CN112852871A - Cas9 system for efficiently editing silkworm genome and application thereof - Google Patents
Cas9 system for efficiently editing silkworm genome and application thereof Download PDFInfo
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Abstract
The invention discloses a Cas9 system for efficiently editing silkworm genome and application thereof, wherein the system comprises a silkworm transgenic expression vector containing an expressed Cas9 gene and virus particles of an expression frame U6-sgRNA of a silkworm nuclear polyhedrosis virus package containing a U6 promoter for regulating sgRNA expression. A bombyx mori nuclear polyhedrosis virus (BmNPV) genome is used as a vector, a Bac-to-Bac transposition is adopted to recombine a U6-sgRNA expression frame to the virus genome to construct a recombinant virus particle, the recombinant virus is infected to a Cas9 cell line or a Cas9 bombyx mori, and a target gene can be effectively edited. The Cas9 system established by the invention does not need a transfection process, has high transfection efficiency and good safety, can stably and efficiently edit target genes, and can be suitable for researches on virus genes, functions of related host genes, baculovirus expression systems and the like.
Description
Technical Field
The invention relates to the technical field of genes, in particular to a Cas9 system for efficiently editing a silkworm genome, and also relates to application of the Cas9 system in editing the silkworm genome or silkworm nuclear polyhedrosis virus genes.
Background
The CRISPR system is a powerful immune weapon evolved in the process of resisting virus invasion by bacteria and archaea, and is characterized in that a guide RNA (ribonucleic acid) specifically recognizes target DNA or RNA through base complementary pairing, and guides effector protein with nuclease activity to cut the target DNA or RNA, so that exogenous nucleic acid is eliminated. The CRISPR/Cas9 system is widely applied to basic research and application research due to the characteristics of single effector protein, simple operation, high editing efficiency and the like. Chinese patent publication No. CN105132460A discloses that the CRISPR/Cas9 system is used for the study of gene function of silkworm cells and the study of antiviral resistance of silkworms, but this method requires liposome transfection, and since silkworm cell lines have lower transfection efficiency of about 30% -40% to foreign genes than mammalian cell lines, this limits the editing efficiency of Cas9 system. At the level of silkworm individuals, gene editing is an important means for researching gene functions and virus resistance, but the creation period of transgenic silkworms is long, time and labor are wasted, and the advantage of gene editing is not obvious.
Therefore, a Cas9 system with high transfection efficiency and good safety is needed. Baculovirus is obligate parasitic to arthropods, is widely applied to expression of foreign proteins at present, is also applied to vaccine production, gene therapy and the like, and has no report of baculovirus used in a Cas9 system at present.
Disclosure of Invention
In order to solve the problems, the recombinant baculovirus of the Cas9 packaged by the bombyx mori nuclear polyhedrosis virus established in the invention has specific and efficient infectivity on bombyx mori cells, can effectively improve the editing efficiency of the Cas9 system, and promotes the application of the Cas9 system in bombyx mori. The invention also aims to provide application of the silkworm nuclear polyhedrosis virus packaging Cas9 system in editing silkworm genome or silkworm nuclear polyhedrosis virus genes; the invention further aims to provide a method for efficiently editing silkworm genomes by using the Cas9 system.
In order to achieve the purpose, the invention provides the following technical scheme:
1. the Cas9 system for efficiently editing the silkworm genome comprises a silkworm transgenic expression vector for expressing a Cas9 gene and a virus particle of an expression frame U6-sgRNA for regulating sgRNA expression by a silkworm nuclear polyhedrosis virus packaged U6 promoter containing a target sequence.
Preferably, the nucleotide sequence of the Cas9 gene is shown as SEQ ID NO.1, and the nucleic acid of the U6-sgRNA expression frame is shown as SEQ ID NO. 2. The silkworm transgenic expression vector containing the Cas9 gene contains an expression frame of regulating the expression of Cas9 by an OpIE2 promoter and an expression frame of regulating the expression of Mcherry by an OpIE2 promoter.
More preferably, the virus particles are prepared by the following method: transferring the recombinant plasmid containing the expression frame U6-sgRNA containing the target sequence into a DH10bac (BmNPV) competent cell, and transposing the recombinant plasmid onto a BmNPV genome under the action of a Tn7 transposon to form a recombinant bacmid BEV-U6-sgRNA; and (3) extracting recombinant bacmid to transfect silkworm cells, collecting virus particles of P1 generation, infecting again, and collecting virus particles of P2 generation.
Preferably, the recombinant plasmid containing the expression cassette U6-sgRNA is constructed by the following method: the sequence shown in SEQ ID NO.2 is inserted into pFastBac-Dual-HSP after being cut by BamHI and EcoRIprmAnd (3) obtaining a vector pFBD-U6-sgRNA on an EGFP-poly vector, and then connecting the sgRNA of the target gene into the pFBD-U6-sgRNA vector through an enzyme cutting site BbsI to obtain a recombinant plasmid of U6-sgRNA containing the target sequence.
Preferably, the sgRNA of the target gene is the sgRNA of the viral gene lef11, the sgRNA of the host gene HSPD1 and the sgRNA of the ATAD 3A; the sgRNA of the virus gene lef11 is a double-stranded DNA sequence formed by annealing SEQ ID NO.14 and SEQ ID NO. 15; the sgRNA of the host gene HSPD1 is a double-stranded DNA sequence formed by annealing SEQ ID NO.16 and SEQ ID NO. 17; the sgRNA of ATAD3A is a double-stranded DNA sequence formed by annealing SEQ ID NO.18 and SEQ ID NO. 19.
2. The application of the Cas9 system for efficiently editing silkworms in editing silkworm genomes or silkworm nuclear polyhedrosis virus genes.
Injecting a silkworm transgenic expression vector containing a Cas9 gene into silkworm eggs, and screening transgenic silkworms to obtain Cas9 transgenic silkworms; and then packaging the bombyx mori nuclear polyhedrosis virus into virus particles containing an expression frame U6-sgRNA expressed by sgRNA regulated by a U6 promoter, and injecting the virus particles into the Cas9 transgenic bombyx mori to obtain the high-efficiency edited bombyx mori genome.
The invention has the beneficial effects that: the invention provides a silkworm nuclear polyhedrosis virus packaging Cas9 system, which can cut virus genes, edit host genes and knock out target genes efficiently and stably. The silkworm nuclear polyhedrosis virus packaging Cas9 system does not need a transfection process, has high transfection efficiency and good safety, can stably and efficiently edit target genes, can be suitable for researches on virus genes, functions of related host genes, baculovirus expression systems and the like, and provides a powerful tool for developing researches on cultivation of efficient antiviral materials.
Drawings
In order to make the object, technical scheme and beneficial effect of the invention more clear, the invention provides the following drawings for explanation:
FIG. 1 is a cellular immunofluorescence map after transfection of pIZ-Mchery-Cas 9 vector;
FIG. 2 is a Cas9 cell line screening fluorescence diagram (A) and a Cas9 protein detection Western Blot diagram (B);
FIG. 3 is a Cas9 positive silkworm screen (A) and Cas9 expression PCR detection map (B);
FIG. 4 is a schematic diagram of the structure of a recombinant bacmid;
FIG. 5 is a flowchart of the operation of a baculovirus-packaged Cas9 system;
FIG. 6 is a schematic diagram of the construction of baculovirus-packaged Cas9 system
FIG. 7 shows the result of gene sequencing targeted to viral gene lef 11;
FIG. 8 shows the results of gene sequencing targeting host genes HSPD1 and ATAD 3A.
Detailed Description
The present invention is further described with reference to the following drawings and specific examples so that those skilled in the art can better understand the present invention and can practice the present invention, but the examples are not intended to limit the present invention.
The experimental procedures, for which specific conditions are not indicated in the examples, are generally carried out according to conventional conditions, for example as described in the molecular cloning protocols (third edition, sambrook et al), or according to the conditions recommended by the manufacturers.
Example 1 establishment of Bombyx mori Cas9 Trace cell line
An addgene vector database (http:// www.addgene.org /), is logged in, downloads the existing insect Cas9 gene and U6 sequence, designs silkworm specific Cas9 gene and U6-sgRNA sequence according to silkworm codon characteristics, the sequences are respectively shown as SEQ ID No.1 and SEQ ID No.2, and then sends the sequences to Gensript company for synthesis.
