagttcg 4080

accacttgct acataaaccc aatcggatga acgatgcagt ccaggatgag gggatacagc 4140

aagcagtcca gtcttcaact aaagcaccca ttagttcaac aaacgaaaat tggataaagt 4200

gggatatttt taaaatatat atttatgtta cagtaatatt gacttttaaa aaaggattga 4260

ttctaatgaa gaaagcagac aagtaagcct cctaaattca ctttagataa aaatttagga 4320

ggcatatcaa atgaacttta ataaaattga tttagacaat tggaagagaa aagagatatt 4380

taatcattat ttgaaccaac aaacgacttt tagtataacc acagaaattg atattagtgt 4440

tttataccga aacataaaac aagaaggata taaattttac cctgcattta ttttcttagt 4500

gacaagggtg ataaactcaa atacagcttt tagaactggt tacaatagcg acggagagtt 4560

aggttattgg gataagttag agccacttta tacaattttt gatggtgtat ctaaaacatt 4620

ctctggtatt tggactcctg taaagaatga cttcaaagag ttttatgatt tatacctttc 4680

tgatgtagag aaatataatg gttcggggaa attgtttccc aaaacaccta tacctgaaaa 4740

tgctttttct ctttctatta ttccatggac ttcatttact gggtttaact taaatatcaa 4800

taataatagt aattaccttc tacccattat tacagcagga aaattcatta ataaaggtaa 4860

ttcaatatat ttaccgctat ctttacaggt acatcattct gtttgtgatg gttatcatgc 4920

aggattgttt atgaactcta ttcaggaatt gtcagatagg cctaatgact ggcttttata 4980

atatgagata atgccgactg tactttttac agtcggtttt ctaatgtcac taacctgccc 5040

cgttagttga aggcattttc tgtcaatgtt ttcttacaaa gaacgctgtg atatactgaa 5100

atttgtccgt atacattttg gaggaatgga tatgttacca aaatacgcgc aagtaaaaga 5160

agaaatcagt tcttggatta atcaaggcaa aatactgccc gatcaaaaaa tccctaccga 5220

aaacgaatta atgcagcaat tcggcgtcag ccggcatacc atccgcaaag cgatcggaga 5280

cctcgtatca caaggtctgc tgtacagcgt gcaaggcgga ggcacctttg tcgcttcacg 5340

tgcctcctgt aatctctgat acagaagacc attatattct gcatccgagc atgatagatt 5400

cggcatttca agcatctatc ggtttgaggc tgggaggagc tacgagttta gaagacagaa 5460

aggcaatgct gccttttgcc atacaggatg tccgaatctt caaaggctgc gaggcatcta 5520

tgtgggcgcg gatcacatat agcgagggca gtacagctgg ggatcgtatg cagaagcttg 5580

atatcgattt atgcaatgaa gagggccagg tttgcgtgcg attgacgagc tattctgcaa 5640

gagtgttaga aacagatcaa gaaggtcctt ctgaagcgaa tgacacgcta ttgttcgaac 5700

atatttggga agaacgtgct gcagagaggc aagagctcat agagtacgac acgtataaag 5760

tcgttgtctg tgatgttgga gaacaaatgg aaagcctcca aaatcatctt gactgtacag 5820

ttctgcagca tgatacagag acaattgatg aacggtttga aggttatgcc atacagttat 5880

ttgaagaaat caaacagcta atgcactcaa aaacaggagg acatacattc attcaagtgg 5940

cggtgcctgc gttggacgag cctcagctat taagcggctt aactggattg ctcaaaacgg 6000

ctgaactgga aaatccaaaa ctgacaggac agttgatcga aatagaaacc gggatgtcag 6060

ctggtgagct attcgaaata ttggaggaaa acagacgcta tccgcgtgac acgcatatcc 6120

gccactggca agggaagcga tttgtaagca agtggaaaga agtgtctggt gagcacctaa 6180

gtgctgacat gccatggaag gataaaggcg tgtatctgat cacaggcggt gcgggcggtc 6240

tgggattcat ctttgcaact gaaatcgcaa accaaacgaa tgatgcggtt gtgatcctga 6300

caggacgctc tccgcttgat gaaaggaaga agaagaagct taaagcctta cagaaacttg 6360

gcatccaagc gatttacaga caagctgatc tggctgacaa acaaacggtt gatgccttat 6420

taaaagaaac ccaaaacgtt tacggacact tagatggc 6458

<210>44

<211>1749

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>44

atgatgaatc accaattttc tagtcttgaa agagatcgca tgctgacaga catgacgaaa 60

aaaacatatg acctatttat tatcggagga ggaattacag gagccggaac agctcttgac 120

gcggcatcaa ggggaatgaa ggtcgcacta agcgaaatgc aggactttgc ggcgggaaca 180

tcaagccgat caacgaagct ggtacatggc ggcttgcgct atttaaaaca atttgaagtg 240

aaaatggtcg ctgaggtagg aaaagagcgg gcgattgtgt atgaaaacgg cccgcatgtc 300

actacaccgg aatggatgct gcttccgttt cataaaggcg gcacattcgg ttcatttaca 360

acatctattg ggttaagggt ttatgacttc cttgcaggtg tgaaaaagtc agaacgaaga 420

agcatgcttt cagcaaaaga aacgctgcaa aaagagcctt tggtgaaaaa agacggctta 480

aaaggcggcg ggtactatgt ggaataccgc actgacgatg cgagactgac catcgaagtc 540

atgaaggaag cggttaaatt cggggcagag cctgtgaatt actccaaagt gaaggagctt 600

ctttacgaaa aaggcaaagc cgtcggcgta ttaattgaag atgtgctgac aaagaaagaa 660

tataaagtgt atgcgaaaaa aattgtcaat gctacaggcc cttgggtcga tcagctcaga 720

gaaaaagacc attcgaaaaa cggaaagcat ttgcagcata caaaaggcat tcaccttgta 780

tttgaccagt ctgtctttcc gctgaaacag gctgtatatt tcgatacacc tgatggccgg 840

atggtatttg cgattcctcg tgaaggcaaa acatacgtgg gaacaacaga cactgtttac 900

aaagaggcgc tggagcatcc gcggatgaca acggaggatc gtgattatgt catcaaatca 960

atcaattaca tgttcccgga actgaatatc actgcgaatg acatcgaatc cagctgggcg 1020

gggctgcgtc cgctgattca tgaagaaggc aaagatcctt ctgaaatttc acggaaagac 1080

gagatttgga catctgactc aggcctgatc accattgccg gcggaaaact gactggatac 1140

agaaaaatgg cggacgacat cgttgatctt gtccgtgatc gcttaaaaga agagggcgaa 1200

aaggattttg gaccatgtaa aacgaaaaac atgccaatct ctggcgggca cgtcggcggt 1260

tcgaaaaatc ttatgtcctt cgttaccgcg aaaacaaaag aaggaattgc agccggttta 1320

tcagaaaaag acgcaaaaca gcttgcgatc agatacggct ctaacgtaga tcgcgtcttt 1380

gaccgggtag aagcgctgaa agatgaggcc gcgaaacgca acattccggt tcatattctt 1440

gctgaggcag aatacagtat agaagaagag atgactgcaa cccctgctga cttctttgtc 1500

cgcagaacgg gacgtttatt ttttgatatc aattgggtaa gaacatataa agatgccgtt 1560

attgatttta tgagcgagcg attccaatgg gatgagcagg cgaaaaacaa acatacagaa 1620

aacctcaaca agcttttaca cgatgcggtc gtaccgcttg agcaataaat cataacgggc 1680

tgtctgcagc ccgttatttc tttttacgtg ccgaaagggg gagatctcag gttatcaagt 1740

gtcagacgc 1749

<210>45

<211>1004

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>45

ggcagacaac attgatgaat tatgggaaaa actgagggac gggcgagact gtattacgga 60

aattcctgca gaccgctggg atcacagtct ttattatgac gaagataaag acaagcctgg 120

aaaaacctac agcaagtggg gcggcttcat gaaggatgta gacaagtttg acccgcagtt 180

tttccatatt tcgccgcgtg aggcaaaatt aatggacccg caagagcgtt tatttttgca 240

gtgcgtgtat gaaacgatgg aagacgcagg ctatacaaga gagcacctcg gccgtaagcg 300

