CN111808876B - Long fragment RNAi vector constructed based on USER enzyme and construction method thereof - Google Patents
Long fragment RNAi vector constructed based on USER enzyme and construction method thereof Download PDFInfo
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Abstract
The invention provides a method for quickly constructing a long-fragment RNAi vector with a hairpin structure based on a one-step method of USER enzyme, and provides a 2301RNAiOS vector which can be used for preparing the long-fragment RNAi vector with the hairpin structure, and a long-fragment RNAi vector for 3LRR gene silencing based on USER enzyme construction. Compared with the existing RNAi vector construction method for generating the hairpin structure, the method can perform one-step construction by utilizing the characteristics of USER enzyme, and the fragments and the vector can quickly obtain the required target vector under the same reaction system; the method is more efficient, the target carrier can be obtained at one time, and the success rate is 100%; meanwhile, the method has no requirement on the length of the inserted fragment and the enzyme cutting site, any fragment can be easily constructed into the 2301RNAiOS vector of the invention, and the method is particularly suitable for constructing a long fragment RNAi vector, and is very efficient and convenient.
Description
Technical Field
The invention relates to the field of RNAi vectors, in particular to a long fragment RNAi vector constructed based on USER enzyme and a construction method thereof.
Background
RNA silencing, also known as RNA interference (RNAi), refers to the phenomenon in which double-stranded RNA-specific endonucleases of the RNaseIII family in eukaryotes, dicer or Dicer-like (DCL for short), cleave double-stranded RNA or partially double-stranded hairpin structures to produce siRNA (small interference RNA) nt long or so, and then form RNA-induced silencing complexes (RNA-induced silencing complex, RISCs for short) that specifically recognize target genes using RNA sequence matching, inhibiting target gene expression at the transcriptional, posttranscriptional or translational levels.
RNAi blocks the expression of specific genes in organisms, thereby allowing cells to exhibit a phenotype of specific gene deletion, a highly evolutionarily conserved regulatory mechanism. RNAi plays a very important role in plants against virus invasion, besides regulating their growth and development. RNAi technology is widely used in gene function research due to its high specificity and rapid action. The technology can be designed to silence only part of members or individual members based on non-conserved regions, or can be designed to silence the whole gene family based on RNAi in conserved regions. Therefore, RNAi can also be used as an effective tool for polyploid plant to regulate gene expression and explore the functional research of the whole genome genes.
It has been considered that hairpin-induced RNAi has better silencing effect and stability than antisense RNA technology in crop genetic breeding. In plants, RNAi is typically achieved by a hairpin structure with double stranded RNA. The vector producing the hairpin structure is typically cloned from the target gene into a set of reverse complement nucleotide sequences, which are linked by an unrelated spacer sequence, and expression is driven by a strong promoter. The construction method of the existing RNAi vector firstly uses a two-step method to construct, a proper enzyme cutting site is required to be found to construct an intermediate vector, then hairpin structural enzymes on the intermediate vector are cut off and connected to a target vector, most RNAi vectors are constructed by adopting the method at present, such as CN106086063, CN106591359A and CN104388455B, the method is time-consuming and labor-consuming, the proper enzyme cutting site is not easy to find sometimes, certain requirements are met for fragments, and long-fragment RNAi vectors (fragments constructed by general RNAi vectors are long fragments in the range of 400bp-600bp and more than 600 bp) are difficult to construct; secondly, a one-step method of homologous recombination is adopted, 15-20bp homology arms exist between fragments, homologous recombination enzymes are utilized for recombination, and the method has poor recombination rate and low success rate because two fragments of hairpin results are complementary fragments, and a target vector is difficult to construct successfully. CN201810046195 discloses construction of plant RNAi vector by Gateway technology, but it still needs to construct multiple enzyme cutting sites, which has high cost and is unfavorable for construction of long fragment RNAi vector. Therefore, a simple and efficient construction method for generating long fragment RNAi vectors with hairpin structures is needed.
The USER enzyme is a mixed enzyme that specifically cleaves uracil, and comprises Uracil DNA Glycosylase (UDG) and endonuclease viii. Wherein UDG catalyzes the cleavage of uracil bases to form a pyrimidine-removing site, but at the same time maintains the integrity of the phosphodiester backbone. The cleavage enzyme activity of endonuclease VIII cleaves the phosphodiester bond backbone at the 3 'and 5' ends of the abasic site, respectively, releasing abasic deoxyribose. The USER enzyme can clone the PCR product directly into the vector without using a cleavage site and a DNA ligase. At present, the USER enzyme cloning technology is generally used for constructing a universal vector for gene knockout and fluorescent fusion protein expression, but a one-step method for constructing a long-fragment RNAi vector by using USER enzyme has not been reported.
The verticillium dahliae (Verticillium dahliae) is a soil-borne vascular bundle fungal disease, has a very wide infection range, can infect more than two hundred commercial crops including woody plants, herbaceous plants and vine plants, and causes great economic loss to China and even the world. In view of its serious harmfulness, it has been a hotspot of research to explore the pathogenic mechanism of verticillium dahliae and the mechanism of interaction between host and pathogenic bacteria. How to improve the disease resistance of host plants to Verticillium dahliae is a urgent problem to be solved. CN105255918B discloses RNAi vectors constructed from a target gene fragment of verticillium dahliae phospholipase, CN104928301B discloses RNAi vectors of key target genes of the disease course of verticillium dahliae thiamine transporter, but long fragment RNAi vectors of 3 LRR genes (genes of domain proteins) of verticillium dahliae have not been constructed, thus being used for host-induced gene silencing (HIGS), and related reports of long fragment RNAi vectors of 3 LRR genes of verticillium dahliae constructed based on a USER enzyme one-step method have not been provided.
Disclosure of Invention
In order to solve the above problems, there is an urgent need for a simple and efficient construction method for generating RNAi vectors with hairpin structures for constructing long fragment RNAi vectors for silencing target genes.
In one aspect, the invention provides a 2301RNAiOS vector that can be used to construct a long fragment RNAi vector with a hairpin structure, wherein the 2301RNAiOS vector comprises a promoter, an enhancer, an intron, a terminator, and USER enzyme cloning sites at both ends of the intron, and the enhancer, the intron, the USER enzyme cloning sites at both ends of the intron, and 2301RNAiOS have nucleotide sequences as shown in Seq ID No.1, seq ID No.2, seq ID No.3, and Seq ID No.4 in the sequence table.
According to the 2301RNAiOS vector constructed by the invention, any target fragment can be constructed into the 2301RNAiOS vector by using a USER enzyme method, and the long fragment RNAi vector with the hairpin structure can be quickly constructed by using a one-step method, so that the length of the inserted fragment and the enzyme cutting site are not required, and the method is very efficient and convenient.
In another aspect, the invention provides a long fragment RNAi vector constructed based on the USER enzyme for gene silencing of any fragment. The long fragment RNAi vector contains a 3 LRR-intron-reverse 3LRR hairpin structure, and the preparation method comprises the steps of carrying out fusion construction on specific fragments in 3LRR sequences of Verticillium dahliae on a pTRV2 vector to obtain a TRV-3LRR vector, and then introducing the 3LRR sequences of the TRV-3LRR vector into a 2301RNAiOS vector; the TRV-3LRR and the TRV-3LRR respectively have nucleotide sequences shown as the Seq ID No.5 and the Seq ID No.6 in the sequence table.
On the other hand, the invention provides a rapid construction method of a long fragment RNAi vector, and a research group surprisingly finds that the RNAi vector containing a hairpin structure of a target gene-intron-reverse target gene can be constructed by a one-step method through a USER enzyme method, so that the method is very time-saving and efficient, and specifically comprises the following steps:
(1) Amplifying an intron sequence in Hurrican by taking Hurrican plasmid as a template, constructing USER enzyme cloning sites at two ends of an intron, and constructing the cloning sites on a 2301.GFP4 carrier to obtain a 2301RNAiOS carrier;
2301 the construction method of gfp4 is as follows: pBinGFP4(Tingli Liu,Tianqiao Song,Xiong Zhang,Hongbo Yuan,Liming Su,Wanlin Li,Jing Xu,Shiheng Liu,Linlin Chen,Tianzi Chen,Meixiang Zhang,Lichuan Gu,Baolong Zhang,Daolong Dou.Unconventionally secreted effectors of two filamentous pathogens target plant salicylate biosynthesis.Nature Communications,2014,5:4686 9) was digested with SacI/HindIII, and the excised fragment was ligated to the SacI/HindIII double digested commercial pCambia2301 vector (Vietnam Beijing). The application is as follows: can be stably or transiently expressed in plants, and the expression level is high because the vector contains the enhancer sequence.
The Hurriane plasmid is constructed and stored in a laboratory, comprises introns, a plurality of enzyme cutting sites such as BamHI, xbaI and the like, is specially used for constructing a hairpin structure, and has a plasmid map shown in figure 2.
(2) The vector 2301RNAiOS was digested and recovered using PacI and Nt.BbvCI enzyme;
(3) Designing amplification primers according to target sequences, respectively adding primer connectors GGTCTTAAU, GGCATTAAU, GGACTTAAU, GGGTTTAAU at the front ends of the primers, amplifying forward insertion fragments and reverse insertion fragments by using high-fidelity enzymes by using target genes as templates, and then cutting and recycling;
(4) Using USER enzyme to connect and transform the fragment recovered in the step (3) and the vector recovered in the step (2) to obtain a long fragment RNAi vector with a hairpin structure of 'target sequence-intron-reverse target sequence';
The Hurrican and 2301.GFP4 have nucleotide sequences shown as Seq ID No.7 and Seq ID No.8 in a sequence table respectively.
The USER enzyme method needs to add a long (9 bp) specific sequence containing deoxyuracil dU complementary to the carrier at the 5' end of the PCR primer. Under the action of USER enzyme, the sequences from the 5 'ends to dU on both sides of the primer are sheared to form a 3' protruding sticky end with 9 bases, and simultaneously PacI and Nt.BbvCI are used for digesting a 2301RNAiOS vector containing a cloning site of the USER enzyme, and the sticky end complementary to the vector is prepared on the vector, and the sticky end is as long as 9 bases and far longer than a common enzyme cutting site, so that the ligation can be completed without the action of DNA ligase.
Further, both the intron sequence and 2301.GFP4 vector described in step (1) were digested with BamHI and XbaI, T4 ligase was ligated overnight, E.coli was transformed, and positive clones were selected to obtain 2301RNAiOS vector.
Further, the amplification primers Intron-F and Intron-R of the Intron sequences described in the step (1) have nucleotide sequences shown as Seq ID No.9 and Seq ID No.10 in the sequence Listing, respectively.
The target sequence and the target gene can be any sequence and any vector.
Further, the target sequence is a 3LRR sequence; the target gene is TRV-3LRR gene.
Further, the primers described in step (3) include forward amplification primers JAGN7910_3LRRF and JAGN7911_3LRRR, reverse amplification primers JAGN7912_3LRRF and JAGN7913_3LRRR; wherein JAGN 7910_3: 3LRRF comprises primer linker GGTCTTAAU, JAGN 7911_3: 3LRRR comprises primer linker GGCATTAAU, JAGN 7912_3: 3LRRR comprises primer linker GGACTTAAU, JAGN 7913_3: 3LRRR comprises primer linker GGGTTTAAU; the JAGN7910_3LRRF, JAGN7911_3LRRR, JAGN7912_3LRRF and JAGN7913_3LRRR have nucleotide sequences as shown by Seq ID No.11, seq ID No.12, seq ID No.13 and Seq ID No.14 in the sequence table.
Further, the ligation conversion condition in the step (4) is that the ligation is carried out at 37 ℃ for 20min and 25 ℃ for 20min.
Further, the RNAi vector obtained in the step (4) is transformed into E.coli, and the colonies growing the next day are verified by using vector primers JAGN7364,7364, JAGN7906,7906, JAGN7907,7907 and JAGN7365,7365, wherein the vector primers JAGN7364,7364, JAGN7906,7906, JAGN7907,7907 and JAGN7365,7365 have nucleotide sequences as shown in Seq ID No.15, seq ID No.16, seq ID No.17 and Seq ID No.18 in the sequence table.
In yet another aspect, the invention provides the use of a USER enzyme for the preparation of a long fragment RNAi vector with a hairpin structure.
Further, the long fragment RNAi vector is an RNAi vector for 3LRR gene silencing.
In yet another aspect, the invention provides the use of 2301RNAiOS vector for constructing long fragment RNAi vector with hairpin structure, characterized in that the RNAi vector is prepared by USER enzyme method.
Further, the long fragment RNAi vector is an RNAi vector for 3LRR gene silencing.
Meanwhile, the invention also provides application of the long fragment RNAi vector constructed based on the USER enzyme in gene transformation. The method can be used for constructing a long fragment RNAi vector for generating hairpin structure RNA, and the long fragment RNAi vector is introduced into plants or animals through a transgenic method to realize the silencing of target genes.
The invention also provides application of the long fragment RNAi vector constructed based on the USER enzyme in preparation of transgenic plants. RNAi vectors can be constructed by the method, and the RNAi vectors are introduced into plant cells by a transgenic method to obtain transgenic plants with target genes silenced. The method can be used for researching gene functions, inhibiting and silencing the expression of target genes and improving crop traits.