The Cas9 gene sequence is cut by Hind III and Xba I enzyme cutting sites pIZ/V5-His, the connection product is transformed into escherichia coli DH5 alpha competent cells, then LB plates containing bleomycin are used for screening positive clones, single colonies of the positive clones are picked up and used after enzyme cutting verification is correct, and the genes are named as pIZ-OpIE2-Cas 9-pA. The Mcherry gene sequence (SEQ ID No.3) is connected to pIZ/V5-His vector through Hind III and Kpn I enzyme cutting sites to obtain pIZ-OpIE2-Mcherry-pA vector, and the vector is used after enzyme cutting verification and sequencing correctness. The OpIE 2-Mchery-pA fragment is amplified through a primer with an Age I enzyme cutting site, the Age I is connected to a pIZ-OpIE2-Cas9-pA vector subjected to the same single enzyme cutting after the single enzyme cutting to obtain a recombinant vector pIZ-Mchery-Cas 9, and then the transformation, the positive cloning and the sequencing verification are carried out through the same method.
The constructed pIZ-Mchery-Cas 9 carrier is transfected into a silkworm cell line BmN-SWU1 by using a liposome transfection reagent, and after transfection is carried out for 48 hours, the expression conditions of the Cas9 and Mchery protein are detected by an immunofluorescence experiment. Washing the cells with 1 × PBS for 5min each time for 3 times, adding 4% paraformaldehyde solution, fixing at room temperature for 15min, and washing the cells with 1 × PBST for 5min each time for 3 times; treating the cells with 0.1% Triton-100 for 10min, and washing the cells with 1 XPBST for 5min each time for 3 times; then blocking the mixture for 1h at 37 ℃ by using immunofluorescence blocking solution (PBS containing 10% of sheep serum and 3% of bovine serum); the murine a-Cas9 antibody was incubated for 1h at 37 deg.C, and the cells were washed with 1 XPBST gently 6 times for 5min each; goat anti-mouse secondary antibody with Alexa488 label and nuclear dye DAPI were added, incubated at 37 ℃ in the dark for 1h, and the cells were washed 6 times with 1 XPBST for 5min each. Finally, the coverslip was removed and photographed under an Olympus confocal scanning laser microscope, and the results are shown in FIG. 1. The result shows that the Cas9 protein and Mchery have co-localization expression, and the constructed system can be used for subsequent experiments. Subsequently, screening was performed using TC-100 medium containing Zeocin at a final concentration of 400. mu.g/mL, and the luminescence of the Mcherry fluorescent protein was observed under an Olympus inverted fluorescence microscope at 2 days, 15 days, and 30 days after transfection, and photographed, and the results are shown in FIG. 2, A. The results show that the red fluorescence is gradually increased along with the selection of antibiotics, and the positive cells account for more than 80% 30 days after transfection. Expression of Cas9 protein was further detected by Western Blot after 0, 2, 15, and 30 days of transfection, and the results are shown in fig. 2, B. The results show that the expression level of Cas9 gradually increased, consistent with the fluorescence results. The results show that the screening and establishment of the silkworm Cas9 cell line are successful.
Example 2 creation of Cas9 transgenic silkworms
Firstly, a pSL1180-IE1-Cas9-SV40 vector is constructed, pSL1180-IE1-Cas9-SV40 and pBac-3Px3-EGFP vector (SEQ ID No.4) are cut by AscI enzyme, a cut IE1-Cas9-SV40 fragment is connected to the pBac-3Px3-EGFP vector, and a recombinant transgenic vector pBac-3Px3-EGFP-IE1-Cas9-SV40, IE1 promoter, 3Px3 promoter and EGFP gene sequences are respectively shown as SEQ ID No.5, SEQ ID No.6 and SEQ ID No. 7.
Through microinjection, the pBac-3Px3-EGFP-IE1-Cas9-SV40 vector is injected into silkworm eggs (G0 generation) within 4h of birth, and is randomly transposed into silkworm genome under the action of piggyBac transposon, so that the transgenic silkworms are constructed. Since 3Px3 is a promoter for eye-specific expression, positive transgenic silkworms were screened by observing the fluorescence of eye EGFP in G1 generation silkworms, and the results are shown in FIG. 3, A. The results showed that EGFP expression was observed in the positive silkworms of G1 generation in all of the silkworms of the transgenic silkworm Egg (Egg), the Ant silkworm (Ant), and the silkworm Moth (Moth). And grinding the positive silkworm moths by liquid nitrogen to extract a genome. Detection of primer Cas9-jiance-F using Cas 9: cctattctgt gctggtggtg g (SEQ ID No.8)
Cas9-jiance-R：caggtagtgc ttgtgctgtt cc(SEQ ID No.9)
Whether the Cas9 gene is inserted into the silkworm genome is detected, and the result is shown in figure 3 and B. The result shows that a specific band can be amplified in the Cas9 positive strain silkworm (305, DZ). Further, the insertion site of the IE1-Cas9-SV40 expression cassette on the Bombyx mori genome was examined. Carrying out enzyme digestion on Cas9 positive silkworm genome by HaeIII, carrying out self-cyclization on the enzyme-digested fragments by T4 ligase, and carrying out forward and reverse PCR primers of a left arm pBacL and a right arm pBacR of a transposon pBac vector:
pBacL-F：5′-atcagtgaca cttaccgcat tgaca-3′(SEQ ID NO.10)；
pBacL-R：5′-tgacgagctt gttggtgagg attct-3′(SEQ ID NO.11)；
pBacR-F：5′-tacgcatgat tatctttaac gta-3′(SEQ ID NO.12)；
pBacR-R：5′-gtactgtcat ctgatgtacc agg-3′(SEQ ID NO.13)；
amplification was performed followed by T-cloning and sequencing validation. The insertion of an IE1-Cas9-SV40 expression frame into a silkworm 11 chromosome nscaf3031 is shown by online alignment of a silkworm database Silkbase (http:// Silkbase.ab.a.u-tokyo.ac.jp/cgi-bin/index. cgi), and the result shows that the transgenic Cas9 silkworm is successfully constructed.
Example 3 construction of baculovirus packaging vector
U6-sgRNA (SEQ ID NO.2) was digested with BamHI and EcoRI and inserted into pFastBac-Dual-HSPprmAn EGFP-poly vector (pFBD vector for short) containing polyhedrin obtained by inserting polyhedrin promoter and polyhedrin gene together on pFastBac-Dual vector through SphI and KpnI, and a recombinant vector pFastBac-Dual-HSP is constructed as shown in SEQ ID No.20prmEGFP-poly-U6-sgRNA, abbreviated as pFBD-U6-sgRNA vector.
Selecting a virus gene lef11 and host genes HSPD1 and ATAD3A as targets, and respectively designing U6-sgRNA sequences. Predicting sgRNA sequences of lef11, HSPD1 and ATAD3A according to a CRI SPRd irect online analysis tool (http:// criprpr. dbcls. jp /), analyzing the off-target efficiency of target sequences in silkworms according to software, and finally selecting the sgRNA sequences with high editing efficiency, wherein the specific sequences are as follows:
sglef11-F：5′-gcacttaggc gggtgtaatt-3′(SEQ ID NO.14)；
sglef11-R：5′-aattacaccc gcctaagtgc-3′(SEQ ID NO.15)；
sgHSPD1-F：5′-ggcacgagca atcgcgaagg-3′(SEQ ID NO.16)；
sgHSPD1-R：5′-ccttcgcgat tgctcgtgcc-3′(SEQ ID NO.17)；
sgATAD3A-F：5′-ggagcaaatg gccaagatta-3′(SEQ ID NO.18)；
sgATAD3A-R：5′-taatcttggc catttgctcc-3′(SEQ ID NO.19)；
the 5 'end and 3' end of sgRNA of three genes are respectively inserted into BbsI enzyme cutting sites (F: AAGT, R: AAAC), the designed sgRNA is sent to Huada gene synthesis primers, double chains are formed by annealing of upstream and downstream primers after synthesis, and the double chains are connected into the pFBD-U6-sgRNA vector expression vector which is subjected to Bbs I enzyme cutting. The ligation product is used for transforming Escherichia coli DH5 alpha competent cells, LB plate containing Zeocin is used for screening positive clones, single colonies of the positive clones are selected, and plasmids of the positive clones after the sequencing is correct are named as pFBD-U6-sglef11, pFBD-U6-sgHSPD1 and pFBD-U6-sgATAD 3A.