tgatgctgag cttggcggaa gcgtcggtgt ttacgtcggt gtcatgtacg aagaatatca 360

gctgtacggg gcacaagagc aggtgcgggg aaggtctctg gctttaacgg gaaatccatc 420

atccatcgcg aaccgggtat cttattattt tgattttcac ggaccgagta ttgctttaga 480

caccatgtgt tcttcttctt taacggctat ccacttggcc tgccaaagcc tgcagcgcgg 540

agaatgtgag gcggcattcg cgggcggcgt gaatgtgtca atccatccaa ataaatattt 600

gatgctcggg cagaacaaat ttatgtccag caaaggccga tgtgaaagct tcggacaggg 660

cggagacggc tatgtgccgg gggaaggcgt tggagcagtg cttttgaagc cattgtctaa 720

agcagttgaa gatggtgatc acatttatgg aatcattaag gggacagcca tcaaccacgg 780

gggaaaaaca aacgggtact cagtgcccaa tccgaatgcc caagcagatg tcatcaaaaa 840

ggcgtttgtg gaagcgaagg ttgatccgcg gacagtcagt tacatcgaag cccacggcac 900

gggaacctct ctcggtgatc ctattgaaat cacagggctg tccaaagtgt ttacgcaaga 960

aaccgatgac aaacaatttt gtgcaataga gtcagccaaa tcaa 1004

<210>46

<211>769

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>46

aggctgatta cgcaatggcc aatgcattta tgaatgcatt cagcgaatat cgaaatggcc 60

aagcagagct gcataagcgt tacggaaaaa ctctttcagt gtgctggccg ctgtggaaag 120

acgggggcat gcaaattgat gcggaaactg caaggatgct gaaaagagaa acaggcatgg 180

tggccatgga aacagatcgt gggattcagg cgctatatca tggctggacg tcaggaaagc 240

cgcaagtgtt agttgcttcc ggtgttactg atcggattcg tgcattttta catgaaacgg 300

gacatggcaa aggccaatct cacaatatca aaaaaagcag tctcaatcag gaggcagaaa 360

aagcagacat gatcggggaa attgatgaag agatactaag agaaaaagcg gagaactatt 420

ttaaacaggt gctttcttcg gtgattaagc tgccagctgg ccagattgat gctgaagctc 480

ctctcgagga ttacgggatc gattctatca tgatcatgca tgtaacgggt caattagaaa 540

aagtattcgg atctttgtcc aagacgctgt tctttgaata ccaggatatt cggtcattaa 600

cccggtattt tattgattcc cgcagagaaa aactgctgga catcttggga tttgaaacgg 660

gaaaaccttc tgttgaaaga aaatcagaac ctgaaaaaca agaaatccca gttatcccaa 720

gaaagtctgg atttcttcct ttgcaagata aagagcggaa gcaggtaag 769

<210>47

<211>6965

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>47

tgcctcctgt aatctctgat acagaagacc attatattct gcatccgagc atgatagatt 60

cggcatttca agcatctatc ggtttgaggc tgggaggagc tacgagttta gaagacagaa 120

aggcaatgct gccttttgcc atacaggatg tccgaatctt caaaggctgc gaggcatcta 180

tgtgggcgcg gatcacatat agcgagggca gtacagctgg ggatcgtatg cagaagcttg 240

atatcgattt atgcaatgaa gagggccagg tttgcgtgcg attgacgagc tattctgcaa 300

gagtgttaga aacagatcaa gaaggtcctt ctgaagcgaa tgacacgcta ttgttcgaac 360

atatttggga agaacgtgct gcagagaggc aagagctcat agagtacgac acgtataaag 420

tcgttgtctg tgatgttgga gaacaaatgg aaagcctcca aaatcatctt gactgtacag 480

ttctgcagca tgatacagag acaattgatg aacggtttga aggttatgcc atacagttat 540

ttgaagaaat caaacagcta atgcactcaa aaacaggagg acatacattc attcaagtgg 600

cggtgcctgcgttggacgag cctcagctat taagcggctt aactggattg ctcaaaacgg 660

ctgaactgga aaatccaaaa ctgacaggac agttgatcga aatagaaacc gggatgtcag 720

ctggtgagct attcgaaata ttggaggaaa acagacgcta tccgcgtgac acgcatatcc 780

gccactggca agggaagcga tttgtaagca agtggaaaga agtgtctggt gagcacctaa 840

gtgctgacat gccatggaag gataaaggcg tgtatctgat cacaggcggt gcgggcggtc 900

tgggattcat ctttgcaact gaaatcgcaa accaaacgaa tgatgcggtt gtgatcctga 960

caggacgctc tccgcttgat gaaaggaaga agaagaagct taaagcctta cagaaacttg 1020

gcatccaagc gatttacaga caagctgatc tggctgacaa acaaacggtt gatgccttat 1080

taaaagaaac ccaaaacgtt tacggacact tagatggcag agtcaggagc atttaaccta 1140

aaaaagcatg acatttcagc ataatgaaca tttactcatg tctattttcg ttcttttctg 1200

tatgaaaata gttatttcga gtctctacgg aaatagcgag agatgataca agaacgtcct 1260

gatcttatta taatataagc aaaaaactca taaaaaggaa aagcattgac ctgaaaactt 1320

atcggtaaag tatgatataa tacaaaaaga ccgattagag gggagagagg acaaatgatg 1380

aatcaccaat tttctagtct tgaaagagat cgcatgctga cagacatgac gaaaaaaaca 1440

tatgacctat ttattatcgg aggaggaatt acaggagccg gaacagctct tgacgcggca 1500

tcaaggggaa tgaaggtcgc actaagcgaa atgcaggact ttgcggcggg aacatcaagc 1560

cgatcaacga agctggtaca tggcggcttg cgctatttaa aacaatttga agtgaaaatg 1620

gtcgctgagg taggaaaaga gcgggcgatt gtgtatgaaa acggcccgca tgtcactaca 1680

ccggaatgga tgctgcttcc gtttcataaa ggcggcacat tcggttcatt tacaacatct 1740

attgggttaa gggtttatga cttccttgca ggtgtgaaaa agtcagaacg aagaagcatg 1800

ctttcagcaa aagaaacgct gcaaaaagag cctttggtga aaaaagacgg cttaaaaggc 1860

ggcgggtact atgtggaata ccgcactgac gatgcgagac tgaccatcga agtcatgaag 1920

gaagcggtta aattcggggc agagcctgtg aattactcca aagtgaagga gcttctttac 1980

gaaaaaggca aagccgtcgg cgtattaatt gaagatgtgc tgacaaagaa agaatataaa 2040

gtgtatgcga aaaaaattgt caatgctaca ggcccttggg tcgatcagct cagagaaaaa 2100

gaccattcga aaaacggaaa gcatttgcag catacaaaag gcattcacct tgtatttgac 2160

cagtctgtct ttccgctgaa acaggctgta tatttcgata cacctgatgg ccggatggta 2220

tttgcgattc ctcgtgaagg caaaacatac gtgggaacaa cagacactgt ttacaaagag 2280

gcgctggagc atccgcggat gacaacggag gatcgtgatt atgtcatcaa atcaatcaat 2340

tacatgttcc cggaactgaa tatcactgcg aatgacatcg aatccagctg ggcggggctg 2400

cgtccgctga ttcatgaaga aggcaaagat ccttctgaaa tttcacggaa agacgagatt 2460

tggacatctg actcaggcct gatcaccatt gccggcggaa aactgactgg atacagaaaa 2520

atggcggacg acatcgttga tcttgtccgt gatcgcttaa aagaagaggg cgaaaaggat 2580

tttggaccat gtaaaacgaa aaacatgcca atctctggcg ggcacgtcgg cggttcgaaa 2640

aatcttatgt ccttcgttac cgcgaaaaca aaagaaggaa ttgcagccgg tttatcagaa 2700

aaagacgcaa aacagcttgc gatcagatac ggctctaacg tagatcgcgt ctttgaccgg 2760

gtagaagcgc tgaaagatga ggccgcgaaa cgcaacattc cggttcatat tcttgctgag 2820

gcagaataca gtatagaaga agagatgact gcaacccctg ctgacttctt tgtccgcaga 2880

acgggacgtt tattttttga tatcaattgg gtaagaacat ataaagatgc cgttattgat 2940

tttatgagcg agcgattcca atgggatgag caggcgaaaa acaaacatac agaaaacctc 3000