Furthermore, the invention also provides application of the long-fragment RNAi vector constructed based on the USER enzyme in preparation of transgenic plants. The long fragment RNAi vector which is constructed by using a USER enzyme method and generates a hairpin structure of 3 LRR-intron-reverse 3LRR is introduced into plant cells by a transgenic method to silence target gene 3LRR, so that a transgenic plant with the target gene 3LRR silenced is obtained. The plant may be any plant susceptible to verticillium dahliae infection, including sunflower, eggplant, peppery, tomato, tobacco, potato, melon, watermelon, cucumber, peanut, kidney bean, mung bean, soybean, sesame, beet, etc.
Further, the plant is cotton. After three LRR structural domains of Verticillium dahliae are knocked out, the pathogenicity to cotton is obviously reduced. The fragments of the three genes are fused together to construct the vector 2301RNAiOS of the invention to form a hairpin structure, and a large amount of small RNAs (sRNAs) of the verticillium dahliae are generated in plants through a host-induced gene silencing (HIGS) technology, so that the resistance of cotton to the verticillium dahliae can be improved.
The invention provides a method for quickly constructing a long-fragment RNAi vector with a hairpin structure based on a one-step method of USER enzyme, and provides a 2301RNAiOS vector which can be used for preparing the long-fragment RNAi vector with the hairpin structure, and a long-fragment RNAi vector for 3LRR gene silencing based on USER enzyme construction. Compared with the existing construction method for RNAi vector producing hairpin structure, the method of the invention can perform one-step construction by utilizing the characteristics of USER enzyme, and the fragment and the vector can rapidly obtain the required target vector under the same reaction system; the method is more efficient, the target carrier can be obtained at one time, and the success rate is 100%; meanwhile, the method has no requirement on the length of the inserted fragment and the enzyme cutting site, any fragment can be easily constructed into the 2301RNAiOS vector of the invention, and the method is particularly suitable for constructing a long fragment RNAi vector, and is very efficient and convenient.
Drawings
FIG. 1 is a schematic diagram showing the structure of 2301.GFP4 vector in example 1
FIG. 2 is a schematic structural diagram of Hurricane plasmid in example 1
FIG. 3 is a schematic structural diagram of 2301RNAiOS vector in example 1
FIG. 4 is a schematic structural diagram of the TRV-3LRR vector in example 2
FIG. 5 shows the vector digestion electrophoresis of 2301RNAiOS in example 3, in which "Marker" is Prime (TsingKs) DL5000DNA MARKER and "digestion" is 2301RNAiOS using PacI and Nt.BbvCI for digestion of plasmids
FIG. 6 shows the result of electrophoresis of 3LRR sequence amplification in example 3, wherein M is Optimazaceae (TsingKs) DL5000 DNA MARKER, lanes 1 and 2 are the forward and reverse inserts, respectively, of 3LRR
FIG. 7 shows the result of the detection of the ligation product of example 3 by transforming E.coli, applying the ligation product to a card-resistant plate, and verifying the colonies grown by electrophoresis using a vector primer, wherein M is maker and the remainder are colonies
FIG. 8 shows the results of the electrophoresis test performed by the restriction enzyme digestion of colonies 4, 6, 15, and 22 of FIG. 5 selected in example 3, wherein lanes 4, 6, 15, and 22 are respectively identified as BamHI/HindIII restriction enzyme digestion tests of positive clones 4, 6, 15, and 22, EV is 2301RNAiOS, and M is maker
FIG. 9 is a map of a long fragment RNAi vector targeting the TRV-3LRR gene obtained in example 3
FIG. 10 shows the results of cloning and sequencing the 3LRR forward sequence in example 4
FIG. 11 shows the results of sequencing the 3LRR reverse sequence clone in example 4
Detailed Description
The present invention will be described in further detail with reference to the following examples, which are intended to facilitate the understanding of the present invention without any limitation thereto. The specific conditions not noted in the examples were carried out according to the conventional conditions or the conditions suggested by the manufacturer, and the reagents or instruments used, not noted by the manufacturer, were conventional products available commercially.
Example 1 construction of 2301R NAiOS vector
The 2301.GFP4 vector used in the present example was provided by the present laboratory construction, and the schematic structure is shown in FIG. 1, and comprises a plurality of cleavage sites such as a promoter, an enhancer, a terminator, bamHI and XbaI; hurrican the plasmid is constructed and stored by the laboratory and comprises introns, bamHI, xbaI and other enzyme cutting sites, and is specially used for constructing a hairpin structure, and the plasmid map is shown in figure 2; bamHI enzyme was purchased from Takara, and XbaI enzyme was purchased from Takara.
The intron sequence in Hurrican was amplified using Hurrican plasmid as template and constructed on 2301.GFP4 vector.
2301 The construction method of gfp4 is as follows: pBinGFP4(Tingli Liu,Tianqiao Song,Xiong Zhang,Hongbo Yuan,Liming Su,Wanlin Li,Jing Xu,Shiheng Liu,Linlin Chen,Tianzi Chen,Meixiang Zhang,Lichuan Gu,Baolong Zhang,Daolong Dou.Unconventionally secreted effectors of two filamentous pathogens target plant salicylate biosynthesis.Nature Communications,2014,5:4686 9) was digested with SacI/HindIII, and the excised fragment was ligated to the SacI/HindIII double digested commercial pCambia2301 vector (Vietnam Beijing). The application is as follows: can be stably or transiently expressed in plants, and the expression level is high because the vector contains the enhancer sequence.
The amplification primers were as follows:
Intron-F：CGCGGATCCGCTGAGGTCTTAATTAATGCCTCAGCCTCCAGGTCCGTCGCTTCTC
Intron-R：TGCTCTAGAGCTGAGGGTTTAATTAAGTCCTCAGCACCTGCACCTATGTTTCTGC
the amplified fragment and 2301.GFP4 vector are digested with BamHI and XbaI, T4 ligase is connected overnight, colibacillus is transformed, positive clone is selected, 2301RNAiOS vector is obtained, and the structure of the vector is shown in figure 3. As can be seen from FIG. 3, the 2301RNAiOS vector prepared in this example contains a promoter, an enhancer, an intron, a terminator, and cloning sites for the USER enzyme at both ends of the intron.
Example 2 TRV-3 construction of LRR vectors
The 3LRR is the gene of three LRR domain proteins in Verticillium dahliae, VDAG _06507 (LRR 6507), VDAG _07687 (LRR 7687), VDAG _09119 (LRR 9119) and the fusion fragments formed by about 200bp are taken, and the size is 644bp.
The preparation method comprises the steps of carrying out fusion construction on specific fragments in 3LRR sequences of Verticillium dahliae onto a pTRV2 vector, carrying out construction by Overlap-PCR, firstly respectively taking DNA of Verticillium dahliae V991 as a template to obtain fragments of LRR6507, LRR7687 and LRR9119, carrying out gel cutting recovery to obtain the three fragments, carrying out amplification by taking the LRR6507 fragment and the LRR7687 fragment as the templates and JAGN7604_F and JAGN7607_R as the primers to obtain an LRR6507-LRR7687 fusion fragment, carrying out gel cutting recovery, then taking the LRR6507-LRR7687 fusion fragment and the LRR9119 fragment as the template, taking JAGN7604_F and JAGN7609_R as the primers, carrying out amplification to obtain fusion fragments of three fragments of LRR6507-LRR7687-LRR9119, naming the fusion fragments as 3LRR, carrying out enzyme cutting and carrying out amplification by using XbaI/BamHI, and constructing the fusion fragments on the vector to be the pTRV2 on the market.
Amplification of LRR6507 fragment:
JAGN7604_F：TGCTCTAGATGACGCCATTCAATCCCT
JAGN7605_R：CGTGGAACGACTTGCTGAT
amplification of LRR7687 fragment:
JAGN7606_F：ATCAGCAAGTCGTTCCACGCTTGGGACCATCTGTCAAATCT
JAGN7607_R：TGTAGGTTGGAGACCGAGGGGTGCCGTCAAAATCGAGCT
amplification of LRR9119 fragment:
JAGN7608_F：CCCTCGGTCTCCAACCTACA
JAGN7609_R：CGCGGATCCCGTCGTCTTCAATGAGCGTCTT
The constructed TRV-3LRR vector map is shown in FIG. 4.
EXAMPLE 3 construction of hairpin Long fragment RNAi vector targeting 3LRR Gene
The USER enzyme used in this example was purchased from NEB, pacI from NEB, nt.BbvCI from NEB, and the high fidelity enzyme from Takara.
The plasmid was digested with PacI and Nt.BbvCI using 2301RNAiOS vector prepared in example 1, and the results of the digestion are shown in FIG. 5, wherein "Marker" is Prime (TsingKs) DL5000 DNA MARKER and "digestion" is 2301RNAiOS with PacI and Nt.BbvCI, and the results of the digestion are correct. Recovered using a DNA purification kit (Axygen).
Designing amplification primers according to the 3LRR sequence, and then adding primer linkers (GGTCTTAAU, GGCATTAAU, GGACTTAAU, GGGTTTAAU) to the front ends of the designed primers to obtain primer sequences as follows:
Amplification of the forward insert:
JAGN7910_3LRRF GGTCTTAAUTGACGCCATTCAATCCCTTG
JAGN7911_3LRRR GGCATTAAUCGTCGTCTTCAATGAGCGTC
Amplification of the reverse insert:
JAGN7912_3LRRF GGACTTAAUCGTCGTCTTCAATGAGCGTC
JAGN7913_3LRRR GGGTTTAAUTGACGCCATTCAATCCCTTG
The plasmid TRV-3LRR is used as a template, and after the forward insert and the reverse insert are amplified by using high-fidelity enzyme, the gel is cut for recovery. Amplification product agarose validation as shown in FIG. 6, M is Optimaceae (TsingKs) DL5000 DNA MARKER, lanes 1 and 2 are the forward and reverse inserts of 3LRR, respectively. As can be seen from FIG. 6, the amplification results were correct.
Adding a reagent into the connection conversion system: 3. Mu.l (150 ng) of PCR product 1 (forward insert of 3 LRR), 3. Mu.l (150 ng) of PCR product 2 (reverse insert of 3 LRR), 3. Mu.l (200 ng) of 2301RNAiOS vector cleavage product, 1. Mu.l of USER enzyme, and a total system of 10. Mu.l.
Reaction conditions: reacting for 20min at 37 ℃; the reaction was carried out at 25℃for 20min (reaction in a PCR apparatus) to obtain a ligation product.
The ligation product was transformed into E.coli by heat transfer and applied to a plate resistant to Cana.
The colonies growing the next day were verified with vector primers (primer sequences as follows).
JAGN7364 CAAGCAATCAAGCATTCTAC
JAGN7906 ACAGAGCAGGAGCTACTGG
JAGN7907 TTCCATAGTCTTGAGTTTTC
JAGN7365 CGGACACGCTGAACTTGTGG
The results of the verification are shown in FIG. 7, where M is maker and the remainder are colonies. As can be seen from FIG. 7, 4, 6, 15, 22 were positive clones.
The plasmids extracted from positive clones 4, 6, 15 and 22 are selected for enzyme digestion verification, the results are shown in FIG. 8, wherein lanes 4, 6, 15 and 22 are respectively identified as BamHI/HindIII enzyme digestion verification of positive clones 4, 6, 15 and 22, EV is 2301RNAiOS, M is maker, and the enzyme digestion verification of positive clones 4, 6, 15 and 22 is correct, and the long fragment RNAi vector of the target 3LRR gene is successfully constructed, and the vector map is shown in FIG. 9.
EXAMPLE 4 clone sequencing to verify the correctness of the forward and reverse sequences of the 3LRR
Clone No. 6 and clone No. 15 of example 2 were selected for sequencing, the correctness was further verified, the 3LRR forward sequence sequencing results are shown in fig. 10, and the 3LRR reverse sequence sequencing results are shown in fig. 11.
As can be seen from FIG. 10, the 3LRR-6 clone inserted in the forward direction had a mutation at position 175, whereas 3LRR-15 was 100% identical to the sequence of interest, and was useful.
As can be seen from FIG. 11, the two clones of the reverse inserted 3LRR are 100% identical to the sequence of interest.
It can be seen that clone No. 6 and clone No. 15 are both sequences of interest.
Example 5 transformation and disease resistance identification of cotton
The long fragment RNAi vector of the 3LRR gene constructed in the example 3 is used for introducing agrobacterium competent cells by a shock transformation method, and the specific operation is as follows: the Agrobacterium competent cells EHA105 were removed at-80℃and thawed on ice; 1 μl of VdH1 RNAi expression vector plasmid is injected into the agrobacterium liquid and placed on ice; injecting the mixed agrobacterium liquid into a electric shock cup, and carrying out electric shock for two seconds under the voltage of 1500 v; adding LB into the electric shock cup, sucking out the mixed solution and injecting the mixed solution into the EP pipe; placing in a shaking table and shaking for 45 minutes at 28 ℃ and 160 revolutions per minute; uniformly coating bacterial liquid on LB+Kan (plasmid resistance) +Rif (agrobacterium resistance) solid culture medium; the colony is coated on LB solid plates added with corresponding antibiotics, and after dark culture for two days at 28 ℃, the grown colony is the agrobacterium which is successfully transformed.