Plasmids pFBD-U6-sglef11, pFBD-U6-sgHSPD1 and pFBD-U6-sgATAD3A were extracted and transformed into DH10Bac competent cells (DH10Bac (BmNPV)) containing BmNPV bacmids, respectively. Based on the principle of Bac-to-Bac baculovirus expression system, the expression frame pFBD-U6-sgRNA is transposed to BmNPV genome under the action of Tn7 transposon to form recombinant bacmid BEV-U6-sgRNA, the structure schematic diagram of the recombinant bacmid is shown in FIG. 4, and the recombinant bacmid in the embodiment is specifically BEV-U6-sglef11, BEV-U6-sgHSPD1 and BEV-U6-sgATAD 3A.
Culturing and screening strains on an LB solid plate containing 50 mu g/mL kanamycin, 7 mu g/mL tetracycline, 7 mu g/mL gentamicin, 40 mu g/mL IPTG and 40 mu g/mL Bluo-gal, selecting a white single colony for PCR amplification verification, selecting a positive colony for amplification culture, extracting recombinant bacmids, transfecting the recombinant bacmids to a housekeeperSilkworm cells, and determining whether the recombinant virus is successfully constructed. Collecting P1 generation virus particles, re-infecting, collecting P2 generation virus particles, passing TCID50The virus titer was determined. The virus particles are used for infecting Cas9 cell lines built in example 1 and Cas9 silkworm individuals built in example 2 respectively, whether a baculovirus packaging Cas9 system is successfully constructed is verified, and the operation flow diagram is shown in FIG. 5. A schematic diagram of the construction of a baculovirus packaging vector based on the Bac-to-Bac baculovirus expression system is shown in FIG. 6.
EXAMPLE 4 application of baculovirus-packaged Cas9 System
In order to verify the function of a baculovirus packaging Cas9 system, BEV-U6-sglef11 is injected into a Cas9 silkworm individual, the quantity of green fluorescence in silkworm cells is observed through a fluorescence microscope after the silkworm cell is infected for 72 hours, the ratio of the quantity of the green fluorescence represents the virus infection efficiency, after the silkworm cell is infected for 72 hours, the virus finishes 2-3 rounds of replication, and basically 70-90% of cells can be infected with the virus to express a target gene. Then, silkworm genomes are extracted, detection primers are designed at the upstream and downstream of the target sites by about 300bp, target fragments are amplified through PCR, the editing efficiency of target genes is detected through T cloning, and the result is shown in figure 7. The results show that the viral gene lef11 was efficiently edited. Obvious fragment deletion occurs near the target site, and deletion phenomena occur in all sequencing results, which indicates that the editing efficiency is very high. Furthermore, we injected BEV-U6-sgHSPD1 or BEV-U6-sgATAD3A into Cas9 silkworm and observed the editing effect of the system on host genes, and the results are shown in FIG. 8. The results show that the host genes HSPD1 and ATAD3A can also be efficiently edited. Most sequencing results show that obvious deletion appears near the target site of the BmATD 3A gene, and the deletion frequency reaches 36.6%; similarly, obvious deletion phenomenon is also generated near the target site of the BmHSPD1 gene, and 30 percent of the deletion phenomenon is generated in the sequencing result. In conclusion, the baculovirus packaging Cas9 system can be effectively applied to gene editing of silkworms.
The above-mentioned embodiments are merely preferred embodiments for fully illustrating the present invention, and the scope of the present invention is not limited thereto. The equivalent substitution or change made by the technical personnel in the technical field on the basis of the invention is all within the protection scope of the invention. The protection scope of the invention is subject to the claims.
Sequence listing
<110> university of southwest
<120> Cas9 system for efficiently editing silkworm genome and application thereof
<160> 20
<170> SIPOSequenceListing 1.0
<210> 1
<211> 4140
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 1
atggacaaga agtactccat tgggctcgat atcggcacaa acagcgtcgg ctgggccgtc 60
attacggacg agtacaaggt gccgagcaaa aaattcaaag ttctgggcaa taccgatcgc 120
cacagcataa agaagaacct cattggcgcc ctcctgttcg actccgggga gacggccgaa 180
gccacgcggc tcaaaagaac agcacggcgc agatataccc gcagaaagaa tcggatctgc 240
tacctgcagg agatctttag taatgagatg gctaaggtgg atgactcttt cttccatagg 300
ctggaggagt cctttttggt ggaggaggat aaaaagcacg agcgccaccc aatctttggc 360
aatatcgtgg acgaggtggc gtaccatgaa aagtacccaa ccatatatca tctgaggaag 420
aagcttgtag acagtactga taaggctgac ttgcggttga tctatctcgc gctggcgcat 480
atgatcaaat ttcggggaca cttcctcatc gagggggacc tgaacccaga caacagcgat 540
gtcgacaaac tctttatcca actggttcag acttacaatc agcttttcga agagaacccg 600
atcaacgcat ccggagttga cgccaaagca atcctgagcg ctaggctgtc caaatcccgg 660
cggctcgaaa acctcatcgc acagctccct ggggagaaga agaacggcct gtttggtaat 720
cttatcgccc tgtcactcgg gctgaccccc aactttaaat ctaacttcga cctggccgaa 780
gatgccaagc ttcaactgag caaagacacc tacgatgatg atctcgacaa tctgctggcc 840
cagatcggcg accagtacgc agaccttttt ttggcggcaa agaacctgtc agacgccatt 900
ctgctgagtg atattctgcg agtgaacacg gagatcacca aagctccgct gagcgctagt 960
atgatcaagc gctatgatga gcaccaccaa gacttgactt tgctgaaggc ccttgtcaga 1020
cagcaactgc ctgagaagta caaggaaatt ttcttcgatc agtctaaaaa tggctacgcc 1080
ggatacattg acggcggagc aagccaggag gaattttaca aatttattaa gcccatcttg 1140
gaaaaaatgg acggcaccga ggagctgctg gtaaagctta acagagaaga tctgttgcgc 1200
aaacagcgca ctttcgacaa tggaagcatc ccccaccaga ttcacctggg cgaactgcac 1260
gctatcctca ggcggcaaga ggatttctac ccctttttga aagataacag ggaaaagatt 1320
gagaaaatcc tcacatttcg gataccctac tatgtaggcc ccctcgcccg gggaaattcc 1380
agattcgcgt ggatgactcg caaatcagaa gagaccatca ctccctggaa cttcgaggaa 1440
gtcgtggata agggggcctc tgcccagtcc ttcatcgaaa ggatgactaa ctttgataaa 1500
aatctgccta acgaaaaggt gcttcctaaa cactctctgc tgtacgagta cttcacagtt 1560
tataacgagc tcaccaaggt caaatacgtc acagaaggga tgagaaagcc agcattcctg 1620
tctggagagc agaagaaagc tatcgtggac ctcctcttca agacgaaccg gaaagttacc 1680
gtgaaacagc tcaaagaaga ctatttcaaa aagattgaat gtttcgactc tgttgaaatc 1740
agcggagtgg aggatcgctt caacgcatcc ctgggaacgt atcacgatct cctgaaaatc 1800