aacaagcttt tacacgatgc ggtcgtaccg cttgagcaat aaatcataac gggctgtctg 3060

cagcccgtta tttcttttta cgtgccgaaa gggggagatc tcaggttatc aagtgtcaga 3120

cgcggcagac aacattgatg aattatggga aaaactgagg gacgggcgag actgtattac 3180

ggaaattcct gcagaccgct gggatcacag tctttattat gacgaagata aagacaagcc 3240

tggaaaaacc tacagcaagt ggggcggctt catgaaggat gtagacaagt ttgacccgca 3300

gtttttccat atttcgccgc gtgaggcaaa attaatggac ccgcaagagc gtttattttt 3360

gcagtgcgtg tatgaaacga tggaagacgc aggctataca agagagcacc tcggccgtaa 3420

gcgtgatgct gagcttggcg gaagcgtcgg tgtttacgtc ggtgtcatgt acgaagaata 3480

tcagctgtac ggggcacaag agcaggtgcg gggaaggtct ctggctttaa cgggaaatcc 3540

atcatccatc gcgaaccggg tatcttatta ttttgatttt cacggaccga gtattgcttt 3600

agacaccatg tgttcttctt ctttaacggc tatccacttg gcctgccaaa gcctgcagcg 3660

cggagaatgt gaggcggcat tcgcgggcgg cgtgaatgtg tcaatccatc caaataaata 3720

tttgatgctc gggcagaaca aatttatgtc cagcaaaggc cgatgtgaaa gcttcggaca 3780

gggcggagac ggctatgtgc cgggggaagg cgttggagca gtgcttttga agccattgtc 3840

taaagcagtt gaagatggtg atcacattta tggaatcatt aaggggacag ccatcaacca 3900

cgggggaaaa acaaacgggt actcagtgcc caatccgaat gcccaagcag atgtcatcaa 3960

aaaggcgttt gtggaagcga aggttgatcc gcggacagtc agttacatcg aagcccacgg 4020

cacgggaacc tctctcggtg atcctattga aatcacaggg ctgtccaaag tgtttacgca 4080

agaaaccgat gacaaacaat tttgtgcaat agagtcagcc aaatcaatct tcaactaaag 4140

cacccattag ttcaacaaac gaaaattgga taaagtggga tatttttaaa atatatattt 4200

atgttacagt aatattgact tttaaaaaag gattgattct aatgaagaaa gcagacaagt 4260

aagcctccta aattcacttt agataaaaat ttaggaggca tatcaaatga actttaataa 4320

aattgattta gacaattgga agagaaaaga gatatttaat cattatttga accaacaaac 4380

gacttttagt ataaccacag aaattgatat tagtgtttta taccgaaaca taaaacaaga 4440

aggatataaa ttttaccctg catttatttt cttagtgaca agggtgataa actcaaatac 4500

agcttttaga actggttaca atagcgacgg agagttaggt tattgggata agttagagcc 4560

actttataca atttttgatg gtgtatctaa aacattctct ggtatttgga ctcctgtaaa 4620

gaatgacttc aaagagtttt atgatttata cctttctgat gtagagaaat ataatggttc 4680

ggggaaattg tttcccaaaa cacctatacc tgaaaatgct ttttctcttt ctattattcc 4740

atggacttca tttactgggt ttaacttaaa tatcaataat aatagtaatt accttctacc 4800

cattattaca gcaggaaaat tcattaataa aggtaattca atatatttac cgctatcttt 4860

acaggtacat cattctgttt gtgatggtta tcatgcagga ttgtttatga actctattca 4920

ggaattgtca gataggccta atgactggct tttataatat gagataatgc cgactgtact 4980

ttttacagtc ggttttctaa tgtcactaac ctgccccgtt agttgaaggc attttctgtc 5040

aatgttttct tacaaagaac gctgtgatat actgaaattt gtccgtatac attttggagg 5100

aatggatatg ttaccaaaat acgcgcaagt aaaagaagaa atcagttctt ggattaatca 5160

aggcaaaata ctgcccgatc aaaaaatccc taccgaaaac gaattaatgc agcaattcgg 5220

cgtcagccgg cataccatcc gcaaagcgat cggagacctc gtatcacaag gtctgctgta 5280

cagcgtgcaa ggcggaggca cctttgtcgc ttcacgctct gctaagtcag cgctgcattc 5340

caataaaacg atcggtgttt tgacaactta catatcagac tatattttcc cgagcatcat 5400

cagaggaatc gagtcctatt taagcgagca ggggtattct atgcttttga caagcacaaa 5460

caacaacccg gacaatgaaa gaagaggctt agaaaacctg ctgtcccagc atattgacgg 5520

actcatcgta gaaccgacaa aaagcgccct tcaaacccca aacatcggct attatctgaa 5580

cttggagaaa aacggcattc cttttgcgat gattaacgcg tcatatgccg agcttgccgc 5640

gccaagtttt accttggatg atgtgaaagg cgggatgatg gcggcggagc atttgctttc 5700

tctcggccac acgcatatga tgggtatttt taaagctgat gacacacaag gcgtgaaacg 5760

gatgaacgga tttatacagg cgcaccggga gcgtgagttg tttccttctc cggatatgat 5820

cgtgacattt acaacggaag aaaaagaatc aaaacttctg gagaaagtaa aagccacact 5880

ggagaaaaac agcaagcaca tgccgacagc cattctttgt tataacgatg aaattgcgct 5940

gaaggtgatt gatatgctga gggagatgga tcttaaagtg ccggaggata tgtctattgt 6000

cgggtacgat gattcacatt tcgcccaaat ctcagaagtg aaactaacct ctgtcaaaca 6060

tccgaaatca gtgcttggaa aagcagccgc caaatatgtc attgactgct tagagcataa 6120

aaagccgaag caagaggatg tcatatttga gcctgagttg atcattcgcc agtccgcacg 6180

aaaactgaat gaataaaggc tgattacgca atggccaatg catttatgaa tgcattcagc 6240

gaatatcgaa atggccaagc agagctgcat aagcgttacg gaaaaactct ttcagtgtgc 6300

tggccgctgt ggaaagacgg gggcatgcaa attgatgcgg aaactgcaag gatgctgaaa 6360

agagaaacag gcatggtggc catggaaaca gatcgtggga ttcaggcgct atatcatggc 6420

tggacgtcag gaaagccgca agtgttagtt gcttccggtg ttactgatcg gattcgtgca 6480

tttttacatg aaacgggaca tggcaaaggc caatctcaca atatcaaaaa aagcagtctc 6540

aatcaggagg cagaaaaagc agacatgatc ggggaaattg atgaagagat actaagagaa 6600

aaagcggaga actattttaa acaggtgctt tcttcggtga ttaagctgcc agctggccag 6660

attgatgctg aagctcctct cgaggattac gggatcgatt ctatcatgat catgcatgta 6720

acgggtcaat tagaaaaagt attcggatct ttgtccaaga cgctgttctt tgaataccag 6780

gatattcggt cattaacccg gtattttatt gattcccgca gagaaaaact gctggacatc 6840

ttgggatttg aaacgggaaa accttctgtt gaaagaaaat cagaacctga aaaacaagaa 6900

atcccagtta tcccaagaaa gtctggattt cttcctttgc aagataaaga gcggaagcag 6960

gtaag 6965

<210>48

<211>1382

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>48

ttctccaagc atcacacaaa accccttttc cttccattag atagcgttta tattatagtg 60

gatgaaaaag aaaacagcaa acgtgcttaa acatttgtca aacgtatctg aattagctaa 120

catgtaagag gatgatagga ggtttcgttc agtgagtgat aaaacaatga aagacataca 180

ggctgaagta gaccgttaca taggccaatt taaagaagga tattttagcc cgctggccat 240

gatggcgagg ctgactgaag aactgggcga gcttgccaga gaagtgaatc accgttatgg 300

agaaaaacca aaaaaagcga ctgaagatga taaaagcatg gaagaggaaa taggcgatgt 360

gctatttgta ttggtttgtt tagccaactc tcttgatatc tctttagagg aagctcacga 420

ccgagtcatg cataaattta atacaagaga taaagatcgc tggaccagaa aagaagaagg 480