Wild-type cotton WC-CK (from Shanxi cotton institute) was then transformed with Agrobacterium, as follows: firstly, carrying out 98% concentrated sulfuric acid delinting on cotton seeds of wild cotton WC-CK to be converted, flushing the cotton seeds with tap water to clean the cotton seeds, airing the cotton seeds, sterilizing the aired cotton seeds with 70% alcohol for a while, pouring the cotton seeds into the cotton seeds, pouring 5-15% hydrogen peroxide to submerge the cotton seeds for 3-4 hours, flushing the cotton seeds on an ultra clean bench with sterile water for 3-4 times, finally soaking the cotton seeds with sterile water overnight, and transferring the cotton seeds after whitewashing to a 1/2MS agar powder culture medium for the next day, wherein the pH value is 7, and culturing the cotton seeds under 28 ℃ illumination; cutting the hypocotyl of the aseptic seedling cultured for 5-7 days into 5mm slices, soaking the slices in agrobacterium liquid which is cultured overnight and is successfully transformed for ten minutes, inoculating the bacteria to an MS culture medium containing KT 0.1mg/L, 2 and 4-D0.1 mg/L, inducing callus at a pH value of 6, and irradiating the callus at a temperature of 28+/-2 ℃ for 200LX for 3 months, and transferring the callus to an MS solid culture medium without hormone for differentiation; the differentiated embryogenic callus is further differentiated on the culture medium, and transferred into soil after buds and roots grow, so as to obtain the transgenic cotton containing the transformation vector.
The disease resistance of cotton is identified by infection of the cotton by the strain. Selecting full cotton seeds, soaking the cotton seeds in 15% sodium hypochlorite for 30min, washing the cotton seeds with sterile water for 2-3 times, soaking the cotton seeds with sterile water for accelerating germination overnight, spreading the cotton seeds in a culture box for preserving moisture, and planting the cotton seeds in a germination box after the buds grow to 3 cm. The cotyledon-grown seedlings were transferred to a plastic box (8-10 cm high) filled with clear water and incubated at 25℃for 16 hours under light and 8 hours in the dark. When true leaves grow out, the clear water is changed into 1/3 MS culture solution, the culture solution is changed once a week, and 1 leaf is inoculated when the true leaves are flattened. Activating Verticillium dahliae V991 strain preserved at-80deg.C with PDA plate for 3-4 days, picking fungus block from colony edge, adding Charles culture solution, shaking culture at 25deg.C and 220rpm for 5 days, filtering, centrifuging 5000g of filtrate for 5min, diluting spores with clear water, counting with blood cell counting plate, and regulating concentration to 1×107 spores/ml. Adding the spore suspension with the adjusted concentration into an empty plastic box, soaking cotton seedlings for 40min, and inoculating. Then, 1/3 of MS culture solution is used for illumination at 25 ℃ for 16 hours, and cotton seedlings are continuously cultured for 8 hours in darkness. Each box is filled with 12 seedlings, each variety is repeated 3 times, and the control cotton seedlings are soaked in clear water for 40min. After 20 hours, the onset was observed. The statistics of the morbidity of wild-type cotton and the transgenic cotton of this example are shown in Table 1 below.
TABLE 1 wild-type cotton and transgenic cotton against verticillium wilt
| Experimental group | Wild cotton | Transgenic cotton of this example |
| 1 | 80% Leaf wilting or death | Leaf-free wilting condition |
| 2 | 90% Leaf wilting or death | 0.1% Leaf wilting |
| 3 | 85% Leaf wilting or death | Leaf-free wilting condition |
As can be seen from Table 1, the transgenic cotton obtained in this example has a very good effect against verticillium dahliae and a very low incidence of verticillium wilt compared to wild-type cotton. It can be seen that fusion of fragments of three LRR genes of verticillium dahliae together to construct the vector 2301RNAiOS of the invention forms a hairpin structure, and resistance of cotton to verticillium dahliae can be significantly improved by host-induced gene silencing (HIGS) technique.
Although the present invention is disclosed above, the present invention is not limited thereto. Various changes and modifications may be made by one skilled in the art without departing from the spirit and scope of the invention, and the scope of the invention should be assessed accordingly to that of the appended claims.
Sequence listing
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tacaggcgat taaagagctg atagcgcgtg acaaaaacca cccaagcgtg gtgatgtgga 1440
gtattgccaa cgaaccggat acccgtccgc aaggtgcacg ggaatatttc gcgccactgg 1500
cggaagcaac gcgtaaactc gacccgacgc gtccgatcac ctgcgtcaat gtaatgttct 1560
gcgacgctca caccgatacc atcagcgatc tctttgatgt gctgtgcctg aaccgttatt 1620
acggatggta tgtccaaagc ggcgatttgg aaacggcaga gaaggtactg gaaaaagaac 1680
ttctggcctg gcaggagaaa ctgcatcagc cgattatcat caccgaatac ggcgtggata 1740
cgttagccgg gctgcactca atgtacaccg acatgtggag tgaagagtat cagtgtgcat 1800
ggctggatat gtatcaccgc gtctttgatc gcgtcagcgc cgtcgtcggt gaacaggtat 1860
ggaatttcgc cgattttgcg acctcgcaag gcatattgcg cgttggcggt aacaagaaag 1920
ggatcttcac tcgcgaccgc aaaccgaagt cggcggcttt tctgctgcaa aaacgctgga 1980
ctggcatgaa cttcggtgaa aaaccgcagc agggaggcaa acaagctagc caccaccacc 2040
accaccacgt gtgaattaca ggtgaccagc tcgaatttcc ccgatcgttc aaacatttgg 2100
caataaagtt tcttaagatt gaatcctgtt gccggtcttg cgatgattat catataattt 2160
ctgttgaatt acgttaagca tgtaataatt aacatgtaat gcatgacgtt atttatgaga 2220
tgggttttta tgattagagt cccgcaatta tacatttaat acgcgataga aaacaaaata 2280
tagcgcgcaa actaggataa attatcgcgc gcggtgtcat ctatgttact agatcgggaa 2340
ttaaactatc agtgtttgac aggatatatt ggcgggtaaa cctaagagaa aagagcgttt 2400
attagaataa cggatattta aaagggcgtg aaaaggttta tccgttcgtc catttgtatg 2460
tgcatgccaa ccacagggtt cccctcggga tcaaagtact ttgatccaac ccctccgctg 2520
ctatagtgca gtcggcttct gacgttcagt gcagccgtct tctgaaaacg acatgtcgca 2580
caagtcctaa gttacgcgac aggctgccgc cctgcccttt tcctggcgtt ttcttgtcgc 2640
gtgttttagt cgcataaagt agaatacttg cgactagaac cggagacatt acgccatgaa 2700
caagagcgcc gccgctggcc tgctgggcta tgcccgcgtc agcaccgacg accaggactt 2760
gaccaaccaa cgggccgaac tgcacgcggc cggctgcacc aagctgtttt ccgagaagat 2820
caccggcacc aggcgcgacc gcccggagct ggccaggatg cttgaccacc tacgccctgg 2880
cgacgttgtg acagtgacca ggctagaccg cctggcccgc agcacccgcg acctactgga 2940
cattgccgag cgcatccagg aggccggcgc gggcctgcgt agcctggcag agccgtgggc 3000
cgacaccacc acgccggccg gccgcatggt gttgaccgtg ttcgccggca ttgccgagtt 3060
cgagcgttcc ctaatcatcg accgcacccg gagcgggcgc gaggccgcca aggcccgagg 3120
cgtgaagttt ggcccccgcc ctaccctcac cccggcacag atcgcgcacg cccgcgagct 3180
gatcgaccag gaaggccgca ccgtgaaaga ggcggctgca ctgcttggcg tgcatcgctc 3240
gaccctgtac cgcgcacttg agcgcagcga ggaagtgacg cccaccgagg ccaggcggcg 3300
cggtgccttc cgtgaggacg cattgaccga ggccgacgcc ctggcggccg ccgagaatga 3360
acgccaagag gaacaagcat gaaaccgcac caggacggcc aggacgaacc gtttttcatt 3420
accgaagaga tcgaggcgga gatgatcgcg gccgggtacg tgttcgagcc gcccgcgcac 3480
gtctcaaccg tgcggctgca tgaaatcctg gccggtttgt ctgatgccaa gctggcggcc 3540
tggccggcca gcttggccgc tgaagaaacc gagcgccgcc gtctaaaaag gtgatgtgta 3600
tttgagtaaa acagcttgcg tcatgcggtc gctgcgtata tgatgcgatg agtaaataaa 3660
caaatacgca aggggaacgc atgaaggtta tcgctgtact taaccagaaa ggcgggtcag 3720
gcaagacgac catcgcaacc catctagccc gcgccctgca actcgccggg gccgatgttc 3780
tgttagtcga ttccgatccc cagggcagtg cccgcgattg ggcggccgtg cgggaagatc 3840
aaccgctaac cgttgtcggc atcgaccgcc cgacgattga ccgcgacgtg aaggccatcg 3900
gccggcgcga cttcgtagtg atcgacggag cgccccaggc ggcggacttg gctgtgtccg 3960
cgatcaaggc agccgacttc gtgctgattc cggtgcagcc aagcccttac gacatatggg 4020
ccaccgccga cctggtggag ctggttaagc agcgcattga ggtcacggat ggaaggctac 4080
aagcggcctt tgtcgtgtcg cgggcgatca aaggcacgcg catcggcggt gaggttgccg 4140
aggcgctggc cgggtacgag ctgcccattc ttgagtcccg tatcacgcag cgcgtgagct 4200
acccaggcac tgccgccgcc ggcacaaccg ttcttgaatc agaacccgag ggcgacgctg 4260
cccgcgaggt ccaggcgctg gccgctgaaa ttaaatcaaa actcatttga gttaatgagg 4320
taaagagaaa atgagcaaaa gcacaaacac gctaagtgcc ggccgtccga gcgcacgcag 4380
cagcaaggct gcaacgttgg ccagcctggc agacacgcca gccatgaagc gggtcaactt 4440
tcagttgccg gcggaggatc acaccaagct gaagatgtac gcggtacgcc aaggcaagac 4500
cattaccgag ctgctatctg aatacatcgc gcagctacca gagtaaatga gcaaatgaat 4560
aaatgagtag atgaatttta gcggctaaag gaggcggcat ggaaaatcaa gaacaaccag 4620
gcaccgacgc cgtggaatgc cccatgtgtg gaggaacggg cggttggcca ggcgtaagcg 4680
gctgggttgt ctgccggccc tgcaatggca ctggaacccc caagcccgag gaatcggcgt 4740
gacggtcgca aaccatccgg cccggtacaa atcggcgcgg cgctgggtga tgacctggtg 4800
gagaagttga aggccgcgca ggccgcccag cggcaacgca tcgaggcaga agcacgcccc 4860
ggtgaatcgt ggcaagcggc cgctgatcga atccgcaaag aatcccggca accgccggca 4920
gccggtgcgc cgtcgattag gaagccgccc aagggcgacg agcaaccaga ttttttcgtt 4980
ccgatgctct atgacgtggg cacccgcgat agtcgcagca tcatggacgt ggccgttttc 5040
cgtctgtcga agcgtgaccg acgagctggc gaggtgatcc gctacgagct tccagacggg 5100
cacgtagagg tttccgcagg gccggccggc atggccagtg tgtgggatta cgacctggta 5160
ctgatggcgg tttcccatct aaccgaatcc atgaaccgat accgggaagg gaagggagac 5220
aagcccggcc gcgtgttccg tccacacgtt gcggacgtac tcaagttctg ccggcgagcc 5280
gatggcggaa agcagaaaga cgacctggta gaaacctgca ttcggttaaa caccacgcac 5340
gttgccatgc agcgtacgaa gaaggccaag aacggccgcc tggtgacggt atccgagggt 5400