attaaagaca aggacttcct ggacaatgag gagaacgagg acattcttga ggacattgtc 1860
ctcaccctta cgttgtttga agatagggag atgattgaag aacgcttgaa aacttacgct 1920
catctcttcg acgacaaagt catgaaacag ctcaagaggc gccgatatac aggatggggg 1980
cggctgtcaa gaaaactgat caatgggatc cgagacaagc agagtggaaa gacaatcctg 2040
gattttctta agtccgatgg atttgccaac cggaacttca tgcagttgat ccatgatgac 2100
tctctcacct ttaaggagga catccagaaa gcacaagttt ctggccaggg ggacagtctt 2160
cacgagcaca tcgctaatct tgcaggtagc ccagctatca aaaagggaat actgcagacc 2220
gttaaggtcg tggatgaact cgtcaaagta atgggaaggc ataagcccga gaatatcgtt 2280
atcgagatgg cccgagagaa ccaaactacc cagaagggac agaagaacag tagggaaagg 2340
atgaagagga ttgaagaggg tataaaagaa ctggggtccc aaatccttaa ggaacaccca 2400
gttgaaaaca cccagcttca gaatgagaag ctctacctgt actacctgca gaacggcagg 2460
gacatgtacg tggatcagga actggacatc aatcggctct ccgactacga cgtggatcat 2520
atcgtgcccc agtcttttct caaagatgat tctattgata ataaagtgtt gacaagatcc 2580
gataaaaata gagggaagag tgataacgtc ccctcagaag aagttgtcaa gaaaatgaaa 2640
aattattggc ggcagctgct gaacgccaaa ctgatcacac aacggaagtt cgataatctg 2700
actaaggctg aacgaggtgg cctgtctgag ttggataaag ccggcttcat caaaaggcag 2760
cttgttgaga cacgccagat caccaagcac gtggcccaaa ttctcgattc acgcatgaac 2820
accaagtacg atgaaaatga caaactgatt cgagaggtga aagttattac tctgaagtct 2880
aagctggtct cagatttcag aaaggacttt cagttttata aggtgagaga gatcaacaat 2940
taccaccatg cgcatgatgc ctacctgaat gcagtggtag gcactgcact tatcaaaaaa 3000
tatcccaagc ttgaatctga atttgtttac ggagactata aagtgtacga tgttaggaaa 3060
atgatcgcaa agtctgagca ggaaataggc aaggccaccg ctaagtactt cttttacagc 3120
aatattatga attttttcaa gaccgagatt acactggcca atggagagat tcggaagcga 3180
ccacttatcg aaacaaacgg agaaacagga gaaatcgtgt gggacaaggg tagggatttc 3240
gcgacagtcc ggaaggtcct gtccatgccg caggtgaaca tcgttaaaaa gaccgaagta 3300
cagaccggag gcttctccaa ggaaagtatc ctcccgaaaa ggaacagcga caagctgatc 3360
gcacgcaaaa aagattggga ccccaagaaa tacggcggat tcgattctcc tacagtcgct 3420
tacagtgtac tggttgtggc caaagtggag aaagggaagt ctaaaaaact caaaagcgtc 3480
aaggaactgc tgggcatcac aatcatggag cgatcaagct tcgaaaaaaa ccccatcgac 3540
tttctcgagg cgaaaggata taaagaggtc aaaaaagacc tcatcattaa gcttcccaag 3600
tactctctct ttgagcttga aaacggccgg aaacgaatgc tcgctagtgc gggcgagctg 3660
cagaaaggta acgagctggc actgccctct aaatacgtta atttcttgta tctggccagc 3720
cactatgaaa agctcaaagg gtctcccgaa gataatgagc agaagcagct gttcgtggaa 3780
caacacaaac actaccttga tgagatcatc gagcaaataa gcgaattctc caaaagagtg 3840
atcctcgccg acgctaacct cgataaggtg ctttctgctt acaataagca cagggataag 3900
cccatcaggg agcaggcaga aaacattatc cacttgttta ctctgaccaa cttgggcgcg 3960
cctgcagcct tcaagtactt cgacaccacc atagacagaa agcggtacac ctctacaaag 4020
gaggtcctgg acgccacact gattcatcag tcaattacgg ggctctatga aacaagaatc 4080
gacctctctc agctcggtgg agacagcagg gctgacccca agaagaagag gaaggtgtga 4140
<210> 2
<211> 615
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
tgattgacct aaaagaatca atacagttta ataaatttat aagtattagg ttatgtagta 60
cacattgttg taaatcactg aattgtttta gatgatttta acaattagta cttattaata 120
ttaaataagt acataccttg agaatttaaa aatcgtcaac tataagccat acgaatttaa 180
gcttggtact tggcttatag ataaggacag aataagaatt gttaacgtgt aagacaaggt 240
cagatagtca tagtgatttt gtcaaagtaa taacagatgg cgctgtacaa accataactg 300
ttttcatttg tttttatgga ttttattaca aattctaaag gttttattgt tattatttaa 360
tttcgtttta attatattat atatctttaa tagaatatgt taagagtttt tgctcttttt 420
gaataatctt tgtaaagtcg agtgttgttg taaatcacgc tttcaatagt ttagtttttt 480
taggtatata tacaaaatat cgtgctctac aagtgggtct tcgagaagac ctgttttaga 540
gctagaaata gcaagttaaa ataaggctag tccgttatca acttgaaaaa gtggcaccga 600
gtcggtgctt ttttt 615
<210> 3
<211> 711
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 3
atggttagca aaggcgaaga ggacaacatg gctatcataa aggaattcat gagattcaag 60
gtccacatgg agggctccgt aaatggtcat gaattcgaga tagaaggcga gggtgaagga 120
cgcccgtacg aaggtacaca aacggcaaaa ctgaaggtca ccaaaggtgg acctcttcca 180
ttcgcgtggg acattctgtc gcctcagttt atgtacggca gtaaggctta cgtaaagcac 240
ccggccgaca tacccgatta cctgaaattg tcgttcccag aaggttttaa gtgggagaga 300
gtcatgaact tcgaagatgg cggtgtggtt acagtaactc aagactcatc tttacaggat 360
ggagagttca tctacaaagt gaaactgaga ggtacaaatt ttccttctga cggaccagtt 420
atgcagaaaa agactatggg ctgggaagct agctccgagc gtatgtatcc tgaagatgga 480
gccctgaaag gcgagattaa gcaaaggctc aaactgaagg acggaggcca ttacgatgca 540
gaagtgaaaa caacttataa ggcgaaaaag ccggtgcagt tgcccggtgc ttacaacgtt 600
aatataaaac tcgacatcac ctcacacaac gaagattaca cgattgttga gcaatatgaa 660
agagccgagg gacgccattc tacaggtgga atggatgagt tgtataagta a 711
<210> 4
<211> 6591
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 4
gtgccaagct ttgtttaaaa tataacaaaa ttgtgatccc acaaaatgaa gtggggcaaa 60
atcaaataat taatagtgtc cgtaaacttg ttggtcttca actttttgag gaacacgttg 120
gacggcaaat ccgtgactat aacacaagtt gatttaataa ttttagccaa cacgtcgggc 180
tgcgtgtttt ttgccgacgc gtctgtgtac acgttgatta actggtcgat taaactgttg 240
aaataattta atttttggtt cttctttaaa tctgtgatga aattttttaa aataacttta 300
aattcttcat tggtaaaaaa tgccacgttt tgcaacttgt gagggtctaa tatgaggtca 360
aactcagtag gagttttatc caaaaaagaa aacatgatta cgtctgtaca cgaacgcgta 420
ttaacgcaga gtgcaaagta taagagggtt aaaaaatata ttttacgcac catatacgca 480
tcgggttgat atcgttaata tggatcaatt tgaacagttg attaacgtgt ctctgctcaa 540
gtctttgatc aaaacgcaaa tcgacgaaaa tgtgtcggac aatatcaagt cgatgagcga 600
aaaactaaaa aggctagaat acgacaatct cacagacagc gttgagatat acggtattca 660
cgacagcagg ctgaataata aaaaaattag aaactattat ttaaccctag aaagataatc 720
atattgtgac gtacgttaaa gataatcatg cgtaaaattg acgcatgtgt tttatcggtc 780
tgtatatcga ggtttattta ttaatttgaa tagatattaa gttttattat atttacactt 840
acatactaat aataaattca acaaacaatt tatttatgtt tatttattta ttaaaaaaaa 900
acaaaaactc aaaatttctt ctataaagta acaaaacttt taaacattct ctcttttaca 960
aaaataaact tattttgtac tttaaaaaca gtcatgttgt attataaaat aagtaattag 1020
cttaacttat acataataga aacaaattat acttattagt cagtcagaaa caactttggc 1080
acatatcaat attatgctct cgacaaataa cttttttgca ttttttgcac gatgcatttg 1140
cctttcgcct tattttagag gggcagtaag tacagtaagt acgttttttc attactggct 1200
cttcagtact gtcatctgat gtaccaggca cttcatttgg caaaatatta gagatattat 1260
cgcgcaaata tctcttcaaa gtaggagctt ctaaacgctt acgcataaac gatgacgtca 1320
ggctcatgta aaggtttctc ataaattttt tgcgactttg gaccttttct cccttgctac 1380
tgacattatg gctgtatata ataaaagaat ttatgcaggc aatgtttatc attccgtaca 1440
ataatgccat aggccaccta ttcgtcttcc tactgcaggt catcacagaa cacatttggt 1500