aaagtagagg agatcaagat gtcaaacgaa acaattaaat tagtcattgc gggaccgcgt 540

ggaagaatgg ggcaggaagc tgttaaattg gcagaacgaa caccacattt tgaccttgta 600

ggggccatag accatacata cgatcagcaa aaattatctg atgtgatgcc tgttgagtca 660

gatgctttca tttacacaga tatccatgcc tgttttacag aaacacaacc ggatgtcttg 720

attgatttaa caacgcccga aatcggaaaa gtacatacaa aaattgcatt agagcacgga 780

gtccgtccag ttgtcggaac aaccggtttc tcagaagctg atttaaaaga gctcacatct 840

ttaacagaag aaaaagggat cggagccatc atcgcgccaa attttgcgct cggtgcgata 900

ctgatgatga aattttcaaa aatggctgcc aactattttg aggatgttga gattattgag 960

cttcatcatg accagaagct tgacgcacca agcggaactg cgcttaaaac agcggaaatg 1020

atttcagaag tccgtaaaga aaagcagcaa ggacatccgg atgaaaaaga aattctccca 1080

ggagcaagag gagcggagca aaacggtatt cgcttgcaca gcgtccgtct tccgggactg 1140

atcgcgcatc aggaggtcat gttcggcatg gatggccaaa cgcttcagat acgccatgat 1200

tcttataacc gtgcttcttt catgtcaggc gttaaactgt cagtcgaaca agtcatgaag 1260

attgatcagc ttgtgtatgg tttagaaaat atcattgatt agacgggggg ataaacaatg 1320

aaaattgctt tgatcgcgca tgacaagaaa aaacaggata tggttcaatt tacgactgcc 1380

ta 1382

<210>49

<211>1130

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>49

cattccgatt ctactcctct gacgaatggg tatattgaaa tcgttgaagc gagagaaaag 60

ctttacggta aagaagcggg cgtggagtat gccgaagttt cttttccaaa cggatgctga 120

tgcttgatca tgatgtgctt gggggcgaac aatgagaaaa ctaaaaatag gaatcacatg 180

ctatccgagc gttggaggct caggtatcat tgcgacagaa ctggggaagc agcttgctga 240

aaagggacat gaaatccatt tcatcacatc aagcattccg tttagactga atacatatca 300

tcccaatatt cattttcatg aagttgaggt taatcaatat gctgttttta agtatcctcc 360

gtatgatttg acattggcaa gcaaaatcgc tgaggtggcg gaacgggaga atttagacat 420

tatccatgct cactatgctc tcccgcatgc cgtttgcgcc tatcttgcaa aacaaatgct 480

gaaacgcaat atcggcattg ttaccacttt gcatggcacg gatatcacgg tgttaggtta 540

tgatccgtct ctaaaagatc tgatccgttt tgctattgag tcatcagaca gggtgacggc 600

cgtctcctca gcgcttgcgg ctgaaacata cgatttaatt aaaccggaga aaaaaattga 660

aacgatttat aactttatag acgagcgcgt gtatctgaag aaaaacacag cggcaattaa 720

agagaaacat ggaattttac cagatgaaaa agtcgtcatc catgtgtcca acttcagaaa 780

agttaaacgc gtgcaggatg tcatccgtgt gttccgcaat atcgctggca aaacgaaagc 840

gaagctgctt ttagtcggag acggtccgga gaaatcgaca gcctgcgagc ttatcagaaa 900

atatggcttg gaagaccaag tcttaatgct tggaaatcaa gaccgtgttg aagatcttta 960

ttctattagc gatttgaagc tgctgctatc tgaaaaagaa agctttggcc ttgtcctgct 1020

tgaagcgatg gcttgcggag tgccttgtat tggaacaaac attggcggta tccctgaggt 1080

tataaaaaac aatgtgagcg gatttttggt ggatgtcgga gatgtttcgg 1130

<210>50

<211>813

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>50

atgaggcgat aggtcttcaa attgaacgtt ttcaatccgg ccctttaggg ggagaccagc 60

aaatcggtgc actgatcgct gccaatgcac tcgatcttgt catttttttg cgcgacccgc 120

tgaccgcgca gccgcatgaa ccggatgtct cggcattaat ccgtttatgt gatgtgtatt 180

ccattccgct cgccacaaat atgggtactg cggaaattct tgtgcgcaca cttgatgaag 240

gtgttttcga attccgtgac cttcttcggg gagaagagcc gaatgtataa tgctgacgtt 300

cttgcttttg gcgcccacag tgatgatgtc gagatcggaa tgggcggcac aatagcgaag 360

tttgtcaaac aggaaaaaaa agtaatgata tgcgatttga cagaagcgga actctcttct 420

aacggtacgg tcagtttgcg taaagaagaa gcagctgaag cagcccgcat attaggcgca 480

gataaaagaa ttcagctaac gcttccagac cgcggcctaa taatgagtga tcaggcaatt 540

cggtcaattg tcactgtcat cagaatctgt cggccaaaag cggtttttat gccgtataaa 600

aaggatcgcc atccggatca cggcaatgcg gctgcactgg tggaagaagc gatcttttcc 660

gccggaatcc ataaatataa agacgaaaaa agccttccgg cgcataaagt cagcaaggtt 720

tactattata tgataaatgg ttttcatcag ccggattttg ttattgatat ctcggataca 780

atagaggcaa agaaacaaag cctcatcgcc tac 813

<210>51

<211>5394

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>51

ttctccaagc atcacacaaa accccttttc cttccattag atagcgttta tattatagtg 60

gatgaaaaag aaaacagcaa acgtgcttaa acatttgtca aacgtatctg aattagctaa 120

catgtaagag gatgatagga ggtttcgttc agtgagtgat aaaacaatga aagacataca 180

ggctgaagta gaccgttaca taggccaatt taaagaagga tattttagcc cgctggccat 240

gatggcgagg ctgactgaag aactgggcga gcttgccaga gaagtgaatc accgttatgg 300

agaaaaacca aaaaaagcga ctgaagatga taaaagcatg gaagaggaaa taggcgatgt 360

gctatttgta ttggtttgtt tagccaactc tcttgatatc tctttagagg aagctcacga 420

ccgagtcatg cataaattta atacaagaga taaagatcgc tggaccagaa aagaagaagg 480

aaagtagagg agatcaagat gtcaaacgaa acaattaaat tagtcattgc gggaccgcgt 540

ggaagaatgg ggcaggaagc tgttaaattg gcagaacgaa caccacattt tgaccttgta 600

ggggccatag accatacata cgatcagcaa aaattatctg atgtgatgcc tgttgagtca 660

gatgctttca tttacacaga tatccatgcc tgttttacag aaacacaacc ggatgtcttg 720

attgatttaa caacgcccga aatcggaaaa gtacatacaa aaattgcatt agagcacgga 780

gtccgtccag ttgtcggaac aaccggtttc tcagaagctg atttaaaaga gctcacatct 840

ttaacagaag aaaaagggat cggagccatc atcgcgccaa attttgcgct cggtgcgata 900

ctgatgatga aattttcaaa aatggctgcc aactattttg aggatgttga gattattgag 960

cttcatcatg accagaagct tgacgcacca agcggaactg cgcttaaaac agcggaaatg 1020

atttcagaag tccgtaaaga aaagcagcaa ggacatccgg atgaaaaaga aattctccca 1080

ggagcaagag gagcggagca aaacggtatt cgcttgcaca gcgtccgtct tccgggactg 1140

atcgcgcatc aggaggtcat gttcggcatg gatggccaaa cgcttcagat acgccatgat 1200

tcttataacc gtgcttcttt catgtcaggc gttaaactgt cagtcgaaca agtcatgaag 1260

attgatcagc ttgtgtatgg tttagaaaat atcattgatt agacgggggg ataaacaatg 1320

aaaattgctt tgatcgcgca tgacaagaaa aaacaggata tggttcaatt tacgactgcc 1380

tacattccga ttctactcct ctgacgaatg ggtatattga aatcgttgaa gcgagagaaa 1440

agctttacgg taaagaagcg ggcgtggagt atgccgaagt ttcttttcca aacggatgct 1500

gatgcttgat catgatgtgc ttgggggcga acaatgagaa aactaaaaat aggaatcaca 1560