gaagccttga ttagccgcta caagatcgta aagagcgaaa ccgggcggcc ggagtacatc 5460
gagatcgagc tagctgattg gatgtaccgc gagatcacag aaggcaagaa cccggacgtg 5520
ctgacggttc accccgatta ctttttgatc gatcccggca tcggccgttt tctctaccgc 5580
ctggcacgcc gcgccgcagg caaggcagaa gccagatggt tgttcaagac gatctacgaa 5640
cgcagtggca gcgccggaga gttcaagaag ttctgtttca ccgtgcgcaa gctgatcggg 5700
tcaaatgacc tgccggagta cgatttgaag gaggaggcgg ggcaggctgg cccgatccta 5760
gtcatgcgct accgcaacct gatcgagggc gaagcatccg ccggttccta atgtacggag 5820
cagatgctag ggcaaattgc cctagcaggg gaaaaaggtc gaaaaggtct ctttcctgtg 5880
gatagcacgt acattgggaa cccaaagccg tacattggga accggaaccc gtacattggg 5940
aacccaaagc cgtacattgg gaaccggtca cacatgtaag tgactgatat aaaagagaaa 6000
aaaggcgatt tttccgccta aaactcttta aaacttatta aaactcttaa aacccgcctg 6060
gcctgtgcat aactgtctgg ccagcgcaca gccgaagagc tgcaaaaagc gcctaccctt 6120
cggtcgctgc gctccctacg ccccgccgct tcgcgtcggc ctatcgcggc cgctggccgc 6180
tcaaaaatgg ctggcctacg gccaggcaat ctaccagggc gcggacaagc cgcgccgtcg 6240
ccactcgacc gccggcgccc acatcaaggc accctgcctc gcgcgtttcg gtgatgacgg 6300
tgaaaacctc tgacacatgc agctcccgga gacggtcaca gcttgtctgt aagcggatgc 6360
cgggagcaga caagcccgtc agggcgcgtc agcgggtgtt ggcgggtgtc ggggcgcagc 6420
catgacccag tcacgtagcg atagcggagt gtatactggc ttaactatgc ggcatcagag 6480
cagattgtac tgagagtgca ccatatgcgg tgtgaaatac cgcacagatg cgtaaggaga 6540
aaataccgca tcaggcgctc ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt 6600
cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca 6660
ggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa 6720
aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat 6780
cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc 6840
cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc 6900
gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt 6960
tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac 7020
cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg 7080
ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca 7140
gagttcttga agtggtggcc taactacggc tacactagaa ggacagtatt tggtatctgc 7200
gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa 7260
accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa 7320
ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac 7380
tcacgttaag ggattttggt catgcattct aggtactaaa acaattcatc cagtaaaata 7440
taatatttta ttttctccca atcaggcttg atccccagta agtcaaaaaa tagctcgaca 7500
tactgttctt ccccgatatc ctccctgatc gaccggacgc agaaggcaat gtcataccac 7560
ttgtccgccc tgccgcttct cccaagatca ataaagccac ttactttgcc atctttcaca 7620
aagatgttgc tgtctcccag gtcgccgtgg gaaaagacaa gttcctcttc gggcttttcc 7680
gtctttaaaa aatcatacag ctcgcgcgga tctttaaatg gagtgtcttc ttcccagttt 7740
tcgcaatcca catcggccag atcgttattc agtaagtaat ccaattcggc taagcggctg 7800
tctaagctat tcgtataggg acaatccgat atgtcgatgg agtgaaagag cctgatgcac 7860
tccgcataca gctcgataat cttttcaggg ctttgttcat cttcatactc ttccgagcaa 7920
aggacgccat cggcctcact catgagcaga ttgctccagc catcatgccg ttcaaagtgc 7980
aggacctttg gaacaggcag ctttccttcc agccatagca tcatgtcctt ttcccgttcc 8040
acatcatagg tggtcccttt ataccggctg tccgtcattt ttaaatatag gttttcattt 8100
tctcccacca gcttatatac cttagcagga gacattcctt ccgtatcttt tacgcagcgg 8160
tatttttcga tcagtttttt caattccggt gatattctca ttttagccat ttattatttc 8220
cttcctcttt tctacagtat ttaaagatac cccaagaagc taattataac aagacgaact 8280
ccaattcact gttccttgca ttctaaaacc ttaaatacca gaaaacagct ttttcaaagt 8340
tgttttcaaa gttggcgtat aacatagtat cgacggagcc gattttgaaa ccgcggtgat 8400
cacaggcagc aacgctctgt catcgttaca atcaacatgc taccctccgc gagatcatcc 8460
gtgtttcaaa cccggcagct tagttgccgt tcttccgaat agcatcggta acatgagcaa 8520
agtctgccgc cttacaacgg ctctcccgct gacgccgtcc cggactgatg ggctgcctgt 8580
atcgagtggt gattttgtgc cgagctgccg gtcggggagc tgttggctgg ctggtggcag 8640
gatatattgt ggtgtaaaca aattgacgct tagacaactt aataacacat tgcggacgtt 8700
tttaatgtac tgaattaacg ccgaattaat tcgggggatc tggattttag tactggattt 8760
tggttttagg aattagaaat tttattgata gaagtatttt acaaatacaa atacatacta 8820
agggtttctt atatgctcaa cacatgagcg aaaccctata ggaaccctaa ttcccttatc 8880
tgggaactac tcacacatta ttatggagaa actcgagctt gtcgatcgac tctagctaga 8940
ggatcgatcc gaaccccaga gtcccgctca gaagaactcg tcaagaaggc gatagaaggc 9000
gatgcgctgc gaatcgggag cggcgatacc gtaaagcacg aggaagcggt cagcccattc 9060
gccgccaagc tcttcagcaa tatcacgggt agccaacgct atgtcctgat agcggtccgc 9120
cacacccagc cggccacagt cgatgaatcc agaaaagcgg ccattttcca ccatgatatt 9180
cggcaagcag gcatcgccat gtgtcacgac gagatcctcg ccgtcgggca tgcgcgcctt 9240
gagcctggcg aacagttcgg ctggcgcgag cccctgatgc tcttcgtcca gatcatcctg 9300
atcgacaaga ccggcttcca tccgagtacg tgctcgctcg atgcgatgtt tcgcttggtg 9360
gtcgaatggg caggtagccg gatcaagcgt atgcagccgc cgcattgcat cagccatgat 9420
ggatactttc tcggcaggag caaggtgaga tgacaggaga tcctgccccg gcacttcgcc 9480
caatagcagc cagtcccttc ccgcttcagt gacaacgtcg agcacagctg cgcaaggaac 9540
gcccgtcgtg gccagccacg atagccgcgc tgcctcgtcc tggagttcat tcagggcacc 9600
ggacaggtcg gtcttgacaa aaagaaccgg gcgcccctgc gctgacagcc ggaacacggc 9660
ggcatcagag cagccgattg tctgttgtgc ccagtcatag ccgaatagcc tctccaccca 9720
agcggccgga gaacctgcgt gcaatccatc ttgttcaatc cccatggtcg atcgacagat 9780
ctgcgaaagc tcgagagaga tagatttgta gagagagact ggtgatttca gcgtgtcctc 9840
tccaaatgaa atgaacttcc ttatatagag gaaggtcttg cgaaggatag tgggattgtg 9900
cgtcatccct tacgtcagtg gagatatcac atcaatccac ttgctttgaa gacgtggttg 9960
gaacgtcttc tttttccacg atgctcctcg tgggtggggg tccatctttg ggaccactgt 10020
cggcagaggc atcttgaacg atagcctttc ctttatcgca atgatggcat ttgtaggtgc 10080
caccttcctt ttctactgtc cttttgatga agtgacagat agctgggcaa tggaatccga 10140
ggaggtttcc cgatattacc ctttgttgaa aagtctcaat agccctttgg tcttctgaga 10200
ctgtatcttt gatattcttg gagtagacga gagtgtcgtg ctccaccatg ttatcacatc 10260
aatccacttg ctttgaagac gtggttggaa cgtcttcttt ttccacgatg ctcctcgtgg 10320
gtgggggtcc atctttggga ccactgtcgg cagaggcatc ttgaacgata gcctttcctt 10380
tatcgcaatg atggcatttg taggtgccac cttccttttc tactgtcctt ttgatgaagt 10440
gacagatagc tgggcaatgg aatccgagga ggtttcccga tattaccctt tgttgaaaag 10500
tctcaatagc cctttggtct tctgagactg tatctttgat attcttggag tagacgagag 10560
tgtcgtgctc caccatgttg gcaagctgct ctagccaata cgcaaaccgc ctctccccgc 10620
gcgttggccg attcattaat gcagctggca cgacaggttt cccgactgga aagcgggcag 10680
tgagcgcaac gcaattaatg tgagttagct cactcattag gcaccccagg ctttacactt 10740
tatgcttccg gctcgtatgt tgtgtggaat tgtgagcgga taacaatttc acacaggaaa 10800
cagctatgac catgattacg aattcccgat ctagtaacat agatgacacc gcgcgcgata 10860
atttatccta gtttgcgcgc tatattttgt tttctatcgc gtattaaatg tataattgcg 10920
ggactctaat cataaaaacc catctcataa ataacgtcat gcattacatg ttaattatta 10980
catgcttaac gtaattcaac agaaattata tgataatcat cgcaagaccg gcaacaggat 11040
tcaatcttaa gaaactttat tgccaaatgt ttgaacgatc ggggaaattc gagctccttg 11100
catgcctgca ggtcaacatg gtggagcacg acacacttgt ctactccaaa aatatcaaag 11160
atacagtctc agaagaccaa agggcaattg agacttttca acaaagggta atatccggaa 11220
acctcctcgg attccattgc ccagctatct gtcactttat tgtgaagata gtggaaaagg 11280
aaggtggctc ctacaaatgc catcattgcg ataaaggaaa ggccatcgtt gaagatgcct 11340
ctgccgacag tggtcccaaa gatggacccc cacccacgag gagcatcgtg gaaaaagaag 11400
acgttccaac cacgtcttca aagcaagtgg attgatgtga taacatggtg gagcacgaca 11460
cacttgtcta ctccaaaaat atcaaagata cagtctcaga agaccaaagg gcaattgaga 11520
cttttcaaca aagggtaata tccggaaacc tcctcggatt ccattgccca gctatctgtc 11580
actttattgt gaagatagtg gaaaaggaag gtggctccta caaatgccat cattgcgata 11640
aaggaaaggc catcgttgaa gatgcctctg ccgacagtgg tcccaaagat ggacccccac 11700
ccacgaggag catcgtggaa aaagaagacg ttccaaccac gtcttcaaag caagtggatt 11760
gatgtgatat ctccactgac gtaagggatg acgcacaatc ccactatcct tcgcaagacc 11820
cttcctctat ataaggaagt tcatttcatt tggagaggac ctcgagaatt ctcaacacaa 11880
catatacaaa acaaacgaat ctcaagcaat caagcattct acttctattg cagcaattta 11940
aatcatttct tttaaagcaa aagcaatttt ctgaaaattt tcaccattta cgaacgatag 12000
ggtacccccg gggtcgacgg atccgctgag gtcttaatta atgcctcagc ctccaggtcc 12060
gtcgcttctc ttccatttct tctcattttc gattttgatt cttatttctt tccagtagct 12120
cctgctctgt gaatttctcc gctcacgata gatctgctta tactccttac attcaacctt 12180
agatctggtc tcgattctct gtttctctgt ttttttcttt tggtcgagaa tctgatgttt 12240
gtttatgttc tgtcaccatt aataataatg aactctctca ttcatacaat gattagtttc 12300
tctcgtctac aaaacgatat gttgcatttt cacttttctt ctttttttct aagatgattt 12360
gctttgacca atttgtttag atctttattt tattttattt tctggtgggt tggtggaaat 12420
tgaaaaaaaa aaaaaacagc ataaattgtt atttgttaat gtattcattt tttggctatt 12480
tgttctgggt aaaaatctgc ttctactatt gaatctttcc tggatttttt actcctattg 12540
ggtttttata gtaaaaatac ataataaaag gaaaacaaaa gttttataga ttctcttaaa 12600
ccccttacga taaaagttgg aatcaaaata attcaggatc agatgctctt tgattgattc 12660
agatgcgatt acagttgcat ggcaaatttt ctagatccgt cgtcacattt tattttctgt 12720
ttaaatatct aaatctgata tatgatgtcg acaaattctg gtggcttata catcacttca 12780
actgttttct tttggctttg tttgtcaact tggttttcaa tacgatttgt gatttcgatc 12840
gctgaatttt taatacaagc aaactgatgt taaccacaag caagagatgt gacctgcctt 12900
attaacatcg tattacttac tactagtcgt attctcaacg caatcgtttt tgtatttctc 12960
acattatgcc gcttctctac tctttattcc ttttggtcca cgcattttct atttgtggca 13020
atccctttca caacctgatt tcccactttg gatcatttgt ctgaagactc tcttggatcg 13080
ttaccacttg tttcttgtgc atgctctgtt ttttagaatt aatgataaaa ctattccata 13140
gtcttgagtt ttcagcttgt tgattctttt gcttttggtt ttctgcagaa acataggtgc 13200
aggtgctgag gacttaatta aaccctcagc tctagatgaa ctagagtccg caaaaatcac 13260
cagtctctct ctacaaatct atctctctct atttttctcc agaataatgt gtgagtagtt 13320
cccagataag ggaattaggg ttcttatagg gtttcgctca tgtgttgagc atataagaaa 13380