ctagcgtgtc cactccgcct ttagtttgat tataatacat aaccatttgc ggtttaccgg 1560
tactttcgtt gatagaagca tcctcatcac aagatgataa taagtatacc atcttagctg 1620
gcttcggttt atatgagacg agagtaaggg gtccgtcaaa acaaaacatc gatgttccca 1680
ctggcctgga gcgactgttt ttcagtactt ccggtatctc gcgtttgttt gatcgcacgg 1740
ttcccacaat ggttaattcg agctcgcccg gggatctaat tcaattagag actaattcaa 1800
ttagagctaa ttcaattagg atccaagctt atcgatttcg aaccctcgac cgccggagta 1860
taaatagagg cgcttcgtct acggagcgac aattcaattc aaacaagcaa agtgaacacg 1920
tcgctaagcg aaagctaagc aaataaacaa gcgcagctga acaagctaaa caatcggggt 1980
accgctagag tcgacggtac cgcgggcccg ggatccaccg gtcgccacca tggtgagcaa 2040
gggcgaggag ctgttcaccg gggtggtgcc catcctggtc gagctggacg gcgacgtaaa 2100
cggccacaag ttcagcgtgt ccggcgaggg cgagggcgat gccacctacg gcaagctgac 2160
cctgaagttc atctgcacca ccggcaagct gcccgtgccc tggcccaccc tcgtgaccac 2220
cctgacctac ggcgtgcagt gcttcagccg ctaccccgac cacatgaagc agcacgactt 2280
cttcaagtcc gccatgcccg aaggctacgt ccaggagcgc accatcttct tcaaggacga 2340
cggcaactac aagacccgcg ccgaggtgaa gttcgagggc gacaccctgg tgaaccgcat 2400
cgagctgaag ggcatcgact tcaaggagga cggcaacatc ctggggcaca agctggagta 2460
caactacaac agccacaacg tctatatcat ggccgacaag cagaagaacg gcatcaaggt 2520
gaacttcaag atccgccaca acatcgagga cggcagcgtg cagctcgccg accactacca 2580
gcagaacacc cccatcggcg acggccccgt gctgctgccc gacaaccact acctgagcac 2640
ccagtccgcc ctgagcaaag accccaacga gaagcgcgat cacatggtcc tgctggagtt 2700
cgtgaccgcc gccgggatca ctctcggcat ggacgagctg tacaagtaaa gcggccgcga 2760
ctctagatca taatcagcca taccacattt gtagaggttt tacttgcttt aaaaaacctc 2820
ccacacctcc ccctgaacct gaaacataaa atgaatgcaa ttgttgttgt taacttgttt 2880
attgcagctt ataatggtta caaataaagc aatagcatca caaatttcac aaataaagca 2940
tttttttcac tgcattctag ttgtggtttg tccaaactca tcaatgtatc ttaaagctta 3000
tcgatacgcg tacggcgcgc ctaggccggc cgaattcgaa tggccatggg acgtcgacct 3060
gaggtaatta taacccgggc cctatatatg gatccaattg caatgatcat catgacagat 3120
ctgacaatgt tcagtgcaga gactcggcta cgcctcgtgg actttgaagt tgaccaacaa 3180
tgtttattct tacctctaat agtcctctgt ggcaaggtca agattctgtt agaagccaat 3240
gaagaacctg gttgttcaat aacattttgt tcgtctaata tttcactacc gcttgacgtt 3300
ggctgcactt catgtacctc atctataaac gcttcttctg tatcgctctg gacgtcatct 3360
tcacttacgt gatctgatat ttcactgtca gaatcctcac caacaagctc gtcatcgctt 3420
tgcagaagag cagagaggat atgctcatcg tctaaagaac tacccatttt attatatatt 3480
agtcacgata tctataacaa gaaaatatat atataataag ttatcacgta agtagaacat 3540
gaaataacaa tataattatc gtatgagtta aatcttaaaa gtcacgtaaa agataatcat 3600
gcgtcatttt gactcacgcg gtcgttatag ttcaaaatca gtgacactta ccgcattgac 3660
aagcacgcct cacgggagct ccaagcggcg actgagatgt cctaaatgca cagcgacgga 3720
ttcgcgctat ttagaaagag agagcaatat ttcaagaatg catgcgtcaa ttttacgcag 3780
actatctttc tagggttaaa aaagatttgc gctttactcg acctaaactt taaacacgtc 3840
atagaatctt cgtttgacaa aaaccacatt gtggccaagc tgtgtgacgc gacgcgcgct 3900
aaagaatggc aaaccaagtc gcgcgagcgt cgactctaga ggatccccgg gtaccgagct 3960
cgaattcgta atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc 4020
cacacaacat acgagccgga agcataaagt gtaaagcctg gggtgcctaa tgagtgagct 4080
aactcacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc 4140
agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt 4200
ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag 4260
ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca 4320
tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt 4380
tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc 4440
gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct 4500
ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg 4560
tggcgctttc tcaatgctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca 4620
agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact 4680
atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta 4740
acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta 4800
actacggcta cactagaagg acagtatttg gtatctgcgc tctgctgaag ccagttacct 4860
tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt 4920
tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga 4980
tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca 5040
tgagattatc aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga agttttaaat 5100
caatctaaag tatatatgag taaacttggt ctgacagtta ccaatgctta atcagtgagg 5160
cacctatctc agcgatctgt ctatttcgtt catccatagt tgcctgactc cccgtcgtgt 5220
agataactac gatacgggag ggcttaccat ctggccccag tgctgcaatg ataccgcgag 5280
acccacgctc accggctcca gatttatcag caataaacca gccagccgga agggccgagc 5340
gcagaagtgg tcctgcaact ttatccgcct ccatccagtc tattaattgt tgccgggaag 5400
ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt gctacaggca 5460
tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag ctccggttcc caacgatcaa 5520
ggcgagttac atgatccccc atgttgtgca aaaaagcggt tagctccttc ggtcctccga 5580
tcgttgtcag aagtaagttg gccgcagtgt tatcactcat ggttatggca gcactgcata 5640
attctcttac tgtcatgcca tccgtaagat gcttttctgt gactggtgag tactcaacca 5700
agtcattctg agaatagtgt atgcggcgac cgagttgctc ttgcccggcg tcaatacggg 5760
ataataccgc gccacatagc agaactttaa aagtgctcat cattggaaaa cgttcttcgg 5820
ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg 5880
cacccaactg atcttcagca tcttttactt tcaccagcgt ttctgggtga gcaaaaacag 5940
gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg gaaatgttga atactcatac 6000
tcttcctttt tcaatattat tgaagcattt atcagggtta ttgtctcatg agcggataca 6060
tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacattt ccccgaaaag 6120
tgccacctga cgtctaagaa accattatta tcatgacatt aacctataaa aataggcgta 6180
tcacgaggcc ctttcgtctc gcgcgtttcg gtgatgacgg tgaaaacctc tgacacatgc 6240
agctcccgga gacggtcaca gcttgtctgt aagcggatgc cgggagcaga caagcccgtc 6300
agggcgcgtc agcgggtgtt ggcgggtgtc ggggctggct taactatgcg gcatcagagc 6360
agattgtact gagagtgcac catatgcggt gtgaaatacc gcacagatgc gtaaggagaa 6420
aataccgcat caggcgccat tcgccattca ggctgcgcaa ctgttgggaa gggcgatcgg 6480
tgcgggcctc ttcgctatta cgccagctgg cgaaaggggg atgtgctgca aggcgattaa 6540
gttgggtaac gccagggttt tcccagtcac gacgttgtaa aacgacggcc a 6591
<210> 5
<211> 638
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 5
ttgcagttcg ggacataaat gtttaaatat atcaatgtct ttgtgatgcg cgcgacattt 60
ttgtaagtta ttaataaaat gcaccgacac gttgcccgac attatcatta aatccttggc 120
gtagaatttg tcgggtccgt tgtccgtgtg cgctagcatg cccgtaacgg accttgagct 180