tgctatccga gcgttggagg ctcaggtatc attgcgacag aactggggaa gcagcttgct 1620

gaaaagggac atgaaatcca tttcatcaca tcaagcattc cgtttagact gaatacatat 1680

catcccaata ttcattttca tgaagttgag gttaatcaat atgctgtttt taagtatcct 1740

ccgtatgatt tgacattggc aagcaaaatc gctgaggtgg cggaacggga gaatttagac 1800

attatccatg ctcactatgc tctcccgcat gccgtttgcg cctatcttgc aaaacaaatg 1860

ctgaaacgca atatcggcat tgttaccact ttgcatggca cggatatcac ggtgttaggt 1920

tatgatccgt ctctaaaaga tctgatccgt tttgctattg agtcatcaga cagggtgacg 1980

gccgtctcct cagcgcttgc ggctgaaaca tacgatttaa ttaaaccgga gaaaaaaatt 2040

gaaacgattt ataactttat agacgagcgc gtgtatctga agaaaaacac agcggcaatt 2100

aaagagaaac atggaatttt accagatgaa aaagtcgtca tccatgtgtc caacttcaga 2160

aaagttaaac gcgtgcagga tgtcatccgt gtgttccgca atatcgctgg caaaacgaaa 2220

gcgaagctgc ttttagtcgg agacggtccg gagaaatcga cagcctgcga gcttatcaga 2280

aaatatggct tggaagacca agtcttaatg cttggaaatc aagaccgtgt tgaagatctt 2340

tattctatta gcgatttgaa gctgctgcta tctgaaaaag aaagctttgg ccttgtcctg 2400

cttgaagcga tggcttgcgg agtgccttgt attggaacaa acattggcgg tatccctgag 2460

gttataaaaa acaatgtgag cggatttttg gtggatgtcg gagatgtttc ggtcttcaac 2520

taaagcaccc attagttcaa caaacgaaaa ttggataaag tgggatattt ttaaaatata 2580

tatttatgtt acagtaatat tgacttttaa aaaaggattg attctaatga agaaagcaga 2640

caagtaagcc tcctaaattc actttagata aaaatttagg aggcatatca aatgaacttt 2700

aataaaattg atttagacaa ttggaagaga aaagagatat ttaatcatta tttgaaccaa 2760

caaacgactt ttagtataac cacagaaatt gatattagtg ttttataccg aaacataaaa 2820

caagaaggat ataaatttta ccctgcattt attttcttag tgacaagggt gataaactca 2880

aatacagctt ttagaactgg ttacaatagc gacggagagt taggttattg ggataagtta 2940

gagccacttt atacaatttt tgatggtgta tctaaaacat tctctggtat ttggactcct 3000

gtaaagaatg acttcaaaga gttttatgat ttataccttt ctgatgtaga gaaatataat 3060

ggttcgggga aattgtttcc caaaacacct atacctgaaa atgctttttc tctttctatt 3120

attccatgga cttcatttac tgggtttaac ttaaatatca ataataatag taattacctt 3180

ctacccatta ttacagcagg aaaattcatt aataaaggta attcaatata tttaccgcta 3240

tctttacagg tacatcattc tgtttgtgat ggttatcatg caggattgtt tatgaactct 3300

attcaggaat tgtcagatag gcctaatgac tggcttttat aatatgagat aatgccgact 3360

gtacttttta cagtcggttt tctaatgtca ctaacctgcc ccgttagttg aaggcatttt 3420

ctgtcaatgt tttcttacaa agaacgctgt gatatactga aatttgtccg tatacatttt 3480

ggaggaatgg atatgttacc aaaatacgcg caagtaaaag aagaaatcag ttcttggatt 3540

aatcaaggca aaatactgcc cgatcaaaaa atccctaccg aaaacgaatt aatgcagcaa 3600

ttcggcgtca gccggcatac catccgcaaa gcgatcggag acctcgtatc acaaggtctg 3660

ctgtacagcg tgcaaggcgg aggcaccttt gtcgcttcac gctctgctaa gtcagcgctg 3720

cattccaata aaacgatcgg tgttttgaca acttacatat cagactatat tttcccgagc 3780

atcatcagag gaatcgagtc ctatttaagc gagcaggggt attctatgct tttgacaagc 3840

acaaacaaca acccggacaa tgaaagaaga ggcttagaaa acctgctgtc ccagcatatt 3900

gacggactca tcgtagaacc gacaaaaagc gcccttcaaa ccccaaacat cggctattat 3960

ctgaacttgg agaaaaacgg cattcctttt gcgatgatta acgcgtcata tgccgagctt 4020

gccgcgccaa gttttacctt ggatgatgtg aaaggcggga tgatggcggc ggagcatttg 4080

ctttctctcg gccacacgca tatgatgggt atttttaaag ctgatgacac acaaggcgtg 4140

aaacggatga acggatttat acaggcgcac cgggagcgtg agttgtttcc ttctccggat 4200

atgatcgtga catttacaac ggaagaaaaa gaatcaaaac ttctggagaa agtaaaagcc 4260

acactggaga aaaacagcaa gcacatgccg acagccattc tttgttataa cgatgaaatt 4320

gcgctgaagg tgattgatat gctgagggag atggatctta aagtgccgga ggatatgtct 4380

attgtcgggt acgatgattc acatttcgcc caaatctcag aagtgaaact aacctctgtc 4440

aaacatccga aatcagtgct tggaaaagca gccgccaaat atgtcattga ctgcttagag 4500

cataaaaagc cgaagcaaga ggatgtcata tttgagcctg agttgatcat tcgccagtcc 4560

gcacgaaaac tgaatgaata aatgaggcga taggtcttca aattgaacgt tttcaatccg 4620

gccctttagg gggagaccag caaatcggtg cactgatcgc tgccaatgca ctcgatcttg 4680

tcattttttt gcgcgacccg ctgaccgcgc agccgcatga accggatgtc tcggcattaa 4740

tccgtttatg tgatgtgtat tccattccgc tcgccacaaa tatgggtact gcggaaattc 4800

ttgtgcgcac acttgatgaa ggtgttttcg aattccgtga ccttcttcgg ggagaagagc4860

cgaatgtata atgctgacgt tcttgctttt ggcgcccaca gtgatgatgt cgagatcgga 4920

atgggcggca caatagcgaa gtttgtcaaa caggaaaaaa aagtaatgat atgcgatttg 4980

acagaagcgg aactctcttc taacggtacg gtcagtttgc gtaaagaaga agcagctgaa 5040

gcagcccgca tattaggcgc agataaaaga attcagctaa cgcttccaga ccgcggccta 5100

ataatgagtg atcaggcaat tcggtcaatt gtcactgtca tcagaatctg tcggccaaaa 5160

gcggttttta tgccgtataa aaaggatcgc catccggatc acggcaatgc ggctgcactg 5220

gtggaagaag cgatcttttc cgccggaatc cataaatata aagacgaaaa aagccttccg 5280

gcgcataaag tcagcaaggt ttactattat atgataaatg gttttcatca gccggatttt 5340

gttattgata tctcggatac aatagaggca aagaaacaaa gcctcatcgc ctac 5394

<210>52

<211>1487

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>52

gccgatgact tggttgtttt gaaccttgat ggagaggtcg tcgaaggctc gcttaaacct 60

tcttcagata cacctaccca tgtttatcta tataaagcct ttccgaatat cgggggaatt 120

gtccataccc attctcaatg ggcgacaagc tgggcgcaat cgggcagaga catccctccg 180

ttaggcacga cccatgctga ttattttgac agtgcgattc catgtactcg agaaatgtac 240

gatgaagaaa tcattcatga ctacgaactg aatacaggaa aagtcatagc ggaaaccttt 300

cagcatcata attacgaaca ggtgccgggt gtgctcgtga ataatcacgg accgttctgc 360

tggggcactg acgccttaaa tgccattcat aacgcagttg tattagaaac ggttgccgaa 420

atggcctatc actccattat gctgaacaag gatgtaaccc caatcaatac agtcctgcat 480

gaaaagcatt tttatcgaaa acacggagca aatgcgtatt atggccagtc atgatacgcc 540

tgtgtcaccg gctggcattc tgattgactt ggacggtact gtattcagag gaaatgagtt 600

gatcgaagga gcaagagaag cgatcaaaac gcttaggaga atgggaaaga aaatcgtctt 660