cccttagtat gtatttgtat ttgtaaaata cttctatcaa taaaatttct aattcctaaa 13440
accaaaatcc agtgacaagc ttggcactgg ccgtcgtttt acaacgtcgt gactgggaaa 13500
accctggcgt tacccaactt aatcgccttg cagcacatcc ccctttcgcc agctggcgta 13560
atagcgaaga ggcccgcacc gatcgccctt cccaacagtt gcgcagcctg aatggcgaat 13620
gctagagcag cttgagcttg gatcagattg tcgtttcccg ccttcagttt agcttcatgg 13680
agtcaaagat tcaaatagag gacctaacag aactcgccgt aaagactggc gaacagttca 13740
tacagagtct cttacgactc aatgacaaga agaaaatctt cgtcaacatg gtggagcacg 13800
acacacttgt ctactccaaa aatatcaaag atacagtctc agaagaccaa agggcaattg 13860
agacttttca acaaagggta atatccggaa acctcctcgg attccattgc ccagctatct 13920
gtcactttat tgtgaagata gtggaaaagg aaggtggctc ctacaaatgc catcattgcg 13980
ataaaggaaa ggccatcgtt gaagatgcct ctgccgacag tggtcccaaa gatggacccc 14040
cacccacgag gagcatcgtg gaaaaagaag acgttccaac cacgtcttca aagcaagtgg 14100
attgatgtga tatctccact gacgtaaggg atgacgcaca atcccactat ccttcgcaag 14160
acccttcctc tatataagga agttcatttc atttggagag aacacggggg actcttgac 14219
<210> 5
<211> 10295
<212> DNA
<213> Artificial sequence (ARTIFICIAL SEQUENCE)
<400> 5
ataaaacatt gcacctatgg tgttgccctg gctggggtat gtcagtgatc gcagtagaat 60
gtactaattg acaagttgga gaatacggta gaacgtcctt atccaacaca gcctttatcc 120
ctctccctga cgaggttttt gtcagtgtaa tatttctttt tgaactatcc agcttagtac 180
cgtacgggaa agtgactggt gtgcttatct ttgaaatgtt actttgggtt tcggttcttt 240
aggttagtaa gaaagcactt gtcttctcat acaaaggaaa acctgagacg tatcgcttac 300
gaaagtagca atgaaagaaa ggtggtggtt ttaatcgcta ccgcaaaaac gatggggtcg 360
ttttaattaa cttctcctac gcaagcgtct aaacggacgt tggggttttg ctagtttctt 420
tagagaaaac tagctaagtc tttaatgtta tcattagaga tggcataaat ataatacttg 480
tgtctgctga taagatcatt ttaatttgga cgattagact tgttgaacta caggttactg 540
aatcacttgc gctaatcaac atgggagata tgtacgatga atcatttgac aagtcgggcg 600
gtcctgctga cttgatggac gattcttggg tggaatcagt ttcgtggaaa gatctgttga 660
agaagttaca cagcataaaa tttgcactac agtctggtag agatgagatc actgggttac 720
tagcggcact gaatagacag tgtccttatt caccatatga gcagtttcca gataagaagg 780
tgtatttcct tttagactca cgggctaaca gtgctcttgg tgtgattcag aacgcttcag 840
cgttcaagag acgagctgat gagaagaatg cagtggcggg tgttacaaat attcctgcga 900
atccaaacac aacggttacg acgaaccaag ggagtactac tactaccaag gcgaacactg 960
gctcgacttt ggaagaagac ttgtacactt attacaaatt cgatgatgcc tctacagctt 1020
tccacaaatc tctaacttcg ttagagaaca tggagttgaa gagttattac cgaaggaact 1080
ttgagaaagt attcgggatt aagtttggtg gagcagctgc tagttcatct gcaccgcctc 1140
cagcgagtgg aggtccgata cgtcctaatc cctagggatt taaggacgtg aactctgttg 1200
agatctctgt gaaattcaga gggtgggtga taccatattc actgatgcca ttagcgacat 1260
ctaaataggg ctaattgtga ctaatttgag ggaatttcct ttaccattga cgtcagtgtc 1320
gttggtagca tttgagtttc gcaatgcacg aattacttag gaagtggctt gacgacacta 1380
atgtgttatt gttagataat ggtttggtgg tcaaggtacg tagtagagtc ccacatattc 1440
gcacgtatga agtaattgga aagttgtcag tttttgataa ttcactggga gatgatacgc 1500
tgtttgaggg aaaagtagag aacgtatttg tttttatgtt caggcggttc ttgtgtgtca 1560
acaaagatgg acattgttac tcaaggaagc acgatgagct ttattattac ggacgagtgg 1620
acttagattc tgtgagtaag gttaccgaat tctctagatg acgccattca atcccttgac 1680
accgggggcg tacacgattg gcctaccaac tgacatcaaa cccgtcccga aaatactgct 1740
agcaacccct gcgattccga aaaagaactt tgacttctgg aaagacatgc cacacgagat 1800
cagtgtacac attttctcct tcctgcgacc caaagagctg gttcaagcct ctcgcatcag 1860
caagtcgttc cacgcttggg accatctgtc aaatctacag tacattgacg tgtcaaacaa 1920
tgagctgcgc agcctggctg gcctcaacaa tctcgttcac ctgcgaggcg tccgcgcgga 1980
taacaataag ctgacatcgc tggacggctt ccacttccac gacgggctcc tgtcgctgcg 2040
agcgcgggac aacctgatcg aggagctcga ttttgacggc acccctcggt ctccaaccta 2100
caaaatttgg aaatctttga tattcacggc aacaacgttt cgactcttcc tactaatttc 2160
gggaatcttg aacggctacg tattcttaac cttgccgaaa acagctttga attgctgcca 2220
tttggcattc tgtcccagat gcccttgaca gaactcaacc tgaagaagaa caagcttgca 2280
aagacgctca ttgaagacga cgggatccgg taccgagctc acgcgtctcg aggcccgggc 2340
atgtcccgaa gacattaaac tacggttctt taagtagatc cgtgtctgaa gttttaggtt 2400
caatttaaac ctacgagatt gacattctcg actgatcttg attgatcggt aagtcttttg 2460
taatttaatt ttctttttga ttttatttta aattgttatc tgtttctgtg tatagactgt 2520
ttgagatcgg cgtttggccg actcattgtc ttaccatagg ggaacggact ttgtttgtgt 2580
tgttatttta tttgtatttt attaaaattc tcaacgatct gaaaaagcct cgcggctaag 2640
agattgttgg ggggtgagta agtactttta aagtgatgat ggttacaaag gcaaaagggg 2700
taaaacccct cgcctacgta agcgttatta cgcccgtctg tacttatatc agtacactga 2760
cgagtcccta aaggacgaaa cgggagaacg ctagccacca ccaccaccac cacgtgtgaa 2820
ttacaggtga ccagctcgaa tttccccgat cgttcaaaca tttggcaata aagtttctta 2880
agattgaatc ctgttgccgg tcttgcgatg attatcatat aatttctgtt gaattacgtt 2940
aagcatgtaa taattaacat gtaatgcatg acgttattta tgagatgggt ttttatgatt 3000
agagtcccgc aattatacat ttaatacgcg atagaaaaca aaatatagcg cgcaaactag 3060
gataaattat cgcgcgcggt gtcatctatg ttactagatc gggaattaaa ctatcagtgt 3120
ttgacaggat atattggcgg gtaaacctaa gagaaaagag cgtttattag aataacggat 3180
atttaaaagg gcgtgaaaag gtttatccgt tcgtccattt gtatgtgcat gccaaccaca 3240
gggttcccct cgggatcaaa gtactttgat ccaacccctc cgctgctata gtgcagtcgg 3300
cttctgacgt tcagtgcagc cgtcttctga aaacgacatg tcgcacaagt cctaagttac 3360
gcgacaggct gccgccctgc ccttttcctg gcgttttctt gtcgcgtgtt ttagtcgcat 3420
aaagtagaat acttgcgact agaaccggag acattacgcc atgaacaaga gcgccgccgc 3480
tggcctgctg ggctatgccc gcgtcagcac cgacgaccag gacttgacca accaacgggc 3540
cgaactgcac gcggccggct gcaccaagct gttttccgag aagatcaccg gcaccaggcg 3600
cgaccgcccg gagctggcca ggatgcttga ccacctacgc cctggcgacg ttgtgacagt 3660
gaccaggcta gaccgcctgg cccgcagcac ccgcgaccta ctggacattg ccgagcgcat 3720
ccaggaggcc ggcgcgggcc tgcgtagcct ggcagagccg tgggccgaca ccaccacgcc 3780
ggccggccgc atggtgttga ccgtgttcgc cggcattgcc gagttcgagc gttccctaat 3840
catcgaccgc acccggagcg ggcgcgaggc cgccaaggcc cgaggcgtga agtttggccc 3900
ccgccctacc ctcaccccgg cacagatcgc gcacgcccgc gagctgatcg accaggaagg 3960
ccgcaccgtg aaagaggcgg ctgcactgct tggcgtgcat cgctcgaccc tgtaccgcgc 4020
acttgagcgc agcgaggaag tgacgcccac cgaggccagg cggcgcggtg ccttccgtga 4080
ggacgcattg accgaggccg acgccctggc ggccgccgag aatgaacgcc aagaggaaca 4140
agcatgaaac cgcaccagga cggccaggac gaaccgtttt tcattaccga agagatcgag 4200
gcggagatga tcgcggccgg gtacgtgttc gagccgcccg cgcacgtctc aaccgtgcgg 4260
ctgcatgaaa tcctggccgg tttgtctgat gccaagctgg cggcctggcc ggccagcttg 4320
gccgctgaag aaaccgagcg ccgccgtcta aaaaggtgat gtgtatttga gtaaaacagc 4380
ttgcgtcatg cggtcgctgc gtatatgatg cgatgagtaa ataaacaaat acgcaagggg 4440
aacgcatgaa ggttatcgct gtacttaacc agaaaggcgg gtcaggcaag acgaccatcg 4500
caacccatct agcccgcgcc ctgcaactcg ccggggccga tgttctgtta gtcgattccg 4560
atccccaggg cagtgcccgc gattgggcgg ccgtgcggga agatcaaccg ctaaccgttg 4620
tcggcatcga ccgcccgacg attgaccgcg acgtgaaggc catcggccgg cgcgacttcg 4680
tagtgatcga cggagcgccc caggcggcgg acttggctgt gtccgcgatc aaggcagccg 4740
acttcgtgct gattccggtg cagccaagcc cttacgacat atgggccacc gccgacctgg 4800
tggagctggt taagcagcgc attgaggtca cggatggaag gctacaagcg gcctttgtcg 4860
tgtcgcgggc gatcaaaggc acgcgcatcg gcggtgaggt tgccgaggcg ctggccgggt 4920
acgagctgcc cattcttgag tcccgtatca cgcagcgcgt gagctaccca ggcactgccg 4980
ccgccggcac aaccgttctt gaatcagaac ccgagggcga cgctgcccgc gaggtccagg 5040
cgctggccgc tgaaattaaa tcaaaactca tttgagttaa tgaggtaaag agaaaatgag 5100
caaaagcaca aacacgctaa gtgccggccg tccgagcgca cgcagcagca aggctgcaac 5160
gttggccagc ctggcagaca cgccagccat gaagcgggtc aactttcagt tgccggcgga 5220
ggatcacacc aagctgaaga tgtacgcggt acgccaaggc aagaccatta ccgagctgct 5280
atctgaatac atcgcgcagc taccagagta aatgagcaaa tgaataaatg agtagatgaa 5340
ttttagcggc taaaggaggc ggcatggaaa atcaagaaca accaggcacc gacgccgtgg 5400
aatgccccat gtgtggagga acgggcggtt ggccaggcgt aagcggctgg gttgtctgcc 5460
ggccctgcaa tggcactgga acccccaagc ccgaggaatc ggcgtgacgg tcgcaaacca 5520
tccggcccgg tacaaatcgg cgcggcgctg ggtgatgacc tggtggagaa gttgaaggcc 5580
gcgcaggccg cccagcggca acgcatcgag gcagaagcac gccccggtga atcgtggcaa 5640
gcggccgctg atcgaatccg caaagaatcc cggcaaccgc cggcagccgg tgcgccgtcg 5700
attaggaagc cgcccaaggg cgacgagcaa ccagattttt tcgttccgat gctctatgac 5760
gtgggcaccc gcgatagtcg cagcatcatg gacgtggccg ttttccgtct gtcgaagcgt 5820
gaccgacgag ctggcgaggt gatccgctac gagcttccag acgggcacgt agaggtttcc 5880
gcagggccgg ccggcatggc cagtgtgtgg gattacgacc tggtactgat ggcggtttcc 5940
catctaaccg aatccatgaa ccgataccgg gaagggaagg gagacaagcc cggccgcgtg 6000
ttccgtccac acgttgcgga cgtactcaag ttctgccggc gagccgatgg cggaaagcag 6060
aaagacgacc tggtagaaac ctgcattcgg ttaaacacca cgcacgttgc catgcagcgt 6120
acgaagaagg ccaagaacgg ccgcctggtg acggtatccg agggtgaagc cttgattagc 6180
cgctacaaga tcgtaaagag cgaaaccggg cggccggagt acatcgagat cgagctagct 6240
gattggatgt accgcgagat cacagaaggc aagaacccgg acgtgctgac ggttcacccc 6300
gattactttt tgatcgatcc cggcatcggc cgttttctct accgcctggc acgccgcgcc 6360
gcaggcaagg cagaagccag atggttgttc aagacgatct acgaacgcag tggcagcgcc 6420
ggagagttca agaagttctg tttcaccgtg cgcaagctga tcgggtcaaa tgacctgccg 6480
gagtacgatt tgaaggagga ggcggggcag gctggcccga tcctagtcat gcgctaccgc 6540
aacctgatcg agggcgaagc atccgccggt tcctaatgta cggagcagat gctagggcaa 6600
attgccctag caggggaaaa aggtcgaaaa ggtctctttc ctgtggatag cacgtacatt 6660
gggaacccaa agccgtacat tgggaaccgg aacccgtaca ttgggaaccc aaagccgtac 6720
attgggaacc ggtcacacat gtaagtgact gatataaaag agaaaaaagg cgatttttcc 6780
gcctaaaact ctttaaaact tattaaaact cttaaaaccc gcctggcctg tgcataactg 6840
tctggccagc gcacagccga agagctgcaa aaagcgccta cccttcggtc gctgcgctcc 6900
ctacgccccg ccgcttcgcg tcggcctatc gcggccgctg gccgctcaaa aatggctggc 6960