tttggcttca aaggttttgc gcacagacaa aatgtgccac acttgcagct ctgcttgtgt 240
acgcgttacc acaaatccca acggcgcagt gtacttgtta tatgtaaata aatctcgata 300
aaggcgcggc gcgcgaatgc agctgatcac gtacgctcct cgtgttccgt tcaaggacgg 360
tgttatcgac ctcagattaa tatttatcgg ccgactgttt tcgtatccgc tcaccaaacg 420
ggtttttgca ttaacattgt atgtcggcgg atgttctgta tctaatttga ataaataaat 480
gataaccgca ttggttttag agggcataat aaaaaaaata ttattatcgt gttcgccatt 540
ggggcagtat aaattgacgt tcatgttgaa tattgtttca gttgcaagtt gacattggcg 600
gcgacacgat cgtgaacaac caaacgacta gggatcta 638
<210> 6
<211> 266
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 6
taattcgagc tcgcccgggg atctaattca attagagact aattcaatta gagctaattc 60
aattaggatc caagcttatc gatttcgaac cctcgaccgc cggagtataa atagaggcgc 120
ttcgtctacg gagcgacaat tcaattcaaa caagcaaagt gaacacgtcg ctaagcgaaa 180
gctaagcaaa taaacaagcg cagctgaaca agctaaacaa tcggggtacc gctagagtcg 240
acggtaccgc gggcccggga tccacc 266
<210> 7
<211> 720
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 7
atggtgagca agggcgagga gctgttcacc ggggtggtgc ccatcctggt cgagctggac 60
ggcgacgtaa acggccacaa gttcagcgtg tccggcgagg gcgagggcga tgccacctac 120
ggcaagctga ccctgaagtt catctgcacc accggcaagc tgcccgtgcc ctggcccacc 180
ctcgtgacca ccctgaccta cggcgtgcag tgcttcagcc gctaccccga ccacatgaag 240
cagcacgact tcttcaagtc cgccatgccc gaaggctacg tccaggagcg caccatcttc 300
ttcaaggacg acggcaacta caagacccgc gccgaggtga agttcgaggg cgacaccctg 360
gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg acggcaacat cctggggcac 420
aagctggagt acaactacaa cagccacaac gtctatatca tggccgacaa gcagaagaac 480
ggcatcaagg tgaacttcaa gatccgccac aacatcgagg acggcagcgt gcagctcgcc 540
gaccactacc agcagaacac ccccatcggc gacggccccg tgctgctgcc cgacaaccac 600
tacctgagca cccagtccgc cctgagcaaa gaccccaacg agaagcgcga tcacatggtc 660
ctgctggagt tcgtgaccgc cgccgggatc actctcggca tggacgagct gtacaagtaa 720
<210> 8
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 8
cctattctgt gctggtggtg g 21
<210> 9
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 9
caggtagtgc ttgtgctgtt cc 22
<210> 10
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 10
atcagtgaca cttaccgcat tgaca 25
<210> 11
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 11
tgacgagctt gttggtgagg attct 25
<210> 12
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 12
tacgcatgat tatctttaac gta 23
<210> 13
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 13
gtactgtcat ctgatgtacc agg 23
<210> 14
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 14
gcacttaggc gggtgtaatt 20
<210> 15
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 15
aattacaccc gcctaagtgc 20
<210> 16
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 16
ggcacgagca atcgcgaagg 20
<210> 17
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 17
ccttcgcgat tgctcgtgcc 20
<210> 18
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 18
ggagcaaatg gccaagatta 20
<210> 19
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 19
taatcttggc catttgctcc 20
<210> 20
<211> 7800
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 20
ttctctgtca cagaatgaaa atttttctgt catctcttcg ttattaatgt ttgtaattga 60
ctgaatatca acgcttattt gcagcctgaa tggcgaatgg gacgcgccct gtagcggcgc 120
attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg ccagcgccct 180
agcgcccgct cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg 240
tcaagctcta aatcgggggc tccctttagg gttccgattt agtgctttac ggcacctcga 300
ccccaaaaaa cttgattagg gtgatggttc acgtagtggg ccatcgccct gatagacggt 360
ttttcgccct ttgacgttgg agtccacgtt ctttaatagt ggactcttgt tccaaactgg 420
aacaacactc aaccctatct cggtctattc ttttgattta taagggattt tgccgatttc 480
ggcctattgg ttaaaaaatg agctgattta acaaaaattt aacgcgaatt ttaacaaaat 540
attaacgttt acaatttcag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg 600
tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac cctgataaat 660
gcttcaataa tattgaaaaa ggaagagtat gagtattcaa catttccgtg tcgcccttat 720
tccctttttt gcggcatttt gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt 780
aaaagatgct gaagatcagt tgggtgcacg agtgggttac atcgaactgg atctcaacag 840
cggtaagatc cttgagagtt ttcgccccga agaacgtttt ccaatgatga gcacttttaa 900
agttctgcta tgtggcgcgg tattatcccg tattgacgcc gggcaagagc aactcggtcg 960
ccgcatacac tattctcaga atgacttggt tgagtactca ccagtcacag aaaagcatct 1020
tacggatggc atgacagtaa gagaattatg cagtgctgcc ataaccatga gtgataacac 1080
tgcggccaac ttacttctga caacgatcgg aggaccgaag gagctaaccg cttttttgca 1140
caacatgggg gatcatgtaa ctcgccttga tcgttgggaa ccggagctga atgaagccat 1200
accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact 1260
attaactggc gaactactta ctctagcttc ccggcaacaa ttaatagact ggatggaggc 1320
ggataaagtt gcaggaccac ttctgcgctc ggcccttccg gctggctggt ttattgctga 1380
taaatctgga gccggtgagc gtgggtctcg cggtatcatt gcagcactgg ggccagatgg 1440
taagccctcc cgtatcgtag ttatctacac gacggggagt caggcaacta tggatgaacg 1500
aaatagacag atcgctgaga taggtgcctc actgattaag cattggtaac tgtcagacca 1560
agtttactca tatatacttt agattgattt aaaacttcat ttttaattta aaaggatcta 1620
ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca 1680
ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg 1740
cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga 1800
tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa 1860
tactgtcctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc 1920
tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg 1980
tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac 2040
ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct 2100
acagcgtgag cattgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc 2160
ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg 2220
gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg 2280
ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct 2340
ggccttttgc tggccttttg ctcacatgtt ctttcctgcg ttatcccctg attctgtgga 2400
taaccgtatt accgcctttg agtgagctga taccgctcgc cgcagccgaa cgaccgagcg 2460
cagcgagtca gtgagcgagg aagcggaaga gcgcctgatg cggtattttc tccttacgca 2520
tctgtgcggt atttcacacc gcagaccagc cgcgtaacct ggcaaaatcg gttacggttg 2580
agtaataaat ggatgccctg cgtaagcggg tgtgggcgga caataaagtc ttaaactgaa 2640
caaaatagat ctaaactatg acaataaagt cttaaactag acagaatagt tgtaaactga 2700
aatcagtcca gttatgctgt gaaaaagcat actggacttt tgttatggct aaagcaaact 2760
cttcattttc tgaagtgcaa attgcccgtc gtattaaaga ggggcgtggc caagggcatg 2820