tttaagcaac cgggggaata tctcccgtgc catgtgcaga aaaaaacttc ttggcgcggg 720

gattgaaacg gacgtaaacg acattgttct gtcatcaagc gtgacagcgg cttttctgaa 780

aaaacattat cgtttttcaa aggtatgggt gcttggggag caaggcttgg ttgacgagct 840

gaggctggcc ggtgtgcaga acgcgagcga accgaaggaa gcggattggc tcgtgatctc 900

ccttcatgaa acgctcacgt acgacgattt aaatcaagcc tttcaagcgg ctgccggcgg 960

cgctcgtatt atcgctacaa acaaagaccg ctcttttccg aacgaagacg gaaatgccat 1020

tgatgtggcc ggaatgatcg gggcaattga gacttctgca caagcgaaga ctgaacttgt 1080

tgtcggaaaa ccgtcatggc tgatggcgga ggctgcctgt acagcaatgg ggctgtccgc 1140

acatgaatgc atgattatag gagacagcat tgaatctgac attgcgatgg ggaagcttta 1200

tggcatgaaa agcgccttag tgctaactgg ttctgcgaaa cagggtgaac agcgtttgta 1260

cacgccggat tatgtgctgg attctattaa ggatgtaacc aaattggctg aggaggggat 1320

tctgatatga atcgtatcgc agctgacgtt cagcgtgctt ttgaaaacgc cggagaaaag 1380

acgttgccta taaaagttga agaaattgtt ctcggtaagc aagcagctga ttcgcttttg 1440

gattatgtaa aacgaaaaaa caatcaacat attgtcattg tctggga 1487

<210>53

<211>1026

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>53

gggctggatt tcgtcaaaaa tgccttcaga cacgcgcaca ccttaagaga ccgatgcacc 60

ggattaagaa tcatcaatga aaacaaaacg ctgatcaacc atggtctata tgaatagccc 120

gcacctcgaa tggaaggggt aacgcagatg aaaaaaatga ctgtctgttt tcttgtgctc 180

atgatgttgc tgacattagt cattgccggg tgttcagcag aaaaatcatc cggcaaatcg 240

ggtgaaactg agctgacctt ttggacattt agcgggcttc atgagcagtt ctatgtggaa 300

atggtgaagg aatggaacaa aaaatatcct gaccgcaaaa ttaagctgaa tacggtcgtt 360

tatccatatg gacaaatgca cgataactta tctatctccc taatagcggg agaaggcgtt 420

cctgatattg cagatgtcga attggcccgt ttttcaaact ttttgaaggg ctctgacata 480

ccgcttgccg acttgactcc gctgattgaa aaggatcgcg ataaattcgt tgaggcgcgg 540

ctgacattgt acagcaaaaa cggaaagctt tacggactcg atacacatgt agggacaacg 600

gtcatgtttt ataacatgga tgtgatgaaa aaagccggcg tcaatcctga cgatattaaa 660

acatgggatg attaccataa agccggacag aaagtgcgca aagtgaccgg gaagccgatg 720

ggaacggtgg aaacaaatga ttccgcaacg ttcttatcta tgatttcaca gcaaaactca 780

ggctattttg ataaaaacgg caagctgatc ctcaataatg acaccaacgt aaaaacactt 840

caatatttaa aagacatgat caatgataaa acgatgattc ctgcgccggg cggcgggcat 900

cacagtgaag aatactacgg ctttatgaac caaggaggag ctgcttcagt tctcatgccg 960

atttggtata tgggaagatt tatcgattat atgcctgatc tgaaagggaa gatagccatc 1020

agaccg 1026

<210>54

<211>682

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>54

cgaaacaacc aacggatgtc atgatcgcag tcggttcggg cacgattcat gatatcgtcc 60

gctttgcggc gtttcaaaga gatttgccgt ttatttctta tccgactgct ccatctgtag 120

acggttttac atcagccggt gcgccgatta ttttatacgg cacgaaaaca accattcaaa 180

cgaaggcccc atctgcgctg ttcgctgatc tggatctatt aaaagcggca ccgcagtcaa 240

tggtggcggc tggctttggt gacatgctcg gtaaaatcac gtctttagca gattgggaaa 300

tatcccggca tcttgccggt gagccttatt cgcctgcagg agctaagatc gttcaggagg 360

cgcttgctgc ctgcattgaa cacacagaag acattgcgat gaaaacggaa actggcatac 420

gggttttgat ggagtcttta cttgtatcgg ggcttgtcat gctggcatta gatcattccc 480

gaccggcatc aggcggcgag catcatattt cacattggat tgaaatggag ttaatggaga 540

aaaaacggcc tcagattctt catggggcaa aggtgggctg tgccgctgtt ttattaactg 600

acacatacag aaagctcgct caggatgacg ggctgaacga attttcacca agccgccggg 660

aagccatcca atcggcttat ca 682

<210>55

<211>5264

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>55

gccgatgact tggttgtttt gaaccttgat ggagaggtcg tcgaaggctc gcttaaacct 60

tcttcagata cacctaccca tgtttatcta tataaagcct ttccgaatat cgggggaatt 120

gtccataccc attctcaatg ggcgacaagc tgggcgcaat cgggcagaga catccctccg 180

ttaggcacga cccatgctga ttattttgac agtgcgattc catgtactcg agaaatgtac 240

gatgaagaaa tcattcatga ctacgaactg aatacaggaa aagtcatagc ggaaaccttt 300

cagcatcata attacgaaca ggtgccgggt gtgctcgtga ataatcacgg accgttctgc 360

tggggcactg acgccttaaa tgccattcat aacgcagttg tattagaaac ggttgccgaa 420

atggcctatc actccattat gctgaacaag gatgtaaccc caatcaatac agtcctgcat 480

gaaaagcatt tttatcgaaa acacggagca aatgcgtatt atggccagtc atgatacgcc 540

tgtgtcaccg gctggcattc tgattgactt ggacggtact gtattcagag gaaatgagtt 600

gatcgaagga gcaagagaag cgatcaaaac gcttaggaga atgggaaaga aaatcgtctt 660

tttaagcaac cgggggaata tctcccgtgc catgtgcaga aaaaaacttc ttggcgcggg 720

gattgaaacg gacgtaaacg acattgttct gtcatcaagc gtgacagcgg cttttctgaa 780

aaaacattat cgtttttcaa aggtatgggt gcttggggag caaggcttgg ttgacgagct 840

gaggctggcc ggtgtgcaga acgcgagcga accgaaggaa gcggattggc tcgtgatctc 900

ccttcatgaa acgctcacgt acgacgattt aaatcaagcc tttcaagcgg ctgccggcgg 960

cgctcgtatt atcgctacaa acaaagaccg ctcttttccg aacgaagacg gaaatgccat 1020

tgatgtggcc ggaatgatcg gggcaattga gacttctgca caagcgaaga ctgaacttgt 1080

tgtcggaaaa ccgtcatggc tgatggcgga ggctgcctgt acagcaatgg ggctgtccgc 1140

acatgaatgc atgattatag gagacagcat tgaatctgac attgcgatgg ggaagcttta 1200

tggcatgaaa agcgccttag tgctaactgg ttctgcgaaa cagggtgaac agcgtttgta 1260

cacgccggat tatgtgctgg attctattaa ggatgtaacc aaattggctg aggaggggat 1320

tctgatatga atcgtatcgc agctgacgtt cagcgtgctt ttgaaaacgc cggagaaaag 1380

acgttgccta taaaagttga agaaattgtt ctcggtaagc aagcagctga ttcgcttttg 1440

gattatgtaa aacgaaaaaa caatcaacat attgtcattg tctgggaggg ctggatttcg 1500

tcaaaaatgc cttcagacac gcgcacacct taagagaccg atgcaccgga ttaagaatca 1560

tcaatgaaaa caaaacgctg atcaaccatg gtctatatga atagcccgca cctcgaatgg 1620

aaggggtaac gcagatgaaa aaaatgactg tctgttttct tgtgctcatg atgttgctga 1680

cattagtcat tgccgggtgt tcagcagaaa aatcatccgg caaatcgggt gaaactgagc 1740

tgaccttttg gacatttagc gggcttcatg agcagttcta tgtggaaatg gtgaaggaat 1800

ggaacaaaaa atatcctgac cgcaaaatta agctgaatac ggtcgtttat ccatatggac 1860