ctacggccag gcaatctacc agggcgcgga caagccgcgc cgtcgccact cgaccgccgg 7020
cgcccacatc aaggcaccct gcctcgcgcg tttcggtgat gacggtgaaa acctctgaca 7080
catgcagctc ccggagacgg tcacagcttg tctgtaagcg gatgccggga gcagacaagc 7140
ccgtcagggc gcgtcagcgg gtgttggcgg gtgtcggggc gcagccatga cccagtcacg 7200
tagcgatagc ggagtgtata ctggcttaac tatgcggcat cagagcagat tgtactgaga 7260
gtgcaccata tgcggtgtga aataccgcac agatgcgtaa ggagaaaata ccgcatcagg 7320
cgctcttccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg 7380
gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga 7440
aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg 7500
gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag 7560
aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc 7620
gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg 7680
ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt 7740
cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc 7800
ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc 7860
actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg 7920
tggcctaact acggctacac tagaaggaca gtatttggta tctgcgctct gctgaagcca 7980
gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc 8040
ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat 8100
cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt 8160
ttggtcatgc attctaggta ctaaaacaat tcatccagta aaatataata ttttattttc 8220
tcccaatcag gcttgatccc cagtaagtca aaaaatagct cgacatactg ttcttccccg 8280
atatcctccc tgatcgaccg gacgcagaag gcaatgtcat accacttgtc cgccctgccg 8340
cttctcccaa gatcaataaa gccacttact ttgccatctt tcacaaagat gttgctgtct 8400
cccaggtcgc cgtgggaaaa gacaagttcc tcttcgggct tttccgtctt taaaaaatca 8460
tacagctcgc gcggatcttt aaatggagtg tcttcttccc agttttcgca atccacatcg 8520
gccagatcgt tattcagtaa gtaatccaat tcggctaagc ggctgtctaa gctattcgta 8580
tagggacaat ccgatatgtc gatggagtga aagagcctga tgcactccgc atacagctcg 8640
ataatctttt cagggctttg ttcatcttca tactcttccg agcaaaggac gccatcggcc 8700
tcactcatga gcagattgct ccagccatca tgccgttcaa agtgcaggac ctttggaaca 8760
ggcagctttc cttccagcca tagcatcatg tccttttccc gttccacatc ataggtggtc 8820
cctttatacc ggctgtccgt catttttaaa tataggtttt cattttctcc caccagctta 8880
tataccttag caggagacat tccttccgta tcttttacgc agcggtattt ttcgatcagt 8940
tttttcaatt ccggtgatat tctcatttta gccatttatt atttccttcc tcttttctac 9000
agtatttaaa gataccccaa gaagctaatt ataacaagac gaactccaat tcactgttcc 9060
ttgcattcta aaaccttaaa taccagaaaa cagctttttc aaagttgttt tcaaagttgg 9120
cgtataacat agtatcgacg gagccgattt tgaaaccgcg gtgatcacag gcagcaacgc 9180
tctgtcatcg ttacaatcaa catgctaccc tccgcgagat catccgtgtt tcaaacccgg 9240
cagcttagtt gccgttcttc cgaatagcat cggtaacatg agcaaagtct gccgccttac 9300
aacggctctc ccgctgacgc cgtcccggac tgatgggctg cctgtatcga gtggtgattt 9360
tgtgccgagc tgccggtcgg ggagctgttg gctggctggt ggcaggatat attgtggtgt 9420
aaacaaattg acgcttagac aacttaataa cacattgcgg acgtttttaa tgtactgaat 9480
taacgccgaa ttaattccta ggccaccatg ttgggcccgg cgcgccaagc ttgcatgcct 9540
gcaggtcaac atggtggagc acgacactct cgtctactcc aagaatatca aagatacagt 9600
ctcagaagac cagagggcta ttgagacttt tcaacaaagg gtaatatcgg gaaacctcct 9660
cggattccat tgcccagcta tctgtcactt catcgaaagg acagtagaaa aggaagatgg 9720
cttctacaaa tgccatcatt gcgataaagg aaaggctatc gttcaagatg cctctaccga 9780
cagtggtccc aaagatggac ccccacccac gaggaacatc gtggaaaaag aagacgttcc 9840
aaccacgtct tcaaagcaag tggattgatg tgatggtcaa catggtggag cacgacactc 9900
tcgtctactc caagaatatc aaagatacag tctcagaaga ccagagggct attgagactt 9960
ttcaacaaag ggtaatatcg ggaaacctcc tcggattcca ttgcccagct atctgtcact 10020
tcatcgaaag gacagtagaa aaggaagatg gcttctacaa atgccatcat tgcgataaag 10080
gaaaggctat cgttcaagat gcctctaccg acagtggtcc caaagatgga cccccaccca 10140
cgaggaacat cgtggaaaaa gaagacgttc caaccacgtc ttcaaagcaa gtggattgat 10200
gtgatatctc cactgacgta agggatgacg cacaatccca ctatccttcg caagaccctt 10260
cctctatata aggaagttca tttcatttgg agagg 10295
<210> 6
<211> 644
<212> DNA
<213> Artificial sequence (ARTIFICIAL SEQUENCE)
<400> 6
tgacgccatt caatcccttg acaccggggg cgtacacgat tggcctacca actgacatca 60
aacccgtccc gaaaatactg ctagcaaccc ctgcgattcc gaaaaagaac tttgacttct 120
ggaaagacat gccacacgag atcagtgtac acattttctc cttcctgcga cccaaagagc 180
tggttcaagc ctctcgcatc agcaagtcgt tccacgcttg ggaccatctg tcaaatctac 240
agtacattga cgtgtcaaac aatgagctgc gcagcctggc tggcctcaac aatctcgttc 300
acctgcgagg cgtccgcgcg gataacaata agctgacatc gctggacggc ttccacttcc 360
acgacgggct cctgtcgctg cgagcgcggg acaacctgat cgaggagctc gattttgacg 420
gcacccctcg gtctccaacc tacaaaattt ggaaatcttt gatattcacg gcaacaacgt 480
ttcgactctt cctactaatt tcgggaatct tgaacggcta cgtattctta accttgccga 540
aaacagcttt gaattgctgc catttggcat tctgtcccag atgcccttga cagaactcaa 600
cctgaagaag aacaagcttg caaagacgct cattgaagac gacg 644
<210> 7
<211> 2300
<212> DNA
<213> Artificial sequence (ARTIFICIAL SEQUENCE)
<400> 7
catgattctg tggataacca gtattaccgc ctttgagtga gctgataccg ctcgccgcag 60
ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg gaagagcgcc caatacgcaa 120
accgcctctc cccgcgcgtt ggccgattca ttaatgcagc tggcacgaca ggtttcccga 180
ctggaaagcg ggcagtgagc gcaacgcaat taatgtgagt tagctcactc attaggcacc 240
ccaggcttta cactttatgc ttccggctcg tatgttgtgt ggaattgtga gcggataaca 300
atttcacaca ggaaacagct atgaccatga ttacgccaag cgcgcaatta accctcacta 360
aagggaacaa aagctggagc tacccgggat ccgcggccgc aagcttctcc aggtccgtcg 420
cttctcttcc atttcttctc attttcgatt ttgattctta tttctttcca gtagctcctg 480
ctctgtgaat ttctccgctc acgatagatc tgcttatact ccttacattc aaccttagat 540
ctggtctcga ttctctgttt ctctgttttt ttcttttggt cgagaatctg atgtttgttt 600
atgttctgtc accattaata ataatgaact ctctcattca tacaatgatt agtttctctc 660
gtctacaaaa cgatatgttg cattttcact tttcttcttt ttttctaaga tgatttgctt 720
tgaccaattt gtttagatct ttattttatt ttattttctg gtgggttggt ggaaattgaa 780
aaaaaaaaaa aacagcataa attgttattt gttaatgtat tcattttttg gctatttgtt 840
ctgggtaaaa atctgcttct actattgaat ctttcctgga ttttttactc ctattgggtt 900
tttatagtaa aaatacataa taaaaggaaa acaaaagttt tatagattct cttaaacccc 960
ttacgataaa agttggaatc aaaataattc aggatcagat gctctttgat tgattcagat 1020
gcgattacag ttgcatggca aattttctag atccgtcgtc acattttatt ttctgtttaa 1080
atatctaaat ctgatatatg atgtcgacaa attctggtgg cttatacatc acttcaactg 1140
ttttcttttg gctttgtttg tcaacttggt tttcaatacg atttgtgatt tcgatcgctg 1200
aatttttaat acaagcaaac tgatgttaac cacaagcaag agatgtgacc tgccttatta 1260
acatcgtatt acttactact agtcgtattc tcaacgcaat cgtttttgta tttctcacat 1320
tatgccgctt ctctactctt tattcctttt ggtccacgca ttttctattt gtggcaatcc 1380
ctttcacaac ctgatttccc actttggatc atttgtctga agactctctt ggatcgttac 1440
cacttgtttc ttgtgcatgc tctgtttttt agaattaatg ataaaactat tccatagtct 1500
tgagttttca gcttgttgat tcttttgctt ttggttttct gcagaaacat aggtgcaggt 1560
ctcgaggaat tcgagctccc gggtacccaa ttcgccctat agtgagtcgt attacgcgcg 1620
ctcactggcc gtcgttttac aacgtcgtga ctgggaaaac cctggcgtta cccaacttaa 1680
tcgccttgca gcacatcccc ctttcgccag ctggcgtaat agcgaagagg cccgcaccga 1740
tcgcccttcc caacagttgc gcagcctgaa tggcgaatgg gacgcgccct gtagcggcgc 1800
attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg ccagcgccct 1860
agcgcccgct cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg 1920
tcaagctcta aatcgggggc tccctttagg gttccgattt agtgctttac ggcacctcga 1980
ccccaaaaaa cttgattagg gtgatggttc acgtagtggg ccatcgccct gatagacggt 2040
ttttcgccct ttgacgttgg agtccacgtt ctttaatagt ggactcttgt tccaaactgg 2100
aacaacactc aaccctatct cggtctattc ttttgattta taaaggattt tgccgatttc 2160
ggcctattgg tttaaaaaat gagctgattt aacaaaaatt taacgcgaat tttacaaata 2220
ttacgcttac atttagtggc acttttcggg gaaatgtgcg cggaacccct atttgtttat 2280
tttttctaaa tacattcaat 2300
<210> 8
<211> 13733
<212> DNA
<213> Artificial sequence (ARTIFICIAL SEQUENCE)
<400> 8
catggtagat ctgagggtaa atttctagtt tttctccttc attttcttgg ttaggaccct 60
tttctctttt tatttttttg agctttgatc tttctttaaa ctgatctatt ttttaattga 120
ttggttatgg tgtaaatatt acatagcttt aactgataat ctgattactt tatttcgtgt 180
gtctatgatg atgatgatag ttacagaacc gacgactcgt ccgtcctgta gaaaccccaa 240
cccgtgaaat caaaaaactc gacggcctgt gggcattcag tctggatcgc gaaaactgtg 300
gaattgatca gcgttggtgg gaaagcgcgt tacaagaaag ccgggcaatt gctgtgccag 360
gcagttttaa cgatcagttc gccgatgcag atattcgtaa ttatgcgggc aacgtctggt 420
atcagcgcga agtctttata ccgaaaggtt gggcaggcca gcgtatcgtg ctgcgtttcg 480
atgcggtcac tcattacggc aaagtgtggg tcaataatca ggaagtgatg gagcatcagg 540
gcggctatac gccatttgaa gccgatgtca cgccgtatgt tattgccggg aaaagtgtac 600
gtatcaccgt ttgtgtgaac aacgaactga actggcagac tatcccgccg ggaatggtga 660
ttaccgacga aaacggcaag aaaaagcagt cttacttcca tgatttcttt aactatgccg 720
gaatccatcg cagcgtaatg ctctacacca cgccgaacac ctgggtggac gatatcaccg 780
tggtgacgca tgtcgcgcaa gactgtaacc acgcgtctgt tgactggcag gtggtggcca 840
atggtgatgt cagcgttgaa ctgcgtgatg cggatcaaca ggtggttgca actggacaag 900
gcactagcgg gactttgcaa gtggtgaatc cgcacctctg gcaaccgggt gaaggttatc 960
tctatgaact cgaagtcaca gccaaaagcc agacagagtc tgatatctac ccgcttcgcg 1020
tcggcatccg gtcagtggca gtgaagggcc aacagttcct gattaaccac aaaccgttct 1080
actttactgg ctttggtcgt catgaagatg cggacttacg tggcaaagga ttcgataacg 1140
tgctgatggt gcacgaccac gcattaatgg actggattgg ggccaactcc taccgtacct 1200
cgcattaccc ttacgctgaa gagatgctcg actgggcaga tgaacatggc atcgtggtga 1260
ttgatgaaac tgctgctgtc ggctttcagc tgtctttagg cattggtttc gaagcgggca 1320
acaagccgaa agaactgtac agcgaagagg cagtcaacgg ggaaactcag caagcgcact 1380
tacaggcgat taaagagctg atagcgcgtg acaaaaacca cccaagcgtg gtgatgtgga 1440
gtattgccaa cgaaccggat acccgtccgc aaggtgcacg ggaatatttc gcgccactgg 1500
cggaagcaac gcgtaaactc gacccgacgc gtccgatcac ctgcgtcaat gtaatgttct 1560