gtaaagacta tattcgcggc gttgtgacaa tttaccgaac aactccgcgg ccgggaagcc 2880
gatctcggct tgaacgaatt gttaggtggc ggtacttggg tcgatatcaa agtgcatcac 2940
ttcttcccgt atgcccaact ttgtatagag agccactgcg ggatcgtcac cgtaatctgc 3000
ttgcacgtag atcacataag caccaagcgc gttggcctca tgcttgagga gattgatgag 3060
cgcggtggca atgccctgcc tccggtgctc gccggagact gcgagatcat agatatagat 3120
ctcactacgc ggctgctcaa acctgggcag aacgtaagcc gcgagagcgc caacaaccgc 3180
ttcttggtcg aaggcagcaa gcgcgatgaa tgtcttacta cggagcaagt tcccgaggta 3240
atcggagtcc ggctgatgtt gggagtaggt ggctacgtct ccgaactcac gaccgaaaag 3300
atcaagagca gcccgcatgg atttgacttg gtcagggccg agcctacatg tgcgaatgat 3360
gcccatactt gagccaccta actttgtttt agggcgactg ccctgctgcg taacatcgtt 3420
gctgctgcgt aacatcgttg ctgctccata acatcaaaca tcgacccacg gcgtaacgcg 3480
cttgctgctt ggatgcccga ggcatagact gtacaaaaaa acagtcataa caagccatga 3540
aaaccgccac tgcgccgtta ccaccgctgc gttcggtcaa ggttctggac cagttgcgtg 3600
agcgcatacg ctacttgcat tacagtttac gaaccgaaca ggcttatgtc aactgggttc 3660
gtgccttcat ccgtttccac ggtgtgcgtc acccggcaac cttgggcagc agcgaagtcg 3720
aggcatttct gtcctggctg gcgaacgagc gcaaggtttc ggtctccacg catcgtcagg 3780
cattggcggc cttgctgttc ttctacggca aggtgctgtg cacggatctg ccctggcttc 3840
aggagatcgg tagacctcgg ccgtcgcggc gcttgccggt ggtgctgacc ccggatgaag 3900
tggttcgcat cctcggtttt ctggaaggcg agcatcgttt gttcgcccag gactctagct 3960
atagttctag tggttggcct acgtacccgt agtggctatg gcagggcttg ccgccccgac 4020
gttggctgcg agccctgggc cttcacccga acttgggggt tggggtgggg aaaaggaaga 4080
aacgcgggcg tattggtccc aatggggtct cggtggggta tcgacagagt gccagccctg 4140
ggaccgaacc ccgcgtttat gaacaaacga cccaacaccc gtgcgtttta ttctgtcttt 4200
ttattgccgt catagcgcgg gttccttccg gtattgtctc cttccgtgtt tcagttagcc 4260
tcccccatct cccggtacct taagcgtaat ctggaacatc gtaatacgcc ggaccagtga 4320
acagaggcgc gtctggtgca aactccttta ttttgaaaac gagagaaacc tcaattagga 4380
tttcctcttc ttcggcagag tctgtgccga tgtaaacgat gggtttgtag aagttctccc 4440
atatgacgcg gttcacaaac gactcgaacg agttggtgta ctcgctgtgg atgttcatga 4500
ttgggcagcc gccgcccttt ttagccagac taattctgta ttcgttgttc atgcccacgt 4560
aggatggctc cacaattctg attacttcgt ggggcacgta gtcttcttcc cacctaagag 4620
cgtgttgagc gaggaacttg tagcacctgt tggggcgtgt gggtttgagg ttggcgacga 4680
ggtacacgtc catcacctct tggtcgttta caatggggaa gctgtcctca acaaaacggg 4740
tccaagtttc acgcaaaaac tctttgccgc tccagttgac gattaacttc atggtatcgg 4800
gtttcacact gcgaatttct ttaaaaaggg taagtttttg gtttttgccc ggtcctaaaa 4860
agggatcttc ggcaaccatg tagttgtcta gaagatccca ttgcttctcc tcttgttcat 4920
gttcgactag gtgcttcttg cgcttggcgt ttttgataag acagcccaag tttttgtaat 4980
atttattgtc gtacacgtaa gtacgcccga tggtgggggt gtatgaataa ttcggcatat 5040
ttataggttt ttttattaca aaactgttac gaaaacagta aaatacttat ttatttgcga 5100
gatggttaat ttgtatcgtt aataaaaaac aaatttgaca tttacaattg ttttattgtt 5160
caataattac aaataggatt gaggcccttg cagttgccag caaacggaca gagcttgtcg 5220
acagttttca ttttttggca gtttggtggt ttatacacat cttgggattt gattaaaaga 5280
tgcatgactt cacccacaca cttggagtcg taattgtaaa aatgtgtcag agccttgatc 5340
aaacatcggt tttcacattt ggtacacgaa accacgtttg caatgtaaca gtttagtata 5400
aacctcttcc tgtacatacc gccgcgcggt acattgttct ttttcaaatc gcccaatatt 5460
ttttgtatgc acggaggcat attaatcttg ttgttcagca tgtcggcgac attatttttt 5520
ccttctacgc attctttcat acaaatgttt cttttgctgc gttttttaaa attggaattt 5580
ttaatttcat taatagtggg cggagccgtt aacagcatgt acaatcgcag tccggaaatt 5640
ttaaccaaaa ccccgccatt gtagaatacc gtgtatggag ttagggtcaa tgcatgctag 5700
gcgatatcag tgatcagatc cagacatgat aagatacatt gatgagtttg gacaaaccac 5760
aactagaatg cagtgaaaaa aatgctttat ttgtgaaatt tgtgatgcta ttgctttatt 5820
tgtaaccatt ataagctgca ataaacaagt taacaacaac aattgcattc attttatgtt 5880
tcaggttcag ggggaggtgt gggaggtttt ttaaagcaag taaaacctct acaaatgtgg 5940
tatggctgat tatgatcctc tagtacttct cgacaagctt ttacttgtac agctcgtcca 6000
tgccgagagt gatcccggcg gcggtcacga actccagcag gaccatgtga tcgcgcttct 6060
cgttggggtc tttgctcagg gcggactggg tgctcaggta gtggttgtcg ggcagcagca 6120
cggggccgtc gccgatgggg gtgttctgct ggtagtggtc ggcgagctgc acgctgccgt 6180
cctcgatgtt gtggcggatc ttgaagttca ccttgatgcc gttcttctgc ttgtcggcca 6240
tgatatagac gttgtggctg ttgtagttgt actccagctt gtgccccagg atgttgccgt 6300
cctccttgaa gtcgatgccc ttcagctcga tgcggttcac cagggtgtcg ccctcgaact 6360
tcacctcggc gcgggtcttg tagttgccgt cgtccttgaa gaagatggtg cgctcctgga 6420
cgtagccttc gggcatggcg gacttgaaga agtcgtgctg cttcatgtgg tcggggtagc 6480
ggctgaagca ctgcacgccg taggtcaggg tggtcacgag ggtgggccag ggcacgggca 6540
gcttgccggt ggtgcagatg aacttcaggg tcagcttgcc gtaggtggca tcgccctcgc 6600
cctcgccgga cacgctgaac ttgtggccgt ttacgtcgcc gtccagctcg accaggatgg 6660
gcaccacccc ggtgaacagc tcctcgccct tgctcaccat ctcgagatcc cgggtattca 6720
gagttctctt cttgtattca ataattactt cttggcagat ttcagtagtt gcagttgatt 6780
tacttggttg ctggttactt ttaattgatt cactttaact tgcactttac tgcagattgt 6840
ttagcttgtt cagctgcgct tgtttatttg cttagctttc gcttagcgac gtgttcactt 6900
tgcttgtttg aattgaattg tcgctccgta gacgaagcgc tctatttata ctccggcgct 6960
cttttcgcga acattcgagg cgcgctctct cgaaccaacg agagcagtat gccgtttact 7020
gtgtgacaga gtgagagagc attagtgcag agagggagac ccaaaaagaa aagagagaat 7080
aacgaataac ggccagagaa atttctcgag ttttcttctg ccaaacaaat gacctaccac 7140
aataaccagt ttgttttggg attctaggga tcccggtccg aagcgcgcgg aattcaaagg 7200
cctacgtcga cgagctcact agtcgcggcc gctttcgaat ctagagcctg cagtctcgac 7260
aagcttgtcg agaagtacta gaggatcata atcagccata ccacatttgt agaggtttta 7320
cttgctttaa aaaacctccc acacctcccc ctgaacctga aacataaaat gaatgcaatt 7380
gttgttgtta acttgtttat tgcagcttat aatggttaca aataaagcaa tagcatcaca 7440
aatttcacaa ataaagcatt tttttcactg cattctagtt gtggtttgtc caaactcatc 7500
aatgtatctt atcatgtctg gatctgatca ctgcttgagc ctaggagatc cgaaccagat 7560
aagtgaaatc tagttccaaa ctattttgtc atttttaatt ttcgtattag cttacgacgc 7620
tacacccagt tcccatctat tttgtcactc ttccctaaat aatccttaaa aactccattt 7680
ccacccctcc cagttcccaa ctattttgtc cgcccacagc ggggcatttt tcttcctgtt 7740
atgtttttaa tcaaacatcc tgccaactcc atgtgacaaa ccgtcatctt cggctacttt 7800
Claims (9)
1. Cas9 system for efficiently editing silkworm genome, which is characterized in that: the Cas9 system comprises a silkworm transgenic expression vector containing an expressed Cas9 gene and virus particles of an expression frame U6-sgRNA of a silkworm nuclear polyhedrosis virus packaged U6 promoter containing a target sequence for regulating sgRNA expression.