aaatgcacga taacttatct atctccctaa tagcgggaga aggcgttcct gatattgcag 1920

atgtcgaatt ggcccgtttt tcaaactttt tgaagggctc tgacataccg cttgccgact 1980

tgactccgct gattgaaaag gatcgcgata aattcgttga ggcgcggctg acattgtaca 2040

gcaaaaacgg aaagctttac ggactcgata cacatgtagg gacaacggtc atgttttata 2100

acatggatgt gatgaaaaaa gccggcgtca atcctgacga tattaaaaca tgggatgatt 2160

accataaagc cggacagaaa gtgcgcaaag tgaccgggaa gccgatggga acggtggaaa 2220

caaatgattc cgcaacgttc ttatctatga tttcacagca aaactcaggc tattttgata 2280

aaaacggcaa gctgatcctc aataatgaca ccaacgtaaa aacacttcaa tatttaaaag 2340

acatgatcaa tgataaaacg atgattcctg cgccgggcgg cgggcatcac agtgaagaat 2400

actacggctt tatgaaccaa ggaggagctg cttcagttct catgccgatt tggtatatgg 2460

gaagatttat cgattatatg cctgatctga aagggaagat agccatcaga ccgtattcaa 2520

ctaaagcacc cattagttca acaaacgaaa attggataaa gtgggatatt tttaaaatat 2580

atatttatgt tacagtaata ttgactttta aaaaaggatt gattctaatg aagaaagcag 2640

acaagtaagc ctcctaaatt cactttagat aaaaatttag gaggcatatc aaatgaactt 2700

taataaaatt gatttagaca attggaagag aaaagagata tttaatcatt atttgaacca 2760

acaaacgact tttagtataa ccacagaaat tgatattagt gttttatacc gaaacataaa 2820

acaagaagga tataaatttt accctgcatt tattttctta gtgacaaggg tgataaactc 2880

aaatacagct tttagaactg gttacaatag cgacggagag ttaggttatt gggataagtt 2940

agagccactt tatacaattt ttgatggtgt atctaaaaca ttctctggta tttggactcc 3000

tgtaaagaat gacttcaaag agttttatga tttatacctt tctgatgtag agaaatataa 3060

tggttcgggg aaattgtttc ccaaaacacc tatacctgaa aatgcttttt ctctttctat 3120

tattccatgg acttcattta ctgggtttaa cttaaatatc aataataata gtaattacct 3180

tctacccatt attacagcag gaaaattcat taataaaggt aattcaatat atttaccgct 3240

atctttacag gtacatcatt ctgtttgtga tggttatcat gcaggattgt ttatgaactc 3300

tattcaggaa ttgtcagata ggcctaatga ctggctttta taatatgaga taatgccgac 3360

tgtacttttt acagtcggtt ttctaatgtc actaacctgc cccgttagtt gaaggcattt 3420

tctgtcaatg ttttcttaca aagaacgctg tgatatactg aaatttgtcc gtatacattt 3480

tggaggaatg gatatgttac caaaatacgc gcaagtaaaa gaagaaatca gttcttggat 3540

taatcaaggc aaaatactgc ccgatcaaaa aatccctacc gaaaacgaat taatgcagca 3600

attcggcgtc agccggcata ccatccgcaa agcgatcgga gacctcgtat cacaaggtct 3660

gctgtacagc gtgcaaggcg gaggcacctt tgtcgcttca cgctctgcta agtcagcgct 3720

gcattccaat aaaacgatcg gtgttttgac aacttacata tcagactata ttttcccgag 3780

catcatcaga ggaatcgagt cctatttaag cgagcagggg tattctatgc ttttgacaag 3840

cacaaacaac aacccggaca atgaaagaag aggcttagaa aacctgctgt cccagcatat 3900

tgacggactc atcgtagaac cgacaaaaag cgcccttcaa accccaaaca tcggctatta 3960

tctgaacttg gagaaaaacg gcattccttt tgcgatgatt aacgcgtcat atgccgagct 4020

tgccgcgcca agttttacct tggatgatgt gaaaggcggg atgatggcgg cggagcattt 4080

gctttctctc ggccacacgc atatgatggg tatttttaaa gctgatgaca cacaaggcgt 4140

gaaacggatg aacggattta tacaggcgca ccgggagcgt gagttgtttc cttctccgga 4200

tatgatcgtg acatttacaa cggaagaaaa agaatcaaaa cttctggaga aagtaaaagc 4260

cacactggag aaaaacagca agcacatgcc gacagccatt ctttgttata acgatgaaat 4320

tgcgctgaag gtgattgata tgctgaggga gatggatctt aaagtgccgg aggatatgtc 4380

tattgtcggg tacgatgatt cacatttcgc ccaaatctca gaagtgaaac taacctctgt 4440

caaacatccg aaatcagtgc ttggaaaagc agccgccaaa tatgtcattg actgcttaga 4500

gcataaaaag ccgaagcaag aggatgtcat atttgagcct gagttgatca ttcgccagtc 4560

cgcacgaaaa ctgaatgaat aacgaaacaa ccaacggatg tcatgatcgc agtcggttcg 4620

ggcacgattc atgatatcgt ccgctttgcg gcgtttcaaa gagatttgcc gtttatttct 4680

tatccgactg ctccatctgt agacggtttt acatcagccg gtgcgccgat tattttatac 4740

ggcacgaaaa caaccattca aacgaaggcc ccatctgcgc tgttcgctga tctggatcta 4800

ttaaaagcgg caccgcagtc aatggtggcg gctggctttg gtgacatgct cggtaaaatc 4860

acgtctttag cagattggga aatatcccgg catcttgccg gtgagcctta ttcgcctgca 4920

ggagctaaga tcgttcagga ggcgcttgct gcctgcattg aacacacaga agacattgcg 4980

atgaaaacgg aaactggcat acgggttttg atggagtctt tacttgtatc ggggcttgtc 5040

atgctggcat tagatcattc ccgaccggca tcaggcggcg agcatcatat ttcacattgg 5100

attgaaatgg agttaatgga gaaaaaacgg cctcagattc ttcatggggc aaaggtgggc 5160

tgtgccgctg ttttattaac tgacacatac agaaagctcg ctcaggatga cgggctgaac 5220

gaattttcac caagccgccg ggaagccatc caatcggctt atca 5264

Claims (2)

1. A method for genetically modifying Bacillus subtilis, comprising the steps of:

construction of starting Strain MK3-MEP 123-. DELTA.dhbB

(1) Overexpression of menA Gene at the yxlA site on the B.subtilis chromosome

Firstly, using chromosome of B.subtilis 168 as a template, and respectively amplifying fragments U (1115bp), A (1057bp), D (1053bp) and G (806bp) by using primers yxlA-menA-U1/yxlA-menA-U2q, yxlA-menA-1q/yxlA-menA-2, yxlA-menA-D1q/yxlA-menA-D2, yxlA-menA-G1 q/yxlA-menA-G2; plasmid pUC57-1.8k-P1 as template and use as primerThe product yxlA-menA-P1/yxlA-menA-P2 contains promoter P_lapSFragment P of (442 bp); amplifying a fragment CR (2069bp) by using a primer yxlA-menA-CR1q/CR2 by using a chromosome of BS168NUm as a template; then, splicing the segment U, P and A into a segment UPA (2614bp) by using a primer yxlA-menA-U1/yxlA-menA-2 through an overlapping PCR method; then the fragment UPA and the fragment D are spliced into a fragment UPAD (3667bp) by using a primer yxlA-menA-U1/yxlA-menA-D2; finally, the fragment UPAD, the fragment CR and the fragment G are spliced into a fragment UPADCRG (6542bp) by using a primer yxlA-menA-U1/yxlA-menA-G2; transforming the UPADCRG fragment into a competent cell of a recipient bacterium BS168NU, and finally obtaining a recombinant strain MK3 with menA gene integrated at a yxlA locus through two-step screening;