gcgacgctca caccgatacc atcagcgatc tctttgatgt gctgtgcctg aaccgttatt 1620
acggatggta tgtccaaagc ggcgatttgg aaacggcaga gaaggtactg gaaaaagaac 1680
ttctggcctg gcaggagaaa ctgcatcagc cgattatcat caccgaatac ggcgtggata 1740
cgttagccgg gctgcactca atgtacaccg acatgtggag tgaagagtat cagtgtgcat 1800
ggctggatat gtatcaccgc gtctttgatc gcgtcagcgc cgtcgtcggt gaacaggtat 1860
ggaatttcgc cgattttgcg acctcgcaag gcatattgcg cgttggcggt aacaagaaag 1920
ggatcttcac tcgcgaccgc aaaccgaagt cggcggcttt tctgctgcaa aaacgctgga 1980
ctggcatgaa cttcggtgaa aaaccgcagc agggaggcaa acaagctagc caccaccacc 2040
accaccacgt gtgaattaca ggtgaccagc tcgaatttcc ccgatcgttc aaacatttgg 2100
caataaagtt tcttaagatt gaatcctgtt gccggtcttg cgatgattat catataattt 2160
ctgttgaatt acgttaagca tgtaataatt aacatgtaat gcatgacgtt atttatgaga 2220
tgggttttta tgattagagt cccgcaatta tacatttaat acgcgataga aaacaaaata 2280
tagcgcgcaa actaggataa attatcgcgc gcggtgtcat ctatgttact agatcgggaa 2340
ttaaactatc agtgtttgac aggatatatt ggcgggtaaa cctaagagaa aagagcgttt 2400
attagaataa cggatattta aaagggcgtg aaaaggttta tccgttcgtc catttgtatg 2460
tgcatgccaa ccacagggtt cccctcggga tcaaagtact ttgatccaac ccctccgctg 2520
ctatagtgca gtcggcttct gacgttcagt gcagccgtct tctgaaaacg acatgtcgca 2580
caagtcctaa gttacgcgac aggctgccgc cctgcccttt tcctggcgtt ttcttgtcgc 2640
gtgttttagt cgcataaagt agaatacttg cgactagaac cggagacatt acgccatgaa 2700
caagagcgcc gccgctggcc tgctgggcta tgcccgcgtc agcaccgacg accaggactt 2760
gaccaaccaa cgggccgaac tgcacgcggc cggctgcacc aagctgtttt ccgagaagat 2820
caccggcacc aggcgcgacc gcccggagct ggccaggatg cttgaccacc tacgccctgg 2880
cgacgttgtg acagtgacca ggctagaccg cctggcccgc agcacccgcg acctactgga 2940
cattgccgag cgcatccagg aggccggcgc gggcctgcgt agcctggcag agccgtgggc 3000
cgacaccacc acgccggccg gccgcatggt gttgaccgtg ttcgccggca ttgccgagtt 3060
cgagcgttcc ctaatcatcg accgcacccg gagcgggcgc gaggccgcca aggcccgagg 3120
cgtgaagttt ggcccccgcc ctaccctcac cccggcacag atcgcgcacg cccgcgagct 3180
gatcgaccag gaaggccgca ccgtgaaaga ggcggctgca ctgcttggcg tgcatcgctc 3240
gaccctgtac cgcgcacttg agcgcagcga ggaagtgacg cccaccgagg ccaggcggcg 3300
cggtgccttc cgtgaggacg cattgaccga ggccgacgcc ctggcggccg ccgagaatga 3360
acgccaagag gaacaagcat gaaaccgcac caggacggcc aggacgaacc gtttttcatt 3420
accgaagaga tcgaggcgga gatgatcgcg gccgggtacg tgttcgagcc gcccgcgcac 3480
gtctcaaccg tgcggctgca tgaaatcctg gccggtttgt ctgatgccaa gctggcggcc 3540
tggccggcca gcttggccgc tgaagaaacc gagcgccgcc gtctaaaaag gtgatgtgta 3600
tttgagtaaa acagcttgcg tcatgcggtc gctgcgtata tgatgcgatg agtaaataaa 3660
caaatacgca aggggaacgc atgaaggtta tcgctgtact taaccagaaa ggcgggtcag 3720
gcaagacgac catcgcaacc catctagccc gcgccctgca actcgccggg gccgatgttc 3780
tgttagtcga ttccgatccc cagggcagtg cccgcgattg ggcggccgtg cgggaagatc 3840
aaccgctaac cgttgtcggc atcgaccgcc cgacgattga ccgcgacgtg aaggccatcg 3900
gccggcgcga cttcgtagtg atcgacggag cgccccaggc ggcggacttg gctgtgtccg 3960
cgatcaaggc agccgacttc gtgctgattc cggtgcagcc aagcccttac gacatatggg 4020
ccaccgccga cctggtggag ctggttaagc agcgcattga ggtcacggat ggaaggctac 4080
aagcggcctt tgtcgtgtcg cgggcgatca aaggcacgcg catcggcggt gaggttgccg 4140
aggcgctggc cgggtacgag ctgcccattc ttgagtcccg tatcacgcag cgcgtgagct 4200
acccaggcac tgccgccgcc ggcacaaccg ttcttgaatc agaacccgag ggcgacgctg 4260
cccgcgaggt ccaggcgctg gccgctgaaa ttaaatcaaa actcatttga gttaatgagg 4320
taaagagaaa atgagcaaaa gcacaaacac gctaagtgcc ggccgtccga gcgcacgcag 4380
cagcaaggct gcaacgttgg ccagcctggc agacacgcca gccatgaagc gggtcaactt 4440
tcagttgccg gcggaggatc acaccaagct gaagatgtac gcggtacgcc aaggcaagac 4500
cattaccgag ctgctatctg aatacatcgc gcagctacca gagtaaatga gcaaatgaat 4560
aaatgagtag atgaatttta gcggctaaag gaggcggcat ggaaaatcaa gaacaaccag 4620
gcaccgacgc cgtggaatgc cccatgtgtg gaggaacggg cggttggcca ggcgtaagcg 4680
gctgggttgt ctgccggccc tgcaatggca ctggaacccc caagcccgag gaatcggcgt 4740
gacggtcgca aaccatccgg cccggtacaa atcggcgcgg cgctgggtga tgacctggtg 4800
gagaagttga aggccgcgca ggccgcccag cggcaacgca tcgaggcaga agcacgcccc 4860
ggtgaatcgt ggcaagcggc cgctgatcga atccgcaaag aatcccggca accgccggca 4920
gccggtgcgc cgtcgattag gaagccgccc aagggcgacg agcaaccaga ttttttcgtt 4980
ccgatgctct atgacgtggg cacccgcgat agtcgcagca tcatggacgt ggccgttttc 5040
cgtctgtcga agcgtgaccg acgagctggc gaggtgatcc gctacgagct tccagacggg 5100
cacgtagagg tttccgcagg gccggccggc atggccagtg tgtgggatta cgacctggta 5160
ctgatggcgg tttcccatct aaccgaatcc atgaaccgat accgggaagg gaagggagac 5220
aagcccggcc gcgtgttccg tccacacgtt gcggacgtac tcaagttctg ccggcgagcc 5280
gatggcggaa agcagaaaga cgacctggta gaaacctgca ttcggttaaa caccacgcac 5340
gttgccatgc agcgtacgaa gaaggccaag aacggccgcc tggtgacggt atccgagggt 5400
gaagccttga ttagccgcta caagatcgta aagagcgaaa ccgggcggcc ggagtacatc 5460
gagatcgagc tagctgattg gatgtaccgc gagatcacag aaggcaagaa cccggacgtg 5520
ctgacggttc accccgatta ctttttgatc gatcccggca tcggccgttt tctctaccgc 5580
ctggcacgcc gcgccgcagg caaggcagaa gccagatggt tgttcaagac gatctacgaa 5640
cgcagtggca gcgccggaga gttcaagaag ttctgtttca ccgtgcgcaa gctgatcggg 5700
tcaaatgacc tgccggagta cgatttgaag gaggaggcgg ggcaggctgg cccgatccta 5760
gtcatgcgct accgcaacct gatcgagggc gaagcatccg ccggttccta atgtacggag 5820
cagatgctag ggcaaattgc cctagcaggg gaaaaaggtc gaaaaggtct ctttcctgtg 5880
gatagcacgt acattgggaa cccaaagccg tacattggga accggaaccc gtacattggg 5940
aacccaaagc cgtacattgg gaaccggtca cacatgtaag tgactgatat aaaagagaaa 6000
aaaggcgatt tttccgccta aaactcttta aaacttatta aaactcttaa aacccgcctg 6060
gcctgtgcat aactgtctgg ccagcgcaca gccgaagagc tgcaaaaagc gcctaccctt 6120
cggtcgctgc gctccctacg ccccgccgct tcgcgtcggc ctatcgcggc cgctggccgc 6180
tcaaaaatgg ctggcctacg gccaggcaat ctaccagggc gcggacaagc cgcgccgtcg 6240
ccactcgacc gccggcgccc acatcaaggc accctgcctc gcgcgtttcg gtgatgacgg 6300
tgaaaacctc tgacacatgc agctcccgga gacggtcaca gcttgtctgt aagcggatgc 6360
cgggagcaga caagcccgtc agggcgcgtc agcgggtgtt ggcgggtgtc ggggcgcagc 6420
catgacccag tcacgtagcg atagcggagt gtatactggc ttaactatgc ggcatcagag 6480
cagattgtac tgagagtgca ccatatgcgg tgtgaaatac cgcacagatg cgtaaggaga 6540
aaataccgca tcaggcgctc ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt 6600
cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca 6660
ggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa 6720
aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat 6780
cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc 6840
cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc 6900
gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt 6960
tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac 7020
cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg 7080
ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca 7140
gagttcttga agtggtggcc taactacggc tacactagaa ggacagtatt tggtatctgc 7200
gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa 7260
accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa 7320
ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac 7380
tcacgttaag ggattttggt catgcattct aggtactaaa acaattcatc cagtaaaata 7440
taatatttta ttttctccca atcaggcttg atccccagta agtcaaaaaa tagctcgaca 7500
tactgttctt ccccgatatc ctccctgatc gaccggacgc agaaggcaat gtcataccac 7560
ttgtccgccc tgccgcttct cccaagatca ataaagccac ttactttgcc atctttcaca 7620
aagatgttgc tgtctcccag gtcgccgtgg gaaaagacaa gttcctcttc gggcttttcc 7680
gtctttaaaa aatcatacag ctcgcgcgga tctttaaatg gagtgtcttc ttcccagttt 7740
tcgcaatcca catcggccag atcgttattc agtaagtaat ccaattcggc taagcggctg 7800
tctaagctat tcgtataggg acaatccgat atgtcgatgg agtgaaagag cctgatgcac 7860
tccgcataca gctcgataat cttttcaggg ctttgttcat cttcatactc ttccgagcaa 7920
aggacgccat cggcctcact catgagcaga ttgctccagc catcatgccg ttcaaagtgc 7980
aggacctttg gaacaggcag ctttccttcc agccatagca tcatgtcctt ttcccgttcc 8040
acatcatagg tggtcccttt ataccggctg tccgtcattt ttaaatatag gttttcattt 8100
tctcccacca gcttatatac cttagcagga gacattcctt ccgtatcttt tacgcagcgg 8160
tatttttcga tcagtttttt caattccggt gatattctca ttttagccat ttattatttc 8220
cttcctcttt tctacagtat ttaaagatac cccaagaagc taattataac aagacgaact 8280
ccaattcact gttccttgca ttctaaaacc ttaaatacca gaaaacagct ttttcaaagt 8340
tgttttcaaa gttggcgtat aacatagtat cgacggagcc gattttgaaa ccgcggtgat 8400
cacaggcagc aacgctctgt catcgttaca atcaacatgc taccctccgc gagatcatcc 8460
gtgtttcaaa cccggcagct tagttgccgt tcttccgaat agcatcggta acatgagcaa 8520
agtctgccgc cttacaacgg ctctcccgct gacgccgtcc cggactgatg ggctgcctgt 8580
atcgagtggt gattttgtgc cgagctgccg gtcggggagc tgttggctgg ctggtggcag 8640
gatatattgt ggtgtaaaca aattgacgct tagacaactt aataacacat tgcggacgtt 8700
tttaatgtac tgaattaacg ccgaattaat tcgggggatc tggattttag tactggattt 8760
tggttttagg aattagaaat tttattgata gaagtatttt acaaatacaa atacatacta 8820
agggtttctt atatgctcaa cacatgagcg aaaccctata ggaaccctaa ttcccttatc 8880
tgggaactac tcacacatta ttatggagaa actcgagctt gtcgatcgac tctagctaga 8940
ggatcgatcc gaaccccaga gtcccgctca gaagaactcg tcaagaaggc gatagaaggc 9000
gatgcgctgc gaatcgggag cggcgatacc gtaaagcacg aggaagcggt cagcccattc 9060
gccgccaagc tcttcagcaa tatcacgggt agccaacgct atgtcctgat agcggtccgc 9120
cacacccagc cggccacagt cgatgaatcc agaaaagcgg ccattttcca ccatgatatt 9180
cggcaagcag gcatcgccat gtgtcacgac gagatcctcg ccgtcgggca tgcgcgcctt 9240
gagcctggcg aacagttcgg ctggcgcgag cccctgatgc tcttcgtcca gatcatcctg 9300
atcgacaaga ccggcttcca tccgagtacg tgctcgctcg atgcgatgtt tcgcttggtg 9360
gtcgaatggg caggtagccg gatcaagcgt atgcagccgc cgcattgcat cagccatgat 9420
ggatactttc tcggcaggag caaggtgaga tgacaggaga tcctgccccg gcacttcgcc 9480
caatagcagc cagtcccttc ccgcttcagt gacaacgtcg agcacagctg cgcaaggaac 9540