2. The Cas9 system for efficiently editing the genome of bombyx mori as claimed in claim 1, wherein: the nucleotide sequence of the Cas9 gene is shown as SEQ ID NO. 1; the nucleotide sequence of the expression frame U6-sgRNA is shown in SEQ ID NO. 2.
3. The Cas9 system for efficiently editing silkworms according to claim 1, wherein: the Cas9 gene is expressed under the control of IE1 promoter.
4. The Cas9 system for efficiently editing silkworms according to claim 1, wherein: the silkworm transgenic expression vector containing the expressed Cas9 gene contains an expression frame of regulating Cas9 expression by an OpIE2 promoter and an expression frame of regulating Mcherry expression by an OpIE2 promoter.
5. The Cas9 system for efficiently editing silkworms according to claim 1, wherein: the virus particle is prepared by the following method: transferring the recombinant plasmid containing the expression frame U6-sgRNA of the target sequence into a DH10bac (BmNPV) competent cell, and transposing the recombinant plasmid onto a BmNPV genome under the action of a Tn7 transposon to form a recombinant bacmid BEV-U6-sgRNA; and (3) extracting recombinant bacmid to transfect silkworm cells, collecting virus particles of P1 generation, infecting again, and collecting virus particles of P2 generation.
6. The Cas9 system for efficiently editing silkworms of claim 5, wherein: the recombinant plasmid containing the target sequence expression frame U6-sgRNA is composed of the following methods: the sequence shown in SEQ ID NO.2 is inserted into pFastBac-Dual-HSP after being cut by BamHI and EcoRIprmAnd (3) obtaining a vector pFBD-U6-sgRNA on an EGFP-poly vector, and then connecting the sgRNA of the target gene into the pFBD-U6-sgRNA vector through an enzyme cutting site BbsI to obtain a recombinant plasmid of U6-sgRNA containing the target sequence.
7. The Cas9 system for efficiently editing silkworms of claim 5, wherein: the sgRNA of the target gene is the sgRNA of the viral gene lef11, the sgRNA of the host gene HSPD1 and the sgRNA of ATAD 3A; the sgRNA of the virus gene lef11 is a double-stranded DNA sequence formed by annealing SEQ ID NO.14 and SEQ ID NO. 15; the sgRNA of the host gene HSPD1 is a double-stranded DNA sequence formed by annealing SEQ ID NO.16 and SEQ ID NO. 17; the sgRNA of ATAD3A is a double-stranded DNA sequence formed by annealing SEQ ID NO.18 and SEQ ID NO. 19.
8. The use of the Cas9 system for efficiently editing silkworms of any one of claims 1 to 7 in editing silkworm genomes or silkworm nuclear polyhedrosis virus genes.
9. A method for efficiently editing the genome of silkworms by using the Cas9 system of any one of claims 1-7, wherein: injecting a silkworm transgenic expression vector containing the expression Cas9 gene into silkworm eggs, and screening transgenic silkworms to obtain Cas9 transgenic silkworms; and then packaging the bombyx mori nuclear polyhedrosis virus into virus particles containing an expression frame U6-sgRNA expressed by sgRNA regulated by a U6 promoter, and injecting the virus particles into the Cas9 transgenic bombyx mori to obtain the high-efficiency edited bombyx mori genome.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113186222A (en) * | 2021-06-16 | 2021-07-30 | 西南大学 | Silkworm gene editing vector mediated by CRISPR/Cas13a and application thereof |
| CN114540421A (en) * | 2022-03-04 | 2022-05-27 | 西南大学 | Controllable editing method for silkworm MSG and PSG expression genes |
Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104673815A (en) * | 2015-02-03 | 2015-06-03 | 西南大学 | Compound type piggyBac recombinant vector as well as preparation method and application of compound type piggyBac recombinant vector |
| CN105132460A (en) * | 2015-09-29 | 2015-12-09 | 西南大学 | Cas9-mediated bombyx mori gene editing carrier and application |
| WO2016109255A1 (en) * | 2014-12-30 | 2016-07-07 | University Of South Florida | Methods and compositions for cloning into large vectors |
| CN107043782A (en) * | 2017-04-10 | 2017-08-15 | 西南大学 | A kind of gene knockout method and its sgRNA fragments and application |
| WO2019025984A1 (en) * | 2017-07-31 | 2019-02-07 | Reflection Biotechnologies Limited | Cellular models of and therapies for ocular diseases |
| CN110799525A (en) * | 2017-04-21 | 2020-02-14 | 通用医疗公司 | Variants of CPF1(CAS12a) with altered PAM specificity |
| CN111549060A (en) * | 2020-05-07 | 2020-08-18 | 西南大学 | Eukaryotic organism CRISPR/Cas9 whole genome editing cell library and construction method |
-
2021
- 2021-01-15 CN CN202110059143.XA patent/CN112852871A/en active Pending
Patent Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2016109255A1 (en) * | 2014-12-30 | 2016-07-07 | University Of South Florida | Methods and compositions for cloning into large vectors |
| CN104673815A (en) * | 2015-02-03 | 2015-06-03 | 西南大学 | Compound type piggyBac recombinant vector as well as preparation method and application of compound type piggyBac recombinant vector |
| CN105132460A (en) * | 2015-09-29 | 2015-12-09 | 西南大学 | Cas9-mediated bombyx mori gene editing carrier and application |
| CN107043782A (en) * | 2017-04-10 | 2017-08-15 | 西南大学 | A kind of gene knockout method and its sgRNA fragments and application |
| CN110799525A (en) * | 2017-04-21 | 2020-02-14 | 通用医疗公司 | Variants of CPF1(CAS12a) with altered PAM specificity |
| WO2019025984A1 (en) * | 2017-07-31 | 2019-02-07 | Reflection Biotechnologies Limited | Cellular models of and therapies for ocular diseases |
| CN111549060A (en) * | 2020-05-07 | 2020-08-18 | 西南大学 | Eukaryotic organism CRISPR/Cas9 whole genome editing cell library and construction method |
Non-Patent Citations (4)
| Title |
|---|
| ZHANQI DONG等: "Establishment of a baculovirus-inducible CRISPR/Cas9 system for antiviral research in transgenic silkworms", 《APPL MICROBIOL BIOTECHNOL》 * |
| 董战旗: "CRISPR/Cas9介导的家蚕抗核型多角体病毒素材创新研究", 《万方学位论文》 * |
| 董战旗等: "对家蚕抗核型多角体病毒的研究策略及研究进展", 《蚕业科学》 * |
| 龙定沛等: "家蚕基因组靶向编辑技术研究进展", 《昆虫学报》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113186222A (en) * | 2021-06-16 | 2021-07-30 | 西南大学 | Silkworm gene editing vector mediated by CRISPR/Cas13a and application thereof |
| CN114540421A (en) * | 2022-03-04 | 2022-05-27 | 西南大学 | Controllable editing method for silkworm MSG and PSG expression genes |
| CN114540421B (en) * | 2022-03-04 | 2024-04-16 | 西南大学 | Controllable editing method for silkworm MSG and PSG expression genes |
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