(2) overexpression of dxs Gene at the yjoB site of the B.subtilis chromosome

Firstly, using chromosome of B.subtilis 168 as a template, and respectively amplifying fragments U (1236bp), s (1947bp), D (808bp) and G (697bp) by using primers yjoB-dxs-U1/yjoB-dxs-U2q, yjoB-dxs-1q/yjoB-dxs-2, yjoB-dxs-D1q/yjoB-dxs-D2 and yjoB-dxs-G1 q/yjoB-dxs-G2; plasmid pUC57-1.8k-P2 was used as a template, and primer P was used₄₃1/P₄₃2 amplification of a plasmid containing promoter P₄₃Fragment P of (232 bp); amplifying a fragment CR (2069bp) by using a primer yjoB-dxs-CR1q/CR2 by using a chromosome of BS168NUm as a template; then, the primer yjoB-dxs-U1/yjoB-dxs-2 is used for splicing the segment U, P and s into a segment UPs (3415bp) by an overlapping PCR method; then splicing the fragment UPs and the fragment D into a fragment UPsD (4223bp) by using a primer yjoB-dxs-U1/yjoB-dxs-D2; finally, splicing the segment UPsD, the segment CR and the segment G into a segment UPsDCRG (6989bp) by using a primer yjoB-dxs-U1/yjoB-dxs-G2; transforming the UPsDCRG fragment into a competent cell of an acceptor bacterium MK3, and finally obtaining a recombinant strain MK3-MEP1 with dxs genes integrated at a yjoB locus through two-step screening;

on the basis of the strain MK3-MEP1, dxr and yacM-yacN are sequentially overexpressed to obtain a recombinant strain MK3-MEP 123; using chromosome of B.subtilis 168 as template, respectively amplifying fragments U (1003bp), D (815bp) and G (605bp) by primers dhbB-U1/dhbB-U2, dhbB-D1q/dhbB-D2 and dhbB-G1 q/dhbB-G2; amplifying a fragment CR (2069bp) with a selection box (cat-araR) by using a primer dhbB-CR1q/CR2 by using a chromosome of a midbody strain BS168NUm as a template; firstly, splicing the fragments U and D into UD (1818bp) by overlap PCR by using primers dhbB-U1/dhbB-D2; then, splicing the three fragments UD, GR and G into UDCRG (4492bp) by using a primer dhbB-U1/dhbB-G2 through an overlapping PCR method; transforming the UDCRG fragment into a competent cell of a receptor bacterium MK3-MEP123, and finally screening to obtain a recombinant strain MK3-MEP 123-delta dhbB with a gene dhbB knocked out;

(II) overexpression of the Glycerol kinase Gene glpK and the Glycerol-3-phosphate dehydrogenase Gene glpD

Constructing a glpK overexpression strain; firstly, using chromosome of starting bacterium B.subtilis MK3-MEP 123-delta dhbB as template, respectively using primers pksJ-glpK-U1/pksJ-glpK-U2q, pksJ-glpK-1q/pksJ-glpK-2, pksJ-glpK-D1q/pksJ-glpK-D2 and pksJ-glpK-G1q/pksJ-glpK-G2 to amplify fragment U, as shown in SEQ ID No.36, 1164bp, and amplified fragment K as shown in SEQ ID No.37 and 1783 bp; the amplified fragment D is shown as SEQ ID No.38 and is 948 bp; the amplified fragment G is shown as SEQ ID No.39 and 990 bp; using chromosome of BS168NUm as template, amplifying fragment CR with primer pksJ-glpK-CR1q/CR2, as shown in SEQ ID No.40, 2069 bp; plasmid pUC57-1.8k-P1 was used as a template, and primer P was used_lapS1/P_lapS2Amplification of a nucleic acid molecule containing promoter P_lapSThe fragment P of the expression cassette is shown as SEQ ID No.41, and is 256 bp; then, splicing the U, P fragment and the K fragment into a UPK fragment (3203bp) by using a primer pksJ-glpK-U1/pksJ-glpK-2 through an overlapping PCR method; splicing the fragment UPK and the fragment D into a fragment UPKD (4151bp) by using a primer pksJ-glpK-U1/pksJ-glpK-D2; finally, the fragment UPKD, the fragment CR and the fragment G are spliced into a fragment UPKDCRG (7210bp) by using a primer pksJ-glpK-U1/pksJ-glpK-G2; transforming UPKDCRG fragment into competent cells of receptor bacteria MK3-MEP 123-delta dhbB, and finally obtaining a recombinant strain BSMK _1 with glpK gene integrated at pksJ locus through two-step screening; the sequence of the UPKDCRG fragment is shown in SEQ ID No. 42;

firstly, using chromosome of starting bacterium B.subtilis MK3-MEP 123-delta dhbB as template, respectively using primers pks L-glpD-U1/pks L-glpD-U2 q, pks L-glpD-1 q/pks L-glpD-2, pks L-glpD-D1 q/pks L-glpD-D2 and pks L-glpD-G1 q/pks L-glpD-G2 to amplify fragment U, as shown in SEQ ID No.43 and 1118bp, and amplifying fragment D as shown in SEQ ID No.44 and 1749 bp;the amplified fragment D is 1004bp shown as SEQ ID No.45, the amplified fragment G is 769bp shown as SEQ ID No.46, the chromosome of BS168NUm is taken as a template, a primer pks L-glpD-CR 1q/CR2 is used for amplifying a fragment CR shown as SEQ ID No.40, 2069bp is used, a plasmid pUC57-1.8k-P1 is taken as a template, and a primer P is used_lapS1/P_lapS2Amplification of a nucleic acid molecule containing promoter P_lapS256bp of fragment P of the expression cassette as shown in SEQ ID No.41, splicing the fragments U, P and D into a fragment UPD (3123bp) by an overlapping PCR method by using primers pks L-glpD-U1/pks L-glpD-2, splicing the fragment UPD and the fragment D into a fragment UPDD (4127bp) by using primers pks L-glpD-U1/pks L-glpD-D2, splicing the fragment UPDD, the fragment CR and the fragment G into a fragment UPDDRG (6965bp) by using primers pks L-glpD-U1/pks L-glpD-G2, transforming the DDRG fragment into competent cells of recipient bacterium BSMK _1, and finally obtaining a DDC recombinant strain BSMK _2 with the glpppPD gene integrated at the pKs L site through two-step screening, wherein the sequence of the UPRG fragment is shown in SEQ ID No. 47;

(III) knocking out methylglyoxal synthetase coding gene mgsA and glycerol-1-phosphate dehydrogenase coding gene araM

Using chromosome of starting bacterium B.subtilis MK3-MEP 123-delta dhbB as template, respectively using primers mgsA-U1/mgsA-U2, mgsA-D1q/mgsA-D2 and mgsA-G1q/mgsA-G2 to amplify fragment U, as shown in SEQ ID No.48, 1382 bp; the amplified fragment D is shown as SEQ ID No.49 and is 1130 bp; the amplified fragment G is shown as SEQ ID No.50 and 813 bp; using chromosome of BS168NUm as template, amplifying fragment CR with selection box (cat-araR) by primer mgsA-CR1q/CR2, as shown in SEQ ID No.40, 2069 bp; firstly, splicing the fragments U and D into UD (2512bp) by overlapping PCR by using primers mgsA-U1/mgsA-D2; then, the three fragments UD, GR and G are spliced into UDCRG (5394bp) by an overlapping PCR method by using primers mgsA-U1/mgsA-G2; transforming the UDCRG fragment into a competent cell of a receptor bacterium BSMK _2, and finally screening to obtain a recombinant strain BSMK _3 with the gene mgsA being knocked out; the sequence of the UDCRG fragment is shown as SEQ ID No. 51;

using chromosome of starting bacterium B.subtilis MK3-MEP 123-delta dhbB as template, respectively using primers araM-U1/araM-U2, araM-D1q/araM-D2 and araM-G1q/araM-G2 to amplify fragment U, as shown in SEQ ID No.52, 1487 bp; the amplified fragment D is shown as SEQ ID No.53 and is 1026 bp; the amplified fragment G is shown as SEQ ID No.54, 682 bp; using chromosome of BS168NUm as template, amplifying fragment CR with primer araM-CR1q/CR2, as shown in SEQ ID No.40, 2069 bp; firstly, splicing fragments U and D into UD (2513bp) by overlapping PCR by using primers araM-U1/araM-D2; then, the three fragments UD, GR and G are spliced into UDCRG (5264bp) by an overlapping PCR method by using a primer araM-U1/araM-G2; transforming the UDCRG fragment into competent cells of receptor bacteria BSMK _3, and finally screening to obtain a recombinant strain BSMK _4 with the knocked-out gene araM; the sequence of the UDCRG fragment is shown as SEQ ID No. 55;

the primer sequences are as follows

2. Use of the strain obtained in claim 1 for the fermentative preparation of menaquinone-7.

Images