gcccgtcgtg gccagccacg atagccgcgc tgcctcgtcc tggagttcat tcagggcacc 9600
ggacaggtcg gtcttgacaa aaagaaccgg gcgcccctgc gctgacagcc ggaacacggc 9660
ggcatcagag cagccgattg tctgttgtgc ccagtcatag ccgaatagcc tctccaccca 9720
agcggccgga gaacctgcgt gcaatccatc ttgttcaatc cccatggtcg atcgacagat 9780
ctgcgaaagc tcgagagaga tagatttgta gagagagact ggtgatttca gcgtgtcctc 9840
tccaaatgaa atgaacttcc ttatatagag gaaggtcttg cgaaggatag tgggattgtg 9900
cgtcatccct tacgtcagtg gagatatcac atcaatccac ttgctttgaa gacgtggttg 9960
gaacgtcttc tttttccacg atgctcctcg tgggtggggg tccatctttg ggaccactgt 10020
cggcagaggc atcttgaacg atagcctttc ctttatcgca atgatggcat ttgtaggtgc 10080
caccttcctt ttctactgtc cttttgatga agtgacagat agctgggcaa tggaatccga 10140
ggaggtttcc cgatattacc ctttgttgaa aagtctcaat agccctttgg tcttctgaga 10200
ctgtatcttt gatattcttg gagtagacga gagtgtcgtg ctccaccatg ttatcacatc 10260
aatccacttg ctttgaagac gtggttggaa cgtcttcttt ttccacgatg ctcctcgtgg 10320
gtgggggtcc atctttggga ccactgtcgg cagaggcatc ttgaacgata gcctttcctt 10380
tatcgcaatg atggcatttg taggtgccac cttccttttc tactgtcctt ttgatgaagt 10440
gacagatagc tgggcaatgg aatccgagga ggtttcccga tattaccctt tgttgaaaag 10500
tctcaatagc cctttggtct tctgagactg tatctttgat attcttggag tagacgagag 10560
tgtcgtgctc caccatgttg gcaagctgct ctagccaata cgcaaaccgc ctctccccgc 10620
gcgttggccg attcattaat gcagctggca cgacaggttt cccgactgga aagcgggcag 10680
tgagcgcaac gcaattaatg tgagttagct cactcattag gcaccccagg ctttacactt 10740
tatgcttccg gctcgtatgt tgtgtggaat tgtgagcgga taacaatttc acacaggaaa 10800
cagctatgac catgattacg aattcccgat ctagtaacat agatgacacc gcgcgcgata 10860
atttatccta gtttgcgcgc tatattttgt tttctatcgc gtattaaatg tataattgcg 10920
ggactctaat cataaaaacc catctcataa ataacgtcat gcattacatg ttaattatta 10980
catgcttaac gtaattcaac agaaattata tgataatcat cgcaagaccg gcaacaggat 11040
tcaatcttaa gaaactttat tgccaaatgt ttgaacgatc ggggaaattc gagctccttg 11100
catgcctgca ggtcaacatg gtggagcacg acacacttgt ctactccaaa aatatcaaag 11160
atacagtctc agaagaccaa agggcaattg agacttttca acaaagggta atatccggaa 11220
acctcctcgg attccattgc ccagctatct gtcactttat tgtgaagata gtggaaaagg 11280
aaggtggctc ctacaaatgc catcattgcg ataaaggaaa ggccatcgtt gaagatgcct 11340
ctgccgacag tggtcccaaa gatggacccc cacccacgag gagcatcgtg gaaaaagaag 11400
acgttccaac cacgtcttca aagcaagtgg attgatgtga taacatggtg gagcacgaca 11460
cacttgtcta ctccaaaaat atcaaagata cagtctcaga agaccaaagg gcaattgaga 11520
cttttcaaca aagggtaata tccggaaacc tcctcggatt ccattgccca gctatctgtc 11580
actttattgt gaagatagtg gaaaaggaag gtggctccta caaatgccat cattgcgata 11640
aaggaaaggc catcgttgaa gatgcctctg ccgacagtgg tcccaaagat ggacccccac 11700
ccacgaggag catcgtggaa aaagaagacg ttccaaccac gtcttcaaag caagtggatt 11760
gatgtgatat ctccactgac gtaagggatg acgcacaatc ccactatcct tcgcaagacc 11820
cttcctctat ataaggaagt tcatttcatt tggagaggac ctcgagaatt ctcaacacaa 11880
catatacaaa acaaacgaat ctcaagcaat caagcattct acttctattg cagcaattta 11940
aatcatttct tttaaagcaa aagcaatttt ctgaaaattt tcaccattta cgaacgatag 12000
ggtacccccg gggtcgacgg atccatggtg agcaagggcg aggagctgtt caccggggtg 12060
gtgcccatcc tggtcgagct ggacggcgac gtaaacggcc acaagttcag cgtgtccggc 12120
gagggcgagg gcgatgccac ctacggcaag ctgaccctga agttcatctg caccaccggc 12180
aagctgcccg tgccctggcc caccctcgtg accaccctga cctacggcgt gcagtgcttc 12240
agccgctacc ccgaccacat gaagcagcac gacttcttca agtccgccat gcccgaaggc 12300
tacgtccagg agcgcaccat cttcttcaag gacgacggca actacaagac ccgcgccgag 12360
gtgaagttcg agggcgacac cctggtgaac cgcatcgagc tgaagggcat cgacttcaag 12420
gaggacggca acatcctggg gcacaagctg gagtacaact acaacagcca caacgtctat 12480
atcatggccg acaagcagaa gaacggcatc aaggtgaact tcaagatccg ccacaacatc 12540
gaggacggca gcgtgcagct cgccgaccac taccagcaga acacccccat cggcgacggc 12600
cccgtgctgc tgcccgacaa ccactacctg agcacccagt ccgccctgag caaagacccc 12660
aacgagaagc gcgatcacat ggtcctgctg gagttcgtga ccgccgccgg gatcactctc 12720
ggcatggacg agctgtacaa gtgatctaga tgaactagag tccgcaaaaa tcaccagtct 12780
ctctctacaa atctatctct ctctattttt ctccagaata atgtgtgagt agttcccaga 12840
taagggaatt agggttctta tagggtttcg ctcatgtgtt gagcatataa gaaaccctta 12900
gtatgtattt gtatttgtaa aatacttcta tcaataaaat ttctaattcc taaaaccaaa 12960
atccagtgac aagcttggca ctggccgtcg ttttacaacg tcgtgactgg gaaaaccctg 13020
gcgttaccca acttaatcgc cttgcagcac atcccccttt cgccagctgg cgtaatagcg 13080
aagaggcccg caccgatcgc ccttcccaac agttgcgcag cctgaatggc gaatgctaga 13140
gcagcttgag cttggatcag attgtcgttt cccgccttca gtttagcttc atggagtcaa 13200
agattcaaat agaggaccta acagaactcg ccgtaaagac tggcgaacag ttcatacaga 13260
gtctcttacg actcaatgac aagaagaaaa tcttcgtcaa catggtggag cacgacacac 13320
ttgtctactc caaaaatatc aaagatacag tctcagaaga ccaaagggca attgagactt 13380
ttcaacaaag ggtaatatcc ggaaacctcc tcggattcca ttgcccagct atctgtcact 13440
ttattgtgaa gatagtggaa aaggaaggtg gctcctacaa atgccatcat tgcgataaag 13500
gaaaggccat cgttgaagat gcctctgccg acagtggtcc caaagatgga cccccaccca 13560
cgaggagcat cgtggaaaaa gaagacgttc caaccacgtc ttcaaagcaa gtggattgat 13620
gtgatatctc cactgacgta agggatgacg cacaatccca ctatccttcg caagaccctt 13680
cctctatata aggaagttca tttcatttgg agagaacacg ggggactctt gac 13733
<210> 9
<211> 55
<212> DNA
<213> Artificial sequence (ARTIFICIAL SEQUENCE)
<400> 9
cgcggatccg ctgaggtctt aattaatgcc tcagcctcca ggtccgtcgc ttctc 55
<210> 10
<211> 55
<212> DNA
<213> Artificial sequence (ARTIFICIAL SEQUENCE)
<400> 10
tgctctagag ctgagggttt aattaagtcc tcagcacctg cacctatgtt tctgc 55
<210> 11
<211> 29
<212> DNA/RNA
<213> Artificial sequence (ARTIFICIAL SEQUENCE)
<400> 11
ggtcttaaut gacgccattc aatcccttg 29
<210> 12
<211> 29
<212> DNA/RNA
<213> Artificial sequence (ARTIFICIAL SEQUENCE)
<400> 12
ggcattaauc gtcgtcttca atgagcgtc 29
<210> 13
<211> 29
<212> DNA/RNA
<213> Artificial sequence (ARTIFICIAL SEQUENCE)
<400> 13
ggacttaauc gtcgtcttca atgagcgtc 29
<210> 14
<211> 29
<212> DNA/RNA
<213> Artificial sequence (ARTIFICIAL SEQUENCE)
<400> 14
gggtttaaut gacgccattc aatcccttg 29
<210> 15
<211> 20
<212> DNA
<213> Artificial sequence (ARTIFICIAL SEQUENCE)
<400> 15
caagcaatca agcattctac 20
<210> 16
<211> 19
<212> DNA
<213> Artificial sequence (ARTIFICIAL SEQUENCE)
<400> 16
acagagcagg agctactgg 19
<210> 17
<211> 20
<212> DNA
<213> Artificial sequence (ARTIFICIAL SEQUENCE)
<400> 17
ttccatagtc ttgagttttc 20
<210> 18
<211> 20
<212> DNA
<213> Artificial sequence (ARTIFICIAL SEQUENCE)
<400> 18
cggacacgct gaacttgtgg 20
Claims (3)
1. The construction method of the long fragment RNAi vector is characterized by comprising the following steps:
(1) Amplifying an intron sequence in Hurrican by taking Hurrican plasmid as a template, constructing USER enzyme cloning sites at two ends of the intron, and constructing the intron on a 2301.GFP4 vector to obtain a 2301RNAiOS vector; the 2301RNAiOS vector comprises a promoter, an enhancer, an intron, a terminator and USER enzyme cloning sites at two ends of the intron, wherein the nucleotide sequences of the enhancer, the intron, the USER enzyme cloning sites at two ends of the intron and the 2301RNAiOS vector are respectively shown as a Seq ID NO.1, a Seq ID NO.2, a Seq ID NO.3 and a Seq ID NO.4 in a sequence table; both the intron sequence and 2301.GFP4 vector were digested with BamHI and XbaI; the nucleotide sequences of the amplification primers Intron-F and Intron-R of the Intron sequences are respectively shown as Seq ID No.9 and Seq ID No.10 in the sequence table;
(2) The vector 2301RNAiOS was digested and recovered using PacI and Nt.BbvCI enzyme;
(3) Designing amplification primers according to target sequences, respectively adding primer connectors GGTCTTAAU, GGCATTAAU, GGACTTAAU, GGGTTTAAU at the front ends of the primers, amplifying forward insertion fragments and reverse insertion fragments by using high-fidelity enzymes by using target genes as templates, and then cutting and recycling; the primers consist of forward amplification primers JAGN7910_3LRRF and JAGN7911_3LRRR, and reverse amplification primers JAGN7912_3LRRF and JAGN7913_3 LRRR; wherein the primer linker of JAGN7910_3LRRF is GGTCTTAAU, the primer linker of JAGN7911_3LRRR is GGCATTAAU, the primer linker of JAGN7912_3LRRF is GGACTTAAU, and the primer linker of JAGN7913_3LRRR is GGGTTTAAU; the target sequence is a 3LRR sequence, and the target gene is a TRV-3LRR gene; the nucleotide sequences of JAGN7910_3LRRF, JAGN7911_3LRRR, JAGN7912_3LRRF and JAGN7913_3LRRR are respectively shown as Seq ID No.11, seq ID No.12, seq ID No.13 and Seq ID No.14 in the sequence table; the nucleotide sequences of the TRV-3LRR and the 3LRR are respectively shown as Seq ID No.5 and Seq ID No.6 in the sequence table;
(4) Using USER enzyme to connect and transform the fragment recovered in the step (3) and the vector recovered in the step (2) to obtain a long fragment RNAi vector with a hairpin structure of 'target sequence-intron-reverse target sequence'; the RNAi vector obtained also needs to be transformed into escherichia coli;
the nucleotide sequences of Hurrican and 2301 GFP4 are respectively shown as Seq ID No.7 and Seq ID No.8 in the sequence table.
2. The method of claim 1, wherein the ligation transformation conditions in step (4) are 37 ℃ for 20min and 25 ℃ for 20min.
3. A long fragment RNAi vector constructed by the construction method of claim 1 or 2.
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| CN106367430A (en) * | 2015-07-21 | 2017-02-01 | 中国科学院微生物研究所 | Efficient gene knockout vector construction method and constructed engineering bacteria |
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